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https://openalex.org/W4378807236	https://aacr.figshare.com/articles/journal_contribution/Figure_S3_from_Targeting_fatty_acid_reprogramming_suppresses_CARM1-expressing_ovarian_cancer/23269428/1/files/41013192.pdf	English	null	Figure S3 from Targeting fatty acid reprogramming suppresses CARM1-expressing ovarian cancer	null	2,023	cc-by	927	Supplementary Figure 3. CARM1 reprograms lipid metabolism. A, The ratio between palmitic acid (16:0) and palmitoleic acid (16:1) in different lipid species determined by lipid profiling in control and CARM1 knockout A1847 cells. B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidic acid; LPE: lysophosphatidylethanolamine; LPS: Lysophosphastidylserine; LPI: A B Lombardi et al Figure S3 Relative Peak Area PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 1.0 3.0 2.0 P = 0.02 P = 0.0008 P = 0.02 P = 0.02 P = 0.002 P = 0.006 P = 3.27e-05 P = 0.0005 P = 0.004 P = 0.04 control KO Relative 16:1/16:0 ratio PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 2.0 4.0 P = 0.003 P = 0.03 P = 1.84e-06 P = 0.01 P = 0.008 P = 0.002 control KO Supplementary Figure 3. CARM1 reprograms lipid metabolism. A, The ratio between palmitic acid (16:0) and palmitoleic acid (16:1) in different lipid species determined by lipid profiling in control and CARM1 knockout A1847 cells. B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidic acid; LPE: lysophosphatidylethanolamine; LPS: Lysophosphastidylserine; LPI: A B Lombardi et al Figure S3 Relative Peak Area PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 1.0 3.0 2.0 P = 0.02 P = 0.0008 P = 0.02 P = 0.02 P = 0.002 P = 0.006 P = 3.27e-05 P = 0.0005 P = 0.004 P = 0.04 control KO Relative 16:1/16:0 ratio PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 2.0 4.0 P = 0.003 P = 0.03 P = 1.84e-06 P = 0.01 P = 0.008 P = 0.002 control KO Lombardi et al Figure S3 A Lombardi et al Figure S3 Relative 16:1/16:0 ratio PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 2.0 4.0 P = 0.003 P = 0.03 P = 1.84e-06 P = 0.01 P = 0.008 P = 0.002 control KO A B Relative Peak Area PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 1.0 3.0 2.0 P = 0.02 P = 0.0008 P = 0.02 P = 0.02 P = 0.002 P = 0.006 P = 3.27e-05 P = 0.0005 P = 0.004 P = 0.04 control KO B Supplementary Figure 3. CARM1 reprograms lipid metabolism. Supplementary Figure 3. CARM1 reprograms lipid metabolism. A, The ratio between palmitic acid (16:0) and palmitoleic acid (16:1) in different lipid species determined by lipid profiling in control and CARM1 knockout A1847 cells. B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidi Supplementary Figure 3. CARM1 reprograms lipid metabolism. A, The ratio between palmitic acid (16:0) and palmitoleic acid (16:1) in different lipid species determined by lipid profiling in control and CARM1 knockout A1847 cells. P value was calculated using a two-tailed Student t test. Data represent mean ± SE biologically independent experiments. Supplementary Figure 3. CARM1 reprograms lipid metabolism. A, The ratio between palmitic acid (16:0) and palmitoleic acid (16:1) in different lipid species determined by lipid profiling in control and CARM1 knockout A1847 cells. B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidic acid; LPE: lysophosphatidylethanolamine; LPS: Lysophosphastidylserine; LPI: A B Lombardi et al Figure S3 Relative Peak Area PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 1.0 3.0 2.0 P = 0.02 P = 0.0008 P = 0.02 P = 0.02 P = 0.002 P = 0.006 P = 3.27e-05 P = 0.0005 P = 0.004 P = 0.04 control KO Relative 16:1/16:0 ratio PC PE PI PS PG LPC PA LPE LPS LPI LPA DG ChE TG 0.0 2.0 4.0 P = 0.003 P = 0.03 P = 1.84e-06 P = 0.01 P = 0.008 P = 0.002 control KO B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidic acid; LPE: lysophosphatidylethanolamine; LPS: Lysophosphastidylserine; LPI: Lysophosphatidylinositol; LPA: Lysophosphatidic acid; DG: Diglyceride; ChE: cholesterol ester; TG: Triglyceride. P value was calculated using a two-tailed Student t test. Data represent mean ± SEM, n = 3 biologically independent experiments. , p ( ) p ( ) p p determined by lipid profiling in control and CARM1 knockout A1847 cells. B, The levels of different lipid species determined by lipid profiling in control or CARM1 knockout A1847 cells. PC: Phosphatidylcholine; PE: phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholine; PA: Phosphatidic acid; LPE: lysophosphatidylethanolamine; LPS: Lysophosphastidylserine; LPI: Lysophosphatidylinositol; LPA: Lysophosphatidic acid; DG: Diglyceride; ChE: cholesterol ester; TG: Triglyceride. P l l l t d i t t il d St d t t t t D t t SEM 3 g y P value was calculated using a two-tailed Student t test. Data represent mean ± SEM, n = 3 biologically independent experiments.
https://openalex.org/W4385699440	https://rrtjournal.biomedcentral.com/counter/pdf/10.1186/s41100-023-00494-z	English	null	Alactic base excess predicts the use of renal replacement therapy in patients with septic shock	Renal replacement therapy	2,023	cc-by	6,334	Abstract Background Alactic base excess (ABE) is a novel biomarker that estimates the renal capability of handling acid–base alterations during the sepsis. Hence, the aim of this study was to evaluate the use of ABE to predict the renal replace- ment therapy (RRT) in patients with septic shock. Methods A total of 164 patients admitted to the intensive care units with a diagnosis of septic shock according to the third international consensus on sepsis and septic shock (Sepsis-3) were included. This study was retrospec- tive, single center, and conducted between January 1, 2016, and December 31, 2020. The individuals were stratified in patients who did [n = 68] or did not [n = 96] receive the RRT. The diagnostic performed of the variables for the clas- sification into patients who required RRT was evaluated by receiver operating characteristic (ROC) analysis and area under curve (AUC) was calculated. Univariate and multivariate logistic regression models were used to identify risk factors for RRT. Results The median age of the patients was 59 years and female sex (51.8%) predominated. ABE (odds ratio [OR] 1.2270, [95% confidence interval [CI] 1.0453–1.4403], p = 0.0124) and urea (OR 1.0114, [95% CI 1.0053–1.0176], p = 0.0002) were associated with risk of RRT. HCO3− (OR 0.6967, [95% CI 0.5771–0.8410], p = 0.0002) was a protective factor of RRT. ABE (AUC = 0.649, p < 0.0008), HCO3− (AUC = 0.729, p < 0.0001), and urea (AUC = 0.76, p < 0.0001) had a cutoff point of ≤ − 5.7 mmol/L, ≤ 19.36 mmol/L and > 75 mg/dL, respectively. Conclusion Although HCO3− is associated with low risk, ABE and urea are independent risk factors for RRT in the patients with septic shock. Keywords Alactic base excess, Renal replacement therapy, Septic shock, Acute kidney injury Renal Replacement Therapy Renal Replacement Therapy Díaz et al. Renal Replacement Therapy (2023) 9:41 https://doi.org/10.1186/s41100-023-00494-z Open Access Introduction Nearly half of the patients admitted to the intensive care unit (ICU) present acute kidney injury (AKI) [1], with sepsis accounting for the majority of cases at just over 50% [2]. About 10% of the patients will require renal replacement therapy (RRT) [3], although will increase up to 50% when the AKI is related to sepsis [4]. The death risk increases in parallel to the severity of AKI with an OR 2.19 (stage 1), OR 3.88 (stage 2), OR 7.18 (stage 3), and up to 80% of patients with RRT die. Seventy-five Correspondence: Karla Gabriela Peniche Moguel gabrielapenichemd@gmail.com 1 Department of Critical Care Medicine, Centro Médico Nacional “Adolfo Ruiz Cortínes”, Instituto Mexicano del Seguro Social, Unidad Médica de Alta Especialidad Hospital de Especialidades No. 14, Avenida Cuauhtémoc S/N Colonia Formando Hogar, C.P 91897 Veracruz, Mexico 2 Department of Department of Health Research, Centro Médico Nacional, IMSS, Hospital de Especialidades No. 14, Veracruz, Mexico Correspondence: Karla Gabriela Peniche Moguel gabrielapenichemd@gmail.com © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Alactic base excess predicts the use of renal replacement therapy in patients with septic shock Jesús S. Sánchez Díaz1, Karla Gabriela Peniche Moguel1, Fabiola Buelna Gaxiola1, José M. Reyes Ruiz2, Orlando R. Pérez Nieto1 and María V. Calyeca Sánchez1 Open Access T © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Díaz et al. Renal Replacement Therapy (2023) 9:41 Page 2 of 8 percent of patients receive continuous RRT and 25% receive intermittent RRT [1, 5]. Of patients present- ing AKI and receiving RRT, up to 15% will be dialysis dependent, even after discharge from the hospital [6].hl December 31, 2020. The research protocol was approved by the local Ethics and Research Committee R-2022- 3001-112 and the Federal Commission for the Protec- tion from Sanitary Risks (COFEPRIS) 17 CI 30 193 067. The study consisted of reviewing the medical records of patients who met the inclusion criteria. This was a non- intervention study, so the informed consent present in the medical records was that of admission to the ICU. The research was carried out based on the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) methodology for observational studies [11]. The hemodynamic status of the patient could influence the RRT type we use; therefore, continuous renal replace- ment therapy (CRRT) could be the best option in the presence of instability. Therefore, the KDIGO (Kidney Disease Improving Global Outcomes) guidelines recom- mend CRRT for patients with hemodynamic instability (Level of Evidence 2B) [7]. AKI requiring RRT is a life- threatening problem, so time to intervention could influ- ence outcomes. On the other hand, hydroelectrolytic disturbances, acid–base imbalance, azo level, and amount of uresis may be useful in deciding the start of RRT. Participant selection and objective Convenience sampling was performed, which included the records of patients admitted to the ICU with a diag- nosis of septic shock according to the third international consensus on sepsis and septic shock (Sepsis-3) [12]. The inclusion criteria were: age > 18 years and diagnosis of septic shock of any etiology. Patients with a history of previous RRT (peritoneal dialysis, intermittent hemodi- alysis, continuous renal replacement therapy), patients with renal transplantation, restrictions for RRT, do not resuscitate order, maximum therapeutic range (medical practice based on the application of extraordinary and disproportionate methods of life support in terminally ill or irrecoverable patients) and patients with pregnancy or puerperium were excluded. Patients with incomplete var- iables in the record were eliminated. RRT was provided at the discretion of the intensivist: TIMING OF INITIA- TION: the clinical judgment guides CRRT initiation, CRRT MODALITY: continuous venovenous hemodia- filtration (CVVHDF) modality was used in all patients, CRRT DOSE: effluent flow rate of 25 ml/kg/h, BLOOD FLOW RATE: typical blood flow rates (150–250 ml/ min) do not affect hemodynamics, PATIENT FLUID REMOVAL: avoid net ultrafiltration rates > 2 ml/kg/h, ANTICOAGULATION: we used systemic anticoagula- tion with unfractionated heparin, CRRT SOLUTIONS: bicarbonate buffered solutions are available, WHEN TO STOP: the decision to discontinue CRRT is based on clinical judgment [13]. The PRSIMAFLEX® system was used. The main objective was to evaluate the asso- ciation between alactic base excess and the use of RRT in patients with septic shock. g p g Recently, Gattinoni et al. [10] introduced the concept of “alactic base excess,” which is related to renal function. This is understood using Stewart’s model for acid–base balance disturbances. As lactate increases, the appar- ent strong ion difference (SIDa) will decrease, resulting in metabolic acidemia (pH decrease). Renal compensa- tory mechanisms are responsible for correcting acidemia; if the above does not occur, impaired renal function is reflected with creatinine > 2 mg/dl and negative alactic base excess, indicating an accumulation of fixed or non- volatile acids (unmeasured anions) other than lactate as the main cause of metabolic acidemia. An alactic base excess of zero or close to zero with creatinine ~ 2 mg/dl suggests that the kidney still can remove fixed or nonvol- atile acids, but cannot fully “compensate” for metabolic acidemia, with lactate as its main cause. Participant selection and objective On the other hand, positive alactic base excess (usually with creati- nine < 2 mg/dl) suggests that either renal mechanisms fully compensate for metabolic acidemia or that other mechanisms contribute to metabolic alkalemia (diuretics, decreased intravascular volume). Thus, negative alactic base excess in septic shock patients helps the physician identify that renal function is impaired. Likewise, nega- tive alactic base excess may be useful in determining the use of RRT in this patient population. Methods The variables obtained were classified as general: sex, age; comorbidities: diabetes mellitus (DM), systemic arterial hypertension (SAH), heart disease, ICU stay, mechanical ventilation (MV), etiology of shock: pulmonary, abdomi- nal, urinary, skin and soft tissue, other; norepinephrine, inotropic, Simplified Acute Physiology Score II (SAPS II) and mortality. Gasometric variables and serum renal Open Access T Thus, the initiation of RRT is based on the time of AKI evolu- tion and the hydrometabolic needs of the patient [8]. The timing of RRT initiation is controversial; in fact, there is no univocal protocol among countries, hospitals, or even among intensivists and nephrologists [9]. Study design A cohort, retrospective, observational, cross-sectional study conducted in the ICU of the Centro Médico Nacional “Adolfo Ruiz Cortines,” Hospital de Especiali- dades No.14 of the Instituto Mexicano del Seguro Social (IMSS) Veracruz, México, from January 1, 2016, to Díaz et al. Renal Replacement Therapy (2023) 9:41 Page 3 of 8 Díaz et al. Renal Replacement Therapy (2023) 9:41 biomarkers were: hydrogen potential (pH), partial pres- sure arterial oxygen/fraction of inspired oxygen (PaO2/ FiO2), arterial pressure of carbon dioxide (PaCO2), bicar- bonate (HCO3−), standard base excess (SBE), alactic base excess (ABE), lactate (Lac), anion gap (AG), sodium- chloride difference (Na–Cl), serum creatinine (SCr) and serum urea (urea). The variables were obtained from the ICU patient admission record. The formula used to cal- culate the SBE was: [14] received inotropic, the median SAPS II was 71 points and mortality 50.6% (Table 1). Table 2 shows the gaso- metric variables and serum renal biomarkers. In the RRT group the median ABE was − 6.0 mmol/L, SCr 3.0 mg/ dL, urea 116.4 mg/dL compared to the patients who did not receive the RRT where ABE was − 3.0 mmol/L, SCr 1.55 mg/dL, urea 61.5 mg/dL with statistically significant difference (p < 0.05). Except for pH and PaO2/FiO2, the other variables showed significant differences. Patients who received the RRT had the highest lactate and AG, as well as, lower HCO3−, SBE, and Na–Cl differences. SBE (mmol/L) = (HCO3−−24.8) + 16.2 ∗(pH −7.4 3f To determine the variables associated with RRT an univariate binary logistic regression analysis was per- formed (Table 3). The variables with highest odds ratio (OR, 95% CI) values were lactate (OR 1.3152, 95% CI 1.1302–1.5305, p = 0.0004), AG (OR 1.0715, 95% CI 1.0074–1.1396, p = 0.0283), SCr (OR 1.1980, 95% CI 1.0597–1.3543, p = 0.0039), and urea (OR 1.0137, 95% CI 1.0076–1.0199, p < 0.0001). Subsequently, variables with statistical significance (p < 0.05) in the univariate analysis were included in the multivariate regression model with stepwise to determine the predictors of RRT (Table 3). ABE (OR 1.2270, 95% CI 1.0453–1.4403, p = 0.0124) and urea (OR 1.0114, 95% CI 1.0053–1.0176, p = 0.0002) were independent risk factors for RRT in the patients. In contrast, HCO3− (OR 0.6967, 95% CI 0.5771–0.8410, p = 0.0002) was a protective factor (Table 3). The formula used to calculate the standard ABE was: [10] ABE (mmol/L) = SBE + lactate Statistical analysis No statistical power calculation was performed before the study because the sample size is indirectly con- ditioned to the administrative information (clinical records) available which is finite. The distribution of quantitative variables was determined using the Shapiro– Wilk test. Normally distributed variables are expressed as mean (± standard deviation, SD) and non-normally distributed variables are presented as the median (inter- quartile range, IQR). Student’s t test or Mann–Whitney U test was used for comparison between patients groups. Qualitative variables were presented as number and per- centage (%) and their association was evaluated with the X2 test or Fisher’s exact test. Receiver operating charac- teristics (ROC) curves were created to evaluate the sensi- tivity and specificity according to the variables associated with RRT [15]. Both univariate and multivariate analy- ses were carried out evaluate the associations of poten- tial risk factors with the risk for RRT. The Odds Ratio (OR) was estimated with 95% confidence intervals (CI). A p-value < 0.05 was defined as a statistical significance. Data were analyzed using the R Studio software (version 1.0.153) and IBM SPSS v.25. To assess the diagnostic value of the hemato- logic parameters ROC curves were generated (Fig. 2a–c). Figure 2a, d shows an ABE with a cut-off point ≤ − 5.7 mmol/L, AUC 0.649 (95% CI 0.57–0.721, p < 0.0008), sensitivity 55.88%, and specificity 71.87%. Figure 2b, d shows HCO3− with a cut-off point of ≤ 19.36 mmol/L, AUC = 0.729 (95% CI 0.654–0.795, p < 0.0001), sensitivity 79.41%, and specificity 56.25%. Urea had an AUC = 0.76 (95% CI 0.688–0.824, p < 0.0001), cutoff point > 75 mg/dL with sensitivity 80.88% and speci- ficity 67.71% (Fig. 2b, d). The comparative analysis of the baseline characteristics according to the cutoff point of ABE reveled that the gender, diabetes mellitus, etiology, pH, PaO2/FiO2, SBE, HCO3−, lactate, AG, Na–Cl, SCr, and urea were statistically significant (Table 4). Results During this period, 180 patients were recruited, of whom 164 were included in the study cohort (Fig. 1). The individuals included in the study were stratified in patients who did [n = 68] or did not [n = 96] receive the renal replacement therapy. The median age was 59 years, female sex (51.8%) predominated, SAH (50%) was the most frequent comorbidity, while pulmonary etiology (41.5%) was the main cause of septic shock, the median ICU stay was 5 days and 4 days of MV, the median norepinephrine was 0.32 mcg/kg/min, 12.8% Discussion Maintaining acid–base balance depends on 3 systems: (1) intra- and extracellular buffers, (2) respiratory compen- sation (carbon dioxide elimination) and (3) renal com- pensation (elimination of non-volatile acids and renal bicarbonate synthesis). These systems are interrelated, starting to work within minutes, where renal compensa- tion is the most effective, but takes the longest time (days) to be completed. Therefore, the deterioration of renal Díaz et al. Renal Replacement Therapy (2023) 9:41 Page 4 of 8 Fig. 1 Flowchart of patients included in the cohort Fig. 1 Flowchart of patients included in the cohort a median of ABE − 6.0 mmol/L (IQR − 9.95 to − 2.55), while in the individuals who did not receive RRT, the median ABE was − 3.0 mmol/L (IQR − 5.05 to − 0.60), where SCr and urea levels were higher, the median lactate was ≥ 4 mmol/L with a significant difference. Smuszkiewicz et al. [18] in 143 patients in shock state demonstrated a relationship between negative ABE and renal function deterioration, as well as increased mor- tality with ABE cutoff point < − 3.63 mmol/L (HR 3.19, 95% CI 1.62–6.27). In our work, mortality was higher in the patients who received RRT (58.8%) compared to the individuals who did not receive RRT (44.8%), but with- out significant difference. This is a recurrent trend in other studies, due to the delay in starting RRT or simply because they were more severe patients. Medina et al. [19] reported, in 414 patients, a higher 90-day mortal- ity (58.5%) in patients who received RRT. The results are comparable to those reported by Prasad et al. [20] (64%), Kao et al. [21] (66.5%), and Gonzalez et al. [22] (68.4%). function leads to the accumulation or lack of elimination of non-volatile acids and, consequently, metabolic aci- demia [16]. Critically ill patients with metabolic acidemia have a mortality rate of 45%, while those without meta- bolic acidemia have a mortality rate of 25%. The origin of metabolic acidemia is important, as lactic acidosis is the most common and the one with the highest mortality; in fact, most of the time, severe metabolic acidemia is an indication to initiate RRT [17]. In this sense, alactic base excess allows rapid differentiation of metabolic acidemia secondary to lactate accumulation from that caused by increased fixed acids, which cannot be eliminated by the lungs. Finally, the resulting acidemia indicates impaired renal function [10]. Gattinoni et al. Discussion An elevated urea level is almost universal during acute kidney injury [23], although in critically ill patients it can be due to multiple causes [24]. Elevated urea level could be an indication to start RRT although, there is not spe- cific cutoff point [8]. Evidence suggests that starting RRT with a high urea level may increase mortality [25, 26]. Not only is the urea level at the start of RRT important, also a reduction of at least 25% during RRT, a value asso- ciated with mortality [27]. Our study demonstrated that ABE (≤ − 5.7 mmol/L) and urea (> 75 mg/dL) are inde- pendent risk factors for RRT. Studies on the timing of RRT have included severe metabolic acidosis as one of the absolute indications; in fact, the variables considered were pH < 7.15 and HCO3− < 18 mEq/L [28, 29]. The main aim of STARRT- AKI trial was to assess whether an accelerated strategy to start RRT would improve outcomes compared with a delayed initiation with absolute indications. The abso- lute indications for RRT included severe metabolic aci- demia defined as pH ≤ 7.2 or HCO3− < 12 mmol/L [30]. In our cohort, we found that a HCO3− with a cutoff point of ≤ 19.36 mmol/L was a protective factor. The “optimal time” to initiate RRT is controversial and the “best vari- able” will remain controversial, but it is certain that med- ical judgment [31] should be taken into account when making this decision. Discussion [10] documented that metabolic aci- demia in septic patients is concomitant with impaired renal function, where negative ABE is related to increased creatinine, decreased diuresis, and the use of RRT. In this study, the patients who received RRT had Díaz et al. Renal Replacement Therapy (2023) 9:41 Page 5 of 8 Table 1 Baseline characteristics of the patients included in the study IQR, interquartile range; DM, diabetes mellitus; SAH, systemic arterial hypertension; mcg/kg/min, microgram/kilogram/minute; MV, mechanical ventilation; ICU, intensive care unit; SAPS II, Simplified Acute Physiology Score II Variable Renal replacement therapy Total n = 164 Yes n = 68 No n = 96 Age, years old (IQR) 59 (46–69) 59 (48–68) 60 (45–74) Gender Female n (%) 85 (51.8) 39 (57.4) 46 (47.9) Male n (%) 79 (48.2) 29 (42.6) 50 (52.1) Comorbidities n (%) DM, Yes 73 (44.5) 34 (50.0) 39 (40.6) SAH, Yes 82 (50.0) 40 (58.8) 42 (43.8) Heart disease, Yes 23 (14.0) 8 (11.8) 15 (15.6) Etiology, n (%) Lung, Yes 68 (41.5) 20 (29.4) 48 (50.0) Abdominal, Yes 64 (39.0) 34 (50.0) 30 (31.3) Urinary, Yes 24 (14.6) 12 (17.6) 12 (12.5) Soft tissues, Yes 3 (1.8) 1 (1.5) 2 (2.1) Other 5 (3.0) 1 (1.5) 4 (4.2) Norepinefrine mcg/kg/min (IQR) 0.32 (0.21–0.48) 0.33 (0.22–0.52) 0.30 (0.21–0.45) Inotropic, Yes n (%) 21 (12.8) 8 (11.8) 13 (13.5) MV days (IQR) 4 (3–8) 5 (3–8) 4 (2–8) ICU stay days (IQR) 5 (3–9) 5 (3–9) 6 (3–8) SAPS II points (IQR) 71 (64–80) 72 (65–80) 70 (63–80) Mortality, YES n (%) 83 (50.6) 40 (58.8) 43 (44.8) Table 1 Baseline characteristics of the patients included in the study An elevated urea level is almost universal during acute kidney injury [23], although in critically ill patients it can be due to multiple causes [24]. Elevated urea level could be an indication to start RRT although, there is not spe- cific cutoff point [8]. Evidence suggests that starting RRT with a high urea level may increase mortality [25, 26]. Not only is the urea level at the start of RRT important, also a reduction of at least 25% during RRT, a value asso- ciated with mortality [27]. Our study demonstrated that ABE (≤ − 5.7 mmol/L) and urea (> 75 mg/dL) are inde- pendent risk factors for RRT. pH, hydrogenion potential; PaO2/FiO2, partial pressure arterial oxygen and fraction of inspired oxygen; mmHg, millimeters of mercury; IQR, interquartile range; SD, standard deviation; PaCO2, arterial carbon dioxide pressure; SBE, standard base excess; HCO3−, bicarbonate; ABE, alactic base excess; AG, anion gap; Na–Cl, sodium- chloride difference; SCr, serum creatinine Discussion Some limitations of our study were the sample size (n = 164) and the fact that it was carried out in a single center; we did not have the variable of urine output; the Table 2 Gasometric variables and serum renal biomarkers Table 2 Gasometric variables and serum renal biomarkers pH, hydrogenion potential; PaO2/FiO2, partial pressure arterial oxygen and fraction of inspired oxygen; mmHg, millimeters of mercury; IQR, interquartile range; SD, standard deviation; PaCO2, arterial carbon dioxide pressure; SBE, standard base excess; HCO3−, bicarbonate; ABE, alactic base excess; AG, anion gap; Na–Cl, sodium- chloride difference; SCr, serum creatinine Significant difference between groups (p<0 05) Variable Renal replacement therapy Total n = 164 Yes n = 68 No n = 96 pH 7.3 (7.2–7.4) 7.3 (7.2–7.3) 7.3 (7.2–7.4) PaO2/FiO2, mmHg (IQR) 96.5 (75.0–137.5) 100 (80–147.5) 93.5 (71–125) PaCO2, mmHg (IQR) 37.2 (35.6–38.7) 33.5 (32–36) 37 (36–41)* SBE, mmol/L (SD) − 8.38 (± 6.44) − 10.79 (± 6.03) * − 6.67 (± 6.19) * HCO3−, mmol/L (SD) 18.07 (± 5.02) 15.83 (± 4.07) * 19.66 (± 5.04) * Lactate mmol/L (IQR) 3.2 (1.95–4.5) 4.0 (2.8–5.9) * 2.6 (1.7–3.8) * ABE, mmol/L (IQR) − 4.2 (− 8.25 to − 1.35) − 6.0 (− 9.95 to − 2.55)* − 3.0 (− 5.05 to − 0.60)* AG, mEq/L (IQR) 14.5 (11.7–17.5) 15.3 (12.4–18.65)* 13.85 (10.25–16.75)* Na–Cl, mEq/L (IQR) 32 (30–35) 31 (28.5–34.5)* 33 (31–36)* SCr, mg/dL (IQR) 2.05 (1.15–3.30) 3.0 (2.10–5.35)* 1.55 (0.90–2.65)* Urea, mg/dL (IQR) 80 (50–126) 116.4 (80–165)* 61.5 (39–94)* Díaz et al. Discussion Renal Replacement Therapy (2023) 9:41 Page 6 of 8 Table 3 Univariate and multivariate analysis to evaluate the importance of the variables in the patients who received the renal replacement therapy pH, hydrogenion potential; PaO2/FiO2, partial pressure arterial oxygen and fraction of inspired oxygen; mmHg, millimeters of mercury; IQR, interquartile range; SD, standard deviation; PaCO2, arterial carbon dioxide pressure; SBE, standard base excess; HCO3−, bicarbonate; ABE, alactic base excess; AG, anion gap; Na–Cl, sodium- chloride difference; SCr, serum creatinine Significant difference between groups (p<0 05) Variable Univariate analysis Multivariate analysis Odds ratio 95% CI p value Odds ratio 95% CI p value pH 0.0407 0.0021–0.8027 0.035 13.212 0.002–106211.039 0.574 PaO2/FiO2 1.0017 0.9961–1.0074 0.547 – – – PaCO2 0.952 0.9196–0.9868 0.007* 1.0300 0.9440–1.1238 0.505 SBE 0.8946 0.8452–0.9468 0.0001* 0.9207 0.7367–1.1507 0.468 HCO3− 0.8338 0.7703–0.9026 < 0.0001* 0.6967 0.5771–0.8410 0.0002* Lactate 1.3152 1.1302–1.5305 0.0004* 1.4338 0.985–2.482 1 ABE 0.9158 0.8601–0.9751 0.0060* 1.2270 1.0453–1.4403 0.0124* AG 1.0715 1.0074–1.1396 0.0283* 0.940 0.866–1.020 0.138 Na–Cl 0.9228 0.8576–0.9930 0.0316* 0.6956 0.2416–1.5829 1 SCr 1.1980 1.0597–1.3543 0.0039* 1.033 0.892–1.198 0.662 Urea 1.0137 1.0076–1.0199 < 0.0001* 1.0114 1.0053–1.0176 0.0002* Table 3 Univariate and multivariate analysis to evaluate the importance of the variables in the patients who received the renal replacement therapy pH, hydrogenion potential; PaO2/FiO2, partial pressure arterial oxygen and fraction of inspired oxygen; mmHg, millimeters of mercury; IQR, interquartile range; SD, standard deviation; PaCO2, arterial carbon dioxide pressure; SBE, standard base excess; HCO3−, bicarbonate; ABE, alactic base excess; AG, anion gap; Na–Cl, sodium- chloride difference; SCr, serum creatinineif Significant difference between groups (p < 0.05) Fig. 2 ROC curves of predicting variables for renal replacement therapy decision to initiate RRT is not protocolized in our ICU and depends on the intensivist in charge of the patient. The results could differ in settings where the decision corresponds to the nephrologist. The strengths are that it is a homogeneous population. Finally, the gasometric resource is possible in most hospitals. Conclusion Our study demonstrated that ABE and urea are inde- pendent risk factors for RRT in the patients with septic shock. Additionally, HCO3− is associated with low risk of RRT. These results may be useful for predicting and monitoring patients with septic shock at high risk of developing RRT. Fig. 2 ROC curves of predicting variables for renal replacement therapy Fig. 2 ROC curves of predicting variables for renal replacement therapy Conclusion decision to initiate RRT is not protocolized in our ICU and depends on the intensivist in charge of the patient. The results could differ in settings where the decision corresponds to the nephrologist. The strengths are that it is a homogeneous population. Finally, the gasometric resource is possible in most hospitals. decision to initiate RRT is not protocolized in our ICU and depends on the intensivist in charge of the patient. Our study demonstrated that ABE and urea are inde- pendent risk factors for RRT in the patients with septic shock. Additionally, HCO3− is associated with low risk of RRT. These results may be useful for predicting and monitoring patients with septic shock at high risk of developing RRT. Our study demonstrated that ABE and urea are inde- pendent risk factors for RRT in the patients with septic shock. Additionally, HCO3− is associated with low risk of RRT. These results may be useful for predicting and monitoring patients with septic shock at high risk of developing RRT. Díaz et al. Ethics approval and consent to participate The research protocol was approved by The Local Ethics and Research Com- mittee R-2022-3001-112 and The Federal Commission for the Protection from Sanitary Risks (COFEPRIS) 17 CI 30 193 067. Funding All data generated or analyzed during this study are included in this published article. Consent for publication This was a non-intervention study, so the informed consent present in the medical records was that of admission to the ICU. Human Subjects Research: According to the WMA Declaration of Helsinki—Ethical Principles for Medical Research involving human subjects in paragraph number 32: Medical research using human identifiable data. The informed consent document is available from the family member responsible for the patient at the time of admission to the intensive care unit, which includes protection of personal data. Competing interests The authors declare that they have no competing interests. Received: 24 February 2023 Accepted: 28 July 2023 Received: 24 February 2023 Accepted: 28 July 2023 Significative difference between groups (p < 0.05) Abbreviations ABE Alactic base excess RRT Renal replacement therapy ICU Intensive care unit COFEPRIS (acronym in Spanish) Federal Commission for the Protection from Sanitary Risks IMSS (acronym in Spanish) Instituto Mexicano del Seguro Social AKI Acute kidney injury OR Odds ratio CRRT Continuous renal replacement therapy Declarations Ethics approval and consent to participate Conclusion Renal Replacement Therapy (2023) 9:41 Page 7 of 8 Table 4 Baseline characteristics, gasometric variables and serum renal biomarkers according to the ABE cutoff point IQR, interquartile range; DM, diabetes mellitus; SAH, systemic arterial hypertension; mcg/kg/min, microgram/kilogram/minute; MV, mechanical i Variable ABE ≤ − 5.7 n = 65 ABE > − 5.7 n = 99 p value Renal replacement therapy, Yes n (%) 38 (58.5) 30 (30.3) < 0.0005* Age, years old (IQR) 62 (17.5) 57 (26) 0.351 Gender 0.019* Female, n (%) 41 (63.1) 44 (44.4) Male, n (%) 24 (36.9) 55 (55.6) Comorbidities n (%) DM, Yes 37 (56.9) 36 (36.4) 0.010* SAH, Yes 34 (52.3) 48 (48.5) 0.632 Heart disease, Yes 12 (18.5) 11 (11.1) 0.185 Etiology, n (%) 0.001* Lung, Yes 15 (23.1) 53 (53.5) Abdominal, Yes 30 (46.2) 34 (34.3) Urinary, Yes 14 (21.5) 10 (10.1) Soft tissues, Yes 2 (3.1) 1 (1) Other 4 (6.2) 1 (1) Norepinefrine mcg/kg/min (IQR) 0.32 (0.32) 0.28 (0.29) 0.077 Inotropic, Yes n (%) 6 (9.2) 15 (15.2) 0.267 MV, days (IQR) 4 (5) 4 (5) 0.170 ICU STAY, days (IQR) 5 (5) 6 (6) 0.062 SAPS II, points (IQR) 70 (13.5) 71 (18) 0.926 Mortality, Yes n (%) 34 (52.3) 49 (49.5) 0.725 pH 7.25 (0.11) 7.34 (0.11) < 0.001* PaO2/FiO2, mmHg (IQR) 105 (65) 90 (58) 0.003* PaCO2, mmHg (IQR) 33 (11) 38 (11) < 0.001* SBE, mmol/L (SD) − 13.6 (5.45) − 4.5 (4.9) < 0.001* HCO3−, mmol/L (SD) 13.6 (4.35) 20.4 (5.2) < 0.001* Lactate, mmol/L (IQR) 3.8 (2.4) 2.8 (2.2) 0.004* AG, mEq/L (IQR) 17 (6.5) 13 (5.7) < 0.001* Na–Cl, mEq/L (IQR) 31 (4.5) 33 (6) < 0.001* SCr, mg/dL (IQR) 2.9 (2.75) 1.8 (1.8) < 0.001* Urea, mg/dL (IQR) 100 (63.75) 64 (70.3) < 0.001* Funding This work did not receive funding. Availability of data and materials All data generated or analyzed during this study are included in this published article. References 1. Hoste EA, Bagshaw SM, Bellomo R, Cely CM, Colman R, Cruz DN, et al. Epi- demiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study. Intensive Care Med. 2015;41(8):1411–23. https://doi.org/10. 1007/s00134-015-3934-7. 2. Kellum JA, Prowle JR. Paradigms of acute kidney injury in the intensive care setting. Nat Rev Nephrol. 2018;14(4):217–30. https://doi.org/10.1038/ nrneph.2017.184. 2. Kellum JA, Prowle JR. Paradigms of acute kidney injury in the intensive care setting. Nat Rev Nephrol. 2018;14(4):217–30. https://doi.org/10.1038/ nrneph.2017.184. Díaz et al. Renal Replacement Therapy (2023) 9:41 Page 8 of 8 Díaz et al. Renal Replacement Therapy (2023) 9:41 3. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA. 2005;294(7):813–8. https://doi.org/10.1001/jama.294.7.813. 3. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA. 2005;294(7):813–8. https://doi.org/10.1001/jama.294.7.813. 22. Gonzalez CA, Pinto JL, Orozco V, Contreras K, García P, Rodríguez P, et al. Early mortality risk factors at the beginning of continuous renal replace- ment therapy for acute kidney injury. Cogent Med. 2018;5:1407485. https://doi.org/10.1080/2331205X.2017.1407485. y 4. Peters E, Antonelli M, Wittebole X, Nanchal R, François B, Sakr Y, et al. A worldwide multicentre evaluation of the influence of deterioration or improvement of acute kidney injury on clinical outcome in critically ill patients with and without sepsis at ICU admission: results from The Inten- sive Care Over Nations audit. Crit Care. 2018;22(1):188. https://doi.org/10. 1186/s13054-018-2112-z. 4. Peters E, Antonelli M, Wittebole X, Nanchal R, François B, Sakr Y, et al. A worldwide multicentre evaluation of the influence of deterioration or improvement of acute kidney injury on clinical outcome in critically ill patients with and without sepsis at ICU admission: results from The Inten- sive Care Over Nations audit. Crit Care. 2018;22(1):188. https://doi.org/10. 1186/s13054-018-2112-z. 23. Mackenzie J, Chacko B. An isolated elevation in blood urea level is not ‘uraemia’ and not an indication for renal replacement therapy in the ICU. Crit Care. 2017;21:275. https://doi.org/10.1186/s13054-017-1868-x. 24. Verdier V, Soulage CO, Koppe L. New clinical evidence for urea toxicity. Nephrol Dial Transplant. 2021;37(1):1–4. https://doi.org/10.1093/ndt/ gfab269. 5. Tolwani A. Continuous renal-replacement therapy for acute kidney injury. N Engl J Med. 2012;367(26):2505–14. https://doi.org/10.1056/NEJMc t1206045. 5. Tolwani A. Continuous renal-replacement therapy for acute kidney injury. N Engl J Med. 2012;367(26):2505–14. https://doi.org/10.1056/NEJMc t1206045. 25. References Gaudry S, Hajage D, Martin-Lefevre L, Lebbah S, Louis G, Moschietto S, et al. Comparison of two delayed strategies for renal replacement therapy initiation for severe acute kidney injury (AKIKI 2): a multicentre, open- label, randomised, controlled trial. Lancet. 2021;397(10281):1293–300. https://doi.org/10.1016/S0140-6736(21)00350-0. 6. Lo LJ, Go AS, Chertow GM, McCulloch CE, Fan D, Ordoñez JD, Hsu CY. Dial- ysis-requiring acute renal failure increases the risk of progressive chronic kidney disease. Kidney Int. 2009;76(8):893–9. https://doi.org/10.1038/ki. 2009.289. 6. Lo LJ, Go AS, Chertow GM, McCulloch CE, Fan D, Ordoñez JD, Hsu CY. Dial- ysis-requiring acute renal failure increases the risk of progressive chronic kidney disease. Kidney Int. 2009;76(8):893–9. https://doi.org/10.1038/ki. 2009.289. 26. Zarbock A, Kellum JA, Schmidt C, Van Aken H, Wempe C, Pavenstädt H, et al. Effect of early versus delayed initiation of renal replacement therapy on mortality in critically Ill patients with acute kidney injury: the ELAIN randomized clinical trial. JAMA. 2016;315(20):2190–9. https://doi.org/10. 1001/jama.2016.5828. 7. Bestle MH, Itenov TS, Berthelsen RE. Renal replacement therapy in critical care: when to start? Curr Anesthesiol Rep. 2019;9:135–43. https://doi.org/ 10.1007/s40140-019-00325-0. 7. Bestle MH, Itenov TS, Berthelsen RE. Renal replacement therapy in critical care: when to start? Curr Anesthesiol Rep. 2019;9:135–43. https://doi.org/ 10.1007/s40140-019-00325-0. 8. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. https://doi.org/10.1159/00033 9789. 8. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. https://doi.org/10.1159/00033 9789. 27. Chávez-Íñiguez JS, Maggiani-Aguilera P, González-Barajas D, Rizo-Topete L, Galindo P, Rifkin B, Chávez-Alonso G, et al. Urea reduction in acute kidney injury and mortality risk. Kidney Blood Press Res. 2023. https://doi. org/10.1159/000530237. 9. Valdenebro M, Martín Rodríguez L, Tarragón B, Sánchez Briales P, Portolés J. Renal replacement therapy in critically ill patients with acute kidney injury: 2020 nephrologist’s perspective. Nefrologia (Engl Ed). 2021;41(2):102–14. https://doi.org/10.1016/j.nefro.2020.07.016. 28. Gaudry S, Hajage D, Schortgen F, Martin-Lefevre L, Pons B, Boulet E, et al. Initiation strategies for renal-replacement therapy in the intensive care unit. N Engl J Med. 2016;375(2):122–33. https://doi.org/10.1056/NEJMo a1603017. 10. Gattinoni L, Vasques F, Camporota L, Meessen J, Romitti F, Pasticci I, et al. Understanding lactatemia in human sepsis. Potential impact for early management. Am J Respir Crit Care Med. 2019;200(5):582–9. https://doi. org/10.1164/rccm.201812-2342OC. 29. Barbar SD, Clere-Jehl R, Bourredjem A, Hernu R, Montini F, Bruyère R, et al. Timing of renal-replacement therapy in patients with acute kidney injury and sepsis. N Engl J Med. 2018;379(15):1431–42. https://doi.org/10.1056/ NEJMoa1803213. 11. Publisher’s Note 15. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology. 1982;143(1):29–36. https://doi.org/10.1148/radiology.143.1.7063747. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 16. Alcázar Arroyo R, Albalate Ramón M, de Sequera Ortíz P. Trastornos del metabolismo ácido-base. En: Lorenzo V., López Gómez JM (Eds). Nefrol- ogía al día. ISSN: 2659-2606. Disponible en: https://www.nefrologiaaldia. org/403. g 17. Kavelak HL. Electrolyte, acid-base, and medication management with renal replacement therapy. Nutr Clin Pract. 2022;37(5):1015–22. https:// doi.org/10.1002/ncp.10880. 18. Smuszkiewicz P, Jawień N, Szrama J, Lubarska M, Kusza K, Guzik P. Admission lactate concentration, base excess, and alactic base excess predict the 28-day inward mortality in shock patients. J Clin Med. 2022;11(20):6125. https://doi.org/10.3390/jcm11206125. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 19. Medina-Liabres KRP, Jeong JC, Oh HJ, An JN, Lee JP, Kim DK, et al. References von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP; STROBE Initiative. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008;61(4):344–9. https://doi.org/ 10.1016/j.jclinepi.2007.11.008. 30. STARRT-AKI Investigators, Canadian Critical Care Trials Group, Australian and New Zealand Intensive Care Society Clinical Trials Group, United Kingdom Critical Care Research Group, Canadian Nephrology Trials Network, Irish Critical Care Trials Group, et al. Timing of initiation of renal- replacement therapy in acute kidney injury. N Engl J Med. 2020;383:240– 51. https://doi.org/10.1056/NEJMoa2000741. 12. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016;315(8):801–10. https://doi. org/10.1001/jama.2016.0287. 31. Sitbon A, Darmon M, Geri G, Jaubert P, Lamouche-Wilquin P, Monet C, et al. Accuracy of clinicians’ ability to predict the need for renal replace- ment therapy: a prospective multicenter study. Ann Intensive Care. 2022;12(1):95. https://doi.org/10.1186/s13613-022-01066-w. 13. See EJ, Bellomo R. How I prescribe continuous renal replacement therapy. Crit Care. 2021;25(1):1. https://doi.org/10.1186/s13054-020-03448-7. 14. Langer T, Brusatori S, Gattinoni L. Understanding base excess (BE): merits and pitfalls. Intensive Care Med. 2022;48:1080–3. https://doi.org/10.1007/ s00134-022-06748-4. Publisher’s Note Mortality predictors in critically ill patients with acute kidney injury requiring continuous renal replacement therapy. Kidney Res Clin Pract. 2021;40(3):401–10. https://doi.org/10.23876/j.krcp.20.205. 20. Prasad B, Urbanski M, Ferguson TW, Karreman E, Tangri N. Early mortal- ity on continuous renal replacement therapy (CRRT): the prairie CRRT study. Can J Kidney Health Dis. 2016;22(3):36. https://doi.org/10.1186/ s40697-016-0124-7. 21. Kao CC, Yang JY, Chen L, Chao CT, Peng YS, Chiang CK, et al. Factors asso- ciated with poor outcomes of continuous renal replacement therapy. PLoS ONE. 2017;12(5):e0177759. https://doi.org/10.1371/journal.pone. 0177759.
https://openalex.org/W2751727372	https://ruj.uj.edu.pl/xmlui/bitstream/handle/item/44732/stachyra_rak_redkiewicz_madeja_gawarecka_chojnacki_swiezewska_masnyk_chmielewski_sirko_gora-sochacka_effective_usage_of_cationic_derivatives_2017.pdf?sequence=1&isAllowed=y	English	null	Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus	Virology journal	2,017	cc-by	6,863	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus Anna Stachyra1†, Monika Rak2†, Patrycja Redkiewicz1, Zbigniew Madeja2, Katarzyna Gawarecka1, Tadeusz Chojnacki1, Ewa Świeżewska1, Marek Masnyk3, Marek Chmielewski3, Agnieszka Sirko1 and Anna Góra-Sochacka1* Stachyra et al. Virology Journal (2017) 14:168 DOI 10.1186/s12985-017-0838-x Stachyra et al. Virology Journal (2017) 14:168 DOI 10.1186/s12985-017-0838-x Abstract Background: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides – PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. Methods: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. Results: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn- glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. Conclusion: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent. Keywords: Adjuvant, Vaccine delivery, Lipofection, Immunization, Humoral response Keywords: Adjuvant, Vaccine delivery, Lipofection, Immunization, Humoral response induce both humoral and cellular responses highly spe- cific to the chosen antigen, of which the structure and posttranslational modification are like those in natural infection. The mechanism by which a DNA vaccine works has been presented in several recently published reviews, for example [1–3]. Briefly, a DNA vaccine which consists of an animal expression vector carrying a se- quence encoding a selected antigen and DNA carrier is injected into the tissue, resulting in somatic and/or antigen-presenting cell (APC) transfection. Then, vac- cine DNA translocates to the cell nucleus where the transgene is transcribed by host enzymes followed by translation in the cytoplasm. After processing, the anti- genic peptides bind to MHC class I and class II and are presented by APC to activate naïve T cells. Alternatively, Open Access Open Access Preparation of PTAI compositions p p Four types of PTAI preparation with different length polyprenoid chains were used in this work: (i) PTAI-11 containing 11 isoprenoid subunits, (ii) PTAI-15 contain- ing 15 isoprenoid subunits, (iii) PTAI-6-8 and (iv) PTAI- 10-14, containing a mixture of polyprenols with chain lengths from 6 to 8 subunits and from 10 to 14 subunits, respectively (Fig. 1). The PTAI were obtained as previ- ously described [10, 11], (patent PL 2012, B1 211824, Pa- tent Office of the Republic of Poland). PTAI and helper lipids (DOPE, DC-cholesterol and DOPC were dissolved in 99.8% ethanol). They were mixed in appropriate ratios in order to achieve the following molar ratios in the mixture: The following five compositions containing PTAI and helper lipids in the indicated molar ratios were prepared: PTAI-6,7,8 + DOPE – 1,5:1; PTAI- 11 + DOPE + DC-cholesterol – 1:1:1; PTAI-11/DOPE/ DC-cholesterol/DOPC - 1:1:1:1, PTAI-15/DOPE/DC- cholesterol/DOPC - 1:1:1:1 and PTAI-10-14/DOPE/ DC – 1:1:1. Then the obtained mixtures were mixed with endotoxin tested DMEM F-12 HAM cell culture medium without additions of serum and antibiotics and intensely vortexed for 3 min. Ethanol content in the A broad prospect for applications of cationic lipids as DNA vaccine carriers and adjuvants is the basis for ex- tensive studies aimed at elaboration of effective and safe lipid vehicles [5, 6]. Numerous studies have confirmed the positive effect of lipid adjuvants [7, 8]; however, so far the ultimate lipid component of vaccines remains to be discovered. This might be the result of a broad diver- sity of vaccines and their administration protocols, di- vergent diseases and responsive mechanisms of the organisms and, finally, numerous side effects caused by vaccination. An additional level of complexity in the search for optimal vaccine carriers and adjuvants is added by the diversity of immunization mechanisms. All these issues result in a complex relationship between the com- ponents of the vaccine and their safety and efficacy. Cationic derivatives of polyprenols, named either PTAI (trimethylpolyprenylammonium iodides) or AP (amino- prenols), have been already recognized as lipofectants and their usage in in vitro cell transfection described [9–11]. Fig. 1 Structure of trimethylpolyprenylammonium iodides (PTAI). Background In the past two decades considerable development of DNA vaccine technology has been observed. This tech- nology has emerged as a promising alternative to tra- ditional vaccines and can be applied for therapy of human and animal infections, cancers, allergies or auto- immune disorders. The main advantages of DNA vac- cines are: (i) rapid and relatively inexpensive mass production and (ii) simplicity of development, modifi- cation, formulation and preparation. DNA vaccines can * Correspondence: annag@ibb.waw.pl †Equal contributors 1Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland Full list of author information is available at the end of the article Stachyra et al. Virology Journal (2017) 14:168 Page 2 of 9 vaccine was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. Comparison of the results obtained with vaccine mix- tures containing several compositions of PTAI is pre- sented and discussed. secreted antigenic proteins can be processed to activate B cells for antibody production. In comparison to live at- tenuated or inactivated vaccines the immunogenicity of DNA vaccines, especially in humans and large animals, is usually lower. Improvement of both the expression level of an antigen and the efficiency of transfection are essential for elevation of DNA immunization efficacy. Thus various modifications of expression cassettes (e.g. codon optimization, introducing of various regulatory elements, other changes in the antigen coding sequence), biological and biochemical adjuvants, different carriers and vaccination routes and other strategies were widely investigated [4]. Vaccine plasmid p The K3 plasmid is a recombinant expression vector pCI (Promega) carrying under control of the cytomegalo- virus promoter the cDNA encoding the full length hemagglutinin (without cleavage site between subunits HA1 and HA2) from the highly pathogenic H5N1 influ- enza virus strain A/swan/Poland/305-135 V08/2006 (clade 2.2.) [12]. After propagation in Escherichia coli (DH5α strain), plasmid DNA was isolated using the NucleoBond® PC 10000 EF endotoxin-free giga purifica- tion kit (Macherey-Nagel) which ensures endotoxins level < 0.1EU/μg DNA. Purified DNA was dissolved in PBS buffer, pH 7.4. One of the commonly used methods of delivery of DNA vaccines is the application of lipofectants, i.e. cat- ionic lipids as DNA vehicles, which are able to sponta- neously interact with negatively charged nucleic acids forming lipoplexes (lipid–nucleic acid complexes). Non- specific interactions with the surface of cellular mem- branes stimulate the uptake of lipoplexes into the cell via endocytosis, while the presence of helper lipids (e.g. neutral lipids, such as DOPE [1,2-dioleoyl-sn-glycero-3- phosphatidylethanolamine], DC [{3ß-[N-(N′,N′-dimethy laminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC [1,2-dioleoyl-sn-glycero-3-phosphatidylcholine]) modifies the properties of lipoplexes and facilitates DNA release from endosomes. Chicken immunization The groups of chickens, broilers in the first experiment and laying hens in the second, were immunized with two doses of the DNA vaccines containing the K3 plas- mid and different PTAI compositions (Table 1). In the first experiment four different PTAI compositions: (i) PTAI-6-8/DOPE, (ii) PTAI-11/DOPE/DC, (iii) PTAI-11/ DOPE/DC/DOPC and (iv) PTAI-15/DOPE/DC/DOPC mixed with 125 μg of K3 were tested along with the positive (K3 mixed with Lipofectin® Transfection Re- agent) and negative (pCI mixed with one of the carriers) controls. The PTAI:DNA ratios (w/w) in vaccine com- positions K3-PTAI 6-8/DOPE, K3-PTAI-11/DOPE/DC, K3-PTAI-11/DOPE/DC/DOPC and K3-PTAI-15/DOPE/ DC/DOPC were 0.9:1, 0.8:1, 1.3:1 and 1.6:1, respect- ively, while calculated charge ratios PTAI+:DNA−or (PTAI + DC)+:DNA−were 1:2.3, 1:1.7, 1:1.1 and 1:1.1, respectively. Efficacy of the applied vaccines was assayed by ELISA and HI tests which allowed estimation of the level of induced specific anti-HA antibodies in the col- lected sera. Independently of the carrier, most chickens immunized with K3 developed a specific antibody against HA, while those vaccinated with the empty vec- tor (pCI, group 6) clearly showed no anti-H5 response (Fig. 2a). All groups immunized with K3 showed an evi- dent increase of humoral response after booster immunization. However, only in the groups vaccinated with K3-PTAI-6-8/DOPE (group 1) and K3-PTAI-11/ DOPE/DC (group 2) did 100% of chickens produce spe- cific anti-H5 antibodies. In the K3-PTAI-11/DOPE/DC/ DOPC (group 3) and K3-PTAI-15/DOPE/DC/DOPC (group 4) one out of ten and three out of seven immu- nized chickens, respectively, gave negative results. The strongest responses to vaccination were observed in the K3-PTAI-11/DOPE/DC group. This group had not only the best individual chicken responses in ELISA (Fig. 2a) One vaccine dose for chickens contained either 125 μg or 62.5 μg of plasmid DNA in a final volume of 160 μl or 100 μl, respectively, while one vaccine dose for mice con- tained 10 μg of plasmid DNA in a final volume of 50 μl. Two types of chicken: broilers (Ross 308) and laying hens (Rosa 1) were immunized intramuscularly in the breast muscle twice on the 7th and the 21st day of life. Blood samples were taken from the wing vein 14, 21 and 28 days after initial immunization. Mice (BALB/c) were immunized intramuscularly twice on the 35th and 49th day of life. Blood was collected at days 14, 21 and 28 days after initial immunization. Chicken immunization The group sizes (n = 5-10 for the tested groups and n = 2-5 for the control groups) are indicated in the respective figures. Details of the PTAI compositions and vaccine doses used in the three immunization experiments are indi- cated in Table 1. Preparation of vaccine mixtures and animal vaccination Preparation of vaccine mixtures and animal vaccination The vaccine mixtures were prepared by combination of the K3 plasmid with the prepared PTAI compositions in different PTAI:DNA ratios (w/w), depending on the DNA dose used in the experiment which is indicated separately. Before injections PTAI compositions were mixed with plasmid DNA and incubated for 30 min at room temperature. The mixtures of K3 plasmid DNA (amounts corresponding to the K3 amounts used with PTAI) and Lipofectin® Transfection Reagent (Invitro- gen™) served as pertinent controls. These mixtures were prepared by mixing DNA with this carrier at a ratio (w:v) of 6:1, as described previously [12]. Briefly, Lipo- fectin was initially diluted in PBS, mixed with DNA and incubated for at least 10 min at room temperature, ac- cording to manufacturer protocol. A non-parametric Mann–Whitney U test, which is a component of Statistica 12 software (StatSoft, Poland), was used to evaluate the statistical differences between groups. A value of p < 0.05 was considered significant. Preparation of PTAI compositions PTAI with diverse length of polyprenoid chains were investigated, respectively n = 6-8 (mixture), n = 11, n = 15 and n = 10-14 (mixture) In this work we investigated if PTAI with diverse length polyprenyl chains (6–15 units) can be used as ef- fective carriers of DNA vaccines. In immunization ex- periments we used two animal models, chickens and mice. Animals were immunized with the DNA vaccine encoding hemagglutinin (HA) from influenza virus H5N1 which was developed and successfully used by us previously [12–14]. The immunogenicity of the DNA Fig. 1 Structure of trimethylpolyprenylammonium iodides (PTAI). PTAI with diverse length of polyprenoid chains were investigated, respectively n = 6-8 (mixture), n = 11, n = 15 and n = 10-14 (mixture) Page 3 of 9 Page 3 of 9 Stachyra et al. Virology Journal (2017) 14:168 mixtures was: PTAI-11/DOPE/DC cholesterol/DOPC – 21,3%, PTAI-15/DOPE/DC cholesterol/DOPC – 15,6%, PTAI-6-8/DOPE – 11,4%, PTAI-11/DOPE/DC-cholesterol – 20,7%, PTAI-10-14/DOPE/DC-cholesterol – 20,7%. The compositions were stored at 4 °C up to 5 days. the highest serum dilution at which a result higher than the estimated value: background + 3SD (three times the value of the standard deviation) was obtained. HI test were performed according to the OIE standard proced- ure using the commercially available antigen from low pathogenic H5N2 strain, A/chicken/Belgium/150/1999 (DG Deventer, Netherlands) which share 96% protein se- quence similarity with vaccine antigen. Serially diluted sera were incubated with antigen for 25 min, and then incubated with chicken erythrocytes for 30 min. HI titer was defined as the reciprocal of the highest dilution of sera that completely inhibited hemagglutination. ELISA assay and HI test aratio for PTAI:DNA (w:w) or for Lipofectin:DNA (v:w) in vaccine doses is indicated; bin control group empty pCI vector was used with PTAI-6-8 (n = 3) or Lipofectin (n = 2); n.a not applicable of the same DNA dose with Lipofectin®, the reference carrier (Fig. 3a). This observation is partially supported by statistical analysis, the results of the HI test (Fig. 3b) and the results of endpoint titer evaluation in individual sera collected on day 28 post immunization (Fig. 4), where the highest titers were observed in the K3-62.5/ PTAI(0.8) group (geometric mean 1.3 × 105). Moreover, the K3-62.5/PTAI groups, regardless of the PTAI:DNA ratio, had no low responders (chickens with low end- point titer of anti-HA antibodies in the serum), in con- trast to the K3-62.5/L group immunized with the reference carrier. but also was significantly different from all other groups in the HI test, had the highest scores for individual chickens, and the highest median and geometric mean of HI in comparison to the other groups (Fig. 2b). g g Based on one-dilution ELISA and HI tests the PTAI- 11/DOPE/DC composition was selected for further trials in the second experiment. Vaccine doses were prepared by mixing PTAI-11/DOPE/DC with 125 μg or 62.5 μg of K3 plasmid DNA (Table 1). Three vaccine compositions were used: (i) K3-125/PTAI (0.8), consisting of 125 μg of DNA per vaccine dose and a PTAI-11:DNA (w/w) ratio of 0.8:1, (ii) K3-62.5/PTAI (1.6), consisting of 62.5 μg of DNA and a PTAI:DNA (w/w) ratio of 1.6:1 and (iii) K3-62.5/PTAI (0.8), consisting of 62.5 μg of DNA and a PTAI-11:DNA (w/w) ratio of 0.8:1. Charge ratios calcu- lated as (PTAI + DC)+:DNA−for these three vaccine compositions were 1:1.7, 1:0.8, and 1:1.7, respectively. Simultaneously, vaccine compositions containing Lipo- fectin® instead of PTAI-11/DOPE/DC (K3-125/L and K3-62.5/L, containing 125 μg and 62.5 μg DNA, respect- ively) and vaccine samples without any carrier (K3-125 and K3-62.5) were prepared. As expected, the anti-H5 levels in the groups immunized without any carrier were lower and much more variable than in the groups im- munized with the carriers. Therefore no end-point titer ELISA was performed in the groups immunized without carrier. Interestingly, the PTAI compositions containing the lower dose of plasmid DNA (62.5 μg) seemed to be slightly superior to the vaccine containing the mixture ELISA assay and HI test The ELISA assay detecting anti-H5 IgY in chicken sera and anti-H5 IgG in mouse sera as well as HI test were performed as described previously [12, 13]. Purified re- combinant H5 HA (A/swan/Poland/305-135 V08/2006) derived from a baculovirus system (Oxford Expression Technologies, UK) was used for plates coating in ELISA assay. Anti-H5 IgY were detected with goat anti-chicken IgY (Fc-specific)-HRP (Thermo Scientific) while anti-H5 IgG were detected with goat anti-mouse IgG-AP (Sigma- Aldrich). For one dilution ELISA sera were diluted 1:200 (chickens) or 1:100 (mice). For end-point ELISA sera were serially diluted and the end-point titer of the anti- H5 antibodies was determined as the inverse value of Stachyra et al. Virology Journal (2017) 14:168 Page 4 of 9 Table 1 Details of the tested vaccine mixtures Animal model Group PTAI compositions or Lipofectin Molar ratio PTAI: helper lipids DNA dose Ratioa Chicken-broilers 1 PTAI-6-8 with DOPE 1.5:1 125 μg 0.9:1 2 PTAI-11 with DOPE/DC 1:1:1 125 μg 0.8:1 3 PTAI-11 with DOPE/DC/DOPC 1:1:1:1 125 μg 1.3:1 4 PTAI-15 with DOPE/DC/DOPC 1:1:1:1 125 μg 1.6:1 5 Lipofectin n.a 125 μg 1:6 6 PTAI-6-8 with DOPE or Lipofectinb 1.5:1 125 μg 0.9:1 or 1:6 Chicken-layers 1 PTAI-11 with DOPE/DC 1:1:1 125 μg 0.8:1 2 PTAI-11 with DOPE/DC 1:1:1 62.5 μg 1.6:1 3 PTAI-11 with DOPE/DC 1:1:1 62.5 μg 0.8:1 4 Lipofectin n.a 125 μg 1:6 5 Lipofectin n.a 62.5 μg 1:6 6 None n.a 125 μg n.a 7 None n.a 62.5 μg n.a 8 Lipofectin n.a 125 μg 1:6 Mice 1 Lipofectin n.a 10 μg 1:6 2 PTAI-10-14 with DOPE/DC 1:1:1 10 μg 0.8:1 3 PTAI-10-14 with DOPE/DC 1:1:1 10 μg 0.8:1 Vaccine mixtures were prepared by mixing the K3 plasmid with PTAI compositions or Lipofectin. Groups 6 and 7 (chicken-layers) were immunized with vaccine containing the K3 plasmid in PBS, pH 7.4 without any carriers. aratio for PTAI:DNA (w:w) or for Lipofectin:DNA (v:w) in vaccine doses is indicated; bin control group empty pCI vector was used with PTAI-6-8 (n = 3) or Lipofectin (n = 2); n.a not applicable Vaccine mixtures were prepared by mixing the K3 plasmid with PTAI compositions or Lipofectin. Groups 6 and 7 (chicken-layers) were immunized with vaccine containing the K3 plasmid in PBS, pH 7.4 without any carriers. Mouse immunization The efficacy of PTAI as a DNA vaccine carrier was also tested in mice (Table 1). Animals were intramuscularly vaccinated with two doses of DNA vaccine. One group had a vaccine consisting of a mixture of K3 (10 μg) and PTAI composition (PTAI-10-14/DOPE/DC) with a PTAI:DNA ratio (w/w) of 0.8:1 and calculated charge ratio [(PTAI + DC)+:DNA−] 1:1.7, while the second group had a vaccine consisting of a mixture of K3 (10 μg) and Lipofectin®. In the negative control K3 was replaced with empty pCI vector. The ELISA results using collected sera demonstrated that the tested PTAI composition is an effective DNA carrier (Fig. 5). How- ever, in this experiment no significant differences be- tween the tested PTAI composition and the control lipid carrier were observed. For example, in both groups the Stachyra et al. Virology Journal (2017) 14:168 Page 5 of 9 Fig. 2 Humoral response of broilers to vaccination. Birds were immunized with a mixture of K3 DNA (125 μg) with the PTAI composition as indicated (PTAI-6-8/DOPE, PTAI-11/DOPE/DC, PTAI-11/DOPE/DC/DOPC, PTAI-15/DOPE/DC/DOPC) or with Lipofectin (L); pCI – group vaccinated with empty pCI vector with PTAI or Lipofectin. a ELISA results for individual chickens; raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; blood collected at indicated day post initial immunization, sera diluted 200-fold. () group showed significantly higher ELISA values than K3-PTAI-11/DOPE/DC/DOPC. () group showed significantly higher ELISA values than K3-PTAI-11/DOPE/DC/DOPC and K3-PTAI-15/ DOPE/DC/DOPC (p < 0.05). b HI titers in sera of individual chickens on day 28 post immunization. Geometric means for each group are indicated at the top of the graph. (*) The K3-PTAI-11/DOPE/DC group showed a significantly higher HI titer than all other groups (p < 0.05) Fig. 2 Humoral response of broilers to vaccination. Birds were immunized with a mixture of K3 DNA (125 μg) with the PTAI composition as indicated (PTAI-6-8/DOPE, PTAI-11/DOPE/DC, PTAI-11/DOPE/DC/DOPC, PTAI-15/DOPE/DC/DOPC) or with Lipofectin (L); pCI – group vaccinated with empty pCI vector with PTAI or Lipofectin. a ELISA results for individual chickens; raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; blood collected at indicated day post initial immunization, sera diluted 200-fold. () group showed significantly higher ELISA values than K3-PTAI-11/DOPE/DC/DOPC. () group showed significantly higher ELISA values than K3-PTAI-11/DOPE/DC/DOPC and K3-PTAI-15/ DOPE/DC/DOPC (p < 0.05). Mouse immunization b HI titers in sera of individual chickens on day 28 post immunization. Geometric means for each group are indicated at the top of the graph. () The K3-PTAI-11/DOPE/DC group showed a significantly higher HI titer than all other groups (p < 0.05) highest levels of anti-HA specific antibodies were ob- served 1 week after the booster and in both groups low responders were present. adjuvants is also favored. For example, the cationic lipid formulation Vaxfectin® (Vical Inc.) has been shown to effectively enhance immune response after DNA vaccin- ation, not due to increased gene transfer efficiency, but rather by direct modulation of immune pathways [15, 16]. This adjuvant which is a mixture of a cationic lipid (GAP- DMORIE) and a neutral phospholipid (DPyPE) was also tested with protein and inactivated vaccines with promi- sing results. Discussion The promising potential of DNA vaccines stimulates the search for efficient, safe and cost-effective carriers of nu- cleic acids suitable for in vivo application. Such carriers should fulfill several additional criteria. First of all, they should enhance or facilitate the transfection process dur- ing vaccination that may translate to higher antigen ex- pression and immunological response. Secondly, they also should form DNA complexes that encapsulate and protect the DNA vaccines. Their ability to act as Recently, PTAI were demonstrated by us to allow effective transfection of plasmid DNA complexes into cells [10, 11]. The aim of this study was evaluation of PTAI as an efficient carrier of DNA vaccines. Several ex- perimental DNA vaccines consisting of K3 plasmid and Page 6 of 9 Stachyra et al. Virology Journal (2017) 14:168 Fig. 3 Comparison of humoral response of layers after immunization with different K3 vaccine mixtures. Vaccine doses composed of K3 plasmid alone (125 μg or 62.5 μg) or K3 plasmid with carrier PTAI-11/DOPE/DC (denoted as PTAI) or Lipofectin (L) in different w/w ratios indicated in brackets; pCI-125 – group vaccinated with empty pCI vector with Lipofectin. a ELISA results for individual chickens; raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; blood collected at indicated day post initial immunization, sera diluted 200-fold. () group showed significantly higher ELISA values than K3-62.5. (*) group showed significantly higher ELISA values than K3-62.5 and K3-62.5/L (p < 0.05). b HI titers in sera of individual chickens on day 28 post immunization. Geometric means for each group are indicated at the top of the graph. () The K3-62.5/PTAI (0.8) group showed a significantly higher HI titer than K3-62.5 (p < 0.05) Fig. 3 Comparison of humoral response of layers after immunization with different K3 vaccine mixtures. Vaccine doses composed of K3 plasmid alone (125 μg or 62.5 μg) or K3 plasmid with carrier PTAI-11/DOPE/DC (denoted as PTAI) or Lipofectin (L) in different w/w ratios indicated in brackets; pCI-125 – group vaccinated with empty pCI vector with Lipofectin. a ELISA results for individual chickens; raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; blood collected at indicated day post initial immunization, sera diluted 200-fold. () group showed significantly higher ELISA values than K3-62.5. () group showed significantly higher ELISA values than K3-62.5 and K3-62.5/L (p < 0.05). Discussion b HI titers in sera of individual chickens on day 28 post immunization. Geometric means for each group are indicated at the top of the graph. () The K3-62.5/PTAI (0.8) group showed a significantly higher HI titer than K3-62.5 (p < 0.05) PTAI compositions with different PTAI:DNA ratios were studied in two animal models, chickens (broiler and layer) and mice. As a reference vaccine we used a mixture of the vaccine plasmid with the transfection reagent Lipofectin®, which is a 1:1 (w/v) liposome for- mulation of the cationic lipid DOTMA (N-[1-(2,3-dio- leyloxy) propyl]-n,n,n-trimethylammonium chloride) and DOPE. Previously, such a vaccine composition has been shown by us to be highly immunogenic and a dose of 125 μg of DNA protected chickens against challenge with homologous and heterologous H5N1 virus [12]. In the first animal experiment we used mixtures con- taining a high concentration of DNA (125 μg/dose) and variants of PTAI with various length polyprenyl chains mixed with various combinations of helper lipids: DOPE, DOPE/DC or DOPE/DC/DOPC. All compositions gave shown by us to be highly immunogenic and a dose of 125 μg of DNA protected chickens against challenge with homologous and heterologous H5N1 virus [12]. In the first animal experiment we used mixtures con- taining a high concentration of DNA (125 μg/dose) and variants of PTAI with various length polyprenyl chains mixed with various combinations of helper lipids: DOPE, DOPE/DC or DOPE/DC/DOPC. All compositions gave Stachyra et al. Virology Journal (2017) 14:168 Page 7 of 9 Fig. 4 End-point titers of anti-H5 HA antibody in sera collected on the 28th day post immunization. The titer of anti-H5 antibodies was determined as the inverse value of the highest serum dilution at which a result higher than the estimated value: background + 3SD (three times the value of the standard deviation) was obtained. If there was no need for dilutions higher than the standard 200-fold dilution (readout < 1 for the 200-fold dilution), the levels were determined by multiplying the OD value read out in the ELISA test by 200, namely by the inverse value of the serum dilution used in the ELISA test. Geometric means for each group are indicated at the top of the graph. () The K3-62.5/PTAI (0.8) group showed a significantly higher anti-HA titer than K3-62.5/L (p < 0.05) Fig. 4 End-point titers of anti-H5 HA antibody in sera collected on the 28th day post immunization. Discussion () The K3-62.5/PTAI (0.8) group showed a significantly higher anti-HA titer than K3-62.5/L (p < 0.05) positive results in ELISA and HI tests; however, a high variability of humoral response was apparent in the K3- 125/PTAI-11/DOPE/DC/DOPC and K3-125/PTAI-15/ DOPE/DC/DOPC groups, resulting in significantly lower antibody levels than other formulations. Interestingly, in both cases we observed a slow process of precipitation in the vaccine mixture after blending the vaccine plas- mid with the PTAI composition, which might be respon- sible for the observed variability of the individual immune responses. The highest HI titers (as well as the highest median and geometric mean of the HI titer) were in the K3-125/PTAI-11/DOPE/DC group (Fig. 2a and b). Therefore, this PTAI composition was further examined using the lower dose of the plasmid (62.5 μg) in two DNA:PTAI ratios and compared with control carrier and no carrier. The PTAI composition consisting of a PTAI:DNA (w/w) ratio of 0.8:1 seemed to be more effective in stimulating specific anti-HA antibodies than the controls. Presumably, it was more effective than the control lipid in provoking endocytosis and further DNA release, which might lead to higher production of the positive results in ELISA and HI tests; however, a high variability of humoral response was apparent in the K3- 125/PTAI-11/DOPE/DC/DOPC and K3-125/PTAI-15/ DOPE/DC/DOPC groups, resulting in significantly lower antibody levels than other formulations. Interestingly, in both cases we observed a slow process of precipitation in the vaccine mixture after blending the vaccine plas- mid with the PTAI composition, which might be respon- sible for the observed variability of the individual immune responses. The highest HI titers (as well as the highest median and geometric mean of the HI titer) were in the K3-125/PTAI-11/DOPE/DC group (Fig. 2a and b). Therefore, this PTAI composition was further examined using the lower dose of the plasmid (62.5 μg) in two DNA:PTAI ratios and compared with control carrier and no carrier. The PTAI composition consisting of a PTAI:DNA (w/w) ratio of 0.8:1 seemed to be more effective in stimulating specific anti-HA antibodies than the controls. Presumably, it was more effective than the control lipid in provoking endocytosis and further DNA release, which might lead to higher production of the target antigen. It is also worth mentioning that we observed some differences between the two chicken experiments, revealing some inconsistencies in the gen- eral effectiveness of immunizations in both the PTAI and Lipofectin groups. Discussion The titer of anti-H5 antibodies was determined as the inverse value of the highest serum dilution at which a result higher than the estimated value: background + 3SD (three times the value of the standard deviation) was obtained. If there was no need for dilutions higher than the standard 200-fold dilution (readout < 1 for the 200-fold dilution), the levels were determined by multiplying the OD value read out in the ELISA test by 200, namely by the inverse value of the serum dilution used in the ELISA test. Geometric means for each group are indicated at the top of the graph. () The K3-62.5/PTAI (0.8) group showed a significantly higher anti-HA titer than K3-62.5/L (p < 0.05) Fig. 4 End-point titers of anti-H5 HA antibody in sera collected on the 28th day post immunization. The titer of anti-H5 antibodies was determined as the inverse value of the highest serum dilution at which a result higher than the estimated value: background + 3SD (three times the value of the standard deviation) was obtained. If there was no need for dilutions higher than the standard 200-fold dilution (readout < 1 for the 200-fold dilution), the levels were determined by multiplying the OD value read out in the ELISA test by 200, namely by the inverse value of the serum dilution used in the ELISA test. Geometric means for each group are indicated at the top of the graph. () The K3-62.5/PTAI (0.8) group showed a significantly higher anti-HA titer than K3-62.5/L (p < 0.05) Fig. 4 End-point titers of anti-H5 HA antibody in sera collected on the 28th day post immunization. The titer of anti-H5 antibodies was determined as the inverse value of the highest serum dilution at which a result higher than the estimated value: background + 3SD (three times the value of the standard deviation) was obtained. If there was no need for dilutions higher than the standard 200-fold dilution (readout < 1 for the 200-fold dilution), the levels were determined by multiplying the OD value read out in the ELISA test by 200, namely by the inverse value of the serum dilution used in the ELISA test. Geometric means for each group are indicated at the top of the graph. Funding 5. Korsholm KS, Andersen PL, Christensen D. Cationic liposomal vaccine adjuvants in animal challenge models: overview and current clinical status. Expert Rev Vaccines. 2012;11:561–77. g This work was funded by Innovative Economy Program, Grant No. WND-POIG.01. 01.02-00-007/08 from the National Centre for Research and Development and by Polish National Cohesion Strategy Innovative Economy Grant UDA- POIG 01.03.01-14-036/09 „Application of polyisoprenoid derivatives as drug carriers and metabolism regulators” co-financed by the European Union within the European Regional Development Fund. Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. 6. Christensen D, Korsholm KS, Rosenkrands I, Lindenstrøm T, Andersen P, Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines. 2007;6:785–96. 7. Carroll TD, Matzinger SR, Barry PA, McChesney MB, Fairman J, Miller CJ. Efficacy of influenza vaccination of elderly rhesus macaques is dramatically improved by addition of a cationic lipid/DNA adjuvant. J Infect Dis. 2014;209:24–33. 8. Firouzmand H, Badiee A, Khamesipour A, Heravi Shargh V, Alavizadeh SH, Abbasi A, Jaafari MR. Induction of protection against leishmaniasis in susceptible BALB/c mice using simple DOTAP cationic nanoliposomes containing soluble Leishmania antigen (SLA). Acta Trop. 2013;128:528–35. Competing interests The authors declare that they have no competing interests. Part of presented results is a subject of patent PL 2012, B1 211824, Patent Office of the Republic of Poland and patent applications: PL 2014 WO/2016/032348, PCT/PL2015/ 000093, P-410063 Patent Office of the Republic of Poland. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations h l 1. Iurescia S, Fioretti D, Rinaldi M. Strategies for improving DNA vaccine performance. Methods Mol Biol. 2014;1143:21–31. 1. Iurescia S, Fioretti D, Rinaldi M. Strategies for improving DNA vaccine performance. Methods Mol Biol. 2014;1143:21–31. 1. Iurescia S, Fioretti D, Rinaldi M. Strategies for improving DNA vaccine performance. Methods Mol Biol. 2014;1143:21–31. DC: DC-cholesterol; HA: Hemagglutinin; PTAI: Cationic derivatives of polyprenols 2. Coban C, Kobiyama K, Jounai N, Tozuka M, Ishii KJ. DNA vaccines: a simple DNA sensing matter? Hum Vaccin Immunother. 2013;9:2216–21. Discussion A possible explanation may be the different chicken lines used (with different genotype and phenotype) and relatively small number of indivi- duals in groups. Additionally, we decided to use a similar vaccine com- position K3/PTAI-10-14/DOPE/DC in mice experi- ments. From a potential commercial perspective, the administration of a mixture of polyprenols with different length chains instead of a single polyprenol (with a de- fined length polyprenol chain) is economically attractive. A mixture is easier to obtain due to the lack of a chro- matographic separation step after plant material isola- tion. Thus, the PTAI-10-14 mix could be a simpler and cheaper replacement for the highly effective PTAI-11 lipid. Previously, for the same reasons, the PTAI-6-8 mixture was chosen to be tested in the first chicken ex- periment. The experiment with mice confirmed that the Fig. 5 Humoral response of mice after immunization. Vaccine was composed of K3 and one of carrier PTAI-10-14-DOPE/DC-cholesterol or Lipofectin. Raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; Blood was collected at the indicated day post initial immunization, sera diluted 100-fold; pCI – group vaccinated with empty pCI vector with PTAI Fig. 5 Humoral response of mice after immunization. Vaccine was composed of K3 and one of carrier PTAI-10-14-DOPE/DC-cholesterol or Lipofectin. Raw data (◊), median (—) and the 10th and 90th percentiles (□) are shown for each group; Blood was collected at the indicated day post initial immunization, sera diluted 100-fold; pCI – group vaccinated with empty pCI vector with PTAI Page 8 of 9 Stachyra et al. Virology Journal (2017) 14:168 Page 8 of 9 Page 8 of 9 Page 8 of 9 PTAI-10-14 mixture could be used as an efficient DNA carrier for DNA vaccination purposes. Authors’ contributions ASt performed the immunization experiments, analyzed the data and contributed to manuscript preparation. MR performed PTAI formula design and preparation, data analysis and participated in preparing the final version of the manuscript. PR performed the immunization experiments and analyzed the data. ZM supervised PTAI design and preparation, analyzed the data. KG involved in PTAI synthesis. TCh designed the synthetic part of the experiments. EŚ supervised the synthetic part of the experiments. MM and MCh contributed unique chemicals. ASi supervised the study, participated in data analysis, discussion of the results and revised the manuscript. AG-S - designed experiments and wrote the paper. All authors have approved the final manuscript. Efficient delivery of plasmid DNA into host cells is a challenge of great importance for the development of DNA vaccines. Early DNA studies in large animal models and the first human trials resulted in little induc- tion of immune response in comparison to mouse models. A possible explanation may be differences in the DNA uptake of target cells [17]. Intramuscular injection, which is the most widely used way of DNA immunization is very susceptible to inter-individual variation related to differences in cell transfection level at the injection site, as most of the intramuscularly-injected bare DNA does not actually transfect cells and is phagocytosed [18]. This lowers general vaccine efficacy in the population and dis- rupts the results of clinical trials. Improvements in ef- ficient and less accidental DNA transfer which results in high and more equal expression in cells make DNA vac- cines more effective. Besides mechanical devices and phys- ical stimulus, various chemical or biomaterial carriers (like the cationic lipids used in this work) are widely investi- gated and represent the most promising strategy for DNA vaccination [19]. Publisher’s Note In this work, cationic derivatives of polyprenols (PTAI) were tested in vivo as carriers of DNA vaccines. Several variants of formulations containing a mixture of differ- ent PTAI with helper lipids were able to induce high im- mune responses against the target antigen in chickens and mice. Such carriers, which can be obtained from common plant material, are a promising approach for DNA vaccine optimization, especially given that there is a great need for systems that make use of natural mate- rials and processing conditions. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgments We dedicate this work to the memory of Professor Włodzimierz Zagórski- Ostoja, who was actively involved in its initial stages. 3. Li L, Saade F, Petrovsky N. The future of human DNA vaccines. J Biotechnol. 2012;162:171–82. 4. Stachyra A, Góra-Sochacka A, Sirko A. DNA vaccines against influenza. Acta Biochim Pol. 2014;61:515–22. Ethics approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Experiments involving chickens immunization were approved by the Second Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, Permit Number 17/ 2009. Experiments of mouse immunization were approved by the Fourth Local Ethical Committee for Animal Experiments at the National Medicines Institutes, Permit Number 03/2014. All efforts were made to minimize suffering. Author details 1 1Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland. 2Department of Cell Biology, Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Gronostajowa 7, 30-387 Kraków, Poland. 3Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland. 1Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland. 2Department of Cell Biology, Jagiellonian University, Faculty of Biochemistry, Biophysics and 3 Received: 19 May 2017 Accepted: 28 August 2017 Received: 19 May 2017 Accepted: 28 August 2017 Availability of data and materials All data generated or analyzed during this study are included in this published article. All data generated or analyzed during this study are included in this published article. Page 9 of 9 Stachyra et al. Virology Journal (2017) 14:168 9. Madeja Z, Rak M, Wybieralska E, Rózański I, Masnyk M, Chmielewski M, Lysek R, Chojnacki T, Jankowski W, Ciepichal E, et al. New cationic polyprenyl derivative proposed as a lipofecting agent. Acta Biochim Pol. 2007;54:873–6. 10. Rak M, Ochałek A, Bielecka E, Latasiewicz J, Gawarecka K, Sroka J, Czyż J, Piwowarczyk K, Masnyk M, Chmielewski M, et al. Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology. J Gene Med. 2016;18:331–42. 11. Grecka E, Statkiewicz M, Gorska A, Biernacka M, Grygorowicz MA, Masnyk M, Chmielewski M, Gawarecka K, Chojnacki T, Swiezewska E, Malecki M. Prenyl ammonium salts–new carriers for gene delivery: a B16-F10 mouse melanoma model. PLoS One. 2016;11:e0153633. 12. Stachyra A, Gora-Sochacka A, Sawicka R, Florys K, Saczynska V, Olszewska M, Pikula A, Smietanka K, Minta Z, Szewczyk B, et al. Highly immunogenic prime-boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus. Trials Vaccinology. 2014;3:40–6. 13. Stachyra A, Redkiewicz P, Kosson P, Protasiuk A, Góra-Sochacka A, Kudla G, Sirko A. Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens. Virol J. 2016;13:143. 14. Stachyra A, Góra-Sochacka A, Zagórski-Ostoja W, Król E, Sirko A. Antibody response to DNA vaccine against H5N1 avian influenza virus in broilers immunized according to three schedules. Acta Biochim Pol. 2014;61:593–6. 15. Reyes L, Hartikka J, Bozoukova V, Sukhu L, Nishioka W, Singh G, Ferrari M, Enas J, Wheeler CJ, Manthorpe M, Wloch MK. Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization. Vaccine. 2001;19:3778–86. 16. Vilalta A, Shlapobersky M, Wei Q, Planchon R, Rolland A, Sullivan S. Analysis of biomarkers after intramuscular injection of Vaxfectin-formulated hCMV gB plasmid DNA. Vaccine. 2009;27:7409–17. 17. Grunwald T, Ulbert S. Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: vaccine-platforms for the battle against infectious diseases. Clin Exp Vaccine Res. 2015;4:1–10. 18. Dupuis M, Denis-Mize K, Woo C, Goldbeck C, Selby MJ, Chen M, Otten GR, Ulmer JB, Donnelly JJ, Ott G, McDonald DM. Stachyra et al. Virology Journal (2017) 14:168 17. Grunwald T, Ulbert S. Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: vaccine-platforms for the battle against infectious diseases. Clin Exp Vaccine Res. 2015;4:1–10. 16. Vilalta A, Shlapobersky M, Wei Q, Planchon R, Rolland A, Sullivan S. Analysis of biomarkers after intramuscular injection of Vaxfectin-formulated hCMV gB plasmid DNA. Vaccine. 2009;27:7409–17. Availability of data and materials Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. J Immunol. 2000;165:2850–8. 19. Farris E, Brown DM, Ramer-Tait AE, Pannier AK. Micro- and nanoparticulates for DNA vaccine delivery. 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https://openalex.org/W1982566777	https://lirias.kuleuven.be/bitstream/123456789/477085/1/1-s2.0-S0370269314008776-main%281%29.pdf	English	null	Single-neutron orbits near 78 Ni: Spectroscopy of the<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:mi>N</mml:mi><mml:mo>=</mml:mo><mml:mn>49</mml:mn></mml:math>isotope 79 Zn	Physics letters. B	2,015	cc-by	5,974	Single-neutron orbits near 78Ni: Spectroscopy of the N = 49 isotope 79Zn Box 35, FIN-40014 Jyväskylä, Finland q Institut für Kernphysik, Technische Universität Darmstadt, D-64289 Darmstadt, Germany r AB Department, CERN 1211, Geneva 23, Switzerland s IPHC, CNRS/IN2P3, Université de Strasbourg, F-67037 Strasbourg, France t Università degli studi di Padova and INFN Sezione di Padova, Padova I-35131, Italy u Institut für Kernphysik, Universität zu Köln, D-50937 Köln, Germany v GANIL, CEA/DSM-CNRS/IN2P3, F-14076 Caen, France Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain a Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain c School of Engineering, University of the West of Scotland, Paisley, P d d Scottish Universities Physics Alliance (SUPA), United Kingdom Contents lists available at ScienceDirect Contents lists available at ScienceDirect Physics Letters B 740 (2015) 298–302 Physics Letters B 740 (2015) 298–302 Single-neutron orbits near 78Ni: Spectroscopy of the N = 49 isotope 79Zn R. Orlandi a,b,c,d,e,∗, D. Mücher f, R. Raabe b, A. Jungclaus a, S.D. Pain g, V. Bildstein f, R. Chapman c,d, G. de Angelis h, J.G. Johansen i, P. Van Duppen b, A.N. Andreyev c,d,j,e, S. Bottoni b,k, T.E. Cocolios m, H. De Witte b, J. Diriken b, J. Elseviers b, F. Flavigny b, L.P. Gaffney n,b, R. Gernhäuser f, A. Gottardo h, M. Huyse b, A. Illana a, J. Konki l,o,p, T. Kröll q, R. Krücken f, J.F.W. Lane c,d, V. Liberati c,d, B. Marsh r, K. Nowak f, F. Nowacki s, J. Pakarinen l,o,p, E. Rapisarda b, F. Recchia t, P. Reiter u, T. Roger b,v, E. Sahin h, M. Seidlitz u, K. Sieja s, J.F. Smith c,d, J.J. Valiente Dobón h, M. von Schmid q, D. Voulot r, N. Warr u, F.K. Wenander r, K. Wimmer f a Instituto de Estructura de la Materia, IEM-CSIC, Madrid, E-28006, Spain b KU Leuven, Instituut voor Kern- en Stralingsfysica, B-3001 Heverlee, Belgium c School of Engineering, University of the West of Scotland, Paisley, PA1 2BE, United Kingdom d Scottish Universities Physics Alliance (SUPA), United Kingdom e Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195, Japan f Physik Department E12, Technische Universität München, D-85748 Garching, Germany g Physics Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA h Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Legnaro, Legnaro, I-35020, Italy i Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus, Denmark j Department of Physics, University of York, Heslington, YO10 5DD, United Kingdom k Dipartimento di Fisica, Università di Milano and INFN Sezione di Milano, I-20133, Italy l PH Department, CERN 1211, Geneva 23, Switzerland m School of Physics and Astronomy, University of Manchester, Manchester, M13 9PL, United Kingdom n Oliver Lodge Laboratory, University of Liverpool, Liverpool L69 9ZE, United Kingdom o Helsinki Institute of Physics, University of Helsinki, P.O. Box 64, FIN-00014 Helsinki, Finland p Department of Physics, University of Jyväskylä, P.O. a r t i c l e i n f o Additional data (ap- proximately 20 hours), collected using a thick (∼1.7 mg/cm2) DPE target, permitted to conﬁrm weak coincidences observed in the thin-target data. The region of isotopes near 78Ni is the focus of intense exper- imental and theoretical research (cf., for example, [7–12] and ref- erences therein). Whether 78Ni can be considered a doubly-magic spherical nucleus depends ultimately on the size of the Z = 28 and N = 50 shell gaps. To date, however, scarce information is available on 78Ni and on its immediate neighbors, and contrasting predic- tions have been made [12,13] about its magicity. The properties of nuclei lying close to 78Ni also impact strongly on astrophysical models of stellar nucleosynthesis and evolution. A recent example is related to the measurement of the 82Zn bind- ing energy and its implications on the composition of neutron- star crust [14]. Furthermore, a sensitivity study on the effect of neutron-capture rates on the A ∼80 and A ∼130 r-process peaks [15] revealed that 78Zn and 79Zn are among the few isotopes which can cause the largest change (>15%) in the overall abun- dance pattern, affecting the abundances of masses as high as A ∼195. The reaction was studied using the segmented T-REX array of Si telescopes [23], and eight triple-cluster HPGe detectors of Miniball [24], which surrounded the T-REX scattering chamber. The coin- cident detection of light charged particles and γ rays led to the identiﬁcation of states which could not be resolved using only the proton data. Furthermore, the charged-particle data constrained the placement of states in the level scheme which would have been ambiguous from the γ -ray data alone. Single-nucleon transfer reactions are a very sensitive technique to populate single-particle states and to investigate the structure of the isotopes produced [16–19]. Performing such reactions on 78Ni will reveal the energies of the single-particle orbits governing the properties of 78Ni and its neighbors. The necessary experiments however still require years of developments in radioactive-ion- beam production. Revealing insights about the structure of 78Ni can nonetheless be gained by studying close-lying isotopes. More- over, an accurate description of the evolution of nuclear structure across neighboring nuclei is an implicit test for theoretical predic- tions of the properties of 78Ni. γ In Fig. 1 (a), the 79Zn excitation energy deduced from reaction kinematics is shown for proton singles and protons in coincidence with all detected γ rays. a r t i c l e i n f o Article history: Received 5 October 2014 Received in revised form 18 November 2014 Accepted 3 December 2014 Available online 9 December 2014 Editor: D.F. Geesaman Keywords: Nuclear structure γ -Ray transitions Transfer reactions N = 50 shell closure Single-neutron states in the Z = 30, N = 49 isotope 79Zn have been populated using the 78Zn(d, p)79Zn transfer reaction at REX-ISOLDE, CERN. The experimental setup allowed the combined detection of protons ejected in the reaction, and of γ rays emitted by 79Zn. The analysis reveals that the lowest excited states populated in the reaction lie at approximately 1 MeV of excitation, and involve neutron orbits above the N = 50 shell gap. From the analysis of γ -ray data and of proton angular distributions, characteristic of the amount of angular momentum transferred, a 5/2+ conﬁguration was assigned to a state at 983 keV. Comparison with large-scale-shell-model calculations supports a robust neutron N = 50 shell-closure for 78Ni. These data constitute an important step towards the understanding of the magicity of 78Ni and of the structure of nuclei in the region. g © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. * Corresponding author at: Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki, 319-1195, Japan. E-mail address: orlandi.riccardo@jaea.go.jp (R. Orlandi). g/ /j p y 014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licen p // g/ /j p y 0370-2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativec SCOAP3. R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 299 Fig. 1. (Color online.) a) 79Zn excitation energy deduced from proton kinematics for all the transfer protons (black solid line) and from the protons in coincidence with any γ ray (green, solid ﬁll). b) 79Zn excitation energy in coincidence with the 983-keV γ ray, corrected for γ -ray eﬃciency. c) Same as b), but in coincidence either with the 236-keV (blue) or the 1859-keV γ transitions (red). Shell structure characterizes several many-body systems of fermions moving in a common potential, such as atomic elec- trons, metal clusters and nuclei. Angular momentum quantization induces a bunching of the single-particle states, resulting in shells separated by energy gaps. a r t i c l e i n f o Three main peaks can be seen, cen- tered respectively around 1.2, 2.5 and 3.3 MeV. Due to kinematic compression and to the detection threshold, only transfer protons corresponding to the lowest-energy peak could be detected both at forward and backward angles, and meaningfully compared to DWBA calculations. At this beam energy, the transferred neutrons should populate mainly states or groups of states corresponding to low-ℓorbits, mostly above the N = 50 gap, namely d5/2, s1/2 and d3/2. The g7/2 orbit above the gap (ℓ = 4) and the neutron–hole states based on p or f conﬁguration are likely to be populated very weakly. As an illustration, the 79Zn case can be compared to the study of the N = 81 isotone 131Sn via the 130Sn(d, p)131Sn re- action [17], in which only neutron orbits above the N = 82 gap were populated. In this Letter, the ﬁrst spectroscopic study of the Z = 30, N = 49 isotope 79Zn is presented. In this nucleus, neutrons can oc- cupy orbits which lie both below and above the N = 50 shell gap. Prior to this work, the available information about 79Zn was lim- ited to its ground-state half life, 0.995(19) s [20]. The beta decay of 79Zn [8] supports a Jπ = 9/2+ ground-state conﬁguration, in line with the shell-model expectation that the odd neutron (hole) oc- cupies the g9/2 orbit, and with N = 49 systematics. In the present work, the 9/2+ assignment for the ground state has been adopted. 79 In this Letter, the ﬁrst spectroscopic study of the Z = 30, N = 49 isotope 79Zn is presented. In this nucleus, neutrons can oc- cupy orbits which lie both below and above the N = 50 shell gap. Prior to this work, the available information about 79Zn was lim- ited to its ground-state half life, 0.995(19) s [20]. The beta decay of 79Zn [8] supports a Jπ = 9/2+ ground-state conﬁguration, in line with the shell-model expectation that the odd neutron (hole) oc- cupies the g9/2 orbit, and with N = 49 systematics. In the present work, the 9/2+ assignment for the ground state has been adopted. In this work, excited states in 79Zn have been populated using the 78Zn(d, p)79Zn reaction in inverse kinematics at REX-ISOLDE, CERN (Q value = 1.796 MeV [21]). a r t i c l e i n f o In the nuclear medium, such shell gaps are revealed by nuclei with neutron and proton numbers corre- sponding to closed-shell conﬁgurations. The properties of these so-called magic nuclei and of their neighbors, which were cardi- nal to the development of the nuclear shell model, could only be reproduced when the role played by the nuclear spin–orbit inter- action was recognized [1]. In recent years, experiments with radioactive ion beams have shown that in some neutron-rich nuclei well-established shell clo- sures can vanish, and new magic numbers appear [2,3]. The chal- lenge to explain and predict the size of shell gaps away from beta stability has led to considerable progress in nuclear physics, both experimentally and theoretically. Despite some remarkable steps forward in describing the evolution of shell structure, e.g. the in- clusion of the tensor interaction [4] and three-body forces [5,6], rare-isotope data are still essential to test and guide theoretical advances. Nuclei away from the valley of beta stability with magic numbers of neutrons and protons, and isotopes in their vicinity, have become new cornerstones for the development of a reliable theoretical picture of all nuclei. Fig. 1. (Color online.) a) 79Zn excitation energy deduced from proton kinematics for all the transfer protons (black solid line) and from the protons in coincidence with any γ ray (green, solid ﬁll). b) 79Zn excitation energy in coincidence with the 983-keV γ ray, corrected for γ -ray eﬃciency. c) Same as b), but in coincidence either with the 236-keV (blue) or the 1859-keV γ transitions (red). with a UCx target. 78Zn atoms were laser ionized using the RILIS set up [22], mass separated, and post-accelerated by the REX- LINAC to 2.83 MeV per nucleon. The 78Zn beam impinged on a thin (105(10) μg/cm2) deuterated polyethylene (DPE) target. In ad- dition to 78Zn, which made up ∼75% of the total intensity, the beam also contained 78Rb (∼20%) and 78Ga (∼5%). Exploiting the fact that without laser ionization only 78Zn disappeared from the beam cocktail, the contribution from the contaminants could be identiﬁed and subtracted oﬄine by collecting data with the laser periodically turned on and off (in total, approximately 100 hours with and 35 hours without laser ionization). From the analysis of elastically scattered deuterons, the estimated average 78Zn beam intensity was 7.8(7) · 105 particles per second. a r t i c l e i n f o Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. discussed below, the analysis of proton angular distributions are indeed consistent with transfer of neutrons to the d5/2 and s1/2 or- bits. The green curve in the same ﬁgure shows that, in coincidence with γ -rays, the intensity of the ﬁrst peak decreases signiﬁcantly more than that of the two higher-lying peaks. This observation suggests the presence, in the excitation-energy range of the ﬁrst peak, of one or more states which are too long lived (more than few nanoseconds) for their decay(s) to be detected in ﬂight. The analysis of the 236- and 1859-keV single-γ -ray-gated exci- tation energy spectra, shown in Fig. 1 (c), is key to positioning the 236-keV transition in the level scheme. A peak at ∼3.20(15) MeV can be seen in both spectra (blue and red in the ﬁgure), but only the 236-keV gate shows also a peak at 2.65(15) MeV, where the large uncertainty is due to the Si-detector resolution (the thin red peak appearing at 2.6 MeV in the 1859-keV gated spectrum is not statistically signiﬁcant). Their observed coincidence implies that the 236-keV lies below the 1859-keV γ ray (otherwise no peak could appear also at 2.65 MeV excitation energy). Hence, the 236-keV transition must be the decay of a state with energy at least as low as the difference between the 3.2 MeV excitation energy and the 1859-keV γ ray, i.e. 1.34(15) MeV. In Fig. 1 (c), however, no peak appears around or below 1.3 MeV, which means that the state at ∼1.34 MeV is not (or too weakly) populated in the direct reaction and only fed from higher-lying states. The prompt character of the 236-keV transition implies that it has E1 or M1 multipolarity. The position of the 1185-keV transition was deduced from its γ -gated excitation energy and the coincidence with the 236-keV γ ray. The γ rays emitted by 79Zn were identiﬁed by requiring a coincidence with the protons ejected in the neutron-transfer re- actions. The Doppler-corrected γ -ray energy spectrum is shown in Fig. 2 (a). Fig. 1 (b) and (c) are examples of excitation-energy spec- tra of 79Zn gated by individual γ rays. Similarly, Fig. a r t i c l e i n f o The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 4. Partial level scheme of 79Zn deduced in this work. The width of the arrows is proportional to the relative transition intensities. With the exception of the isomeric state ‘X’ at 1.10(15) MeV, all energies are in keV, with the error given in brackets. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. ing to a neutron–hole in the f5/2 orbit. Interestingly, comparable B(E2; 5/2+ →9/2+) strengths have been measured in neighbor- ing N = 49 isotones 81Ge and 83Se (respectively, 0.0383(20) and ≈0.13 W.u. [25]). The direct and indirect feeding to the 983-keV state, which makes its ground-state decay the most intense transition in the spectrum, supports a 5/2+ assignment. The DWBA analysis dis- cussed below conﬁrms the 5/2+ assignment deduced from the γ -ray analysis. In the case of the 1424-keV state, the prompt character of the 441-keV transition, the coincidence between the 983- and 441-keV γ rays and the unobserved crossover ground-state transition favor a spin 3/2+ or 5/2+. Few additional weak transitions, of 888, 1774 and 2321 keV, were tentatively placed in the level scheme: their γ -ray-gated excitation energy spectra in fact exhibit a peak respec- tively at 2.35(15), 3.30(15) and 3.45(15) MeV, compatible with a direct feeding to either the 1424- or the 983-keV states. For the 888-keV transition, this placement is also supported by the ob- served coincidence with the 441-keV transition. Fig. 3. a r t i c l e i n f o 78Zn (T1/2 = 1.47(15) s) was produced in collisions of 1.4 GeV protons from the CERN PS Booster In this work, excited states in 79Zn have been populated using the 78Zn(d, p)79Zn reaction in inverse kinematics at REX-ISOLDE, CERN (Q value = 1.796 MeV [21]). 78Zn (T1/2 = 1.47(15) s) was produced in collisions of 1.4 GeV protons from the CERN PS Booster Fig. 1 (a) reveals that the lowest-lying states which are strongly populated via transfer are found near 1 MeV. This observation is important, since the position of the excited states based on neutron orbits above the N = 50 shell reﬂects the gap size. As R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 300 Fig. 4. Partial level scheme of 79Zn deduced in this work. The width of the arrows is proportional to the relative transition intensities. With the exception of the isomeric state ‘X’ at 1.10(15) MeV, all energies are in keV, with the error given in brackets. See text for details. Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. The strongest peaks are labeled by their energy. The peak labeled “c” is due to a small amount of 78Ga in the beam, due to in-ﬂight 78Zn β decay. b) Same spectrum gated on 79Zn excitation energy in the 0.8–1.7 MeV range, where only the 441- and 983-keV transitions can be seen. See text for details. Fig. 3. Coincidence γ -ray spectra gated, respectively, on the 983-keV (a) or the 1859-keV (b) transitions, obtained using thick-target data. Fig. 4. Partial level scheme of 79Zn deduced in this proportional to the relative transition intensities. W state ‘X’ at 1.10(15) MeV, all energies are in keV, w See text for details. ing to a neutron–hole in the f5/2 orbit. B(E2; 5/2+ →9/2+) strengths have bee ing N = 49 isotones 81Ge and 83Se (res ≈0.13 W.u. [25]). The direct and indirect feeding to t makes its ground-state decay the most spectrum, supports a 5/2+ assignment. cussed below conﬁrms the 5/2+ assign γ -ray analysis. In the case of the 1424-keV state, the 441-keV transition, the coincidence betw γ rays and the unobserved crossover gro a spin 3/2+ or 5/2+. Few additional wea and 2321 keV, were tentatively placed i d Fig. 2. a) Doppler-corrected 79Zn γ -ray spectrum, gated by all transfer protons. a r t i c l e i n f o The large amount of feeding from higher lying states favors a positive parity, but a negative parity cannot be excluded. Illustrative angular distributions are shown in Fig. 5 (a) and (b). In both cases, the excitation energy of 79Zn was restricted to the lowest peak of Fig. 1 (a). The data in Fig. 5 (a) were not gated on any γ -ray line, and are poorly ﬁtted by any single ℓtrans- fer. These data are instead well described by a sum of ℓ = 0 and ℓ = 2 distributions. The simultaneous ﬁt of two distribu- tions yields a signiﬁcantly smaller reduced χ 2 (2.1) than obtained by ﬁtting a single ℓ(χ 2 = 17.8 and 18, respectively). The scal- ing factors were left as free parameters in the ﬁt and found to be, respectively, 0.41(3)(10) and 0.51(4)(12) (where the ﬁrst error is statistical, and the second systematic). If, in addition to ℓ = 0 and 2, also a third, ℓ = 1 distribution is added to the simultane- ous ﬁt, the latter has a scaling factor compatible with zero while the former are substantially unchanged, indicating that mostly d and s orbits were populated in this energy range. It is instructive to compare the 5/2+-state energy, 983 keV, with state-of-the-art shell-model calculations [12]. The large shell- model space includes the proton orbits p1/2, p3/2, f72 and f5/2, and the neutron orbits p1/2, p3/2, f5/2, g92 and d5/2, which lies above the N = 50 shell gap. Details about the interaction can be found in Refs. [12,34]. As it can be seen in Fig. 6, these calcu- lations reproduce very well the measured energies of the lowest lying 5/2+-state N = 49 isotones. For 79Zn they predict an energy of 1029 keV, strikingly close to the measured 983 keV. This lowest calculated 5/2+ state (SF = 0.53) is formed by the promotion of one neutron from the g9/2 to the d5/2 orbit. The calculated B(E2) transition strength to the ground state is only 4 e2 fm4 (0.2 W.u.). The equivalent lifetime, 240 ps, is compatible with the observed tail of the 983-keV transition. The angular distribution changes instead considerably by requir- ing the coincident detection of the 983-keV γ ray. This is shown in Fig. 5 (b), together with DWBA calculations for transfer to, re- spectively, pure ℓ = 0 or ℓ = 2 states. a r t i c l e i n f o For the neutron bound-state potential, the radius and diffuseness parameters were 1.25 and 0.65 fm, respectively. Exper- imental spectroscopic factors (SF) were determined from the ratio of experimental and DWBA cross sections, which were calculated for SF = 1. The low beam energy leads to appreciable differences in the DWBA distributions calculated using different optical-model- potential parameterizations. Additional ﬁts were therefore also per- formed using different parameters from Refs. [30–33], and varying the bound-state radius parameter from 1.20 to 1.30. The system- atic uncertainties amount to approximately 20–25% variation in the calculated SFs. It should be remarked however that the arguments based on the analysis of angular distributions do not depend on the chosen parameterization. Refs. [28,29]. For the neutron bound-state potential, the radius and diffuseness parameters were 1.25 and 0.65 fm, respectively. Exper- imental spectroscopic factors (SF) were determined from the ratio of experimental and DWBA cross sections, which were calculated for SF = 1. The low beam energy leads to appreciable differences in the DWBA distributions calculated using different optical-model- potential parameterizations. Additional ﬁts were therefore also per- formed using different parameters from Refs. [30–33], and varying the bound-state radius parameter from 1.20 to 1.30. The system- atic uncertainties amount to approximately 20–25% variation in the calculated SFs. It should be remarked however that the arguments based on the analysis of angular distributions do not depend on the chosen parameterization. The ℓ = 0 strength revealed by the proton data (not gated by any γ ray) of Fig. 5 (a) testiﬁes the presence of an isomeric 1/2+ state near 1 MeV, with SF = 0.41(3)(10). The proton-singles data were also split in three smaller ranges in excitation energy and compared to DWBA calculations similar to those in Fig. 5 (a). The weighted average of the ℓ = 0 strengths measured in the sub-sets indicates that the 1/2+ state lies at approximately 1.05(15) MeV. It seems likely that this s1/2 neutron state corresponds in fact to the 1.10(15)-MeV state deduced from gamma-ray gated excitation energy spectra (labeled X in Fig. 4). If the strongly populated 1/2+ state coincides indeed with the level at 1.1 MeV, then the prompt observation of the 236-keV γ -ray limits the possible spin of the 1.34(15)-MeV state to (1/2) or (3/2) (X +236 in Fig. 4). a r t i c l e i n f o 2 (b) shows that only the 441- and 983-keV lines are still clearly visible if the 79Zn excitation energy is restricted to the lowest peak of Fig. 1 (a) (from 0.8 to 1.7 MeV). Such spectra, together with observed γ coincidences, such as those presented in Fig. 3, proved essen- tial to place the main transitions in the level scheme. No other γ ray was observed below 0.8-MeV excitation energy. It should also be noted that the γ -ray detection eﬃciency drops drastically below 120 keV. Fig. 3 shows that both the 441–983-keV and the 236–1859-keV pairs were each found to be coincident. The partial level scheme of 79Zn deduced in this work is pre- sented in Fig. 4. According to measured intensities and coincidence relations, the 983-keV state decays directly to the ground state, and it is fed by the 441-keV transition. Since the 983-keV tran- sition is prompt, despite a low energy tail, its multipolarity can only be dipole or quadrupole. If the tail is due to the lifetime of the 983-keV state, and not to unobserved feeding, it suggests a lifetime of the order of few hundred picoseconds, with mul- tipolarity E2 or M2. A slow E2 (0.05 < B(E2) < 0.2 W.u.) seems much more likely. An M2 character would in fact require this low- lying and strongly populated state to be a 5/2−state, correspond- From the unobserved coincidence with the 983-keV transition it follows that the 236-keV γ ray feeds an isomeric state lying approximately at 1.10(15) MeV. Proton angular distributions, which are characteristic of the angular momentum transfer, ℓ, provide a strong argument to identify this 1.10(15)-keV state with a 1/2+ state. The measured proton angular distributions were compared to DWBA calculations performed with the codes FRESCO [26] and TWOFNR [27] using global optical model potentials from R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 301 Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. a r t i c l e i n f o The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). and feed the 983-keV state. In summary, approximately 75% of the Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy 0 85 155 M V d l d DWBA l l i f ℓ 0 2 d f Fig. 6. (Color online.) Experimental and calculated N = 50 gap sizes (ﬁlled and open diamonds) and ﬁrst-excited 5/2+-state energies (ﬁlled and open circles) for N = 49 isotones. The measurement from this work is highlighted in blue. The gap sizes were determined from two-neutron separation energies calculated from mass ex- cesses from [21] (and [14] for 82Zn). Fig. 5. (Color online.) a) Proton angular distributions for the 79Zn excitation energy range 0.85–1.55 MeV and scaled DWBA calculations for ℓ = 0, 2 and a sum of the two. b) Same proton data, gated also on the 983-keV γ -ray line, and DWBA calculations for ℓ = 0 or 2. and feed the 983-keV state. In summary, approximately 75% of the measured ℓ = 2 strength in this energy range results in the direct or indirect feeding of the state at 983 keV, again consistent with a 5/2+ assignment. Refs. [28,29]. a r t i c l e i n f o A pure ℓ = 2 distribution (transfer to a d5/2 or d3/2 neutron state) yields a better ﬁt and conﬁrms the 5/2+ assignment of the 983-keV state deduced from γ -ray data. Jπ = 3/2+ can in fact be excluded since it would lead to an isomeric M3 ground-state transition. Fig. 6 shows that the calculations also reproduce reasonably well the evolution of the N = 50 gap deduced from 2-neutron sep- aration energies [14,21], with a minimum at Z = 32 and a larger gap in zinc than in germanium, although the experimental gap size in these two isotones is 450 keV smaller. The calculations predict a doubly-magic 78Ni with shell gaps of 4.7 MeV for neutrons and 5.0 MeV for protons, and a ﬁrst 2+-state lying at nearly 4 MeV. If at least the relative increase in gap size between zinc and nickel is correct, the N = 50 gap in 78Ni can be expected to be at least as large as 4.2 MeV. A similar distribution is observed when gating the same excita- tion energy region by the 441-keV transition: the data, not shown here, are best ﬁtted by ℓ = 2, and yield SF = 0.05(1)(2). The comparison between Fig. 1 (b) and the intensity strength of the 441-keV transition suggests that the 983-keV state is also fed by additional low-energy undetected γ -ray transition(s) from one or more states between 1.0 and 1.4 MeV. The measured angular dis- tributions imply however that these states have ℓ = 2 character In conclusion, this spectroscopic study of 79Zn has permitted the identiﬁcation of key states near 1 MeV of excitation energy, R. Orlandi et al. / Physics Letters B 740 (2015) 298–302 302 based on the occupancy of single-particle states above the N = 50 shell gap. The agreement between the current measurement and recent large-scale shell-model calculations supports the picture of a robust N = 50 shell closure for 78Ni. This newly acquired knowl- edge about neutron single-particle states in 79Zn will also be im- portant to constraining neutron-capture rates on 78Zn, which have been shown to impact the ﬁnal r-process abundance pattern dur- ing freeze-out periods. [9] Z.Y. Xu, S. Nishimura, G. Lorusso, F. Browne, P. Doornenbal, et al., Phys. Rev. Lett. 113 (2014) 032505, http://dx.doi.org/10.1103/PhysRevLett.113.032505. [10] T. Marchi, G. de Angelis, T. Baugher, D. Bazin, J. Berryman, EPJ Web Conf. 66 (2014) 02066, http://dx.doi.org/10.1051/epjconf/20146602066. Acknowledgements [15] R.A. Surman, M.R. Mumpower, G.C. Mclaughlin, R. Sinclair, W.R. Hix, K.L. Jones, in: P.E. Garrett, B. Hadinia (Eds.), Proceedings of the 14th International Sympo- sium on Capture Gamma-Ray Spectroscopy and Related Topics, Guelph, 2013, World Scientiﬁc, 2013, p. 304. This work was supported by the European Commission through the Marie Curie Actions Contracts Nos. PIEFGA-2011-30096 (R.O.) and PIEFGA-2008-219175 (J.P.), by the Spanish Ministerio de Ciencia e Innovación under contracts FPA2009-13377-C02 and FPA2011-29854-C04, by the Spanish MEC Consolider – Ingenio 2010, Project No. CDS2007-00042 (CPAN), by FWO-Vlaanderen (Belgium), by GOA/2010/010 (BOF KU Leuven), by the Interuni- versity Attraction Poles Programme initiated by the Belgian Sci- ence Policy Oﬃce (BriX network P7/12), by the European Union Seventh Framework Programme through ENSAR, contract no. RII3-CT-2010-262010, and by the German BMBF under contracts 05P09PKCI5, 05P12PKFNE, 05P12RDCIA and 06DA9036I. R.O., R.C., J.F.W.L., V.L. and J.F.S. also acknowledge support from STFC, Grant Nos. PP/F000944/1, ST/F007590/1, and ST/J000183/2. The authors thank A.M. Moro for valuable discussions. [16] K.L. Jones, et al., Nature 465 (2010) 454–457, http://dx.doi.org/10.1038/ nature09048. [17] R.L. Kozub, et al., Phys. Rev. Lett. 109 (2012) 172501, http://dx.doi.org/10. 1103/PhysRevLett.109.172501. [18] J.S. Thomas, et al., Phys. Rev. C 76 (2007) 044302, http://dx.doi.org/10.1103/ PhysRevC.76.044302. [19] D.K. Sharp, et al., Phys. Rev. C 87 (2013) 014312, http://dx.doi.org/10.1103/ PhysRevC.87.014312. [20] K.-L. Kratz, H. Gabelmann, P. Möller, B. Pfeiffer, H.L. Ravn, A. Wöhr, ISOLDE Collaboration, Z. Phys. A 340 (1991) 419. [21] M. Wang, G. Audi, A.H. Wapstra, F.G. Kondev, M. MacCormick, X. Xu, B. Pfeiffer, et al., Chin. Phys. C 36 (2012) 1603, http://dx.doi.org/10.1088/1674-1137/36/ 12/003. [22] V. Fedoseyev, G. Huber, U. Köster, J. Lettry, V.I. Mishin, H. Ravn, V. Sebastian, ISOLDE Collaboration, Hyperﬁne Interact. 127 (2000) 409, http://dx.doi.org/ 10.1023/A:1012609515865. [23] V. Bildstein, R. Gernhäuser, T. Kröll, R. Krücken, K. Wimmer, P. Van Duppen, M. Huyse, N. Patronis, R. Raabe, for the T-REX Collaboration, Eur. Phys. J. A 48 (2012) 85, http://dx.doi.org/10.1140/epja/i2012-12085-6. a r t i c l e i n f o [11] L. Coraggio, A. Covello, A. Gargano, N. Itaco, Phys. Rev. C 89 (2014) 024319, http://dx.doi.org/10.1103/PhysRevC.89.024319. [12] K. Sieja, F. Nowacki, Phys. Rev. C 85 (2012) 051301(R), http://dx.doi.org/10. 1103/PhysRevC.85.051301. [13] M.-G. Porquet, O. Sorlin, Phys. Rev. C 85 (2012) 014307, http://dx.doi.org/10. 1103/PhysRevC.85.014307. / y [14] R.N. Wolf, et al., Phys. Rev. Lett. 110 (2013) 041101, http://dx.doi.org/10.1103/ PhysRevLett.110.041101. References [24] N. Warr, J. Van de Walle, et al., Eur. Phys. J. A 49 (2013) 40, http://dx.doi.org/ 10.1140/epja/i2013-13040-9. [1] M. Goeppert Mayer, Phys. Rev. 75 (1949) 1969, http://dx.doi.org/10.1103/ PhysRev.75.1969; O. Haxel, J.H.D. Jensen, H.E. Suess, Phys. Rev. 75 (1949) 1766, http://dx.doi.org/ 10.1103/PhysRev.75.1766.2. [25] P. Hoff, B. Fogelberg, Nucl. Phys. A 368 (1981) 210, http://dx.doi.org/ 10.1016/0375-9474(81)90683-7. [26] I.J. Thompson, Comput. Phys. Rep. 7 (1988) 167. [2] O. Sorlin, M.-G. Porquet, Prog. Part. Nucl. Phys. 61 (2008) 602, http://dx.doi.org/ 10.1016/j.ppnp.2008.05.001. [27] J.A. Tostevin, University of Surrey version of the code TWOFNR (of M. Toyama, M. Igarashi and N. Kishida) and code FRONT (Priv. Comm.). [3] R. Kanungo, Phys. Scr. T 152 (2013) 014002, http://dx.doi.org/10.1088/0031- 8949/2013/T152/014002. [28] H. An, C. Cai, Phys. Rev. C 73 (2006) 054605, http://dx.doi.org/10.1103/ PhysRevC.73.054605. [4] T. Otsuka, T. Suzuki, M. Honma, Y. Utsuno, N. Tsunoda, K. Tsukiyama, M. Hjorth-Jensen, Phys. Rev. Lett. 104 (2010) 012501, http://dx.doi.org/10.1103/ PhysRevLett.104.012501. [29] A. Koning, J. Delaroche, Nucl. Phys. A 713 (2003) 231, http://dx.doi.org/10.1016/ S0375-9474(02)01321-0. [30] J.M. Lohr, W. Haeberli, Nucl. Phys. A 232 (1974) 381, http://dx.doi.org/10.1016/ 0375-9474(74)90627-7. [5] A.P. Zuker, Phys. Rev. Lett. 90 (2003) 042502, http://dx.doi.org/10.1103/ PhysRevLett.90.042502. [31] Y. Han, Y. Shi, Q. Shen, Phys. Rev. C 74 (2006) 044615, http://dx.doi.org/ 10.1103/PhysRevC.74.044615. [6] G. Hagen, M. Hjorth-Jensen, G.R. Jansen, R. Machleidt, T.P. Papenbrock, Phys. Rev. Lett. 109 (2012) 032502, http://dx.doi.org/10.1103/PhysRevLett.109. 032502. [32] F.G. Perey, Phys. Rev. 131 (1963) 745, http://dx.doi.org/10.1103/PhysRev. 131.745. [33] F.D. Becchetti, G.W. Greenlees, Phys. Rev. 182 (1969) 1190, http://dx.doi.org/ 10.1103/PhysRev.182.1190. [7] Y. Tsunoda, T. Otsuka, N. Shimizu, M. Honma, Y. Utsuno, Phys. Rev. C 89 (2014) 031301(R), http://dx.doi.org/10.1103/PhysRevC.89.031301. [34] S.M. Lenzi, F. Nowacki, A. Poves, K. Sieja, Phys. Rev. C 82 (2010) 054301, http://dx.doi.org/10.1103/PhysRevC.82.054301. [8] A. Korgul, K.P. Rykaczewski, J.A. Winger, S.V. Ilyushkin, C.J. Gross, et al., Phys. Rev. C 86 (2012) 024307, http://dx.doi.org/10.1103/PhysRevC.86.024307.
https://openalex.org/W4234734205	https://www.researchsquare.com/article/rs-47527/latest.pdf	English	null	Effect of vitamin A on intestinal mucosal injury in pediatric patients receiving hematopoietic stem cell transplantation and chemotherapy: a quasai-randomized trial	Research Square (Research Square)	2,020	cc-by	4,672	Effect of vitamin A on intestinal mucosal injury in pediatric patients receiving hematopoietic stem cell transplantation and chemotherapy: a quasai- randomized trial Ploy Pattanakitsakul Mahidol University Faculty of Medicine Ramathibodi Hospital Nalinee Chongviriyaphan Mahidol University Faculty of Medicine Ramathibodi Hospital Samart Pakakasama Mahidol University Faculty of Medicine Ramathibodi Hospital Nopporn Apiwattanakul (  np36@hotmail.com ) Faculty of Medicine Ramathibodi Hospital Mahidol University https://orcid.org/0000-0003-1833-5127 Research note Keywords: vitamin A supplementation, citrulline, hematopoietic stem cell transplantation, mucosal injury, pediatrics Posted Date: September 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-47527/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Ploy Pattanakitsakul Mahidol University Faculty of Medicine Ramathibodi Hospital Nalinee Chongviriyaphan Mahidol University Faculty of Medicine Ramathibodi Hospital Samart Pakakasama Mahidol University Faculty of Medicine Ramathibodi Hospital Nopporn Apiwattanakul (  np36@hotmail.com ) Faculty of Medicine Ramathibodi Hospital Mahidol University https://orcid.org/0000-0003-1833-5127 Research note Keywords: vitamin A supplementation, citrulline, hematopoietic stem cell transplantation, mucosal injury, pediatrics Posted Date: September 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-47527/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 2nd, 2020. See the published Research note Posted Date: September 22nd, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 2nd, 2020. See the published version at https://doi.org/10.1186/s13104-020-05307-8. Page 1/13 Abstract Objective: Vitamin A is involved in maintenance of gut mucosal integrity and normal immune function. However, it is unclear whether these functions of vitamin A have any beneficial effects in patients undergoing hematopoietic stem cell transplantation (HSCT). In this study, we aimed to examine the potential protective effect of vitamin A supplementation on gastrointestinal (GI) mucosal integrity in HSCT recipients using plasma citrulline as a surrogate marker of intestinal integrity. Results: We performed a quasi-randomized trial in 30 pediatric patients undergoing HSCT. Half (n = 15) of the patients received a single high dose of vitamin A (200,000 IU) before the conditioning regimen was given, and half (n = 15) did not. Clinical data of patients who developed post-transplant complications were recorded for 60 days after HSCT. There were no significant differences in mean plasma citrulline levels on day 7 after HSCT between the treatment and control groups (5.8 vs. 5.9 µmol/L, respectively). The incidence of mucositis and other complications were not different between the two groups within 60 days of HSCT. Vitamin A supplementation prior to HSCT in pediatric patients had no clinical benefit in protecting GI mucosal integrity. Study design This study was a preliminary, single center, prospective, quasi-randomized, clinical trial. Eligible patients were assigned to one of two groups by 1:1 alternation: 15 patients received a single oral dose of 200,000 IU of vitamin A before the conditioning chemotherpay; the other 15 patients did not receive vitamin A supplementation (Supplementary Fig. 1). Baseline characteristics including age, sex, body weight, height, diagnosis, donor type and conditioning chemotherapy regimens were collected. Blood samples were collected before administration of vitamin A, on stem cell infusion day (day 0), and 7 days after HSCT. Plasma was separated by centrifugation and stored at −80°C until analysis. Plasma retinol and citrulline levels were analyzed using high-performance liquid chromatography (HPLC) and cation-exchange HPLC techniques, respectively. All of the patients were followed for a period of 60 days after HSCT. Clinical data including number and consistency of stools, fever and oral mucositis were recorded thrice daily (every 8 hours). Participants We prospectively enrolled patients aged 1–18 years who underwent HSCT from May 2017 to August 2018 at the Bone Marrow Transplantation Unit, Ramathibodi Hospital, Thailand. Patients were excluded if they had received vitamin A supplementation in the previous 3 months before enrollment or had renal insufficiency (GFR < 50 mL/min), which causes false elevation of citrulline levels [10]. HSCT was performed on the basis of underlying diseases according to our institutional protocols. All participants received conditioning chemotherapy, supportive care and antimicrobial prophylaxis after receiving stem cells. This trial was approved by the Institutional Ethics Committee, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (ID 03-60-18). All patients and/or their parents provided written informed consent. The study was registered at the Thai Clinical Trials Registry (TCTR20191207002) and partly adhered to CONSORT guidelines. Introduction Infection is a major cause of morbidity and mortality during the neutropenic phase in patients undergoing hematopoietic stem cell transplantation (HSCT) [1, 2]. Approximately 40% of stem cell transplant patients develop mucositis following the intensive chemotherapy. In addition to prolonged severe neutropenia, mucosal barrier injury in the gastrointestinal (GI) tract poses additional risks of local bacterial infection and septicemia during the pre-engraftment period [3-9]. Mucositis that occurs following conditioning chemotherapy in these patients is found throughout the GI tract and is generally not directly detectable. Thus, many studies have used plasma citrulline as a noninvasive biomarker of intestinal mucosal damage. Citrulline is an amino acid mainly produced by enterocytes of the small bowel. Decreased levels of plasma citrulline are correlated with significant epithelial small bowel loss [10-12]. Vitamin A plays an essential role in maintaining gut mucosal integrity and immune function[13]. In previous studies of newly diagnosed children with malignancy, vitamin A-deficient patients tended to have more serious febrile episodes, oral mucositis and infections compared with children who had normal vitamin A status [14, 15]. Furthermore, a study in vitamin A-deficient rats revealed that vitamin A supplementation had a protective effect on the intestinal mucosal barrier following intensive chemotherapy [16, 17]. Currently, the World Health Organization (WHO) recommends a single high dose of vitamin A supplementation for severely malnourished children (100,000 IU in 6–11 month-old infants; 200,000 IU in children ≥ 12–71 months of age), especially in Southeast Asia and Africa, where deficiency is a public health problem [18]. Reports have also suggested that vitamin A supplementation can reduce mortality in Page 2/13 Page 2/13 childhood measles and diarrhea [19]. To date, the potential protective effect of vitamin A supplementation on mucosal injury in HSCT patients remains unclear. We conducted a preliminary study by quasi-randomized trial to examine whether vitamin A could protect against GI mucosal injury after chemotherapy in pediatric patients undergoing HSCT. childhood measles and diarrhea [19]. To date, the potential protective effect of vitamin A supplementation on mucosal injury in HSCT patients remains unclear. We conducted a preliminary study by quasi-randomized trial to examine whether vitamin A could protect against GI mucosal injury after chemotherapy in pediatric patients undergoing HSCT. Results The baseline characteristics of the eligible patients are shown in Table 1. The mean age was 9.6 years in the vitamin A group and 7.8 years in the control group. Forty percent of the patients had underlying diseases of malignancy. Two-thirds of the patients underwent haploidentical HSCT. The baseline plasma vitamin A level was similar between the vitamin A (44.1 ± 21.4 µg/dL) and control (47.9 ± 19.3 µg/dL) groups. Five patients in the vitamin A group and two patients in the control group had a vitamin A level < 30 µg/dL (Table 2). No patients had clinical signs of vitamin A deficiency at the time of enrollment. Figure 1A shows that the plasma vitamin A level increased from baseline, peaked at day 0 and decreased at day 7; however, these changes were not different between the two groups. Statistical analysis Between-group comparisons were analyzed using the Mann Whitney U-test or the Student’s t-test for continuous data, and the χ2 test or Fisher’s exact test for categorical data. Comparisons of plasma vitamin A and citrulline levels between the two groups were performed by mixed linear regression. Statistical significance was defined as a p value < 0.05. Statistical analysis of data was performed using the Stata version 15.1 (StataCorp LLC, College Station, TX, USA). Outcomes The primary outcome of this study was GI mucosal damage after HSCT indicated by plasma citrulline level. Secondary outcomes, assessed up to 60 days after HSCT, were post-HSCT complications including Page 3/13 incidence of febrile neutropenia, oral mucositis, infections, diarrhea, graft-versus-host disease (GVHD), hemorrhagic cystitis, time of neutrophil engraftment, length of hospital stay, and mortality. Incidence of vitamin A toxicity (headache, vomiting, seizure, change in mental status, visual disturbance) was also recorded. incidence of febrile neutropenia, oral mucositis, infections, diarrhea, graft-versus-host disease (GVHD), hemorrhagic cystitis, time of neutrophil engraftment, length of hospital stay, and mortality. Incidence of vitamin A toxicity (headache, vomiting, seizure, change in mental status, visual disturbance) was also recorded. Definitions Diarrhea was defined as passage of ≥ 3 watery stools in a 24-hour period. Bacterial infection was defined as presence of pathogenic bacteria in cultures of blood, cerebrospinal fluid or > 105 CFU/mL in cultures of urine. Pneumonia was diagnosed by imaging study. Febrile neutropenia was defined as a single oral temperature measurement > 38.3°C, or a temperature > 38°C sustained over a 1-hour period with absolute neutrophil count < 500 cells/µL or expected to decrease to < 500 cells/ µL during the next 48 hours. Neutrophil engraftment was defined as an absolute neutrophil count > 500 cells/ µL for 3 consecutive days after HSCT. Vitamin A deficiency and insufficiency were defined as plasma vitamin A levels < 20 and 30 µg/dL, respectively. Incidence of vitamin A toxicity None of the enrolled patients had clinical features of vitamin A toxicity. Vitamin A supplementation in patients with normal vitamin A status at baseline did not lead to any adverse events. Moreover, plasma vitamin A at day 7 after HSCT did not reach a level considered toxic (> 100 µg/dL) [20] in either group (Fig. 1A). Complications after HSCT There was no significant difference in the incidence of febrile neutropenia, oral mucositis, invasive bacterial infections, diarrhea, hemorrhagic cystitis, graft-versus-host disease or death between the two groups (Table 2). The severity of oral mucositis graded by the WHO scale [5] was also not different between the two groups (Table 2). Two patients died during the study period, both belonging to the control group. The first patient was a 5- year-old girl with underlying neuroblastoma who underwent haploidentical HSCT and developed toxic leukoencephalopathy 18 days later. The second patient was an 8-year-old girl with underlying acute myeloblastic leukemia who also underwent haploidentical HSCT and developed massive GI bleeding 48 days after HSCT. The cause of bleeding could not be determined. The mean durations of febrile neutropenia and diarrhea tended to be shorter in the vitamin A group, but these differences were not statistically significant (Table 2). Effect of vitamin A supplementation on plasma citrulline levels Low plasma citrulline (< 20 μmol/L) indicates intestinal mucosal injury [12]. In this study, plasma citrulline decreased following HSCT, with mean plasma citrulline levels reaching their lowest points at day 7 in both groups. There was no statistically significant difference in the change in citrulline levels between the two groups (p = 0.09; Figure 1B). Page 4/13 Page 4/13 Discussion This preliminary trial on vitamin A supplementation in pediatric patients undergoing HSCT did not show a beneficial effect of vitamin A on intestinal mucosal integrity. We found a prevalence of vitamin A insufficiency of 23% in our study population. Conditioning chemotherapy can markedly impair intestinal mucosal integrity, as indicated by a reduction in plasma citrulline level. While vitamin A supplementation in this study did not rescue this effect, it did show a tendency of beneficial effects in terms of reduced duration of diarrhea and febrile neutropenia. There are several possible explanations for why supplementation of vitamin A, proven to be an essential nutrient for the maintenance of gut mucosal integrity and immune function [13, 21], did not show a significant beneficial effect on mucosal integrity in our pediatric patients undergoing HSCT. We found that the prevalence of vitamin A deficiency in this study was lower than a previous study [15]. Therefore, the efficacy of a single high dose of vitamin A might not have been observable. Another possibility is that the dose of 200,000 IU vitamin A in this study was not sufficient to rescue the mucosal integrity, as evidenced by the patients in both groups showing similar decreases in plasma citrulline levels following HSCT. The fact that no patients who received vitamin A supplementation had a toxic level of vitamin A after supplementation implied that the dose could be safely increased. Hussey and Klein [22] showed that high-dose vitamin A supplementation (400,000 IU) led to markedly diminished mortality and morbidity in Page 5/13 Page 5/13 in children hospitalized with severe measles in South Africa. However, 92% of the children in their study had hyporetinemia (serum vitamin A level < 20 µg/dL). Since one animal study showed that exogenous retinoic acid significantly increased expression of the gut-homing molecules, CCR9 and α4β7, and accumulation of CD4+ and CD8+ expression in the gut mucosa, which could potentially increase the risk of gut GVHD in patients who receive exogenous vitamin A [23]. However, the most recent study conducted by Gjærde et al. [24] showed that pretransplant vitamin A level was not associated with incidence of acute GVHD or gastrointestinal GVHD. Our result supported this study that vitamin A supplementation did not affect the incidence of acute GVHD in patients following HSCT. Conclusion This preliminary study showed that vitamin A supplementation in the preconditioning period had no clinical benefit on GI mucosal integrity in pediatric patients undergoing HSCT. Several factors could be involved in modulating the effects of vitamin A on GI mucosal integrity, including the status of other vitamins and minerals and the gut microbiota. The dose and appropriate timing for administration of vitamin A supplementation should be investigated further. Discussion The discrepancy between the animal study and the studies in human may be due to the fact that all-trans-retinoic acid was injected directly into peritoneum in the animal study. This may not represent what actually occurs in human body. Of note, we observed that plasma vitamin A levels tended to increase at day 0 in patients in both groups compared with baseline levels. This result might reflect an improved nutritional status of the patients after being hospitalized. The patients’ diets were well controlled by hospital dieticians, and may have elevated their vitamin A levels. This might have played a role in masking the potentially beneficial effects of vitamin A supplementation on our outcomes of interest. However, plasma vitamin A levels decreased at day 7 after HSCT. This may have been related to low nutritional intake and decreased intestinal absorption due to presence of mucositis or higher vitamin A utilization [15]. Although plasma citrulline levels were lowest at day 7 after HSCT, reflective of ongoing intestinal mucosal damage, plasma vitamin A levels were still in the normal range. We speculate that the intestinal mucosal integrity in our patients was also influenced by other factors, including zinc level [25], inflammation, infection [26], gut microbiota [27, 28] and host immune response [13]. Another possibility is that a higher plasma concentration of vitamin A was needed to rescue the intestinal damage. Given that the serum vitamin A and citrulline levels decreased from day 0 to day 7, it is worth exploring further whether vitamin A supplementation around this time period could improve intestinal integrity. It would also be interesting to investigate whether plasma vitamin A levels continue to decrease after day 7 post-HSCT. If so, supplementation of vitamin A at this time point may show benefits in improved outcomes of HSCT. Limitations Page 6/13 The results of this study are preliminary. A larger sample size is needed to examine the potential benefits of vitamin A in the reduction of duration of febrile neutropenia and diarrhea in this patient population. The results of this study are preliminary. A larger sample size is needed to examine the potential benefits of vitamin A in the reduction of duration of febrile neutropenia and diarrhea in this patient population. This study was not a true randomized clinical trial. However, preliminary results from this study could be a basis for a randomized clinical trial with appropriate sample size. This study was not a true randomized clinical trial. However, preliminary results from this study could be a basis for a randomized clinical trial with appropriate sample size. A dose-response effect of vitamin A was not performed. Ethics approval and consent to participate This study was performed in accordance with the Declaration of Helsinki. The study protocol was approved by the Research Ethical Committee of Ramathibodi Hospital, Mahidol University (ID 03-60-18). All subjects and/or parents provided written informed consents. Consent for publication Not applicable. Availability of data and materials All data generated and analyzed during this study are included in this published article (in the supplementary information files). Declarations Ethics approval and consent to participate Abbreviations WHO: World Health Organization WHO: World Health Organization GI: gastrointestinal HSCT: hematopoietic stem cell transplantation TPN: total parenteral nutrition TPN: total parenteral nutrition Acknowledgments The authors gratefully acknowledge the nurses and staff of the Stem Cell Transplant Unit for cooperating with this study. We also thank Michelle Kahmeyer-Gabbe, PhD, from Edanz Group (https://en-author- services.edanzgroup.com/) for editing a draft of this manuscript. References 1. Elting LS, Cooksley C, Chambers M, Scott B Cantor, Ellen Manzullo, Edward B Rubenstein. The burdens of cancer therapy: clinical and economic outcomes of chemotherapy‐induced mucositis. Competing interests The authors declare no conflicts of interest. The authors declare no conflicts of interest. This study was funded by a Ramathibodi Hospital Research Grant (RF_60079), Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand. Author Contributions Page 7/13 Page 7/13 Contributor role Authors Conceptualization N. Apiwattanakul, N. Chongviriyaphan, S. Pakakasama Methodology P. Pattanakitsakul, N. Apiwattanakul Software NA Validation P. Pattanakitsakul, N. Apiwattanakul Formal analysis P. Pattanakitsakul, N. Apiwattanakul Investigation P. Pattanakitsakul Resources NA Data Curation P. Pattanakitsakul, N. Apiwattanakul Writing — original draft preparation P. Pattanakitsakul, Writing — review and editing N. Apiwattanakul, N. Chongviriyaphan, S. Pakakasama Visualization P. Pattanakitsakul Supervision N. Apiwattanakul Project administration N. Apiwattanakul Funding acquisition P. Pattanakitsakul Cancer. 2003;98(7):1531–9. 2. Sahin U, Toprak SK, Atilla PA, Atilla E, Demirer T. An overview of infectious complications after allogeneic hematopoietic stem cell transplantation. J Infect Chemother. 2016;22(8):505–14. 3. Blijlevens NM, Logan RM, Netea MG. Mucositis: from febrile neutropenia to febrile mucositis. Journal Antimicrob Chemother. 2009;63(suppl_1):i36–i40. 4. Knox JJ, Puodziunas AL, Feld R. Chemotherapy-induced oral mucositis. Drugs Aging. 2000;17(4):257–67. 5. Naidu MUR, Ramana GV, Rani PU,Mohan IK, Suman A, Roy P. Chemotherapy-induced and/or radiation therapy-induced oral mucositis—complicating the treatment of cancer. Neoplasia. 2004;6(5):423. 6. Sonis ST. Oral mucositis in cancer therapy. J Support Oncol. 2004;2(6 Su 6. Sonis ST. Oral mucositis in cancer therapy. J Support Oncol. 2004;2(6 Suppl 3):3–8. 7. van der Velden WJ, Herbers AH, Netea MG, Blijlevens NMA. Mucosal barrier injury, fever and infection in neutropenic patients with cancer: introducing the paradigm febrile mucositis. Br J Haematol. 2014;167(4):441–52. 8. Herbers A, De Haan A, van der Velden W, Donnelly JP, Blijlevens NMA. Mucositis not neutropenia determines bacteremia among hematopoietic stem cell transplant recipients. Transpl Infect Dis. 2014;16(2):279–85. 9. Gibson RJ, Bowen JM. Biomarkers of regimen-related mucosal injury. Cancer Treat Rev. 2011;37(6):487–93. 10. Crenn P, Messing B, Cynober L. Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction. Clin Nutr. 2008;27(3):328–39. 11. Kuiken NS, Rings EH, Blijlevens NM, Tissing WJE. Biomarkers and non-invasive tests for gastrointestinal mucositis. Support Care Cancer. 2017;25(9):2933–41. 12. Fragkos KC, Forbes A. Citrulline as a marker of intestinal function and absorption in clinical settings: A systematic review and meta-analysis. United European Gastroenterol J. 2018;6(2):181–91. 12. Fragkos KC, Forbes A. Citrulline as a marker of intestinal function and absorption in clinical settings: A systematic review and meta-analysis. United European Gastroenterol J. 2018;6(2):181–91. 13. Stephensen CB. Vitamin A, infection, and immune function. Ann Rev Nut 13. Stephensen CB. Vitamin A, infection, and immune function. Ann Rev Nutr. 2001;21(1):167–92. 13. Stephensen CB. Vitamin A, infection, and immune function. Ann Rev Nutr. 2001;21(1):167–92. 14. Wessels G, Hesseling P, Stefan D, Labadarios D. The effect of vitamin A status in children treated for cancer. Pediatr Hematol Oncol. 2008;25(4):283–90. 14. Wessels G, Hesseling P, Stefan D, Labadarios D. The effect of vitamin A status in children treated for cancer. Pediatr Hematol Oncol. 2008;25(4):283–90. 15. High KP, Legault C, Sinclair JA, Cruz J, Hill K, Hurd DD. Low plasma concentrations of retinol and α- tocopherol in hematopoietic stem cell transplant recipients: the effect of mucositis and the risk of infection. 1. Elting LS, Cooksley C, Chambers M, Scott B Cantor, Ellen Manzullo, Edward B Rubenstein. The burdens of cancer therapy: clinical and economic outcomes of chemotherapy‐induced mucositis. 1. Elting LS, Cooksley C, Chambers M, Scott B Cantor, Ellen Manzullo, Edward B Rubenstein. The burdens of cancer therapy: clinical and economic outcomes of chemotherapy‐induced mucositis. Page 8/13 Cancer. 2003;98(7):1531–9. Cancer. 2003;98(7):1531–9. A randomized, controlled trial of vitamin A in children with severe measles. New Engl J Med. 1990;323(3):160–4. 23. Chen X, Dodge J, Komorowski R, Drobyski WR. A critical role for the retinoic acid signaling pathway in the pathophysiology of gastrointestinal graft-versus-host disease. Blood. 2013;121(19):3970-80. 23. Chen X, Dodge J, Komorowski R, Drobyski WR. A critical role for the retinoic acid signaling pathway in the pathophysiology of gastrointestinal graft-versus-host disease. Blood. 2013;121(19):3970-80. 24. Gjaerde LK, Andersen NS, Friis LS, Kornblit B, Petersen SL, Schjodt I, et al. Pretransplantation vitamin A plasma levels and risk of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2020;55(7):1457-9. 25. Hu CH, Song ZH, Xiao K, Song J, Jiao LF, Ke YL. Zinc oxide influences intestinal integrity, the expressions of genes associated with inflammation and TLR4-myeloid differentiation factor 88 signaling pathways in weanling pigs. Innate Immun. 2014;20(5):478–86. 25. Hu CH, Song ZH, Xiao K, Song J, Jiao LF, Ke YL. Zinc oxide influences intestinal integrity, the expressions of genes associated with inflammation and TLR4-myeloid differentiation factor 88 signaling pathways in weanling pigs. Innate Immun. 2014;20(5):478–86. 26. Haines RJ, Beard Jr RS, Eitner RA, Chen L, Wu MH. TNFα/IFNγ mediated intestinal epithelial barrier dysfunction is attenuated by microRNA-93 downregulation of PTK6 in mouse colonic epithelial cells. PloS ONE. 2016;11(4). 26. Haines RJ, Beard Jr RS, Eitner RA, Chen L, Wu MH. TNFα/IFNγ mediated intestinal epithelial barrier dysfunction is attenuated by microRNA-93 downregulation of PTK6 in mouse colonic epithelial cells PloS ONE. 2016;11(4). 27. Hibberd MC, Wu M, Rodionov DA, Li X, Cheng J, Griffin NW, et al. The effects of micronutrient deficiencies on bacterial species from the human gut microbiota. Sci Transl Med. 2017;9(390):eaal4069. 28. Lee H, Ko G. Antiviral effect of vitamin A on norovirus infection via modulation of the gut microbiome. Sci Rep. 2016;6(1):1–9. 28. Lee H, Ko G. Antiviral effect of vitamin A on norovirus infection via modulation of the gut microbiome. Sci Rep. 2016;6(1):1–9. Cancer. 2003;98(7):1531–9. Am J Clin Nutr. 2002;76(6):1358–66. 15. High KP, Legault C, Sinclair JA, Cruz J, Hill K, Hurd DD. Low plasma concentrations of retinol and α- tocopherol in hematopoietic stem cell transplant recipients: the effect of mucositis and the risk of infection. Am J Clin Nutr. 2002;76(6):1358–66. 16. Elli M, Aydin O, Bilge S, Bozkurt A, Dagdemir A, Pinarli FG, et al. Protective effect of vitamin A on ARA- C induced intestinal damage in mice. Tumori. 2009;95(1):87–90. 16. Elli M, Aydin O, Bilge S, Bozkurt A, Dagdemir A, Pinarli FG, et al. Protective effect of vitamin A on ARA- C induced intestinal damage in mice. Tumori. 2009;95(1):87–90. 17. Yüncü M, Eralp A, Koruk M, Sari I, Bağci C, Inalöz S. Effect of vitamin A against methotrexate-induced damage to the small intestine in rats. Med Princ Pract. 2004;13(6):346–52. 17. Yüncü M, Eralp A, Koruk M, Sari I, Bağci C, Inalöz S. Effect of vitamin A against methotrexate-induced damage to the small intestine in rats. Med Princ Pract. 2004;13(6):346–52. 18. Guideline: Vitamin A supplementation in infants and children 6–59 months of age. Geneva: World Health Organization 2011. 18. Guideline: Vitamin A supplementation in infants and children 6–59 months of age. Geneva: World Health Organization 2011. Page 9/13 Page 9/13 https://www.ncbi.nlm.nih.gov/books/NBK185172/pdf/Bookshelf_NBK185172.pdf https://www.ncbi.nlm.nih.gov/books/NBK185172/pdf/Bookshelf_NBK185172.pdf 19. Mayo-Wilson E, Imdad A, Herzer K, Yakoob MY, Bhutta ZA. Vitamin A supplements for preventing mortality, illness, and blindness in children aged under 5: systematic review and meta-analysis. BMJ. 2011;343:d5094. 19. Mayo-Wilson E, Imdad A, Herzer K, Yakoob MY, Bhutta ZA. Vitamin A supplements for preventing mortality, illness, and blindness in children aged under 5: systematic review and meta-analysis. BMJ. 2011;343:d5094. 20. Olson JA. Recommended dietary intakes (RDI) of vitamin A in humans. Am J Clin Nutr. 1987;45(4):704–16. 20. Olson JA. Recommended dietary intakes (RDI) of vitamin A in humans. Am J Clin Nutr. 1987;45(4):704–16. 21. Dagdemir A, Yildirim H, Aliyazicioglu Y, Kanber Y, Albayrak D, Acar S. Does vitamin A prevent high- dose-methotrexate-induced D-xylose malabsorption in children with cancer? Support Care Cancer. 2004;12(4):263–7. 21. Dagdemir A, Yildirim H, Aliyazicioglu Y, Kanber Y, Albayrak D, Acar S. Does vitamin A prevent high- dose-methotrexate-induced D-xylose malabsorption in children with cancer? Support Care Cancer. 2004;12(4):263–7. 22. Hussey GD, Klein M. A randomized, controlled trial of vitamin A in children with severe measles. New Engl J Med. 1990;323(3):160–4. 22. Hussey GD, Klein M. Table 2. Incidence and duration of secondary outcomes within 60 days of hematopoietic stem cell transplantation in the two groups of study patients Median (min–max). SD: standard deviation; TPN: total parenteral nutrition. Figures Tables Table 1. Baseline characteristics of the two groups of study patients Table 1. Baseline characteristics of the two groups of study patients Page 10/13 Characteristics Study groups Vitamin A supplementation (n = 15) Non-vitamin A supplementation (n = 15) Age, mean years ± SD 9.6 ± 4.3 7.8 ± 2.5 Male, n (%) 10 (66.7) 7 (46.7) Underlying disease, n (%) Malignant disease Nonmalignant disease 7 (46.7) 8 (53.3) 5 (33.3) 10 (66.7) Type of HSCT, n (%) Haploidentical Non-haploidentical 10 (66.7) 5 (33.3) 10 (66.7) 5 (33.3) Weight for height, mean % ± SD 102.4 ± 18.63 97.9 ± 26.07 Baseline plasma retinol (µg/dL), mean ± SD 44.1 ± 21.4 47.9 ± 19.3 Plasma retinol < 30 µg/dL, n (%) 5 (33.3) 2 (13.3) Baseline plasma citrulline (μmol/L), mean ± SD 25.7 ± 10.8 29.2 ± 8 SD: standard deviation; HSCT: hematopoietic stem cell transplantation. D: standard deviation; HSCT: hematopoietic stem cell transplantation. SD: standard deviation; HSCT: hematopoietic stem cell transplantation. Table 2. Incidence and duration of secondary outcomes within 60 days of hematopoietic stem cell transplantation in the two groups of study patients Table 2. Incidence and duration of secondary outcomes within 60 days of hematopoietic stem cell transplantation in the two groups of study patients Page 11/13 Outcomes Study groups P value Vitamin A supplementation (n = 15) Non-vitamin A supplementation (n = 15) Febrile neutropenia, n (%) 11 (73.3) 11 (73.3) 1 Oral mucositis, n (%) 14 (93.3) 15 (100) 1 Oral mucositis severity grade 3–4, n (%) 6 (40) 9 (60) 0.27 Diarrhea, n (%) 14 (93.3) 14 (93.3) 1 Hemorrhagic cystitis, n (%) 5 (33.3) 5 (33.3) 1 Graft-versus-host disease, n (%) 6 (40) 7 (46.7) 1 Bacterial infection, n (%) 1 (6.7) 2 (13.3) 1 Viral infection, n (%) 10 (66.7) 6 (40) 0.14 Day of neutrophil engraftment , mean days ± SD 14 ± 2.8 14.2 ± 3.9 0.87 Duration of febrile neutropenia , days 5 (2–10) 6.5 (1–21) 0.8 Duration of mucositis, mean days ± SD 9 ± 3.8 9 ± 4.7 0.96 Duration of diarrhea* , days 11.5 (2–44) 15.5 (2–37) 0.42 Duration of need of TPN, days ± SD 13 ± 7.1 11 ± 6.6 0.42 Duration of hospitalization after day 0*, days 29 (18–83) 24 (15–54) 0.13 Mortality, n (%) 0 2 (13.3) 0.48 Study groups 0 Figures Median (min–max). SD: standard deviation; TPN: total parenteral nutrition. Figure 1 Levels of plasma retinol (A) and citrulline (B) at baseline, and at days 0 and 7 following hematopoietic stem cell transplantation in the two study groups. The values represent median with interquartile range. Figures Page 12/13 Page 12/13 Page 12/13 Supplementary Files his is a list of supplementary files associated with this preprint. Click to dow CONSORT2010ChecklistVitaminAGIintegrity.doc Fig.S1.TIF supplementarydataJuly212020.xlsx Page 13/13
https://openalex.org/W4221036233	https://zenodo.org/records/6397059/files/219-228.pdf	Quechua	null	O'SMIRLIK DAVRIDA DEVIANT XULQ – ATVORNING NAMOYON BO'LISHI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,369	JOURNAL OF NEW CENTURY INNOVATIONS JOURNAL OF NEW CENTURY INNOVATIONS Mirvaliyeva Maxliyo Yoqubjon qizi NamDU o'qituvchisi Nabijonova Muslimaxon Baxodirjon qizi NamDU talabasi +998 90 693 94 77 Mirvaliyeva Maxliyo Yoqubjon qizi NamDU o'qituvchisi Nabijonova Muslimaxon Baxodirjon qizi NamDU talabasi +998 90 693 94 77 Annotatsiya: Ushbu maqolada deviant xulq – atvorning o’smirlarda paydo bo’lishi, tarbiyasi qiyin o’smirlarni o’rganish tamoyillari shu bilan bir qatorda deviant xulq – atvor haqida ma'lumot berilgan. Kalit so’zlar: O’smirlik davri , deviantlik , intellektual qiziqishlar , emansipatsiya . O’smirlarda deviant xulq – atvorning paydo bo’lishidan avval , deviantlik nima ekanligini bilib olishimiz zarur . Deviant xulq - atvor – jamiyatda o’rnatilgan axloq me'yorlariga mos kelmaydigan insoniy faoliyat yoki xatti – harakat ijtimoiy hodisa bo’lib , o’ģrilik , ichkilikbozlik , guyohvandlik , o’z joniga qasd qilish va boshqa ko’plab shu kabi holatlar ushbu xulq – atvor xususiyatlari hisoblanadi. Biz “oģishgan xulq” atamasini 5 yoshdan kichik bo’lmagan bolalarga qo’llash mumkin, qat’iy ma'noda esa – 9 yoshdan keyin qo’llaniladi. Deviant axloqni ko’rib chiqayotib, biz yaqqol salbiy assotsiatsiyani uyģotuvchi so’zni qo’llashga majburmiz : deviant, addikt , noijtimoiy va ijtimoiy xulqqa qarshi va boshqalar. Shu bilan birgalikda , aniq shaxs bilan ishlashda biz ongli ravishda hamda kamsituvchi ohangli atamalardan foydalanishdan , shuningdek , tamģa osishdan tiyilmoģimiz kerak. Chunki kimnidir noto’ģri nom bilan chaqirsak , unga nisbatan adolatsizlik qilgan bo’lamiz. Shuni esda tutishimiz kerakki , birinchidan , axloqiy muammolar o’ta keng tarqalgan . Ikkinchidan , oģishgan xulq chegaralarini aniqlash ko’pincha murakkab. Jamiyatdagi o’zgarishlar me'yorlarning , albatta , axloqiy deviatsiya WSR journal.com Volume – 1_ march 2022 219 Volume – 1_ march 2022 219 WSR journal.com JOURNAL OF NEW CENTURY INNOVATIONS turlarining o’zgarishiga olib keladi. Biroq me'yorlarning o’zi va ulardan oģishish har qanday ijtimoiy jamiyatning ajralmas qismi. Ijtimoiy darajada oģishgan xulq – bu faqat jamiyat va shaxs orasidagi o’zaro munosabatning muhim bo’lgan shakllaridan biri. O’gishgan xulqni ijtimoiy hodisa sifatida “tugatish" dargumon. Har bir holat va vaziyatlarda bu xulqning ko’rinishlari insonlarda namoyon bo’ladi , xoh u maktab bo’lsin , xoh u jamoat joyi hamma yerda oģishgan xulqni ko’rishimiz mumkin. Bundan tashqari , maxsus ko’rib chiqishda deviatsiyalar jamiyat uchun me’yoriy va foydali ekanini isbotlash mumkin , chunki unda istiqbolli o’zgarishlarni raģbatlantiradi. Individual darajada deviant axloq anchagina muammoli ko’rinadi , chunki shaxsning o’zi va atrofdagi odamlar hayoti uchun real zarar , deviant shaxsning ijtimoiy muhit bilan janjali , uning ijtimoiy moslashmaganligi kabi salbiy fenomenlar bilan boģliq. Xususan , oģishgan xulq shaxsiy darajada – bu shaxsning deviant usul va hayot tarzi shaklida yuzaga chiquvchi ijtimoiy nuqtai nazardir. Ma'lumki , ko’pchilik odamlar istasalar jamiyatga nisbatan o’z nuqtai nazarlarini o’zgartirishlari mumkin. JOURNAL OF NEW CENTURY INNOVATIONS oshadi. R. Mertonning anomiya nazariyasiga ko’ra , deviant harakatlar avvalo , jamiyat tomonidan qabul qilingan va o’rnatilgan qiymatlarga erisha olmagan paytda yuzaga keladi. Ijtimoiylashish nazariyasida , odamlar oģish harakatlarining ba'zi birlari raģbatlantirish va e'tiborsiz qoldirish doirasida sodir bo’ladi. Stigmatizatsiya nazariyasida deviant xulq atvorning paydo bo’lishi shaxsni shunchaki ijtimoiy nuqtai nazardan aniqlanishi va unga nisbatan repressiv yoki tuzatish choralarini qo’llash bilan mumkin bo’ladi , deb ishoniladi. Bugungi kunda ko’plab insonlarda deviantlikni ko’rishimiz mumkin. Bu xulq - atvorga ma'lum bir doiradagi omillar sabab bo’ladi. Hozirgi kunda bolada , shaxsda xulq oģishlikni keltirib chiqaruvchi omillar quyidagilar : oshadi. R. Mertonning anomiya nazariyasiga ko’ra , deviant harakatlar avvalo , jamiyat tomonidan qabul qilingan va o’rnatilgan qiymatlarga erisha olmagan paytda yuzaga keladi. Ijtimoiylashish nazariyasida , odamlar oģish harakatlarining ba'zi birlari raģbatlantirish va e'tiborsiz qoldirish doirasida sodir bo’ladi. Stigmatizatsiya nazariyasida deviant xulq atvorning paydo bo’lishi shaxsni shunchaki ijtimoiy nuqtai nazardan aniqlanishi va unga nisbatan repressiv yoki tuzatish choralarini qo’llash bilan mumkin bo’ladi , deb ishoniladi. Bugungi kunda ko’plab insonlarda deviantlikni ko’rishimiz mumkin. Bu xulq - atvorga ma'lum bir doiradagi omillar sabab bo’ladi. Hozirgi kunda bolada , shaxsda xulq oģishlikni keltirib chiqaruvchi omillar quyidagilar :  Biologik omillar – bu asosan shaxsning nerv sistemasi , irsiyati , nasliy kasalliklari bilan boģliq omildir.  Biologik omillar – bu asosan shaxsning nerv sistemasi , irsiyati , nasliy kasalliklari bilan boģliq omildir.  Ijtimoiy omillar – bu bola yoki shaxsning ijtimoiy maqomi , roli , shaxslararo munosabatlardagi o’rni bilan belgilanadigan sabab hisoblanadi.  Psixologik omillar – bu shaxsning temperamenti , ruhiyati bilan uzviy boģliq mezon sanaladi.  Psixologik omillar – bu shaxsning temperamenti , ruhiyati bilan uzviy boģliq mezon sanaladi. Yuqoridagi omillarni bilgan holda , tarbiya o’smirlar hayotida qanday ahamiyatga ega ekanligini ko’rib chiqamiz. Tarbiya ģoyat murakkab psixologik jarayondir. Shuning uchun ham uluģ o’zbek pedagogi va ma'rifatparvari Abdulla Avloniy o’zining “ Turkiy Guliston yoxud axloq “ asarida shunday deb iyozganlar. “ Alxosil , tarbiya bizlar uchun yo hayot – yo najot – yo halokat ,yo saodat – yo falokat masalasidir “. ( Toshkent , “ O’qituvchi “ nashriyoti. 1967 – yil 27 – bet. ) Birinchi Prezidentimiz Islom Abduģaniyevich Karimov ham o’zlarining nutqlarida Abdulla Avloniyning bu fikrlari ma'nosini bir karra quyidagicha ta'kidlab o’tgan edilar: “ Men Abdulla Avloniyning “ Tarbiya biz uchun yo hayot – yo mamot , yo najot - yo halokat , yo saodat – yo falokat masalasidir “. – degan fikrini ko’p mushohada qilaman. Mirvaliyeva Maxliyo Yoqubjon qizi NamDU o'qituvchisi Nabijonova Muslimaxon Baxodirjon qizi NamDU talabasi +998 90 693 94 77 D i t tti h k tl ’ b tid ikki t b ’li di lbi ij bi Deviant xatti – harakatlar o’z navbatida ikki turga bo’linadi : salbiy va ijobiy. Deviant xulqning salbiy turlari: 1. Belgilangan yashash joyisiz yashash ,ko’chalarda tilanchilik , voyaga yetmagan bolalar va o’spirinlar haqida gap ketganda uysiz qolish. 1. Belgilangan yashash joyisiz yashash ,ko’chalarda tilanchilik , voyaga yetmagan bolalar va o’spirinlar haqida gap ketganda uysiz qolish. 2. Hayvonlar va odamlarga nisbatan shafqatsizlik , bu qonun ustuvorligida , lekin boshqalarning tashvishi va qo’rquviga sabab bo’ladi. 3. Yomon odatlarga moyillik – alkogol , giyohvand moddalar. Bu shuningdek , kompyuter va qimor o’yinlariga , fohishalikka , o’ģrilikka va qonunga muvofiq turli darajadagi javobgarlik uchun javobgarlikka tortishni o’z ichiga oladi . Ijobiy turi gepiraktivlik , daho bilan chegaradosh iste'dod , vazminlik - qonun baxtiga zid bo’lmagan harakatlardir. Ijobiy turi gepiraktivlik , daho bilan chegaradosh iste'dod , vazminlik - qonun baxtiga zid bo’lmagan harakatlardir. Deviant xulq – atvor haqidagi olimlarning fikriga keladigan bo’lsak , E. Dyurkgaymning fikriga ko’ra , jamiyat darajasida tartibga solish nazorati zaiflashishi bilan xatti – harakatlardagi oģish ehtimoli sezilarli darajada Deviant xulq – atvor haqidagi olimlarning fikriga keladigan bo’lsak , E. Dyurkgaymning fikriga ko’ra , jamiyat darajasida tartibga solish nazorati zaiflashishi bilan xatti – harakatlardagi oģish ehtimoli sezilarli darajada WSR journal.com Volume – 1_ march 2022 220 Volume – 1_ march 2022 WSR journal.com JOURNAL OF NEW CENTURY INNOVATIONS JOURNAL OF NEW CENTURY INNOVATIONS Buyuk ma'rifatparvarning bu so’zlari asrimiz boshida millatimiz uchun qanchalar muhim va dolzarb bo’lgan bo’lsa hozirgi kunda biz uchun ham shunchalik , balki undan ham ko’ra muhim sanaladi. Chunki ta'lim – tarbiya ong mahsuli , lekin WSR journal.com Volume – 1_ march 2022 221 Volume – 1_ march 2022 221 WSR journal.com JOURNAL OF NEW CENTURY INNOVATIONS ayni vaqtda ong darajasini va uning rivojini ham belgilaydigan omildir. Ta'lim – tarbiya tizimini o’zgartirmasdan turib , ong darajasini o’zgartirib bo’lmaydi. Ongni , tafakkurni o’zgartirmasdan turib esa biz ko’zlagan oliy maqsad – ozod va obod jamiyatni barpo etib bo’lmaydi “. ( “Barkamol avlod orzusi. Toshkent. O’zbekiston milliy ensiklopediyasi” Davlat ilmiy nashriyoti. 2000 – yil Z – b ) Mustaqillikka erishganimizdan so’ng milliy qadriyatlarimizning tiklanishi va xalqimiz azaldan e'zozlab kelayotgan milliy urf – odatlarimiz an'analarimiz by borada katta ahamiyatga ega. Lekin ba’zi oilada tarbiyaviy faoliyatning susayishi natijasida oila a'zolarining xulqida yomon odatlar ya'ni ichkilikbozlik, chekish , narkomaniya , turli diniy oqimlar ta'siriga berilish , ma'naviy buzuqlik yo’liga kirish kabi holatlar kuzatiladi. Hozirgi kunda barcha oilalarning shu qatorda yosh oilalarning ham muhim vaziffalaridan biri – jamiyatda komil insonni shakklantirish va tarbiyalashdir. Chunki shaxsning kamol topishi va tarbiyalanishi oilada , ota – ona va boshqalarning ko’magi yordamida amalga oshadi. Xalqimizda ajoyib bir maqol bor: “ Qush uyasida ko’rganini qiladi “. Oilada qanday muhit bo’lsa , bola shu muhitni o’zida shakllantiradi. Ota – onalar va farzandlar o’rtasidagi nizolar bolalar , qolaversa , o’smirlar ongiga ham salbiy ta'sir ko’rsatadi. Bunga yaqqol misol qilib , bir do’stimizni olishimiz mumkin. U doim darsga kech qolardi. Vazifalarni ham o’rnida bajarmas, ustozlarga gap qaytarar , o’zidan yomon qahramonni yasab olardi. Ustozlar va barcha katta yoshdagi insonlar gapirishsa ham ta'sir qilmasdi. Keyin uni yaxshilab kuzatishdi va asosiy muammo oilada ekanligiga guvoh bo’lishdi , ya'ni uning otasi bilan orasi yaxshi emasdi. Qandaydir masala yuzasidan tortishib , ota o’z bolasiga e'tibor bermaydigan bo’lib qolgan ekan. Bu vaziyatdan ko’rishimiz mumkin bo’lgan holat: o’smir yoshdagi bu bola boshqalarning e’tibori orqali otasidan olmagan mehrni va e’tiborni olmoqchi bo’ladi. Bundan ko’rinib turibdiki , biz hozirda asosiy e'tiborimizni oilaga qaratishimiz kerak. Inson birinchi qadamni , hamma narsani oiladan oladi. Yoshlar va o’smirlarning bo’sh vaqtini mustaqil va mazmunli tashkil etilmasa , xulqi oģishgan bolalar ko’payadi. Ota – onalar Ustozlar va barcha katta yoshdagi insonlar gapirishsa ham ta'sir qilmasdi. Keyin uni yaxshilab kuzatishdi va asosiy muammo oilada ekanligiga guvoh bo’lishdi , ya'ni uning otasi bilan orasi yaxshi emasdi. JOURNAL OF NEW CENTURY INNOVATIONS Qandaydir masala yuzasidan tortishib , ota o’z bolasiga e'tibor bermaydigan bo’lib qolgan ekan. Bu vaziyatdan ko’rishimiz mumkin bo’lgan holat: o’smir yoshdagi bu bola boshqalarning e’tibori orqali otasidan olmagan mehrni va e’tiborni olmoqchi bo’ladi. Bundan ko’rinib turibdiki , biz hozirda asosiy e'tiborimizni oilaga qaratishimiz kerak. Inson birinchi qadamni , hamma narsani oiladan oladi. Yoshlar va o’smirlarning bo’sh vaqtini mustaqil va mazmunli tashkil etilmasa , xulqi oģishgan bolalar ko’payadi. Ota – onalar ichkilikka ruju qo’ysa yoki or – nomusni yiģishtirib qo’yib buzuqichilik qilsa ham shunday bolalar ko’payib boraveradi. Insonning biologik , fiziologik , psixologik WSR journal.com Volume – 1_ march 2022 222 Volume – 1_ march 2022 WSR journal.com 222 JOURNAL OF NEW CENTURY INNOVATIONS o’sishining eng murakkab o’zgarishlar , qayta qurishlar , umid , intilishlar davri va shu bilan birga oģir inqirozlar salbiy holatlar , kechinmalar davri hamdir. Ma'lumki , affektiv qo’zgaluvchanlik ko’p jihatdan o’smirlik davrida to’ģri keladi. Bu holat me’yorda ham kuzatiladi. Uning vujudga kelishi bu yosh fazasining vegetativ – endokrin jihatdan qayta qurishi bilan uzviy boģliqdir. Bu yosh davri ham psixologik tomondan o’zgarishlar bilan xarakterlanadi. Biologik o’zgarishlar endokrin sistemasining qayta ko’rilishi bo’lsa , psixologik o’zgarish portlash va agressiyaga moyil bo’lgan qo’zģaluvchanlikdir. Bu holat ko’pincha ijtimoiy adaptatsiyaning buzilishi jumladan qonunbuzarlikning sabablaridan biridir. Ammo psixik soģlom atrof – muhitning qulay sharoitlarida tarbiyalanayotgan o’smirlarda affektiv qo’zģaluvchanlik adaptatsiyaning buzilishiga olib kelmaydi. Statiskaning ko’rsatishicha , har bir o’ninchi jinoyat voyaga yetmaganlar tomonidan sodir qilinar ekan. O’smirlar psixologiyasini chuqur va har tomonlama bilish , ularni o’qitish va tarbiyalash samaradorligining eng muhim shartlaridan biri. O’smirlarning axloqiy bilimlari bilan ularning axloq tajribasi uzviy boģliq bo’lsada , bu boģlanishning bir necha eng yirik variantlari mavjud: o Asosiy axloqiy normativlar nomigagina beriladi , biroq tegishli tajriba yo’q yoki kam , shuning uchun to’ģri xulq – atvor zarurligiga ham ishonch ham yo’q. o Ijodiy tajriba mavjud , biroq axloqiy xatti – harakat va uning mohiyatiga qo’yiladigan talablar haqida zarur bilim yo’q , yetishmaydi. o Maktabda egallangan bilim va tajriba odamlar bilan shaxsiy muomalada bir – biriga ta'sir ko’rsatish natijasiida hosil qilingan bilim va tajribaga qarama – qarshi bo’lib qoladi. o Nihoyat , axloq qoidalariga xilof xulq – atvor o’smirning axloqiy tasavvuri va e'tiqodi bilan uzviy boģliq bo’lgan hollar ham mavjud. o Nihoyat , axloq qoidalariga xilof xulq – atvor o’smirning axloqiy tasavvuri va e'tiqodi bilan uzviy boģliq bo’lgan hollar ham mavjud. O’quvchining axloqiy rivojlanishi asosan uning tevarak – atrofidagi kishilarga o’z – o’ziga to’ģri munosabatni shakllantirish bilan qo’shilib ketadi. JOURNAL OF NEW CENTURY INNOVATIONS “ Axir tarbiya avvalo , bizning munosabatlarimizdan , masalan , o’z ishiga vijdonan WSR journal.com Volume – 1_ march 2022 223 Volume – 1_ march 2022 WSR journal.com 223 JOURNAL OF NEW CENTURY INNOVATIONS munosabatda bo’lish , o’z vazifasiga ma'suliyat bilan yondashish , is burchiga fidokorona munosabat , kishilarga ziyraklik bilan munosabat , muqaddas O’zbekistonga – ona Vatanga sadoqat bilan munosabat, uning dushmanlariga murosasizlikdan iborat emasmi ?”. Pedagogik jihatdan oz holimga tashlab qo’yilgan o’smirlarning shaxsiy muomala doirasi xususiyatlariga qarab , ko’pincha ularning kishilarga munosabatlari ham aniqlanadi. Bu yorqin xususiyatlardan biri kattalarga , shu jumladan o’qituvchilarga ham ishonmaslik va yoqtirmay munosabatda bo’lishdir. Biroq , , , ular kattalar bilan muomala qilishga ehtiyoj sezadi. Shu bilan birga , ular mustasnosiz hamma kattalarga salbiy munosabatda bo’ladilar. O’smirlarda individual yondashishning eng murakkab sharti , ularning psixologik xususiyatlarini mukammal bilishdir. Ask holda , individual yondashish haqidagi har qanday gap quruq safsata bo’lib qoladi. Shaxsni chuqur va har tomonlama bilish uni alohida ajratilgan individ sifatida emas , balki jamoa bilan aloqada bilish demakdir. O’smirni o’rganishning quyida gap boradigan ajralmas va o’zaro bir – biri bilan boģliqdir. Ular quyidagilardan iborat: Birinchidan: o’smirning individual xususiyatlari ko’p sonli va xilma – xil boģlanish va munosabatlar sistemasida boģliqdir. Bu o’smirning biror bir xususiyatlari yoki xarakter xususiyati ham namoyon bo’la olmaydi va qayd etilmaydi. Masalan , o’smirda o’tkir va sinchkov aqlni payqaymiz. Lekin biz uning aqli nimaga yo’naltirliganligini , uning sinchkovligi kasbini egallashda namoyon bo’lish – bo’lmasligini , uning o’quvchilar bilan o’zaro munosabatiga ta'sir etish – etmasligini va boshqa holatlarni biz bilib olmasak , bu xususiyatning ahamiyati qolmaydi. Ikkinchidan: bu prinsip – tamoyil - obyektivlik. Inson haqida xato o’ta tajribali mutaxassis bo’lmasin , qandaydir bir kishining fikriga qarab xulosa chiqarib bo’lmaydi. Ya'ni har qanday o’smir bo’lmasin , ular haqida noto’ģri fikr qoldirish xatodir. Uchinchidan: uchinchi tamoyil – ulģayishni o’rganishning dinamikligi. Inson umr bo’yi o’zgarib boradi. Yosh ulģayadi. Yosh ulģaygan sari o’zgarish ,asosan WSR journal.com Volume – 1_ march 2022 224 Volume – 1_ march 2022 WSR journal.com 224 JOURNAL OF NEW CENTURY INNOVATIONS JOURNAL OF NEW CENTURY INNOVATIONS , sustlashadi , albatta jismoniy , ba'zi hollarda esa ruhiy holatdagi o’zgarish juda jadallik bilan ro’y beradi. Ochilgan yangi ijobiy potensialdan foydalanish va muqarrarlashib borayotgan kamchilikka nisbatan o’z vaqtida chora ko’rish uchun bu o’zgarish ko’ra bilish o’ta muhimdir. O’smir haqida yagona va bir umrga qayd etilgan fikr bo’lmasligi lozim. Afsuski , ba'zan amalda tarbiyasi qiyin o’smirlarni nisbatan shunday holga duch kelamiz. To’rtinchidan: to’rtinchi tamoyil – prinsip o’rganishning dialektikasidir. Pedagog o’smirning muhim asosiy sifatlariga ko’ra bilish , ularga tasodifiy , ikkinchi darajali abraham topayotgan sifatlardan ajrata bilishi , o’quvchi shaxsidagi qarama – qarshiliklarni seza bilishi hamda ularni hal qilishning pedagogik maqsadga muvofiq yo’llarni dasturlashtirishi lozim. Beshinchidan: beshinchi tamoyil – prinsip o’smirlarni o’rganish doirasidan chiqib , ham o’smirni o’rganish , ham unga tarbiyaviy nuqtai nazardan yondashish tamoyili sanaladi. Ya'ni darsdan tashqari holatlarda ham o’smirni kuzatish va qiziqishlarini bilishdir. Oltinchidan: oltinchi tamoyil – prinsip o’rganish professionalizm ya’ni o’smir psixologiyasini chuqur bilish va uni to’ģri tushuna olishdan iborat. Hayot tajribasi , pedagogik praktika va o’smirlar bilan muomala ularni tushunishda juda katta yordam beradi. Lekin by psixologiya va pedagogikani chuqur o’rganish zaruriyatini inkor etmaydi. Yettinchidan: yettinchi tamoyil – prinsip maqsad ko’zlaganlikdir. O’smirni o’rganish umumiy tarzda uch vazifasini ko’zda tutadi: o’smirni o’qitish va tarbiyalash uchun uning eng qulay xususiyati va imkoniyatlarini aniqlash , uni o’qitish va tarbiyalashda foydalanadigan vositalarning samaradorligini baholash. O’smirlar bilan olib boriladigan ishning barcha bosqichlarida by vazifalar pedagog uchun har xil ahamiyat kasb etadi. O’smir davriga xos bo’lgan ya'ni o’smir xulq – atvorining , muhitning ayniqsa yaqinlarining o’zaro ta’siriga bo’lgan xulq – atvor reaksiyasi ham muhim ahamiyat kasb etadi. O’ziga xos bo’lgan o’smirning xulq – atvorning reaksiyalari quyidagilarga taalluqlidir. Volume – 1_ march 2022 225 WSR journal.com JOURNAL OF NEW CENTURY INNOVATIONS JOURNAL OF NEW CENTURY INNOVATIONS 1. Emansipatsiya reaksiyasi. U kattalarning qaramoģidan , nazoratidan va homiyligidan ozod bo’lishga intilish , harakat qilishda namoyon bo’ladi. Buni asosan , o’smirning uyidan qochib ketishida ko’rishimiz mumkin. 1. Emansipatsiya reaksiyasi. U kattalarning qaramoģidan , nazoratidan va homiyligidan ozod bo’lishga intilish , harakat qilishda namoyon bo’ladi. Buni asosan , o’smirning uyidan qochib ketishida ko’rishimiz mumkin. 1. Emansipatsiya reaksiyasi. U kattalarning qaramoģidan , nazoratidan va homiyligidan ozod bo’lishga intilish , harakat qilishda namoyon bo’ladi. Buni asosan , o’smirning uyidan qochib ketishida ko’rishimiz mumkin. va 2. Tengqurlari bilan guruhlashgan reaksiya. O’smirlarning tengqurlari bilan guruhlarga birlashish va jipslashish – instinktlik darajasiga xos jarayondir. Odatda ikki guruh o’smirlarini ajratib ko’rsatishimiz mumkin. Bular: bir jinsga asoslanib , unda lider mavjuddir va guruhning barcha a'zolari vazifalari , ularning guruhdagi o’rni qat’iy belgilab qo’ygan bo’ladi. Keyingisi rollarni qat'iy taqsim etilmagani va unda doimiy liderning yo’qligi bilan izohlanadi. 3. Qiziqish – xobbi reaksiyasi. O’smirlik davri uchun qiziqish – xobbi o’ziga xos xususiyatni mujassamlashtiradi. Xuddi bola uchun o’yin zarur xususiyatga ega bo’lganidek o’smirning shaxs sifatida shakllanishi uchun qiziqish muhim ahamiyat kasb etadi. 4. Intellektual- estetik qiziqishlar predmetga ( musiqa , tasviriy san’at , radiotexnika , adabiyot va boshqalar ) nisbatan chuqur qiziqish bilan ifodalanadi. Atrofdagilar uchun bu ahamiyatsizdir , lekin o’smirlar uchun bu ularning ahamiyati beqiyosdir. 5. Jismoniy qiziqishlarga o’z kuchi, chidamliligi , chaqqonligi va mohirligini oshirishga bo’lgan harakatlar kiradi. 5. Jismoniy qiziqishlarga o’z kuchi, chidamliligi , chaqqonligi va mohirligini oshirishga bo’lgan harakatlar kiradi. 6. Liderlikka qiziqish boshqalarga rahbarlik qilishga intilish uchun xizmat qiladi. Bunday qiziqish kuchli bo’lgan o’smirlar turli guruhlarda manfaatlarga erishish uchun to’ģri yo’naltirilgan sharoitlarda ijtimoiy foydali faoliyat ko’rsatish mumkin. 7. Yiģishga qiziqish turli xildagi kolleksiyalarni yiģishda namoyon bo’ladi. Har bir kolleksiya muayyan muhim ahamiyatga ega bo’lishini his qilish tufayli ushbu qiziqishning asosini moddiy farovonlikka erishishga yo’naltirilgan harakat tashkil etadi. 8. Dastlabki qiziqishlar. Atrofdagilarning diqqat markazida bo’lishga turtki bo’ladi. Bu yerda eng muhimi badiiy havaskorlik Yoko sport musobaqalarida qatnashish yoki oxirgi modadagi kiyim vositasida o’zini namoyon qiladi. WSR journal.com Volume – 1_ march 2022 226 Volume – 1_ march 2022 WSR journal.com JOURNAL OF NEW CENTURY INNOVATIONS ishlashda yuqorida ko’rib chiqqanimizdek , pedagog va psixologlarning o’rni katta ekanligiga yana bir bor amin bo’lishimiz mumkin. JOURNAL OF NEW CENTURY INNOVATIONS JOURNAL OF NEW CENTURY INNOVATIONS 9. Jo’shqin qiziqish. Doimo , jo’shqinlikning oziga xosligi bilan asoslanadi va katta o’yin , qimorga va garovga intilishda namoyon bo’ladi. Bunday yutuq albatta jozibador bo’lsa ham , lekin qaltis tavakkal qilish his tuyģusi muayyan lazzat baxsh etadi. 9. Jo’shqin qiziqish. Doimo , jo’shqinlikning oziga xosligi bilan asoslanadi va katta o’yin , qimorga va garovga intilishda namoyon bo’ladi. Bunday yutuq albatta jozibador bo’lsa ham , lekin qaltis tavakkal qilish his tuyģusi muayyan lazzat baxsh etadi. 10. Informatsion- kommunikatsion qiziqish yangi yengil axborotlarga ega bo’lishga intilishda mavjud bo’ladi. Ushbu xobbi tasodifan uchrab qolgan suhbatdosh bilan bo’lib o’tadigan ko’p soatli quruq gapga moslangan suhbatda namoyon bo’ladi. Shuningdek sarguzasht , detektiv filmlarni ko’rish orqali ham vujudga kelishi mumkin. Yuqoridagi o’smir reaksiyalari muayyan sharoitda vujudga kelib , ular o’smirlik davrining kechishida o’zining ma'lum bir hissasini qo’shadi. Yuqoridagi o’smir reaksiyalari muayyan sharoitda vujudga kelib , ular o’smirlik davrining kechishida o’zining ma'lum bir hissasini qo’shadi. Tarbiyasi qiyin o’smirlarni bir necha shartli guruhlarga ajratish mumkin:  Orsiz , subutsiz o’smirlar ;  Orsiz , subutsiz o’smirlar ;  Mustaqil fikrga ega bo’lmaganlar ;  Mustaqil fikrga ega bo’lmaganlar ;  Shaxsiy talab va ehtiyojlarini qondirish uchun qonunbuzarlik yo’liga kirgan o’smirlar ;  Injiq tabiatli o’smirlar. Shu kabi o’smirlarni to’ģri yo’liga boshlash uchun faqat ijobiy harakatlar o’smirlardagi buzuq xatti – harakatlarning o’zgarishiga va oldini olishga yordam beradi. Bunday tuzatish shakllariga quyidagilar kiradi:  Kundalik hayot uchun qulay muhit yaratish.  Axborot mazmuni – deviant va to’ģri xulq – atvor o’rtasidagi qanday aniqlash kerakligini tushuntiradigan ma'ruzalar , munozaralar , darslar tashkil etish.  “ Qarama – qarshi “ terapiya – bu odam odatdagi hayotda hech qachon qaror qilmaydigan harakat , masalan , parashut qilish yoki o’zini ijodda ifoda etish.  Jamiyatning axloqiy me’yorlaridan chetga chiqmaydigan ifoda usullarini rivojlantirish. Biz mana shunday o’smirlarni nafaqat , ular balki , yoshlarni tarbiyalashda ham birgalikda yelkadosh bo’lib harakat qilishimiz kerak. Bunday o’smirlar bilan Biz mana shunday o’smirlarni nafaqat , ular balki , yoshlarni tarbiyalashda ham birgalikda yelkadosh bo’lib harakat qilishimiz kerak. Bunday o’smirlar bilan Volume – 1_ march 2022 WSR journal.com Foydalanilgan adabiyotlar ro’yxati  Karimov. I.A “ Barkamol avlod - O’zbekiston taraqqiyotining poydevori “ O’zbekiston Respublikasi Oliy Majlisining IX sessiyasida so’zlagan nutq 1997  “ Barkamol avlod orzusi. Toshkent. O’zbekiston Milliy ensiklopediyasi “ davlat ilmiy nashriyoti. 2000 -yil Z – b  Abdulla Avloniy “ Turkiy Guliston yoxud axloq “ . Toshkent. “ O’qituvchi “ nashriyoti. 1967 – yil 27 – bet  Abdulla Avloniy “ Turkiy Guliston yoxud axloq “ . Toshkent. “ O’qituvchi “ nashriyoti. 1967 – yil 27 – bet  Ģoziyev. E.G. “ Tarbiyasi qiyin o’smirlar psixologiyasi “. Lektorga yordam. Toshkent- 1984  Ģoziyev. E.G. “ Tarbiyasi qiyin o’smirlar psixologiyasi “. Lektorga yordam. Toshkent- 1984  D. Abduvahobova. “ Xulqi oģishgan bolalar psixologiyasi “ . A . Qodiriy nomli Jizzax Davlat Pedagogika Instituti.  Moskva davlat ijtimoiy universiteti. Bolalar va o’smirlarning deviant xatti – harakatlari: muammolar va yechimlar. Moskva shahar ilmiy – amaliy konferensiyasi materiallari. – M: Uyushma , 1996 – yil. Volume – 1_ march 2022 WSR journal.com
https://openalex.org/W4233648211	https://www.qeios.com/read/26G219/pdf	English	null	Patient-Controlled Epidural Analgesia	Definitions	2,020	cc-by	77	Open Peer Review on Qeios Open Peer Review on Qeios Patient-Controlled Epidural Analgesia National Cancer Institute Qeios ID: 26G219 · https://doi.org/10.32388/26G219 Qeios · Definition, February 2, 2020 Source National Cancer Institute. Patient-Controlled Epidural Analgesia. NCI Thesaurus. Code C123385. A method of analgesia that uses an epidural catheter and a pump to allow the patient to administer predetermined doses of analgesic into the epidural space as needed to maintain adequate pain control. Qeios ID: 26G219 · https://doi.org/10.32388/26G219 1/1
https://openalex.org/W3007438208	https://europepmc.org/articles/pmc7819934?pdf=render	English	null	The possibility of endoscopic treatment of cN0 submucosal esophageal cancer: results from a surgical cohort	Surgical endoscopy/Surgical endoscopy and other interventional techniques	2,020	cc-by	6,431	Abstract Background We analyzed the pathological characteristics and recurrence pattern of cN0 submucosal esophageal cancer after esophagectomy and conducted risk stratification to determine the feasibility of performing endoscopic resection for cN0pT1b esophageal squamous cell malignancies. Methods We retrospectively enrolled 167 patients who underwent right-sided transthoracic esophagectomy and extended thoracic/abdominal two-field lymphadenectomy. Patients with pathologically confirmed lymph node metastasis or tumor recurrence constituted the high-risk group for endoscopic submucosal resection, and the remainder were defined as low risk. Factors affecting lymphatic metastasis and long-term recurrence were identified by univariate and multivariate analyses. Results Postoperative pathology showed that five patients (5/167; 3%) had lymph node metastases. Follow-up ranged from 12–60 months, with a median of 29 months. A total of 17 patients (10.2%) had recurrences during follow-up, including three patients with pathologic nodal metastasis (pN +) found at surgery. Invasion depth, differentiation, and tumor size differed significantly in high-risk patients. Overall 3-year survival rates were 94.2% (low-risk) and 40.9% (high-risk) (p < 0.01). Twenty-one patients with sm1 cancer, high tumor differentiation, and tumor length < 2 cm had no lymph node metastasis or lymphovascular invasion, and none of these patients experienced recurrence. Results Postoperative pathology showed that five patients (5/167; 3%) had lymph node metastases. Follow-up ranged from 12–60 months, with a median of 29 months. A total of 17 patients (10.2%) had recurrences during follow-up, including three patients with pathologic nodal metastasis (pN +) found at surgery. Invasion depth, differentiation, and tumor size differed significantly in high-risk patients. Overall 3-year survival rates were 94.2% (low-risk) and 40.9% (high-risk) (p < 0.01). Twenty-one patients with sm1 cancer, high tumor differentiation, and tumor length < 2 cm had no lymph node metastasis or lymphovascular invasion, and none of these patients experienced recurrence. Conclusions Endoscopic submucosal resection alone may be feasible for patients with small (≤ 2 cm) clinically N0 sub- mucosal esophageal squamous cell carcinoma with low invasion depth (sm1) and higher differentiation, but prospective studies are required for confirmation. Other patients require surgical resection with extended two-field thoracic/abdominal lymphadenectomy. Keywords Esophageal · Squamous cell carcinoma · Endoscopic submucosal resection · Clinical N0 pathologic T1b words Esophageal · Squamous cell carcinoma · Endoscopic submucosal resection · Clinical N0 path Superficial esophageal cancer is defined as esophageal carcinoma with tumor invasion limited to the submucosa. and Other Intervent and Other Intervent Surgical Endoscopy (2021) 35:593–601 https://doi.org/10.1007/s00464-020-07420-y Bo Ye1 · Xiaobin Zhang1 · Yuchen Su1 · Shuguang Hao2 · Haohua Teng3 · Xufeng Guo1 · Yu Yang1 · Yifeng Sun1 · Teng Mao1 · Zhigang Li1 Received: 9 October 2019 / Accepted: 10 February 2020 / Published online: 18 February 2020 © The Author(s) 2020 1 Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Huaihaixi Road 241, Shanghai 200030, People’s Republic of China Bo Ye and Xiaobin Zhang have contributed equally to this study. * Zhigang Li dr_lizhigang@163.com 3 Department of Pathology, Shanghai Chest Hospital, Shanghai JiaoTong University, Huaihaixi Road 241, Shanghai 200030, People’s Republic of China Materials and methods is an alternative treatment for T1b patients unwilling to undergo radical resection [3]. A recent study by the Japan Clinical Oncology Group (JCOG0508 study) [9] of patients with T1b (sm1–2) N0M0 thoracic esophageal SCC confirmed the efficacy of endoscopic resection combined with selective chemoradiotherapy, with results comparable to those following surgery [9, 10]. However, patient selection in the JCOG0508 study was based on a main tumor depth of invasion of csm1–2 by endoscopic ultrasonography or pathological diagnosis from endo- scopically resected specimens, which do not accurately reflect the depth of invasion, especially the possibility of sm3 involvement. Additionally, the sample size was not large (n = 86; pT1b). Larger numbers of surgical cases are needed to fully explain which patients are suitable for non-surgical treatment. is an alternative treatment for T1b patients unwilling to undergo radical resection [3]. A recent study by the Japan Clinical Oncology Group (JCOG0508 study) [9] of patients with T1b (sm1–2) N0M0 thoracic esophageal SCC confirmed the efficacy of endoscopic resection combined with selective chemoradiotherapy, with results comparable to those following surgery [9, 10]. However, patient selection in the JCOG0508 study was based on a main tumor depth of invasion of csm1–2 by endoscopic ultrasonography or pathological diagnosis from endo- scopically resected specimens, which do not accurately reflect the depth of invasion, especially the possibility of sm3 involvement. Additionally, the sample size was not large (n = 86; pT1b). Larger numbers of surgical cases are needed to fully explain which patients are suitable for non-surgical treatment. We retrospectively analyzed data from 2023 consecutive patients with primary esophageal SCC who were treated at Shanghai Chest Hospital from January 2014 to Decem- ber 2017. Of these, 229 (11.32%) patients had pathologi- cally confirmed esophageal SCC with submucosal invasion (pT1b). After excluding 62 patients preoperatively diagnosed with lymph node metastasis (cN1), we analyzed data for 167 patients diagnosed with cN0pT1b esophageal SCC (Fig. 1). All 167 patients underwent R0 resection. Our focus in this study was patients with stage cN0pT1b esophageal SCC, therefore, we excluded data for stage cN0cT1b patients. The study protocol was approved by the Shanghai Chest Hospital ethics committee, which waived the requirement for informed consent because of the retrospective design. This retrospective study analyzed data for patients with pathologically confirmed T1b esophageal SCC with clinical N0 submucosal lesions treated with esophagec- tomy in our center over a 3-year period. Fig. 1 Flow chart showing the patient selection process for the study Abstract Because of the wide use of endoscopic screening techniques, approximately 36% of patients with esophageal squamous cell carcinoma (SCC) are diagnosed with superficial lesions in Japan [1]. Improving overall treatment efficacy has become a key in enhancing overall outcomes in patients with esophageal SCC. Because the rates of lymph node metastasis with intramucosal (T1a) esophageal carcinomas are extremely low, endoscopic submucosal resection alone has become the standard treatment [1–3]. In contrast, T1b esophageal carcinomas with submucosal invasion have a much higher lymph node metastasis rate of approximately 12–54% [4–7]. Therefore, the standard treatment for submu- cosal lesions consists of radical resection of the esophageal cancer plus lymph node dissection. Bo Ye and Xiaobin Zhang have contributed equally to this study. * Zhigang Li dr_lizhigang@163.com 1 Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Huaihaixi Road 241, Shanghai 200030, People’s Republic of China 2 Department of Thoracic Surgery, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, People’s Republic of China 3 Department of Pathology, Shanghai Chest Hospital, Shanghai JiaoTong University, Huaihaixi Road 241, Shanghai 200030, People’s Republic of China Bo Ye and Xiaobin Zhang have contributed equally to this study. 2 Department of Thoracic Surgery, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, People’s Republic of China Surgical treatment of esophageal cancer is traumatic for patients and associated with high postoperative com- plication rates [8]. Endoscopic submucosal resection (0121 594 Surgical Endoscopy (2021) 35:593–601 cN0 diagnostic criteria Patients were diagnosed with cN0 disease if carotid/ abdominal ultrasound, endoscopic ultrasound, contrast- enhanced thoracoabdominal CT, and other imaging modalities found no evidence of lymph nodes > 1 cm diam- eter. Other diagnostic criteria for cN0 disease included an absence of central liquefaction/necrosis, peripheral enhancement, or disappearance of the fatty gap near the lymph nodes. No patients underwent endoscopic ultra- sound-guided biopsy to confirm cN0 status. Surgical treatment Decisions on appropriate therapy for superficial esopha- geal SCC were discussed by a multidisciplinary tumor board. No patients received preoperative induction ther- apy, and all patients underwent thoracic and abdominal lymph node dissection, including dissection of the lymph nodes near bilateral recurrent laryngeal nerves. Materials and methods We compared the different clinical features of high-risk and low-risk patients, where high-risk patients were those with surgi- cal lymph node metastasis or later recurrence, and the remainder were defined as low-risk patients. The latter are considered possible endoscopic treatment candidates who can avoid esophagectomy and extensive lymph node dissection. All patients underwent thoracic and abdominal lymph node dissection. We retrospectively reviewed and recorded patients’ general information, surgical procedures, postop- erative hospitalization details, follow-up duration, and recur- rence and survival rates after discharge from the hospital. Preoperative assessment included contrast-enhanced thoracoabdominal computed tomography (CT), cervi- cal ultrasound, and upper gastrointestinal endoscopy. All patients underwent endoscopic ultrasound, and only 53 patients underwent positive emission tomography. Flow chart showing the t selection process for the Fig. 1 Flow chart showing the patient selection process for the study 1 3 Surgical Endoscopy (2021) 35:593–601 595 during each follow-up examination, and we used the Cla- vien–Dindo system to evaluate complications [11]. Statistical analysis All statistical analyses were performed using SPSS software (IBM SPSS Statistics, V21, macOS X; IBM Corp., Armonk, NY). Categorical data were analyzed with the Chi-square test. For continuous data, we used the independent t test or Mann–Whitney U test to analyze age, depth of invasion, and tumor length. We also performed multivariate logistic regression analysis using a backward stepwise procedure and adding all of the significant risk factors from the univariate analysis to identify the significant predictors for the high- risk group. Survival was defined as the time from the first postoperative day to the last day of follow-up or death. We used Kaplan–Meier survival curves to calculate recurrence- free and overall survival for all patients, and p < 0.05 was considered statistically significant. Postoperative pathological diagnosis We analyzed data for 167 patients, namely, 131 men (81.4%) and 36 women (21.6%) with a mean (± standard deviation) age of 62.9 ± 8.8 years. Of these patients, 22 (13.2%) had lesions in the upper third, 111 (66.5%) in the middle third, and 34 (20.4%) in the lower third of the esophagus. Preop- erative T stages included T1a in 30 patients (18%), T1b in 106 (63.5%), T2 in 31 (18.5%), and T3 in no patients; all patients were preoperative N stage 0 (N0). Pathological sections for all clinically diagnosed T1b esophageal SCC samples were retrospectively analyzed by two senior pathologists. T1b (submucosal invasion) was defined as invasion into the submucosal layer but not reaching the muscularis propria. The specific pathologi- cal characteristics that we analyzed were tumor length, location, differentiation, and depth of invasion. Depth of invasion was categorized as: sm1, invasion of the upper third of the submucosal layer or ≤ 200 μm from the mucosal muscularis in the specimens resected by endo- scopic resection; sm2, invasion reaching the middle third of the submucosal layer; or sm3, invasion reaching the lower third of the submucosal layer. Other parameters were lymphovascular invasion, perineural invasion, total num- ber of lymph nodes dissected, and number and location of positive lymph nodes. No patients received preoperative induction therapy. All patients underwent esophageal SCC resection via a right transthoracic approach, with 151 (90.4%) undergoing McK- eown (three-incision) esophagectomy and 16 (9.58%) under- going Ivor–Lewis operations; 136 patients (81.4%) under- went minimally invasive video-assisted thoracoscopy, and 31 (18.6%) underwent open surgery. Complications of any type occurred in 41.3% (69/167) of the patients. The percentage of patients experiencing Clavien–Dindo complications ≥ grade 3 was 12% (20/167). The main complications were anastomotic leak (confirmed radiographically) in 28 patients (16.7%), respiratory insuffi- ciency in 10 (6%) (defined as patients requiring reintubation or a non-invasive ventilator support), and recurrent laryngeal nerve paralysis (confirmed using laryngoscopy 1-week post- operatively) in 19 (11.4%) patients (Table 1). We recorded the concordance rate between the two sen- ior pathologists, and differences in opinion regarding the pathological diagnosis for any sample were resolved by discussion among all of the department’s pathologists. Follow‑up The average hospital stay was 18.5 days (range, 7–190 days). No patients died in-hospital or within 90 days postoperatively (Table 1), and 5 patients with lymph node metastasis received two cycles of adjuvant chemotherapy. Patients were followed at the out-patient clinic or by tel- ephone 1, 3, 6 months, and then every 6 months, post- operatively. Esophagogastroduodenoscopy, and CT of the thorax, abdomen, and neck were routinely performed Postoperative pathological examination showed submu- cosal invasion (T1b) in all patients. The numbers of patients 1 3 Surgical Endoscopy (2021) 35:593–601 596 Table 1 Patients’ demographic and clinical characteristics (n = 167) n number Variable n(%) Age (years) ± standard deviation 62.91 ± 8.80 Sex Male 131(78.44) Female 36(21.56) Tumor location in the esophagus Lower third 34(20.36) Middle third 111(66.47) Upper third 22(13.17) Tumor length (mm) 20.36 ± 10.18 Surgical approach McKeown 151(90.42) Ivor–Lewis 16(9.58) Minimally invasive esophagectomy 136(81.44) Open 31(18.56) Complications Anastomotic leak 28(16.77) Respiratory failure 10(5.99) Recurrent laryngeal nerve paralysis 19(11.38) In-hospital Stay (days) (mean, range) 18.50(7–190) Death within 90 days postoperatively 0(0) in the inferior mediastinum/abdomen, and 1 (5.8%) in the pleural cavity (pleural effusion).f f Univariate analysis showed that degree of differentiation, invasion depth, and tumor length differed significantly in the two groups (p < 0.05 for all comparisons) (Table 2). Multi- variate analysis showed that degree of differentiation, inva- sion depth, and tumor length were independently associated with the likelihood of being in the high-risk group (Table 3). The median postoperative follow-up duration for all patients was 29 months. The median follow-up was 36.4 months (range, 13.5–51.8 months) in the high-risk group and 31.2 months (range, 10.4–57.6 months) in the low-risk group. Overall 3-year survival rates were 94.2% (low-risk group) and 40.9% (high-risk group) (hazard ratio: 9.418, 95% confidence interval: 3.819–23.222; p < 0.01) (Fig. 2). Nineteen patients died after 90 days; 10 in the high-risk group and 9 in the low-risk group. In the high-risk group, 9 patients died of recurrence, and 1 died of abdominal infec- tion. In the low-risk group, 1 patient died of suicide, 1 of heart disease, 3 of lung infection, 1 secondary to anasto- mosis stenosis and malnutrition, and 3 of unknown causes. Follow‑up Regarding the diagnostic thresholds for endoscopic submucosal resection, a separate analysis showed that 21 patients with sm1 tumors, high tumor differentiation, and tumor length < 2 cm had no lymph node metastasis or lym- phovascular invasion, and none experienced recurrence. Of the remaining 146 patients, 5 (5/146, 3.4%) experienced lymph node metastasis (p < 0.01), 17 (17/146, 11.6%) expe- rienced recurrence (p < 0.01), and 10 (10/146, 6.8%) expe- rienced lymphovascular invasion (Table 4). All of these 146 patients had at least one of the following findings: > sm1 tumors, low tumor differentiation, and tumor length > 2 cm. with sm1, sm2, and sm3 invasion were 96/167 (57.5%), 48/167 (28.7%), and 23/167 (13.8%). The median number of dissected lymph nodes was 23 (range, 10–44). Postoperative pathology showed that five patients (5/167, 3%) had lymph node metastases, which were located at the thoracic paratra- cheal (n = 3, 60%), paracardiac (n = 1, 20%), and subcarinal (n = 1, 20%) sites. Of the patients with sm1, sm2, and sm3 invasion, 0, 2/48 (4.2%), and 3/23 (13.04%), respectively, had lymph node metastases (p < 0.05, for all comparisons). Lymphovascular invasion was detected in 10 patients, but none had postoperative lymph node metastasis or recurrence. f The concordance rate between the two senior pathologists analyzing the tissue samples was 89.5% (205/229 histologi- cal diagnosis agreements). p p y p The 19 patients (19/167, 11.4%) with pathologically confirmed lymph node metastasis or tumor recurrence were classified as the high-risk group, namely, 5 patients with positive lymph node metastasis and 14 patients with recur- rent disease, with the remaining 148 patients (148/167, 88.6%) classified as the low-risk group. During follow- up, 17 patients (17/167, 10.2%) developed recurrence at a median of 683.5 days, namely, 14/162 (8.6%) in the pN0 group and 3/5 (60%) in the pN+ group. Of the 14 patients with recurrent tumors in the pN0 group, 12 were classified as sm2 or sm3, 13 had moderate/low differentiation, and 13 had a diameter ≥ 2 cm. Of the 17 patients with tumor recurrence (14 in the pN0 group and 3 in the pN+ group), 9 (52.9%) had recurrent tumors in the neck/superior mediastinum, 5 (29.4%) in the carina/left and/or right bronchus, 3 (17.6%) Discussion Endoscopic submucosal dissection can achieve R0 resection of some localized superficial lesions; however, because of the high rate of lymph node metastasis 1 3 3 Surgical Endoscopy (2021) 35:593–601 597 Values are presented as number, number(percent), or mean ± standard deviation a Mann–Whitney U test; LND number of lymph nodes dissected, LVI lymphovascular invasion, sm1 inva- sion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscu- laris, sm2 invasion reaching the middle third of the submucosal layer, sm3 invasion reaching the lower third of the submucosal layer Variable Low-risk group (n = 148) High-risk group (n = 19) p valuea Sex 0.067 Male 113(76.35) 18(94.74% Female 35(23.65) 1(5.26) Age 63.02 ± 7.3 62.05 ± 8.52 0.946 Surgical approach McKeown 135(91.22) 16(84.21) 0.329 Ivor–Lewis 13(8.78) 3(15.79) Minimally invasive esophagectomy 122(82.43) 14(73.68) 0.356 Open 26(17.57) 5(26.32) Number of LND 24.48 ± 7.90 23.11 ± 6.60 0.504a Tumor location in the esophagus 0.904 Upper third 18(12.16) 3(15.79) Middle third 97(65.54) 12(63.16) Lower third 33(22.3) 4(21.05) Differentiation < 0.001 Well 70(47.3) 2(10.53) Moderate 41(27.7) 5(26.32) Poor 37(25) 12(63.16) Depth of invasion < 0.001 sm1 93(62.84) 3(15.79) sm2 43(29.05) 5(26.32) sm3 12(8.11) 11(57.89) LVI 10 (6.76) 0 0.243 Tumor length (mm) 17.18 ± 10.53 23.71 ± 6.85 < 0.001a < 20 mm 81(54.73) 1(5.26) ≥ 20 mm 67(45.27) 18(94.73) Low-risk group (n = 148) High-risk group (n = 19) p valuea Low-risk group (n = 148) High-risk group (n = 19) p valuea Values are presented as number, number(percent), or mean ± standard deviation a Mann–Whitney U test; LND number of lymph nodes dissected, LVI lymphovascular invasion, sm1 inva- sion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscu- laris, sm2 invasion reaching the middle third of the submucosal layer, sm3 invasion reaching the lower third of the submucosal layer Table 3 Multivariate analysis of factors predictive of low risk (nega- tive lymph node metastasis and recurrence-free survival) following endoscopic submucosal resection for esophageal squamous cell car- cinoma Table 3 Multivariate analysis of factors predictive of low risk (nega- tive lymph node metastasis and recurrence-free survival) following endoscopic submucosal resection for esophageal squamous cell car- cinoma Table 3 Multivariate analysis of factors predictive of low risk (nega- tive lymph node metastasis and recurrence-free survival) following endoscopic submucosal resection for esophageal squamous cell car- cinoma thresholds for endoscopic treatment in these select T1b patients. Discussion The high incidence of complications after surgery for esophageal cancer remains a challenge. In most large-scale studies, the incidence of postoperative complications was approximately 40% [8]. Although advances in minimally invasive resection of esophageal SCC have improved patients’ postoperative quality of life and decreased the incidence of pulmonary complications, overall mortal- ity rates have not changed dramatically [12, 13]. Radical treatment for esophageal SCC includes complete removal of the primary tumor and thorough lymph node dissection or inactivation. Discussion Our study indicated that the degree of tumor differentiation, invasion depth, and lesion size were risk fac- Fig. 2 Overall survival in low- and high-risk patients with clinical N0 pathological T1b esophageal squamous cell carcinoma after endoscopic sub- mucosal resection. OS overall survival Table 4 Differences between G1S<20 and G2–3S>20 patients n number, G1 high differentiation, G2–3S>20 low differentiation and tumor length > 20 mm, LVI lymphovascular invasion, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis, S<20 tumor length < 20 mm Sm1 G1S<20 (n = 21) Others (n = 146) Lymph node metastases 0 5 LVI 0 10 Recurrence 0 17 Fig. 2 Overall survival in low- and high-risk patients with clinical N0 pathological T1b esophageal squamous cell carcinoma after endoscopic sub- mucosal resection. OS overall survival Fig. 2 Overall survival in low- and high-risk patients with clinical N0 pathological T1b esophageal squamous cell carcinoma after endoscopic sub- mucosal resection. OS overall survival rall survival in gh-risk patients al N0 pathological geal squamous cell after endoscopic sub- section. OS overall Table 4 Differences between G1S<20 and G2–3S>20 patients n number, G1 high differentiation, G2–3S>20 low differentiation and tumor length > 20 mm, LVI lymphovascular invasion, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis, S<20 tumor length < 20 mm Sm1 G1S<20 (n = 21) Others (n = 146) Lymph node metastases 0 5 LVI 0 10 Recurrence 0 17 can be used both in the primary tumor diagnosis and in the treatment regime for superficial lesions. Table 4 Differences between G1S<20 and G2–3S>20 patients n number, G1 high differentiation, G2–3S>20 low differentiation and tumor length > 20 mm, LVI lymphovascular invasion, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis, S<20 tumor length < 20 mm Sm1 G1S<20 (n = 21) Others (n = 146) Lymph node metastases 0 5 LVI 0 10 Recurrence 0 17 i To our knowledge, our study is the largest single-center study of patients with cN0pT1b esophageal SCC (n = 167). Because the overall lymph node metastasis rate in our patients (cN0pT1b) was 3% (5/167), R0 resection in patients with cN0pT1b lesions based only on clinical N0 findings is insufficient. Discussion The biological features of clinical N0 submucosal esopha- geal SCC are key to determining the feasibility of endoscopic treatment. However, less is known about the biological fea- tures of cN0pT1b esophageal SCC. Some studies evaluated small numbers of patients with esophageal adenocarcinoma [15, 16], while others included larger numbers and evaluated patients undergoing endoscopic submucosal resection for either T1a or T1b superficial esophageal adenocarcinoma [4, 17–20]. Two studies evaluating larger numbers of T1b patients did not evaluate cN0pT1b patients, specifically [5, 7]. More importantly, these studies did not include subgroup analyses. Because radical endoscopic treatment targets cN0 patients, oncological studies in patients with cN0 lesions can help determine the feasibility of endoscopic treatment. CI confidence interval, OR odds ratio, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis Variable OR 95% CI p value Differentiation 0.138 0.085–0.524 < 0.001* Depth of invasion (sm1) 0.246 0.115–0.634 < 0.001* Tumor length (< 20 mm) 1.085 1.142–1.231 < 0.001* CI confidence interval, OR odds ratio, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis with submucosal esophageal carcinoma, no consensus has been reached regarding endoscopic submucosal resection of submucosal esophageal cancers [14]. Our retrospective analysis of patients with clinical N0 submucosal esophageal SCC identified a low-risk subgroup and provided diagnostic with submucosal esophageal carcinoma, no consensus has been reached regarding endoscopic submucosal resection of submucosal esophageal cancers [14]. Our retrospective analysis of patients with clinical N0 submucosal esophageal SCC identified a low-risk subgroup and provided diagnostic Esophageal endoscopic ultrasonography (EUS) is gener- ally considered the best tool for staging superficial esoph- ageal lesions. In this study, all patients underwent EUS 1 3 598 Surgical Endoscopy (2021) 35:593–601 598 Surgical Endoscopy (2021) 35:593–601 can be used both in the primary tumor diagnosis and in the treatment regime for superficial lesions. To our knowledge, our study is the largest single-center study of patients with cN0pT1b esophageal SCC (n = 167). Because the overall lymph node metastasis rate in our patients (cN0pT1b) was 3% (5/167), R0 resection in patients with cN0pT1b lesions based only on clinical N0 findings is insufficient. Therefore, it is extremely important to identify additional patients at high risk of lymph node metastasis and recurrence. Discussion Additionally, prospective studies are needed to clarify which patients could benefit from endoscopic resec- tion followed by esophagectomy.i tumor differentiation, and tumor length < 2 cm experienced lymph node metastasis, and no patients experienced recur- rence, indicating these measures as diagnostic thresholds for endoscopic submucosal resection. Therefore, simple endo- scopic submucosal resection may be sufficient for superficial lesions with sm1 invasion, high differentiation, and tumor length < 2 cm, which were the diagnostic thresholds for extended endoscopic treatment in these select T1b patients identified in our study. In contrast, surgical resection with extended thoracic and abdominal lymph node dissection remains the optimal treatment for high-risk patients with submucosal esophageal SCC. Postoperative recurrences, particularly micrometastases, provide important clues for analyzing the scope of tumor invasion. Although our patients underwent radical thoracic and abdominal lymph node dissection, 8.4% (14/167) of patients experienced disease recurrence in the pN0 group. In this study, we classified patients with lymph node metas- tasis and/or distant recurrence as the high-risk group, and all others as the low-risk group. Univariate and multivari- ate analyses showed that degree of differentiation, invasion depth, and tumor length differed significantly between these two groups. Only 21/167 patients (12.6%) in our study met all three conditions for our diagnostic thresholds for endo- scopic submucosal resection (i.e., sm1 invasion, high dif- ferentiation, and tumor length < 2 cm). These 21 patients experienced no lymph node metastasis or recurrence, there- fore, endoscopic submucosal dissection with negative resec- tion margins may be an appropriate treatment for inoperable low-risk patients meeting all three of the above conditions. Postoperative pathology showed that five patients had lymph node metastases: three in the left/right recurrent laryngeal nerve/superior mediastinum, one in the gastric car- dia/peritoneum, and one in the carina/left or right bronchus. The three patients with recurrent laryngeal nerve/superior mediastinal metastasis constituted 60% of our patients with metastasis, which is a concern in the West, where dissecting these lymph nodes is rarely performed, even in patients with advanced disease. Rates of lymph node recurrence near the gastric cardia and around the celiac trunk were lower than in the superior mediastinum, showing that surgical treatment effectively controlled metastasis to these regions. Therefore, esophagectomy plus thorough thoracic and abdominal lymph node dissection, followed by small T-shaped field adjuvant radiation therapy in the neck and superior mediastinum, may be a more reasonable option for high-risk patients. Discussion Therefore, it is extremely important to identify additional patients at high risk of lymph node metastasis and recurrence. Our study indicated that the degree of tumor differentiation, invasion depth, and lesion size were risk fac- tors for pathological lymph node metastasis and recurrence. Subgroup analysis showed that lymph node metastasis rates were 0, 1.4% (1/72), and 1.2% (1/82) in patients with sm1, highly differentiated, and smaller (< 2 cm) tumors, respec- tively; findings similar to those observed in patients with intramucosal (T1a) esophageal SCC [6]. Although lymph node involvement has been reported in up to 27% of patients with sm1 tumors, results may have been affected by the num- ber of included patients [15]. Therefore, patients with sm1, highly differentiated, and small (< 2 cm) tumors are more likely to benefit from endoscopic R0 resection, alone. Our separate analysis showed that no patients with sm1, high n number, G1 high differentiation, G2–3S>20 low differentiation and tumor length > 20 mm, LVI lymphovascular invasion, sm1 invasion of the upper third of the submucosal layer or reaching the submucosal layer > 200 µm from the muscularis, S<20 tumor length < 20 mm evaluation preoperatively; however, the diagnostic accuracy of T1b was only 63.5%, preoperatively. Diagnosing the N stage of superficial esophageal cancer appears to be more feasible. We found that the rate of lymph node metastasis in all T1b patients was as high as 29% (67/229) when cN+ was included. However, after preoperative CT and ultrasound evaluation, the postoperative pathological metastasis rate decreased to 3% (5/167) in the cN0 population. Thus, appro- priate N staging can provide sufficiently accurate lymph node results and guide subsequent treatment. It is difficult to obtain a reference value for T staging. Endoscopic resection 1 3 3 Surgical Endoscopy (2021) 35:593–601 599 cancer treated by endoscopic resection followed by chemo- radiotherapy. Therefore, endoscopic resection followed by chemoradiotherapy is now an important treatment strategy for T1b patients with clinical N0 esophageal SCC [10]. As a limitation, the JCOG0508 study involved only 86 (48.9%) pT1b patients and no sm3 patients. Our study involved 167 patients with clinical N0 pathologic T1b esophageal SCC confirmed surgically and 23 (23/167, 13.8%) sm3 patients. Our study is a good supplement to the JCOG0508 study, but studies involving more patients are needed to confirm our results. Compliance with ethical standards 10. Kurokawa Y, Muto M, Minashi K, Boku N, Fukuda H; Gastroin- testinal Oncology Study Group of Japan Clinical Oncology Group (JCOG) (2009) A phase II trial of combined treatment of endo- scopic mucosal resection and chemoradiotherapy for clinical stage I esophageal carcinoma: Japan Clinical Oncology Group Study JCOG0508. Jpn J Clin Oncol 39:686–689 Disclosures Dr. Bo Ye, Dr. Xiaobin Zhang, Dr. Yuchen Su, Dr. Shu- guang Hao, Dr. Haohua Teng, Dr. Xufeng Guo, Dr. Yu Yang, Dr. Yifeng Sun, Dr. Teng Mao, and Dr. Zhigang Li have no conflicts of interest to disclose. Disclosures Dr. Bo Ye, Dr. Xiaobin Zhang, Dr. Yuchen Su, Dr. Shu- guang Hao, Dr. Haohua Teng, Dr. Xufeng Guo, Dr. Yu Yang, Dr. Yifeng Sun, Dr. Teng Mao, and Dr. Zhigang Li have no conflicts of interest to disclose. 11. Clavien PA, Barkun J, De Oliveira ML, Vauthey JN, Dindo D, Schulick RD, de Santibañes E, Pekolj J, Slankamenac K, Bassi C, Graf R, Vonlanthen R, Padbury R, Cameron JL, Makuuchi M (2009) The Clavien-Dindo classification of surgical complica- tions: five-year experience. Ann Surg 250:187–196 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. i 12. Takeuchi H, Miyata H, Gotoh M, Kitagawa Y, Baba H, Kimura W, Tomita N, Nakagoe T, Shimada M, Sugihara K, Mori M (2014) A risk model for esophagectomy using data of 5354 patients included in a Japanese nationwide web-based database. Ann Surg 260:259–266 13. Discussion Additionally, no cN0 patients had lymph nodes 1 3 600 Surgical Endoscopy (2021) 35:593–601 cancer invading muscularis mucosa and submucosal layer. Dis Esophagus 26:496–502 biopsied by endoscopic ultrasonography/fine needle aspira- tion, which could also have affected accurate clinical lymph node staging. 4. Gockel I, Sgourakis G, Lyros O, Polotzek U, Schimanski CC, Lang H, Hoppo T, Jobe BA (2011) Risk of lymph node metastasis in submucosal esophageal cancer: a review of surgically resected patients. Expert Rev Gastroenterol Hepatol 5:371–384 In conclusion, careful clinical stratification of diagno- sis and treatment is required in the treatment of patients with cN0 submucosal esophageal SCC. Simple endoscopic submucosal resection may be sufficient for superficial lesions with sm1 invasion, high differentiation, and tumor length < 2 cm, which were the diagnostic thresholds for extended endoscopic treatment in the select T1b patients identified in our study. More study is required, specifically randomized studies, to make any firm conclusions about endoscopic mucosal resection in this subset of patients. In contrast, surgical resection with extended thoracic and abdominal lymph node dissection remains the optimal treat- ment for high-risk patients with submucosal esophageal SCC. If lymph node metastasis is present, postoperative adjuvant treatment targeting the neck and superior medi- astinum may prevent tumor recurrence. 5. Qi X, Li M, Zhao S, Luo J, Shao Y, Zhang Z, Chen Y (2016) Prevalence of metastasis in T1b esophageal squamous cell carci- noma: a retrospective analysis of 258 Chinese patients. J Thorac Dis 8:966–976 6. Sgourakis G, Gockel I, Lyros O, Lanitis S, Dedemadi G, Polotzek U, Karaliotas C, Lang H (2012) The use of neural networks in identifying risk factors for lymph node metastasis and recom- mending management of t1b esophageal cancer. Am Surg 78:195–206 7. Eguchi T, Nakanishi Y, Shimoda T, Iwasaki M, Igaki H, Tachi- mori Y, Kato H, Yamaguchi H, Saito D, Umemura S (2006) His- topathological criteria for additional treatment after endoscopic mucosal resection for esophageal cancer: analysis of 464 surgi- cally resected cases. Mod Pathol 19:475–480 8. Wright CD, Kucharczuk JC, O’Brien SM, Grab JD, Allen MS; Society of Thoracic Surgeons General Thoracic Surgery Data- base (2009) Predictors of major morbidity and mortality after esophagectomy for esophageal cancer: a Society of Thoracic Sur- geons General Thoracic Surgery Database risk adjustment model. Discussion J Thorac Cardiovasc Surg 137:587–595 Acknowledgements We thank Jane Charbonneau, DVM, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. g 9. Minashi K, Nihei K, Mizusawa J, Takizawa K, Yano T, Ezoe Y, Tsuchida T, Ono H, Iizuka T, Hanaoka N, Oda I, Morita Y, Tajika M, Fujiwara J, Yamamoto Y, Katada C, Hori S, Doyama H, Oyama T, Nebiki H, Amagai K, Kubota Y, Nishimura K, Kob- ayashi N, Suzuki T, Hirasawa K, Takeuchi T, Fukuda H, Muto M (2019) Efficacy of endoscopic resection and selective chemora- diotherapy for stage I esophageal squamous cell carcinoma. Gas- troenterology 157:382–390 Funding This study received funding from the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support. Funding This study received funding from the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support. Compliance with ethical standards Biere SS, van Berge Henegouwen MI, Maas KW, Bonavina L, Rosman C, Garcia JR, Gisbertz SS, Klinkenbijl JH, Hollmann MW, de Lange ES, Bonjer HJ, van der Peet DL, Cuesta MA (2012) Minimally invasive versus open oesophagectomy for patients with oesophageal cancer: a multicentre, open-label, ran- domised controlled trial. Lancet 379:1887–1892 Discussion In this study, the overall incidence of postoperative com- plications was 41.3%, and involved anastomotic leak in 16.7% and respiratory insufficiency in 6% of the patients. No patients died within 90 days postoperatively, and the 3-year overall survival rate for the low- and high-risk groups, inclusive, was 86.9%. Therefore, surgery yielded good early outcomes and long-term survival indicating that, for patients with high-risk submucosal esophageal SCC, endoscopic treatment, even when followed by adjuvant chemoradio- therapy, was not as effective in controlling tumors as there was a surgical resection with extended thoracic and abdomi- nal lymph node dissection, and postoperative cervical and upper mediastinal adjuvant radiotherapy. These survival results support using our diagnostic thresholds for extended endoscopic treatment in our defined low-risk T1b group. No consensus has yet been reached regarding treatment after endoscopic submucosal resection in patients with N0 high-risk submucosal esophageal SCC (> sm1, low degree of differentiation and tumor length > 2 cm). The adequacy of adjuvant chemoradiotherapy as a salvage treatment has not been well-documented [3, 9, 21]; however, our results suggest that salvage adjuvant chemoradiotherapy may be inadequate. First, most of the metastatic lymph nodes in our patients were distributed near the superior mediastinum or recurrent laryngeal nerve (3/5, 60%) and gastric cardia/left gastric artery (1/5, 20%). These findings were consistent with metastases of the submucosal esophageal SCC upward and downward following the long axis of the esophagus [22]. After endoscopic submucosal dissection, adjuvant radio- therapy alone in the area of the primary lesion cannot com- pletely cover both areas, and extensive irradiation in multiple fields may increase toxicity. Furthermore, adjuvant chemo- therapy has a limited role in controlling lymph node metasta- ses in esophageal SCC. These findings suggest that surgical esophageal resection plus radical thoracic and abdominal lymph node dissection may be more effective in controlling these tumors. However, the results of the JCOG0508 study showed excellent long-term outcomes for T1b esophageal i This study has several limitations, namely, its single- center, retrospective design, and the lack of accurate clinical staging, which meant we were unable to compare outcomes of surgical vs non-surgical treatments in our patients with cN0pT1b esophageal SCC. Additionally, the number of sm1 patients was low. Another limitation is the cN0 classifica- tion without staging using positron emission tomography, therefore, some cN0 patients may have been understaged in our study. 21. Mochizuki Y, Saito Y, Tsujikawa T, Fujiyama Y, Andoh A (2011) Combination of endoscopic submucosal dissection and chemora- diation 7therapy for superficial esophageal squamous cell carci- noma with submucosal invasion. Exp Ther Med 2:1065–1068 22. Tanaka T, Matono S, Nagano T, Shirouzu K, Fujita H, Yamana H (2012) Esophagectomy with extended lymphadenectomy for sub- mucosal esophageal cancer: long-term outcomes and prognostic factors. Ann Surg Oncol 19:750–756 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 14. Pennathur A, Farkas A, Krasinskas AM, Ferson PF, Gooding WE, Gibson MK, Schuchert MJ, Landreneau RJ, Luketich JD (2009) Esophagectomy for T1 esophageal cancer: outcomes in 100 patients and implications for endoscopic therapy. Ann Thorac Surg 87:1048–1054 1. Takubo K, Aida J, Sawabe M, Kurosumi M, Arima M, Fujishiro M, Arai T (2007) Early squamous cell carcinoma of the oesopha- gus: the Japanese viewpoint. Histopathology 51:733–742 15. Nentwich MF, von Loga K, Reeh M, Uzunoglu FG, Marx A, Izbicki JR, Bogoevski D (2014) Depth of submucosal tumor infiltration and its relevance in lymphatic metastasis formation for T1b squamous cell and adenocarcinomas of the esophagus. J Gastrointest Surg 18:242–249 2. Tachibana M, Hirahara N, Kinugasa S, Yoshimura H (2008) Clin- icopathologic features of superficial esophageal cancer: results of consecutive 100 patients. Ann Surg Oncol 15:104–116 3. Yoshii T, Ohkawa S, Tamai S, Kameda Y (2013) Clinical outcome of endoscopic mucosal resection for esophageal squamous cell 1 3 Surgical Endoscopy (2021) 35:593–601 601 16. Lee L, Ronellenfitsch U, Hofstetter WL, Darling G, Gaiser T, Lip- pert C, Gilbert S, Seely AJ, Mulder DS, Ferri LE (2013) Predict- ing lymph node metastases in early esophageal adenocarcinoma using a simple scoring system. J Am Coll Surg 217:191–199 g p g y g 17. Aadam AA, Abe S (2018) Endoscopic submucosal dissection for superficial esophageal cancer. Dis Esophagus. https://doi. org/10.1093/dote/doy021 22. Tanaka T, Matono S, Nagano T, Shirouzu K, Fujita H, Yamana H (2012) Esophagectomy with extended lymphadenectomy for sub- mucosal esophageal cancer: long-term outcomes and prognostic factors. Ann Surg Oncol 19:750–756 g y 18. Barret M, Prat F (2018) Diagnosis and treatment of superficial esophageal cancer. Ann Gastroenterol 31:256–265 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 19. Probst A, Aust D, Märkl B, Anthuber M, Messmann H (2015) Early esophageal cancer in Europe: endoscopic treatment by endo- scopic submucosal dissection. Endoscopy 47:113–121 20. Sgourakis G, Gockel I, Lang H (2013) Endoscopic and surgical resection of T1a/T1b esophageal neoplasms: a systematic review. World J Gastroenterol 19:1424–1437 1 3
https://openalex.org/W3043651498	https://zenodo.org/records/59396/files/THE_EFFECTS_OF_THE_PEDAGOGICAL_EXPERIENCE_ON_THE_QUALITY_OF_TEACHER_EDUCATION.pdf	English	null	From Smart Apes to Human Brain Boxes. A Uniquely Derived Brain Shape in Late Hominins Clade	Frontiers in earth science	2,020	cc-by	3,720	1. INTRODUCTION ( ) Abstract. Faculties should ensure integration of science and teaching: science that constantly evolves following the changes in scientific thought and teaching that incorporates these changes in their own organization. So there is a need of including the students, who are prepare for teachers, in the process of pedagogical experience during their study. Pedagogical experience is a completed with a pedagogical practice and hospitation on one side and theoretical knowledge on the other side. Pedagogical experience is a kind of activities that students are involved in the educational process. There have been changes in higher edu cation as a result of many changes in the mod ern life. It takes modern higher education in stitutions to meet the requirements in terms of education of teachers. Faculties should ensure integration of science and practical teaching: a science that constantly evolves following the changes in scientific thought and teaching that incorporates these changes in their own orga nization.Studying the faculties that educates teachers involves professional training in terms of scientific and academic achievements on the one hand and the study of pedagogical and didactic-methodological content, strate gies and models of educational work, on the other hand (Koteva-Mojsovska-Tatiana 2008.) Considering the importance of these activities, we made a research to determine the effects of pedagogi cal experience of students in the fourth year of studies at the Pedagogical Faculty in Skopje. This research is guided by two assumptions: 1. Pedagogical experience as an integral part of studies has positive effects on the quality of initial teacher education and educators; 2. The organization of pedagogical internship does not fully satisfy the educational-applicative needs of students and objectives of the internship. The future teachers acquire knowledge and skills that represent the profession they have chosen. Theoretical knowledge is nec essary but not always sufficient in preparing the appropriate staff. Theoretical knowledge needs to be supported by appropriate practical skills and knowledge. The practical training is able to provide adequate practical support of theoretical knowledge. “The future teachers through practical experience have the chance to be involved in the process of practical teaching. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Dr. Suzana Nikodinovska Bancotovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: suzi.niko.bancot@gmail.com Received: November, 15.2015. Revised: December, 06.2015. Accepted: December, 09.2015. Original Research UDK 371.26-057.875:[378.6:37(497.7)”2014/2015” 159.947.5.072-057.875(497.7)”2014/2015” Received: November, 15.2015. Revised: December, 06.2015. Accepted: December, 09.2015. Original Research UDK 371.26-057.875:[378.6:37(497.7)”2014/2015” 159.947.5.072-057.875(497.7)”2014/2015” THE EFFECTS OF THE PEDAGOGICAL EXPERIENCE ON THE QUALITY OF TEACHER EDUCATION Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Dr. Suzana Nikodinovska Bancotovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: suzi.niko.bancot@gmail.com Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Dr. Suzana Nikodinovska Bancotovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: suzi.niko.bancot@gmail.com Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Dr. Suzana Nikodinovska Bancotovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: suzi.niko.bancot@gmail.com 1. INTRODUCTION ( ) Evidence suggests that teacher learn ing is enhanced in school practice contexts as professional communities - where teachers get along and have a regular professional dia logue with one another, with strong leadership and adequate teaching resources” (Caena, F., 2014.).The future teachers through practical experience have the chance to be involved in the process of practical teaching. During the survey, we have found that the students have good theoretical knowledge about the educational process and are successful in selecting topics for discus sion with the competent persons in institutions. But they are not initiative enough and they are not sufficiently active in the process that have no direct obligation to implement and to record. Because of that, students have to be well prepared and instructed for all activities through the practice that will relate to their overall engagement as teachers.It shows that there is a necessity to redefine the structure, objectives, content and organization of the internship. p Keywords: pedagogical expirience, pedagogical internship, teacher education, practical teaching, edu cator, teacher. Corresponding Author Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Corresponding Author Dr. Tatjana Koteva-Mojsovska, “Ss Cyril and Methodius” University, Faculty of Pedagogy “St Kliment Ohridski”, Skopje, Republic of Macedonia E-mail: tanja.koteva@yahoo.com Pedagogical experience is a completed with a pedagogical practice and demonstration classes on one side and theoretical knowledge www.ijcrsee.com 41 (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Educatio Vol. 3, No.2, 2015. on the other side. Pedagogical experience is the kind of activities that students are involved in the educational process. Students activities are organized and systematized according to previously prepared plan. Pedagogical experi ence includes the involvement of students in the organization and implementation of regular lessons or educational activities in the partner institution. Also, students involved in outside educational activities taking place in the part ner institution in that period. In the implemen tation of pedagogical practice, students can gain performance of the overall organization of life and work in the kindergarten/school. In some educational system, it is called pro fessional integration (Sacilotto-Vasylenko, M., 2010.). Students realize activities/classes which previously are instructed by the men tor. 1. INTRODUCTION ( ) The experience is implemented in order to complete the process of practical training, with appropriate involvement in the educational ac tivities of the kindergarten/school. During the pedagogical experience, students are included in all educational activities, such as the vari ous sections, parent meetings, events, orga nizing leisure activities, lessons and so on. During the pedagogical experience, students develop different kinds of skills such as an ob servation, thinking, understanding, planning, reflection, discussion, evaluation, etc. The study used surveys, interviews, and analysis of documents. The questionnaire was designed for students - trainees, and in it there were issues of open and closed. Besides the questionnaire, with students and some teach ers/educators and we conducted the unstruc tured interview in order to get relevant infor mation on issues that were the focus of our interest. In order to get a fuller picture of acti vation of the students as well as their commit ment to the problems of educational practice, we realized and protocol analysis of the docu ments which they led during the internship. In seniority had adequate working conditions in which each student was constantly active. This means that one mentor tutor/teacher worked with three students. According to the number of mentors, it was the smallest possible num ber of students in a group or separately. Our interest through research focused on several issues: the competence of students for practical application of knowledge, their interest in different aspects of the education al process, creative and innovative potential of students in accordance with the acquired knowledge, opportunities for individual acti vation of students in the educational process, as well as topics of conversation that students realize with educators/teachers during their practical activities, preparation of mentors for mentoring students - trainees. At the end of the questionnaire, we are interested in the opinion of students and their suggestions and proposals for improving the practical teach ing in initial education of future teachers and mentors. 2. MATERIALS AND METHODS In the academic year 2014/15. we con ducted research with students of the fourth year of studies at the Faculty of Pedagogy. The object of the research was testing the effi ciency of the pedagogical internship. The sur vey was conducted in order to determine the effects of pedagogical experience. The survey covered 40 students in the fourth year of stud ies at institutes of pre-school education and el ementary school teaching in Macedonian and Albanian language. This research is guided by two assumptions: Pedagogical experience has positively effected to the systemizing theoretical stu dents knowledge. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. The results are shown in the following Table 1: Table 1. Student needs for intervention in the activities during internship The results are shown in the following Table 1: Table 1. Student needs for intervention in the activities during internship The results are shown in the following Table 1: Table 1. Student needs for intervention in the activities during internship From the table, it is evident that most of the students have need of intervention in the evaluation of the educational process (85%) and in the cooperation with parents (87.5%). This certainly can be justified by the fact that they showed no initiative for the imple mentation of these key issues. The interview with students showed that teachers/educators avoided situations in which students should realize their competencies. include practice only by the available activi ties which are offered by partner educational institution. The responses received are read in the second table. Table 2. Initiative of students for new activities dur ing the seniority ing the seniority p In this study, we are interested in the opinion of students regarding the effects of pedagogical experience. Namely, we wanted to know whether the seniority helped system atize the knowledge acquired through theo retical part of initial teacher education. That is the answer we received in addition to the positive effects of pedagogical experience. In fact, most of the respondents, or 82.5% of the total, prefer this answer: the pedagogical experience helped me to systematize my the oretical knowledge. For the answer: The se niority partially helped me to systematize my theoretical knowledge, opted 6 respondents or 15% of the total while only one respondent stated that seniority did not help him to sys tematize theoretical knowledge. This suggests that pedagogical experience as an integral part of initial education has an important role in shaping the educational staff.fi From said table, it is evident that stu dents do not show initiative for activities that are important for their service. Through inter views with them, we found that this is because they do not know whether they have the right to initiate and content of the program outside the school/kindergarten in a given period. 3. RESULTS AND DISSCUSION Data analysis resulted in interesting findings related to the pedagogical experi ence of students in the fourth year of study. But in this paper are shown results in specific answers to the respondents. Accordingly that regarding the competence of students-trainees for practical application of theoretical knowl edge we are interested in whether and what kind of intervention by the mentor is needed. (Никодиновска-Банчотовска, С., 2008.). p 1. Pedagogical experience as an inte gral part of studies has positive ef fects on the quality of initial teacher education and educators; 2. The organization of pedagogical internship does not fully satisfy the educational-applicative needs of students and objectives of the in ternship. www.ijcrsee.com 42 (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. As far as the activation of students in activities necessary to realize the pedagogi cal internship, we were interested in whether and to what extent (fully, partially or not at all) they are activated in the process of their imple mentation (Table 3). The responses confirmed some information obtained through other questions in the questionnaire and through conducted interview. Table 3. Activation of students during internship Table 3. Activation of students during internship In terms of efficiency, also we were in terested in whether students during internship shown initiative for the organization and im plementation of activities they need. In this case, we wanted to know whether the students (respondents) have initiated activities that are important for their profiling, but which are not currently active within the school/kindergar ten and are not placed in the current period. Or www.ijcrsee.com 43 (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. and the monitoring of various kinds of activi ties are placed on the lowest level of priority in the opinion of students respondents. In an interview conducted with these respondents said they had no opportunity to be in contact with the director, what confirms the statement that in the partner institutions do not adapt ac tivities and events based on student needs and goals of the pedagogical experience. In addi tion to this is the interview with the teachers and educators in which it is determined that one part of them received some training for mentoring students, but it was focused on the interpretation of the manual that applies to all three parts of teaching practice with students (pedagogical practice, attending and pedagog ical experience). The interview also showed that instruction is not adapted by teachers and educators to student’s needs and the needs arising from the objectives of the pedagogical experience. The period for implementation of the activities was also short (3 weeks). In connection with the activation of students in different types of activities, it is evident that all students (100%) are fully in volved in activities that had an obligation to implement them (preparation, implemen tation, and records per hour). Interesting for us is the inclusion of students in the teaching process. Namely, in this type of activities stu dents showed 100% full activation. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. But this response, however, refers only to the classes which was assigned to realize them, for a total of 6 hours from 15 days pedagogical experi ence. Because students have to take the full role of the teacher during the days of intern ship, this situation is not satisfactory. This is probably due to the majority of students which must rotate in a group or department versus the number of partner educational institutions that perform an internship and versus the num ber of mentors, mentors, and tutors. In other situations, a small number of students interns shown its activation. These situations actually relate to activities which in the current period are not provided within the school/kindergar ten. It coincides with the data obtained by the issue of the initiative of the side of students. ( ) The question that referred to the themes that have been the subject of discussion be tween students and tutor/teacher was from the open source. In this respect, the results show that the most common topics of conversa tion were: the organization of school hours, the content of pedagogical Documentation and ways of its guidance, treatment and mo tivating children/students, the organization of the day in kindergarten, working conditions, monitoring developmental achievements of the children, the openness of kindergarten/ school, etc. Students who realized experience in kindergarten were interested in adopting children. Students who realized experience in primary schools showed interest in contempo rary learning strategies and parental involve ment in education. On the questions What are your general expressions from the whole educational ac tivity in the school. The responses have been ranked according to percentage obtained from analysis: y 1. methods of the work- 22 answers or 55% 2. adequately arranged interior and ex terior- 11 answers or 27,5% , 3. ways of planning and programming the educational work – 4 answers or 10 % 4. wholesome realization of the educa tional process- 3 answers or 7,5%. p , In terms of types of activities during the realization of the practice, we are interested in the opinion of students about the priority on the following activities: Maintenance of educational activities and classes; Chat with educator/teacher; conversation with the direc tor; talk with counselor/psychologist, activi ties for monitoring and insight into pedagogi cal documentation. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. Among other activities, a huge part of students (38 students or 95%) give an accent on the involvement in maintain ing educational activities and classes. In 90% of cases (36 students/participants), they have decided to talk to the teacher as an important activity, while 29 respondents or 72.5% of the surveyed students give priority to the conver sation with the counselor/psychologist. Inter estingly, the conversation with the director The survey was completed with propos als for improving the quality of pedagogical experience obtained by the students who are preparing for teachers and educators. In fact, they said that the internship should be longer; should have fewer students together in the same educational group/grade; there is a need to keep a diary of realization of all activities for an internship in the kindergarten/school etc. Also, students propose a final report that will summarize the experiences and knowl edge gained through internship and it will be a theme for discussing with their teachers in the initial education. www.ijcrsee.com 44 (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. Conflict of interests A h d l Conflict of interests Authors declare no conflict of interests. Conflict of interests Authors declare no conflict of interests. 4. CONCLUSION proach to the development of future teachers and educators.i Hence, it can conclude that these find ings resulting from analysis of the data con firmed the two hypotheses laid the basis for research or: From the analysis and interpretation of the survey results, one can conclude that the pedagogical experience has positively effect to the systemizing theoretical students’ knowl edge. The processing of the data showed that students have good theoretical knowledge about educational processes and are success ful in selecting topics for discussion with the competent persons in institutions. But on the other hand, they are not enough initiative and are not sufficiently active in processes that have no direct obligation to implement and recorded. This part is interesting that there is not enough initiative and motivation and by teachers / educators in practice. Regarding the organization of pedagogical experience, we concluded that in the current period in partner educational institutions there is not adjust on the educational process according to the needs of students and the purposes of the internship. We estimate that this part of the implementa tion of the internship should be governed by a written cooperation agreement between the Faculty and its partner educational institu tions. 1. Pedagogical experience as an inte gral part of studies have positive ef fects on the quality of initial teacher education and educators; ; 2. The organizational structure of the pedagogical experience does not fully satisfy educational needs of students and objectives of the intern ship. p Considering the role of the pedagogical experience on the quality of the education of future teachers and educators, it is necessary redefining its structure, objectives, content and organization of the internship. ACKNOWLEDGMENTS For this research we express acknowl edgment to the students of the Pedagogical Faculty in Skopje that were involved in the process, as well as to the professional staff in educational institutions that enabled us to ob serve the process and to do interview during the pedagogical experience of the students. It can also conclude that during the se niority students demonstrate competence in the practical implementation of knowledge. Only in some activities that we appreciate as more complex and sensitive requiring an in tervention in terms of their planning, realiza tion, and understanding. It is justified given that the internship process is implemented for the duration of the studies. (IJCRSEE) International Journal of Cognitive Research in Science, Engineering and Education Vol. 3, No.2, 2015. REFERENCES Given that students are not distributed by one of them in a group or department they are not able to realize all the activities or classes under a program for them. Accordingly, over the course of an internship does not realize a sufficient number of classes and activities in accordance with the students needs involved in the process. n addition to all can be con cluded that the students did not take the full role of the teacher / educator during the intern ship, which could jeopardize the smooth pro cess of practicing the theoretical knowledge. This means students to be well prepared and instructed for all activities through the prac tice that will relate to their overall engagement as teachers. Namely the realization of practi cal teaching students only in terms of main tenance of educational activities and lessons will not provide the complete experience of student-teacher activities, and fully experi ence the educational atmosphere in the institu tion. That would mean and unprofessional ap Caena, F. (2014). Teacher Competence Frameworks in Europe: policy-as-discourse and policy-as- practice. European Journal of Education, 49(3), 311-331.ii Career Entry profile for Newly Qualified Teachers (Standards for the Award of Qualified Teacher Status), 1997. London: Teacher Training Agen cy. y DeRoche, E. F. (1987). An administrator’s guide for evaluating programs and personnel: An effec tive schools approach. Publication Sales.i Koteva-Mojsovska, T. (2008). Profiling Modern Teach er - Reality and Needs Contemporary Intentions in Education, University “Ss. Cyril and metho dius” Skopje, Faculty of Pedagogy “Ss. Kliment Ohridski” Skopje. pj McMillan, J. H., & Wergin, J. F. (1998). Understanding and Evaluating Educational Research. Prentice- Hall, Inc., One Lake Street, Upper Saddle River, NJ 07458. Moyles, J. R. (1988). Self-evaluation: a primary teach er’s guide. Delmar Pub.i g National Standards for Qualified Teacher Status (1998). London: Teacher Training Agency, G. Britain. g g y, Sacilotto-Vasylenko, M. (2009). Improving policy and www.ijcrsee.com 45 practice of teacher induction into the profes sion. ADVANCING QUALITY CULTURES FOR TEACHER EDUCATION IN EUROPE: Ten sions and Opportunities, 121. Schratz, M. (2009). What is a ‘European teacher’. EN TEP the first ten years after Bologna, 97-102. Никодиновска-Банчотовска, С, (2008). Професио- налните компетенции на наставниците, Академски печат, Скопје. www.ijcrsee.com www.ijcrsee.com 46
https://openalex.org/W4226381033	https://ujvas.com.ua/index.php/journal/article/download/103/125	English	null	Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs	Ukrainian journal of veterinary and agricultural sciences	2,021	cc-by	5,415	Citation: Citation: Mysak, A. R., & Ivashkiv, B. B. (2021). Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(3), 62–67. Citation: Mysak, A. R., & Ivashkiv, B. B. (2021). Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(3), 62–67. Mysak, A. R., & Ivashkiv, B. B. (2021). Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(3), 62–67. nancy and the appointment of adequate treatment, prediction of biological behavior, and survival of animals are based on the results of histological examination (Thompson et al., 2010; Blackwood et al., 2012; Camus et al., 2016; Hergt et al., 2017). Abstract According to foreign researchers, mastocytoma is one of the most common (7–12 %) skin tumors in dogs. The study aimed to study the features of the clinical manifestation of cutaneous mast cells according to the criteria of the clinical TNM classification. We analyzed 24 cases of cutaneous mastocytoma in dogs during 2016–2020, of diagnostic and therapeutic measures in the Department of Surgery and Clinic of the Small Pets Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv. According to the criteria of the internation- al clinical TNM classification at the time of admission to the clinic in 7 (29.2 %) dogs established stage I of the tumor, 10 (41.7 %) – II, 6 (25 %) – III, and 1 4.1 %) animals – stage IV. Based on cytological evaluation of biopsy material selected from tumors and regional lymph nodes of low-grade mastocytes, 17 (70.8 %) animals were found, and in 7 (29.2 %) cases, neoplasia was characterized as high-grade. 2021 Mysak A., and Ivashkiv B. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords: dog, mast cell tumor, clinical signs, cytological examination, mast cell classification. Contents 1. Introduction ……..……….. 2. Materials and methods …... 3. Results and discussion …... 4. Conclusions ……………… References ………………….. 62 63 63 66 66 Citation: Mysak, A. R., & Ivashkiv, B. B. (2021). Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(3), 62–67. Contents 1. Introduction ……..……….. 2. Materials and methods …... 3. Results and discussion …... 4. Conclusions ……………… References ………………….. 62 63 63 66 66 Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs A. R. Mysak, B. B. Ivashkiv Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, Pekarska Ukraine J Veterinary and Agricultural Sciences http://ujvas.com.ua Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv Volume 4 Number 3 original article UDC 616-006: 636.7 doi: 10.32718/ujvas4-3.11 Features of the clinical manifestation of the cutaneous form of mastocytoma in dogs A. R. Mysak, B. B. Ivashkiv Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, Pekarska Str., 50, Lviv, 79010, Ukraine Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv 3. Results and discussion During 2016–2020, diagnostic tests and treatment of 24 dogs (13 dogs and 11 bitches, different breeds, aged 4 to 16 years) with skin mastocytoma were performed. In 22 (91.7 %) studied dogs, neoplasia was single and only in 2 (8.3 %) cases – multiple nodular lesions. The most common sites of MCTs were: torso (45.8 %), limbs (37.5 %), less common head and neck, axillary, inguinal, and perineal- perianal areas (16.7 %). At the initial examination, at the time of admission to the clinic, among these animals found stage I of the tumor process (T1aN0M0, T1aN1aM0) in 7 (29.2 %) dogs, 10 (41.7 %) animals – II (T2aN0M0, T2aN1aM0, T2aN1bM0, T2bN1bM0), in 6 (25 %) – III (T3aN0M0, T3aN1bM0, T3bN1aM0) and in 1 (4.1 %) ani- mals – stage IV (T3b, in N1bM1). A detailed analysis of the epidemiological situation, the results of clinical trials of dogs, and the dynamics of development of MCTs in them was presented by us in a previous study (Ivashkiv et al., 2020). However, due to the importance of establishing a pathomorphological diagnosis of clinical data: symptoms of neoplasia, its location, growth rate and doubling of tumor size, germination in subcutaneous tissue, lesions and spread (metastasis) to regional lymph nodes, internal organs, etc., we in this the article focuses only on clinical trial data. In particular, in the studied dogs, stage I neoplasia had the appearance of a small, dense, well-demarcated, mobile tu- mor node located in the thickness of the skin. Stage II tu- mors were 3–5 cm in size, and stage III tumors were 6–10 cm or more. In some dogs, eczema-like skin lesions similar to nodular panniculitis occurred. In 10 (41.7 %) of the stud- ied animals, the growth rate of neoplasms was extremely rapid because, in 56.5 ± 1.91 days, the tumors doubled in size. In 7 (29.2 %) dogs, the doubling time of the primary tumor reached, on average, four months (122.1 ± 10.6 days), and in 5 (20.8 %) dogs, this period lasted for two years. Information on tumor development in 2 (8.3 %) dogs was unknown. For most mast cells, in contrast to other solid tumors, the increase in their size was provoked, as a rule, by episodic inflammation and edema in the tumor and sur- rounding tissues, which is associated with mast cell activa- tion and degranulation syndrome. 1. Introduction Mast cell tumors (MCTs) of skin neoplasms, the propor- tion of mast cells can be 7 to 21 % MCTs are characterized by excessive proliferation of atypical mast cells. Also, by their activation and degranulation (mast cell activation syn- drome), active substances, mediators, and specific IgE anti- bodies. Under such conditions, the growth and development of mast cells are usually masked by inflammation, diffuse edema, allergic reactions, and other pathological processes that cause a variety of appearances and symptoms of these neoplasms and cause periodic fluctuations in tumor size. It is also noted that the clinical course of mastocytoma is usu- ally accompanied by periods of exacerbation and remission, so it is difficult to assess biological behavior and predict the prognosis. According to the generalized results of studies for identifying MCTs in dogs, cytological examination of the biopsy obtained by fine-needle aspiration tumor biopsy (FNA) is sufficient. However, the final diagnosis of masto- cyte and its classification according to the degree of malig- , ) A meta-analysis of current scientific sources shows that due to advanced technologies to study MCTs' molecular genetic and immunobiological properties, many researchers have identified many changes and atypicalities in subcellular structures that may indicate malignant neoplasia. However, as the present shows, approaches to the pathomorphological criteria for mast cell verification and their evaluation sys- tems are constantly being refined and improved and are often the subject of debate. Thus, for a long time in veteri- nary oncology, the three-stage histological classification of MCTs by Patnaik et al. (1984). According to this classifica- tion, mastocytomas are divided into high- (G1), moderate- (G2), and low-differentiated (G3). The most characteristic morphological features of grade I mast cells are considered, as a rule, their limited skin location the presence of well- Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 62 differentiated with noticeable metachromatic cytoplasmic granules. At the same time, low degree of cell differentia- tion, aggressive growth, moderate or high mitotic activity, mitotic atypia, the presence of single or no cytoplasmic granules are the main signs of grade III neoplasia. Grade II mastocytomas are an intermediate variable between the two previous groups in this context. 1. Introduction Accordingly, when as- sessing the potential biological behavior of MCTs, the high- est probability of survival is inherent in animals with the established category G1 and the worst prognosis (survival up to one year) for G3. According to numerous researchers, neoplasia with moderate differentiation (G2) is the most problematic in predicting their biological behavior, as pathomorphologists may include neoplasms of both G1 and G3 in doubtful or vague histological signs. Therefore, it is difficult to predict the aggressiveness of the further devel- opment of such tumors. The 2-3 smears were made from the obtained aspirates, dried in the air, fixed with methyl alcohol for 5 minutes, stained according to Romanovsky-Gimza, and studied under a microscope using an immersion system. The criterion for verifying mastocytoma was detecting homologous round tumor cells with azurophilic granules in the cytoplasm in cytological preparations. y g All dogs, after cytological confirmation of MCTs, un- derwent surgical treatment. Removal of tumors was per- formed under general anesthesia following the rules of ablastics and antiblastics. 3. Results and discussion And to a lesser extent, the growth of tumor mass. It was noted that the rapidly pro- gressing course of the disease was characteristic of mast cells localized in the pelvic limbs, groin, perineum, neck, namely areas freely available for permanent injury and, above all, self-injury (combing, licking, gnawing). Many researchers also report the aggressive development of MCTs with rapid metastasis to regional lymph nodes with the lo- calization of lesions in the muzzle, including the lips, peri- neum, foreskin, scrotum p In 2011, a team of scientists from different countries proposed a two-stage classification of MCTs, dividing mas- tocytomas into high-grade and low-grade. According to researchers, the two-stage classification (Kiupel et al., 2010) eliminates the prognostic uncertainties of the Patnaik sys- tem, has high predictive value, minimal variability, and high rate (96.8 %) consistency in pathomorphological assess- ment. However, it does not provide 100 % accuracy in pre- dicting the biological behavior of MCTs. In recent years, many papers have been published indicating that the histo- logical method of cell phenotyping should be considered the basis for the final diagnosis and classification of neo- plasms. However, regardless of the evaluation system used, the prognosis should be based on a combined ap- proach. According to the literature, prognostic information should take into account clinical signs and anatomical location of the tumor (Thompson et al., 2010; Garrett, 2014), growth rate, size, general appearance (Mullins et al., 2006; Pierini et al., 2019), clinical-stage (Stefanello et al., 2015; Sabattini et al., 2015; Warland et al., 2015), recur- rences, metastases (Ferrari et al., 2018; Horta et al., 2018; Cruz et al., 2020), immunohistochemical and molecular markers (Sabattini et al., 2015; Sledge et al., 2016; Kiupel & Camus, 2019). Based on this, our research aimed to establish the features of the clinical manifestation of the cutaneous form of mastocytoma in dogs. 2. Materials and methods It was found that more than 37 % of biopsies obtained by “blind” biopsy were un- suitable or uninformative for cytological studies due to in- sufficient or poor (unpresentable) cellular content of the obtained material, and this required a repeat procedure. q p p Furthermore, such a biopsy poses a serious risk of dis- similation of tumor cells in cases of penetrating the tumor node or getting a needle into a vessel. The use of sonogra- phy allowed to establish the boundaries, shape, and size of the tumor, its echotexture, tissue perfusion (Doppler). Ac- cordingly, it provides visual control of the targeted selection of material from specific foci of neoplasia. In addition, so- nography also provided additional clinical information on the state of neoplasia in real-time. For example, sonographic visualization of solid hypervascular foci with a central type of vascularization, against the background of diffuse infiltra- tion of neoplasia into the subcutaneous tissue, with a pro- nounced perinodular inflammatory response of surrounding tissues is evidence of mastocytoma malignancy. Undoubted- ly, such data are essential to the cytological evaluation of the biopsy and interpretation of the diagnosis. Following the generally accepted recommendations for verifying neoplasms at the stage of preoperative examina- tion, fine-needle biopsy (FNA) of tumors and regional lym- phatic and cytological examination of aspirates was per- formed. It was noted that the result of such a study largely depends on the quality of the biopsy. Unlike other skin tu- mors, the clinical manifestation of mastocytoma is often accompanied by inflammation, diffuse edema, and other pathological processes, which usually cause structural changes in the tumor site and surrounding tissues and create Fig. 1. Performing a targeted fine-needle biopsy under ultrasound control Fig. 1. Performing a targeted fine-needle biopsy under ultrasound control Thus, it can be noted that despite the more painstaking preparation and time spent on fine-needle biopsy under ultrasound control, this manipulation allowed us to obtain quality material for cytological verification of mastocytoma. into low- and high-malignant tumors. The granularity of the cytoplasm was assessed as intensely granular and lightly granular, sometimes mixed (with the simultaneous presence of the first and second). Nuclear pleomorphism was assessed as present if no rounded nuclear forms were present and absent if only rounded or ovoid forms were noted. Collagen fibrils, mitotic figures, and binuclear or multinucleated cells were assessed as present or absent. 2. Materials and methods The study included 24 dogs with mastocytoma of the skin. During 2016–2020 underwent an entire course of diag- nostic and therapeutic measures in the Department of Sur- gery and Clinic of the Small Pets Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv. The animals were of different breeds and genders, aged 4 to 16 years. Upon admission to the clinic, cancer patients were examined according to clinical (TNM) classi- fication of tumors (Owen, 1980), using clinical, instrumen- tal, and laboratory research methods. The cytological examination was performed to verify neoplasia at the stage of preoperative examination. Selection of material from tumor-like skin formations and regional lymph nodes was performed by fine-needle biopsy (FNA) in two variants. When the puncture was performed by “blind” method and by targeted biopsy under ultrasound control “ESAOTE MyLab 40” 5.0–7.5 and 15 MHz, respectively). Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 Ukrainian Journal of Veterinary and Agri g 63 The results of the study of the local process showed that in more than 87 % of cases, the characteristic signs of mas- tocytoma were: swelling and inflammation in the tumor and surrounding tissues, alopecia, erythema and skin ulcers, periodic fluctuations in tumor size, pain in palpation, itchy skin in the tumor area. Examination of regional lymph nodes in 11 (45.8 %) dogs with cancer did not reveal any changes in the latter. In 8 (33.3 %) animals, the regional lymph nodes are slightly enlarged but not painful, mobile, not compacted, with a smooth surface. At the same time, in 5 (20.8 %) dogs with tumors of considerable size (more than 10 cm in diameter), lymph nodes were 2–3 cm in size, pas- sive, dense consistency. In one of these animals, the regional lymph node palpation was complicated by its growth and fixation to the surrounding tissues. difficulties in the biopsy. According to the results of com- parative studies of biopsy material obtained by the "blind" method of fine-needle biopsy and target biopsy performed under ultrasound control (Fig. 1), we have substantiated the significant advantages of the latter. 2. Materials and methods Anisocariasis was de- fined as >50 % of nucleus size fluctuations. According to the research results, the above morphological parameters are easily identifiable by cytological evaluation of aspirates. They have high specificity (94–97.3 %) for histological evaluation of masocytes and are essential for developing treatment strategies and tactics. According to numerous literature sources, screening for verification of mastocytoma is the detection of atypical mast cells by cytological examination of tumor biopsies. Thus, our study's establishment of the primary diagnosis was based on the cytological identification of mast cells of large round cells with purple granules in the basophilic cytoplasm. It should be noted that the cytological study also focused on the morphological structure of mast cells. In particular, they were evaluated for intracytoplasmic granules, nuclear pleo- morphism, mitotic figures, bi- or multinucleated, and an- isocariasis. The two-level classification criteria according to the Kiupel system, according to which MCTs are divided icultural Sciences 2021 Vol 4 N 3 For MCTs of low malignancy, the most typical cytologi- cal feature was detecting round or oval mast cells, 1.5–3 Ukrainian Journal of Veterinary and Agricultural Sc 64 For cytograms of MCTs, the high degree of malignancy (Fig. 2) was characterized by visualization in the studied smears of atypical mast cells located in dense clusters or layers. The cytoplasm of most cells with fuzzy contours and low content of light purple granules. In some cells, intra- cytoplasmic granules are absent altogether. Cells with irreg- ularly shaped nuclei and binuclear and, less frequently, multinucleated mast cells were often found in the smears studied. At the high magnification of the microscope in some cytological smears, the number of detected mitoses could reach five figures. times larger than neutrophils, which were located in groups with different cell numbers (Fig. 2). The nuclei of such mast cells are round or oval, clearly delineated. Visualization of the nucleus is problematic because it is poorly stained and covered with granules that absorb large amounts of dye and shade it. The cytoplasm of most cells is densely filled with dark purple granules, as a result, such cells had an intense blue-violet color. At the same time, cells with a lower con- tent of granules in the cytoplasm acquired a light purple hue. 2. Materials and methods Single binuclear and giant mast cells were rarely found in the smears, and single cells with high magnification (400 times) were found in the cytograms of individual tu- mors in a state of mitosis. Fig. 2. Cytogram of mastocytoma with a low degree of malignancy. Clusters of cells of homogeneous structure. Mixed-grained mast cells (most cells are highly granular and fewer less granular) with minimal anisocariasis; According to Romanovsky- Gimza. EP. 10, lens 100 Fig. 2. Cytogram of mastocytoma with a low degree of malignancy. Clusters of cells of homogeneous structure. Mixed-grained mast cells (most cells are highly granular and fewer less granular) with minimal anisocariasis; According to Romanovsky- Gimza. EP. 10, lens 100 Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 65 mast cells (most cells are highly granular and fewer less granular) with minimal anisocariasis; According to Romanovsky- Gimza. EP. 10, lens 100 Fig. 3. Cytogram of mastocytoma of high malignancy. Mast cells with a small number of intracytoplasmic granules and slight anisocariasis; nuclear pleomorphism - the presence of non-rounded nuclear forms (1); the presence of dinuclear (2) or multinucleated cells. According to Romanovsky-Gimza. EP. 10, lens 100 1 2 Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 65 , Fig. 3. Cytogram of mastocytoma of high malignancy. Mast cells with a small number of intracytoplasmic granules and slight anisocariasis; nuclear pleomorphism - the presence of non-rounded nuclear forms (1); the presence of dinuclear (2) or multinucleated cells. According to Romanovsky-Gimza. EP. 10, lens 100 1 2 Fig. 3. Cytogram of mastocytoma of high malignancy. Mast cells with a small number of intracytoplasmic granules and slight anisocariasis; nuclear pleomorphism - the presence of non-rounded nuclear forms (1); the presence of dinuclear (2) or multinucleated cells. According to Romanovsky-Gimza. EP. 10, lens 100 65 65 It is now known that an important prognostic indicator for the diagnosis and evaluation of the biological behavior of MCTs is the detection of metastases in regional lymph nodes and internal organs. Based on the research results, cytological criteria for diagnosing lymphogenic metastases have been developed. Many researchers have found that metastasis to regional lymph nodes in dogs with cutaneous MCTs correlates with reduced survival time and a high risk of spreading to remote areas. Reports at the stage of pre- operative examination for neoadjuvant therapy and lym- phadenectomy. 2. Materials and methods or not.According to the results of our stud- ies, a correlation was established between the clinical mani- festation of MCTs and cytology data was found. Cytological evaluation of aspirates of regional lymph nodes from seven dogs and lymphoid cells and neutrophils revealed an accu- mulation of atypical polymorphic mast cells in the form of nests of 3–5 cells, which is evidence of tumor metastasis. According to the clinical (TNM) classification in all these dogs, stage III and IV tumors were large, and regional lymph nodes were enlarged. Cytological smears of eight dogs with clinical stage I and II neoplasia showed many neutrophils and lymphoid elements: large lymphocytes, prolymphocytes, lymphoblasts and, at the same time, the presence of single mast cells. Considering the physical ex- amination data, ulcers on the skin surface in the tumor area, lymphadenomegaly, it was possible to predict reactive lym- phadenitis. Cytological examination of aspirates from lymph nodes that were not enlarged pathomorphological changes was not found. Dobson, J. M. (2013). Breed-predispositions to cancer in pedigree dogs. ISRN Vet Sci., 2013, 941275. DOI: 10.1155/2013/941275. Ferrari, R., Marconato, L., Buracco, P., et al. (2018). The impact of extirpation of non‐palpable/normal‐sized regional lymph nodes on staging of canine cutaneous mast cell tumours: A multicen- tric retrospective study. Vet Comp Oncol., 16, 505–510. DOI: 10.1111/vco.12408. Garrett, L. (2014). Canine mast cell tumors: diagnosis, treatment, and prognosis. Vet Med (Auckl), 2014(5), 49–58. DOI: 10.2147/VMRR.S41005. Gieger, T. L., Theon, A. P., Werner, J. A., McEntee, M. C., Rassnick, K. M., & DeCock, H. E. V. (2003). Biologic behav- ior and prognostic factors for mast cell tumors of the canine muzzle: 24 Cases (1990-2001). Journal of Veterinary Internal Medicine, 17(5), 687–692. DOI: 10.1892/0891- 6640(2003)017<0687:BBAPFF>2.3.CO;2. Goralskyj, L. P., Homych, V. T., & Kononskyj, O. I. (2005). The main base of histological techniques and morphological meth- ods in normal and pathological conditions. Zhytomyr: “Poliss- ja” (in Ukrainian). Grüntzig, K., Graf, R., Hässig, M., et al. (2015). The Swiss Canine Cancer Registry: a retrospective study on the occurrence of tumours in dogs in Switzerland from 1955 to 2008. J Comp Pathol, 152(2–3), 161–171. DOI: 10.1016/j.jcpa.2015.02.005. Hergt, F., von Bomhard, W., Kent, M. S., & Hirschberger, J. (2017). Use of a 2-tier histologic grading system for canine cu- taneous mast cell tumors on cytology specimens. Vet Clin Pathol., 45(3), 477–483. DOI: 10.1111/vcp.12387. Hillman, L. A., Garrett, L. D., De Lorimier, L. P., Charney, S. C., Borst, L. 2. Materials and methods B., & Fan, T. M. (2010). Biological behavior of oral and perioral mast cell tumors in dogs: 44 cases (1996-2006). J Am Vet Med Assoc., 237(8), 936–942. DOI: 10.2460/javma.237.8.936. Horta, R. S., Lavalle, G. E., Monteiro, L. N., Souza, M. C. C., Cassali, G. D., & Araújo, R. B. (2018). Assessment of Canine Mast Cell Tumor Mortality Risk Based on Clinical, Histologic, Immunohistochemical, and Molecular Features. Vet Pathol., 55(2), 212–223. DOI: 10.1177/0300985817747325. 4. Conclusions According to the results of preoperative clinical exami- nation of 24 studied dogs and cytological evaluation of bi- opsy material selected from tumors and regional lymph nodes of low-grade mastitis, 17 (70.8 %) animals and 7 (29.2 %) cases of neoplasia were characterized as high- grade. Ivashkiv, B. B., Mysak, A. R., & Pritsak, V. V. (2020). Clinical characteristics of mastocytoma in dogs. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotech- nologies. Series: Veterinary sciences, 22(98), 144–153. DOI: 10.32718/nvlvet9825 (in Ukrainian). Kiupel, M., & Camus, M. (2019). Diagnosis and Prognosis of Canine Cutaneous Mast Cell Tumors. Vet Clin North Am Small Anim Pract., 49(5), 819–836. DOI: 10.1016/j.cvsm.2019.04.002. Conflict of interest The authors declare that there is no conflict of interest. Kiupel, M., Webster, J. D., Bailey, K. L., Best, S., DeLay, J., Detri- sac, C. J., Fitzgerald, S. D., Gamble, D., Ginn, P. E., Gold- schmidt, M. H., Hendrick, M. J., Howerth, E. W., Janovitz, E. B., Langohr, I., Lenz, S. D., Lipscomb, T. P., Miller, M. A., Mis- dorp, W., Moroff, S., Mullaney, T. P., Neyens, I., O'Toole, D., Ramos-Vara, J., Scase, T. J., Schulman, F. Y., Sledge, D., Smed- ley, R. C., Smith, K., Snyder, P., Southorn, E., Stedman, N. L., Steficek, B. A., Stromberg, P. C., Valli, V. E., Weisbrode, S. E., Yager, J., Heller, J., & Miller, R. (2010). Proposal of a 2-tier his- tologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol, 48(1), 147–155. DOI: 10.1177/0300985810386469. References (2016). Cytological grading of canine cutaneous mast cell tumours. Vet Comp Oncol., 14(3), 245–251. DOI: 10.1111/vco.12090. Misdorp, W. (2004). Mast cells and canine mast cell tumours. A review. Veterinary Quarterly, 26(4), 156–169. DOI: 10.1080/01652176.2004.9695178. Sfiligoi, G., Rassnick, K. M., Scarlett, J. M., Northrup, N. C., Gieger, T. L. (2005). Outcome of dogs with mast cell tumors in the inguinal or perineal region versus other cutaneous loca- tions: 124 cases (1990-2001). J Am Vet Med Assoc., 226(8), 1368–1374. DOI: 10.2460/javma.2005.226.1368. Moore, A. S., Frimberger, A. E., Taylor, D., & Sullivan, N. (2020). Retrospective outcome evaluation for dogs with surgically ex- cised, solitary Kiupel high-grade, cutaneous mast cell tumours. Vet Comp Oncol., 18(3), 402–408. DOI: 10.1111/vco.12565. Shaw, T., Kudnig, S. T., & Firestone, S. M. (2018). Diagnostic accuracy of pre-treatment biopsy for grading cutaneous mast cell tumours in dogs. Vet Comp Oncol, 16(2), 214–219. DOI: 10.1111/vco.12346. Mullins, M. N., Dernell, W. S., Withrow, S. J., Ehrhart, E. J., Thamm, D. H., & Lana, S. E. (2006). Evaluation of prognostic factors associated with outcome in dogs with multiple cutane- ous mast cell tumors treated with surgery with and without ad- juvant treatment: 54 cases (1998-2004). J Am Vet Med Assoc, 228(1), 91–95. DOI: 10.2460/javma.228.1.91. Sledge, D. G., Webster, J., & Kiupel, M. (2016). Canine cutaneous mast cell tumors: A combined clinical and pathologic approach Sledge, D. G., Webster, J., & Kiupel, M. (2016). Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection. Vet J., 215, 43–54. DOI: 10.1016/j.tvjl.2016.06.003. to diagnosis, prognosis, and treatment selection. Vet J., 215, 43–54. DOI: 10.1016/j.tvjl.2016.06.003. Murphy, S., Sparkes, A. H., Blunden, A. S., Brearley, M. J., & Smith, K. C. (2006). Effects of stage and number of tumours on prognosis of dogs with cutaneous mast cell tumours. The Veteri- nary Record, 158(9), 287–291. DOI: 10.1136/vr.158.9.287. Smiech, A., Slaska, B., Lopuszynski, W., Jasik, A., Bochyńska, D., & Dąbrowski, R. (2018). Epidemiological assessment of the risk of canine mast cell tumours based on the Kiupel two-grade malignancy classification. Acta Veterinaria Scandinavica, 60, 70. DOI: 10.1186/s13028-018-0424-2. Northrup, N. C., Howerth, E. W., Harmon, B. G., Brown, C. A., Carmicheal, K. P., Garcia, A. P., Latimer, K. S., Munday, J. S., Rakich, P. M., Richey, L. J., Stedman, N. L., & Gieger, T. L. (2005). References Baginski, H., Davis, G., & Bastian, R. P. (2014). The prognostic value of lymph node metastasis with grade 2 MCTs in dogs: 55 cases (2001-2010). J Am Anim Hosp Assoc., 50(2), 89–95. DOI: 10.5326/JAAHA-MS-5997. y, R. C., Smith, K., Snyder, P., Southorn, E., Stedman, N. L., teficek, B. A., Stromberg, P. C., Valli, V. E., Weisbrode, S. E., Blackwood, L., Murphy, S., Buracco, P. et al. (2012). European con- sensus document on mast cell tumours in dogs and cats. Vet Comp Oncol, 10(3), e1–e29. DOI: 10.1111/j.1476-5829.2012.00341.x. Yager, J., Heller, J., & Miller, R. (2010). Proposal of a 2-tier his- tologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol, 48(1), 147–155. DOI: 10.1177/0300985810386469. Camus, M. S., Priest, H. L., Koehler, J. W., Driskell, E. A., Rakich, P. M., Ilha, M. R., & Krimer, P. M. (2016). Cytologic Criteria for Mast Cell Tumor Grading in Dogs With Evaluation of Clin- ical Outcome. Vet Pathol., 53(6), 1117–1123. DOI: 10.1177/0300985816638721. Krick, E. L., Billings, A. P., Shofer, F.S., Watanabe, S., & Sorenmo, K. U. (2009). Cytological lymph node evaluation in dogs with mast cell tumours: association with grade and sur- vival. Vet Comp Oncol., 7(2), 130–138. DOI: 10.1111/j.1476- 5829.2009.00185.x. Christopher, M. M., & Ku, C. K. (2018). Likelihood of Neoplasia for Diagnoses Modified by Probability Terms in Canine and Feline Lymph Node Cytology: How Probable Is Probable? Front Vet Sci., 5, 246. DOI: 10.3389/fvets.2018.00246. Lapsley, J., Hayes, G., Janvier, V., Newman, A., Peters-Kennedy, J., Balkman, C., Sumner, J., & Johnson, P. (2020). Influence of locoregional lymph node aspiration cytology vs sentinel lymph node mapping and biopsy on disease stage assignment in dogs with integumentary mast cell tumors. Veterinary Surgery, 50. DOI: 10.1111/vsu.13537. Cruz, V. S., Borges, J. C. A., Nepomuceno, L. L., Gonçalves, P. A. M., Prado, Y. C. L., Bianchi, C., Fioravanti, M. C. S., & Araújo, E. G. (2020). Histological classification and expression of markers of canine mast cell tumors. Vet World., 13(8), 1627–1634. DOI: 10.14202/vetworld.2020.1627-1634. London, C. A., & Seguin, B. (2003). Mast cell tumours in the dog. Veterinary Clinics of North America Small Animal Practice, 33(3), 473–489. DOI: 10.1016/S0195-5616(03)00003-2. Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 66 London, C. A., & Thamm, D. H. (2013). Mast cell tumors. In Withrow and MacEwen's small animal clinical oncology (pp. 335–355). WB Saunders. Scarpa, F., Sabattini, S., & Bettini, G. References Variation among pathologists in the histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference. J Vet Diagn Invest., 17(6), 561–564. DOI: 10.1177/104063870501700606. Stefanello, D., Buracco, P., Sabattini, S., Finotello, R., Giudice, C., Grieco, V., Iussich, S., Tursi, M., Scase, T., Di Palma, S., Bet- tini, G., Ferrari, R., Martano, M., Gattino, F., Marrington, M., Mazzola, M., Elisabetta Vasconi, M., Annoni, M., & Marcona- to, L. (2015). Comparison of 2- and 3-category histologic grad- ing systems for predicting the presence of metastasis at the time of initial evaluation in dogs with cutaneous mast cell tu- mors: 386 cases (2009-2014). J Am Vet Med Assoc, 246(7), 765–769. DOI: 10.2460/javma.246.7.765. Owen, L. N. (1980). TNM Classification of Tumors in Domestic Animals. Geneva: World Health Organization. URL: https://apps.who.int/iris/handle/10665/68618. Tham, D.M., & Vail, D. M. (2007). Mast cell tumors. In: Withrow S. J. & Vail D. M. (Eds). Small Animal Clinical Oncology. St. Louis: Saunders Elsevier, 402–424. Patnaik, A. K., Ehler, W., & MacEwen, E. (1984). Canine Cutane- ous Mast Cell Tumor: Morphologic Grading and Survival Time in 83 Dogs. Veterinary pathology, 21, 469–474. DOI: 10.1177/030098588402100503. , Thompson, J. J., Pearl, D. L., Yager, J. A., Best, S. J., Coomber, B. Thompson, J. J., Pearl, D. L., Yager, J. A., Best, S. J., Coomber, B. L., & Foster, R. A. (2010). Canine subcutaneous mast cell tu- mor: characterization and prognostic indices. Vet Pathol., 48(1), 156–168. DOI: 10.1177/0300985810387446. Pierini, A., Lubas, G., Gori, E., Binanti, D., Millanta, F., & Mar- chetti, V. (2019). Epidemiology of Breed-Related Mast Cell Tumour Occurrence and Prognostic Significance of Clinical Features in a Defined Population of Dogs in West-Central Ita- ly. Vet Sci., 6(2), 53. DOI: 10.3390/vetsci6020053. 48(1), 156–168. DOI: 10.1177/0300985810387446. Villamil, J. A., Henry, C. J., Bryan, J. N. et al. (2011). Identifica- tion of the most common cutaneous neoplasms in dogs and evaluation of breed and age distributions for selected neo- plasms. J Am Vet Med Assoc., 239(7), 960–965. DOI: 10.2460/ javma.239.7.960. Pizzoni, S., Sabattini, S., Stefanello, D., Dentini, A., Ferrari, R., Dacasto, M., Giantin, M., Laganga, P., Amati, M., Tortorella, G., & Marconato, L. (2017). Features and prognostic impact of distant metastases in 45 dogs with de novo stage IV cutaneous mast cell tumours: A prospective study. Vet Comp Oncol., 16(1), 28–36. DOI: 10.1111/vco.12306. Warland, J., Brioschi, V., Owen, L., & Dobson, J. (2015). Canine mast cell tumours: decision-making and treatment. References In Pract., 37(7), 315–332. DOI: 10.1136/inp.h3440. Romansik, E.M., Reilly, C.M, Kass PH, Moore PF, London CA. Mitotic index is predictive for survival for canine cutaneous mast cell tumors. Vet Pathol., 44(3), 335–341. DOI: 10.1354/vp.44-3-335. Weishaar, K. M., Thamm, D. H., Worley, D. R., & Kamstock, D. A. (2014). Correlation of nodal mast cells with clinical out- come in dogs with mast cell tumour and a proposed classifica- tion system for the evaluation of node metastasis. J Comp Pathol., 151(4), 329–338. DOI: 10.1016/j.jcpa.2014.07.004. Sabattini, S., Scarpa, F., Berlato, D., & Bettini, G. (2015). Histo- logic grading of canine mast cell tumor: is 2 better than 3? Vet Pathol, 52(1), 70–73. DOI: 10.1177/0300985814521638. Welle, M. M., Rohrer Bley, C., Howard, J., & Rufenacht, S. (2008). Canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment. Veterinary Dermatology, 19(6), 321–339. DOI: 10.1111/j.1365-3164.2008.00694.x. Sapierzyński, R. (2018). Badanie histopatologiczne w onkologii weterynaryjnej. Część III. Guzy z komórek tucznych u psów Życie Weterynaryjne, 93(3), 169–174. Ukrainian Journal of Veterinary and Agricultural Sciences, 2021, Vol. 4, N 3 67 g 67
https://openalex.org/W3046620113	https://essuir.sumdu.edu.ua/bitstream/123456789/79258/1/Trifu_Is_It_Necessary_a_Renewal.pdf	English	null	Is It Necessary a Renewal of the Theory of the Firm Based on Pandemic realities?	Business ethics and leadership	2,020	cc-by	4,029	Is It Necessary a Renewal of the Theory of the Firm Based on Pandemic realities? http://doi.org/10.21272/bel.4(2).96-101.2020 http://doi.org/10.21272/bel.4(2).96-101.2020 Alexandru Trifu, ORCID: https://orcid.org/0000-0001-9891-4836 PhD, Professor, Faculty of Economics, Department of Economic Sciences, University "Petre Andrei” of Iasi, Iasi, Romania Abstract This paper deals with the study of the reverse impact made by the state regulation of entrepreneurial activity on economic development indicators and with substantiation of the expediency to develop new business management theories. According to the analysis of studies on "firm" definition and the peculiarities of its operation, these issues have been actively studied since Adam Smith when the firm was an important component of the marketplace, and until the legislative definition and certification of its operation. The article considers the firm a legal entity, an open economy institution operating under corporate law. The study is conducted based on a critical review of the literature in this field, including information and news of the pandemic period, the synthesis of which allows to draw conclusions about the possibility and need to update the theoretical basis for organizational management, based on the deduction method. According to the analysis results, today, the firm operates in conditions of increased competition and an open environment. Its main functions are the production of goods or services, making a profit and meeting the needs of the population, government agencies or other firms, and the basis of the activities of legislation and regulations approved by government agencies. One of the main problems in modern firms' functioning is the coronavirus pandemic, which is the essential structural crisis in the economy after World War II. It caused rising unemployment, significant loss of profits, and bankruptcy of small and medium-sized enterprises. Thus, one of the most stable and efficient sectors of the pandemic economy is the IT business and the catering sector. The study shows the need to transform organizations' existing management strategies in terms of their adaptation to the realities of the modern world economic system. The study results can be useful for managers and heads of enterprises in improving the efficiency of firms during the crisis. Keywords: Lockdown, Pandemic, Profit, Public Authorities Support, Theory of the Firm, Wellbeing. JEL Classification: D21, E71, M38. This work is licensed under a Creative Commons Attribution 4.0 International License Cite as: Trifu, A. (2020). Is It Necessary a Renewal of the Theory of the Firm Based on Pandemic realities? Business Ethics and Leadership, 4(2), 96-101. http://doi.org/10.21272/bel.4(2).96-101.2020. © The Author, 2020. This article is published with open access at Sumy State University. © The Author, 2020. This article is published with open access at Sumy State University. Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 Exposure of the Issue The literature in this important domain of our lives and economic activities, that is the complex Theory of the Firm, is very large and diverse, starting around 1930s with the work of Ronald Coase (1937), laureate of the 1991 Nobel Prize in Economic Sciences. In this paper, we are interested in recent achievements in the field, particularly, regarding psychological aspects as well as the relationships between entrepreneurs/CEOs/managers and employees/workers that ensure the well-functioning of the firm and sustainability on its specific market (Sautet, 2006). Peter Klein (2016) is one interesting author in what we intend to highlight, because he successfully and to a large extent included the theory of the entrepreneurs into the main theoretical ground of the Theory of the Firm and, at the same time, emphasized the specific influence of the particularities of the theory of entrepreneurs on a better understanding of the multidimensional corpus of the Theory of the Firm (Klein, 2016). Also, the work of P. Aghion, Dewatripont, Legros, Zingales (2016) is an attempt to refine the existing theory and it is, also, an example for the increasing use of the language and style of contemporary formal economics (that is activities embedded in a legal framework, with continuous relationships between public authorities and the other economic and social actors). Paraphrasing a well-known scientific title, we can say that entrepreneurs/CEOs need firms (small, medium, corporate) and, vice versa, firms need good managing theories and decisions. The lockdown was the main sanitary decision to curb the spread of the new coronavirus, and the new modality of working from home, where it was possible, was successfully used in order to maintain the safety of employees and, at the same time, to not hamper the output and to sustain the business activity. The entrepreneurs (from the French word entrepreneur for a person who engages in a business) are considered even a factor of production within a standard economic system (along with land, workforce, capital). In fact, we may consider them along with CEOs and managers as "propellers" for the economic system, the main human resource for the existence of economic and social activities. In the present situation, we are interested in the ways of re-establishing economic life and in the modalities of managing entities/organizations after the obvious huge slowing down of all social and economic activities. Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 We are living in times when the authorities and public sector (administration and, especially, health care system) are in charge in determining measures, in the form of state of emergency, or of a state of alert, to manage the people lives and social-economic activities. Economies and societies/groups are stick together and, of course, policies are needed to be issued for all domains of activities, for both public and private sectors. These new realities, the necessity of problem-solving, even life and death, involve a rethinking of what to do to meet the urgent demands, but also demands to spin the economic life. Introduction The problem to be analyzed in the following rows started from the general concept of Management which surrounds us in every respect of our lives and activities. Browsing the literature in the field and, at the same time, the feedback coming from the practice, taking into account the unexpected and even invisible challenges, we agree that for now and for medium-run, the knowledge, abilities, skills, specific psychological traits as positive thinking and empathy, humans will remain the most adequate and functional beings to manage [18]. Developing and adapting these theories of management, entrepreneurship, or leadership to the new realities and challenges, it is possible and we are saying, even mandatory, to find new theoretical approaches, capable to fix these realities and to lead to functional strategic framework of management and of the other components. The basic approaches remain, including the decisive action of decision-making, both in SMEs and TNCs (Hebert at al., 2009; Zimmerer et al., 2008). The pandemic came and alters the structural relationships, especially when it’s about strategic management and development (Barney et al., 2006). That is even the theoretical construction of management theories, these ones pliable to the best applications for the workforce and company’s culture, based on knowledge and skills (Grant, 1996). 96 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 We are living in times when the authorities and public sector (administration and, especially, health care system) are in charge in determining measures, in the form of state of emergency, or of a state of alert, to manage the people lives and social-economic activities. Economies and societies/groups are stick together and, Exposure of the Issue Therefore, we looked for some appropriate organization managing theories and theories of entrepreneurship that relate to the scope of the present paper. In this respect, we have chosen theories X, Y, and Z and, on the other hand, the Cochran and Kirzner theories of entrepreneurship. Theory X, Y was stated and sustained by Douglas McGregor (2006), a management professor at MIT. In his capital work of 1960, The Human Side of Enterprise, the author focused on how managers/CEOs perceive and address employee’s motivation, under the form of two manifestations: Theory X and Theory Y. The first one (X) assumes that the monetary factor is paramount for employee motivation and, we may say, the support for pursuing happiness. This aspect is doubled by the security need, as in the basic needs level of the Maslow Pyramid of Needs (McLeod, 2020): 97 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 Figure 1. Maslow Pyramid of Needs Source: McLeod 2020 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 Figure 1. Maslow Pyramid of Needs Source: McLeod, 2020 Theory Y assumes that it is an authentic possibility to align personal goals and organization goals, with people/employees being considered creative and participative to the organization actions, and with the esteem and self-actualization levels (Figure 1) being seen as ongoing needs for people, but never completely satisfied. Theory Y assumes that it is an authentic possibility to align personal goals and organization goals, with people/employees being considered creative and participative to the organization actions, and with the esteem and self-actualization levels (Figure 1) being seen as ongoing needs for people, but never completely satisfied. Theory Z, on the other hand, stresses the need/intention to help workers to become somehow generalists in their domain of expertise, rather than specialists. It was introduced by management professor Ouchi (1993) who presented some assumptions about employees, such as: developing co-operative relationships with co- workers, i.e. an imperative desire for affiliation, and that workers expect reciprocity and support from the company. This the aspect that we want to highlight working people seek support from their firm to secure and maintain a balance between work and personal life, in the latter being included family, culture, habits and even the pursuit of happiness. Methodology The approach of the literature review in this field, along with the information and news from the pandemic period, were the basic elements of this paper’s scientific construction. The synthesis of all these elements led to the issuance of our own opinion, through deduction method, regarding the possibility and the necessity of renewing the theory on the matter of organization management. The ongoing process of checking realities on which to base a renewal of the theory. The realities during the pandemic time, with priority on a decisive policy of lockdown, have shown that for ensuring the general restart of economies, for the elimination of technical unemployment and for diminishing the general unemployment level it is mandatory that economic and social activities return to a reasonable intensity. What we saw that thee cities were silent; the quasi-majority of businesses were closed because the lockdown was the main measure taken by the authorities in order to stop the spread out of the new coronavirus. The healthcare was the priority, but, at the same rank, it was necessary to envisage solutions to stop the failing down of the economies because anyone felt that the pandemic was and is still bad for economy and society. So, what we also see is that the European Union is prepared to support the member states’ economies with a huge recovery fund of more than 540 billion euros , under the German and French initiative, in a so-called "Hamilton moment- agreement”1 between the wealthiest and most powerful countries of the EU. This is meant to be a breakthrough moment in the lockdown situation of entire Europe (McCaffrey, 2020). Under the German and French initiative, the European Union has managed a concrete solution, but there are other solutions to come up which will use the general EU budget to transfer money to those countries or regions that need it most. In this respect, the management of European Commission has drafted a plan of more than a trillion Euros to support economies (with the main focus on technical unemployment) towards a gradual re- opening of Europe that will provide goods and services to its citizens again and, finally, produce profit (some see this as the pressure of the business sector on political decision-makers – but without profit there is no investment and without investment there are no goods and services). Methodology Furthermore, one month ago, the leaders of EU signed off more than 500 billion euros fund to keep people in work and businesses afloat (our emphasis). The logic and reasonable solution was to pump and transfer money, as quickly as possible, and which might even amount to a package of 2.4 trillion euros, for the economies or parts of Europe that needed it most, either under the form of grants, or under the form of loans. So, in this period, we experience (or we expect to experience) the impact of regulations from EU institutions and/or national authorities to the social and economic environment. These solutions are designed for the entrepreneurs who desperately intend to re-open the businesses and for employees and customers that will re- establish the supply-demand game on the market, in the benefit of all parts involved in. The initial question asked by the European authorities was "Sink or Swim"? The answer is clear: all member states need to act as one, either sink together, or float/swim together (McCaffrey, 2020). On the one side there are the public authorities with support funds, on the other there are the abilities, skills, knowledge of entrepreneurs, CEOs/managers, who comprehend how to use those funds for achieving their goals, for re-starting their activities and for ensuring a reasonable level of revenues and wellbeing of employees and people in general. The nexus to abilities, skills and knowledge is another part of the ongoing research on entrepreneurship and the theory of the firm. It can be categorized into several taxonomies or theoretical sequences, that have come up in the last 5-6 years (McMullen, 2015; McCaffrey, M., 2015 and others). They focus on the two positive parts of a SWOT analysis, Strengths and Opportunity, along with Alertness on possible internal and external disturbance-factors and on the Judgment approach in selecting, monitoring and co-ordination of all assets and human resources or, in multi-person firms, the delegation of decision authority to other owners or employees. It is these categories that serve best for the renewal of the theory of the firm. And we have a clear connection to praxis: The necessity for new ideas and sequential theories, is mirrored in a forecast of the International Labour Organization, indicating that approx.195 million jobs will be lost around the world in the next three months (McCaffrey, 2020). Exposure of the Issue As professor Ouchi said, and other authors appreciated, Theory Z is not the last word in relational management, but it seems to be a right support to it, which can be beneficial in cases as in the period we are going through. In the essence, Theory Z allows to develop, among others by rotation and training of the employees, a more dedicated, loyal and permanent workforce, which benefits most of the companies. And it is exactly these employees/people, on which the management of the companies can rely for re- establishing the business and for the re-use of the workforce temporary sent into technical unemployment [20]. Regarding the two Theories of entrepreneurship, we consider them pliable on what has happened in this period and conducive to re-building the theoretical framework in the field. The Cochran Theory, or the cultural theory of entrepreneurship, is a sociological approach of this issue. Cochran (1965) considers the entrepreneurs as an emerging personality of society. The dynamics in the activity of an entrepreneur is depending on social factors, which result from changes and actions of the business and the business environment. The theory looks at the culturally determined inter-connection between the potential and the attitude of entrepreneurs with the expectations by the sanctioning groups, i.e. by those who facilitate new entrepreneurial ventures. Enlarging the significance of Cochran Theory, we consider the role of entrepreneurs defined by those groups, i.e. members of board of directors, other top officials and, further, by the groups with which the owner/entrepreneur interacts closely within the firm/corporation, in achieving the proposed goals. The Kirzner theory (1973; 2009), called The Alertness Theory of Entrepreneurship, is based on the idea of spontaneous learning and which is perfectly adaptable to the actual situation, like in the "Crusoe example”. The rationale is that the entrepreneurs/CEOs/managers must promptly respond to the challenges and changes produced in short-term within economy and society. In a word, both owners and decision-makers must stay alert and monitor the development of facts and phenomena and the regulations issued by executive and legislative powers [19]. 98 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 1The term was used to show the parallel with the legendary fiscal union forged by U.S. treasury secretary Alexander Hamilton after the American Revolution that helped prevent states from being bankrupted by their war debts. Methodology In order to understand the Conclusions, we consider that the aspect proves that the coronavirus crisis is more devastating on employment than the global recession ten years ago. It remains to be seen whether the instruments provided by the E.U. will make this loss of jobs a temporary one. But, as said, the financial instruments provided must be complemented by the entrepreneurial 99 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 resources exhibited above and of which we think that they must be embedded into a renewed theory of the firm. Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 resources exhibited above and of which we think that they must be embedded into a renewed theory of the firm. resources exhibited above and of which we think that they must be embedded into a renewed theory of the firm. The practice in Economics is developing faster than the theories, especially that is necessary de recover the losses in these last three months and new modalities of chain distribution, new instruments for delivery (such as drones), automated deliveries companies, especially Chinese e-commerce and others, prove that we cannot speak about the end of Economics, but rather that the thinking in this domain to become a sound support for these realities and necessities. Our lives have changed, but we must know how to manage the medical danger and to succeed a desired level of satisfaction, even happiness. Conclusions In general, the theory of the firm includes entrepreneurial concepts and concepts of organization management. Its value for practice lies with being adaptive and with prompting responses to all influences or changes, and with ensuring innovative actions as well as the proper functioning of all components, actors and factors involved in entrepreneurial and management activities. Regarding the impact of the new coronavirus, it is obvious that it has changed and is changing all around us, all of our known world, but not at the same time and not to the same extent. It is essential to develop a quick answer to the coronavirus challenge and to its effects on other environmental factors in order to preserve the health of people and maintain the due course of society. A major contribution comes from innovative thinking, swift and smart decisions, training the employees/workers to perform activities that will re-establish the functioning of the firms and, step by step, the growth of their revenues and welfare. We think that the modality of a tryptic action type which includes alertness-innovation-smart decisions will help to understand the functional internal environment and provide quick responses and adaptations to external factors and regulations. For praxis, this is the way to efficient action; for scholarly purposes it has to be transposed into a flexible sequence that can be embedded into the Theory of the Firm, as a whole. These findings impact also on higher education institutions, which are involved, more or less explicitly, in relationships of students that are similar to those between CEOs/decision-makers and other persons., Students should be supplied with solid knowledge, but especially with abilities and skills to understand facts that surround them, to respond quickly and to operate efficiently and effectively within an entity/firm/organization. References 1. Aghion, P. et al. (eds.). (2016). The impact of incomplete contracts on economics, New York: Oxford University Press. Available at: www.global.oup.com. 1. Aghion, P. et al. (eds.). (2016). The impact of incomplete contracts on economics, New York: Oxford University Press. Available at: www.global.oup.com. 2. Barney, J. B. & Hesterly, W. S. (2006). Strategic management and competitive advantage: Concepts and cases, Upper Saddle River, NJ: Pearson/Prentice Hall. Available at: www.theacademicessays.com. 2. Barney, J. B. & Hesterly, W. S. (2006). Strategic management and competitive advantage: Concepts and cases, Upper Saddle River, NJ: Pearson/Prentice Hall. Available at: www.theacademicessays.com. pp y 3. Bylund, Per. (2018). The Economic Theory of the Firm, accessed June 9, 2020. Available at: www.mises.org/library/. 3. Bylund, Per. (2018). The Economic Theory of the Firm, accessed June 9, 2020. Available at: www.mises.org/library/. 4. Coase, R. (first published 1937). The Nature of the Firm, in Economica, 4(16), 386-405. Accessed May 15, 2020. Available at: www.onlinelibrary.wiley.com. 4. Coase, R. (first published 1937). The Nature of the Firm, in Economica, 4(16), 386-405. Accessed May 15, 2020. Available at: www.onlinelibrary.wiley.com. y y 5. Cochran, T. C. (1965). The entrepreneur in economic change, Explorations in Economic History, 3(1), 25-38. Available at: www.entrepreneurshiptheories.blogspot.com. 5. Cochran, T. C. (1965). The entrepreneur in economic change, Explorations in Economic History, 3(1), 25-38. Available at: www.entrepreneurshiptheories.blogspot.com. 6. Grant, Robert M. (1996). Toward a Knowledge-Based Theory of the Firm, accessed June 5, 2020, Available at: www.researchgate.net/publication/. 6. Grant, Robert M. (1996). Toward a Knowledge-Based Theory of the Firm, accessed June 5, 2020, Available at: www.researchgate.net/publication/. 7. Hebert, R. F. & Link, A. N. (2009). A history of entrepreneurship, New York: Routledge. Available at: www.1lib.eu/book. 7. Hebert, R. F. & Link, A. N. (2009). A history of entrepreneurship, New York: Routledge. Available at: www.1lib.eu/book. 8. Kirzner, Israel M. (2009). Competition and Entrepreneurship, abstract on www.papers.ssrn.com accessed May 20, 2020, paper written 1973. 8. Kirzner, Israel M. (2009). Competition and Entrepreneurship, abstract on www.papers.ssrn.com accessed May 20, 2020, paper written 1973. 9. Klein, Peter G. (2016). Why entrepreneurs need firms and the theory of the firm needs entrepreneurship theory. Revista de Administração, 51, 323-326, accessed May 5, 2020. Available at: www.sciencedirect.com. 10. Klein, Peter G. (2016). My contributions to entrepreneurship theory, in Audretsch & Lehman (eds.) The Routledge companion to the makers of modern entrepreneurship, Routledge. Available at: www.mises.org/wire/. g 11. McCaffrey, D. (2020). References Coronavirus Briefings, months of April and May, www.euronews.com accessed May 3 and June 1st, 2020. 100 Business Ethics and Leadership, Volume 4, Issue 2, 2020 ISSN (online) – 2520-6311; ISSN (print) – 2520-6761 12. McCaffrey, M. (2015). Economic policy and entrepreneurship alertness or judgement? In P.L. Bilund & M. McCaffrey (eds). The next generation of Austrian Economics. Essays in honor of Joseph T. Salerno, 183-199. Available at: www.papers.ssrn.com. p p 13. McGregor, D. (2006). The Human Side of Enterprise, Bew York:McGraw-Hill Companies, Inc. Available at: www.amazon.com. 14. McLeod, S. (2020). Maslow’s Hierarchy of Needs, accessed May 10, 2020. Available at: www.simplypsychology.org. 15. McMullen, J. S. (2015) Entrepreneurial judgment as empathic accuracy: A sequential decision-making approach to entrepreneurial action. Journal of Institutional Economics, 11(3), 651-681. Available at: www.cambridge.org/core/. 16. Sautet, F. (2006). An Entrepreneurial Theory of the Firm, Foundations of the Market Economy, published by Routledge, New York. Available at: www.amazon.com. 17. Zimmerer, T. W. & Scarborough, N. M. (2008). Essentials of entrepreneurship and small business management, Upper Saddle River, NJ: Pearson/Prentice Hall. Available at: www.pearson.com/us/. 18 *Management theories Concepts surrounding recommended management strategies g p p management, Upper Saddle River, NJ: Pearson/Prentice Hall. Available at: www.pearson.com/us/. 18. Management theories. Concepts surrounding recommended management strategies, www.corporatefinanceinstitute.com/resources/, accessed June 10, 2020. g pp p 18. Management theories. Concepts surrounding recommended management strategies, www.corporatefinanceinstitute.com/resources/, accessed June 10, 2020. 19. Top 14 Theories of Entrepreneurship (with Criticism),www.businessmanagementideas.com. Accessed May 18, 2020. 20. **Reading: Ouchi’s Theory Z, Introduction in Business, accessed May 18, 2020, Available at: www.courses.lumenlearning.com. 101
https://openalex.org/W2328904387	https://sciendo.com/pdf/10.1515/atd-2015-0014	English	null	Medical Education in the Field of Communication as a Value and a Means of Health Quality Improvement	Acta Technologica Dubnicae	2,014	cc-by	7,749	* Jana Goriup, University of Maribor, Maribor, Slovenia; jana.goriup@um.si Kleopatra Kodrič, University of Maribor, Maribor, Slovenia; kleokodric@gmail.com Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 DOI: 10.1515/atd-2015-0014 DOI: 10.1515/atd-2015-0014 Medical Education in the Field of Communication as a Value and a Means of Health Quality Improvement Jana Goriup – Kleopatra Kodrič Vita brevis, ars longa, accasio valucris, experientia periculosa, idicium, difficile. Nec vero satis est, medicum suum fecisse officium, nisi suum quoque aegrotus, sum astantes faciant, sintque: externarite comparata. Life is short, art is long, opportunity is fleeting, experience is dangerous, judgement is hard. It is not enough if the doctor does his job, when it is necessary, but the patients have to contribute their part as well and so do other life circumstances. (first Hipocrates Aforism) Abstract: The article discusses the issues of communication between a doctor and a patient as one of the main approaches within the patients' medical treatment. This approach, which includes the field of communication in medicine as a value, more and more appears as obligatory not only in the field of profession itself, but also in the field of the patients’ needs. Communication covers a huge part of our social as well as intimate lives. Our social functioning is based on communication. The transfer and preserving of values is based on communication. Especially in the field of medicine, the function of communication appears to be one of the key elements to allow a better and complete treatment of the patient. The article discusses studies which have shown that communication skills have to be developed, it is advisable to introduce them into the educational programmes for med students and medical staff. Researches have shown that students, who were taught the field of communication skills, later as doctors or medical staff obtained a lot more information about patients than those who were not given such education. Key words: communication, doctor, patient, education, values. Key words: communication, doctor, patient, education, values. 64 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 1 Introduction Communication is the central and most basic process in society. Communication intertwines the broadest segment of society, i.e. from the public sphere to the most basic cell of society – the family. Apart from all the important processes taking place in our lives and the public sphere, communication (between family members, within a team, at work, between doctors and patients) and solving problems as current issues have become the central thematic chapters in the field of health. Brajša (1982) defines conflicts as a relationship crisis or crisis in development. The one who does not admit the existence of the development crisis denies development. There is no development without conflicts (Brajša, 1982, p. 52). The doctor-patient relationship and the question of trust are among the key issues that must be dealt with. Communication allows people to interact and co–operate. Communication in the field of medicine represents a two-way flow of messages between the doctor and the nurse on one side and the patient on the other side, with empathy and trust being very important here. The essence of the doctor's empathy is his/her ability to listen. Doctors who are able to recognize their patients’ feelings are more successful in their treatment. When getting in touch with a patient, the doctor takes responsibility not only for the treatment but also for the patient as an individual. An individual approach based on mutual communication must be applied. The essence of building trust is communication, however, it must not be stereotypical, routine, phlegmatic or impersonal. The relationship between the doctor and the patient is based on an interactive process and has to be equal. The doctor has the role of an experienced professional and the patient is his/her active co-operator in the process of treatment. While building the relationship with a patient, The Medical Deontology Codex (http://www.zdravniskazbornica.si/zzs.asp?FolderId=386) can be helpful for the doctors. The Codex is meant to be used as a guide for doctors when assessing his way of treatment. In the postmodern society, the relationship and communication between the doctor and the patient are often perceived as negative, especially due to the deficient and (sometimes) simple health care organization. Doctors do not have enough time for one patient (7 minutes on average!!!) and patients become objects for the doctor. Thus, medicine is going through a crisis of mistrust based on the depersonalisation of the patient. Acta Technologica Dubnicae volume 4, 2014, issue 1 neither a method nor a technique and it cannot be “easily learnt”. However, sympathy and empathy are crucial dimensions within the relationship between the doctor and the patient. neither a method nor a technique and it cannot be “easily learnt”. However, sympathy and empathy are crucial dimensions within the relationship between the doctor and the patient. In their offices, doctors are confronted with their daily routine work because of many patients, but especially due to the enormous lack of time for them. Doctors often advise their patients (by routine) to go to another doctor, i.e. to see a specialist. The result of such a relationship is unsatisfied and disappointed patients who wander from one office to another, from clinic to clinic and whose expectations have not been fulfilled (Gottschlich, 1998, p. 35). A respect towards patients’ autonomy and dignity are the essential features of doctors’ empathetic communication. Ule (2003) states that the results of research on general medicine study programmes show that doctors do not possess enough knowledge about communication. During their studies, they are only given general instructions on how to interview patients. These instructions are referred to diagnostic methods. Most doctors are enabled to competently find the history of physical symptoms, however, they are not trained nor motivated to connect these to patients’ psychological and/or social symptoms (for example: fears, conflicts in their surroundings, etc.). Thus, the author notices that a lot of students finish their medicine studies without adequate communication skills. In the paper entitled Communication Skills for Medicine (2004) the authors Margaret Lloyd and Robert Bor discuss what should good communication be like, reminding that communication is the core of health care. Special attention should be paid to the research of Peter Magguire in Manchester who, together with students of medicine, interviewed patients about the students' ability to perform an interview. The patients were attracted by those interviewers who showed warmth and attention as well as those they could easily communicate with. Those students introduced themselves to the patients, showed self– awareness, listened to the patients, reacted to their comments and asked questions in an appropriate way without repeating themselves. This is also the answer to the question why good communication is important. It allows a better care of patients. Doctors who communicate with their patients well and in an appropriate way, set a detailed diagnosis and can also see a patient's emotional instability. 2 Communication and education of doctors In the process of communication with patients, doctors often face demanding situations since clinical practice requires high degree of empathy on the side of doctors. On the other hand, doctors are also advised not to show patients their feelings and emotions. They must realize that empathic communication is 65 Acta Technologica Dubnicae volume 4, 2014, issue 1 patient’s task it to respond to the doctor's findings and initiatives, passively or actively depending on the situation. patient’s task it to respond to the doctor's findings and initiatives, passively or actively depending on the situation. Despite the above mentioned, there is still some bad communication present between the doctor and the patient. A research (Lloyd and Bor, 2004), focused on the patients’ reactions to the surgeon's demands, has been realised in Florida. Comparing the patients’ reactions to the surgeon's demands, the complaints mainly referred to the doctors' behaviour, i.e. they were not listening, did not inform the patients and did not show any interest or understanding towards them. In countries where patients complain less about their doctors they openly express their dissatisfaction regarding the way their doctors communicate with them. The authors' answer to the question whether communication skills can be learnt is that this is impossible if med students copy the communication model of their teachers. A good communication model needs effective elementary schooling and education in the field of communication. Realizing this fact, some medical educational institutions have already reacted to the need for good elementary communication and have already included a subject based exclusively on gaining knowledge and skills in the field of communication into the process of professional (medical) education. In the 1970s of the last century, studies were performed, by students of psychiatry, too, in the field of teaching communication between doctors and patients to find out about the problems in the doctor-patient communication. The most important two reasons standing out were that the doctors did not obtain all necessary information about the patient and they “forgot” to ask the patient some essential questions about their problems, since they did not notice any non- verbal messages of the patient: in brief, they showed no interest (Lloyd and Bor, 2004, pp. 4-5). In the course of this study, the students were divided into two groups, the control group and the feedback group. Their task was to obtain information about the patient and his/her anamnesis within 15 minutes while being recorded. An additional task of the feedback group was to handle two more patients. Again, this procedure was recorded. After the interviews, they discussed their mistakes with their mentor. Acta Technologica Dubnicae volume 4, 2014, issue 1 Thanks to the possession of that skill, patients trust the doctor and follow the doctor's instructions during treatment. It is also evident that communication has a positive impact on patients’ physical condition, e.g. their blood pressure. Research has shown that patients whose communication with their doctors was successful, had less problems with high blood pressure or were successful at stabilising it (Lloyd and Bor, 2004, pp. 3-4). Each patient must be aware of his role and tasks in the process of diagnostics, during treatment and has to take responsibility for this process. One of the 66 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 the group which had attended additional education in the field of communication with patients, were better at communication. Finally, we can confirm the fact that good communication can be learnt and it helps to build a good relationship with the patient (Lloyd and Bor, 2004, p. 5). Doctor-patient communication is a process in the organization where the doctor works. He/she guarantees the flow of information from its source to the user, i.e. the patient or family member (Bezenšek and Barle, 2007, pp. 175-176). Such a communication exceeds economic, social, cultural or interpersonal differences and produces trust, respect and co-operation. However, good communication does not necessarily mean that the doctor and the patient will agree in everything and it does not mean that there will be no dissatisfaction, mistakes, faults, complaints or even conflicts. These can also be the consequence of unrealistic expectations of the patients and their “blind trust” in medical technology as well as a lack of knowledge or understanding such procedures. Another reason could be the doctor's routine approach towards the patients, a lack of motivation for work or professional disinterest. This kind of complaints may be reduced by good communication. Greene says that the majority of cultures do not use compulsive communication anymore, even “under-cover” compulsion, as he names it, is out and nowadays not used. The fact that it is not effective in motivation anymore is very important (Greene, 1991, p. 28). p When, in the process of communication, the doctor should get closer to the patient as a co-speaker and to show respect towards him/her. The doctor should try to listen to the patients and find out what they want, what troubles and motivates them. Ule (2003) accentuates that communication is based on mutual respect and she also exposes that communication is a form of treatment (as well). In each communication relationship there is an unfelt and priceless treatment potential hiding, however, there are unexpected dark sides which can arise as well and these arise due to the doctor's relationship towards him/herself or his/her own wounds which are hidden many times. Research has shown that being isolated from communication makes people sick. For such people seeing the doctor means running away from isolation and, of course, such patients wish to talk to the doctor even more. Acta Technologica Dubnicae volume 4, 2014, issue 1 Afterwards, both groups made an additional interview with the patients, which showed that the feedback group was more successful in communication, since they had obtained three times more information about the family anamnesis of the patients than the control group. Similar studies were realized with the medical staff, too, and most of them showed that students, who had been taught the field of communication with the patient, in the end had much more success in communication with patients. An interesting question, which arose in this, was whether the gained knowledge in the field of communication would fade after some time or it would remain. The question was answered and confirmed by the same study four to six years later, when the interviewed people, who had been the same students at that time, were already doctors. Again, the hypothesis confirmed that the feedback group students, i.e. 67 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 significantly influences the course of treatment. Surely, each doctor-patient relationship has its own characteristics. Unfortunately, patients do not have many positive communication experiences nowadays. From the doctor's point of view, the communication is mostly set into some conversational routine to find out the diagnosis and for further treatment. Research (Gottschlich, 1998, p. 10 cited in Ule, 2003) confirms that, due to this conversational routine, doctors do not obtain essential information which would be helpful for a higher quality treatment (Ule, 2003, pp. 131-132). Some researchers (Fox, 1993, Hak, 1994, Roth, 1969, cited in Bezenšek and Barle, 2007, pp. 102) say that the way of communication between patients and doctors varies according to individual social groups. Based on their findings, communication is of a higher quality between the members from the middle and higher social classes, not due to the social status of the social groups nor their status in the community, but due to their similar cultural background since doctors originate from these social classes and therefore the cultural similarity is obvious. l ( i d i žič 2004 22 23) h h b significantly influences the course of treatment. Surely, each doctor-patient relationship has its own characteristics. Unfortunately, patients do not have many positive communication experiences nowadays. From the doctor's point of view, the communication is mostly set into some conversational routine to find out the diagnosis and for further treatment. Research (Gottschlich, 1998, p. 10 cited in Ule, 2003) confirms that, due to this conversational routine, doctors do not obtain essential information which would be helpful for a higher quality treatment (Ule, 2003, pp. 131-132). significantly influences the course of treatment. Surely, each doctor-patient relationship has its own characteristics. Unfortunately, patients do not have many positive communication experiences nowadays. From the doctor's point of view, the communication is mostly set into some conversational routine to find out the diagnosis and for further treatment. Research (Gottschlich, 1998, p. 10 cited in Ule, 2003) confirms that, due to this conversational routine, doctors do not obtain essential information which would be helpful for a higher quality treatment (Ule, 2003, pp. 131-132). pp Some researchers (Fox, 1993, Hak, 1994, Roth, 1969, cited in Bezenšek and Barle, 2007, pp. 102) say that the way of communication between patients and doctors varies according to individual social groups. Acta Technologica Dubnicae volume 4, 2014, issue 1 Based on their findings, communication is of a higher quality between the members from the middle and higher social classes, not due to the social status of the social groups nor their status in the community, but due to their similar cultural background since doctors originate from these social classes and therefore the cultural similarity is obvious. Ule (cited in Janežič, 2004, pp. 22-23) accentuates that treatment has been connected with communication in different forms for ages. She exposes that communication is based on mutuality and mutual engagement of the doctor and the patient as in this way the dualism between the doctor and the patient is exceeded. However, here we have to consider that illness, as well as the process of treatment itself is not only the patient's problem but the doctor's as well. Ule is convinced that the doctor's words represent the means for helping and treating the patient, however they can also be harmful. This way the doctor and the patient become partners in the process of treatment. In this process communication plays a significant role as both the patient and the doctor act as partners in the process of communication complementing and assessing each other based on their experiences and knowledge. Often, we are not aware enough that conversation means a lot more to the patient than just medicine. Kind and friendly words let him/her hope to recover (Janežič, 2004, pp. 22-23). Ule (cited in Janežič, 2004, pp. 22-23) accentuates that treatment has been connected with communication in different forms for ages. She exposes that communication is based on mutuality and mutual engagement of the doctor and the patient as in this way the dualism between the doctor and the patient is exceeded. However, here we have to consider that illness, as well as the process of treatment itself is not only the patient's problem but the doctor's as well. Ule is convinced that the doctor's words represent the means for helping and treating the patient, however they can also be harmful. This way the doctor and the patient become partners in the process of treatment. In this process communication plays a significant role as both the patient and the doctor act as partners in the process of communication complementing and assessing each other based on their experiences and knowledge. Acta Technologica Dubnicae volume 4, 2014, issue 1 That is why the author emphasises that family doctors should “play” the role of a communication therapist (Ule, 2003, p. 131). If the patient sees that the doctor will not give any clear instruction and answers to his questions about his/her health problems, he feels helpless, numb and becomes inactive in the process of treatment. Their reactions are unmotivated and it seems that they accept their situation. That is why they do not ask questions nor express their doubt. They become “obedient”, manageable, and especially in the process of treatment, they co-operate. On the contrary, the patients’ style of communication influences the doctor who should pay attention to the patient also on the level of his/her own perception of the disease. Research confirms that the patient’s perception of the illness 68 Acta Technologica Dubnicae volume 4, 2014, issue 1 motivating communication field. It is of elementary importance for a hurt modern human being. It is the communication between the doctor and the patient which has a main, central position, no matter if this position is opposed and underestimated in modern medicine. This is the reason why the relationship between the doctor and the patient is determined to fail in advance. The main problem here is the identity of both, what is the reason why doctors’ communication is intended just for diagnosing, noticing and standardising the illness (Ule, 2003, p. 131). Acta Technologica Dubnicae volume 4, 2014, issue 1 Often, we are not aware enough that conversation means a lot more to the patient than just medicine. Kind and friendly words let him/her hope to recover (Janežič, 2004, pp. 22-23). Unfortunately, communication is often a one-way process because patients (mostly) do not speak much or are silent, it is only the doctor who speaks. Kersnik (cited in Janežič, 2004, p. 23) believes that doctors should know and effectively use both the verbal and the non-verbal forms of communication. Most of all he/she should be able to lead a consultation with the patient, say bad news, ease the patient's and his/her relatives' acceptance of terminal phases, talk about mental signs without embarrassment, rationally use medicine for the treatment of mental problems and consult psychic help. The author reminds us that patients must feel that the doctor pays attention to them and is ready for consultation. However, such interaction requires the possession of communication skills (Janežič, 2004, pp. 23-24). Unfortunately, communication is often a one-way process because patients (mostly) do not speak much or are silent, it is only the doctor who speaks. Kersnik (cited in Janežič, 2004, p. 23) believes that doctors should know and effectively use both the verbal and the non-verbal forms of communication. Most of all he/she should be able to lead a consultation with the patient, say bad news, ease the patient's and his/her relatives' acceptance of terminal phases, talk about mental signs without embarrassment, rationally use medicine for the treatment of mental problems and consult psychic help. The author reminds us that patients must feel that the doctor pays attention to them and is ready for consultation. However, such interaction requires the possession of communication skills (Janežič, 2004, pp. 23-24). As our discussion includes the field of communication between the doctor and the patient, it is necessary to expose the fact which, by Ule (2003), is named 69 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 and intensity of the doctor's interest in his health or health problem. At the same time, non–verbal communication can indicate how much and whether the doctor appreciates the information obtained from the patient. If a doctor manages and considers all the above mentioned, the patient is more satisfied. Surely, the doctor should be aware of the most important signs of non–verbal communication, e.g. face gestures, smiling, mimes, body language, hand position etc. and intensity of the doctor's interest in his health or health problem. At the same time, non–verbal communication can indicate how much and whether the doctor appreciates the information obtained from the patient. If a doctor manages and considers all the above mentioned, the patient is more satisfied. Surely, the doctor should be aware of the most important signs of non–verbal communication, e.g. face gestures, smiling, mimes, body language, hand position etc. At this point, the author is right, claiming that a doctor should not use the mentioned elements in his/her communication, neither should he use them just as an example for the purpose of his communication with the patient. A successful, good quality communication process can be characterised by “the ability to communicate with patients in an ethical and effective way it has become a fundamental skill expected from each specialist.” (Leo, 2011, pp. 720- 725) Every interpersonal contact is mutual, which means that all we say or even do not express (through our appearance, movements, face expressions, dressing style, etc.) influences the other person. This person can react by showing appreciation, thoughts or even rejection. Our words and the facial expression can make one feel comfortable or, on the contrary, we can even cause some discomfort. When two people meet, their communication is always interactive. We sometimes feel uncomfortable because of the voice of the other person while communicating or even because the other person has no eye–contact with us. Despite all these facts, the conviction that communication is something usual, known to everybody and everyone knows how to communicate, prevails. Words are the essential means of conversation. Communication is always a process of awareness whereas silence expresses a dysfunction in communication. Especially in the field of sociology of health, there is lots of prejudice regarding communication, i.e. communication is unimportant due to other, more important activities and the patient is an unintellectual being who will not understand anything. 3 Patients’ needs in communication with the doctor 3 Patients’ needs in communication with the doctor In the article Working toward the best doctor–patient communication (2011), Leo L. F., a psychiatrist and a representative of the Clinical Centre of the Pediatric Section in Singapore, presents the results of an analysis made in 1989. It revealed that doctor-patient communication is a one–way process as the doctor mainly gives instructions instead of allowing to run the communication mutually. Doctors should motivate patients to play an active role in the conversation, i.e. during the medical consultation including the discussion about a patient’s physical condition. When patients notice that the doctor is listening to them, they starts to appreciate and consider them more. Such a process is very important for doctors as well since in mutual communication they can notice how the patient understands and accepts the whole medical process. If patients are involved in the process of communication in such a way, they can monitor and view their own treatment in a different way. The author accentuates that doctors are expected to explain the patients their health condition in a comprehensible way. He also consults an appropriate vocabulary that would allow and at the same time simplify and unify the process of doctor-patient communication. The same author exposes the function of the bio-psycho-social factor to assure a more qualitative communication with the patient. Doctors usually do not pay enough attention to the psycho–sociological views of treating the patient. The study by Baberg et al. (Baberg, 2001, cited in Leo, 2011, p. 721) notices that patients’ problems are not only of medical but of psychological– emotional nature as well. This is why the author emphasises that doctor-patient communication does not include only questions from the field of symptomatology but social, emotional and behavioural questions as well. In this way, both the doctor and the patient are more satisfied since the patient can co– operate actively in the process of treatment and thus help to establish a partnership with the doctor. In order to make the doctor-patient communication effective, non–verbal communication is essential. However, the non–verbal way of the doctor's communication is often disregarded. If non-verbal communication is suitable, indicators such as body language, eye contact and the colour of the voice indicate the patient the level 70 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 patients. The doctor is a formal leader who is hidden. In this case, he/she must be confident, supportive and should have an influence on other group members (anonymous alcoholics). Here his/her role is to remove the limits within the organisation. The higher the doctor’s position in the hierarchy of the organisation is, the stronger his influence on the flow of information is. Such a doctor has a lot of social power which is very important for his/her work as well as life environment (Bezenšek and Barle, 2007, p. 189). patients. The doctor is a formal leader who is hidden. In this case, he/she must be confident, supportive and should have an influence on other group members (anonymous alcoholics). Here his/her role is to remove the limits within the organisation. The higher the doctor’s position in the hierarchy of the organisation is, the stronger his influence on the flow of information is. Such a doctor has a lot of social power which is very important for his/her work as well as life environment (Bezenšek and Barle, 2007, p. 189). When being in contact with patients, doctors are responsible both for the treatment of a particular illness or disease and patients as individuals. Gadžijev (cited in Janežič, 2004, p. 17) says that he/she is often told by young doctors that, during their studies, they were not taught how to communicate appropriately with patients and their family members. A doctor must be aware of the fact that doctor/patient communication is a complex process, therefore he/she should be interested in the context of patient's life as well (family situation, work circumstances, status, relationships). He/she should also learn about the patient's personal characteristics (self–image, social and cultural capacity, emotional strength, ability to control difficult situations, ability of self–defence, etc.). Furthermore, he/she has to learn about the history of the patient's disease, obtain sufficient information for setting the appropriate diagnosis and understand the patient’s problems. A bad doctor can be characterised as somebody who treats patients as numbers, and does not listen to them. Research has shown that not being treated as an individual with feelings and emotions, patients mostly perceive as unacceptable Thus, the lack of trust endangers the doctor-patient relationship. Patients are the ones who are forced to trust (Bezenšek and Barle, 2007, p. 103). Acta Technologica Dubnicae volume 4, 2014, issue 1 However, appropriate communication can allow a faster and more correct diagnosis as well as more satisfaction of the patients (Rakovec–Felser, 2002, pp. 206-207). A problem in doctor-patient communication is presented by the fact that doctors play several very important roles when organizing their work. These roles can be expressed in the process of communication. Thus, the doctor can function as “receptionist” who controls the flow of communication through a communication network. Such a position can be either formal or informal. He/she can also have the function of a moderator or some bridge connecting individual communication sub–systems. It is important that the doctor as the transmitter of the information flow is among other team members, individuals and groups, however, he is not a part of sub–systems which connect them otherwise. A doctor can also be a leader of opinion, and as such plays an informal role of influencing opinions, values, behaviour and decisions of the 71 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 Nevertheless, there are some communication competencies that every doctor should possess, i.e.: Special features of communication, especially ethical principles, respecting the patient's personality, considering the obligation of secrecy, the patient's rights, his/her culture and the principle of co-operation which includes the principle of partnership without a hierarchy as well. Generally, it is necessary to get rid of the former relationship between a high–standing, all–knowing doctor and a totally dependent weak patient. Medical work should be based on interaction and it is the doctor who should take care of this. Thus, the relationship between the doctor and the patient should be warm, human and characterised by a certain level of sympathy on the side of the doctor. Certainly, the patient above all needs good treatment and not sympathy, but there is also some emotional affection needed which should not be an obstacle for a doctor (Trček, 1994, pp. 189-199). Si th t f d t ti t i ti i l it it Since the nature of doctor-patient communication is complex – it unites professional distance and the intimacy of the patient's problems, it is hard to set and define exact rules of communication. What is more, communication in this profession is influenced by various factors and, sometimes, by special, unusual situations. Nevertheless, there are some communication competencies that every doctor should possess, i.e.: p - giving exact and sufficient information, - careful and active listening and reacting to the patient's resistance, - correct or appropriate interpreting of the diagnosis, giving clear and comprehensible instructions to the patient, professional behaviour (especially when having a physical contact during health examination), - respecting the patients’ decisions and believes, obtaining credibility and trust. - respecting the patients’ decisions and believes, obtaining credibility and trust. There is no doubt that doctors must pay attention to each patient, treat them a subjects with own believes, fears, feelings and not as just one of several case There is no doubt that doctors must pay attention to each patient, treat them as subjects with own believes, fears, feelings and not as just one of several cases. Each patient is a different individual and, therefore, needs a different approach on the side of the doctor. The problem is that doctors do not have a universal recipe for successful communication and building a high quality relationship with their patients. 4 Classification of doctor-patient relationships Židanik (cited in Janežič, 2004, p. Acta Technologica Dubnicae volume 4, 2014, issue 1 19) states that the doctor-patient relationship develops through three phases as follows: Acta Technologica Dubnicae volume 4, 2014, issue 1 The actors of a communication process establish mutual connections, their own world and also try to keep these connections (Ule, 2009, p. 16). The question of good communication with patients and of appropriate information delivery is of a great importance in medicine and health care. Philip Lay (cited in Payne and Walker, 2002, pp. 99-100) found that good communication is a very important component for patients. This is especially true when we want the patient to consider appropriately the doctor's advice regarding taking medicine, as inappropriate use of medicine can have bad consequences. The use of technical slang in medicine is a common communication problem as well. Communication is a part of treatment, it is based on mutuality and mutual engagement, especially calming the other person. That is why effective communication in medicine depends on several factors. The most important factor here is to respect the patient and consider his/her views and opinions. During the process of communication, eye–contact is necessary and the doctor has to take his/her time to explain things in an appropriate, simple and comprehensible way (Prebil, Mohar and Fink, cited in Kapler, 2011, p. 31). 72 Acta Technologica Dubnicae volume 4, 2014, issue 1 Special features of communication, especially ethical principles, respecting the patient's personality, considering the obligation of secrecy, the patient's rights, his/her culture and the principle of co-operation which includes the principle of partnership without a hierarchy as well. Generally, it is necessary to get rid of the former relationship between a high–standing, all–knowing doctor and a totally dependent weak patient. Medical work should be based on interaction and it is the doctor who should take care of this. Thus, the relationship between the doctor and the patient should be warm, human and characterised by a certain level of sympathy on the side of the doctor. Certainly, the patient above all needs good treatment and not sympathy, but there is also some emotional affection needed which should not be an obstacle for a doctor (Trček, 1994, pp. 189-199). Since the nature of doctor-patient communication is complex – it unites professional distance and the intimacy of the patient's problems, it is hard to set and define exact rules of communication. What is more, communication in this profession is influenced by various factors and, sometimes, by special, unusual situations. 4 Classification of doctor-patient relationships Židanik (cited in Janežič, 2004, p. 19) states that the doctor-patient relationship develops through three phases as follows: - initial contact – i.e. defeating the first rejection up to the beginning of the therapeutic relationship – an active search for the doctor and making appointments with the recommended doctor, - initial contact – i.e. defeating the first rejection up to the beginning of the therapeutic relationship – an active search for the doctor and making appointments with the recommended doctor, - evaluation of the doctor – i.e. the phase of development or checking if the doctor is interested in the patient and the patient compares the doctor with other doctors and, - the phase of balancing – i.e. the establishment of a mutual relationship – the doctor is adequate according to the patient's expectations. - the phase of balancing – i.e. the establishment of a mutual relationship – the doctor is adequate according to the patient's expectations. 73 Acta Technologica Dubnicae volume 4, 2014, issue 1 Here Ule notices that the relationship between the patient and the doctor is ambivalent, it is expanded from the patient's uncertainty to the doctor's routine. This is the point where several mistakes of communication between the doctor and the patient emerge from. In medical practice, Wolf and Flis (Ule, cited in Janežič, 2004, p. 19) distinguish between three models of relationships between the doctor and the patient as follows: - paternalistic model - contractual model and - partner model. The paternalistic model is based on the doctor's ability and conviction that only he/she is able to help the patient with his/her health problem. In this model the patient is passive whereas the doctor is dominant. Simultaneously, this means that trust in the doctor-patient relationship is very important for treatment. The paternalistic relationship is mostly established when the patient's life is in danger, i.e. in case of accidents, epidemy, surgery, etc. In this model of co– operation the patient is passive and, therefore, totally depends on the doctor. In the contractual model, the patient asks for medical service and meets a doctor who is the supplier of such medical service. This relationship is to be found in medical centres where various therapeutic and diagnostic services are offered (surgeries, sanatoriums, spas, rehabilitation centres, etc.). Acta Technologica Dubnicae volume 4, 2014, issue 1 society in which the process of communication takes place. For instance, when meeting someone, a Slovene usually shakes hands whereas an Indian bows. If a doctor experiences major cultural differences at his work and in communication, he/she violates the rules and principles of the partner in case that he/she is not aware of them. This is not only uncomfortable but the doctor is also expected to expand his knowledge and to understand the messages in communication, i.e. be able to describe and foresee how the others will behave and to understand their reactions. The most frequent obstacles in communication between the doctor and foreigners are ethnocentrism, stereotypes and prejudice. Communication between people with the same or similar cultural background is definitely easier (Bezenšek and Barle, 2007, pp. 193-194). Certain social and cultural factors come to expression when doctors meet people with other habits and from other cultural and social environment. In such cases, people behave in the way they were taught. Thus, communicating with foreign patients does not only mean understanding a foreign language but it also requires open-mindedness and a capacity to accept differentness. Belonging to a certain social class influences the way how somebody accepts, understands and reacts in a certain situation. Thus, the factors of our environment are connected not only to social but also to cultural factors. The technical aspects of communication are important as well, especially nowadays. As much information as possible must be processed in a short time. Media and IT–systems play a crucial role here. The latter have a great influence on the possibility of diagnosing and clinical treatment and they are more and more included in the work of nurses and doctors (Filipič, 1998, pp. 221-222). In the report by the Association of American Medical Colleges entitled Contemporary Issues in Medicine: Communication in Medicine (Report III), in the chapter discussing the importance of effective communication in medicine one can find a research by Barbara Korsch et al., published in the late 1960s, which inspired several studies about communication between doctors and patients. The resolution, confirmed by two committees, i.e. the Liaison Committee on Medical Education and the Committee of the Accreditation of Canadian Medical Schools in 1995, confirms this need for more and more knowledge in the field of communication in medicine in North America. Communication skills are essential for the effectiveness of doctors’ work. 4 Classification of doctor-patient relationships This model is more or less a part of marketing mechanism, which mainly occurs in labs, RTG, in cases of minor surgeries, in the field of aesthetic and plastic surgery. Janežič (2004, pp. 19-20) believes that a partner relationship between the patient and the doctor is the one which allows the patient and the doctor to be equal partners in the process of treatment. Such a relationship is important especially in the treatment of chronic diseases. In this relationship the doctor is an experienced expert and the patient is his/her partner in the process of treatment. Such a relationship can also include the patient's family members who take part in the process of treatment. Ule (cited in Janežič, 2004, p. 20) notices that doctors often combine models of relationships, especially the contractual and the paternalistic relationship, since these do not require as much effort as the partner relationship does. Gadžijev (cited in Janežič, 2004, p. 21) notices, that in communication with patients, doctors often try to hide their insufficient communication skills by presenting various papers and brochures including complications. Patients often welcome such an approach as they are given information about his disease. But it is not enough, doctors should pay more attention to their patients, they should be given a sufficient amount of information about their disease, diagnosis, treatment and possible complications. Successful communication requires knowledge of the partner's culture on the doctor’s side, i.e. the partner in the process of communication (e.g. the patient, family members, etc.). This mostly depends on the norms and values of the 74 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 Acta Technologica Dubnicae volume 4, 2014, issue 1 Their skills have to be assessed and developed by instruction. They refer to their responsibilities including communication with patients, the patients' families, colleagues and other medical staff. Similar orders from the past, obviously were not sufficient in order to change the curriculum. Theory and practice of developing communication skills need systematic attention, elementary knowledge of communication has to be included in the medical curriculum and communication skills have to be assessed. The development and extension of a humanistic approach, which is focused on the patient, have to be accented (Association of American Medical Colleges, 1999, pp. 5-6). 75 Acta Technologica Dubnicae volume 4, 2014, issue 1 5 Conclusion The analysis presented allows us to conclude that the global problem of communication in medicine or the doctor-patient communication is in the centre of attention of medicine and health care. Thus, personal, sincere communication between the doctor and the patient on an interpersonal level is an important process stretching to the field of science and social values as well. Research shows that there is still a lot to do in order to improve the relationship between the patient and the doctor in the field of communication. A survey among the Slovene public (Toš and Malnar, 2002, cited in Bezenšek and Barle, 2007, pp. 125-126) confirmed a high level of trust in doctors as two thirds of the respondents stated that their doctor does everything for them. According to the interviewees, the profession of the doctor enjoys a high level of reputation as well. Furthermore, it is necessary to accentuate the fact that the results showed great correlation between the feelings of happiness and the health of people. The contentment of people referring to their life and feelings of happiness is much worse when their health condition is bad (Bezenšek and Barle, 2007, p. 126). It is a fact that there is a shift as for healthy lifestyle. Programs supporting better health of individuals gain more and more attention. Thus, expectations regarding the doctor-patient relationship are high. This means that the paternalistic or the contractual model of relationship should grow into the partner model or co– operation between the doctor and the patient which would guarantee an increased quality of values in the society. References Associaton of American Medical Colleges. Contemporary Issues in Medicine: Communicaton in Medicine. Report III, 1999, 1-29. Communicaton in Medicine. Report III, 1999, 1-29. BACH, S. and GRANT, A. Communication and interpersonal skills for nurses. Southernhay East: Learning Matters, 2009. BALAŽIC J d KORNHAUSER P M d k k d l šk š BACH, S. and GRANT, A. Communication and interpersonal skills for nurses. Southernhay East: Learning Matters, 2009. Ž BALAŽIC, J. and KORNHAUSER, P. Medicinska etika: deontološka vprašanja pri delu zdravnika. Ljubljana: Medicinska fakulteta, Inštitut za sodno medicine, 2003. BEZENŠEK, J. and BARLE, A. Poglavja iz sociologije medicine. Maribor: Medicinska fakulteta, 2007. BRAJŠA, P. Človek, spolnost, zakon. Ljubljana: Delavska enotnost, 1982. FILIPIČ, I. (1998): Komunikacija v zdravstveni negi. Obzornik zdravstvene 1998 32 221 225 BRAJŠA, P. Človek, spolnost, zakon. Ljubljana: Delavska enotnost, 1982. FILIPIČ I (1998) K ik ij d i i Ob ik d BRAJŠA, P. Človek, spolnost, zakon. Ljubljana: Delavska enotnost, 1982. FILIPIČ, I. (1998): Komunikacija v zdravstveni negi. Obzornik zdravstvene nege, 1998, 32, 221-225. FILIPIČ, I. (1998): Komunikacija v zdravstveni negi. Obzornik zdravstvene nege, 1998, 32, 221-225. GOTTSCHLICH, M. Sprachloses Leid: Wege zu einer kommunikativen Medizin: die heilsame Kraft des Wortes. Wien: New York: Springer, 1998. GREENE, H. R. Nov način komunikacije: praktični nasveti za boljše poslovno in družinsko sporazumevanje. Ljubljana: Samozaložba, 1991. 76 Acta Technologica Dubnicae volume 4, 2014, issue 1 JANEŽIČ, M. Komunikacija med bolniki v informacijski družbi. Diploma thesis. Ljubljana: Fakulteta za družbene vede, 2004. KAPLER, T. Komunikacija v zdravstvu. Zaključna projektna naloga. Koper: Fakulteta za management, 2011. NEO, L. F. (2011): Working towards the best doctor – patient communication. Rewiew Article. Singapore Med J, 2011, 52(10), 720-725. LLOYD, M. and BOR, R. Communication skills for Medicine 2e., Philadelphia: Elsevier's Health Science Rights Department in Philadelphia, 2004. PAYNE, S. and WALKER, J. Psihologija v zdravstveni negi. Ljubljana: Educy, 2002. RADLEY, A. Making Sense of Illness. Sage, London, 1995. RAKOVEC– FELSER, Z. Zdravstvena psihologija. Maribor: Visoka zdravstvena šola, 2002. TRČEK, J. Medosebno komuniciranje in kontaktna kultura. Radovljica: Didakta, 1994. ULE, M. Psihologija komuniciranja in medosebnih odnosov. Ljubljana: Fakulteta za družbene vede, 2009. http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 (6.11.2010) http://med.over.net/forum5/read.php?358,6396482,6398632 (12.11.2010) http://med.over.net/forum5/read.php?13,537007.. (12.11.2010) http://www.dpbs.si/upload/63577491ee_KAKO_DOBRO_KOMUNICIRATI_Z _ZDRAVNIKOM.pdf JANEŽIČ, M. Komunikacija med bolniki v informacijski družbi. Diploma thesis. Ljubljana: Fakulteta za družbene vede, 2004. KAPLER, T. Komunikacija v zdravstvu. Zaključna projektna naloga. Koper: Fakulteta za management, 2011. NEO, L. F. (2011): Working towards the best doctor – patient communication. Rewiew Article. Singapore Med J, 2011, 52(10), 720-725. LLOYD, M. and BOR, R. Communication skills for Medicine 2e., Philadelphia: Elsevier's Health Science Rights Department in Philadelphia, 2004. PAYNE, S. and WALKER, J. Psihologija v zdravstveni negi. Ljubljana: Educy, 2002. RADLEY, A. Making Sense of Illness. Sage, London, 1995. RAKOVEC– FELSER, Z. Zdravstvena psihologija. Maribor: Visoka zdravstvena šola, 2002. TRČEK, J. Medosebno komuniciranje in kontaktna kultura. Radovljica: Didakta, 1994. RČEK, J. Medosebno komuniciranje in kontaktna kultura. Radovljica: Didakta, 994. ULE, M. Psihologija komuniciranja in medosebnih odnosov. Ljubljana: Fakulteta za družbene vede, 2009. Fakulteta za družbene vede, 2009. http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 (6.11.2010) http://med.over.net/forum5/read.php?358,6396482,6398632 (12.11.2010) http://med.over.net/forum5/read.php?13,537007.. (12.11.2010) http://www.dpbs.si/upload/63577491ee_KAKO_DOBRO_KOMUNICIRATI_Z ZDRAVNIKOM.pdf http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 (6.11.2010) http://med.over.net/forum5/read.php?358,6396482,6398632 (12.11.2010) http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 http://www zdravniskazbornica si/zzs asp?FolderId 386 (6 11 2010) http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 (6.11.2010) http://www.zdravniskazbornica.si/zzs.asp?FolderId=386 (6.11.2010) http://med.over.net/forum5/read.php?358,6396482,6398632 (12.11.2010) http://www.dpbs.si/upload/63577491ee_KAKO_DOBRO_KOMUNICIRATI_Z _ZDRAVNIKOM.pdf 77
https://openalex.org/W3199137132	https://findresearcher.sdu.dk/ws/files/192272716/s41380_021_01285_w.pdf	English	null	The relationship between dose and serotonin transporter occupancy of antidepressants—a systematic review	Molecular psychiatry	2,021	cc-by	10,322	Download date: 24. Oct. 2024 Citation for pulished version (APA): Sørensen, A., Ruhé, H. G., & Munkholm, K. (2022). The relationship between dose and serotonin transporter occupancy of antidepressants—a systematic review. Molecular Psychiatry, 27(1), 192-201. https://doi.org/10.1038/s41380-021-01285-w University of Southern Denmark The relationship between dose and serotonin transporter occupancy of antidepressants—a systematic review Sørensen, Anders; Ruhé, Henricus G.; Munkholm, Klaus Citation for pulished version (APA): Sørensen, A., Ruhé, H. G., & Munkholm, K. (2022). The relationship between dose and serotonin transporter occupancy of antidepressants—a systematic review. Molecular Psychiatry, 27(1), 192-201. https://doi.org/10.1038/s41380-021-01285-w Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only • You may download this work for personal use only. Y t f th di t ib t th t i l it f y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk Molecular Psychiatry www.nature.com/mp REVIEW ARTICLE OPEN The relationship between dose and serotonin transporter occupancy of antidepressants—a systematic review Anders Sørensen 1✉, Henricus G. Ruhé 2,3 and Klaus Munkholm4,5 © The Author(s) 2021 Anders Sørensen 1✉, Henricus G. Ruhé 2,3 and Klaus Munkholm4,5 © The Author(s) 2021 Brain imaging techniques enable the visualization of serotonin transporter (SERT) occupancy as a measure of the proportion of SERT blocked by an antidepressant at a given dose. We aimed to systematically review the evidence on the relationship between antidepressant dose and SERT occupancy. We searched PubMed and Embase (last search 20 May 2021) for human in vivo, within- subject PET, or SPECT studies measuring SERT occupancy at any dose of any antidepressant with highly selective radioligands ([11C]- DASB, [123I]-ADAM, and [11C]-MADAM). We summarized and visualized the dose-occupancy relationship for antidepressants across studies, overlaying the plots with a curve based on predicted values of a standard 2-parameter Michaelis–Menten model ﬁtted using the observed data. We included seventeen studies of 10 different SSRIs, SNRIs, and serotonin modulators comprising a total of 294 participants, involving 309 unique occupancy measures. Overall, following the Michaelis–Menten equation, SERT occupancy increased with a higher dose in a hyperbolic relationship, with occupancy increasing rapidly at lower doses and reaching a plateau at approximately 80% at the usual minimum recommended dose. All the studies were small, only a few investigated the same antidepressant, dose, and brain region, and few reported information on factors that may inﬂuence SERT occupancy. The hyperbolic dose-occupancy relationship may provide mechanistic insight of relevance to the limited clinical beneﬁt of dose-escalation in antidepressant treatment and the potential emergence of withdrawal symptoms. The evidence is limited by non-transparent reporting, lack of standardized methods, small sample sizes, and short treatment duration. Future studies should standardize the imaging and reporting procedures, measure occupancy at lower antidepressant doses, and investigate the moderators of the dose- occupancy relationship. Molecular Psychiatry; https://doi.org/10.1038/s41380-021-01285-w Molecular Psychiatry; https://doi.org/10.1038/s41380-021-01285-w Molecular Psychiatry; https://doi.org/10.1038/s41380-021-01285-w 1Nordic Cochrane Centre, Rigshospitalet, Copenhagen, Denmark. 2Department of Psychiatry, Radboudumc, Nijmegen, The Netherlands. 3Donders Institute for Brain, Cognition and Behavior, Radboud University, Nijmegen, The Netherlands. 4Centre for Evidence-Based Medicine Odense (CEBMO) and Cochrane Denmark, Department of Clinical Research, University of Southern Denmark, Odense, Denmark. 5Open Patient data Exploratory Network (OPEN), Odense University Hospital, Odense, Denmark. ✉email: anders ks90@gmail com Received: 7 June 2021 Revised: 11 August 2021 Accepted: 25 August 2021 Search strategy and selection criteria In our protocol, we planned to conduct a meta-analysis of occupancy data for each drug and dose and to report the summary mean occupancy (with 95% conﬁdence interval) by presenting the summary estimates of occupancy as a function of dose in graphs for each drug and brain region, provided the studies were sufﬁciently similar. However, too few studies investigated the same drug, brain region, and dose using the same ligand, design, and duration of drug-intake to make a meta-analysis meaningful. We, therefore, presented occupancy for each dose, drug, and brain region in tables as means and standard deviations (SDs). Studies investigating SERT occupancy at any given dose of any approved antidepressant administered orally in humans were eligible. We included studies using the ligands [11C]-DASB, [123I]- ADAM, and [11C]-MADAM, which all have a 1000:1 afﬁnity for SERT over the dopamine transporter (DAT) and the norepinephrine transporter (NET) [21, 22]. In addition, these ligands have all been extensively validated via kinetic modeling of arterial input sampling [23–26]. We excluded studies using the ligands [11C] (+)-McN and [123I]-ß-CIT due to their non-selectivity for SERT over other receptors: [123I]-ß-CIT has nearly equal afﬁnity for SERT and DAT, while [11C](+)-McN is considered “likely selective” with an afﬁnity for SERT over NET of between 10:1 and 100:1 [21]. We considered studies using a within-subject design only, not studies using a between-subject design, as our focus was occupancy, which is optimally calculated in a within-subject design; the study design minimizes variance and is, therefore, more likely than a between-subject design to provide reliable estimates of the relationship between antidepressant dose and occupancy. We visualized the dose-occupancy relationship by plotting occupancy against dose for antidepressants that were adminis- tered at four or more different doses across studies; if several occupancy measures were available per study, we prioritized, those with the shortest lag time between administration of the last dose of the antidepressant and imaging and those with the longest antidepressant treatment duration. Based on those data, to provide a visual reference for the data points, we ﬁtted a 2-parameter Michaelis–Menten model implemented by the drm R package using the formula p p p y We searched PubMed and Embase (last search 20 May 2021). The search terms for Embase were: antidepressant.mp. or exp anti- depressant agent/ OR (antidepressant* or SSRI or SNRI or “selective serotonin reuptake inhibitor“ or TCA or tricyclic or “serotonin- norepinephrine reuptake inhibitor“).af. INTRODUCTION insight into important clinical aspects of antidepressant treatment relevant to both the effectiveness of the treatment but also symptoms that may arise during tapering or discontinuation of the treatment. The efﬁcacy of antidepressant treatment for depression is modest compared with placebo [2, 3] and many patients may need additional treatment or treatment adjustments. Common treatment strategies in patients with depression for whom antidepressant treatment has not been effective include increasing the antidepressant dose or switching to a different antidepressant; such strategies may, however, also largely be ineffective [4–7]. Although a relation between SERT occupancy and clinical improvement in depressive symptoms has not been shown [1], knowledge about the relationship between antide- pressant dose and SERT occupancy, as well as overlapping actions on SERT between different antidepressants, may be able to provide some mechanistic insight into the apparently limited effectiveness of the above treatment strategies. Approximately half of the patients stopping or reducing the dose of antidepres- sants experience withdrawal symptoms [8], which, among others, may include ﬂu-like symptoms, anxiety, emotional lability, low- ering of mood, and irritability [9, 10]. As one postulated Many antidepressants are deﬁned by their high afﬁnity for the serotonin transporter (SERT). Brain imaging studies using positron emission tomography (PET) and single photon emission computed tomography (SPECT) techniques have enabled the in vivo visualiza- tion of neurotransmitter receptor occupancy, and thus of the proportion of SERT blocked, at a given dose of a drug. By using radioligands that bind to the available SERT receptors, PET and SPECT techniques provide an estimate of the expression of SERT in a particular brain region, usually given as the non-displaceable binding potential (BPND)—the ratio of the speciﬁcally bound radioligand to that of non-displaceable radioligand. When anti- depressants with an afﬁnity for SERT are administered, the SERT availability for binding of the radioligand decreases, depending on the binding of the antidepressant to SERT. By visualizing the binding potential in the drug free state and subsequently after administra- tion of an antidepressant, PET and SPECT imaging can provide an estimate of the antidepressant occupancy of SERT. p p y As one postulated working mechanism of many antidepressants is serotonin reuptake inhibition via blockade of SERT [1], PET and SPECT imaging studies may potentially provide mechanistic Received: 7 June 2021 Revised: 11 August 2021 Accepted: 25 August 2021 A. Sørensen et al. METHODS We conducted a systematic review of in vivo PET and SPECT studies that measured SERT occupancy of antidepressants in humans using a within-subject design. We registered a protocol at the Open Science Framework before undertaking the review, which can be accessed at: https://osf.io/b6hau/. We reported the review according to the PRISMA guidelines [20]. Data items and data extraction Data were extracted by two researchers independently using a standardized and piloted extraction form. We extracted the following data items: author, year, antidepressant name, antide- pressant dose, occupancy, brain region, antidepressant plasma- or serum concentration, ED50, duration of intake of the antidepressant, time lag between administration of last dose and time of scanning, type of scan, ligand, method used to quantify SERT binding potential, characteristics of the study (availability of a study protocol, assessment of drug adherence, time of day of scan, fasting regimen used), and characteristics of the participants (age, sex, diagnosis, smoking status, alcohol use, and other medications used). While several individual studies have investigated the relationship between dose and SERT occupancy of antidepressants [17–19], this evidence has not been systematically reviewed, which we, therefore, aimed to do. Our primary objective was to determine the relation- ship between the dose and SERT occupancy measured with highly selective radioligands for antidepressants. In studies that repeated measurements of SERT occupancy over time after discontinuation, we additionally aimed to determine the SERT occupancy decline rate and relate it to the plasma concentration decline rate. We contacted study authors whenever data was not available in the articles. When study authors did not reply to supply the data, we extracted it from graphs where possible using a web plot digitizer. This was needed in one study [17]. INTRODUCTION 2 potentially eligible titles were retrieved and read by two researchers independently (AS and KM). Reasons for exclusion were noted. Disagreements were resolved by discussion, which, in case of unresolved issues, involved a third researcher. mechanism underlying antidepressant withdrawal symptoms is a rapid decrease in SERT occupancy arising when antidepressants are tapered or stopped [11, 12], a clearer understanding of the SERT occupancy at speciﬁc, especially lower, doses may provide essential mechanistic information for understanding the occur- rence of withdrawal symptoms. Given the paucity of evidence for speciﬁc tapering regimens [13, 14], this could perhaps offer insights into better approaches to mitigate withdrawal symptoms. The potential utility of the dose/occupancy relationship in guiding tapering to mitigate withdrawal symptoms has received recent interest by multiple groups [11, 12, 15], and was considered in a recent Cochrane review on approaches to antidepressant discontinuation [14] and in a recent iteration of a major guideline on the management of patients with mood disorders [16]. Data synthesis We described the study characteristics and presented key study characteristics in tables. We assessed, for each study, several factors that may affect the relationship between antidepressant dose and occupancy. These were speciﬁcation of dosing regimen (duration, dose, assessment of adherence, time of last intake), standardization of laboratory methods (time of day, fasting regimen, precision of scanning results, lag time between dose administration and scanning), clinical characterization of partici- pants (clinical diagnosis, age, gender, co-medication, smoking, alcohol use), method used to quantify binding potential, use of reference region, and the type of radioligand. Search strategy and selection criteria AND exp serotonin trans- porter/ AND (occupancy or “binding potential” or availabilit or block* or chang* or inhibit* or binding ratio or reduc* or quantiﬁc* or alter*).af. AND (PET.mp. or exp positron emission tomography/ + SPECT.mp. or exp single photon emission computed tomography/). The search strategy for PubMed is available in the study protocol. In addition to electronic searches, we scanned the references of retrieved articles and relevant review articles. f x; K; Vm ð Þ ¼ Vmx K þ x where Vm is the horizontal asymptote (expressing maximum occupancy), x is the drug dose and the parameter K is the dose where the occupancy is halfway between 0 and Vm [27]. For these models, the lowest dose and occupancy were ﬁxed at 0 and the maximum dose at the highest dosing in any of the included studies. We then plotted the predicted dose-occupancy curve from the model as an overlay to the data plots of occupancy and dose. In our protocol, we planned to investigate the time-course of the occupancy decline rate as it relates to the antidepressant plasma concentration decline rate, after taking the last dose, by calculating pooled correlation coefﬁcients from the studies that Molecular Psychiatry RESULTS Our literature search identiﬁed 793 abstracts. Screening of titles and abstracts identiﬁed 100 records for which we obtained a full- text report. After further removing duplicates (N = 16), excluding review articles (N = 5), studies not measuring SERT occupancy by antidepressants (N = 23), studies administering the drug intrave- nously (N = 4), an animal study (N = 1), studies using a between- subjects design (N = 4), studies using the non-selective ligands [11C](+)-McN or [123I]-ß-CIT (N = 20), studies of drugs not approved for use in depression or that have been withdrawn (N = 8), and studies not providing occupancy expressed as a mean (N = 3), a total of 16 articles (reporting on 17 unique studies) were included [17–19, 28–40]. In one article [29], outcomes and methods were elaborated in two other publications [41, 42], which we included accordingly. PRISMA ﬂowchart of study selection process is available in Supplementary Figure 1. Excluded studies, with reasons, are available in Supplementary Table 1. , , pp y We contacted the authors of 12 articles to obtain missing data, four of whom were able to provide the requested data [31, 33, 35, 38] (previously unreported data is marked (†) in the tables). y The 17 studies investigated 10 different antidepressants using three different ligands ([11C]-DASB (N = 11), [11C]-MADAM (N = 2), and [123I]-ADAM (N = 4)) and comprised a total of 294 participants (Table 1). In two studies the participants participated in two different scans at different doses after a washout period [30, 33], resulting in a total of 309 unique measurements of occupancy. Five studies provided data on the time-course of SERT occupancy as it decreases over time after a single dose [30, 35, 39, 40] or repeated (7–10 days) [36, 40] administration of antidepressants in healthy volunteers. Three of these studies compared the decline rates of occupancy and plasma concentra- tion (Fig. 2) [30, 36, 39]. For escitalopram [30], citalopram [36], and sertraline [30], but not paroxetine [30] and ﬂuvoxamine [39], SERT occupancy appeared to decrease at a slower rate than the plasma concentration (Fig. 2). The designs of the studies investigated were highly hetero- geneous, involving 16 different regions of interest, 11 different durations of drug-intake, 15 different periods of time lag between administration of last dose and time of scanning, two different health statuses (depressed patients or healthy controls), and four different modeling-approaches. Study selection process Titles and abstracts were screened for eligibility by two researchers independently (AS and KM). Full-text versions of Molecular Psychiatry A. Sørensen et al. 3 desvenlafaxine in 8 participants (one study) [33], and ﬂuvoxamine in six participants (one study) [39]. measured occupancy at several different time points. However, for any drug and dose, no more than one study provided such data, and a meta-analysis was therefore not possible. We, therefore, presented those data narratively instead. p p y Occupancy at different doses and brain regions is presented in Table 2 as the range of means (SD) for each drug. Four studies calculated and reported the dose corresponding to 50% of maximal occupancy (ED50). ED50 values in mg were 3·4 for citalopram [17], 9·1 for sertraline [17], 5 for paroxetine [17], 2·7 for ﬂuoxetine [17], 5·8 for venlafaxine [17], 7·9 for duloxetine [40], 8·5 for vortioxetine [42], and 14·4 for desvenlafaxine [33]. Figure 1 illustrates the dose-occupancy relationship for antidepressants that were administered at four or more different doses (citalopram, desvenlafaxine, duloxetine, escitalopram, ﬂuoxetine, paroxetine, venlafaxine, and vortioxetine). The relationship between dose and SERT occupancy was ﬁtted according to a 2-parameter Michaelis–Menten model for all antidepressants separately: occupancy increased hyperbolically with increasing antidepressant dose in the lower dose-range, reaching a plateau at an occupancy of approximately 80% at roughly the usual minimum recommended dose for depression (Fig. 1). The relation- ship between dose and occupancy appeared largely similar across drugs (Fig. 1). For desvenlafaxine and escitalopram, the relationship between dose and occupancy appeared relatively consistent across brain regions; only for escitalopram, the plateau appeared to be reached at a slightly lower occupancy in the putamen, compared with other brain regions, as shown in Table 2 and Fig. 1. The parameter estimates for the Michaelis–Menten model for each antidepressant and brain region of interest (RoI) are provided in Supplementary Table 2. RESULTS An arterial input function was used in one study [33]. The included studies also varied in duration of drug-intake after the (drug-free) baseline scan until imaging of the drug-occupancy, with six studies administering the drug just once and 10 studies using a repeated dose regimen (ranging from 4 days to 10 weeks). Three studies measured occupancy after both single and repeated dosing in the same subjects [28, 31, 41], all of which showed increased occupancy after repeated dosing compared with a single dose. For duloxetine at 20 mg, occupancy increased from 65% to 78% after four days [28]. For citalopram at 20 mg and escitalopram at 10 mg, occupancy increased from 73% and 74% to 80% and 77%, respectively, after 25 days [31]. For vortioxetine at 2·5, 10, and 60 mg, occupancy increased from 27%, 44%, and 70%, to 35%, 63%, and 93%, respectively, after nine days [41]. ( g g y ) All included studies controlled for adherence to the antide- pressant used in the study by measuring plasma- or serum concentrations. Nine of the included studies provided no information on whether participants were taking other drugs and stated no such restrictions to study entry. Eight studies excluded participants taking other possibly interacting medica- tions (e.g., other psychotropic drugs or substances with high afﬁnity for SERT) [19, 31, 32, 37–40, 42]. A fasting regimen was mentioned in one study [35]: requiring a minimum of 4 h fasting prior to imaging. Two studies mentioned having standardized the time of day of drug administration and scanning [19, 35]. One study reported overall alcohol consumption during the study period [35], and seven studies controlled for present or past alcohol problems or abuse [17, 28, 30, 33, 34, 39, 40]. Five of 17 studies included non-smokers only, while the remaining studies did not mention smoking status [19, 30, 35, 39, 40]. None of the studies provided information on a pre-published study protocol. SERT occupancy of escitalopram was investigated in 55 participants (six studies) [18, 19, 30, 31, 35, 36], citalopram in 77 participants (eight studies) [17–19, 31, 34, 36–38], vortioxetine in 46 participants (two studies presented in one article) [41], paroxetine in 27 participants (three studies) [17, 30, 32], duloxetine in 25 participants (two studies) [28, 40], sertraline in 18 participants (two studies) [17, 30], venlafaxine in 18 participants (one study) [17], ﬂuoxetine in 18 participants (one study) [17], DISCUSSION For this ﬁrst systematic review of the evidence of the relationship between dose and SERT occupancy of antidepressants we identiﬁed 17 studies investigating 10 different SSRIs, SNRIs and serotonin modulators in a total of 294 participants, comprising 309 unique measurements of SERT occupancy with highly speciﬁc ligands. Overall, occupancy increased with higher dose but in a hyperbolic pattern: occupancy increased rapidly at lower doses and reached an apparent plateau at approximately 80%––at the usual minimum recommended dose––modeled by the Michaelis–Menten equation. Generally, the studies were small, only a few studies investigated the same antidepressant at the same dose and in the same RoI, and few studies reported information on relevant factors that may inﬂuence drug metabo- lization and hence bioavailability of antidepressants (e.g. use of potentially interacting drugs, smoking status [43], and alcohol consumption [44]). The Michaelis–Menten curves we reconstructed based on the ﬁndings in individual studies showed that overall, there was no substantial increase of SERT occupancy with SSRI and SNRI doses above the usual minimum recommended doses for depression. Molecular Psychiatry A. Sørensen et al. Table 1. Study characteristics of included studies. Study Drug N Total N males Dose range (mg) Ligand Lag (hours) Duration (days) Age (mean ± SD years) Diagnosis RoI Ref. model Meyer et al. (2004) [17] Cit 18 NAa 1–60 DASB 6–13 28 NAa Mix of healthy and MDDb Str, BT, ACC, PFC, mid, BC Logan Ven 18 2.4–225 Flu 18 1–60 Ser 14 10–200 Par 14 5–60 Klein et al. (2006) [18] Cit 10 20 10–20 ADAM 6 1 NAc Healthy Mid SRTM Esc 15 5–20 Lundberg et al. (2007) [19] Cit 8 16 20 MADAM 6 1 NA Healthy ACC, FC, TC, ins, hip, put, rap SRTM Esc 8 10 Baldinger et al. (2014) [31] Cit 9 6 20 DASB 6 1 + 25 42.3 ± 7.8 MDD Accu, ins, amy, cau, put, tha, str, mid MRTM2 Esc 10 10 Klein et al. (2007) [36] Cit 9 9 20 ADAM 6 + 54 10 28 ± 3 Healthy Mid SRTM Esc 6 10 Smith et al. (2011) [38] Cit 7 4 20–40 DASB NA 56–70 65 ± 5 MDD Str, tha Logan, MRTM2 Houle et al. (2000) [34] Cit 3 NA 40 DASB 3 1 NA Healthy Str, mid, tha NA Herold et al. (2006) [37] Cit 13 11 10 ADAM 6–7 7 NA MDD Mid Logan Kim et al. DISCUSSION (2017) [35] Esc 12 12 5–30 DASB 3 + 24 + 46 1 23 ± 2.7 Healthy Put, DRN, cau, tha MRTM2 Arakawa et al. (2016) [30] Esc 8 8 10–20 DASB 4 + 24 + 48 1 29.1 ± 4.6 Healthy Tha SRTM Ser 4 50 Par 4 20 Catafau et al. (2006) [32] Par 9 NAd 20 ADAM NA 39 NA MDD Mid, tha, str Tissue ratio method Takano et al. (2006a) [39] Flv 6 6 50 DASB 5 + 26 + 53 1 24.3 ± 4.8 Healthy FC, tha, str, hip, amy MRTM2 Takano et al. (2006b) [40] Dul 15 15 5–60 DASB 6 + 25 + 49 + 53 + 78 1 + 7 24.1 ± 2.4 Healthy Tha MRTM2 Abanades et al. (2011) [28] Dul 10 10 20 DASB 6, 4 1 + 4 40.2 ± 11 Healthy Mid, str, tha SRTM Areberg et al. (2012) [41] Vor 35 35 2.5–20 DASB 7 13 NAa Healthy Rap SRTM Areberg et al. (2012) [41, 42] Vor 11 11 2.5–60 MADAM 7 1 + 9 NAa Healthy Rap SRTM Frankle et al. (2018) [33] Des 15 8 25–150 DASB 24e 3 27 ± 9 Healthy Mid, tha, amy, str SRTM N number of participants, lag (hours) time lag in hours between drug administration and scanning, SD standard deviation, RoI brain region of interest, Ref. model reference model for quantifying binding potential, cit citalopram, ven venlafaxine, ﬂu ﬂuoxetine, ser sertraline, par paroxetine, esc escitalopram, ﬂv ﬂuvoxamine, dul duloxetine, vor vortioxetine, des desvenlafaxine, NA not applicable, MDD major depressive disorder, str striatum, BT bilateral thalamus, ACC anterior cingulate cortex, PFC prefrontal cortex, mid midbrain, BC bilateral cuneus, FC frontal cortex, TC temporal cortex, ins insula, hip hippocampus, put putamen, rap raphe nuclei, accu accumbens, amy amygdala, tha thalamus, cau caudate, DRN dorsal raphe nucleus, SRTM simpliﬁed reference tissue model, MRTM-2 multilinear reference tissue model 2. aInformation on sex and age provided only for 77 of 82 participants in Meyer 2004 (33 females, 44 males, mean age (SD) 35 (9), and only for both groups combined in Areberg 2012 (46 males, mean age 28 years (21–41). bHealthy participants received low doses, unhealthy participants received high doses. cMean age data includes four dropouts (26.8 years for all 29 participants). dParticipant characteristics include one dropout (6 males, 4 females, mean age (SD) 36 (10.8), range 20–53). eU i t thi i A. DISCUSSION Sørensen et al. 4 Molecular Psychiatry A. Sørensen et al. 5 Table 2 continued Table 2. Serotonin transporter occupancy at different antidepressant doses. antidepressant doses. DISCUSSION There is no ﬁrm evidence that clinical efﬁcacy [1, 17, 45], nor adverse effects are dependent on SERT occupancy; the studies included in the present review that measured the relationship between clinical effect and SERT occupancy did not ﬁnd signiﬁcant correlations [17, 32, 37, 38, 48]. Table 2 continued Even at, and below, the lowest manufactured dose of the antidepressants we included, there is considerable SERT occupancy. For all drugs except vortioxetine, 50% occupancy occurred at doses quite below half of the lowest manufactured dose. Given this hyperbolic relationship between dose and SERT occupancy, even relatively small dose changes at the lower dose range will have large effects on SERT occupancy and thus presumably on synaptic serotonin levels—with progressively increasing magnitude as the dose decreases linearly towards zero. This ﬁnding may particularly have implications for discontinuation and tapering, as a linear tapering regimen, involving stopping at the lowest manufactured dose, or even half of it would correspond to increasingly larger reductions in occupancy, which might be related to the occurrence of withdrawal symptoms. Assuming that withdrawal symptoms are predominantly related to unblocking of SERT, a linear and gradual unblocking of SERT, which has been suggested to mitigate withdrawal symptoms [11, 45], would require a hyperbolic dose reduction regimen, necessitating smaller dose decrements than possible with currently manufactured antidepressants. Eventually, this assumption should be tested in a blinded RCT, where the primary hypothesis would be that discontinuation via hyperbolic tapering should be more successful, with less withdrawal symptoms than stopping via the lowest manufactured dose (or half of it). t a stopp g a t e o est a u actu ed dose (o a o t). Conversely, even large dose reductions above the occupancy plateau appear to be associated with only relatively minor decreases in SERT occupancy, and dose reductions at higher doses may therefore potentially be less likely to result in marked withdrawal symptoms although, similar to the lack of evidence for a relationship with efﬁcacy, there is no unequivocal evidence of a correlation between changes in SERT occupancy and withdrawal symptoms. As SSRIs, SNRIs, and serotonin modulators are not selective to SERT [49–52], dose reductions in the higher dose- range, while not markedly reducing SERT occupancy, could potentially also result in changes in other transmitter systems that could potentially be associated with withdrawal symptoms. DISCUSSION Citalopram Dose in mg N RoI (number of occupancy measures) Occupancy (mean ± SD) Duration of drug- intake (days) 1 (17) 2 str (2) 16 ± 6% 28 2.5 (17) 2 str (2) 42 ± 1% 28 5 (17) 2 str (2) 67 ± 18% 28 10 (17, 18, 37) 21 overall 61–76 ± 9% str (3) 76 ± 9% 28 mid (22) 61–65 ± 10% 7, 1 20 (17–19, 31, 36, 38) 39 overall 64 ± 13–91 ± 5 str (17) 74 ± 5–77 ± 10 1, 28 mid (23) 64 ± 13–86 ± 4 10, 25 ACC (17) 75 ± 16–80 ± 21 1, 25 FC (8) 75 ± 14 1 TC (8) 66 ± 19 1 ins (17) 71 ± 12–73 ± 9 25, 1 hip (8) 78 ± 17 1 put (17) 68 ± 5–76 ± 4 1, 25 rap (8) 76 ± 9 1 amy (9) 91 ± 5 25 cau (9) 80 ± 5 25 tha (13) 74 ± 7–77 ± 6 56–70, 25 accu (9) 84 ± 4 25 30 (38)† 1 overall 67–70 str (1) 67 56–70 tha (1) 70 56–70 40 (17, 34, 38)a 6 overall 80 ± 5–85 ± 4 str (8) 73 ± 6–85 ± 4 56–70, 28 mid (3) 80 ± 5 1 tha (5) 80 ± 7–80 ± 5 56–70, 1 60 (17) 2 str (2) 87 ± 6 28 Escitalopram 5 (18, 35)a 9 overall 50 ± 1–67 ± 7 mid (5) 60 ± 6 1 put (4) 51 ± 1 1 DRN (4) 56 ± 2 1 cau (4) 67 ± 7 1 tha (4) 50 ± 1 1 10 (18, 19, 30, 31, 35, 36) 37 overall 59 ± 5–88 ± 9 mid (20) 64 ± 6–81 ± 5 1, 10 ACC (18) 65 ± 11–75 ± 8 1, 25 FT (8) 67 ± 12 1 TC (8) 63 ± 23 1 ins (18) 59 ± 15–69 ± 9 1, 25 hip (8) 59 ± 23 1 put (22) 59 ± 5–72 ± 4 1, 25 rap (8) 69 ± 13 1 amy (10) 88 ± 9 25 cau (14) 69 ± 5–78 ± 4 1, 25 tha (18) 60 ± 3–75 ± 4 1, 25 str (10) 73 ± 4 25 accu (10) 81 ± 4 25 DRN (4) 74 ± 8 25 20 (18, 30, 35)a 10 Overall 65–81 mid (5) 75 ± 5 1 put (1) 65 1 DRN (1) 81 1 cau (1) 77 1 tha (5) 72–78 ± 3 1, 1 Citalopram Dose in mg N RoI (number of occupancy measures) Occupancy (mean ± SD) Duration of drug- intake (days) 30 (35)a 3 overall 62 ± 3–79 ± 6 put (3) 62 ± 3 1 DRN (3) 79 ± 6 1 cau (3) 71 ± 11 1 tha (3) 64 ± 7 1 Sertraline 10 (17) 3 str (3) 49 ± 13 28 25 (17) 2 str (2) 72 ± 4 28 50 (17, 30) 7 overall 74 ± 6–85 ± 7 1, 28 str (3) 85 ± 7 28 tha (4) 74 ± 6 1 100 (17) 4 str (4) 86 ± 3 28 150 (17) 1 str (1) 87 28 200 (17) 1 str (1) 84 28 Paroxetine 5 (17) 2 str (2) 52 ± 16 28 10 (17) 1 str (1) 60 28 20 (17, 30, 32) 21 overall 45 ± 21–93 ± 8 str (17) 61 ± 11–82 ± 10 39, 28 BT (7) 75 ± 16 28 ACC (7) 76 ± 15 28 PFC (7) 80 ± 18 28 tha (13) 45 ± 21–63 ± 10 1, 39 mid (16) 66 ± 10–93 ± 8 39, 28 BC (7) 67 ± 29 28 40 (17) 2 str (2) 90 ± 2 28 60 (17) 1 str (1) 91 28 Fluoxetine 1 (17) 2 str (2) 30 ± 6 28 2.5 (17) 2 str (2) 41 ± 13 28 4 (17) 1 str (1) 67 28 5 (17) 2 str (2) 65 ± 6 28 10 (17) 2 str (2) 73 ± 1 28 20 (17) 4 overall 69 ± 9–85 ± 9 28 str (4) 76 ± 8 28 BT (4) 69 ± 9 28 ACC (4) 80 ± 14 28 PFC (4) 85 ± 9 28 mid (4) 82 ± 9 28 BC (4) 81 ± 6 28 40 (17) 4 str (4) 83 ± 9 28 60 (17) 1 str (1) 82 28 Fluvoxamine 50 (39) 6 overall 71 ± 2–76 ± 3 FC (6) 75 ± 9 28 tha (6) 72 ± 4 28 str (6) 71 ± 2 28 hip (6) 76 ± 3 28 amy (6) 72 ± 13 28 Venlafaxine 2.4 (17) 2 str (2) 25 ± 13 28 5 (17) 2 str (2) 40 ± 1 28 10 (17) 1 str (1) 63 28 18.75 (17) 2 str (2) 66 ± 3 28 37.5 (17) 3 str (3) 76 ± 10 28 75 (17) 4 overall 71 ± 10–92 ± 5 28 str (4) 84 ± 2 28 BT (4) 71 ± 10 28 ACC (4) 85 ± 13 28 Molecular Psychiatry A. DISCUSSION Sørensen et al. 6 Table 2 continued Citalopram Dose in mg N RoI (number of occupancy measures) Occupancy (mean ± SD) Duration of drug- intake (days) PFC (4) 91 ± 11 28 mid (4) 91 ± 8 28 BC (4) 92 ± 5 28 150 (17) 2 str (2) 90 ± 1 28 225 (17) 2 str (2) 87 ± 4 28 Duloxetine 5 (40) 3 tha (3) 44 ± 9 1 20 (28, 40) 13 overall 71 ± 5–85 ± 4 tha (13) 71 ± 5–74 ± 7 1, 4 mid (10) 85 ± 4 4 str (10) 75 ± 8 4 40 (40) 3 tha (3) 81 ± 5 1 60 (40) 3 tha (3) 85 ± 3 7 Vortioxetine 2.5 (41, 42) 16 RN (16) 35 ± 10–49 ± 12 9, 13 5 (41) 11 RN (11) 51 ± 10 13 10 (42) 4 RN (4) 63 ± 23 9 20 (41) 12 RN (12) 90 ± 6 13 60 (42) 3 RN (3) 93 ± 9 9 Desvenlafaxine 25 (33) 4 overall 55 ± 5–71 ± 13 mid (4) 68 ± 8 3 tha (4) 55 ± 5 3 amy (4) 71 ± 13 3 str (4) 60 ± 7 3 50 (33) 4 overall 70 ± 8–90 ± 9 mid (4) 85 ± 7 3 tha (4) 70 ± 8 3 amy (4) 90 ± 9 3 str (4) 77 ± 7 3 100 (33) 3 overall 78 ± 2–96 ± 5 mid (3) 87 ± 6 3 tha (3) 78 ± 2 3 amy (3) 96 ± 5 3 str (3) 87 ± 1 3 150 (33) 4 overall 90 ± 3–97 ± 4 mid (4) 94 ± 6 3 tha (4) 91 ± 10 3 amy (4) 97 ± 4 3 str (4) 90 ± 3 3 above the lower range of the licensed dose in general, whether using a ﬁxed dosing regimen [4, 5], a ﬂexible dosing regimen [46], or as a second-step strategy in patients not responding to standard dosing [6, 45]. Adverse events, in contrast, may have an ascending dose-response curve [4, 47], presumably due to co- afﬁnity to other receptors. Molecular Psychiatry DISCUSSION However, given that the shape of the receptor occupancy curve is hyperbolic for most dose–response relationships, consequent to the law of mass action [53, 54], hyperbolic dose reduction is likely pharmacologically meaningful regardless of the speciﬁc receptor systems contributing to withdrawal symptoms. Where a dose was only investigated in one study, the mean (SD) occupancy of that individual study is presented; where a dose was investigated in multiple studies, the range of means (SD) from those studies is presented. Duration of drug-intake for the individual studies is presented in the same order as the occupancy range. The observation that escitalopram, citalopram, and sertraline SERT occupancy appear to decrease at a slower rate compared with the plasma concentration of the drugs may indicate that plasma half-life does not accurately reﬂect the rate at which SERT occupancy declines. This apparently delayed decrease in SERT occupancy compared with the plasma concentration decline could be speculated to contribute to the delayed withdrawal effects, which have been observed clinically. N number of participants, RoI brain region of interest (number of occupancy measures in the different regions), SD standard deviation, str striatum, mid midbrain, ACC anterior cingulate cortex, FC frontal cortex, TC temporal cortex, ins insula, hip hippocampus, put putamen, rap raphe nuclei, amy amygdala, cau caudate, tha thalamus, accu accumbens, DRN dorsal raphe nucleus, BC bilateral cuneus. aFull occupancy data is unique to this review. Several issues related to the study designs must be taken into consideration when interpreting our ﬁndings since these could contribute to the heterogeneity of ﬁndings between studies. The included studies that measured occupancy after both single and repeated dosing all found that occupancy increased with longer treatment, the absolute occupancy being in the range of 3 to 23% higher after repeated dosing compared with single dosing [28, 31, 41]. This suggests that occupancy data from single-dose studies may underestimate the occupancy occurring in patients who take the drugs continuously for extended periods. A possible reason This ﬁnding is in accordance with one dose-escalation occupancy study of paroxetine using the β-CIT, a ligand not included in the present review, that found no increase in SERT occupancy, nor a clinical effect as a result of paroxetine dose escalation from 20 to 50 mg, despite observed increases in paroxetine serum concen- tration [45]. DISCUSSION Finally, occupancies in smaller brain regions, like the amygdala, are likely determined with less certainty compared with larger brain regions due to issues of radiotracer reliability in brain regions that are small relative to the resolution of SPECT. for this ﬁnding is that after the ﬁrst administration of an antidepressant these lipophilic drugs dissolve into the entire body, which only becomes saturated after repeated dosing. Additionally, studies varied in the duration between the last dose administration and time of scanning (ranging from three to 24 h), which could underlie some of the heterogeneity of the observed SERT occupancy, although this is expected to cause less variability after longer treatment when a steady-state of the antidepressant plasma concentration will be established. It is also not clear whether our ﬁndings are generalizable to all patients on antidepressants, especially for all ages and both sexes, as most included studies investigated healthy individuals in the age range from 25 to 40 years, of whom most were male. Few studies provided precise information on all factors relevant to measuring occupancy, and accurate participant characteristics were sometimes difﬁcult to determine since some studies included dropouts and later excluded participants when reporting age and sex characteristics. Tobacco smoking [43], alcohol consumption [44], fasting [55], and con- comitant drug use [56, 57] may inﬂuence antidepressant metabo- lization and thus the bioavailability of the drugs, which, in turn, could affect the relationship between antidepressant dose and occupancy, but information on those factors was not available in most studies. Sample sizes were generally small; ﬁve drugs were investigated in less than 20 participants each, and some doses were investigated in just one to three participants. Consideration should also be given to the fact that PET and SPECT are known to detect signals of unspeciﬁc radioligand binding, i.e. noise that does not represent target occupancy. Therefore, a reference region assumed devoid of speciﬁc SERT binding must be used as control, assuming that signals detected in this brain region do not represent speciﬁc binding to SERT. Cerebellum was earlier suggested as the optimal Our study is the ﬁrst to review the body of evidence of the relationship between dose and SERT occupancy of antidepressants. DISCUSSION It is also in agreement with the observation that there is a limited beneﬁt associated with increasing doses of SSRIs Molecular Psychiatry Molecular Psychiatry Molecular Psychiatry A. Sørensen et al. upancy and dose relationship for antidepressants administered at four or more doses. The dose-occupancy relatio sants that were administered at four or more different doses (citalopram, desvenlafaxine, duloxetine, escitalopram, ﬂ venlafaxine, and vortioxetine), ﬁtted according to a 2-parameter Michaelis–Menten model as f x; K; Vm ð Þ ¼ Vmx Kþx, where asymptote (expressing maximum occupancy), x is the drug dose and the parameter K is the dose where the occupancy and Vm. The parameter estimates Vm and K for each model are provided in Supplementary Table 2. For each individ represented by uniquely colored dots; the size of the dots is proportional to the number of occupancy measures. Dashe sent the usual minimum recommended dose. A. Sørensen et al. sychiatry 7 Fig. 1 Occupancy and dose relationship for antidepressants administered at four or more doses. The dose-occupancy relationship for antidepressants that were administered at four or more different doses (citalopram, desvenlafaxine, duloxetine, escitalopram, ﬂuoxetine, paroxetine, venlafaxine, and vortioxetine), ﬁtted according to a 2-parameter Michaelis–Menten model as f x; K; Vm ð Þ ¼ Vmx Kþx, where Vm is the horizontal asymptote (expressing maximum occupancy), x is the drug dose and the parameter K is the dose where the occupancy is halfway between 0 and Vm. The parameter estimates Vm and K for each model are provided in Supplementary Table 2. For each individual ﬁgure, studies are represented by uniquely colored dots; the size of the dots is proportional to the number of occupancy measures. Dashed vertical lines represent the usual minimum recommended dose. Molecular Psychiatry A. Sørensen et al. 8 Fig. 2 Time-course of occupancy and plasma/serum concentration. The relationship between serotonin transporter occupancy and plasma serum concentration as it decreases over time after dose administration of escitalopram, citalopram, sertraline, paroxetine, and ﬂuvoxamin mg milligram, N number of participants, SERT Serotonin transporter, H hours after drug administration, Occ serotonin transporter occupanc ng/mL nanograms per milliliter, nmol/L nanomoles per litre. Fig. 2 Time-course of occupancy and plasma/serum concentration. The relationship between serotonin transporter occupancy and plasma/ serum concentration as it decreases over time after dose administration of escitalopram, citalopram, sertraline, paroxetine, and ﬂuvoxamine. DISCUSSION mg milligram, N number of participants, SERT Serotonin transporter, H hours after drug administration, Occ serotonin transporter occupancy, ng/mL nanograms per milliliter, nmol/L nanomoles per litre. for this ﬁnding is that after the ﬁrst administration of an antidepressant these lipophilic drugs dissolve into the entire body, which only becomes saturated after repeated dosing. Additionally, studies varied in the duration between the last dose administration and time of scanning (ranging from three to 24 h), which could underlie some of the heterogeneity of the observed SERT occupancy, although this is expected to cause less variability after longer treatment when a steady-state of the antidepressant plasma concentration will be established. It is also not clear whether our ﬁndings are generalizable to all patients on antidepressants, especially for all ages and both sexes, as most included studies investigated healthy individuals in the age range from 25 to 40 years, of whom most were male. Few studies provided precise information on all factors relevant to measuring occupancy, and accurate participant characteristics were sometimes difﬁcult to determine since some studies included dropouts and later excluded participants when reporting age and sex characteristics. Tobacco smoking [43], alcohol consumption [44], fasting [55], and con- comitant drug use [56, 57] may inﬂuence antidepressant metabo- lization and thus the bioavailability of the drugs, which, in turn, could affect the relationship between antidepressant dose and occupancy, but information on those factors was not available in most studies. Sample sizes were generally small; ﬁve drugs were investigated in less than 20 participants each, and some doses were investigated in just one to three participants. Consideration should also be given to the fact that PET and SPECT are known to detect signals of unspeciﬁc radioligand binding, i.e. noise that does not represent target occupancy. Therefore, a reference region assumed devoid of speciﬁc SERT binding must be used as control, assuming that signals detected in this brain region do not represent speciﬁc binding to SERT. Cerebellum was earlier suggested as the optimal reference region, but later studies revealed that cerebellum is not completely devoid of speciﬁc SERT binding. Therefore, the binding potential will be slightly overestimated if this issue is not speciﬁcally controlled for, which only one study did [35]. Since all included studies used cerebellum as reference region, this will have resulted in a small and systematic bias. DISCUSSION Along the same line, we included studies regardless of whether co-medication was allowed, which may have contributed to heterogeneity in results between studies; due to the scarcity of data it was not possible to explore the potential effect of co-medication. unresolved. Third, our literature search may have missed some studies that did measure occupancy but used different terms due to inconsistent nomenclature for occupancy and binding potential, especially in the ﬁeld’s earlier stages. However, our search strategy was created with this in mind, and we systematically scanned the reference lists of all included studies. Fourth, our plot overlays ﬁtted using the Michalis-Menten model did not consider the sample size of individual studies; an overlay plot based on individual patient data would have been preferable, but we did not obtain those data. Along this line, the dose ranges were limited for some drugs, which means that it is uncertain whether the observed plateau for those drugs at approximately 80% occupancy represents the highest possible level of the plateau, e.g. for escitalopram the highest dose was 30 mg and for duloxetine 60 mg. Lastly, in order to investigate differences in occupancy between single- and repeated dosing regimens, which appear to yield different occupancy levels, we synthesized the evidence using both dosing regimens but due to few studies, it was not possible to construct ﬁtted curves stratiﬁed by dosing regimen. Along the same line, we included studies regardless of whether co-medication was allowed, which may have contributed to heterogeneity in results between studies; due to the scarcity of data it was not possible to explore the potential effect of co-medication. 3. Cipriani A, Furukawa TA, Salanti G, Chaimani A, Atkinson LZ, Ogawa Y, et al. Comparative efﬁcacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis. Lancet 2018;391:1357–66. 4. Furukawa TA, Cipriani A, Cowen PJ, Leucht S, Egger M, Salanti G. Optimal dose of selective serotonin reuptake inhibitors, venlafaxine, and mirtazapine in major depression: a systematic review and dose-response meta-analysis. Lancet Psy- chiatry 2019;6:601–9. 5. Braun C, Adams A, Rink L, Bschor T, Kuhr K, Baethge C. In search of a dose- response relationship in SSRIs-a systematic review, meta-analysis, and network meta-analysis. Acta Psychiatr Scand 2020;142:430–42. 6. Rink L, Braun C, Bschor T, Henssler J, Franklin J, Baethge C. DATA AVAILABILITY 23. Ginovart N, Wilson AA, Meyer JH, Hussey D, Houle S. Positron emission tomo- graphy quantiﬁcation of [(11)C]-DASB binding to the human serotonin trans- porter: modeling strategies. J Cereb Blood Flow Metab 2001;21:1342–53. All data (including template data collection forms, data extracted from included studies, and data used for all analyses) are available on the Open Science Framework at: https://doi.org/10.17605/OSF.IO/RKYFS. 24. Acton PD, Choi SR, Hou C, Plössl K, Kung HF. Quantiﬁcation of serotonin trans- porters in nonhuman primates using [(123)I]ADAM and SPECT. J Nucl Med 2001;42:1556–62. Conclusion PET d SP 18. Klein N, Sacher J, Geiss-Granadia T, Attarbaschi T, Mossaheb N, Lanzenberger R, et al. In vivo imaging of serotonin transporter occupancy by means of SPECT and [123I]ADAM in healthy subjects administered different doses of escitalopram or citalopram. Psychopharmacol (Berl) 2006;188:263–72. PET and SPECT studies provide a mechanistic background for understanding the limited effect of dose-escalation of antidepres- sants and for the potential emergence of withdrawal symptoms even with small dose reductions in the lower dose range. The evidence base is limited by few, small studies of short treatment duration and sub-optimal, non-uniform reporting, which should be improved in the future. Such improvements could lead to a better understanding of factors inﬂuencing SERT occupancy and the association with treatment efﬁcacy, adverse effects, and withdrawal symptoms after dose-reductions or stopping of antidepressants. 19. Lundberg J, Christophersen JS, Petersen KB, Loft H, Halldin C, Farde L. PET measurement of serotonin transporter occupancy: a comparison of escitalopram and citalopram. Int J Neuropsychopharmacol 2007;10:777–85. 20. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 2009;6:e1000097. 21. Meyer JH. Imaging the serotonin transporter during major depressive disorder and antidepressant treatment. J Psychiatry Neurosci 2007;32:86–102. 22. Chalon S, Tarkiainen J, Garreau L, Hall H, Emond P, Vercouillie J, et al. Pharma- cological characterization of N,N-dimethyl-2-(2-amino-4-methylphenyl thio)ben- zylamine as a ligand of the serotonin transporter with high afﬁnity and selectivity. J Pharm Exp Ther 2003;304:81–7. Implications for future research p Our review points to a need for larger occupancy studies of antidepressants administered also at low doses, investigation of moderators of the dose-occupancy relationship, standardization of methods, assessment of associations with clinical effects, and more transparent reporting. Future studies should also measure other transporters (e.g., NET and DAT) than SERT to uncover the full biological effects of the drugs as many antidepressants also act on non-serotonergic systems. As many patients take antidepressants for years, studies should also study patients (before and) after long treatment duration. Finally, the theoretical link between unblocking of SERT and withdrawal symptoms should ideally be investigated directly, for example by measuring occupancy with repeated measurements during a period of dose reduction while recording the occurrence of potential withdrawal symptoms. The feasibility of conducting such studies, however, would be challenged by likely high costs associated with long follow-up, an unknown event rate, and issues associated with performing repeated PET procedures. 11. Horowitz MA, Taylor D. Tapering of SSRI treatment to mitigate withdrawal symptoms. Lancet Psychiatry 2019;6:538–46. 12. Ruhe HG, Horikx A, van Avendonk MJP, Groeneweg BF, Woutersen-Koch H. Tapering of SSRI treatment to mitigate withdrawal symptoms. Lancet Psychiatry 2019;6:561–2. 13. Maund E, Stuart B, Moore M, Dowrick C, Geraghty AWA, Dawson S, et al. Managing antidepressant discontinuation: a systematic review. Ann Fam Med 2019;17:52–60. 14. Van Leeuwen E, van Driel ML, Horowitz MA, Kendrick T, Donald M, De Sutter AI, et al. Approaches for discontinuation versus continuation of long-term anti- depressant use for depressive and anxiety disorders in adults. Cochrane Database Syst Rev 2021;4:CD013495. 15. Shapiro BB. Subtherapeutic doses of SSRI antidepressants demonstrate con- siderable serotonin transporter occupancy: implications for tapering SSRIs. Psy- chopharmacol (Berl) 2018;235:2779–81. 16. Malhi GS, Bell E, Bassett D, Boyce P, Bryant R, Hazell P, et al. The 2020 Royal Australian and New Zealand College of Psychiatrists clinical practice guidelines for mood disorders. Aust N Z J Psychiatry 2021;55:7–117. 17. Meyer JH, Wilson AA, Sagrati S, Hussey D, Carella A, Potter WZ, et al. Serotonin transporter occupancy of ﬁve selective serotonin reuptake inhibitors at different doses: an [11C]DASB positron emission tomography study. Am J Psychiatry 2004;161:826–35. DISCUSSION Dose increase versus unchanged continuation of antidepressants after initial antidepressant treatment failure in patients with major depressive disorder: a systematic review and meta- analysis of randomized, double-blind trials. J Clin Psychiatry. 2018;79:17r11693. https://doi.org/10.4088/JCP.17r11693. 7. Bschor T, Kern H, Henssler J, Baethge C. Switching the antidepressant after non- response in adults with major depression: a systematic literature search and meta- analysis. J Clin Psychiatry. 2018;79:16r10749. https://doi.org/10.4088/JCP.16r10749. 8. Davies J, Read J. A systematic review into the incidence, severity and duration of antidepressant withdrawal effects: are guidelines evidence-based? Addict Behav 2019;97:111–21. 9. Fava GA, Gatti A, Belaise C, Guidi J, Ofﬁdani E. Withdrawal symptoms after selective serotonin reuptake inhibitor discontinuation: a systematic review. Psy- chother Psychosom 2015;84:72–81. 10. Nielsen M, Hansen EH, Gøtzsche PC. What is the difference between dependence and withdrawal reactions? A comparison of benzodiazepines and selective ser- otonin re-uptake inhibitors: SSRi and dependence. Addiction 2012;107:900–8. DISCUSSION This not only provides a complete picture of the evidence across many different antidepressants but also allows for a more detailed assessment of the relationship between dose and SERT occupancy by integrating data from different studies for the same antidepres- sants. Importantly, it also allowed for an assessment of the limitations in the evidence base. In addition to these strengths, our study has several limitations. First, we presented ﬁndings from studies using different reference tissue models together; these could inﬂuence occupancy measures. However, one study calculated occupancy using three different methods, and found only minor differences between SRTM, MRTM-2, and logan [38], potentially indicating that the reference tissue model does not substantially bias SERT occupancy. Second, comparison of occupancy data from different brain regions is potentially not meaningful, as SERT is not equally distributed throughout the brain [1]. It is therefore possible that the reported occupancy measures do not reﬂect those areas which are most important for patients, clinically. Whether blocking of SERT in some brain regions is of particular importance regarding treatment effect, adverse events, or withdrawal symptoms remains Molecular Psychiatry A. Sørensen et al. 9 unresolved. Third, our literature search may have missed some studies that did measure occupancy but used different terms due to inconsistent nomenclature for occupancy and binding potential, especially in the ﬁeld’s earlier stages. However, our search strategy was created with this in mind, and we systematically scanned the reference lists of all included studies. Fourth, our plot overlays ﬁtted using the Michalis-Menten model did not consider the sample size of individual studies; an overlay plot based on individual patient data would have been preferable, but we did not obtain those data. Along this line, the dose ranges were limited for some drugs, which means that it is uncertain whether the observed plateau for those drugs at approximately 80% occupancy represents the highest possible level of the plateau, e.g. for escitalopram the highest dose was 30 mg and for duloxetine 60 mg. Lastly, in order to investigate differences in occupancy between single- and repeated dosing regimens, which appear to yield different occupancy levels, we synthesized the evidence using both dosing regimens but due to few studies, it was not possible to construct ﬁtted curves stratiﬁed by dosing regimen. 1. Spies M, Knudsen GM, Lanzenberger R, Kasper S. The serotonin transporter in psychiatric disorders: insights from PET imaging. Lancet Psychiatry 2015;2:743–55. 2. Munkholm K, Paludan-Müller AS, Boesen K. Considering the methodological limitations in the evidence base of antidepressants for depression: a reanalysis of a network meta-analysis. BMJ Open 2019;9:e024886. REFERENCES 25. Yang B-H, Wang S-J, Chou Y-H, Su T-P, Chen S-P, Lee J-S, et al. Evaluation of reference tissue model and tissue ratio method for 5-HTT using [123I] ADAM tracer. Comput Methods Prog Biomed 2008;92:294–8. 1. Spies M, Knudsen GM, Lanzenberger R, Kasper S. The serotonin transporter in psychiatric disorders: insights from PET imaging. Lancet Psychiatry 2015;2:743–55. 2. Munkholm K, Paludan-Müller AS, Boesen K. Considering the methodological limitations in the evidence base of antidepressants for depression: a reanalysis of a network meta-analysis. BMJ Open 2019;9:e024886. 1. Spies M, Knudsen GM, Lanzenberger R, Kasper S. The serotonin transporter in psychiatric disorders: insights from PET imaging. Lancet Psychiatry 2015;2:743–55. 26. Lundberg J, Odano I, Olsson H, Halldin C, Farde L. Quantiﬁcation of 11C-MADAM binding to the serotonin transporter in the human brain. J Nucl Med 2005;46:1505–15. 2. Munkholm K, Paludan-Müller AS, Boesen K. Considering the methodological limitations in the evidence base of antidepressants for depression: a reanalysis of a network meta-analysis. BMJ Open 2019;9:e024886. 27. Ritz C, Streibig JC. Nonlinear regression with R. Vol. 2018. Springer Science; 2008. Molecular Psychiatry A. Sørensen et al. 10 28. Abanades S, van der Aart J, Barletta JAR, Marzano C, Searle GE, Salinas CA, et al. Prediction of repeat-dose occupancy from single-dose data: characterisation of the relationship between plasma pharmacokinetics and brain target occupancy. J Cereb Blood Flow Metab 2011;31:944–52. 51. Shang Y, Gibbs MA, Marek GJ, Stiger T, Burstein AH, Marek K, et al. Displacement of serotonin and dopamine transporters by venlafaxine extended release capsule at steady state: a [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)-tropane single photon emission computed tomography imaging study. J Clin Psychopharmacol 2007;27:71–5. 29. Areberg J, Mahableshwarkar A, Sanchez C. Vortioxetine: exploratory analysis of the relation between target engagement and integrated clinical database ana- lysis of MADRS single items. Int J Neuropsychopharmacol 2014;17:58. 52. Tatsumi M, Groshan K, Blakely RD, Richelson E. Pharmacological proﬁle of anti- depressants and related compounds at human monoamine transporters. Eur J Pharmacol 1997;340:249–58. 112–3 30. Arakawa R, Tateno A, Kim W, Sakayori T, Ogawa K, Okubo Y. Time-course of serotonin transporter occupancy by single dose of three SSRIs in human brain: a positron emission tomography study with [(11)C]DASB. Psychiatry Res Neuroi- maging 2016;251:1–6. 53. Aronson JK, Ferner RE. The law of mass action and the pharmacological concentration-effect curve: resolving the paradox of apparently non-dose-related adverse drug reactions. Br J Clin Pharmacol 2016;81:56–61. 2015/10/26 ed 31. ADDITIONAL INFORMATION 41. Areberg J, Luntang-Jensen M, Søgaard B, Nilausen DØ. Occupancy of the ser- otonin transporter after administration of Lu AA21004 and its relation to plasma concentration in healthy subjects. Basic Clin Pharm Toxicol 2012;110:401–4. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41380-021-01285-w. 42. Stenkrona P, Halldin C, Lundberg J. 5-HTT and 5-HT(1A) receptor occupancy of the novel substance vortioxetine (Lu AA21004). A PET study in control subjects. Eur Neuropsychopharmacol 2013;23:1190–8. Correspondence and requests for materials should be addressed to Anders Sørensen. 43. Oliveira P, Ribeiro J, Donato H, Madeira N. Smoking and antidepressants phar- macokinetics: a systematic review. Ann Gen Psychiatry 2017;16:17. ACKNOWLEDGEMENTS 34. Houle S, Ginovart N, Hussey D, Meyer JH, Wilson AA. Imaging the serotonin transporter with positron emission tomography: Initial human studies with [11C] DAPP and [11C]DASB. Eur J Nucl Med 2000;27:1719–22. nk Marinos Ioannides for assistance with screening and data extrac 35. Kim E, Howes OD, Kim B-H, Chon M-W, Seo S, Turkheimer FE, et al. Regional differences in serotonin transporter occupancy by escitalopram: an [11C]DASB PK-PD study. Clin Pharmacokinet 2017;56:371–81. AUTHOR CONTRIBUTORS All authors contributed to the study. AS and KM planned the project and wrote the research protocol. AS did the literature search and data extraction with KM and Marinos Ioannides as second and independent researchers. AS wrote the ﬁrst draft of the manuscript with input from KM. HGR contributed to the interpretation of the data and provided extensive critical revisions of the manuscript. All three authors revised the manuscript and approved its ﬁnal version. 36. Klein N, Sacher J, Geiss-Granadia T, Mossaheb N, Attarbaschi T, Lanzenberger R, et al. Higher serotonin transporter occupancy after multiple dose administration of esci- talopram compared to citalopram: an [123I]ADAM SPECT study. Psychopharmacol (Berl) 2007;191:333–9. 37. Herold N, Uebelhack K, Franke L, Amthauer H, Luedemann L, Bruhn H, et al. Imaging of serotonin transporters and its blockade by citalopram in patients with major depression using a novel SPECT ligand [123I]-ADAM. J Neural Transm (Vienna) 2006;113:659–70. REFERENCES Baldinger P, Kranz GS, Haeusler D, Savli M, Spies M, Philippe C, et al. Regional differences in SERT occupancy after acute and prolonged SSRI intake investigated by brain PET. Neuroimage 2014;88:252–62. 54. Holford N. Pharmacodynamic principles and the time course of delayed and cumulative drug effects. Transl Clin Pharmacol 2018;26:56–9. 2018/06/18 ed. 55. Lammers LA, Achterbergh R, Mathôt RAA, Romijn JA. The effects of fasting on drug metabolism. Expert Opin Drug Metab Toxicol 2020;16:79–85. 32. Catafau AM, Perez V, Plaza P, Pascual J-C, Bullich S, Suarez M, et al. Serotonin transporter occupancy induced by paroxetine in patients with major depression disorder: a 123I-ADAM SPECT study. Psychopharmacol (Berl) 2006;189:145–53. 56. Sandson NB, Armstrong SC, Cozza KL. An overview of psychotropic drug-drug interactions. Psychosomatics 2005;46:464–94. 1 33. Frankle WG, Robertson B, Maier G, Paris J, Asmonga D, May M, et al. An open- label positron emission tomography study to evaluate serotonin transporter occupancy following escalating dosing regimens of (R)-(-)-O-desmethylvenla- faxine and racemic O-desmethylvenlafaxine. Synapse. 2018;72. https://doi.org/ 10.1002/syn.22021. 57. Schellander R, Donnerer J. Antidepressants: clinically relevant drug interactions to be considered. Pharmacology 2010;86:203–15. COMPETING INTERESTS 38. Smith GS, Kahn A, Sacher J, Rusjan P, Van Eimeren T, Flint A, et al. Serotonin transporter occupancy and the functional neuroanatomic effects of citalopram in geriatric depression. Am J Geriatr Psychiatry 2011;19:1016–25. AS has nothing to disclose. KM has nothing to disclose. HGR reports Speaking Fees from Janssen, grants from ZonMW (GGG), HersenStichting NL, and is Chair of the Multidisciplinary Task-Force Discontinuation Antidepressants and vice-Chair of the Multidisciplinary Guideline for Unipolar Depression in the Netherlands, all outside the submitted work. 39. Takano A, Suhara T, Ichimiya T, Yasuno F, Suzuki K. Time course of in vivo 5-HTT transporter occupancy by ﬂuvoxamine. J Clin Psychopharmacol 2006;26:188–91. 40. Takano A, Suzuki K, Kosaka J, Ota M, Nozaki S, Ikoma Y, et al. A dose-ﬁnding study of duloxetine based on serotonin transporter occupancy. Psychopharmacology 2006;185:395–9. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints 44. Weathermon R, Crabb DW. Alcohol and medication interactions. Alcohol Res Health 1999;23:40–54. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 45. Ruhe HG, Booij J, Weert HCV, Reitsma JB, Fransen EJF, Michel MC, et al. Evidence why paroxetine dose escalation is not effective in major depressive disorder: a randomized controlled trial with assessment of serotonin transporter occupancy. Neuropsychopharmacology 2009;34:999–1010. 46. Furukawa TA, Salanti G, Cowen PJ, Leucht S, Cipriani A. No beneﬁt from ﬂexible titration above minimum licensed dose in prescribing antidepressants for major depression: systematic review. Acta Psychiatr Scandinavica 2020;141:401–9. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. 47. Safer DJ. Raising the minimum effective dose of serotonin reuptake inhibitor antidepressants: adverse drug events. J Clin Psychopharmacol 2016;36:483–91. 48. Lanzenberger R, Kranz GS, Haeusler D, Akimova E, Savli M, Hahn A, et al. Pre- diction of SSRI treatment response in major depression based on serotonin transporter interplay between median raphe nucleus and projection areas. Neuroimage 2012;63:874–81. 49. Moriguchi S, Takano H, Kimura Y, Nagashima T, Takahata K, Kubota M, et al. Occupancy of norepinephrine transporter by duloxetine in human brains mea- sured by positron emission tomography with (S,S)-[18F]FMeNER-D2. Int J Neu- ropsychopharmacol 2017;20:957–62. 50. Owens MJ, Morgan WN, Plott SJ, Nemeroff CB. Neurotransmitter receptor and transporter binding proﬁle of antidepressants and their metabolites. J Pharm Exp Ther 1997;283:1305–22. © The Author(s) 2021 © The Author(s) 2021 Molecular Psychiatry
https://openalex.org/W4225749396	https://air.uniud.it/bitstream/11390/1221210/1/2022_CONAVI_Nebbiolo.pdf	English	null	Increasing the yield of Nebbiolo grapevines grown in acid soils: a comparison of different techniques	Bio web of conferences/BIO web of conferences	2,022	cc-by	3,757	1 Introduction than the cluster during flower development but not during fruit set. Since clusters are unimportant sinks until bloom, shoot topping before or during bloom may improve fruit set because more assimilates are diverted into the developing clusters just prior to fruit set [10]. Moreover, hedging stimulates cross-transfer of assimilates to clusters on both sides of the shoot, and induces even young leaves to switch from upward to downward export [9]. Grapevines, as many other plants, can be grown in several conditions of soil, but as pH becomes too acid there are many implications for the productivity. Generally speaking, the vine does not perform well at pH values lower than 5,0 and stunted shoot growth as well as poor root growth may be symptoms [1]. The plants cultivated in acid soils suffer both phosphorus deficiency and aluminum toxicity stresses [2]. More than 80% of P in the soil is unavailable for plant uptake because of colloid adsorption, precipitation, or conversion to the organic form. P deficiency is the result of high fixation by aluminum and iron oxides and hydroxides [3]. Conradie [4] reported that the active absorption of P starts about three weeks after bud burst and stops at veraison, and a second peak period occurs from about five weeks after harvest until leaf fall. Phosphorous is the second most frequently limiting macronutrient for plant growth [5]. Reproductive development is more sensitive than vegetative development to P deficiencies [6]; phosphate deficiency is detrimental to inflorescence formation, and Skinner et al. [7] found that P fertilization promoted an improvement of berry-set, which resulted in heavier clusters and thus increased yield. In acid soils, clays are often saturated with a mixture of hydrogen and aluminum. Toxic levels of Al in the soils can be reduced by liming [11]. In addition to an increase in soil pH and the neutralization of exchangeable Al3+, other beneficial effects of liming soil are an increased Ca and Mg base saturation and P availability. The neutralization of exchangeable Al3+ by liming increases the response of plants to additions of P. This may be explained by better solubility and thereby better availability of P from Ca-phosphates compared to Al, Mn and Fe-phosphates in acid soils [12]. * Corresponding author: paolo.sivilotti@uniud.it Increasing the yield of Nebbiolo grapevines grown in acid soils: a comparison of different techniques Roberto Merlo1, Leone Braggio1, Giacomo Nunin2, Paolo Sivilotti2,* 1Uva Sapiens s.r.l., Via Rialto 3/4, 31010 Farra di Soligo, Treviso, Italy. 2University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Via delle Scienze 206, 33100 Udine, Italy s.r.l., Via Rialto 3/4, 31010 Farra di Soligo, Treviso, Italy. Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Via delle Scienze 206, 33100 Udine, Abstract. The detrimental effect of soil acidity on the performance of vines can be attributed to both P deficiency and Al toxicity stresses. This study aimed to investigate the effect of different agronomical techniques applied in a vineyard of Nebbiolo to solve the problem of low productivity due to low soil pH. The effect of liming, phosphorus fertilization and pre-bloom shoot trimming on yield and bud fruitfulness was followed over a period of 3 years. Moreover, in the last year of study grape analyses were performed to assess treatments’ effect on grape quality. Results confirmed that it is possible to alleviate in part the low productivity due to low soil pH. Overall, pre-bloom shoot trimming, P fertilization, and the combination of these two treatments have been the most effective options. Regarding the differences between treatments, only the combination of pre-bloom shoot trimming and P fertilisation significantly affected bud fruitfulness and yields in comparison to untreated. Little or no effect has been observed among treatments in terms of grape composition at harvest. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 BIO Web of Conferences 44, 02006 (2022) https://doi.org/10.1051/bioconf/20224402006 CO.NA.VI. 2020 Increasing the yield of Nebbiolo grapevines grown in acid soils: a comparison of different techniques Roberto Merlo1, Leone Braggio1, Giacomo Nunin2, Paolo Sivilotti2,* 1Uva Sapiens s.r.l., Via Rialto 3/4, 31010 Farra di Soligo, Treviso, Italy. 2University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Via delle Scienze 206, 33100 Udine, Italy 1 Introduction An experiment was set up in a vineyard of Nebbiolo planted in Piedimont (Sperino, Biella) on acid soil, with the aim to find the best techniques to apply in order to obtain an increase of the yield compatible with the standard of Nebbiolo in other viticultural areas. Cluster fall and poor berry set are the major contributors to low yields due to high soil acidity; the applied techniques were intended to eliminate or lessen these phenomena. The carbohydrate nutrition of the inflorescence at pre- bloom stage can markedly affect flower and cluster development and fruit set [8]. Leaves that have recently become source, initially export their assimilates to the growing shoot tip and unfolding leaves [9]. According to Hale and Weaver [8], shoot tips are more powerful sinks BIO Web of Conferences 44, 02006 (2022) https://doi.org/10.1051/bioconf/20224402006 BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 CO.NA.VI. 2020 2.1 Plant material and experimental design The study was conducted on Vitis vinifera L. cv. Nebbiolo clone CVT 423 (“Picotendro”) grafted onto Riparia Gloire rootstock during the seasons 2014, 2015 and 2016, at Proprietà Sperino Soc. Agr. (Lessona, Alto Piemonte, Italy). The vineyard is located on the pre-alpine plateau of Orolungo, in the district of Ori; the vines were planted in 2001 with a vine spacing of 2,2 m between rows and 0,9 m between vines (5050 vines/ha), and were trained using a Guyot training system. Table 2. details and labels of the treatments under comparison. Table 2. details and labels of the treatments under comparison. Labels Treatments detail UNT Control untreated Li 10 Lime 10 tonns/ha Li 20 Lime 20 tonns/ha PBST Pre-bloom shoot trimming P Phosphorus (P2O5) 100kg/ha Li 10 + PBST Lime 10 tonns/ha + Pre-bloom shoot trimming Li 10 + P Lime 10 tonns/ha + P2O5 100kg/ha P + PBST P2O5 100kg/ha + Pre-bloom shoot trimming The vineyard site is characterized by a well-drained acidic soil, consisting of silt and marine sands on quaternary fluvio-glacial sediments. The physical and chemical analyses of the soil are shown in Tab. 1. The soil present a low concentration of P, K, and organic matter, and the low CEC accounts for a scarce availability of nutrients for plant absorption. Moreover the low pH and the high concentration of aluminum surely contribute to further reduce the availability of phosphorous, detrimental condition for bud fertility and consequently for grapevine production. Table 1. Physical and chemical analyses of the soil of the vineyard of Nebbiolo. Physical properties Coarse fraction (%) 5,6 2-mm sieved fraction Sand (%) 17,2 Silt (%) 61,7 Clay (%) 21,1 Soil classification SICLLO Chemical properties pH 4,0 CEC (meq/100g) 10,4 P (ppm) 4 K (ppm) 73 Ca (ppm) 1530 Al (mg/kg) 60,6 Organic matter (%) 0,9 Table 1. Physical and chemical analyses of the soil of the vineyard of Nebbiolo. 2.2 Yield parameters and bud fruitfulness Shoot and cluster number was evaluated on all 5 vines of each plot during both pre- and post-bloom time; shoots originated from the cane and from the left spur on the grapevine head were examined separately. At harvest time yield and cluster number were evaluated for each plot, and the average cluster weight computed by rating the yield by the number of clusters. The bud fruitfulness was calculated rating the number of clusters by the number of shoots developed on the canes (shoots and clusters originated on the spurs were removed from the calculation). 2.3 Statistical analysis Data were analyzed through full-factorial two-ways ANOVA; levels of significance in tables and graphs are given as ns, , , ** for not significant, significant at p<0,05, p<0,01, and p<0,001, respectively. When ANOVA was significant, differences among seasons were separated using Tukey’s test; values followed by the same letter within a column are not significantly different at p<0,05. Regarding the differences between treatments, orthogonal comparisons were performed using contrast analysis between each treatment and the untreated, and the significance of the differences (p<0,05) are reported. 3 Results Analysis of variance highlighted significant differences between UNT and treated vines in terms of number of clusters at pre and post-bloom, bud fruitfulness and yield per vine (Tab. 3). In most cases, the applied techniques caused an increase in bud fruitfulness with the exception of Li10, Li20 and Li10+P treatments. PBST+P treatment with an increase of 20,6% was the most effective on bud fruitfulness, while P addition, PBST and Li10+PBST increased this parameter to a lesser extent. Liming did not improve bud fruitfulness when applied alone or together with P. Mean pairwise comparisons demonstrated that only PBST+P treatment significantly increase bud fruitfulness in comparison to untreated (Tab. 3). A completely randomized experimental design was arranged within the vineyard, with 8 treatments, 3 replicates for each treatment and 5 vines/replicate. The treatments are described in detail in Tab. 2. A completely randomized experimental design was arranged within the vineyard, with 8 treatments, 3 replicates for each treatment and 5 vines/replicate. The treatments are described in detail in Tab. 2. Pre-bloom shoot trimming (PBST) was performed ca 10 days before flowering time, while lime and phosphorous were added during winter time at 10 tonns/ha (Li 10), 20 tonns/ha (Li20) or 100 kg/ha (P). Pre-bloom shoot trimming (PBST) was performed ca 10 days before flowering time, while lime and phosphorous were added during winter time at 10 tonns/ha (Li 10), 20 tonns/ha (Li20) or 100 kg/ha (P). 2 2 https://doi.org/10.1051/bioconf/20224402006 BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 BIO Web of Conferences 44, 02006 (2022) Table 3. Effect of treatments and year on the different parameters examined in this study. Factor Level No. of clusters at pre-bloom No. of clusters at post-bloom Cluster fall (%) Bud fruitfulness Cluster weight (g) Yield (g/vine) 100 Berry weight (g) Treatment UNT 6,57 6,36 3,06 0,59 228 1496 167 Li10 6,05 5,93 1,99 0,50 224 1327 162 Li20 6,67 6,19 5,73 0,57 231 1437 161 PBST 6,71 6,38 4,40 0,62 246 1543 162 P 6,76 6,73 0,61 0,67 231 1562 152 Li10 + PBST 7,42 7,27 2,08 0,69 243 1825 165 Li10 + P 6,88 6,39 8,56 0,58 234 1612 161 PBST + P 8,69 8,05 6,65 0,71 242 1951 159 sign. 3 Results F a * * ns ** ns * ns contrasts significance UNT vs Li10 ns ns ns ns ns UNT vs Li20 ns ns ns ns ns UNT vs PBST ns ns ns ns ns UNT vs P ns ns ns ns ns UNT vs (Li10 + PBST) ns ns ns ns ns UNT vs (Li10 + P) ns ns ns ns ns UNT vs (PBST +P) ** ns * Year 2014 6,10 b b 5,83 b 3,91 0,48 b 224 b 1337 b 154 b 2015 7,38 a 6,96 a 5,45 0,70 a 183 c 1301 b 171 a 2016 7,39 a 7,16 a 2,89 0,67 a 297 a 2138 a 158 b sign. F * * ns * * * * Interaction T x Y sign. F ns ns ns ns ns ns ns aData were analyzed through full-factorial two-way ANOVA (ns, not significant; , p < 0,05; , p < 0,01; **, p < 0,001), and when differences were significant, the season means were separated using Tukey’s test (p < 0,05), while in case of the treatments contrast analysis between each treatment and the untreated was used (p < 0,05). bDifferent letters identify significantly different means. Li 10, lime 10 tons/ha; Li 20, lime 20 tons/ha; PBST, pre-boom shoot trimming; P, phosphorus. Li 10 was the only treatment showing a decrease in l t b ( 7 9%) b f fl i hil i th PBST+P treatment, which accounted for an increase of 32 4% d t th t l At t bl l t Table 3. Effect of treatments and year on the different parameters examined in this study. PBST+P treatment, which accounted for an increase of 32,4% compared to the control. At post-bloom, cluster number was positively affected by all treatments with the exception of lime addition. PBST+P and PBST+Li10 treatments showed lower levels of cluster fall, recording respectively 26,6% and 14,3% more clusters compared Li 10 was the only treatment showing a decrease in cluster number (-7,9%) before flowering, while in the other cases higher cluster number was obtained. Addition of P along with PBST caused the higher positive variation, followed by Li10+PBST and Li10+P. In comparison to the control, the only significant difference was found for PBST+P treatment, which accounted for an increase of 32,4% compared to the control. 3 Results Liming did not promote a positive effect on yields. As for other parameters, the treatment PBST+P resulted in significantly higher yield as compared to untreated. The season significantly modified all parameters of the study (Tab. 3). As regard the number of clusters and the bud fruitfulness, in the first season (2014) the values were significantly lower, while no differences between the other two seasons could be appreciated. As opposite, the average cluster weight was significantly the lowest in 2015, intermediate in 2014 and the highest in 2016. Significantly higher yield was shown in the last season, while similar values were ascertained in the other two seasons. The lowest value of average cluster weight in 2015 was also related with the lower significant value of berry weight. Since in the present vineyard the soil pH was really low, we decided to apply significant amounts of lime (10 or 20 tonns/ha), but no significant increase of yield was obtained. On the contrary, a slight reduction of yield can be shown in case of the lower lime rate over three years of treatments. To date, soil has a huge buffering capacity counteracting the effects of all kinds of amendments [16]. Moreover, the root system could end deeper than the liming treatment, where the pH value drops and the Al3+ concentration becomes relatively important [17]. Thus, both conditions could help to explain the poor effect of lime on increasing yield. Another technique applied in the present experiment was pre-bloom shoot thinning, with the aim to increase the berry-set and so the average cluster weight. During the first part of the seasons, the strongest sinks for the plant are represented by the shoot tips. Considering a rapidly growing shoot as a whole, the translocation in the terminal portion is acropetal only: movement of photosynthates from the two or three youngest exporting leaves is almost exclusively to the shoot tips. While the removal of mature leaves reduces set in grapes [18], topping and pinching are known to increase set if done during flowering [10]. Moreover, hedging stimulates cross-transfer of assimilates to clusters on both sides of the shoot and induces even young leaves to switch from upward to downward export [9]. This is probably why shoot topping during bloom often improves fruit set. In the present trial, PBST promoted a timid increase of yield, due to the slight increase of berry-set (expected) and so cluster weight. 3 Results At post-bloom, cluster number was positively affected by all treatments with the exception of lime addition. PBST+P and PBST+Li10 treatments showed lower levels of cluster fall, recording respectively 26,6% and 14,3% more clusters compared 3 BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 BIO Web of Conferences 44, 02006 (2022) https://doi.org/10.1051/bioconf/20224402006 Al reduces root growth and affects nutrient translocation by plants. Morlat and Jacquet [14] confirmed that the root density was noticeably lower in case of sandy acid soils, because of both strong acidity and high concentration of manganese, which creates unfavorable conditions for rooting. Himelrick [15] reported that P and Ca levels were always higher in the plants grown in soils with higher pH, while Mg, Fe, Cu, and Zn levels showed an opposite trend. with the control. Again, P+PBST was the only treatment reporting significant higher values as compared to untreated vines. with the control. Again, P+PBST was the only treatment reporting significant higher values as compared to untreated vines. Cluster fall shows the reduction in cluster number that occurred between pre and post-bloom. Applied agronomical techniques did not impact cluster fall. Control untreated presented 3,06% of cluster fall. In general, a reduction of cluster fall was seen in Li10, P and Li 10 + PBST. In the present experiment, the yield/vine was really low, basically because of the poor fertility impaired by the acid pH. As described in the results, different techniques were applied with the aim to increase yield, exploiting different mechanisms. Berry and cluster weight were not significantly affected by treatments (Tab. 3). In general, although mean results did not differ significantly, the treated vines showed a tendency towards lower values of berry weight and higher values of cluster weight. Contrast analysis confirmed that data collected were not significantly different against untreated. As reported by Singleton et al. [11], one possibility to attenuate the toxic effect of aluminum in the soil is the application of lime, as CaCO3 reacts with carbonic acid to form calcium bicarbonate, a more soluble and leachable intermediate. The calcium bicarbonate ionizes so that its calcium ions are able to replace hydrogen and aluminum ions on the exchange sites of clay particles. This means an increased base saturation, increased pH, and a loss of hydrogen ions. Yield was significantly affected by treatments. PBST+P and Li10+PBST increased yield more than the other techniques. 3 Results Still, not significant difference from the UNT was found, and probably another solution providing an increase of the cluster number needed to be found in order to get a better result. 4 Discussion Ann. Bot., 106, 183-184 (2010). 17. B.R. Meyer, E. Le Roux, M.J. Renan, M. Peisach. Nucl. Instrum. Methods Phys. Res. B 3, 557-560 (1984). 5 Conclusion 18. M. Sternad Lemut, P. Sivilotti, L. Butinar, J. Laganis, U. Vrhovšek Aust. J. Grape Wine Res. 21, 439-450 (2015). The three techniques applied alone, did not promote sensible modifications in the yield parameters as expected. Differently, more interesting effects were ascertained when the techniques were combined. In detail, the combination of Li10 with P or with PBST, was profitable to promote an increase of yield, mainly in the second treatment. At last, the combination of PBST and P fertilization was the most interesting technique, that allowed maintaining the higher number of grapes on the vines without reducing the berry-set. The results of the experiment here presented highlight that acid soils are not properly suitable for the cultivation of grapes, since the low pH negatively affects the yield. Anyway, the application of suitable techniques, such as pre-bloom shoot thinning and P fertilization could partially offset the problem by increasing the bud fruitfulness and percentage of berry-set. These techniques applied together provided the only significant increase of the yield observed in the present experiment over three years of trial, and thus they represent a sustainable viticultural solution for the winegrowers of this area. In future experiments, analyses of leaf concentration of P, Al, and Ca would be important in order to understand the effects of different treatments on plant mineral absorption, and explain why and how an increase of yield can be obtained. 4 Discussion There are several processes involved in the formation of acid soils. The majority of acid soils originate in areas characterized by heavy rainfall; in fact, the acidification process is strictly dependent on the amount of water that pass through the soil profile. Exchangeable basic cations (Ca2+, Mg2+, K+) of clay minerals are leached out by water; they are so substituted with hydrogen and aluminum ions, which neutralize negative charges of colloids. More than 95% of mineral soils’ acidity is attributed to hydrogen and aluminum. Other important causes of soil acidification are root and microbial respiration, mineralization and nitrification processes, organic acids release from vegetation, abundancy of soil organic matter and plant roots. As reviewed in the introduction, plants growing in acid soils suffer both P deficiency and aluminum toxicity stresses. The plants developed different morphological and physiological mechanisms to cope with soil P limitation, such as the development of root hairs and association of the roots with vesicular-arbuscular mycorrhizae. Moreover, bound inorganic P can be released by the exudation of organic acids (improved with citrate), which allows for the displacement of P from Al3+, Fe3+ and Ca2+ phosphates [13]. In addition to P fixation, The third technique considered in the present trial, was the application of a high rate of phosphorous, aiming to increase the bud fruitfulness and so the number of clusters growing on the shoots. Skinner and Matthews [6] reported that withholding P at bloom and veraison, a significant 4 BIO Web of Conferences 44, 02006 (2022) CO.NA.VI. 2020 BIO Web of Conferences 44, 02006 (2022) https://doi.org/10.1051/bioconf/20224402006 11. P.L. Singleton, D.C. Edmeades, R.E. Smart. New Zeal. J. Agric. Res. 30, 517–522 (1987). decrease in berry number, cluster weight and the number of cluster primordial occurred. Initiation, differentiation and maintenance of reproductive primordia are sensitive to P supply throughout the season, being the maintenance of initiated clusters more sensitive than initiation of clusters [6]. In the present experiment, also P treatment contributed with a timid increase of yield, caused by a slightly higher number of clusters remained post- flowering, partially compensated by a reduction in berry weight. g 12. L. Čoga, M.H. Ćustić, I. Pavlović. Agric. Conspec. Sci. 74, 39–43 (2009). 13. C.L. Ward, A. Kleinert, K.C. Scortecci, V.A. Benedito, A.J. Valentine. J. Plant Physiol. 168, 459–465 (2011). 14. R. Morlat, A. Jacquet. Vitis 32, 35–42 (1993). 15. D.G. Himelrick. HortScience 26, 269–271 (199 16. S. J. Zheng S.J. References 1. W.J. Conradie. S. Afr. J. Enol. Vitic 4, 39–44 (1983). 1. W.J. Conradie. S. Afr. J. Enol. Vitic 4, 39–44 (1983). 2. M. Jemo, R.C. Abaidoo, C. Nolte, W.J. Horst. J. Plant Physiol. 164, 442–451 (2007). 3. R.L. Parfitt. J. Soil Sci. 40, 359–369 (1989). 4. W.J. Conradie. S. Afr. J. Enol. Vitic. 2, 7–13 (1981). 5. D.P. Schachtman, R.J. Reid, S.M. Ayling. Plant Physiol. 116, 447–453 (1998). 6. P.W. Skinner, M.A. Matthews. Sci. Hortic. 38, 49– 60 (1989). 7. P.W. Skinner, J.A. Cook, M.A. Matthews. Vitis 27, 95–10 (1988). 8. C.R. Hale, R.J. Weaver. Hilgardia 33, 89–131 (1962). 9. Keller, M. The science of grapevines. (Elsevier Inc. 2010). 10. B.G. Coombe. Am. J. Enol. Vitic. 10, 85–100 (1959). 5 5
https://openalex.org/W2149572885	https://scientiamarina.revistas.csic.es/index.php/scientiamarina/article/download/621/634/633	English	null	Peter Wangersky (1927-2007)	Scientia marina	2,007	cc-by	1,390	Peter Wangersky (1927-2007) Peter John Wangersky passed away on January 7, 2007 at the age of 79 after a brief bout with cancer. His wife, Eleanor, and their three sons were at his side. full Department of Oceanography. Pete later served two terms as chairman. He served on the Dalhousie faculty until his retirement in 1993 when he and Eleanor moved to Victoria, British Columbia, where Pete accepted an adjunct professorship at the University of Victoria. Pete was born in Woonsocket, Rhode Island and spent his childhood there and in Lynn, Massachusetts. He entered Brown University in 1943 at age 16 under a wartime accelerated pro- gram, majoring in chemistry. His university career was interrupted when he was drafted into the U.S. military where he served until the end of the war. He then returned to Brown under the GI Bill to com- plete his undergraduate degree. After Brown, he worked briefly at the Scripps Institute of Oceanography and the U.S. Fish and Wildlife Service. He then went to Yale University for gradu- ate studies, earning his doctorate in zoology under the polymath zoologist G. Evelyn Hutchinson, who probably was the inspiration for his own wide-rang- ing scientific interests. He taught briefly at the University of Miami, where he met and married Eleanor, and then at the Bingham Oceanography Laboratory at Yale University. In 1965 he accepted an offer to join the Dalhousie University Institute of Oceanography in Canada, and was joined there by Gordon Riley, who became the chairman. Under their leadership the Institute grew and evolved into a Pete was a very broad and diverse researcher; he was a theoretical ecologist as well as a chemical oceanographer long before either of these ‘interdis- ciplinary’ fields became accepted as intellectually respectable. He published in fields as disparate as the organic chemistry of seawater, algal physiology, and mathematical modeling of population dynamics. Through his own early publications and his influ- ence as editor, he was a main stimulus for develop- ment of the field of organic chemistry of seawater. In theoretical ecology, his paper on lag effects in population cycles, published in 1956, when he was a graduate student, is still widely cited. He also held two patents on chemostat design. He loved to go to sea and participated in numerous oceanographic cruises. SCIENTIA MARINA 71(4) December 2007, 823-824, Barcelona (Spain) ISSN: 0214-8358 SCIENTIA MARINA 71(4) December 2007, 823-824, Barcelona (Spain) ISSN: 0214-8358 OBITUARY Peter Wangersky (1927-2007) Peter Wangersky (1927-2007) Selected bibliography (from more than 100 publications) Selected bibliography (from more than 100 publications) Selected bibliography (from more than 100 publications) ment. Ann. Rev. Ecol. Syst., 7: 161-176. Wangersky, P.J. – 1978. Production of dissolved organic matter. In: O. Kinne (ed.), Marine Ecology, Vol. IV. . pp. 115-220. J. Wiley and Sons, New York. Wangersky, P.J. – 1952. Isolation of ascorbic acid and rhamnosides from sea water. Science, 115: 685. y Wangersky, P.J. and C.P. Wangersky. – 1980. The Manna effect: A model of phytoplankton patchiness in a regenerative system. Int. Rev. ges. Hydrobiol., 65: 681-690. Wangersky, P.J. and W.J. Cunningham. – 1957. Time lags in prey- predator population models. Ecology, 38: 136-139. g y Wangersky, P.J. – 1982. Model ecosystems: The limits of pre- dictability. Thalassia Jugosl., 18: 1-10. p p p gy Wangersky, P.J. and G.E. Hutchinson. – 1958. Manganese deposi- tion and deep water movement in the Caribbean. Nature, 181: 108-109. y Johnson, B.D. and P.J. Wangersky. – 1985. Sea water filtration: Particle flow and impaction considerations. Limnol. Oceanogr., 30: 966-971. Wangersky, P.J. and R.R.L. Guillard. – 1960. Low molecular weight organic base from the dinoflagellate Amphidinium car- teri. Nature, 185: 689-690. Wangersky, P.J. – 1986. Biological control of trace metal residence time and speciation: A review and synthesis. Mar. Chem., 18: 269-297. Rosholt, J.N., C. Emiliani, J. Geiss, F.F. Kozy and P.J. Wangersky. – 1963. Pa231/230Th dating and 18O/16O temperature analysis of core A-254-BR-C. J. Geophys. Res., 67: 2907-2911. g p core A-254-BR-C. J. Geophys. Res., 67: 2907-2911. Parrish, C.C. and P.J. Wangersky. – 1987. Particulate and dissolved lipid classes in cultures of Phaeodactylum tricornutum grown in cage culture turbidistats with a range of nitrogen supply rates. Mar. Ecol. Progr. Ser., 35: 119-128. Riley, G.A., P.J. Wangersky, and D.Van Hemert. – 1964. Organic aggregates in tropical and subtropical surface waters of the North Atlantic Ocean. Limnol. Oceanogr., 9: 546-550. g Wangersky, P.J. – 1965. The organic chemistry of sea water. Am. Sci., 53: 358-374. g Wangersky, P.J. – 1993. Dissolved organic carbon methods: a crit- ical review. Mar. Chem., 41: 61-74. Lombardi, A.T. and P.J. Wangersky. – 1995. Particulate lipid class composition of three marine phytoplankters Chaetoceros gra- cilis, Isochrysis galbana (Tahiti) and Dunaliella tertiolecta grown in batch culture. Hydrobiologia, 306: 1-6. Wangersky, P.J. – 1967. Sodium chloride content and compaction in deep-sea cores. J. Geol., 75: 332-335. p Wangersky, P.J. – 1972. The control of seawater pH by ion pairing. Limnol. Peter Wangersky (1927-2007) Pete served as advisor to about 25 PhD stu- dents at Dalhousie and was a friend and casual men- tor to almost all of the other oceanography graduate students during his tenure. He was known for his breadth of knowledge, wisdom, willingness to help anyone seeking advice and his warm personality. 824 • J. SHARP et al. Assisted by Eleanor, he helped to create a colle- gial atmosphere at the Department of Oceano- graphy. Students, staff and friends were all invited to stop by the Wangersky home on Sunday afternoons for coffee, sweets and stimulating conversation. He was a quick and voracious reader with eclectic tastes. His favorite hobbies included photography and fishing. similar duties for Marine Ecology Progress Series in 1998. In response to a request from Limnology and Oceanography in 1998, he volunteered to help for- eign authors with English usage, and received many such calls over the years. He also organized and edit- ed two volumes of The Springer-Verlag series “Handbook of Environmental Chemistry”; Marine Chemistry (2000) and Estuaries (2006), and served on the advisory board for the series. He was a 50-year member of Sigma Xi. In 1974 Pete took over as the second editor, from volume 2, of the journal Marine Chemistry, initially as a temporary fill-in, and then served as editor-in- chief for the next twenty years. His stewardship of the journal took it from a new, relatively unproven forum, to the well-established authority that it is today. He was noted for encouraging a wide variety of research subjects and new ideas. A special issue of the journal was published in 1997, containing papers based on presentations by former students and close associates at a symposium organized in honor of his retirement. Pete was a true pioneer of many disciplines, and his mind remained active and razor-sharp, spinning off more research ideas, spanning many fields, right until his death. Oceanography, marine chemistry, and population ecology have each lost one of their most innovative and productive workers. His ashes were blended into seawater chemistry at Canadian time-series station PAPA on June 11, 2007. JONATHAN SHARP, SIFFORD PEARRE, DONALD GORDON, ERIC MILLS and CARL BOYD SCI. MAR., 71(4), December 2007, 823-824. ISSN: 0214-8358 JONATHAN SHARP, SIFFORD PEARRE, DONALD GORDON, ERIC MILLS and CARL BOYD In retirement he continued on the editorial boards of Scientia Marina (Barcelona) and Environmental Science and Pollution Research, although he gave up Selected bibliography (from more than 100 publications) Oceanogr., 17: 1-6. Jenkins, W.J., M.A. Beg, W.B. Clarke, P.J. Wangersky, and H. Craig. – 1972. Excess 3He in the Atlantic Ocean. Earth Planet. Sci. Lett., 16: 122-126. Chen, W. and P.J. Wangersky. – 1996. Production of dissolved organic carbon in phytoplankton cultures as measured by high- temperature catalytic oxidation and ultraviolet photo-oxidation methods. J. Plankton Res., 18: 1201-1211. Wangersky, P.J. – 1972. Evolution and the niche concept. Trans. Connecticut Acad. Arts Sci., 44: 369-376. Dafner, E.V. and P.J. Wangersky. – 2002. A brief overview of mod- ern directions in marine DOC studies Part II.-Recent progress in marine DOC studies. J. Environ. Monitoring, 4: 55-69. Wangersky, P.J. – 1976. Particulate organic carbon in the Atlantic and Pacific Oceans. Deep Sea Res., 23: 457-465. and Pacific Oceans. Deep Sea Res., 23: 457-465. p , Wangersky, P.J. – 1976. The surface film as a physical environ- p Wangersky, P.J. – 1976. The surface film as a physical en SCI. MAR., 71(4), December 2007, 823-824. ISSN: 0214-8358
https://openalex.org/W4294770284	https://iiste.org/Journals/index.php/RJFA/article/download/59479/61405	English	null	A Critical Analysis of the Core Determinants of Central Bank Digital Currency in Zambia	null	2,022	cc-by	7,277	Abstract In this study, we analyze the main determinant of a Central Bank Digital Currency (CBDC) in Zambia using a critical literature review. There are many determinants of Central Bank Digital Currency but this critical literature review only focuses on the main ones namely, how the introduction of a CBDC can improve the efficiency of payments systems platforms, make a stronger national defense against AML/CFT, ensure non- discriminatory access to domestic financial services and whether the introduction of a CBDC can mitigate the high cost of currency processing in Zambia. The methodology follows a critical review of CBDC literature from central Banks that have already launched, performing proof of concepts or piloting CBDCs in their jurisdictions. The literature also considers publications and working papers from global standards setting organizations such as the IMF, World Bank, WEF, ITU, ECB and many others. The study focuses on the Zambian jurisdiction and reviewed whether these main determinants may warrant the introduction of a CBDC in Zambia. Keywords: Central Bank Digital Currencies (CBDC), Payments Efficiency, Financial Inclusion, AML/CFT, Cost of Currency processing, Zambia DOI: 10.7176/RJFA/13-16-01 Publication date:August 31st 2022 Publication date:August 31st 2022 A Critical Analysis of the Core Determinants of Central Bank Digital Currency in Zambia Kombe Kaponda1* Austin Mwange2 Oswald K. Mungule3 1. Department of Business Studies, School of Business, ZCAS University, Dedan Kimathi Road, P.O. Box 35243, Lusaka, Zambia - 10101, DBS Scholar, *kapondak@gmail.com 2. Department of Business Studies, School of Social Sciences, ZCAS University, Dedan Kimathi Road, P.O. Box 35243, Lusaka, Zambia - 10101, austinmwange1@gmail.com; austin.mwange@zcasu.edu.zm 3. Department of Business Studies, School of Business, University of Lusaka, off Alick Nkhata Road, P.O. Box 36711, Lusaka, Zambia - 10101, swldmngl993@gmail.com 1. Introduction This study has looked at the history of currency and payment systems platforms in the pre and post- independence era in Zambia. It brings out the major risks posed by virtual currencies on the payments systems landscape and highlights the value and the properties that a Central Bank Digital Currency (CBDC) brings to mitigate the risks of these growing virtual currencies risks (BOZ, 2017). The study was beneficial to the government of the Republic of Zambia, the ministry of finance, the monetary authorities, financial regulators, Technology regulators and financial conduct authorities in Zambia and the sub-Saharan region. Furthermore, the research brought out potential benefits of a CBDC and evaluated how CBDC can bring about innovation in the payment systems landscape, lower the cost of cross border payments, increase financial inclusion and provide insights on how to safeguard, ensure stability, a secure, stable and sound financial system and to foster sustainable economic development in the country (BOZ, 2020). The study also looked at other major prevailing determinants for introducing a CBDC in Zambia and how it can improve the efficiency of payments systems, make a stronger national defense against AML/CFT, demonstrate whether a CBDC can ensure non- discriminatory access to domestic financial services and how they can mitigate the high costs associated with processing cash in circulation. www.iiste.org www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 www.iiste.org 2. Growing Usage of Virtual Currencies in Zambia This problem can be addressed by adopting some form of centralized currency in form of a CBDC that will deal with the risk of suffocating the kwacha and could facilitate instant, inclusive, interoperable and secure and trusted payment platforms that will be regulated, supervised and monitored (FSB, 2020). Figure 1.1 below shows the growing landscape of financial technologies in Zambia including virtual platforms. Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 www.iiste.org These VC’s poses a great risk to the money supply and the currency in circulation. There is a problem because these virtual currencies are not recognized as legal tender by the monetary authorities and still not yet legal tender in the country and the Central Bank of Zambia has to date just issued a cautionary statement advising citizens to trade and transact in virtual currencies at their own risk (BOZ, 2018). This problem of virtual assets continues to grow as the assets are actively traded but not issued by the monetary authority, not regulated and supervised and are obtained by mining, peer-to-peer exchange which is a process by which new virtual currencies are entered into circulation using high end computing resources. This problem distorts monetary policies, financial stability and money supply losing kwacha to virtual currencies and risk of suffocating of the kwacha by growing illegal market which also poses the risk of money laundering and terrorism financing. This problem can be addressed by adopting some form of centralized currency in form of a CBDC that will deal with the risk of suffocating the kwacha and could facilitate instant, inclusive, interoperable and secure and trusted payment platforms that will be regulated, supervised and monitored (FSB, 2020). Figure 1.1 below shows the growing landscape of financial technologies in Zambia including virtual platforms. 2. Growing Usage of Virtual Currencies in Zambia Cryptocurrencies sometimes referred to as Virtual currencies or crypto assets and are a form of Financial Technology (FinTech) innovation and digital asset that has the potential to disrupt the way financial services currently operate globally (BIS, 2019). These VC’s are already having an impact on the worldwide financial systems as a payments system platform and their unique properties make them function as a form of electronic money and forms of digital assets which can be used for payments and investments. They bring about secure, always available, peer-to-peer and decentralized payment methods that have some distinct advantages over the traditional methods of payments (Auer, Raphael; Böhme, Rainer, 2021). According to the financial stability board, FinTech is defined as the financial technology that is applied to financial services, resulting in new business models, applications, processes, products and services with an associated disruptive effect on financial markets and institutions (FSB, 2019). This definition emphasizes the focus on technology-driven innovations that could potentially reshape how the financial services industry operates in developing countries and Zambia in particular as it evolves into several platforms as depicted in Figure 1.1. Given the wide range of innovations across financial services, the existing regulatory architecture should be assessed to determine and evaluate the cost of processing currency as a determinant for a CBDC. 1 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 These VC’s poses a great risk to the money supply and the currency in circulation. There is a problem because these virtual currencies are not recognized as legal tender by the monetary authorities and still not yet legal tender in the country and the Central Bank of Zambia has to date just issued a cautionary statement advising citizens to trade and transact in virtual currencies at their own risk (BOZ, 2018). This problem of virtual assets continues to grow as the assets are actively traded but not issued by the monetary authority, not regulated and supervised and are obtained by mining, peer-to-peer exchange which is a process by which new virtual currencies are entered into circulation using high end computing resources. This problem distorts monetary policies, financial stability and money supply losing kwacha to virtual currencies and risk of suffocating of the kwacha by growing illegal market which also poses the risk of money laundering and terrorism financing. 2. Growing Usage of Virtual Currencies in Zambia Figure 1.1: Zambia's Financial Technologies Ecosystem 2021 (Source: Authors Construct) Figure 1.1: Zambia's Financial Technologies Ecosystem 2021 (Source: Authors Construct) 2.1 Zambian Virtual Currency Landscape y p There is growing evidence from global cryptocurrency platforms (ChainAlysis, 2021) shows that there is a lot of virtual currencies trading and activity going on in the SADC region, particularly Zambia and citizens are currently trading in these virtual currencies peer-to-peer using several online platforms such as https://paxful.com/ https://www.remitano.com, https://www.coinmama.com, https://www.localbitcoins.com and many other available global online platforms according to (CryptoCompare, 2021). Regrettably, the illiteracy surrounding the virtual currency is not just affecting the general public because it also affects those who are assigned with the responsibility of regulating the financial services landscape and those fighting cybercrime which may have its origins in the use or misapplication of virtual currencies. The regulators are currently playing catch up and only issuing cautioning statements to the citizens on the risks of using virtual currencies instead of exploring through research and development in order to get the most out of this technology. The digital financial services DFS and the specific virtual currency platforms form part of the overall financial services infrastructure in Zambia which is under the regulation of the ICT and telecommunications regulator called Zambia Information and Communications Authority (ZICTA). The communications platforms are composed of the terrestrial networks owned by Internet Service Providers (ISP’s) and connect base stations and communications towers in different locations around the country. This is an essential and fundamental underlying infrastructure that facilitates and allows for the use of virtual assets or currencies as a medium of exchange. Figure 1.2 below shows the trading of virtual assets in the Zambian jurisdiction on live online platforms such as localcitcoin.com. 2 2 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 Figure 1.2: Evidence of Virtual Asset Trading (Source: (Localbitcoins, 2021)) Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 Figure 1.2: Evidence of Virtual Asset Trading (Source: (Localbitcoins, 2021)) 2.3 Motivations for Isuing Central Bank Digital Currency 2.3 Motivations for Isuing Central Bank Digital Currency Research by (Reuters, 2019); (Taskinsoy, 2019) points out some of the advantages of issuing CBDC and the following as some of the motivation for issuing CBDC in any jurisdiction. These motivations differ depending on the status of an economy being higher income country (HICs), developing country or a low-Income Countries (LIC) and below are the motivations for Central Bank Digital Currency (CBDC). 1. To make the payment system landscape more efficient and resilient. 1. To make the payment system landscape more efficient and resilient. 1. To make the payment system landscape more efficient and resilient. 2. To retain monetary sovereignty for any jurisdiction and to mitigate the risk of privately issued virtual currencies and global stable coins adoption and improved distribution of government-to-peer (G2P) direct payments to households such as welfare or social cash transfers. p y 3. To make a stronger national defense against AML/CFT and other semi-anonymous nature of virtual currency transactions. The defense is against a host of infamous activities and illicit activities, such as money laundering, terrorism financing, illegal arms dealing, human, child, sex trafficking, and tax evasion from the authorities. 4. Ensure non-discriminatory access to domestic financial services and facilitate access for all citizens in a selected jurisdiction. j 5. The high cost of currency, physical currency processing approximated an average of 21% of the Central Banks' Budgets (BIS, 2019). g ( ) 6. Improving monetary policy effectiveness to implement targeted policy or tap more granular payment data to enhance macroeconomic projections. 7. Enhancing monetary policy transmission by an interest bearing CBDC and improving monetary policy effectiveness, supervisory policies, access granular real-time payments with interest-bearing CBDC, breaking the policy rate zero lower bound. g p y 8. To enhance financial inclusion and financial digitization to facilitate fast, instant efficient and tracible payments platforms. p y p 9. Improving traction of local currency as means of payments in jurisdictions that are attempting to reduce dollarization. 10. Distributing fiscal stimulus distributing to the unbanked including incorporating helicopter or hot money features to increase spending velocity. This provides efficient transmission of social benefits, welfare and all other social services for the citizens that are provided by the government. 10. Distributing fiscal stimulus distributing to the unbanked including incorporating helicopter or hot money features to increase spending velocity. 2.2 Payment Systems Providers in Zambia 2.2 Payment Systems Providers in Zambia y y Research by the Bank for international settlement Reviewed that the Bank of Zambia is responsible for the designation and licensing of Payment Systems, Payment System Participants and payment system businesses in the Zambian jurisdiction (BIS.Org, 2019). Table 1.1 below shows the landscape of the designated payment system entities in the country and the type of payment system platform used. The country has 18 commercial banks that utilize various payment systems platforms such as the high value payments using Real-Time Gross Settlement System (RTGS) locally called the Zambia Interbank Payment and Settlement System (ZIPS). Other payment systems platforms used the commercial banks in Zambia include the Cheque Image Clearing (CIC), Direct Debit and Credit Clearing (DDACC) via the Zambia Electronic Clearing House (ZECHL), the point of sale (POS) and the electronic fund's transfer. The table also shows the growing number of low value payments provided by the non-bank financial institutions (NBFI), payment systems providers (PSP), financial services providers and now financial technologies (Fintech) service providers. The table also shows that there is currently no regulations for virtual currencies that are being traded online. Further research by the BIS also recommends that any CBDC consideration should interoperate with other existing digital and mobile financial services such as the platforms in table 1 1 below as t e p at o s tab e . be ow. Table 1.1: Payment Systems Provider Platforms and state of Regulation (Source: Authors Construct) Item # # of Payment Systems Providers Type of Business Type of Payment Systems Platform Location Regulated/ Framework 1 3 Mobile Network Operators (MNO) Mobile Financial Services (Mobile Payment Services and Money Transmission Services) Local Yes 2 18 Commercial Banks CIC, DDACC & ZIPSS/RTGS Local and Internatio nal Yes 3 48 NBFI, PSP, FSP, FinTech E-money Issuer and Money Transfer Services Local Yes 4 12 NBFI, PSP, FSP, FinTech Mobile Payment Services and Money Transmission Services Local Yes 5 Not Known (Online) Virtual currencies and Virtual currencies service providers Virtual currencies Transmission and Virtual Assets Service Providers (VASP) Internatio nal and Online No p able 1.1: Payment Systems Provider Platforms and state of Regulation (Source: Authors Construct) 3 Research Journal of Finance and Accounting www.iiste.org www.iiste.org g ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 2.3 Motivations for Isuing Central Bank Digital Currency This provides efficient transmission of social benefits, welfare and all other social services for the citizens that are provided by the government. p y g This study focused on four main motivations which are summarized in Figure 2.1 below and the literature review ocused on these four motivations. p y g This study focused on four main motivations which are summarized in Figure 2.1 below and the literature review focused on these four motivations. Figure 2.1: Summary Motivation of the Study (Source: Authors Construct). To make payment systems more efficient To make stronger nation defense against AML/CFT To ensure non- discriminatory access to domestic financial services (Financial Inclusion) To mitigate the high cost of currency processing Figure 2.1: Summary Motivation of the Study (Source: Authors Construct). 3.2 CBDC to provide a stronger defence against AML/CFT Global bodies such as the Financial Action Task Force (FATF) also agree that these new technologies, products, and related services have the potential to spur financial innovation and efficiency and improve financial inclusion, but they also create new opportunities for criminals and terrorists to launder their proceeds or finance their illicit activities (FATF, 2014); (FATF, 2019). Consistent with the risk-based approach which underpins the FATF Standards, understanding and responding to identified money laundering and terrorist financing (ML/TF) risks is at the heart of what the FATF does. However, FATF recently provided direction on the treatment of crypto assets by amending their global recommendation on new technologies (FATF, 2019). These amended recommendations now require jurisdictions around the world to regulate cryptocurrencies and cryptocurrency service providers (CASPs) for anti-money laundering and combatting the financing of terrorism (AML/CFT) and member countries and affiliates including Zambia should comply. FAFT also reviews that cryptocurrencies may create conditions for regulatory arbitrage while posing risks to the financial sector and all its participants that operate in a highly regulated environment, which assists in ensuring a sound and safe financial system. Cryptocurrencies are still operating within a regulatory vacuum as no globally harmonized approach or position has been reached yet as many regulators around the globe have not yet sufficiently addressed the phenomenon of crypto assets and have not yet settled on a collective approach. Further, cryptocurrencies are mainly operating in unregulated environments and currently, there is no specific regulations in the region and Zambia in particular. Many financial regulators including the Central Bank of Zambia (BOZ) are currently playing catch up and only issuing cautioning statements (BOZ, 2018); (SEC, 2018) to citizens on the risk of these cryptocurrencies instead of exploring exhaustive research and development to get the most out of these technologies. Globally, central banks have raised concerns of the cryptocurrencies' spillover effects to the formal financial services industry and the eventual effects of these currencies on guiding monetary policies and how to repair and mitigate these eventual risks (SARB, 2014); (Borri & Shakhnov, 2019); (Kosc, Sakowski , & Ślepaczuk, 2019). Meanwhile, the technology keeps growing, AML/CFT concerns also keep increasing and the technology aware generation is actively using it and the various activities associated with this innovation can no longer remain outside of the regulatory perimeter. Consequently, some countries have issued communications declaring restrictions or a downright ban on the use of crypto assets. g 3.1 Payment Systems Efficiency y y y The Bank of international settlements (BIS, 2017), describes a payment system as any financial system that is used to settle financial transactions through the transfer of monetary value from one party to another. This includes transfers between institutions, instruments, people, rules, procedures, standards, and technologies that make the exchange of value possible. BIS further reviews that payment systems need to be efficient, secure and reliable as they act as a lifeblood of modern monetary economies, payment systems are the financial markets circulation system. An economic modeling journal on the optimal online-payment security system and the role of liability sharing (Berg & Kim, 2022), further reviews that payment systems need to exhibit strong properties of security, reliability, immutability, scalability, anonymity, acceptability, customer base, flexibility, convertibility, efficiency, ease of integration with applications and ease of use. Emerging economies such as Zambia can make use of CBDC as payment systems platforms to allow for an efficient, secure and reliable payment system for enhanced economic development and to reduce the cost of exchanging goods and services. Economic efficiency also implies an economic state in which every resource is optimally allocated to serve each individual or entity in the best way while minimizing waste and inefficiency. When an economy is economically efficient, any changes made to assist one entity would harm another. An efficient system infrastructure is also a very important and 4 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 essential tool for the effective implementation of monetary policy, and the smooth functioning of money and capital markets. For an economy to attain an efficient payment system platform, needs specific business policies which need improvement by monitoring and measuring different aspects of payment systems, payment conversions, successful transactions, fraud, and provided actionable data that can be used to make targeted changes in the payment systems ecosystem (BIS, 2017). The study further highlights the following key performance indicators measuring efficiency including payments conversion rates, conversion rates by the financial services providers (FSP), conversion rates by the payment methods and card type, reported fraud rates on all DFS and mobile financial services and measuring of downtime of payment systems platforms. Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 essential tool for the effective implementation of monetary policy, and the smooth functioning of money and capital markets. g 3.1 Payment Systems Efficiency For an economy to attain an efficient payment system platform, needs specific business policies which need improvement by monitoring and measuring different aspects of payment systems, payment conversions, successful transactions, fraud, and provided actionable data that can be used to make targeted changes in the payment systems ecosystem (BIS, 2017). The study further highlights the following key performance indicators measuring efficiency including payments conversion rates, conversion rates by the financial services providers (FSP), conversion rates by the payment methods and card type, reported fraud rates on all DFS and mobile financial services and measuring of downtime of payment systems platforms. 3.4 CBDC to mitigate the High Cost of Currency Processing g g y g Literature from the Reserve bank of South Africa (SARB, 2021) estimates that the cost of handling currency makes up about 0.3% of the total GDP in the country. The accumulative cost of processing fiat currency in circulation (CiC) also includes the actual cost of printing the currency, currency management and the cost of cash processing machines such as the BPS M7, DLR7000 high-speed banknote processing machine, BPS, C5 and C4 desktop processing machines, glory FS-300 and FS-120 machines, Kisan smart K3 machines and Nota packs. The process also includes the costs associated to currency recycling that makes use of currency shredding machines such as online bracketing (OBS) machines that perform the mutilated and soiled notes management and reissuing back for public circulation. The cumulative cost of cash processing is therefore associated with the simple model (Ramos , Garratt, & Arauz, 2021) that assumes that we let the total cash in circulation (CiC) include all the costs of processing fiat currency. Figure 1.8 below shows a summary of the complete currency processing cycle from the Bank of Zambia. processing cycle from the Bank of Zambia. Figure 3.2: Cost of Processing Cash (Source: (BoZ,2020)) Despite some considerable improvements in the process in the recent past and sometimes accelerated by the need to prevent the person-to-person transmission of COVID-19, several mobile financial services have been adopted to reduce the use of cash. This cumulative cost is mainly attributed to the following costs that are associated with processing fiat currency from printing and minting, distribution through commercial Banks, day to day use by all economic agents and the recycling of soiled or unfit notes. Below is the summary currency circle: 1. The cost of printing currency including the recurring cost of cash templates, security features to prevent counterfeit notes 2. The cost of currency management that makes use of BPS M7 high-speed banknote processing machines, DLR7000 high-speed banknote processing machines, BPS, C4 desktop banknote processing machine, d kt t l FS 300 d FS 120 hi d ki t K3 hi Figure 3.2: Cost of Processing Cash (Source: (BoZ,2020)) Figure 3.2: Cost of Processing Cash (Source: (BoZ,2020)) Despite some considerable improvements in the process in the recent past and sometimes accelerated by the need to prevent the person-to-person transmission of COVID-19, several mobile financial services have been adopted to reduce the use of cash. 3.2 CBDC to provide a stronger defence against AML/CFT implementation of Central Bank Digital Currency lessons learnt and key insights reviews that any CBDC initiative should supplement all the other digital financial services that have contributed to financial inclusion (Morales-Resendiz, Raúl; Ponce, Jorge; Picardo, Pablo; Chen, Bobby; Velasco, Andrés; Guiborg, Gabriela; Sanz, León, 2021). CDBC should incorporate open standards, open protocols and application programmable interfaces (API) to allow interoperability with other digital and mobile financial services to meet the financial inclusion agenda. g The thesis further reviews that financial inclusion can be measured in three dimensions including the access to financial services, the usage of financial services and the quality of the products and the service delivery that are offered in a market. The study postulates that the degree of financial inclusion is determined by three dimensions mainly the usage of digital financial services, the barriers and the access to digital financial systems. Another study from the World bank (Douglas & Claudia Ruiz, 2017) also reviews that to effectively measure financial inclusion, jurisdictions should qualify the number of automated teller machines per specific number of adults, the number of bank branches per specific number of adults, the deposit accounts with commercial banks per specific number of adults, the borrowers from commercial banks per specific number of adults and sometimes consider the digital insurance transactions in a specific area. All these measures may be inherited by a central bank digital currency making financial inclusion a compelling motivation to issue CBDC. 3.2 CBDC to provide a stronger defence against AML/CFT Figure 3.1 below depicts the development challenges, the macroeconomic challenges and the cross CBDC border challenges and how these could affect the weaker capacity to address AML/CFT risks. Figure 3.1: CAML/CFT Challenges to CBDC (Source: VoxEU) Figure 3.1: CAML/CFT Challenges to CBDC (Source: VoxEU) 3.3 Non-discriminatory access to Domestic Financial Services (Financial Inclusion) y ( ) Some publications by the Alliance for Financial Inclusion, digital financial services working group (DFSWG, 2019) access to domestic financial services is also commonly known as financial inclusion as the availability and equality of opportunities to access financial services by citizens. It also refers to a process by which individuals, households, firms and businesses can access appropriate, affordable, and timely financial products and services for an improved quality of life. These services include banking, loan, mobile micro loans, equity, and insurance products which every individual should have an opportunity to access. According to the top-level findings on the Zambian Finscope survey of 2020 (FinScope, 2020), access to financial services through financial inclusion increased by 10.1 percentage points to 69.4% from 59.3% in 2015. This increase was mainly attributed to the growth in the use of mobile financial services and other mobile financial platforms. Further research on the 5 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 implementation of Central Bank Digital Currency lessons learnt and key insights reviews that any CBDC initiative should supplement all the other digital financial services that have contributed to financial inclusion (Morales-Resendiz, Raúl; Ponce, Jorge; Picardo, Pablo; Chen, Bobby; Velasco, Andrés; Guiborg, Gabriela; Sanz, León, 2021). CDBC should incorporate open standards, open protocols and application programmable interfaces (API) to allow interoperability with other digital and mobile financial services to meet the financial inclusion agenda. Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 implementation of Central Bank Digital Currency lessons learnt and key insights reviews that any CBDC initiative should supplement all the other digital financial services that have contributed to financial inclusion (Morales-Resendiz, Raúl; Ponce, Jorge; Picardo, Pablo; Chen, Bobby; Velasco, Andrés; Guiborg, Gabriela; Sanz, León, 2021). CDBC should incorporate open standards, open protocols and application programmable interfaces (API) to allow interoperability with other digital and mobile financial services to meet the financial inclusion agenda. 4. Central Bank Digital Currency Model g y The literature reviewed that there are several models for CBDC including the single-tier, two-tier and the hybrid setups. The single-tier setup has no hierarchy of distribution of CBDC and it is more suitable for the direct model, where the central bank issues digital currencies to all its users, the financial institutions and citizens, after conducting the KYC and AML checks before onboarding. This study adopted the two-tier model proposed by the Bank for International settlement in which the CBDC is issued from the Central Bank using the commercial banks as the second tier and may take the form of a retail, a wholesale and interoperable cross border multiple Central Bank Digital Currency (mCBDC). This model was ideal for this study because the study looks at the CBDC determinants that apply a two-tier CBDC model (BIS, 2021), which cements the benefits of tiered architectures for the distribution of retail CBDC through commercial banks, financial services providers and payment service providers alike. The two-tier model is a simplified abstraction of reality that incorporates the central Bank and all the financial institutions, banks, non-bank financial institutions (NBFIs), payment systems providers (PSPs) and all other financial services providers (FSPs). These in turn service the individuals, households and organizations that could have a CBDC account with FSPs. The model assumes that the CBDC can coexist with cash and serve as legal tender and be used as the medium of exchange, a unit of account and store of value for all agents in the Zambian financial landscape. The study adopted the framework of (Lagos & Wright, 2005) which has the same structure of a centralized market (CM) and a decentralized market (DM) and considered other theoretical investigations on the impact of CBDC in order to firm up on the two-tier model. The study also assumes that Zambia already has a competitive banking industry comprised of 18 commercial Banks (BOZ, 2021) and a growing number of NBFI, payment systems providers (PSP) and financial technologies (Fintech) organizations that are providing various payment systems platforms in the country. 3.4 CBDC to mitigate the High Cost of Currency Processing This cumulative cost is mainly attributed to the following costs that are associated with processing fiat currency from printing and minting, distribution through commercial Banks, day to day use by all economic agents and the recycling of soiled or unfit notes. Below is the summary currency circle: 1. The cost of printing currency including the recurring cost of cash templates, security features to prevent counterfeit notes 2. The cost of currency management that makes use of BPS M7 high-speed banknote processing machines, DLR7000 high-speed banknote processing machines, BPS, C4 desktop banknote processing machine, currency desktop sorters, currency glory FS-300 and FS-120 machines and kisan smart K3 machines 6 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 3. The costs of currency distribution that uses armored bulletproof cash trucks 4. Cost of Security services for Transporting cash to all sub chests 4. Cost of Security services for Transporting cash to all sub chests 5. The cost of currency electronic management and Security systems including connectivity and maintenance to sub chests, technology, ICT and CCTV Equipment at main sites and sub chested 5. The cost of currency electronic management and Security systems including connectivity and maintenance to sub chests, technology, ICT and CCTV Equipment at main sites and sub chested 7. The cost of currency recycling of removing soiled notes from circulation, currency shredding machines, online Bricketing System (OBS) and equipment and the soiled notes management and reissuing 7. The cost of currency recycling of removing soiled notes from circulation, currency shredding machines, online Bricketing System (OBS) and equipment and the soiled notes management and reissuing 4. Central Bank Digital Currency Model The access to a CBDC does not imply that the central bank provides retail services to all holders of CBDC, and for simplicity, the study assumes that only banks and NBFIs can trade CBDC directly with the central bank, while households and firms use a new type of electronic money issuance from NBFI, which can be assumed as a CBDC exchange that can be used to buy and sell CBDC in exchange for deposits. This is an alternative where individuals, households and firms can directly trade CBDC with the central bank but this is not the main responsibility of the central bank and the heavy lifting should be left to the commercial players for the central bank to concentrate on its main mandate (BIS, 2021). Figure 2.4 below shows the two-tier CBDC model and how the variables interact with each other. The main variables in this model include cash in circulation (CiC), internet penetration (IP), mobile subscription rates (MS), narrow money (M0,M1), broad money (M2,M3) and the nominal exchange rate (NER) for cross border transactions. Figure 3.4: Two-tier CBDC Model (Source: Authors Construct) To simplify the exposition, the study assumes that banks and NBFIs do not themselves use the services of Figure 3.4: Two-tier CBDC Model (Source: Authors Construct) gu e 3. : wo e C C ode (Sou ce: u o s Co s uc ) To simplify the exposition, the study assumes that banks and NBFIs do not themselves use the services of g simplify the exposition, the study assumes that banks and NBFIs do not themselves use the services of 7 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 Research Journal of Finance and Accounting Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 www.iiste.org CBDC exchanges, given their direct access to the central bank and their ability to transact in wholesale debt markets to acquire eligible collateral. CBDC account holders can trade CBDC among themselves, in exchange for assets which might include bank deposits, goods and services. Banks, in addition to having CBDC accounts are also likely to minimize CBDC holdings, have access to reserve accounts at the central bank, and no other economic agents have access to reserve accounts. 4. Central Bank Digital Currency Model g Research further shows that there is a growing body of literature studies on CBDC determinants and their implications for payment systems, monetary policy implementation, and financial stability across the globe. Most notable is the study on the dynamic stochastic general equilibrium (DSGE) model which was used to study the impact of Central Bank Digital Currencies (CBDCs) on the financial sector (Gross & Schiller, 2021),(Kumhof & Noone, 2021). Another study by (Barrdear, John; Kumhof, Michael, 2021) estimates that CBDC issuance could increase GDP by as much as 3%, mostly by lowering the real interest rates of developing economies like Zambia. Other recent papers have studied the implications of an interest-bearing CBDC in micro- founded models of money and banking. This is a model where banks’ deposits in form of CBDC yield interest and act as an incentive for the public to use formal banking services and increase the much-needed financial inclusion and hence making the banking sector perfectly competitive (Todd & Sanches, 2018). The model also shows that a CBDC crowds out bank intermediation according to literature by (Brunnermeiera & Niepelt, 2019) which further argues that if the central bank can channel funds back to the commercial banks, CBDC does not necessarily disintermediate banks as the interest-bearing CBDC may incentivize individuals keeping the CBDC in commercial banks, unlike individual electronic wallets which should have limits on the amounts of CBDC they should hold to avoid the risk of disintermediation. In another model with a monopoly bank, (Andolfatto, 2020) reviews that a CBDC could lead to more financial inclusion, a higher deposit rate, and more bank deposits. (Chiu, Davoodalhosseini, Jiang, & Zhu, 2019), further show that a CBDC could limit banks’ market power, increasing the deposit rate, bank deposits, and bank lending. Quantitatively, the lending and output can be increased up to 3.55% and 0.50%, respectively. In a model where independence of a central bank and scarcity of assets backing bank deposits are considered, (Williamson S. , 2019) derives conditions under which an interest- bearing CBDC can increase social welfare in a narrow banking arrangement. It is further argued by (Dong & Xiao, 2021), that some forms of CBDC can help implement a negative interest rate which can boost economic activity for a developing economy like Zambia by encouraging banks and other entities to lend or invest excess funds rather than pay penalties on funds in bank accounts. 4. Central Bank Digital Currency Model Literature from another paper by (Monnet & Keister, 2020) rightly argues that a CBDC can lead to less bank risk-taking and higher output and welfare and that CBDC can provide the central bank with more information about the state of the commercial banks, thereby increasing the effectiveness of the regulatory policy and improving financial stability. 5. Conclusion The study conducted a critical analytical review of the main determinant of Central Bank Digital Currency in Zambia. The literature reviewed determinates of issuing CBDC that are driven by the need for efficient payment systems platforms, stronger national defense against AML/CFT, non-discriminatory access to domestic financial services and for the CBDC to mitigate the high cost of processing cash. The research further showed that a decision to issue CBDC in a developing economy like Zambia could depend on these determinants. This critical literature review considered published literature from working series papers from global standards-setting organizations including the IMF, World Bank, WEF, ITU, ECB and many other international organizations. These determinants are applied to a developing jurisdiction like Zambia to ascertain the feasibility of a Central Bank Digital currency. (2019). Big Tech in Finance - Opportunities and Risks,. Bank for International Settlement. Bank f nternational Settlement. Retrieved 05 06, 2020, from https://www.bis.org/publ/arpdf/ar2019e3.pdf Arner, Douglas W; Auer, Raphael ; Frost, Jon. (2020). Stablecoins - Risks, Potential and Regulation. BIS Working Papers no. 905, 905. doi:http://dx.doi.org/10.2139/ssrn.3979495 lfatto, D. (2020). Assessing the Impact of Central Bank Digital Currency on Private Banks. The Econom Journal. doi:https://doi.org/10.1093/ej/ueaa073 RELEASE-ON-CRYPTOCURRENCIES.pdf BOZ. (2018). Press Releases on Cryptocurrencies. Bank of Zambia. Retrieved 10 04, 2020, from https://www.boz.zm/PRESS-RELEASE-ON-CRYPTOCURRENCIES.pdf p p BOZ. (2020). 2020 BOZ Position Paper on CBDC. Bank of Zambia. Lusaka: Bank of Zambia. Retrieved 04 04, 2021, from https://www.boz.zm/annual-reports.htm p BOZ. (2020). 2020 BOZ Position Paper on CBDC. Bank of Zamb 2021, from https://www.boz.zm/annual-reports.htm , p p Brunnermeiera, M. K., & Niepelt, D. (2019). CBDC On the Equivalence of private and public money. Journal of Monetary Economics, 106. doi:https://doi.org/10.1016/j.jmoneco.2019.07.004 ChainAlysis. (2021). Cryptocurrencies Trade in SADC. Chainalysis. Retrieved 11 30, 2021, from https://www.chainalysis.com/ CryptoCompare. (2021). Crypto Compare SADC Trading Volumes. Crypto Compare. Retrieved 12 07, 2021, from https://www.cryptocompare.com/ WG. (2019). Digital Financial Services and Financial Inclusion. AFI Working Group Paper Seri Retrieved 03 20, 2022, from https://www.afi-global.org/working-groups/dfs/ Douglas, P., & Claudia Ruiz, O. (2017). Financial Inclusion Strategies. WB Reference Frameworks. Retrieved 03 13, 2022, from Douglas, P., & Claudia Ruiz, O. (2017). Financial Inclusion Strategies. WB Reference Frameworks. Retrieved 03 13, 2022, from https://documents1.worldbank.org/curated/en/801151468152092070/pdf/787610WP0P144500use0only090 0A9RD899.pdf s://documents1.worldbank.org/curated/en/801151468152092070/pdf/787610WP0P144500use0only090 RD899.pdf EPC. (2019). Improving the Efficiency of the Handling of cash. Working paper Series on Cash Cycle Models. Retrieved 03 07, 2022, from https://www.europeanpaymentscouncil.eu/sites/default/files/KB/files/EPC037- 13%20Improving%20the%20Efficiency%20of%20the%20Handling%20of%20Cash%20- %20Cash%20Cycle%20Models.pdf FATF. (2014). Virtual Currencies: Key Definitions and Potential AML/CFT Risks. FATF. Retrieved 10 from https://www.fatf-gafi.org/documents/documents/virtual-currency-definitions-aml-cft-risk.html p g g y FATF. (2019). Guidance for Risk-Based Approach to Virtual Assets. Financial Action Task Force. Financial Action Task Force. Retrieved 05 06, 2020, from http://www.fatf- gafi.org/media/fatf/documents/recommendations/RBA-VA-VASPs.pdf FATF. (2020). FATF Paper to the G20 Finance Ministers and Central Bank Governors . FATF Working Paper Series. Retrieved 04 05, 2022, from https://www.fatf- gafi.org/media/fatf/documents/recommendations/Virtual-Assets-FATF-Report-G20-So-Called- Stablecoins.pdf FATF. (2020). FATF Paper to the G20 Finance Ministers and Central Bank Governors . FATF Working Paper Series. Retrieved 04 05, 2022, from https://www.fatf- gafi org/media/fatf/documents/recommendations/Virtual Assets FATF Report G20 So Called p FATF. (2022). FATF International Standards on AML/CFT. Retrieved 03 20, 2022, from https://www.fatf- gafi.org/media/fatf/documents/recommendations/pdfs/FATF%20Recommendations%202012.pdf: https://www.fatf- gafi.org/media/fatf/documents/recommendations/pdfs/FATF%20Recommendations%202012.pdf p gafi.org/media/fatf/documents/recommendations/pdfs/FATF%20Recommendations%202012.pdf FinScope. (2020). Finscope 2020 Topline Findings. Lusaka: Zambia Statistics Agency. Retrieved 01 02, 2021, from https://www.boz.zm/finscope 2020 survey topline findings.pdf p p _ _ y_ p _ g p FSB. (2019). Financial Stability Board (FSB), 2019. Financial Stability Board (FSB). Financial Stability Board (FSB). Retrieved 05 06, 2020, from https://www.fsb.org/2019/02/fsb-report-assesses-fintech-developments- and-potential-financial-stability-implications/ p y p Keister, Todd; Sanches, Daniel. (2019). Should Central Banks Issue Digital Currency? WP 19-26 Ferderal Reserve Bank of Philadelphia, 19(26). 6. References Arner, Douglas W; Auer, Raphael ; Frost, Jon. (2020). Stablecoins - Risks, Potential and Regulation. BIS Working Papers no. 905, 905. doi:http://dx.doi.org/10.2139/ssrn.3979495 Arner, Douglas W; Auer, Raphael ; Frost, Jon. (2020). Stablecoins - Risks, Potential and Regulation. BIS Working Papers no. 905, 905. doi:http://dx.doi.org/10.2139/ssrn.3979495 g p p g Auer, Raphael; Böhme, Rainer. (2021). CBDC the Quest for Minimally Invasive Technology. BIS Working Papers, No 948, 948. Retrieved 06 26, 2022, from https://www.bis.org/publ/work948.pdf p , , , , p g p p Barrdear, John; Kumhof, Michael. (2021). The Macroeconomics of Central Bank Digital Currencies. Journal of Economic Dynamics and Control. doi:https://doi.org/10.1016/j.jedc.2021.104148 p , , p g p p Barrdear, John; Kumhof, Michael. (2021). The Macroeconomics of Central Bank Digital Currencies. Journal of Economic Dynamics and Control. doi:https://doi.org/10.1016/j.jedc.2021.104148 y p g j j Berg, N., & Kim, J.-Y. (2022). Optimal Online-payment Security System and the Role of Liability Sharing. Economic Modelling. doi:https://doi.org/10.1016/j.econmod.2022.105805 y p g j j Berg, N., & Kim, J.-Y. (2022). Optimal Online-payment Security System and the Role of Liability Sharing. Economic Modelling. doi:https://doi.org/10.1016/j.econmod.2022.105805 8 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 ww.iiste.org BIS. (2020). BIS Innovation Hub on CBDC. BIS Working Papers. Retrieved 09 19, 2021, from https://www.bis.org/about/bisih/topics/cbdc.htm BIS.Org. (2019). Payment Systems in Zambia. BIS Working Papers. Retrieved 01 26, 2022, from https://www.bis.org/cpmi/paysys/zambia.pdf Boar, Codruta; Wehrli, Andreas. (2021). Results of the third BIS survey on CBDC. BIS Papers No 114 - Monetary and Economic Department. Retrieved 02 08, 2021, from https://www.bis.org/publ/bppdf/bispap114.pdf p g p pp p p p Borri , N., & Shakhnov, K. (2019). Regulation Spillovers Across cryptocurrency Markets. Finance Research. doi:https://doi.org/10.1016/j.frl.2019.101333 p g j BOZ. (2017). The Prohibition and Prevention of Money Laundering Act. Lusaka: Bank of Zambia. Retrieved 06 12, 2022, from https://www.boz.zm/53_TheProhibitionandPreventionofMoneyLaunderingAct.pdf 018). (B. o. Zambia, Editor) Retrieved 12 07, 2019, from Bank of Zambia: https://www.boz.zm/PRESS- LEASE-ON-CRYPTOCURRENCIES.pdf RELEASE-ON-CRYPTOCURRENCIES.pdf doi:https://doi.org/10.21799/frbp.wp.2019.26 Ś f p ( ) p g p p Kosc, K., Sakowski , P., & Ślepaczuk, R. (2019). Momentum and Contrarian Effects on the Cryptocurrency Market. Physica A - Statistical Mechanics and its Applications, 523. doi:https://doi.org/10.1016/j.physa.2019.02.057 p g j p y McGough, C. M. (2017). Evaluating the Impact of AML/CFT Regulations. Duke University Working Papers. Retrieved 03 23, 2022, from https://sites.duke.edu/djepapers/files/2016/10/caitlinmcgough-dje.pdf Mohammadreza, S., & Hosseini, D. (2018). Central Bank Digital Currency and Monetary Policy. 9 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) Vol.13, No.16, 2022 www.iiste.org ww.iiste.org doi:http://dx.doi.org/10.2139/ssrn.3011401 Morales-Resendiz, Raúl ; Ponce, Jorge; Picardo, Pablo; Chen, Bobby; Velasco, Andrés ; Guiborg, Gabriela; Sanz, León. (2021). Implementing a Retail CBDC - Lessons learned and Key Insights. Latin American Journal of Central Banking, 2(1). Retrieved 07 30, 2021, from https://www.sciencedirect.com/science/article/pii/S2666143821000028 León. (2021). Implementing a Retail CBDC - Lessons learned and Key Insights. Latin American Journal of Central Banking, 2(1). Retrieved 07 30, 2021, from https://www.sciencedirect.com/science/article/pii/S2666143821000028 Morales-Resendiz, Raúl; Ponce, Jorge; Picardo, Pablo; Chen, Bobby; Velasco, Andrés; Guiborg, Gabriela; Sanz, León. (2021). Implementing a Retail CBDC - Lessons learned and Key Insights. Latin American Journal of Central Banking, 2(1). Retrieved 07 30, 2021, from https://www.sciencedirect.com/science/article/pii/S2666143821000028 p p Ramos , D. F., Garratt, R., & Arauz, A. (2021). The Rise and Fall of Ecuador's CBDC. Latin American Journal of Central Banking, 2(2). doi:https://doi.org/10.1016/j.latcb.2021.100030 Reuters. (2019). France Creates G7 Cryptocurrency Task Force. Paris. Retrieved 08 06, 2020, from https://www.reuters.com/article/us-facebook-crypto-france/france-creating-g7-cryptocurrency-taskforce- says-central-banker-idUSKCN1TM0SO SARB. (2014). Position Paper on Vitual Currencies. South African Reserve Bank, National Payment System Department (NPSD). Pretoria: South African Reserve Bank. Retrieved 05 06, 2020, from https://www.resbank.co.za/RegulationAndSupervision/NationalPaymentSystem(NPS)/Legal/Documents/Po sition%20Paper/Virtual%20Currencies%20Position%20Paper%20%20Final_02of2014.pdf SARB. (2021). SARB Feasibility Study for a general-purpose retail CBDC. South African Reserve Bank (SARB) . South African Reserve Bank (SARB). Retrieved 09 19, 2020, from https://www.resbank.co.za/content/dam/sarb/publications/media-releases/2021/cbdc- /Feasibility%20study%20for%20a%20general- purpose%20retail%20central%20bank%20digital%20currency.pdf y y g rpose%20retail%20central%20bank%20digital%20currency.pdf purpose%20retail%20central%20bank%20digital%20currency.pdf SEC. (2018). Notice on Cryptocurrencies and Related Digital Products/Assets. SEC. Retrieved 10 04, 2020, from http://www.seczambia.org.zm/wp-content/uploads/2018/02/Public-Notice-on-Cryptocurrencies.pdf p p % % % % g % y p (2018). Notice on Cryptocurrencies and Related Digital Products/Assets. SEC. Retrieved 10 04, 202 rom http://www.seczambia.org.zm/wp-content/uploads/2018/02/Public-Notice-on-Cryptocurrencies.pdf Taskinsoy, J. (2019). Facebook’s Project Libra - Will Libra Sputter Out or Spur Central Banks to Introduce Their Own Unique Cryptocurrency Projects? Social Science Research Network (SSRN). doi:http://dx.doi.org/10.2139/ssrn.3423453 UNODC. (2020). Global Programme against Money Laundering. Working Paper Series. Retrieved 03 20, 2022, from https://www.unodc.org/unodc/en/money-laundering/global-programme-against-money- laundering/.html Wincor. (2018). Understanding the cost of handling cash in Asia Pacific. RELEASE-ON-CRYPTOCURRENCIES.pdf Asian Banker Research. Retrieved 03 06, 2022, from https://retailbanking.theasianbanker.com/assets/media/dl/whitepaper/Understandingthecostofhandlingcashin AsiaPacific.pdf Wincor. (2018). Understanding the cost of handling cash in Asia Pacific. Asian Banker Research. Retrieved 03 06, 2022, from 10
https://openalex.org/W4221065171	https://air.unimi.it/bitstream/2434/920913/2/Donation%20after%20Circulatory%20Death%20Liver%20Transplantation%20in%20Paediatric%20Recipients.pdf	English	null	Donation after Circulatory Death Liver Transplantation in Paediatric Recipients	Transplantology	2,022	cc-by	9,210	Citation: Parente, A.; Tirotta, F.; Ronca, V.; Schlegel, A.; Muiesan, P. Donation after Circulatory Death Liver Transplantation in Paediatric Recipients. Transplantology 2022, 3, 91–102. https://doi.org/10.3390/ transplantology3010009 Keywords: paediatric liver transplant; donation after cardiac death; non-heart-beating donor Review Donation after Circulatory Death Liver Transplantation in Paediatric Recipients Alessandro Parente 1 , Fabio Tirotta 1, Vincenzo Ronca 1,2 , Andrea Schlegel 3,4,,† and Paolo Muiesan 3,,† e 1 , Fabio Tirotta 1, Vincenzo Ronca 1,2 , Andrea Schlegel 3,4,,† and Paolo Muiesan 3,,† Alessandro Parente 1 , Fabio Tirotta 1, Vincenzo Ronca 1,2 , Andrea Schlegel 3,4,,† and 1 Liver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH, UK; aleparen@gmail.com (A.P.); fabio.tirotta.chirurgia@gmail.com (F.T.); v.ronca@bham.ac.uk (V.R.) 1 Liver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH, UK; aleparen@gmail.com (A.P.); fabio.tirotta.chirurgia@gmail.com (F.T.); v.ronca@bham.ac.uk (V.R.) g g 2 Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherap U i it f Bi i h Bi i h B15 2TT UK Ce e o e G s o es ese c , s e o o ogy o e p University of Birmingham, Birmingham B15 2TT, UK y g g 3 General and Liver Transplant Surgery Unit, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and University of Milan, 20122 Milan, Italy p gg y y 4 Swiss HPB and Transplant Center, Department of Visceral Surgery and Transplantation, University Hospital Zurich, 8091 Zurich, Switzerland Correspondence: schlegel.andrea@outlook.de (A.S.); muiesan@ † These authors contributed equally as senior authors. Abstract: Waiting list mortality together, with limited availability of organs, are one of the major challenges in liver transplantation (LT). Especially in the paediatric population, another limiting factor is the scarcity of transplantable liver grafts due to additional concerns regarding graft size matching. In adults, donation after circulatory death (DCD) liver grafts have been used to expand the donor pool with satisfactory results. Although several studies suggest that DCD livers could also be used in paediatric recipients with good outcomes, their utilisation in children is still limited to a small number of reports. Novel organ perfusion strategies could be used to improve organ quality and help to increase the number of DCD grafts utilised for children. With the current manuscript, we present the available literature of LT using DCD grafts in paediatric recipients, discussing current challenges with the use of these livers in children and how machine perfusion technologies could be of impact in the future. 2. DCD Liver Transplantation: Deﬁnitions and Overview of Current Literature The outcomes of a successful DCD transplantation, as opposed to donation after brain death (DBD), rely on particular events occurring during the whole process of organ retrieval. In most countries, controlled DCD Maastricht category III is the most frequent form of donation, where withdrawal of life-sustaining therapies (WLST) is planned in a controlled environment. After WLST, the donor warm ischemia time (dWIT) starts and subsequently the vital parameters drop until circulatory arrest. The no-touch period is variable between countries [20]. After conﬁrmation of death, donor surgery is commenced and organs are retrieved. Deﬁning dWIT is crucial as it has been shown to impact outcomes [5,21]. Recent guidelines developed from the International Liver Transplantation Society have summarised the deﬁnitions of dWIT: (1) total dWIT is deﬁned as time from donor WLST to cold perfusion; (2) functional dWIT is deﬁned as the timepoint where saturation (SpO2) drops below 70% and/or mean arterial blood pressure (MAP) drops below <50 mm Hg until the start of cold organ ﬂush [22]. During the recent consensus conference in Venice, a new uniform deﬁnition for functional dWIT was introduced, corresponding to the guidelines practised in Spain, where this time starts at a saturation below 80% and a MAP below 60 mmHg [22] In the early reports, DCD livers have been linked to inferior long-term outcomes when compared to DBD [23,24], related to donor risk, graft selection, management of recipients and additional ischemic damage caused by dWIT [25]. Despite the general classiﬁcation of DCD livers as marginal, the overall survival of grafts and patients in adults have signiﬁcantly improved over the past decades and appear now comparable to DBD livers, provided donor and recipient risk factors are limited [7]. DCD liver transplantation is a common practice in the Western world, mainly in the adult population. A recent report from the UNOS database showed a 10-fold lower utilisation of DCD livers in paediatric recipients compared to adults [10]. This is related to a more conservative approach in the donor selection for paediatric recipients with a need for optimal grafts. A recent study has shown interesting data regarding paediatric DCD liver procurement and utilisation [26], where the authors have analysed the Organ Procurement and Transplantation Network (OPTN) and data set contained US recipients undergoing transplantation of livers from DCD donors from 1993 to 2018. Some 17071 DCD liver donors were identiﬁed. 1. Introduction Liver transplantation (LT) is the only effective treatment for end-stage liver disease or fulminant liver failure. However, organ shortage is still one of the main challenges for the transplant community worldwide, especially in the paediatric population, where the disparity between the number of donors and recipients appears even higher compared to adults. In fact, paediatric recipients, and in particular small children, face a unique challenge in receiving organs due to the graft size mismatch [1]. In this context, it has been shown that up to 30% of organs were declined based on size mismatch [1]. In the United States (US), the mortality of paediatric recipients awaiting LT remains high, counting 1 in 10 infants and 1 in 20 older children [2]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. In response to the increasing number of listed candidates and the growing demand for available grafts, donation after circulatory death (DCD) has been pursued during the last decades to recruit more organs, particularly in adult recipients. Indeed, in some countries, including Spain, the Netherlands and the UK, DCD transplantations are routinely performed and account for up to 40% of overall donations [3,4]. However, DCD livers are known to induce more complications, including primary non-function (PNF), acute kidney injury (AKI) and biliary complications [5]. Despite the frequent utilisation of DCD livers in adult recipients, their use in the paediatric population remains limited. Since the very ﬁrst cases performed by Starzl et al. in 1968 [6], with further encouraging reports in the early Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/transplantology Transplantology 2022, 3, 91–102. https://doi.org/10.3390/transplantology3010009 Transplantology 2022, 3 92 2000s [7], the experience with DCD grafts in paediatric recipients is controversial and the literature is limited to a few case reports and small series [8–19]. 2000s [7], the experience with DCD grafts in paediatric recipients is controversial and the literature is limited to a few case reports and small series [8–19]. p With this article, we provide an overview of the currently available literature in paediatric DCD liver transplantation and discuss future directions. 2. DCD Liver Transplantation: Deﬁnitions and Overview of Current Literature Groups were divided into paediatric aged 0–12 years (n = 856, 5%) and adolescent aged 13–17 years (n = 757, 4.4%), which were compared to donors aged 18–40 years (n = 6504, 38.1%). Overall, there were 7293 (42.7%) livers recovered with a total of 5124 (30%) DCD livers transplanted. With a thorough analysis, they have demonstrated that paediatric DCD allografts were retrieved at a signiﬁcantly lower rate (n = 234/856, 27.3%) compared to donors aged 18–40 (3664/6504, 56.3%), but once procured, these paediatric organs are transplanted at a similar rate to those from the 18–40-year-old cohort. Interestingly, the highest rate of recovery was recorded in the 13–17-year-old group (n = 486/757, 64.2%). The authors argued that this could be related to the misperception of poor outcomes when smaller organs are transplanted. However, they have shown that the 10-year relative risks for graft failure and patient death were similar between paediatric and adult DCD donors. The authors concluded that paediatric DCD liver grafts could be an underestimated source of viable organs. 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience The authors ﬂushed the biliary tree during organ procurement with preservation solution and injected tissue plasminogen activator (TPA) into the donor hepatic artery during the back table procedure to decrease the rates of biliary complications. Additionally, no retransplantations or major complications, including vascular thrombosis, PNF or ischemic cholangiopathy (IC) were reported. However, one DCD and three DBD recipients experienced anastomotic biliary strictures. The authors explained their excellent outcomes with a strict donor selection, and a minimised CIT, which was kept as low as 300 min. Of note, the median PELD score of the DCD recipient was higher compared to the DBD group. This trend towards sicker recipients could have played a role in the DCD graft selection within the strict criteria identiﬁed by the authors, in order to minimise additional risk factors. This growing experience with the utilisation of DCD liver grafts has been recently reported by other authors from the US [10]. Hwang et al. presented an update of the UNOS data on paediatric DCD LT between 1993 and 2017. The entire paediatric cohort of 11646 recipients included 57 (0.49%) transplantations with controlled DCD livers. Living donors were excluded from the analysis. Only three grafts were left lateral segments (5.26%), whereas 54 (94.74%) were whole grafts. Comparing the two cohorts of DCD and DBD livers, some risk parameters appeared different. In fact, DCD recipients were older (7.7 vs. 5.2 years) and with a signiﬁcantly higher BMI (20.4 vs. 18.3 kg/m2), when compared to DBD recipients. Additionally, DCD recipients were more often on ventilatory support. The most common indications for transplantation were biliary atresia (21% in the DCD group, 28% in the DBD group) followed by acute hepatic necrosis (19% in the DCD group, 10% in the DBD group). There were no signiﬁcant differences regarding donor parameters, including age, BMI or CIT. The graft survival was comparable in the two groups with similar rates of PNF (3.5% DCD vs. 4.2% DBD), vascular thrombosis (7% DCD vs. 4.4% DBD), biliary complications (0% DCD vs. 1.3% DBD) and acute rejection (3.5% DCD vs. 1.6% DBD). Interestingly, patient survival in the DCD group was superior compared to the DBD group (p < 0.05). The authors have further demonstrated a signiﬁcantly better graft survival in paediatric DCD liver recipients compared to the adult DCD cohort (p < 0.05). 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience In the US, there were 57 cases described in three studies overall. Abt et al. analysed almost 5000 paediatric LT, performed between 1995 and 2005 and provided the ﬁrst compre- Transplantology 2022, 3 93 hensive report of DCD grafts in paediatric recipients [8]. Importantly, only 19 livers from the overall cohort of 4991 (0.4%) were DCDs transplanted in children. Most grafts (84.2%) were procured from donors younger than 18 years. The mean donor warm ischemia time (dWIT) was 11.6 min, and the mean cold ischemia time (CIT) was 8.1 h [8]. Of note, the exact deﬁnition of dWIT is not further mentioned in the manuscript. The authors reported three graft failures, including one PNF (5.3%) and two retransplantations (10.5%) in the DCD group, but the causes were not disclosed in the manuscript [8]. The overall results of DCD and DBD liver transplants were comparable. One-year graft survival in DCD and DBD grafts was 89.2% and 75.6%, respectively. Over the prolonged follow-up of 5 years, graft survival rates were 65.8% in DBD and 79.3% in DCD transplantation. The authors concluded that, despite the small numbers of DCD livers used for paediatric recipients with accurate selection the results were comparable to DBD livers (Table 1). p ( ) In 2014, authors from the University of California Los Angeles (UCLA) compared the outcomes of paediatric DCD liver recipients with primary DBD liver transplants in a single center matched analysis [9]. Recipients who received partial grafts or multiorgan transplants were excluded. The DCD selection criteria were strict, with donor age < 45 years, Body-Mass-Index (BMI) < 30 kg/m2, CIT < 8 h, donor hospital stay <5 days, serum transaminase levels less than twice the normal range and a total dWIT < 30 min. The median total dWIT and median CIT were 24 and 300 min, respectively. While most recipient characteristics were similar among the groups, the median paediatric end-stage liver disease (PELD) score was 19 points for the DCD group and 11 points in DBD recipients (p = 0.48). The most common indications were biliary atresia, acute liver failure, neonatal hepatitis and malignant neoplasms. Importantly, at a prolonged follow-up of 10-years, none of the grafts or patients was lost in both groups. 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience Such ﬁndings were based on the selective donor acceptance in line with the paediatric recipients (Table 1). Transplantology 2022, 3 94 Table 1. Summary of all reported cases of paediatric LT with DCD grafts. Results are presented in mean deviation standard or median and IQR, based on how each study reported. Author and Year Country Donor Age (yr) Total dWIT (min) Functional dWIT (min) CIT (h) Graft Type Recipient Number Recipient Age Recipient Gender Complications Status at Last FU Abt 2006 US 12.2 (14.4) 11.6 (5.3) NA 8.1 Whole 19 8.7 yr 12M/7F 2 re-LT 1 PNF NA Muiesan 2006 Bartlett 2010 UK 23 (10–64) NA 16 (11–29) 7 (5.5–8.4) 4: whole 8: reduced 1: split LLS 1: auxiliary 14 7 yr (8 mo–16 yr) 7M/7F 2 pleural effusion 1 sepsis 4 ACR 2 ductopenic rejection 100% survival Perera 2009 Gozzini 2010 Gelas 2012 UK 14.2 (11–20) NA 15 (10–22) 7.4 (6–8.4) 2: Whole 5: reduced 7 6 (0.1–15) yr 2M/5F 4: ACR 3: IC 1: CR 100% survival Hong 2014 US 2.4 (0.3–6) 24 NA 5 (4–7) Whole 7 28.4 mo (9.6–59.2) 3M/4F 1 AS 100% survival Van Rijn 2017 Netherlands 5 (3–9) 25 (20–31) NA 8 (7–9) Whole 3 8.6 (6–13) yr NA 2 HAT 1 PVT 66% survival 33% death Hwang 2018 US 11.2 ± 12.3 16.9 ± 6.8 NA 7.9 ± 5.2 54: whole 3: LLS 57 7.7 ± 6.8 NA 4 vascular thrombosis 2 ACR 2 PNF 1 CR 75% graft survival at 4 years Liu 2018 China NA 5 NA 7.5 Whole 4 4.5 yr 2M/2F 1 NASH 1 Biloma 1 EBV infection 100% survival Werner 2019 Netherlands 13 34 NA 6.4 h + 2 h HMP Whole 1 16 F Relaparotomy for bleeding 100% survival ACR: acute cellular rejection; AS: anastomotic stricture; CIT: cold ischemia time; CR: chronic rejection; DCD: donation after circulatory death; dWIT: donor warm ischemia time; HAT: hepatic artery thrombosis; HMP: hypothermic machine perfusion; IC: ischemic cholangiopathy; LLS: left lateral segment; LT: liver transplantation; Mo: months; NA: not available; NASH: non-alcoholic steatohepatitis; PNF: primary non-function; PVT: portal vein thrombosis. Yr: years. Deﬁnition of dWIT: in the reports from the US and the Netherlands it was deﬁned as total dWIT, from withdrawal of life-sustaining therapies until the time of aortic perfusion with cold preservation solution. 4. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The European Experience In Europe, there have been seven studies reporting a total of 25 cases. The ﬁrst paediatric DCD liver transplantation was reported by Muiesan et al. in 2003 [11], from King’s College Hospital, UK. The authors retrieved a liver from a 27-year-old controlled DCD donor with the super-rapid technique [12]. The functional dWIT, deﬁned as the interval between systolic blood pressure <50 mmHg and the time of aortic perfusion with cold preservation solution, was 21 min. The liver was reduced ex-situ obtaining a right-lobe auxiliary graft, which was implanted after performing an extended right hepatectomy in the recipient. The indication for this auxiliary transplantation with a DCD liver was an acute liver failure. The CIT was 8.8 h and the outcome was excellent after 2 months of follow-up. p Three years later, the same group reported a case series of seven paediatric transplants from controlled DCD Maastricht category III donors [13]. The functional dWIT was 14.6 min (range 11–18) and the CIT was kept below 8 h (mean CIT: 7.3 h, range 6.2–8.8). The graft types included four reduced and one left lateral segment (LLS), one left lobe and the above- mentioned extended auxiliary right graft (segments IV to VIII). At a mean follow-up of 20 months [10–36], patient and graft survival rates were 100% [12]. The same group updated the case series in 2010, showing the outcomes of 14 children in total [13]. All DCD donors were in the controlled Maastricht III category, with a median fuctional dWIT of 15 min (IQR: 10–29 min) and a median CIT of 7 h (IQR: 5.5–8.4 h). The most common recipient diagnoses were extrahepatic biliary atresia, acute liver failure, factor VII deﬁciency, and neonatal sclerosing cholangitis. Four livers were whole organs, eight grafts were reduced (7 LLS and 1 left lobe), one liver was a formal LLS split and one was the previously mentioned auxiliary partial orthotopic graft. Some early post-operative complications were reported, which included pleural effusion (n = 2), sepsis (n = 2), wound infection (n = 1) and a frontal intracerebral haematoma following preoperative insertion of an intra-cranial pressure monitor. Long-term complications included ductopenic rejection in two recipients and sirolimus-related chronic hypoalbuminemia in one recipient. Remarkably, after a median follow-up of three years, the overall survival was 100% and there were no vascular or biliary complications [14]. 4. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The European Experience The reasons for this success were attributed to the strict donor selection, with a short functional dWIT (kept <30 min), a short CIT (<8 h) and a rapid donor liver procurement with extensive ﬂush of the biliary tree by an experienced team [27]. p y y p Subsequently, three reports from Birmingham, UK, showed the results of seven chil- dren transplanted with DCD livers. In their ﬁrst series presented in 2009, two DCD grafts were reduced for two children with acute liver failure, at the age of 10 weeks and 6 years. Both patients had a good recovery. However, the ﬁrst recipient experienced mild cholestasis after 6 months, whereas the second was affected by multiple intra-hepatic strictures three years after transplantation [15]. Consequently, the same group described four additional cases, where graft and patient survival, with a mean follow-up of 19 months, was 100%. One child was treated for two episodes of acute rejection and two children developed mild ischemic cholangiopathy, treated conservatively [16]. In 2012, a neonate with acute liver failure due to hemochromatosis was transplanted with an ABO-incompatible reduced-size DCD graft [17]. The authors explained the success of this transplant case with the strict donor selection and limitation of functional dWIT and CIT to less than 30 min and <8 h [17]. In 2017, a study from the Netherlands [18] described the long-term outcome after transplantation of 20 livers, procured from controlled paediatric DCD donors. Three grafts (15%) were transplanted in paediatric recipients. Unfortunately, all three paediatric DCD recipients were lost due to vascular complications. Two recipients developed a hepatic artery thrombosis and one had a portal vein thrombosis. Of note, the donor risk was higher with longer functional dWIT (24 min) and CIT (8 h) when compared to the British and American series presented earlier [9–11,13]. p y y p Subsequently, three reports from Birmingham, UK, showed the results of seven chil- dren transplanted with DCD livers. In their ﬁrst series presented in 2009, two DCD grafts were reduced for two children with acute liver failure, at the age of 10 weeks and 6 years. Both patients had a good recovery. However, the ﬁrst recipient experienced mild cholestasis after 6 months, whereas the second was affected by multiple intra-hepatic strictures three years after transplantation [15]. Consequently, the same group described four additional cases, where graft and patient survival, with a mean follow-up of 19 months, was 100%. 3. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The US Experience In the UK reports, functional dWIT was deﬁned as the interval between systolic blood pressure < 50 mmHg and the time of aortic perfusion with cold preservation solution. The single report from China did not specify a deﬁnition of dWIT. with DCD grafts. Results are presented in mean deviation standard or median and IQR, based on how each ACR: acute cellular rejection; AS: anastomotic stricture; CIT: cold ischemia time; CR: chronic rejection; DCD: donation after circulatory death; dWIT: donor warm ischemia time; HAT: hepatic artery thrombosis; HMP: hypothermic machine perfusion; IC: ischemic cholangiopathy; LLS: left lateral segment; LT: liver transplantation; Mo: months; NA: not available; NASH: non-alcoholic steatohepatitis; PNF: primary non-function; PVT: portal vein thrombosis. Yr: years. Deﬁnition of dWIT: in the reports from the US and the Netherlands it was deﬁned as total dWIT, from withdrawal of life-sustaining therapies until the time of aortic perfusion with cold preservation solution. In the UK reports, functional dWIT was deﬁned as the interval between systolic blood pressure < 50 mmHg and the time of aortic perfusion with cold preservation solution. The single report from China did not specify a deﬁnition of dWIT. ACR: acute cellular rejection; AS: anastomotic stricture; CIT: cold ischemia time; CR: chronic rejection; DCD: donation after circulatory death; dWIT: donor warm ischemia time; HAT: hepatic artery thrombosis; HMP: hypothermic machine perfusion; IC: ischemic cholangiopathy; LLS: left lateral segment; LT: liver transplantation; Mo: months; NA: not available; NASH: non-alcoholic steatohepatitis; PNF: primary non-function; PVT: portal vein thrombosis. Yr: years. Deﬁnition of dWIT: in the reports from the US and the Netherlands it was deﬁned as total dWIT, from withdrawal of life-sustaining therapies until the time of aortic perfusion with cold preservation solution. In the UK reports, functional dWIT was deﬁned as the interval between systolic blood pressure < 50 mmHg and the time of aortic perfusion with cold preservation solution. The single report from China did not specify a deﬁnition of dWIT. Transplantology 2022, 3 95 6. DCD Liver Transplantation: Overview on Risk Scores and Benchmarking There are several scores that have been developed in LT based on a relatively limited number of parameters, and they express the total risk up to a risk threshold [28–31]. However, their applicability in DCD liver transplants is limited, as they include the variable DCD as overall risk factors without considering some key parameters involved in the DCD injury, e.g., dWIT. j y g In 2011, authors from UCLA [32] identiﬁed six multivariate factors predictive for graft failure in DCD livers, three from the recipients (diagnosis of hepatitis C virus with malignancy, non–hepatitis C virus with malignancy, or hepatitis C virus only, re-graft, and BMI > 30 and three from the donors (hepatitis B core antibody positivity, mean arterial pressure lower than 60 mm Hg for longer than 20 min after WLST and CIT > 6 h). The team from Kings College has identiﬁed among 261 DCD LT six donor, graft and recipient factors to predict DCD graft failure by the DCD-RI. The score also included 18 (6.9%) paediatric DCD donors and 15 (5.7%) DCD paediatric recipients as there was no difference in adult and paediatric DCD graft survival. The DCD-RI combined functional dWIT, duration of donor hepatectomy, MELD > 25, CIT, indication for transplantation and retransplantation [33]. According to the calculated DCD-RI score, three risk classes could be deﬁned of low (DCD-RI < 1), standard (DCD-RI 2–4) and high risk (DCD-RI > 5) having graft survival at 5 years of 86%, 78% and 34%, respectively. However, it needs to be highlighted that the DCD cases were not equally distributed to the three risk classes of this score. The UK-DCD-Risk score was recently developed to deﬁne futile donor-recipient combinations in DCD liver transplantation [34]. Seven parameters (donor age, donor BMI, functional dWIT, CIT, recipient age, recipient MELD and retransplantation) have been found to correlate with graft survival. Although most parameters in the UK-DCD-Risk score are the clinically relevant factors, also described in the paediatric DCD population, the speciﬁc role of this model in this recipient category remains unexplored. In addition to the identiﬁcation of futile DCD constellations, the best possible outcome is of importance to assess the impact of novel treatment modalities. In this context, the tool of Benchmarking was recently developed for DCD liver transplantation. 4. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The European Experience One child was treated for two episodes of acute rejection and two children developed mild ischemic cholangiopathy, treated conservatively [16]. In 2012, a neonate with acute liver failure due to hemochromatosis was transplanted with an ABO-incompatible reduced-size DCD graft [17]. The authors explained the success of this transplant case with the strict donor selection and limitation of functional dWIT and CIT to less than 30 min and <8 h [17]. In 2017, a study from the Netherlands [18] described the long-term outcome after transplantation of 20 livers, procured from controlled paediatric DCD donors. Three grafts (15%) were transplanted in paediatric recipients. Unfortunately, all three paediatric DCD recipients were lost due to vascular complications. Two recipients developed a hepatic artery thrombosis and one had a portal vein thrombosis. Of note, the donor risk was higher with longer functional dWIT (24 min) and CIT (8 h) when compared to the British and American series presented earlier [9–11,13]. Transplantology 2022, 3 96 5. Paediatric Liver Transplantation from Donation after Circulatory Death Donors: The Chinese Experience Lately, a case series has been published by authors from China [19]. Five children affected by progressive familial intrahepatic cholestasis underwent LT, four received a whole DCD graft and one a DBD liver. The CIT ranged from 370 to 550 min with a very short dWIT of 5 min, where the type of dWIT was not further speciﬁed. One of the DCD recipients developed an ischemic cholangiopathy with the clinical picture of recurrent cholangitis with biloma [19]. None of the patients or grafts were lost during the follow-up period of two years [19]. 7. The Potential Role of Machine Perfusion in DCD Liver Transplantation for Paediatric Recipients Machine perfusion has gained wide interest during the last decades. In adult LT, there is evidence that the different perfusion techniques have an impact on the rate of early allograft dysfunction (EAD), post-transplant complications and graft survival [35–37]. In the setting of DCD liver transplantation, hypothermic oxygenated perfusion (HOPE) was shown to protect recipients from the development of overall and liver-speciﬁc compli- cations, including ischemic cholangiopathy and graft loss [38–42]. Van Rijn et al. have recently demonstrated in the ﬁrst randomised controlled trial (RCT) a signiﬁcant reduction in ischemic cholangiopathy with a two-hour dual HOPE in DCD liver grafts [43]. In 2019, the Groningen group [44] published the ﬁrst case of paediatric DCD liver transplantation with the HOPE technique. A graft from a 13-year-old DCD donor was retrieved with the super rapid technique, having a total dWIT (from withdrawal of life support to in situ cold perfusion) of 34 min. Subsequently, after 6.4 h of CIT, the graft underwent pressure-controlled dual HOPE using the Liver Assist (Organ Assist, Groningen, The Netherlands) for 2 h. This DCD liver was used for a 16-year-old recipient affected by progressive familial intrahepatic cholestasis type 2. The surgery was complicated by a relaparotomy due to post-transplant bleeding on day 4. The further recovery and follow up of the recipient were uneventful with no additional complications at the one-year follow- up. Importantly, there were no clinical signs of vascular or biliary complications and a routine liver biopsy was normal. This report showed that HOPE can be safely performed in paediatric livers from DCD donors with successful outcomes. The hypothermic perfusion techniques were found to protect the recipients from complications through mitochondrial protection [35,41–43,45,46]. In fact, the underlying mechanism of protection through HOPE is directly linked to mitochondria. Cold oxygena- tion of ischemic tissue reduces previously accumulated Succinate and recharges ATP. The subsequent IRI—cascade of inﬂammation, which occurs once the organ undergoes nor- mothermic reperfusion (either on a device or in vivo during implantation), is signiﬁcantly reduced [47]. Based on the reduced succinate concentration, the initial reoxygenation is less toxic to mitochondrial complex I, which produces less reactive oxygen species (ROS), which is the main instigator of downstream inﬂammation in the entire organ. As a direct consequence, the release of danger signals, including mitochondrial DNA and damage- associated mitochondrial patterns (DAMPs), is reduced [48–50]. 6. DCD Liver Transplantation: Overview on Risk Scores and Benchmarking Among 2219 controlled DCD LT, benchmark values were identiﬁed for the adult transplant population as follows: PNF ≤2.5%, intensive care unit stay ≤3, in hospital stays ≤16 days, post-operative bleeding ≤10.3%, renal replacement therapy ≤9.6%, any complications ≤95%, ischemic cholangiopathy ≤16.8% anastomotic strictures ≤28.4% biliary leak ≤8.3%, one-year graft loss ≤14.4%, retransplantation ≤6.9%, one-year mortal- ity ≤9.6% [5]. Of note, paediatric LT from DCD donors reported today were found within these values, particularly the donor risk (i.e., dWIT) was within 15 min asystolic and 30 min total dWIT and also the main outcome parameters, for example, biliary complications and graft survival [5]. The benchmark tool provides a large comparator cohort to assess the risk and outcome accepted and achieved in a speciﬁc cohort. Transplantology 2022, 3 97 7. The Potential Role of Machine Perfusion in DCD Liver Transplantation for Paediatric Recipients This leads to a reduced activation of the innate immune system and decreases the inﬂammatory response in the recipient. Morphological features of such HOPE protections are a reduced number of biliary complications, a decreased graft stiffness with lower acute rejection rates and less intrahepatic vascular resistance [50]. Particularly the rejection and vascular resistance appears beneﬁcial for paediatric grafts with their higher risk of vascular complications and immune system activation. Such interesting mechanisms of protection of the liver graft could pave the way for routine utilisation of HOPE in paediatric DCD and, as such, expand the donor pool. p In addition to ex situ preservation technologies, in situ normothermic regional per- fusion (NRP) provided clinical evidence of adequate outcomes in adult LT with DCD grafts [51–53] in terms of biliary complications and graft loss. Italy, Spain and France procure DCD livers routinely with normothermic regional perfusion (NRP) in the donor. Such grafts are obtained based on the macroscopic appearance during 2–4 h of NRP, biopsy results and the level of parameters of injury in circulating donor blood (liver enzymes and lactates). Outcomes after transplantation of such adult DCD grafts appeared good with very low IC rates, although the grafts are of better quality with shorter dWIT [5,51–53]. To date, no reports are available with regards to the utilisation of NRP in DCD livers for paediatric recipients, but this option has been explored in DCD heart transplantation with good outcomes. [54] g Furthermore, machine perfusion could also promote paediatric LT allowing a split transplantation. This interesting concept of splitting livers during machine perfusion Transplantology 2022, 3 98 was recently explored by different authors who demonstrated feasibility with both nor- mothermic and hypothermic perfusion techniques [55–59]. Two case series [58,60] have shown the feasibility of splitting liver grafts during HOPE. Spada et al. have reported a mono-segmental (SII) split procedure during HOPE, which was successfully transplanted in a 3.7 kg neonate with acute liver failure, whereas the extended right lobe was trans- planted in a 9-year-old patient [57]. Unfortunately, the SII recipient developed a portal vein thrombosis 2 weeks after LT and underwent re-transplantation. Thorne et al. reported a classical splitting procedure with a successful transplant of LLS in a paediatric recipient [58]. Although both the grafts were retrieved from DBD, the procedure could potentially be applied to DCD grafts, allowing an expansion of organ availability. 7. The Potential Role of Machine Perfusion in DCD Liver Transplantation for Paediatric Recipients However, the liver splitting process itself can release inﬂammatory cytokines as a result of the parenchyma transection, as shown in experimental models [60,61], and this could potentially contribute to an elevated inﬂammatory response in the recipient [60]. In this regard, machine perfu- sion itself could be a compelling tool to mitigate and downregulate the recipient’s innate immune response. However, such protection from post-transplant IRI and inﬂammation has been demonstrated for the HOPE technique in the clinical setting and in experimental models [43,49,50]. Recent advances in the optimisation of donor stability with extracorporeal membrane oxygenation (ECMO), which is used to support cardiopulmonary failure, could represent another important modality to expand the donor pool [62,63]. Although no cases of livers donated from DCD donors supported by ECMO have been reported for paediatric recipients, one DBD case was successfully transplanted [48]. In addition, the availability of a mobile ECMO team could enable the use of this technology in smaller hospitals, as recently demonstrated by authors in Spain [64]. 8. Summary and Future Perspective The overall number of paediatric DCD liver recipients remains limited with 86 cases across four countries (Figure 1). Of note, the largest case series of 57 recipients [10] illus- trates a survival comparable to DBD grafts. Additionally, all other case series reported good outcomes with the loss of only one patient. The data shown in the literature and summarised in this review underline that with meticulous donor selection, adequate out- comes can be achieved with DCD grafts in paediatric recipients. The ideal DCD donor is considered as young and healthy, with short dWIT (<30 min), and short CIT (<8 h), good graft perfusion at procurement and rapid donor hepatectomy performed by an ex- perienced donor surgeon to avoid additional liver injury through unnecessary prolonged hepatectomy and delayed cooling. Indeed, throughout all reports, the mean or median dWIT is less than 30 min, although it needs to be highlighted that the dWIT followed heterogenous deﬁnitions throughout the here-discussed papers. Interestingly, although in a small number of studies, split livers from DCD were used with success, the additional liver transection may prolong the cold ischemia and the division of hepatocytes increases the enzyme and cytokine release with potentially higher reperfusion injury or even systemic inﬂammatory response syndrome (SIRS), similarly to features seen in patients who un- derwent the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) procedure [60,65]. p We acknowledge that, due to the heterogeneity of the data reported across four countries, it is difﬁcult to recommend the routine utilisation of DCD grafts for children, and this is further hampered by small numbers of cases. However, the use of a DCD graft in children where no living donor is available and who cannot wait any longer can be a lifesaving procedure. In summary, current literature suggests that DCD livers may be used in paediatric recipients with satisfactory results, provided the overall risk is limited. With novel machine perfusion technologies, the use of DCD could be extended even to split grafts, expanding the donor pool and potentially decreasing waitlist mortality in the paediatric population. Presently, a cautious and strict donor selection seems to be the most important factor that Transplantology 2022, 3 99 99 can inﬂuence overall outcomes. With larger studies and more evidence available in the future, the utilisation of DCD liver grafts for children will be increased. Presently, a cautious and strict donor selection seems to be the most important Conﬂicts of Interest: The authors declare that there is no conﬂict of interest regarding the publication of this article. Figure 1 Total number of DCD paediatric LT per reported countries Figure 1. Total number of DCD paediatric LT per reported countries. Figure 1 Total number of DCD paediatric LT per reported countries Figure 1. Total number of DCD paediatric LT per reported countries. We acknowledge that, due to the heterogeneity of the data reported across four coun- tries, it is difficult to recommend the routine utilisation of DCD grafts for children, and this is further hampered by small numbers of cases. However, the use of a DCD graft in children where no living donor is available and who cannot wait any longer can be a life- Author Contributions: A.P., A.S. and P.M. conceived the study; A.P. wrote the paper; A.P., F.T. and V.R. collected and analysed the data; A.S. and P.M. critically revised the paper. All authors have read and agreed to the published version of the manuscript. g y g saving procedure. In summary current literature suggests that DCD livers may be used in paediatric Funding: This research received no external funding. In summary, current literature suggests that DCD livers may be used in paediatric recipients with satisfactory results, provided the overall risk is limited. With novel ma- chine perfusion technologies, the use of DCD could be extended even to split grafts, ex- panding the donor pool and potentially decreasing waitlist mortality in the paediatric Acknowledgments: Figure 1 created with BioRender.com, accessed on 2 January 2022. The authors have nothing to disclose and did not receive any speciﬁc funding dedicated to this work. y, gg y p recipients with satisfactory results, provided the overall risk is limited. With novel ma- chine perfusion technologies, the use of DCD could be extended even to split grafts, ex- panding the donor pool and potentially decreasing waitlist mortality in the paediatric Acknowledgments: Figure 1 created with BioRender.com, accessed on 2 January 2022. The authors have nothing to disclose and did not receive any speciﬁc funding dedicated to this work. panding the donor pool and potentially decreasing waitlist mortality in the paediatric population. Presently, a cautious and strict donor selection seems to be the most important Conﬂicts of Interest: The authors declare that there is no conﬂict of interest regarding the publication of this article. panding the donor pool and potentially decreasing waitlist mortality in the paediatric population. 8. Summary and Future Perspective temic inflammatory response syndrome (SIRS), similarly to features seen in patients who underwent the Associating Liver Partition and Portal Vein Ligation for Staged Hepatec- tomy (ALPPS) procedure [60,65]. can inﬂuence overall outcomes. With larger studies and more evidence available in the future, the utilisation of DCD liver grafts for children will be increased. temic inflammatory response syndrome (SIRS), similarly to features seen in patients who underwent the Associating Liver Partition and Portal Vein Ligation for Staged Hepatec- tomy (ALPPS) procedure [60,65]. References Hong, J.C.; Venick, R.; Yersiz, H.; Kositamongkol, P.; Kaldas, F.M.; Petrowsky, H.; Farmer, D.G.; Agopian, V.; McDiarmid, S.V.; Hiatt, J.R.; et al. Liver transplantation in children using organ donation after circulatory death: A case-control outcomes analysis of a 20-year experience in a single center. JAMA Surg. 2014, 149, 77–82. [CrossRef] y p g g 10. Hwang, C.S.; Levea, S.L.; Parekh, J.R.; Liang, Y.; Desai, D.M.; MacConmara, M. Should more donation be used in pediatric transplantation? Pediatr. Transplant. 2018, 23, e13323. [CrossRef] p p p 11. Muiesan, P.; Girlanda, R.; Baker, A.; Rela, M.; Heaton, N. Successful segmental auxiliary liver transplantation from a non-heart- beating donor: Implications for split-liver transplantation. Transplantation 2003, 75, 1443–1445. [CrossRef] g p p p p 12. Casavilla, A.; Ramirez, C.; Shapiro, R.; Nghiem, D.; Miracle, K.; Bronsther, O.; Randhawa, P.; Broznick, B.; Fung, J.J.; Starzl, T. Experience with liver and kidney allografts from non-heart-beating donors. Transplantation 1995, 59, 197–203. [CrossRef] 13. Muiesan, P.; Jassem, W.; Girlanda, R.; Steinberg, R.; Vilca-Melendez, H.; Mieli-Vergani, G.; Dhawan, A.; Rela, M.; Heaton, N. Segmental Liver Transplantation from Non-Heart Beating Donors-An Early Experience with Implications for the Future. Am. J. Transplant. 2006, 6, 1012–1016. [CrossRef] p 14. Bartlett, A.; Vara, R.; Muiesan, P.; Mariott, P.; Dhawan, A.; Mieli-Vergani, G.; Rela, M.; Heaton, N. A single center experience of donation after cardiac death liver transplantation in pediatric recipients. Pediatr. Transplant. 2010, 14, 388–392. [CrossRef] 15. Perera, M.T.P.R.; Gozzini, S.; Mayer, D.; Sharif, K.; Bennett, J.; Muiesan, P.; Mirza, D.F. Safe use of segmental liver grafts from donors after cardiac death (DCD) in children with acute liver failure. Transpl. Int. 2009, 22, 757–760. [CrossRef] 16. Gozzini, S.; Perera, M.T.P.R.; Mayer, D.A.; Mirza, D.F.; Kelly, D.A.; Muiesan, P.; Sharif, K. Liver transplantation in children using non-heart-beating donors (NHBD). Pediatr. Transplant. 2010, 14, 554–557. [CrossRef] 17. Gelas, T.; Hartley, J.; Mirza, D.F.; Muiesan, P.; Bowen, C.; Sharif, K. Segmental ABO-incompatible liver graft from a donor after cardiac death in neonatal acute liver failure. Pediatr. Transplant. 2011, 16, E53–E57. [CrossRef] 18. Van Rijn, R.; Hoogland, P.E.R.; Lehner, F.; Van Heurn, E.L.W.; Porte, R.J. Long-term results after tran grafts from donation after circulatory death donors. PLoS ONE 2017, 12, e0175097. [CrossRef] g y 19. Liu, Y.; Sun, L.; Zhu, Z.J.; Wei, L.; Qu, W.; Zeng, Z.G. Liver Transplantation for Progressive Familial Intrahepatic Cholestasis. Ann. Transplant. 2018, 23, 666–673. [CrossRef] 20. References 1. Hsu, E.K.; Shaffer, M.L.; Gao, L.; Sonnenday, C.; Volk, M.L.; Bucuvalas, J.; Lai, J.C. Analysis of Liver Offers to Pediatric Candidates on the Transplant Wait List. Gastroenterology 2017, 153, 988–995. [CrossRef] p gy , , [ ] 2. Perito, E.R.; Roll, G.; Dodge, J.L.; Rhee, S.; Roberts, J.P. Split Liver Transplantation and Pediatric Waitlis States: Potential for Improvement. Transplantation 2019, 103, 552–557. [CrossRef] 2. Perito, E.R.; Roll, G.; Dodge, J.L.; Rhee, S.; Roberts, J.P. Split Liver Transplantation and Pediatric Waitlist Mortality in the United States: Potential for Improvement. Transplantation 2019, 103, 552–557. [CrossRef] 3 J h R J B db L L M i K N b J O d i d l i i h UK Th l d d A 3. Johnson, R.J.; Bradbury, L.L.; Martin, K.; Neuberger, J. Organ donation and transplantation in the UK-T from the UK national transplant registry. Transplantation 2014, 97, S1–S27. [CrossRef] 4. De La Rosa, G.; Fondevila, C.; Navasa, M. Liver transplantation in Spain. Liver Transplant. 2016, 22, 1259–1264. [CrossRef] 5. Schlegel, A.; van Reeven, M.; Croome, K.; Parente, A.; Dolcet, A.; Widmer, J.; Meurisse, N.; De Carlis, R.; Hessheimer, A.; Jochmans, I.; et al. A multicentre outcome analysis to deﬁne global benchmarks for donation after circulatory death liver transplantation. J. Hepatol. 2021, 76, 371–382. [CrossRef] p p 6. Starzl, T.E.; Groth, C.G.; Brettschneider, L.; Moon, J.B.; Fulginiti, V.A.; Cotton, E.K.; Porter, K.A. Extended survival in 3 cases of orthotopic homotransplantation of the human liver. Surgery 1968, 63, 549–563. [CrossRef] p p g y 7. Abt, P.; Desai, N.; Crawford, M.; Forman, L.; Markmann, J.; Olhoff, K. Survival following liver transplantation from nonheart beating donors. Ann. Surg. 2004, 239, 87–92. [CrossRef] g g 8. Abt, P.; Kashyap, R.; Orloff, M.; Jain, A.; Tsoulfas, G.; Bozorgzadeh, A.; Olthoff, K. Pediatric Liver With Allografts From DCD Donors: A Review of UNOS Data. Transplantation 2006, 82, 1708–1711. g g Abt, P.; Kashyap, R.; Orloff, M.; Jain, A.; Tsoulfas, G.; Bozorgzadeh, A.; Olthoff, K. Pediatric Liver and Ki ; Orloff, M.; Jain, A.; Tsoulfas, G.; Bozorgzadeh, A.; Olthoff, K. Pediatric Liver and Kidney Transplantation , ; y p, ; , ; J , ; , ; g , ; , With Allografts From DCD Donors: A Review of UNOS Data. Transplantation 2006, 82, 1708–1711. [CrossR fts From DCD Donors: A Review of UNOS Data. Transplantation 2006, 82, 1708–1711. [CrossRef] 9. Abbreviations BMI Body Mass Index CIT Cold Ischemia Time DAMPs Damage Associated Molecular Patterns DBD Donation after Brain Death DCD Donation after Cardiac Death dWIT Donor Warm Ischemia Time ECMO Extracorporeal membrane oxygenation HMP Hypothermic Machine Perfusion HOPE Hypothermic Oxygenated Liver Perfusion IC Ischemic Cholangiopathy LLS left lateral segment LT Liver Transplantation NRP Normothermic Regional Perfusion PNF Primary non-function ROS Reactive Oxygen Species TPA Tissue plasminogen activator WIT Warm Ischemia Time WLST Withdrawal of life-sustaining therapies UNOS United Network for Organ Sharing 100 Transplantology 2022, 3 References Lomero, M.; Gardiner, D.; Coll, E.; Haase-Kromwijk, B.; Procaccio, F.; Immer, F.; Gabbasova, L.; Antoine, C.; Jushinskis, J.; Lynch, N.; et al. Donation after circulatory death today: An updated overview of the European landscape. Transpl. Int. 2019, 33, 76–88. [CrossRef] 21. Taner, C.B.; Bulatao, I.G.; Willingham, D.L.; Perry, D.K.; Sibulesky, L.; Pungpapong, S.; Aranda-Michel, J.; Keaveny, A.P.; Kramer, D.J.; Nguyen, J.H. Events in procurement as risk factors for ischemic cholangiopathy in liver transplantation using donation after cardiac death donors. Liver Transplant. 2012, 18, 100–111. [CrossRef] p 22. Kalisvaart, M.; Croome, K.P.; Hernandez-Alejandro, R.; Pirenne, J.; Cortés-Cerisuelo, M.; Miñambres, E.; Abt, P.L. Donor Warm Ischemia Time in DCD Liver Transplantation-Working Group Report From the ILTS DCD, Liver Preservation, and Machine Perfusion Consensus Conference. Transplantation 2021, 105, 1156–1164. [CrossRef] p 23. Mateo, R.; Cho, Y.; Singh, G.; Stapfer, M.; Donovan, J.; Kahn, J.; Fong, T.L.; Sher, L.; Jabbour, N.; Aswad, S.; et al. Risk Factors for Graft Survival After Liver Transplantation from Donation After Cardiac Death Donors: An Analysis of OPTN/UNOS Data. Am. J. Transplant. 2006, 6, 791–796. [CrossRef] [PubMed] p 24. De Vera, M.E.; Lopez-Solis, R.; Dvorchik, I.; Campos, S.; Morris, W.; Demetris, A.J.; Fontes, P.; Marsh, J.W. Liver Transplantation Using Donation After Cardiac Death Donors: Long-Term Follow-Up from a Single Center. Am. J. Transplant. 2009, 9, 773–781. [CrossRef] [PubMed] 25. Taner, C.B.; Bulatao, I.G.; Perry, D.K.; Sibulesky, L.; Willingham, D.L.; Kramer, D.J.; Nguyen, J.H. Asystole to cross-clamp period predicts development of biliary complications in liver transplantation using donation after cardiac death donors. Transpl. Int. 2012, 25, 838–846. [CrossRef] [PubMed] 101 Transplantology 2022, 3 26. Little, C.J.; Dick, A.A.S.; Perkins, J.D.; Hsu, E.K.; Reyes, J.D. Livers From Pediatric Donation After Circulatory Death Donors Represent a Viable and Underutilized Source of Allograft. Liver Transplant. 2020, 26, 1138–1153. [CrossRef] [PubMed] p g 27. Giorgakis, E.; Khorsandi, S.E.; Jassem, W.; Heaton, N. Minimization of Ischemic Cholangiopathy in Donation after Cardiac Death Liver Transplantation: Is It Thrombolytic Therapy or Warm Ischemic Time Stringency and Donor Bile Duct Flush? Am. J. Transpl. 2018, 18, 274–275. [CrossRef] 28. Feng, S.; Goodrich, N.; Bragg-Gresham, J.; Dykstra, D.; Punch, J.; DebRoy, M.; Greenstein, S.; Merion, R. Characteristics Associated with Liver Graft Failure: The Concept of a Donor Risk Index. Am. J. Transplant. 2006, 6, 783–790, Erratum in 2018, 18, 3085. [CrossRef] 29. Dutkowski, P.; Oberkoﬂer, C.E.; Slankamenac, K.; Puhan, M.A.; Schadde, E.; Müllhaupt, B.; Geier, A.; Clavien, P.A. References Are there better guidelines for allocation in liver transplantation? A novel score targeting justice and utility in the model for end-stage liver disease era. Ann. Surg. 2011, 254, 745–753. [CrossRef] g , , [ ] 30. Rana, A.; Hardy, M.A.; Halazun, K.J.; Woodland, D.C.; Ratner, L.E.; Samstein, B.; Guarrera, J.V.; Brown, R.S.J.; Emond, J.C. Survival outcomes following liver transplantation (SOFT) score: A novel method to predict patient survival following liver transplantation. Am. J. Transpl. 2008, 8, 2537–2546. [CrossRef] p J p , , [ ] 31. Collett, D.; Friend, P.J.; Watson, C.J. Factors Associated With Short- and Long-term Liver Graft Survival in the United Kingdom: Development of a UK Donor Liver Index. Transplantation 2017, 101, 786–792. [CrossRef] [PubMed] 32. Hong, J.C.; Yersiz, H.; Kositamongkol, P.; Xia, V.W.; Kaldas, F.M.; Petrowsky, H.; Farmer, D.G.; Lipshutz, G.; Markovic, D.; Hiatt, J.R.; et al. Liver transplantation using organ donation after cardiac death: A clinical predictive index for graft failure-free survival. Arch. Surg. 2011, 146, 1017–1023. [CrossRef] [PubMed] g 33. Khorsandi, S.E.; Giorgakis, E.; Vilca-Melendez, H.; O’Grady, J.; Heneghan, M.; Aluvihare, V.; Suddle, A.; Agarwal, K.; Menon, K.; Prachalias, A.; et al. Developing a donation after cardiac death risk index for adult and pediatric liver transplantation. World J. Transpl. 2017, 7, 203–212. [CrossRef] [PubMed] p 34. Schlegel, A.; Kalisvaart, M.; Scalera, I.; Laing, R.W.; Mergental, H.; Mirza, D.F.; Perera, T.; Isaac, J.; Dutkowski, P.; Muiesan, P. The UK DCD Risk Score: A new proposal to deﬁne futility in donation-after-circulatory-death liver transplantation. J. Hepatol. 2018, 68, 456–464. [CrossRef] 35. Dutkowski, P.; Polak, W.G.; Muiesan, P.; Schlegel, A.; Verhoeven, C.J.; Scalera, I.; DeOliveira, M.L.; Kron, P.; Clavien, P.A. First comparison of hypothermic oxygenated perfusion versus static cold storage of human donation after cardiac death liver transplants: An international-matched case analysis. Ann. Surg. 2015, 262, 764–771. [CrossRef] [PubMed] p y g 36. Nasralla, D.; Coussios, C.C.; Mergental, H.; Akhtar, M.Z.; Butler, A.J.; Ceresa, C.D.L.; Chiocchia, V.; Dutton, S.J.; García-Valdecasas, J.C.; for the Consortium for Organ Preservation in Europe; et al. A randomized trial of normothermic preservation in liver transplantation. Nature 2018, 557, 50–56. [CrossRef] [PubMed] p 37. Mergental, H.; Laing, R.W.; Kirkham, A.J.; Perera, M.T.P.R.; Boteon, Y.L.; Attard, J.; Barton, D.; Curbishley, S.; Wilkhu, M.; Neil, D.A.H.; et al. Transplantation of discarded livers following viability testing with normothermic machine perfusion. Nat. Commun. 2020, 11, 2939. [CrossRef] 38. Schlegel, A.; Graf, R.; Clavien, P.A.; Dutkowski, P. References [CrossRef] p 49. Kron, P.; Schlegel, A.; Muller, X.; Gaspert, A.; Clavien, P.-A.; Dutkowski, P. Hypothermic Oxygenated Perfusion: A Simple and Effective Method to Modulate the Immune Response in Kidney Transplantation. Transplantation 2019, 103, e128–e136. [CrossRef] 50. Schlegel, A.; Kron, P.; Graf, R.; Clavien, P.A.; Dutkowski, P. Hypothermic Oxygenated Perfusion (HOPE) downregulates the immune response in a rat model of liver transplantation. Ann. Surg. 2014, 260, 931–937. [CrossRef] p p g 51. Watson, C.J.E.; Hunt, F.; Messer, S.; Currie, I.; Large, S.; Sutherland, A.; Crick, K.; Wigmore, S.J.; Fear, C.; Cornateanu, S.; et al. In situ normothermic perfusion of livers in controlled circulatory death donation may prevent ischemic cholangiopathy and improve graft survival. Am. J. Transplant. 2018, 19, 1745–1758. [CrossRef] p g J p 52. Hessheimer, A.J.; Coll, E.; Torres, F.; Ruíz, P.; Gastaca, M.; Rivas, J.I.; Gómez, M.; Sánchez, B.; Santoyo, J.; Ramírez, P.; et al. Normothermic regional perfusion vs. super-rapid recovery in controlled donation after circulatory death liver transplantation. J. Hepatol. 2018, 70, 658–665. [CrossRef] [PubMed] p 53. Muller, X.; Mohkam, K.; Mueller, M.; Schlegel, A.; Dondero, F.; Sepulveda, A.; Savier, E.; Scatton, O.; Bucur, P.; Salame, E.; et al. Hypothermic Oxygenated Perfusion versus Normothermic Regional Perfusion in Liver Transplantation From Controlled Donation After Circulatory Death: First International Comparative Study. Ann. Surg. 2020, 272, 751–758. [CrossRef] [PubMed] p 53. Muller, X.; Mohkam, K.; Mueller, M.; Schlegel, A.; Dondero, F.; Sepulveda, A.; Savier, E.; Scatton, O.; Bucur, P.; Salame, E.; et al. Hypothermic Oxygenated Perfusion versus Normothermic Regional Perfusion in Liver Transplantation From Controlled Donation 54. Tchana-Sato, V.; Ledoux, D.; Vandendriessche, K.; Van Cleemput, J.; Hans, G.; Ancion, A.; Cools, B.; Amabili, P.; Detry, O.; Massion, P.B.; et al. First report of a successful pediatric heart transplantation from donation after circulatory death with distant procurement using normothermic regional perfusion and cold storage. J. Heart Lung Transplant. 2019, 38, 1112–1115. [CrossRef] [PubMed] [ ] 55. Brockmann, J.G.; Vogel, T.; Coussios, C.; Friend, P.J. Liver splitting during normothermic organ preservation. Liver Transplant. 2017, 23, 701–706. [CrossRef] 56. Stephenson, B.T.F.; Bonney, G.K.; Laing, R.W.; Bhogal, R.H.; Marcon, F.; Neil, D.A.H.; Perera, M.T.P.R.; Afford, S.C.; Mergental, H.; Mirza, D.F. Proof of concept: Liver splitting during normothermic machine perfusion. J. Surg. Case Rep. 2018, 2018, rjx218. [CrossRef] 57. Spada, M.; Angelico, R.; Grimaldi, C.; Francalanci, P.; Safﬁoti, M.C.; Rigamonti, A.; Pariante, R.; Bianchi, R.; Vici, C.D.; Candusso, M.; et al. References Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation. J. Hepatol. 2013, 59, 984–991. [CrossRef] p p 39. Schlegel, A.; de Rougemont, O.; Graf, R.; Clavien, P.A.; Dutkowski, P. Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts. J. Hepatol. 2013, 58, 278–286. [CrossRef] 40. Dutkowski, P.; Schlegel, A.; De Oliveira, M.; Müllhaupt, B.; Neff, F.; Clavien, P.A. HOPE for human liver grafts obtained from donors after cardiac death. J. Hepatol. 2014, 60, 765–772. [CrossRef] p 41. Schlegel, A.; Muller, X.; Kalisvaart, M.; Muellhaupt, B.; Perera, M.T.P.; Isaac, J.R.; Clavien, P.A.; Muiesan, P.; Dutkowski, P. Outcomes of DCD liver transplantation using organs treated by hypothermic oxygenated perfusion before implantation. J. Hepatol. 2018, 70, 50–57. [CrossRef] [PubMed] p 42. Muller, X.; Schlegel, A.; Würdinger, M.; Wendt, M.; Kron, P.; Eshmuminov, D.; Müllhaupt, B.; Clavien, P.A.; Dutkowski, P. Can hypothermic oxygenated perfusion (HOPE) rescue futile DCD liver grafts? HPB 2019, 21, 1156–1165. [CrossRef] 43. van Rijn, R.; Schurink, I.; de Vries, Y.; van den Berg, A.; Cortes Cerisuelo, M.; Murad, S.D.; Erdmann, J.I.; Gilbo, N.; de Haas, R.J.; Heaton, N.; et al. Hypothermic Machine Perfusion in Liver Transplantation—A Randomized Trial. N. Engl. J. Med. 2021, 384, 1391–1401. [CrossRef] [PubMed] 44. Werner, M.J.M.; van Leeuwen, O.B.; de Jong, I.E.M.; Bodewes, F.A.J.A.; Fujiyoshi, M.; Luhker, O.C.; Scheenstra, R.; de Vries, Y.; de Kleine, R.H.J.; Porte, R.J. First report of successful transplantation of a pediatric donor liver graft after hypothermic machine perfusion. Pediatr. Transpl. 2019, 23, e13362. [CrossRef] [PubMed] p p 45. Panconesi, R.; Flores Carvalho, M.; Mueller, M.; Dutkowski, P.; Muiesan, P.; Schlegel, A. Mitochondrial Reprogramming—What Is the Beneﬁt of Hypothermic Oxygenated Perfusion in Liver Transplantation? Transplantology 2021, 2, 149–161. [CrossRef] 46. Schlegel, A.; Muller, X.; Mueller, M.; Stepanova, A.; Kron, P.; de Rougemont, O.; Muiesan, P.; Clavien, P.A.; Galkin, A.; Meierhofer, D.; et al. Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation. EBioMedicine 2020, 60, 103014. [CrossRef] 47. Boteon, Y.L.; Laing, R.W.; Schlegel, A.; Wallace, L.; Smith, A.; Attard, J.; Bhogal, R.H.; Neil, D.A.H.; Hübscher, S.; Perera, M.T.P.R.; et al. Combined Hypothermic and Normothermic Machine Perfusion Improves Functional Recovery of Extended Criteria Donor Livers. Liver Transplant. 2018, 24, 1699–1715. [CrossRef] 102 Transplantology 2022, 3 48. Kron, P.; Schlegel, A.; Mancina, L.; Clavien, P.A.; Dutkowski, P. Hypothermic oxygenated perfusion (HOPE) for fatty liver grafts in rats and humans. J. Hepatol. 2018, 68, 82–91. References The New Horizon of Split-Liver Transplantation: Ex Situ Liver Splitting during Hypothermic Oxygenated Machine Perfusion. Liver Transplant. 2020, 26, 1363–1367. [CrossRef] 58. Thorne, A.M.; Lantinga, V.; Bodewes, S.; de Kleine, R.H.J.; Nijkamp, M.W.; Sprakel, J.; Hartog, H.; Polak, W.G.; Porte, R.J.; de Meijer, V.E. Ex Situ Dual Hypothermic Oxygenated Machine Perfusion for Human Split Liver Transplantation. Transpl. Direct 2021, 4, e666. [CrossRef] 59. Attard, J.A.; Osei-Bordom, D.C.; Boteon, Y.; Wallace, L.; Ronca, V.; Reynolds, G.; Perera, M.T.P.R.; Oo, Y.H.; Mergental, H.; Mirza, D.F.; et al. Ex situ Normothermic Split Liver Machine Perfusion: Protocol for Robust Comparative Controls in Liver Function Assessment Suitable for Evaluation of Novel Therapeutic Interventions in the Pre-clinical Setting. Front. Surg. 2021, 8, 5. [CrossRef] 60. Schlegel, A.; Lesurtel, M.; Melloul, E.; Limani, P.; Tschuor, C.; Graf, R.; Humar, B.; Clavien, P.A. ALPPS: From human to mice highlighting accelerated and novel mechanisms of liver regeneration. Ann. Surg. 2014, 260, 839–847. [CrossRef] 61. Shi, H.; Yang, G.; Zheng, T.; Wang, J.; Li, L.; Liang, Y.; Xie, C.; Yin, D.; Sun, B.; Sun, J.; et al. A preliminary study of ALPPS procedure in a rat model. Sci. Rep. 2015, 5, 17567. [CrossRef] 62. Angelico, R.; Perera, M.T.P.R.; Manzia, T.M.; Parente, A.; Grimaldi, C.; Spada, M. Donation after Circulatory Death in Paediatric Liver Transplantation: Current Status and Future Perspectives in the Machine Perfusion Era. BioMed Res. Int. 2018, 2018, 1756069. [CrossRef] 63. Isnardi, D.; Olivero, F.; Lerda, R.; Guermani, A.; Cornara, G. Extracorporeal Membrane Oxygenation as a Bridge to Organ Donation: A Case Report. Transplant. Proc. 2013, 45, 2619–2620. [CrossRef] 64. Pérez-Villares, J.M.; Lara-Rosales, R.; Fernández-Carmona, A.; Fuentes-Garcia, P.; Burgos-Fuentes, M.; Baquedano-Fernández, B. Mobile ECMO team for controlled donation after circulatory death. Am. J. Transplant. 2018, 18, 1293–1294. [CrossRef] [PubMed] 65. Linecker, M.; Kambakamba, P.; Reiner, C.S.; Nguyen-Kim, T.D.L.; Stavrou, G.A.; Jenner, R.M.; Oldhafer, K.J.; Björnsson, B.; Schlegel, A.; Györi, G.; et al. How much liver needs to be transected in ALPPS? A translational study investigating the concept of less invasiveness. Surgery 2016, 161, 453–464. [CrossRef] [PubMed]
https://openalex.org/W2120747289	https://biomedical-engineering-online.biomedcentral.com/counter/pdf/10.1186/s12938-015-0088-3	English	null	On the design of a DEA-based device to pot entially assist lower leg disorders: an analytical and FEM investigation accounting for nonlinearities of the leg and device deformations	BioMedical engineering online	2,015	cc-by	9,991	© 2015 Pourazadi et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access On the design of a DEA‑based device to potentially assist lower leg disorders: an analytical and FEM investigation accounting for nonlinearities of the leg and device deformations Shahram Pourazadi, Sadegh Ahmadi and Carlo Menon* Shahram Pourazadi, Sadegh Ahmadi and Carlo Menon* *Correspondence: cmenon@sfu.ca MENRVA Group, School of Engineering Science, Faculty of Applied Science, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada On the design of a DEA‑based device to potentially assist lower leg disorders: an analytical and FEM investigation accounting for nonlinearities of the leg and device deformations Open Access RESEARCH Pourazadi et al. BioMed Eng OnLine (2015) 14:103 DOI 10.1186/s12938-015-0088-3 On the design of a DEA‑based device to potentially assist lower leg disorders: an analytical and FEM investigation accounting for nonlinearities of the leg and device deformations Open Access RESEARCH Pourazadi et al. BioMed Eng OnLine (2015) 14:103 DOI 10.1186/s12938-015-0088-3 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 DOI 10.1186/s12938-015-0088-3 Background Venous dysfunction in human lower extremities causes disorders that impact the life quality of considerable amount of individuals. Disorders such as orthostatic hypoten- sion, edema and deep vein thrombosis are closely tied to the venous system failure to pump the blood back to the heart. Typically certain body mechanisms including vein valve and muscle contraction help the blood return. Sometimes these mechanisms fail to operate properly and lead to decrease in blood flow and an increase in blood volume accumulated in the lower extremities. In particular, any malfunction in capillary filtra- tion causes abnormal balance between blood in capillaries and fluids in interstitial tis- sue. While standing, venous pressure in the lower leg increases which in turn results in an increase of oncotic pressure. This results in greater pressure gradient in the capil- laries and thus more filtration. An increase in filtration causes accumulation of more fluid in interstitial tissue which results in edema [1]. Also, due to a higher compliance of venous, veins inflate while standing and result in venous pooling. Venous pooling can cause orthostatic intolerance and blood clots. It can also lead to chronic varicose veins, in which the valve mechanism in veins does not operate properly and blood tends to accumulate upstream. In order to prevent and cure these disorders, different methods have been suggested [2]. The common way to treat venous pooling is to apply a compression to the lower extremities [3]. Compression to the lower part of the leg mimics the natural muscle contraction and helps blood return. It also increases the pressure of interstitial fluid and decreases the superficial venous pressure. Thus, the leakage of fluids to intersti- tial space is reduced [4]. In this regard, compression stocking (CS) is one of the recom- mended devices that apply mechanical compressions to the lower leg area. CS reduces venous hypertension and helps the leg muscle pump, which improves filtration, prevents skin breakdown, and eventually results in reduction of edema and venous pooling [5, 6]. Another mechanical device that prevents these disorders is intermittent pneumatic compression (IPC). IPC increases blood flow and improves blood return by simulating normal leg muscle activity [7]. The efficacy of CSs has been questioned [8, 9] and IPCs are typically bulky and therefore not suitable for ambulatory use. Dielectric elastomer actuators (DEAs) are introduced as active compression bandages (ACBs) to address the shortcomings of the previous devices [10]. Abstract The paper presents the actual pressure in the different regions of the calf for different values of the ACB’s stretch ratio when it is either electrically activated or Page 2 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 not activated, and when compliancy of the leg is either considered or not considered. For example, results of the performed simulation show that about 10 % variation in compression in the ankle region is expected when the ACB initially applies 6 kPa and the compressibility of the calf is first considered and then not considered. Such a varia- tion reduces to 5 % when the initial pressure applied by the ACB reduced by half. Conclusions: Comparison with non-linear FEM simulations show that the analytical models used in this work can closely estimate interaction between an active com- pression bandage and a human calf. In addition, compliancy of the leg should not be neglected when either designing a compression band or predicting the compressive force it can exert. The methodology proposed in this work can be extended to other types of elastic compression bandages and garments for biomedical applications. Keywords: Dielectric elastomer actuator, Active compression bandage, Lower leg disorders Calf compliance Finite element modeling Keywords: Dielectric elastomer actuator, Active compression bandage, Lower leg disorders, Calf compliance, Finite element modeling Abstract Background: One of the recommended treatments for disorders associated with the lower extremity venous insufficiency is the application of external mechanical com- pression. Compression stockings and elastic bandages are widely used for the purpose of compression therapy and are usually designed to exert a specified value or range of compression on the leg. However, the leg deforms under external compression, which can lead to undesirable variations in the amount of compression applied by the compression bandages. In this paper, the use of an active compression bandage (ACB), whose compression can be regulated through an electrical signal, is investigated. The ACB is based on the use of dielectric elastomer actuators. This paper specifically inves- tigates, via both analytical and non-linear numerical simulations, the potential pressure the ACB can apply when the compliancy of the human leg is taken into account. The work underpins the need to account for the compressibility of the leg when designing compression garments for lower extremity venous insufficiency. Methods: A mathematical model is used to simulate the volumetric change of a calf when compressed. Suitable parameters for this calf model are selected from the litera- ture where the calf, from ankle to knee, is divided into six different regions. An analyti- cal electromechanical model of the ACB, which considers its compliancy as a function of its pre-stretch and electricity applied, is used to predict the ACB’s behavior. Based on these calf and ACB analytical models, a simulation is performed to investigate the inter- action between the ACB and the human calf with and without an electrical stimulus applied to the ACB. This simulation is validated by non-linear analysis performed using a software based on the finite element method (FEM). In all simulations, the ACB’s elas- tomer is stretched to a value in the range between 140 and 220 % of its initial length. Results: Using data from the literature, the human calf model, which is examined n this work, has different compliancy in its different regions. For example, when a 28.5 mmHg (3.8 kPa) of external compression is applied to the entire calf, the ankle shows a 3.7 % of volume change whereas the knee region undergoes a 2.7 % of vol- ume change. Background In particular, DEAs can apply a variable Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 3 of 18 (time-dependent) mechanical compression using an input electrical stimulus. Variable compression was shown to be more efficient than a static compression in different stud- ies [11, 12]. Moreover, DEAs are lightweight, portable and very fast with experimental time responses as short as 2 ms [13]. DEAs are a category of electro-active polymers (EAPs) which have drawn much atten- tion due to their simplicity, stability, fast response, high stretch ratio, high applicable force, and low cost [14]. Numerous studies are presented in literature that discuss the DEA applications and characterize them in various shapes and configuration such as roll [14], planar [15], cylindrical [16] and spherical [17, 18] actuators. A simple DEA unit consists of a dielectric elastomer that is sandwiched between a pair of compliant electrodes. Once voltage bias is applied to the DEA electrodes, the dielectric elastomer expands as a result of electrostatic Maxwell pressure across the dielectric. Considerable actuation strains and energy densities are achieved with DEAs which makes them inter- esting to be used for development of new emerging technologies [14]. The quantity of external compression is an important factor in treating the previously mentioned disorders on the lower extremities. For instance, it was shown that an exter- nal compression of 35–40 mmHg was required at the ankle to prevent edema in patients with severe venous disease [19]. Also [20] suggested that in order to optimize the venous flow in the lower leg, a graded compression of 18 mmHg at the ankle, 14 mmHg at mid- calf, and 8 mmHg at the knee is required. Thus, the bandages used for compression ther- apy should be designed to apply the proper range of compression. Typically, the amount of pressure exerted by elastic bandages is a function of the calf geometry. In practice, the calf is compressible and its volume and geometry change upon compression. Thus, the amount of compression that elastic bandages exert differs depending on the volume and shape of the calf. In this regard, Stenger et al. suggested that volumetric changes of the leg should be considered prior to the design of elastic bandages [3].Therefore, it is interesting to study and identify how profound the calf compressibility would affect the behavior of the DEA bandage. Background In this paper, an analytical study and a numerical simulation based on the finite ele- ment method (FEM) are proposed to simulate the interaction between the ACB and the calf. The results of this study assist predicting how calf compressibility affects perfor- mance of an ACB and the use of this technology in future clinical studies. Mechanical properties of the calf The human calf compresses upon application of pressure. The geometry and size of the calf change considerably with the application of an external compression. The relation- ship between changes in volume to changes in external pressure is often determined by its compliance (C): (1) C = δV δP C = δV δP (1) The compliance is a nonlinear function of external pressure and different regions of the calf show different values. There are several works in the literature that studied and measured the calf compliance with different methodologies [21–28]. The common clini- cal method of calf compliance measurement is to apply external pressure proximal to the The compliance is a nonlinear function of external pressure and different regions of the calf show different values. There are several works in the literature that studied and measured the calf compliance with different methodologies [21–28]. The common clini- cal method of calf compliance measurement is to apply external pressure proximal to the Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 4 of 18 knee with congestion cuffs and then monitor the volume changes in the calf area using plethysmography [21–25]. In this method it is assumed that the volume change in the limb is equivalent to volume change of the underlying venous vessel. This assumption however, is argued to be not very accurate [28]. Moreover, this method is believed to not precisely predict the compliance of the calf as the external pressure is applied proxi- mal to the knee and not to the calf itself. A more sophisticated method was however used to accurately measure the calf compliance by monitoring cross sectional changes of the calf under application of an external pressure to the calf area [27, 28]. Specifically Thirsk et al. [28] proposed a procedure that removed possible artifacts, such as involun- tary muscle contractions, from the measurements to obtain accurate measurements. In that work, compliance was measured in six regions of the leg, from the ankle to the knee (labeled as region 6 to 1 from now onward as it is shown in Fig. 1a, in 3 human subjects. The calf compliance was presented in the plots of calf cross sectional area change versus external pressure change. In this work, the compliance data acquired by Thirsk et al. [28] was digitalized from their published report. Analytical modeling of the DEA on a simulated human calf In this section, an analytical model of the cylindrical DEA [10, 16] is combined to the model of the human calf described by (2). The total pressure applied by the ACB has two components, the mechanical pressure which is the pressure resulting from the mechani- cal stress in the ACB before actuating the ACB, and the actuation pressure, which is the pressure variation after actuating the ACB. The ACB is assumed to be made out of a flat DEA with length L [11]. This flat DEA can be bent to form a cylinder, whose external radius is R = L 2π. The ACB, consisting of this DEA cylinder, is stretched radially as it is wrapped around the calf and the following stretch ratio λ can be used: (3) = r R = r R (3) (3) where r is the deformed radius of the ACB when it conforms the calf. Since the geom- etry of calf is conical, the ACB undergoes different stretch ratios along the height of the calf. In the analytical modeling of this work, it was assumed that the conical geometry of each calf region was consisted of small cylindrical geometries with finite height. The simulation procedure, which is explained in this section, is done for each of these small cylindrical geometries and the final values of radius and pressure was averaged over the entire calf region. As the stretch ratio of the ACB changes the amount of compression that is exerted on the calf is also changed. The amount of this compression is obtained using the following equation [16]. (4) Pm = a b −µ1 α1 −−α1 + µ2(α2 −−α2) (2 −1) d (4) where λa and λb are stretch ratios of inner and outer radii of the cylindrical DEA respec- tively, and μ1, μ2, α1, α2 are the ogden parameters for the DEA material. As this com- pression from the ACB is applied, the calf radius changes. Assuming Rc as the radius of the undeformed calf (as shown in Fig. 2), λc is defined as the calf stretch ratio: where λa and λb are stretch ratios of inner and outer radii of the cylindrical DEA respec- tively, and μ1, μ2, α1, α2 are the ogden parameters for the DEA material. As this com- pression from the ACB is applied, the calf radius changes. Mechanical properties of the calf The values of percentage change in the cross section area of the calf were divided by 2 to obtain the radial strain. This operation is considered to be a valid conversion assuming a uniform geometrical shape of the calf cross section [29]. True stress–strain data points were calculated from the pressure-radius change data set for each of the six regions of the calf. A curve fitting was then used to fit a best func- tion for the relation between the true radial stress (P) in “Pa” on the calf and the true radial strain (εr). The stress–strain curves for all calf regions are depicted in Fig. 1b. For all regions the best fitted curves were found to be an exponential function of the form presented in Eq. (2). The curves in Fig. 1b are continuation of curves that yield 0 strain at 0 pressure, thus a single exponential term cannot accurately follow this behavior and a second term was added in Eq. (2) to address this issue. The corresponding constant parameters are provided in Table 1 (2) εr = a · eb·P + c · ed·P. εr = a · eb·P + c · ed·P. (2) Fig. 1 Human calf. a Conical geometry of the calf and its six different regions. Each region is assumed to be 35 mm apart in height [28]. b Fitted curves to calf compliance data obtained from [28] at the calf’s different regions Fig. 1 Human calf. a Conical geometry of the calf and its six different regions. Each region is assumed to be 35 mm apart in height [28]. b Fitted curves to calf compliance data obtained from [28] at the calf’s different regions Page 5 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Table 1 Constant parameters of the calf compliance relation expressed in Eq. 1 Region a b c d 6 0.013392 7.898E−05 −0.01600 −0.001137 5 0.009989 4.967E−05 −0.01465 −0.001386 4 0.008678 7.532E−05 −0.00913 −0.000828 3 0.01373 3.492E−05 −0.01686 −0.001133 2 0.01188 5.511E−05 −0.01466 −0.001392 1 0.012223 2.731E−05 −0.01513 −0.001263 Table 1 Constant parameters of the calf compliance relation expressed in Eq. 1 Analytical modeling of the DEA on a simulated human calf Assuming Rc as the radius of the undeformed calf (as shown in Fig. 2), λc is defined as the calf stretch ratio: (5) c = r Rc = R Rc (6) c = 1 −εr. (5) c = r Rc = R Rc c = r Rc = R Rc (5) (6) c = 1 −εr. c = 1 −εr. (6) Page 6 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Substituting Eq. (2) in Eq. (6) and using Eq. (5), the calf radius after application of Pm can be obtained: Substituting Eq. (2) in Eq. (6) and using Eq. (5), the calf radius after application of Pm can be obtained: (7) r = Rc −Rc(aebPm + cedPm). r = Rc −Rc(aebPm + cedPm). (7) In order to find the equilibrium state for r and Pm, the set of coupled Eqs. (4) and (7) should be solved. However, this set of equations is nonlinear and neither r nor Pm can be written in terms of each other explicitly and a closed form analytical solution cannot be obtained. Hence, an iterative procedure is used to solve Eqs. (4) and (7) for the final calf radius r and external pressure Pm applied by ACB. In the first iteration of this procedure, the calf is undeformed and the ACB is stretched to Rc. Using Eq. (4) a compression Pm is obtained. Then using Eq. (7) a new calf radius r is calculated. In the next iteration the Pm is again obtained using the new calf radius and consequently a newer calf radius is calculated. These iterations are continued until the value of r changes less than 0.01 % of the initial radius Rc. Once the bandage is wrapped and an equilibrium radius is reached for the calf and the ACB, the actuation phase is modeled and analyzed. The actuation pressure is calculated from the following equation [16]: (8) Pa = ε0εrV 2 2ln2(b/a) b2 −a2 a2b2 . (8) As discussed earlier the total ACB pressure on the calf is composed of Pa and Pm and is given by: As discussed earlier the total ACB pressure on the calf is composed of Pa and Pm and is given by: (9) Pt = Pm −Pa Pt = Pm −Pa (9) As it can be realized for Eq. (9), the ACB total pressure is reduced upon actuation. Analytical modeling of the DEA on a simulated human calf Thus, the radius of the calf is increased which in turn results in an increase of the ACB mechanical pressure. So we need to solve Eqs. (7) and (9) in order to find the final equi- librium state of the calf radius r and ACB total pressure Pt. Again, this set of equation is Unstretched DEA R r RC Stretched DEA on deformable calf Undeformed calf cross section Compressed and deformed calf cross section Fig. 2 Changes of the ACB radius on compressible human calf r RC Stretched DEA on deformable calf Undeformed calf cross section section Compressed and deformed calf cross section Stretched DEA on deformable calf Unstretched DEA Fig. 2 Changes of the ACB radius on compressible human calf Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 7 of 18 solved similar to the previously mentioned iterative procedure. The steps of this iterative procedure are summarized in Fig. 3. The developed analytical model gives us the ability to predict the behavior of the ACB on the flexible calf. Moreover, it provides us with a tool to design the ACB parameters such that desired compressions on the lower leg can be achievable. Simulations by the analytical model were carried out for the six regions of the calf and the results are dis- cussed in the following sections. FEM analysis Finite element modeling showed promising results for cylindrical and conical DEA geometries in the literature [10, 16]. Pourazadi et al. [10] used FEM to analyze the behav- ior of the ACB on a lower human leg geometry. In that work, the calf was assumed to be incompressible and the volume change effects in the lower leg region were neglected. However, the calf volume changes when external pressure is applied. To study the behav- ior of ACB on the compressible human calf, ANSYS software (Ansys Mechanical APDL 2011 ANSYS Inc V14.0) was used. The ACB was originally modeled as a cylinder (as shown in Fig. 4) and a 2nd-order Ogden model was used to approximate its hyperelastic behavior. Ogden parameters from [10] were used. A truncated cone with specific dimensions (as shown in Fig. 4) was modeled for each calf region. A total of six regions were studied. The cone height for all regions was 35 mm. Every calf region had a different material property and required dif- ferent nonlinear model parameters. A Hyper-elastic Response Function model was used to model the calf material in each region. In this model, the material’s compressibility is specified by d1 parameter—which is the inverse value of the bulk modulus. Further Fig. 3 Flowchart of the analytical modeling of the ACB on the flexible calf Fig. 3 Flowchart of the analytical modeling of the ACB on the flexible calf Page 8 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Fig. 4 Geometry of the calf and ACB in FEM simulations details of this material model, which is a built-in model in Ansys, can be found in Ansys documentation [30]. After creating the ACB and calf regions in the model, a fine FEM mesh was needed to be generated to obtain high-accuracy results. By selecting a finer mesh, although the accuracy of results was improved, the simulation time was rapidly increased as well. Therefore, an optimum mesh size was needed to be determined to achieve accurate results within a reasonable simulation time. As there was rotational symmetry for both the ACB and the calf geometries, only 1/4th of the geometries were modeled (as seen in Fig. 4). For the ACB, the geometry was meshed into 3 segments along the thickness, 15 segments along the circumference, and 20 segments along the height of the 1/4th cylin- der. FEM analysis The calf geometry was also meshed into 18 × 18 × 20 segments respectively along its radial, circumferential and axial directions. Also, a total of 300 contact elements and 360 target elements were used in the model. To include the electro-active properties of the ACB in simulations, Solid226 (a higher order 3-D 20-node solid element with elec- trostatic material option) was selected. Also, Solid186 (a higher order 3-D 20-node solid element) was chosen for the calf geometry. For the contact areas of the ACB Conta174 (a 3-D 8-node surface-to-surface contact element) and for the calf’s contact area Targe170 (a 3-D target element) was used. Moreover, a frictionless contact was assumed between the solid geometries. In reality, an ACB bandage is wrapped around the calf and then is actuated. To avoid modeling a complicated contact problem and to reduce the calculation time, an alterna- tive procedure which gave identical results was considered. The simulation steps were: (1) the sides of the ACB were fixed (to keep the height constant at 35 mm) and the ACB was stretched radially to the size of the largest radius of the calf region; (2) the stretched ACB was then displaced to perfectly locate on the calf region; (3) the radial constraint was removed to release the ACB and make contact with the calf region. The sides of both the ACB and calf segment were kept fixed and the contact pressure and volume changes were reported as outputs. The ratio of the average calf radius (mean of smaller and larger Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 9 of 18 calf radii) to the initial ABC’s inner radius was defined as the stretch ratio (λ) which was chosen to vary from 1.4 to 2.2 in this study; (4) the ACB was actuated by applying an electrical potential difference of 11.3 kV between the top and bottom surfaces of the ACB. The pressure difference and volume changes were the outputs of this step. Simula- tion steps are shown in Fig. 5. In the FEM setup explained above, there were 900 ACB elements, 6480 calf elements, and 660 contact elements. The total computation time for an ACB sample in contact with the calf region 6 varied from 5.5 to 9.5 h for stretch ratios of 1.4 and 2.2, respec- tively. FEM analysis It was found that the most time-consuming step is the 3rd step as the ACB makes initial contact with the calf region. A desktop PC with AMD FX™-8350 eight-core (4.7 GHz) processor and 16 GB of RAM was used. All CPU cores were fully utilized using ANSYS High Performance Computing (HPC) feature. To study the effect of mesh size on the accuracy of results, a second run was performed by selecting 4 × 20 × 25 = 2000 ACB elements, 20 × 20 × 25 = 10,000 calf elements, 500 contact elements, and 500 target elements. If the ACB sample was deformed to a stretch ratio of 2.2 the simula- tion finished after 42.5 h and the mechanical pressure after a full contact in step 3 was 6091.2 Pa. However, the coarser mesh size that was selected originally gave a nearly sim- ilar mechanical pressure, 6102.8 Pa. The relative error for such large pressures was less Fig. 5 Deformation plots showing the position of the ACB and the calf region at the end of each FEM load step. a ACB is radially stretched to the maximum radius of the calf region; b ACB is positioned at the top of the calf region; c ACB is released to make contact with the calf region. Deformation plot after electrically actu- ating the ACB is similar to the plot c since the deformations after electrical actuation of the ACB is negligible. At each load step, wireframe lines show the geometries before deformation starts and solid shapes show the deformed geometries Fig. 5 Deformation plots showing the position of the ACB and the calf region at the end of each FEM load step. a ACB is radially stretched to the maximum radius of the calf region; b ACB is positioned at the top of the calf region; c ACB is released to make contact with the calf region. Deformation plot after electrically actu- ating the ACB is similar to the plot c since the deformations after electrical actuation of the ACB is negligible. At each load step, wireframe lines show the geometries before deformation starts and solid shapes show the deformed geometries Fig. 5 Deformation plots showing the position of the ACB and the calf region at the end of each FEM load step. Results and discussion Different sizes of ACBs were investigated to study the effect of the initial length of the ACB and also cover a wide range of compressions on the calf. An ACB thickness of 1 mm with a dielectric constant of 7.41 was used for the simulations. Ogden parameters similar to [10] were used to simulate the hyperelastic behavior of the elastomer. Also, the ACB was actuated with 11.3 kV DC voltage for all simulations. The initial lengths of the bandage were determined in order for the ACB to undergo a certain range of stretch ratio (i.e. 1.4–2.2) considering the non-deformed shape of the calf. However, as discussed earlier, the calf deforms under compression and thus the actual stretch ratio differs from the initial desired stretch ratio. In this work, the results are presented in terms of the initial desired stretch ratio for better consistency. Table 2 shows the values of the ACB lengths for the different calf regions and initial stretch ratios. The simulation results of the analytical and FEM modeling that were explained in “Analytical modeling of the DEA on a simulated human calf” and “FEM analysis” are fol- lowed in this section. Figure 6 shows the final volume of the different calf regions for dif- ferent initial stretch ratios of the ACB after the ACB was wrapped and actuated. In this figure, the analytical and FEM results are represented with solid lines and circle mark- ers, respectively. The maximum relative difference between the analytical and the FEM simulations is 2.4 % which is happening at region 6 for λ = 2.2. The detailed list of the volumes is listed in Additional file 1. i Figure 7 shows the relative change in the volume of the different calf regions (Ev), defined by Eq. (10) versus different initial stretch ratios of the ACB. (10) Ev = V0 −VA V0 × 100 (10) where V0 is the initial calf volume and VA is the calf volume after wrapping and actuation of the ACB that is given in Fig. 6. V0 is also plotted on the secondary axis of Fig. 7. The analytical simulations in Fig. 7 suggest that after wrapping and actuating the ACB the calf underwent a maximum of approximately 4.4 % volume change at the ankle region. FEM analysis a ACB is radially stretched to the maximum radius of the calf region; b ACB is positioned at the top of the calf region; c ACB is released to make contact with the calf region. Deformation plot after electrically actu- ating the ACB is similar to the plot c since the deformations after electrical actuation of the ACB is negligible. At each load step, wireframe lines show the geometries before deformation starts and solid shapes show the deformed geometries Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 10 of 18 than 0.19 % with coarser mesh size but the calculation time was about 4.5 times shorter. Therefore, the coarser mesh size was chosen and simulations were performed for five different stretch ratios (range of 1.4–2.2 with 0.2 increments) and for all six calf regions. Results are discussed in the next section. Results and discussion where V0 is the initial calf volume and VA is the calf volume after wrapping and actuation of the ACB that is given in Fig. 6. V0 is also plotted on the secondary axis of Fig. 7. The analytical simulations in Fig. 7 suggest that after wrapping and actuating the ACB the calf underwent a maximum of approximately 4.4 % volume change at the ankle region. Table 2 Initial ACB lengths in centimetre Calf region \Stretch ratio 1.4 1.6 1.8 2 2.2 1 28.2 24.6 21.9 19.7 17.9 2 27.5 24.1 21.4 19.2 17.5 3 26.0 22.7 20.2 18.2 16.5 4 23.7 20.7 18.4 16.6 15.1 5 20.9 18.3 16.3 14.7 13.3 6 17.8 15.6 13.8 12.4 11.3 Table 2 Initial ACB lengths in centimetre Table 2 Initial ACB lengths in centimetre Page 11 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Fig. 6 Analytical (solid lines) and FEM (circle markers) results for volume variations in various regions of the calf and different stretch ratios Fig. 7 Relative change in the calf volume after wrapping and actuating the ACB. Each color represents a stretch ratio. The secondary axis on the right hand side shows the calf region initial volume indicated by the solid line with circle markers Fig. 6 Analytical (solid lines) and FEM (circle markers) results for volume variations in various regions of the calf and different stretch ratios Fig. 6 Analytical (solid lines) and FEM (circle markers) results for volume variations in various regions of the calf and different stretch ratios Fig. 7 Relative change in the calf volume after wrapping and actuating the ACB. Each color represents a stretch ratio. The secondary axis on the right hand side shows the calf region initial volume indicated by the solid line with circle markers Fig. 7 Relative change in the calf volume after wrapping and actuating the ACB. Each color represents a stretch ratio. The secondary axis on the right hand side shows the calf region initial volume indicated by the solid line with circle markers For a certain stretch ratio, the maximum relative volume change took place in region 6 while the minimum was in region 4. The effect of volume changes on the performance of the DEA bandage is shown in Figs. 8, 9, 10, 11 and 12. Results and discussion Figure 8 shows the difference between the FEM and the analytical simulations for the total pressure on regions 1 and 6 for the two cases of considering a volume change (VC) and no volume change (NVC). The maximum difference between results from the analytical and FEM methods is 2.9 % at region 6 at λ = 1.4. The FEM simulation results shown in Figs. 6 and 8 verifies that the analytical model can closely estimate the ACB behavior on the human calf. A table of total pressure results is included in Additional file 2 for further reference. i Figure 9 shows the analytical results for the total pressure in all calf regions and illus- trates the effect of considering volume change (VC) on the total pressure of the ACB. In this figure, each column group represents a specific initial stretch ratio for all sec- tions of the calf. For each column, the bottom stack represents the total pressure after Page 12 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 0 20 40 60 80 100 120 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 Difference between analytical and FEM results (Pa) Pt (kPa) λ Region6-VC Region6-NVC Region1-VC Region1-NVC Error_VC6 Error-NVC6 Error-VC1 Error-NVC1 Fig. 8 FEM (circle markers) and analytical (solid lines) results of the total pressure for regions 1 and 6 Fig. 9 The ACB total pressure in all calf regions for different stretch ratios 0 20 40 60 80 100 120 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 Difference between analytical and FEM results (Pa) Pt (kPa) λ Region6-VC Region6-NVC Region1-VC Region1-NVC Error_VC6 Error-NVC6 Error-VC1 Error-NVC1 Fig. 8 FEM (circle markers) and analytical (solid lines) results of the total pressure for regions 1 and 6 Fig. 9 The ACB total pressure in all calf regions for different stretch ratios considering VC in the calf and the upper stack is the difference between considering VC and no volume change (NVC) in the calf. Thus, the combined height of each column represents the total pressure for the NVC case. Figure 9 shows that for a specific stretch ratio, the total pressure increases as we move from the knee to the ankle. It also shows that for a specific calf region the total pressure increases as the stretch ratio increases. Results and discussion However, the change in total pressure between the two cases of VC and NVC (shown by the second stack in each column/yellow color) is not uniformly decreasing or increas- ing and is a function of the compliance of the calf region. This change in total pressure follows the same pattern as the relative volume change in Fig. 7. Since region 4 has the lowest compliance (as shown in Fig. 1b), the change in total pressure is minimal for this region. These changes in the total pressure are also illustrated in Fig. 10. For a con- stant stretch ratio the change in total pressure is the lowest for region 4. The maximum considering VC in the calf and the upper stack is the difference between considering VC and no volume change (NVC) in the calf. Thus, the combined height of each column represents the total pressure for the NVC case. Figure 9 shows that for a specific stretch ratio, the total pressure increases as we move from the knee to the ankle. It also shows that for a specific calf region the total pressure increases as the stretch ratio increases. However, the change in total pressure between the two cases of VC and NVC (shown by the second stack in each column/yellow color) is not uniformly decreasing or increas- ing and is a function of the compliance of the calf region. This change in total pressure follows the same pattern as the relative volume change in Fig. 7. Since region 4 has the lowest compliance (as shown in Fig. 1b), the change in total pressure is minimal for this region. These changes in the total pressure are also illustrated in Fig. 10. For a con- stant stretch ratio the change in total pressure is the lowest for region 4. The maximum Page 13 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Fig. 10 Percentage of change in the ACB total pressure between the two cases of VC and NVC Fig. 10 Percentage of change in the ACB total pressure between the two cases of VC and NVC change in total pressure corresponds to the ankle (region 6) and a 2.2 stretch ratio with 9.7 % change. Results and discussion This results implies that if the volume change in the ankle is considered, a total pressure of 5.82 kPa (43.6 mmHg) is achieved instead of 6.45 kPa (48.4 mmHg) which corresponds to a 630 Pa (4.7 mmHg) reduction in the total pressure. After region 6, region 3 has the highest change in total pressure with 7.1 % of change. Fig. 11 Difference between actual and expected values in mechanical pressure as a result of calf compress- ibility Fig. 12 ACB actuation pressure for different calf regions Fig. 11 Difference between actual and expected values in mechanical pressure as a result of calf compress- ibility Fig. 11 Difference between actual and expected values in mechanical pressure as a result of calf compress- ibility change in total pressure corresponds to the ankle (region 6) and a 2.2 stretch ratio with 9.7 % change. This results implies that if the volume change in the ankle is considered, a total pressure of 5.82 kPa (43.6 mmHg) is achieved instead of 6.45 kPa (48.4 mmHg) which corresponds to a 630 Pa (4.7 mmHg) reduction in the total pressure. After region 6, region 3 has the highest change in total pressure with 7.1 % of change. Fig. 12 ACB actuation pressure for different calf regions Fig. 12 ACB actuation pressure for different calf regions change in total pressure corresponds to the ankle (region 6) and a 2.2 stretch ratio with 9.7 % change. This results implies that if the volume change in the ankle is considered, a total pressure of 5.82 kPa (43.6 mmHg) is achieved instead of 6.45 kPa (48.4 mmHg) which corresponds to a 630 Pa (4.7 mmHg) reduction in the total pressure. After region 6 region 3 has the highest change in total pressure with 7 1 % of change Fig. 12 ACB actuation pressure for different calf regions change in total pressure corresponds to the ankle (region 6) and a 2.2 stretch ratio with 9.7 % change. This results implies that if the volume change in the ankle is considered, a total pressure of 5.82 kPa (43.6 mmHg) is achieved instead of 6.45 kPa (48.4 mmHg) which corresponds to a 630 Pa (4.7 mmHg) reduction in the total pressure. After region 6, region 3 has the highest change in total pressure with 7.1 % of change. Results and discussion change in total pressure corresponds to the ankle (region 6) and a 2.2 stretch ratio with 9.7 % change. This results implies that if the volume change in the ankle is considered, a total pressure of 5.82 kPa (43.6 mmHg) is achieved instead of 6.45 kPa (48.4 mmHg) which corresponds to a 630 Pa (4.7 mmHg) reduction in the total pressure. After region 6, region 3 has the highest change in total pressure with 7.1 % of change. Page 14 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 As mentioned earlier, the amount of pressure that the ACB exerts before electri- cal activation is denoted by mechanical pressure. The mechanical pressure of the ACB increases as the stretch ratio of the ACB increases. Also this mechanical pressure gen- erally increases as the radius of the calf reduces [10]. Thus for a specific stretch ratio the amount of mechanical pressure is higher at the ankle section compared to other regions of the calf. One of the important rationales for this study is demonstrated in Fig. 11. Often the mechanical bandages are designed to provide a desired mechanical pressure. However, in practice, the amount of mechanical pressure changes due to the compressibility of the calf. In this case, the ACB can be stretched more to apply a higher amount of pressure and counteract the effect of volume change. Figure 11 shows the percent of change in actual mechanical pressure for a range of desired mechanical pres- sures. For example if the bandage is designed to provide a desired mechanical pressure of 4.90 kPa (36.8 mmHg) on the ankle, the actual mechanical pressure would be reduced by 6.7 % which is equivalent to 330 Pa (2.5 mmHg), making the actual mechanical pres- sure 4.57 kPa (34.3 mmHg). Variation of the actual compression from the desired com- pression by elastic bandages is an important fact that can lead to improper treatment of lower leg disorders [3]. Thus, it is proposed that the design mechanical pressure be increased by the percentage suggested in Fig. 11 to compensate for compression varia- tions due to the changes in the calf volume. In Fig. 12 the actuation pressure for the ACB, which is the pressure drop of the ACB once activated with 11.3 kV voltage, is shown for different sections of the calf. Results and discussion A volt- age of 11.3 kV was used in order to conduct a study consistent with [10]. As it was also discussed in [10], the amount of activation pressure increased as the radius of the calf was reduced. As shown in Fig. 12, the actuation pressure changed almost linearly with respect to changes of stretch ratio. The maximum actuation pressure was 287.7 Pa (2.2 mmHg) which corresponded to the ankle region. As mentioned earlier, applying a graded compression that reduces from ankle to knee is one of the recommended treatments for disorders associated with lower leg blood pooling [3]. One way to generate a compression that gradually reduces from ankle to knee is by making independent ACB short modules with different sizes for different calf sections. Each ACB module covers a single region of the calf and can be stretched inde- pendently, allowing for various compression profiles along the calf. Figure 9 provides the required stretch ratios that each ACB at each calf section should have in order to apply a desired compression profile. In this modular design, each single module could be actu- ated individually in order to generate an arbitrary variable compression such as peristal- tic compression. This modular design, although challenging, provides a high degree of freedom to cover a wide range of dynamic compression profiles. Future improvements and limitations This work investigated a recently proposed technology that potentially addresses some of the disadvantages of current compression therapy devices for the lower leg disorders. It should be noted that the investigated technology is still in its early stage of develop- ment, which justifies this study aimed at predicting the ACB behavior when a compress- ible human calf is considered. Page 15 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 The ACB faces some challenges and limitations that would need to be addressed in future studies. Specifically, DEAs are generally actuated with high actuation, which may pose limitations for use in wearable devices. The use of a stack of thin actuators could be used to address this challenge, as voltage could drastically be reduced. The use of a source not able to provide sufficient current to cause possible harm could also be considered. Another aspect that should be considered is the prolonged use of ACB in contact with the human skin. Future studies should investigate the use of a layer of anti-allergic, breathable fabric laid at the interface between ACB and skin, which wicks away the skin sweat and moisture. Such fabrics are currently used in available compression garments [31]. The use of alternative materials for the ACB should also be studied.f Future studies should also consider the effect of the stress relaxation behavior of DEAs and its implications in the use of active compression garments, such as the one investi- gated in this work. It should in fact be noted that the stress relaxation in the ACB’s elas- tomer may potentially present a considerable reduction in the mechanical compression of the ACB over time. The amount of relaxation depends on the material of the elasto- mer and is a function of time, temperature and stress/strain level. Typically, the level of relaxation is very large and considerable in acrylic elastomers, however it is small par- ticularly in silicone elastomers [14]. Typical acrylic elastomers show up to 500 % stress reduction after settling time of about half an hour for stretch ratios as low as 1.5 [32] while silicone elastomers can demonstrate less than 4 % stress reduction after settling time of 2 h for stretch ratios up to 2 [33]. Thus, the level of stress relaxation must be determined based on the material and the level of maximum stretch ratio used when wearing the ACB. Future improvements and limitations Materials with low or negligible stress relaxation should be used in the fabrication of the ACB to lessen the reduction of mechanical compression on the limb due to the relaxation. For the prototype investigated in this work, Fig. 10 shows that the ACB total pressure would reduce of around 4 % for a stretch ratio of 2 due to the sole calf compressibil- ity. This amount of reduction in mechanical compression, as a result of the calf com- pressibility, would be comparable to the 4 % reduction in compression due to the stress relaxation [33]. Thus, it can be concluded that the effect of stress relaxation may consid- erably affect the performance of the ACB. While future investigations should address this aspect by investigating novel materials, simple pragmatic solutions could also be implemented. For instance, the ACB could be stretched more than needed when initially donned in order to compensate for the subsequent relaxation of the material. Alterna- tively, the ACB could be embedded with a suitable tightening mechanism, such as the Boa closure system [34]—this solution would enable the user to tighten the ACB when needed to compensate for the compression reduction due to the relaxation. Conclusion In this paper the behavior of the active compression bandage (ACB) made out of dielec- tric elastomer actuator (DEA) was studied on a simulated human leg. ACBs could poten- tially be used in the treatment of different types of disorders associated with venous pooling in lower extremities such as orthostatic hypotension and edema. External com- pression on the lower leg helps balance the capillary filtration and venous return which Pourazadi et al. BioMed Eng OnLine (2015) 14:103 Page 16 of 18 in turn results in the reduction of venous pooling and normalizing the blood return. The literature suggests that a compression of 35–40 mmHg (4.67–5.33 kPa) at the ankle is required to prevent edema in patients with varicose veins. Also a graded compres- sion of 18 mmHg (2.40 kPa) at the ankle, 14 mmHg (1.87 kPa) at mid-calf and 8 mmHg (1.07 kPa) at knee optimizes the blood flow in lower leg. l Since the human calf is flexible and compressible, the size and shape of the calf changes upon application of external compression. The literature was reviewed to obtain a relation between external pressure and change in volume of the calf (i.e. compliance). Different methods were discussed in this regard and finally a model for the relation between external compression and radial strain of the calf was developed based on [28]. This model was incorporated with analytical models available for the DEA. An iterative process was used to solve the system of equations. Also an FEM model was developed to simulate the contact problem between the ACB and the human calf. Results from the FEM model were used to cross validate the analytical simulations. The maximum rela- tive difference between the FEM and the analytical modeling was as low as 2.9 %. The simulations demonstrated the values of the calf volume change under various compres- sions on different calf regions. For example, the calf underwent a 4.4 % volume change for an external pressure of 44 mmHg (5.82 kPa) at the ankle region. This change in vol- ume can possibly be a considerable issue in analyzing stiff bandages with large young modulus. In this paper it was shown that calf compressibility caused variations in the amount of compression exerted by elastic bandages. Specifically, the actual amount of compres- sion by elastic bandages is different from the expected value. Additional files Additional file 1. Final calf volume values in liter after wrapping and actuating the ACB. Additional file 2. Total pressure values in kPa from the simulations for different stretch ratios and different calf regions. Conclusion In the case of using the ACB, consideration of ankle flexibility resulted in approximately 9.7 % variations in the total pressure of the ACB for a case when the initial mechanical pressure of the ACB was 50 mmHg (6.70 kPa). However, results suggest that the effect of calf compressibil- ity on the total pressure of the ACB was less than 5 % for initial mechanical pressures of less than 23 mmHg (3.07 kPa), 24.5 mmHg (3.27 kPa) and 27.5 mmHg (3.67 kPa) at the knee, mid-calf and ankle regions. Generally, the ACB with a specific value of stretch ratio, exerted higher mechanical pressure and resulted in higher actuation pressure for calf regions which had smaller sizes. The study in this work suggested that, the size and the compressibility of the calf should be taken into account in order to create a suffi- ciently accurate design of the elastic bandages that are supposed to exert a specific range of compressions. Additional file 1. Final calf volume values in liter after wrapping and actuating the ACB. Additional file 2. Total pressure values in kPa from the simulations for different stretch ratios and different calf regions. References 1. Koeppen BM, Stanton BA. Berne and Levy Physiology. Amsterdam: Elsevier; 2009. 2. Lewis T. A lecture on vasovagal syncope and the carotid sinus mechanism. Br Me g y 3. Stenger MB, Brown AK, Lee SMC, Locke JP, Platts SH. Gradient compression garments as a countermeasure to post- fli h h i i l A i S E i M d 2010 81 883 7 3. Stenger MB, Brown AK, Lee SMC, Locke JP, Platts SH. Gradient compression garments as a countermeasure to post- fl spaceflight orthostatic intolerance. Aviat Space Environ Med. 2010;81:883–7. 4. Burnand KG, Layer GT. Graduated elastic stockings. Br Med J. 1986;293(6541):224–5.fi . Burnand KG, Layer GT. Graduated elastic stockings. Br Med J. 198 5. Pierson S, Pierson D, Swallow R, Johnson G. Efficacy of graded elastic compression in the lower leg. JAMA. 1983;249:242–3. 6. Jones NAG, Webb PJ, Rees RI, Kakkar VV. A physiological study of elastic compression stockings in venous disorders of the leg. Br J Surg. 1980;67:569–72. g g 7. Walker L, Lamont S. The use of antiembolic stockings. Part 1: a literature review. Br J Nurs. 2007;2:1408–13. 8. Ginsberg JS, Hirsh J, Julian J, Vander Laandevries M. Prevention and treatment of postphlebitic syndrome: results of a 3-part study. Arch Intern Med. 2001;161:3–7. 8. Ginsberg JS, Hirsh J, Julian J, Vander Laandevrie a 3-part study. Arch Intern Med. 2001;161:3–7. a 3-part study. Arch Intern Med. 2001;161:3–7. 9. Protheroe CL, Dikareva A, Menon C, Claydon VE. Are compression stockings an effective treatment for orthostatic presyncope? PLoS One. 2011;6:e28193. 9. Protheroe CL, Dikareva A, Menon C, Claydon VE. Are compr presyncope? PLoS One. 2011;6:e28193. p y p 10. Pourazadi S, Ahmadi S, Menon C. Towards the development of active compression bandages using dielectric elasto- S S 10. Pourazadi S, Ahmadi S, Menon C. Towards the development of active compression bandages using dielectric elasto- mer actuators. Smart Mater Struct. 2014;23:065007. mer actuators. Smart Mater Struct. 2014;23:065007. 11. Silbersack Y, Taute B-M, Hein W, Podhais ment. J Bone Jt Surg. 2004;86:809–12. 11. Silbersack Y, Taute B-M, Hein W, Podhaisky H. Prevention of deep-vein thrombosis after total hip and knee replace- ment. J Bone Jt Surg. 2004;86:809–12. ment. J Bone Jt Surg. 2004;86:809–12. g 12. Pedegana LR, Burgess EM, Moore AJ, Carpenter ML. Prevention of thromboembolic disease by external pneumatic compression in patients undergoing total hip arthroplasty. Clin Orthop Relat Res. 1977;128:190–3.i 13. Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Received: 21 February 2015 Accepted: 12 October 2015 Authors’ contributions SP conducted literature survey and extracted required data from literature, participated in design of the study, devel- oped the mathematical modeling and simulations, and drafted the manuscript. SA conducted finite element modeling and analysis, participated in discussions and drafting the manuscript. CM supervised and coordinated the project, contributed to design of the study, discussions and analysis and participated in manuscript revisions. All authors read and approved the final manuscript. Page 17 of 18 Page 17 of 18 Pourazadi et al. BioMed Eng OnLine (2015) 14:103 References Pelrine R, Kornbluh R, Joseph J, Heydt R, Pei Q, Chiba S. High-field deformation of elastomeric dielectrics for actua- tors. Mater Sci Eng. 2000;11:89–100. 14. Carpi F, De Rossi D, Kornbluh R, Pelrine R, Sommer-Larse P. Dielectric elastomers as electromechanical transducers. Amsterdam: Elsevier; 2008. 15. Carpi F, Chiarelli P, Mazzoldi A, De Rossi D. Electromechanical characterisation of dielectric elastomer planar actua- tors: comparative evaluation of different electrode materials and different counterloads. Sensors Actuators A Phys. 2003;107:85–95. 16. Ahmadi S, Camacho Mattos A, Barbazza A, Soleimani M, Boscariol P, Menon C. Fabrication and performance analysis of a DEA cuff designed for dry-suit applications. Smart Mater Struct. 2013;22:035002. 17. Soleimani M, Menon C. Preliminary investigation of a balloon-shape actuator based on electroactive elastomers. Smart Mater Struct. 2010;19:047001. 18. Artusi M, Potz M, Aristizabal J, Menon C, Cocuzza S, Debei S. Electroactive elastomeric actuators for the implementa- tion of a deformable spherical rover. IEEE/ASME Trans Mechatron. 2011;16:50–7. 19. Stemmer R. Ambulatory elasto-compressive treatment of the lower extremities particularly with elastic stockings Der Kassenarzt. 1969;9:1–8. 20. Sigel B, Edelestein A, Savitch L, Hasty J, Felix W. Types of compression for reducing venous stasis. Arch Surg. 1975;110:171–5. 21. Halliwill JR, Minson CT, Joyner MJ, John R. Measurement of limb venous compliance in humans: technical consider tions and physiological findings. J Appl Physiol. 1999;87:1555–63. tions and physiological findings. J Appl Physiol. 1999;87:1555–6 yi y 22. Monahan KD, Dinenno FA, Seals DR, Halliwill JR. Smaller age-associated reductions in leg venous compliance in i 22. Monahan KD, Dinenno FA, Seals DR, Halliwill JR. Smaller age-associated reductions in leg venous compliance in endurance exercise-trained men. Am J Physiol Heart Circ Physiol. 2001;281:H1267–73. g endurance exercise-trained men. Am J Physiol Heart Circ Physiol. 2001;281:H1267–73. endurance exercise-trained men. Am J Physiol Heart Circ Physio 23. Monahan KD, Ray CA. Gender affects calf venous com Am J Physiol Heart Circ Physiol. 2004;286:H895–901. 23. Monahan KD, Ray CA. Gender affects calf venous compliance at rest and during baroreceptor unloading in humans Am J Physiol Heart Circ Physiol. 2004;286:H895–901. Am J Physiol Heart Circ Physiol. 2004;286:H895–901 24. Lipp A, Sandroni P, Ahlskog JE, Maraganore DM, Shults CW, Low PA. Calf venous compliance in multiple system atrophy. J Physiol Heart Circ Physiol. 2007;55905:260–5. p y y y ; 25. Sielatycki JA, Shamimi-noori S, Pfeiffer MP, Monahan KD. Adrenergic mechanisms do not contribute to age-related decreases in calf venous compliance. J Appl Physiol. 2011;2390:29–34. Acknowledgements Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Cana- di I tit t f H lth R h (CIHR) d th Mi h l S ith F d ti f H lth R h (MSFHR) g This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Cana- dian Institutes of Health Research (CIHR) and the Michael Smith Foundation for Health Research (MSFHR). Competing interests The authors declare that they have no competing interests. 33. Meunier L, Chagnon G, Favier D, Orgéas L, Vacher P. Mechanical experimental characterisation and numerical mod- elling of an unfilled silicone rubber. Polym Test. 2008;27:765–77. 34. Boa closure system. http://www.boatechnology.com. References p y y y 25. Sielatycki JA, Shamimi-noori S, Pfeiffer MP, Monahan KD. Adrenergic mechanisms do not contribute to age-related d i lf li J A l Ph i l 2011 2390 29 34 p y y y 25. Sielatycki JA, Shamimi-noori S, Pfeiffer MP, Monahan KD. Adrenergic m decreases in calf venous compliance. J Appl Physiol. 2011;2390:29–34. 25. Sielatycki JA, Shamimi-noori S, Pfeiffer MP, Monahan KD f decreases in calf venous compliance. J Appl Physiol. 2011;23 26. Binzoni T, Quaresima V, Ferrari M, Hiltbrand E, Cerretelli infrared spectroscopy. J Appl Physiol. 2000;88:369–72. 26. Binzoni T, Quaresima V, Ferrari M, Hiltbrand E, Cerretelli P. Human calf microvascular compliance measured by near- infrared spectroscopy. J Appl Physiol. 2000;88:369–72. 26. Binzoni T, Quaresima V, Ferrari M, Hiltbrand E, Cerretelli infrared spectroscopy. J Appl Physiol. 2000;88:369–72. 27. Zicot M, Parker KH, Caro CG. Effect of positive external pressure on calf volume and local venous haemodynamics. Phys Med Biol. 1977;22:1146–59. f Phys Med Biol. 1977;22:1146–59. y ; 28. Thirsk RB, Kamm RD, Shapiro AH. Changes in venous blood volume produced by external compression of the lower leg Med Biol Eng Comput 1980 18 650 6 y 28. Thirsk RB, Kamm RD, Shapiro AH. Changes in leg. Med Biol Eng Comput. 1980;18:650–6. 28. Thirsk RB, Kamm RD, Shapiro AH. Changes in venous blood volume produced by external compression of the lower leg. Med Biol Eng Comput. 1980;18:650–6. 28. Thirsk RB, Kamm RD, Shapiro AH. Changes in venous blo p g leg. Med Biol Eng Comput. 1980;18:650–6. 29. Whitney BYRJ. Working efficiency. Department of Human Anatomy, University of Oxford; 1952. C(c). 29. Whitney BYRJ. Working efficiency. Department of Human y gfi y p y y 30. ANSYS Workbench. Www.ansys.com. 2011. fi 30. ANSYS Workbench. Www.ansys.com. 2011 y 31. Hoyos A, Prendergast P. Complications of high-definition body sculpting. Berlin: Springer; 2014. 32. Wissler M, Mazza E. Modeling and simulation of dielectric elastomer actuators. Smart Mater Struct. 2005;14:1396. 32. Wissler M, Mazza E. Modeling and simulation of dielectric elastomer actuators. Smart Mater Struct. 2005;14:1396. Page 18 of 18 Pourazadi et al. 33. Meunier L, Chagnon G, Favier D, Orgéas L, Vacher P. Mechanical experimental characterisation and numerical mod- elling of an unfilled silicone rubber. Polym Test. 2008;27:765–77. Pourazadi et al. BioMed Eng OnLine (2015) 14:103 34. Boa closure system. http://www.boatechnology.com. References BioMed Eng OnLine (2015) 14:103 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2102515881	https://ejnmmires.springeropen.com/counter/pdf/10.1186/2191-219X-3-77	English	null	The PET-derived tumor-to-blood standard uptake ratio (SUR) is superior to tumor SUV as a surrogate parameter of the metabolic rate of FDG	EJNMMI research	2,013	cc-by	6,759	The PET-derived tumor-to-blood standard uptake ratio (SUR) is superior to tumor SUV as a surrogate parameter of the metabolic rate of FDG Jörg van den Hoff1,2†, Liane Oehme2, Georg Schramm1, Jens Maus1, Alexandr Lougovski1, Jan Petr1, Bettina Beuthien-Baumann1,2 and Frank Hofheinz1† Abstract Results: For any fixed time point T at sufficiently late times post injection, the Patlak equation predicts a linear correlation between SUR and Km under the following assumptions: (1) approximate shape invariance (but arbitrary scale) of the AIF across scans/patients and (2) low variability of the apparent distribution volume Vr (the intercept of the Patlak Plot). This prediction - and validity of the underlying assumptions - has been verified in the investigated patient group. Replacing tumor SUVs by SURs does improve the linear correlation of the respective parameter with Km from r = 0.61 to r = 0.98. Results: For any fixed time point T at sufficiently late times post injection, the Patlak equation predicts a linear correlation between SUR and Km under the following assumptions: (1) approximate shape invariance (but arbitrary scale) of the AIF across scans/patients and (2) low variability of the apparent distribution volume Vr (the intercept of the Patlak Plot). This prediction - and validity of the underlying assumptions - has been verified in the investigated patient group. Replacing tumor SUVs by SURs does improve the linear correlation of the respective parameter with Km from r = 0.61 to r = 0.98. Conclusions: SUR is an easily measurable parameter that is highly correlated to Km. In this respect, it is clearly superior to SUV. Therefore, SUR should be seriously considered as a drop-in replacement for SUV-based approaches. Keywords: SUV; Tumor-to-blood ratio; PET; PET/CT; Therapy response control; FDG © 2013 van den Hoff et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: j.van_den_hoff@hzdr.de †Equal contributors 1PET Center, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden 01328, Germany 2Department of Nuclear Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden 01307, Germany Abstract Background: The standard uptake value (SUV) approach in oncological positron emission tomography has known shortcomings, all of which affect the reliability of the SUV as a surrogate of the targeted quantity, the metabolic rate of [18F]fluorodeoxyglucose (FDG), Km. Among the shortcomings are time dependence, susceptibility to errors in scanner and dose calibration, insufficient correlation between systemic distribution volume and body weight, and, consequentially, residual inter-study variability of the arterial input function (AIF) despite SUV normalization. Especially the latter turns out to be a crucial factor adversely affecting the correlation between SUV and Km and causing inter-study variations of tumor SUVs that do not reflect actual changes of the metabolic uptake rate. In this work, we propose to replace tumor SUV by the tumor-to-blood standard uptake ratio (SUR) in order to distinctly improve the linear correlation with Km. Methods: Assuming irreversible FDG kinetics, SUR can be expected to exhibit a much better linear correlation to Km than SUV. The theoretical derivation for this prediction is given and evaluated in a group of nine patients with liver metastases of colorectal cancer for which 15 fully dynamic investigations were available and Km could thus be derived from conventional Patlak analysis. Methods: Assuming irreversible FDG kinetics, SUR can be expected to exhibit a much better linear correlation to Km than SUV. The theoretical derivation for this prediction is given and evaluated in a group of nine patients with liver metastases of colorectal cancer for which 15 fully dynamic investigations were available and Km could thus be derived from conventional Patlak analysis. Results: For any fixed time point T at sufficiently late times post injection, the Patlak equation predicts a linear correlation between SUR and Km under the following assumptions: (1) approximate shape invariance (but arbitrary scale) of the AIF across scans/patients and (2) low variability of the apparent distribution volume Vr (the intercept of the Patlak Plot). This prediction - and validity of the underlying assumptions - has been verified in the investigated patient group. Replacing tumor SUVs by SURs does improve the linear correlation of the respective parameter with Km from r = 0.61 to r = 0.98. van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Theory We start with the standard Patlak formula: ct(t) ca(t) = Km × t 0 ca(s)ds ca(t) + Vr (1) (1) (1) where Km is the metabolic trapping rate and Vr is the so- called apparent volume of distribution. This equation is valid for times later than some time point t∗(typically t∗≈ 15 to 20 min post injection (p.i.)). This equation is of course valid independent of the units chosen to specify the tracer concentrations, but in the following we view ct(t), ca(t) as being expressed in SUV units. For the sake of argument, we further assume the following: 1. All input functions exhibit a common shape ba(t), i.e., are approximately shape invariant across all considered investigations and only differ by an investigation-specific scaling factor N: ca(t) ≈ N × ba(t) where N is measured in the same units as ca and the shape function ba(t) is dimensionless. Taking into account that all SUV-based evaluations require that the measurement time is strictly standardized and denoting this fixed time point as T, we choose the arbitrary normalization of ba(t) in such a way that ba(T) = 1. The scaling factor N then becomes identical to the blood concentration at the chosen time point: N = ca(T). The SUV approach, on the other hand, has several known shortcomings [10-12] all of which affect the relia- bility of the SUV as a surrogate of Km (and the metabolic rate of glucose consumption). The following are among these shortcomings: Taking into account that all SUV-based evaluation • Time dependence of the SUV (requiring to strictly standardize the time point after injection chosen for imaging and evaluation), • Susceptibility to errors in scanner calibration, 2. Variability of the apparent volume of distribution, Vr, is small for the considered tumor entity across all considered investigations, and it can thus be taken to be approximately constant: Vr ≈¯Vr = const. • Insufficient correlation between systemic distribution volume and body weight (leading to variants of the SUV approach using lean body mass (SUVlbm) [13] or body surface area (SUVbsa) [14] for normalization), and, consequentially, residual inter-study variability of the arterial input function (AIF) despite SUV normalization. van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 2 of 8 Page 2 of 8 Background In this work, we propose to replace tumor SUVs by the tumor-to-blood uptake ratio, called standard uptake ratio (SUR), in the following: SUR can be expected on theoret- ical grounds to exhibit a much better linear correlation to Km than SUV. The derivation for this prediction is given and evaluated in a group of nine patients with liver metas- tases of colorectal cancer for which 15 fully dynamical investigations were available and Km could thus be derived from conventional Patlak analysis. g Today, in the clinical oncological setting, the standard uptake value (SUV, g/ml), defined as the tracer concentra- tion at a certain time point normalized to injected dose per unit body weight is essentially the only means for quantitative evaluation of [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET). While it is per- fectly possible to quantify the actually targeted parameter, namely the absolute metabolic rate of glucose consump- tion (or, rather, the rate of irreversible FDG accumulation, Km), the conventional approaches such as the Patlak plot [1,2] require to a varying degree dynamic imaging and also determination of the full arterial input function both of which requirements are not compatible with onco- logical whole body imaging. For this reason, different alternative approaches have been investigated in the past which have tried to facilitate the quantification of Km in order to make it more suitable for the oncological set- ting [3-8]. However, none of these approaches have so far gained widespread acceptance since they either rest on quite restrictive assumptions and approximations or still impose demands on data acquisition and evaluation that are significantly higher than for SUV-based approaches. Recently, we have shown that a purely image-based quan- tification of Km might be performed that only requires a dual time point PET measurement [9]. This seems to con- stitute the minimum requirement necessary to actually determine this parameter. While such dual time point pro- tocols could be considered acceptable for clinical routine, they still do somewhat increase complexity of the work flow and data evaluation and will probably only augment but not replace static oncological imaging. Data evaluation The AIF was determined from a roughly cylindrical three- dimensional (3D) region of interest (ROI) centered in the aorta. To exclude partial volume effects, a concentric safety margin of at least 1 cm was used in the transax- ial plane. To compensate for the resulting small transaxial diameter, the ROIs were extended axially (along the aorta) for about 10 cm. This resulted in sufficiently large ROI volumes (mean ± standard deviation 7.3 ± 1.8 cm3) and ensured sufficiently high statistical accuracy of the derived AIF values. Contrary to SUR, the tissue SUV itself (i.e., ct) would only be related linearly to Km if the input functions - when expressed in SUV units - were in fact completely identical instead of only shape invariant across different investiga- tions and target structures. This is directly obvious from Equation 2 when multiplying both sides by ca: ct = SUV ≈AUC × Km + ¯Vr × ca (4) (4) since now, both ca and the area under the input curve would be required to assume investigation-independent fixed values. This trivially is the case for intra-scan com- parisons of different target structures but not at all for inter-subject comparisons (or inter-scan comparisons of the same patient). Actually, ca is known to exhibit substan- tial variability across scans even when expressed in SUV units [13]. Three-dimensional ROIs were defined in 22 lesions, and the respective tissue response functions were computed. ROI definition was performed using the ROVER software (ABX, Radeberg, Germany; [15,16]). Further data anal- ysis was carried out using the R software for statistical computing [17]. For all 22 lesions, Km was derived from conventional Patlak analysis of the full dynamic data later than 20 min p.i. (at which time, all Patlak plots were already linear). The summed late time data between 50 and 60 min p.i. (corresponding to T = 55 min) were used to determine ca and ct. For each ROI mean values of SUV (= ct), SUR (= ct/ca) and Km were computed. Linear regression anal- ysis was performed for SUV vs. Km and SUR vs. Km, respectively. To summarize, the difference between SUV and SUR can be stated as follows : SUR is linearly related to Km under a distinctly weaker assumption (common shape of input function required) than SUV (identical input function required). Study sample Nine male patients with liver metastases of colorectal cancer were included retrospectively (age, 48 to 76 years (mean 62.8); weight, 73 to 100 kg (mean 85.5)). For each patient, one to three dynamic PET scans of 60 min in dura- tion were performed (altogether 15 scans). Scans started immediately after injection of 346 to 430 MBq FDG administrated as bolus over 10 to 20 s. Diabetics were excluded, and no forced diuresis was used. The scans were conducted with an ECAT EXACT HR+ (Siemens/CTI, Knoxville, TN, USA). The acquired data were sorted into 23 to 31 frames with 10 to 20 s in duration during bolus passage, 30 to 150 s in duration until 10 min p.i., and 300 s in duration afterwards. Tomographic images were recon- structed using attenuation weighted OSEM reconstruc- tion (six iterations, 16 subsets, 6-mm FWHM Gaussian filter). (T) = AUC(T) ca(T) = T 0 ca(s)ds ca(T) = T 0 ca(T) ba(s) ds ca(T) = T 0 ba(s)ds . (3) (3) Obviously, is identical to the independent (x-) coor- dinate of the Patlak plot which dimensionally is a time (sometimes called ‘Patlak’s funny time’). The crucial point here is to realize that is not influenced by scale changes of ca(t). If ba(t) can be assumed to have a fixed shape (at least for ‘most of the time’: shape variations during the bolus passage do not sizably influence the integral up to the late time point T), (T) is in fact constant across all considered investigations for any given choice of the measurement time point T. The above equations show that if the used assumptions are adequate, one can expect a linear relation between SUR and Km (in order to improve readability, any dependence on T is no longer stated explicitly here and in the following but has to be kept in mind). The study protocol has been approved by the local Clinical Institutional Review Board and has complied with the Declaration of Helsinki. Theory It then follows immediately from Equation 1 that the tumor-to-blood uptake ratio at time t = T is related to the metabolic rate Km according to Especially the latter point turns out to be a crucial factor adversely affecting the correlation between SUV and Km and, more generally, leading to uncontrolled inter-study variability of tumor SUVs. ct(T) ca(T) = SUR(T) ≈(T) × Km + ¯Vr ct(T) ca(T) = SUR(T) ≈(T) × Km + ¯Vr (2) Page 3 of 8 Page 3 of 8 Page 3 of 8 Page 3 of 8 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 where Results and discussion Results consequence of approximate shape invariance of ca across investigations) and amounts to a standard deviation of 20%. Due to the highly correlated variations of ca and AUC, = AUC/ca exhibits a much smaller variability with a standard deviation of only 3.2% which demon- strates that actually is approximately constant across all conducted investigations. Figure 1 shows the complete time course of, both the AIF - normalized to the time-average of the respective curve, ¯ca = 1 T T 0 ca(s)ds - and (t). As can be seen, the nor- malized AIFs exhibit only a very small variability beyond t ≈1 min, demonstrating that the assumption of approx- imate shape invariance is valid. Consequently, inter-study variability of (t) is small, too. This is especially true at late times which are the relevant ones in the present context. Figure 4 shows the correlation between Km and, respec- tively, SUV (Figure 4A) and SUR (Figure 4B) together with straight-line fits to the data. There is only a rather weak correlation between Km and SUV (r = 0.61) while Km and SUR are strongly correlated (r = 0.98). The axis intercept of the straight-line fit in the latter case is a = 0.69 ± 0.10 which compares reason- ably well with the average apparent volume of distribution in this patient group (as a result of the Patlak analysis) of 0.53 ± 0.11. The slope is m = 76.8 ± 3.7 min which, too, is in accord with the theoretical expectation m ≈ and with Figure 3C from which an average of 82.9 ± 2.6 min follows for . In Figure 2, the inter-study variability of ca is investi- gated in detail for the chosen time point T = 55 min. The degree of variability is not reduced when using either SUVlbm (Figure 2B) or SUVbsa (Figure 2C) instead of conventional (body weight normalized) SUV (Figure 2A): on average, we obtain ca = 2.59 ± 0.58 (SUV), 1.99 ± 0.43 (SUVlbm), and 1.99 ± 0.42 (SUVbsa), respectively, corresponding to a standard deviation of 22% to 23% rel- ative to the respective mean. Moreover, in our patient group, ca and serum glucose concentration (range 4.8 to 10.3 μmol/ml) were found to be essentially uncorrelated (r = −0.14, P < 0.65). Results and discussion Results With the possible exception of sub- jects 2 and 6, it can also be observed that the intra-subject variability is of the same order of magnitude than the inter-subject variability. Data evaluation The prediction following from the above considerations is very simple: SUR should be a distinctly better surrogate parameter of Km than the tissue SUV since it is expected to exhibit a much higher linear correlation to Km. We have tested this prediction in the present study focusing on the potentially most relevant case of investigations in the trunk where ca can be easily derived within the recon- structed image volumes from a suitable region in either the aorta or the left ventricle of the heart. The inter-study variability of ca was determined in SUV units (SUVa), SUVlbm units (SUVa lbm), and SUVbsa units (SUVa bsa), respectively. For better comparability, SUVa bsa was normalized such that the mean SUVa bsa equals the mean SUVa lbm as described in [18]. Additionally, the vari- ability of area under the curve (AUC) and of = AUC/ca was investigated. Page 4 of 8 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 4 of 8 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Discussion EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 5 of 8 scan 3 scan 2 scan 1 C SUVα bsa (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 scan 3 scan 2 scan 1 B SUVα lbm (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 scan 3 scan 2 scan 1 A SUVα (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 Figure 2 Inter-study variability of ca (55 min p.i.) in nine subjects and 15 scans expressed in three SUV units. (A) Conventional SUV (body weight normalized), (B) SUVlbm (lean body mass normalized), and (C) SUVbsa (body surface area normalized). scan 3 scan 2 scan 1 C SUVα bsa (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 scan 3 scan 2 scan 1 B SUVα lbm (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 scan 3 scan 2 scan 1 A SUVα (g/ml) #9 #8 #7 #6 #5 #4 #3 #2 #1 4 3.5 3 2.5 2 1.5 1 0.5 0 Figure 2 Inter-study variability of ca (55 min p.i.) in nine subjects and 15 scans expressed in three SUV units. (A) Conventional SUV (body weight normalized), (B) SUVlbm (lean body mass normalized), and (C) SUVbsa (body surface area normalized). C B A Figure 3 Inter-study variability of ca and of the corresponding AUC. (A) Using SUV units in nine subjects and 15 scans and (B) the corresponding AUC as well as (C) = AUC/ca at 55 min p.i. C B A Figure 3 Inter-study variability of ca and of the corresponding AUC. (A) Using SUV units in nine subjects and 15 scans and (B) the corresponding AUC as well as (C) = AUC/ca at 55 min p.i. C B A Figure 3 Inter-study variability of ca and of the corresponding AUC. (A) Using SUV units in nine subjects and 15 scans and (B) the corresponding AUC as well as (C) = AUC/ca at 55 min p.i. Figure 3 Inter-study variability of ca and of the corresponding AUC. Discussion We could demonstrate a very high linear correlation between SUR and Km in the investigated patient group which is in marked contrast to the rather poor lin- ear correlation between SUV and Km. In fact, the latter correlation is somewhat inferior to results reported in the literature (e.g., [5,19]) which might be traced back to the occurrence of several unusual high ca values Figure 3 compares the variability of ca at T = 55 min with that of AUC and . Variability of AUC closely fol- lows that of ca (which can be understood as a direct A B Figure 1 Inter-study variability of AIF shape and (t) = AUC(t)/ca(t) in the available 15 data sets. The thick red lines represent the group-averaged curves. (A) AIF normalized to the mean value (time-average) of the respective curve. As can be seen, the shape of all AIF curves is very similar beyond t ≈1 min. (B) (t), the ratio of the AUC up to time t divided by ca(t), exhibits only small inter-study variability. Averaging the last two points of these curves (acquisition time from 50 to 60 min p.i.) yields the data presented in more detail below. A B B Figure 1 Inter-study variability of AIF shape and (t) = AUC(t)/ca(t) in the available 15 data sets. The thick red lines represent the group-averaged curves. (A) AIF normalized to the mean value (time-average) of the respective curve. As can be seen, the shape of all AIF curves is very similar beyond t ≈1 min. (B) (t), the ratio of the AUC up to time t divided by ca(t), exhibits only small inter-study variability. Averaging the last two points of these curves (acquisition time from 50 to 60 min p.i.) yields the data presented in more detail below. van den Hoff et al. Discussion were nevertheless able to demonstrate that the resulting Km estimate correlates highly with true Km (r = 0.98 and r = 0.97) while SUV (called DUR in that paper) is found to exhibit a rather unsatisfactory corre- lation to Km (r = 0.73 and r = 0.84). These findings are in full accord with those of the present investiga- tion. The main difference to our work is that Hunter et al. impose the requirement of a suitable control group averaged and analytically modeled input curve plus a sin- gle blood sample in order to estimate the individual area under the input curve. This knowledge is necessary in order to estimate Km in their approach. However, the implied assumption is that =AUC/ca is constant across investigations so that, due to the neglect of the small Vr-dependent term, SUR becomes approximately propor- tional to Km. While these conclusions are implied in the work of Hunter et al., they are not explicitly stated or uti- lized in their work. The present work, on the other hand, demonstrates that there is no need for either a population- averaged AIF (and assumption of a specific shape of the individual AIFs) or blood sampling in order to derive a quantity (SUR) that is linearly correlated to Km. We believe this very distinct linear correlation to be the practi- cally most relevant aspect. Whether conversion from SUR to Km does always make sense is not so obvious as will be discussed below. As is well known, target to reference tissue ratios have been frequently used successfully in SPECT as well as in PET (and still are), e.g. in neuro-receptor imaging, but also in oncology. In view of this fact, it is somewhat surprising that seemingly, SUR has up to now not been investigated systematically as an alternative to SUV in oncological PET. We were only able to locate a single study explicitly comparing prognostic parameters derived from tumor- to-blood ratios and SUVs, respectively, in patients with non-small cell lung cancer but without comparing these parameters to Km [20]. The authors reported superiority of tumor-to-blood ratios over SUV in this study without further investigating the reason for this finding. Other- wise, there is only a single methodological work we know of, namely that by Hunter et al. [5], which proposes a strat- egy that bears some resemblance to the SUR approach. Discussion EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 6 of 8 ﬁt data intercept = 0.69 slope = 77 min r = 0.98 B (ml/min/ml) SUR 4 3 2 1 0 4 3 2 1 0 ﬁt data intercept = 2.65 g/ml slope = 165 g/ml × min r = 0.61 A (ml/min/ml) SUV (g/ml) 4 3 2 1 0 14 12 10 8 6 4 2 0 Figure 4 Correlation between Km and, respectively, lesion SUV (A) and SUR (B) determined at 55 min p.i. Dashed lines represent the least-squares straight-line fits to the data. ﬁt data intercept = 0.69 slope = 77 min r = 0.98 B (ml/min/ml) 4 3 2 1 0 4 3 2 1 0 B Figure 4 Correlation between Km and, respectively, lesion SUV (A) and SUR (B) determined at 55 min p.i. Dashed lines represent the least-squares straight-line fits to the data. Contrary to SUV determination, the SUR approach requires delineation of a second ROI to derive the AIF concentration. This could be seen as an additional source of inter-observer variability and thus constitute a poten- tial disadvantage of the approach in comparison with the SUV usage. While this is true in principle, determination of the arterial concentration does actually contribute only modestly to the observed variability of the SUR vs. Km correlation (which is quite small, anyway). This was veri- fied by repeated, intentionally differing delineations of the corresponding 3D ROI. As long as the prescription to use a substantial safety margin in the transaxial plane and a reasonable large ROI volume (extending the ROI over a sufficiently large axial section of the aorta) is respected, the reproducibility of the results is very high. the slowest exponential, and the described procedure therefore essentially reduces to using an invariant-shaped input function scaled to the actually observed individ- ual late tracer concentration in the blood. The scaled input function is then integrated analytically (using the sum of exponential models) and a formula for Km (called P2 in that paper) derived that is essentially equivalent to our Equation 2. Finally, these authors propose to use ct/AUC for Km estimation, thus neglecting the influence of the term proportional to Vr in Equation 4 completely. Hunter et al. Discussion (A) Using SUV units in nine subjects and 15 scans and (B) the corresponding AUC as well as (C) = AUC/ca at 55 min p.i. Figure 2 Inter-study variability of ca (55 min p.i.) in nine subjects and 15 scans expressed in three SUV units. (A) Conventional SUV (body weight normalized), (B) SUVlbm (lean body mass normalized), and (C) SUVbsa (body surface area normalized). the other hand, is well known to be reasonably well ful- filled. This has already been utilized in the past, e.g., in the context of population-averaged input curves ([5,7]). For completeness, it might also be noted that, in fact, all shape variations leaving the integral T 0 ba(s)ds unchanged do not influence the SUR vs. Km correlation. Strictly speak- ing, it is not really the shape invariance of the input function that has to be assumed but only the invari- ance of the area under the normalized input curve ba(t) up to time t = T. Notably, this implies that the fre- quently observed inter-study variability of the AIF during the early (bolus passage) phase has essentially no influ- ence since it does not contribute much to the integral up to T (see Figure 1). Overall, the actually observed deviations of SUR from the regression line in Figure 4B directly reflect the (quite small) cumulative effect of both Vr and input function shape variability in this patient group. corresponding to the data points exhibiting the largest deviation from the regression line. This finding might hint at unidentified problems such as erroneous scanner cal- ibration at the time of the respective scans although a retrospective investigation did not reveal any proof for this. Incidentally, this also demonstrates the insensitivity of SUR to such possible sources of errors. However, even when taking the literature values as reference instead of our own results, it is still true that the degree of linear cor- relation between SUR and Km is distinctly higher than is the case when using SUV. In theoretical terms, the linear SUR vs. Km correla- tion can be understood as a consequence of a common input function shape and small variability of the appar- ent volume of distribution, Vr. The latter was already demonstrated and discussed in [9]. The former requirement, namely the approximate shape invariance of FDG input curves after bolus injection, on van den Hoff et al. Discussion These authors used a control-group averaged input func- tion (modeled as a sum of three exponentials) plus single venous sampling to fix the unknown amplitudes of the exponentials. By far, the most important contribution to the time integral over the input function stems from van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 7 of 8 Page 7 of 8 (in an unknown way) if the time of measurement is changed. Compared to tissue SUV, the use of SUR eliminates a couple of shortcomings of the former. For one, SUR is a dimensionless quantity that is neither affected by inaccuracies of scanner or dose calibration nor by erro- neous body weight (or related quantities). While this is clearly advantageous, the most relevant improve- ment of SUR over SUV in our view is elimination of a very large part of the variability caused by inter- scan variations of the input function. Given the empir- ically well demonstrated approximately invariant shape of FDG input functions, SUR eliminates the influence of inter-scan variations of tracer supply nearly com- pletely. Quite to the contrary, variations in the scale of ca(t) (if measured in SUV units) directly translate into substantial SUV changes. As Figure 2 demonstrates, the inter-subject as well as the intra-subject variabil- ity of ca is indeed substantial (exhibiting a standard deviation of about 23% from the mean) which directly explains the large fluctuations of the SUVs observed in Figure 4A. It should be stressed, that this variabil- ity of ca persists after conversion to SUV units (or to SUVlbm or SUVbsa, for that matter). In other words, the central objective of SUV-based approaches, namely correction for variations of injected dose and systemic distribution volume of the tracer, is not achieved sat- isfactorily. Consequently, comparing SUV values across investigations makes only limited sense and is prone to potentially serious errors if the objective is to assess changes of glucose metabolism. Inter-study compari- son of SUR values appears much more reasonable in this respect. At least since the advent of PET/CT a purely image-based determination of the blood signal from a large vessel is straightforward even where this would not be quite as easy when using the whole body PET data alone. Discussion Therefore, SUR computation does especially not require any blood sampling and could also easily be performed retrospectively using existing data for systematic performance comparisons of SUV and SUR. Second, the intercept of the regression line approxi- mates the average apparent volume of distribution, ¯Vr, and thus might vary across different tumor entities and tissues. We believe this probably to be a small effect for most tumors and healthy tissues (although this can- not be taken for granted and would need to be proven by further investigations), but for certain tissues and when inflammation is involved (i.e., if the assumption of irreversible trapping is not fulfilled), larger varia- tions of Vr will occur, and consequently, the relation between SUR and Km will be distinctly modified. Obvi- ously, the presence of a sizable dephosphorylation (i.e., a sizable k4 in the corresponding two-compartment model) would actually invalidate the prediction of a lin- ear SUR vs. Km correlation completely. It should be noted, however, that this is not a specific problem of the SUR approach but affects SUV evaluations accord- ingly. In the present study, inspection of the Patlak plots unambiguously demonstrated that the FDG kinet- ics in the liver lesions is compatible with the assumption k4 = 0. Still, in general, it cannot be expected that the regres- sion line in Figure 4B is universally valid (at least not with good accuracy), and quantitative conversion of SUR to Km should probably be avoided in our view (and would not offer additional benefits). Instead, we surmise that it might be worthwhile to com- bine the SUR approach with a dual time point-based determination of Km [9] in order to identify regions in which the SUR vs. Km relation is changed. This possi- bly could, e.g., enable a more sensitive identification of inflammatory processes than is possible with dual time point measurements alone. Discussion Where such dual time point protocols allowing for inde- pendent determination of Km and SUR are considered not compatible with the requirements of clinical routine, SUR alone still offers distinct advantages over SUV-based approaches because a good linear correlation to Km sim- ilar to that shown in Figure 4B can be expected in most tissues and target structures (i.e., those where FDG kinet- ics can be considered to be irreversible): a fractional change of SUR will in general correspond to a concomi- tant change of Km where even modest SUR changes will be meaningful due to the very high degree of linear corre- lation of both quantities (as long as a sufficiently accurate determination of ca within the image data is performed that avoids partial volume and spill over effects). Inter- study SUV differences, instead, are likely to be partially or even completely spurious (i.e., unrelated to Km changes) due to the insufficient correction for inter-study variabil- ity of arterial tracer concentration achieved by the SUV approach. As demonstrated, replacing SUVs by SURs does elim- inate some important sources of SUV-specific system- atic errors which leads to a very good linear correlation between SUR and Km. On the other hand, this does not imply that SURs are necessarily also suitable for quantitative determination of Km in general since the SUR vs. Km correlation shown in Figure 4B need not be universally valid for two reasons. First, according to Equation 3, the slope of the SUR (Km) correlation is identical to the integral over ba(t) up to t = T. Differ- ent injection protocols (duration of injection, bolus vs. ramp etc.) naturally will thus lead to a different (and unknown) slope parameter. However, even if the invari- ant shape assumption can be considered universally valid, the slope remains a function of T and thus will change van den Hoff et al. EJNMMI Research 2013, 3:77 http://www.ejnmmires.com/content/3/1/77 Page 8 of 8 Authors’ contributions 19. Freedman N, Sundaram S, Kurdziel K, Carrasquillo J, Whatley M, Carson J, Sellers D, Libutti S, Yang J, Bacharach S: Comparison of SUV and Patlak slope for monitoring of cancer therapy using serial PET scans. Eur J Nucl Med Mol Imaging 2003, 30:46–53. 19. Freedman N, Sundaram S, Kurdziel K, Carrasquillo J, Whatley M, Carson J, Sellers D, Libutti S, Yang J, Bacharach S: Comparison of SUV and Patlak slope for monitoring of cancer therapy using serial PET scans. Eur J Nucl Med Mol Imaging 2003, 30:46–53. JVDH had the initial idea for SUR, performed part of the data analysis, and wrote part of the manuscript. LO and GS performed part of the data analysis. JM, AL, and JP provided intellectual input and reviewed the manuscript. BBB selected the patient studies and performed the lesion delineation. FH performed part of the data analysis and wrote part of the manuscript. All authors read and approved the final manuscript. g g 20. Mak RH, Digumarthy SR, Muzikansky A, Engelman JA, Shepard JAO, Choi NC, Sequist LV: Role of 18F-fluorodeoxyglucose positron emission tomography in predicting epidermal growth factor receptor mutations in non-small cell lung cancer. Oncologist 2011, 16(3):319–326. Acknowledgements This study was supported in part by the Federal Ministry of Education and Research Germany, BMBF, contract 03\|4021. doi:10.1186/2191-219X-3-77 Cite this article as: van den Hoff et al.: The PET-derived tumor-to-blood standard uptake ratio (SUR) is superior to tumor SUV as a surrogate parameter of the metabolic rate of FDG. EJNMMI Research 2013 3:77. doi:10.1186/2191-219X-3-77 Cite this article as: van den Hoff et al.: The PET-derived tumor-to-blood standard uptake ratio (SUR) is superior to tumor SUV as a surrogate parameter of the metabolic rate of FDG. EJNMMI Research 2013 3:77. Received: 9 September 2013 Accepted: 4 November 2013 Published: 23 November 2013 Received: 9 September 2013 Accepted: 4 November 2013 Published: 23 November 2013 Conclusions 12. Huang S: Anatomy of SUV. Nucl Med Biol 2000, 27(7):643–646. 13. Sugawara Y, Zasadny KR, Neuhoff AW, Wahl RL: Reevaluation of the standardized uptake value for FDG: variations with body weight and methods for correction. Radiology 1999, 213(2):521–525. The tumor-to-blood standard uptake ratio, SUR exhibits a much higher linear correlation to the metabolic rate of FDG, Km, than tumor SUV (or SUVlbm or SUVbsa). This improved correlation can be understood theoretically as a consequence of the approximate shape invariance of FDG input functions. Purely image-based determination of SUR is straightforward in whole body investigations and allows to account for variations in tracer supply across investigations distinctly better than when using SUVs. Therefore, SUR should be seriously considered as a drop- in replacement for SUV-based approaches. 14. Kim CK, Gupta NC, Chandramouli B, Alavi A: Standardized uptake values of FDG: body surface area correction is preferable to body weight correction. J Nucl Med Official Publ, Soc Nucl Med 1994, 35:164. g 15. ROVER: ROVER: ROI visualization, evaluation and image registration. Radeberg: ABX; 2008. http://www.abx.de/rover. 16. Hofheinz F, Pötzsch C, Oehme L, Beuthien-Baumann B, Steinbach J, Kotzerke J, van den Hoff J: Automatic volume delineation in oncological PET: evaluation of a dedicated software tool and comparison with manual delineation in clinical data sets. Nuklearmedizin 2012, 51:9–16. 17. R Development Core Team: R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing; 2011. [ISBN 3-900051-07-0]. 17. R Development Core Team: R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing; 2011. [ISBN 3-900051-07-0]. 18. Kim C, Gupta N: Dependency of standardized uptake values of fluorine-18 fluorodeoxyglucose on body size: comparison of body surface area correction and lean body mass correction. Nucl Med Commun 1996, 17(10):890–894. 18. Kim C, Gupta N: Dependency of standardized uptake values of fluorine-18 fluorodeoxyglucose on body size: comparison of body surface area correction and lean body mass correction. Nucl Med Commun 1996, 17(10):890–894. Competing interests The authors declare that they have no competing interests. References 1. Patlak C, Blasberg R, Fenstermacher J: Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. J Cereb Blood Flow Metab 1983, 3:1–7. 2. Patlak C, Blasberg R: Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data: generalizations. J Cereb Blood Flow Metab 1985, 5(4):584–590. , g p constants from multiple-time uptake data: generalizations. J Cereb Blood Flow Metab 1985, 5(4):584–590. 3. Wong W, Hicks K: A clinically practical method to acquire parametric images of unidirectional metabolic rates and blood spaces. J Nucl Med 1994, 35(7):1206. 4. Zasadny KR, Wahl RL: Enhanced FDG-PET tumor imaging with correlation-coefficient filtered influx-constant images. J Nucl Med 1996, 37(2):371. 5. Hunter GJ, Hamberg LM, Alpert NM, Choi NC, Fischman AJ: Simplified measurement of deoxyglucose utilization rate. J Nucl Med 1996, 37(6):950–954. 6. Sadato N, Tsuchida T, Nakaumra S, Waki A, Uematsu H, Takahashi N, Hayashi N, Yonekura Y, Ishii Y: Non-invasive estimation of the net influx constant using the standardized uptake value for quantification of FDG uptake of tumours. Eur J Nucl Med 1998, 25(6):559–564. 7. Graham M, Peterson L, Hayward R: Comparison of simplified quantitative analyses of FDG uptake. Nucl Med Biol 2000, 27(7):647–655. 8. Sundaram SK, Freedman NM, Carrasquillo JA, Carson JM, Whatley M, Libutti SK, Sellers D, Bacharach SL: Simplified kinetic analysis of tumor 18F-FDG uptake: a dynamic approach. J Nucl Med 2004, 45(8):1328–1333. 9. van den Hoff J, Hofheinz F, Oehme L, Schramm G, Langner J, Beuthien-Baumann B, Steinbach J, Kotzerke J: Dual time point based quantification of metabolic uptake rates in 18F-FDG PET. EJNMMI Res 2013, 3:16. 10. Hamberg L, Hunter G, Alpert N, Choi N, Babich J, Fischman A: The dose uptake ratio as an index of glucose metabolism: useful parameter or oversimplification? J Nucl Med 1994, 35(8):1308. 11. Keyes Jr J: SUV: Standard uptake or silly useless value? J Nucl Med 1995, 36(10):1836–1839. Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com 11. Keyes Jr J: SUV: Standard uptake or silly useless value? J Nucl Med 1995, 36(10):1836–1839.
https://openalex.org/W2336252669	https://icm-experimental.springeropen.com/track/pdf/10.1186/2197-425X-3-S1-A159	English	null	Predictors of poor outcomes in critically ill patients with hematologic malignancy	Intensive care medicine experimental	2,015	cc-by	743	Results Overall, 206 patients (mean age 51.3 ± 13.6 years; 60% male) with HM were admitted to the ICU during the study period. The most common types of HM were acute leukemia (34.5%) and lymphoma (33.5%). Less than half (44.7%) of patients had a stem cell transplant prior to admission. The most frequent ICU admission diagnosis was respiratory failure (39.8%), followed by hemodynamic instability (37.9%). The mean ICU stay was 3 ± 6 days, Objectives d f To identify prognostic variables and outcomes in patients with HM who are admitted to the ICU. Predictors of poor outcomes in critically ill patients with hematologic malignancy M Cornish1, MB Butler2, M Erdogan3, RS Green2,3* From ESICM LIVES 2015 Berlin, Germany. 3-7 October 2015 Methods We performed a structured chart review of all adult patients (age > 18 years) with HM who were admitted to the ICU of a Canadian tertiary care hospital between 2004 and 2014. Outcome measures included ICU length of stay, prolonged ICU stay (> 10 days), ICU readmission, and mortality (ICU, 30-day, 60-day, and 12-month). A logistic regression model was adjusted for potential confounding variables and fitted to determine factors that were predictive of ICU readmission, prolonged ICU stay, and mortality. Introduction with 14.1% of patients requiring prolonged ICU stay. Readmission to the ICU was necessary for 13.1% of patients. During their ICU course, 71.8% of patients required mechanical ventilation and 56.8% required vaso- pressor therapy. Patients with hematologic malignancy (HM) often require admission to the intensive care unit (ICU) due to organ failure through disease progression or complications of treatment. Although there have been improvements in the prognosis of patients with HM, mortality in this patient population remains high. We sought to describe patient characteristics and determinants of outcomes in a Cana- dian tertiary care setting. Of patients admitted to the ICU, 45.6% died during ICU admission, 30-day mortality was 59.2%, 60-day mor- tality was 62.6%, and 12-month mortality was 74.3%. After adjustment, factors predictive of mortality in the ICU were the need for mechanical ventilation (adjusted estimate [AE] 3.38; 95% CI 1.37, 8.37; P = 0.008) and need for vasopressor therapy (AE 3.65; 95% CI 1.67, 7.96; P = 0.001). A surgical procedure was associated with increased survival (AE 0.21; 95% CI 0.05, 0.86; P = 0.031). For mortality in survivors of ICU admission, myeloma was the only significant predictor of 30-day mortality (AE 0.21; 95% CI 0.06, 0.7; P = 0.011), 60-day mortality (AE 0.15; 95% CI 0.04, 0.52; P = 0.003), or 12-month mortality (AE 0.15; 95% CI 0.04, 0.52; P = 0.003). Cornish et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A159 http://www.icm-experimental.com/content/3/S1/A159 Open Access Conclusions Over a 10-year period, ICU mortality in patients with HM was 45.6% and 12-month mortality was 74.3%. Necessity for mechanical ventilation and vasopressor therapy were significant predictors of mortality in the ICU, while myeloma was predictive of longer term mortality. Grant Acknowledgment This study was funded by a Clinician Scientist Award from the Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada. © 2015 Cornish et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Authors’ details 1 1Dalhousie University, Emergency Medicine, Halifax, Canada. 2Dalhousie University, Critical Care Medicine, Halifax, Canada. 3Trauma Nova Scotia, Halifax, Canada. 2Dalhousie University, Critical Care Medicine, Halifax, Canada Full list of author information is available at the end of the article 2Dalhousie University, Critical Care Medicine, Halifax, Canada Full list of author information is available at the end of the article © 2015 Cornish et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2Dalhousie University, Critical Care Medicine, Halifax, Canada Full list of author information is available at the end of the article Page 2 of 2 Page 2 of 2 Cornish et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A159 http://www.icm-experimental.com/content/3/S1/A159 Published: 1 October 2015 Published: 1 October 2015 Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
https://openalex.org/W4205956116	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/DDB2D00C029E906B50D518BE9C6586CA/S0955603600112668a.pdf/div-class-title-information-technology-working-group-div.pdf	English	null	Information Technology working group	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	1,992	cc-by	554	Recommendations 1.Two 3^4 day meetings would be held the summer meeting (Annual Meeting) in July and the winter meeting in January/February. Preliminary soundings seem to suggest that with higher numbers more economical packages can be obtained from conference venues, particularly if conferences are held over weekends. For instance, the Metropole group of hotels (London, Blackpool, Birmingham, Brighton) have agreed to a reduced rate for members if meetings are rotated to these four centres over two years. There are other hotel chains who might be able to offer similar packages. g J y y 2. One meeting per year might be held in London, the other one might be held in appropriate conference locations outside London. 3. Workshops, CME courses and satellite meetings would be incorporated into the programme. There would be a greater emphasis on these activities. g p 4. Named lectures, previously held at Autumn and Spring Meetings (Maudsley Lecture, Mental Health Care Delivery), would be moved to the Summer Meeting. g p g The College Bye-Laws state that, in addition to the Annual General Meeting "not less than three Quarterly General Meetings shall be held in each College year. The date and place of each Quarterly General Meeting shall be fixed by the President or by the Council". This means that four Business Meetings for the membership need to be held each year. It would be possible to change the Bye-Laws and to suggest to the Privy Council that there should be two General Meetings per year. Alternatively, two meetings could be held in conjunction with the two scientific meetings and the other two could be held at the College. They could be held on the same day as Council, possibly in the afternoon followed by an evening reception or a special lecture. A notice would need to appear in the Bulletin informing members of the dates of these meetings. Important motions, as at present, could always be deferred to the Annual General Meeting. g 5. The Maudsley Bequest Lectures could be held the day before the Winter Meeting which could make this a four-day event. These lectures would retain their original remit of being aimed primarily at trainees. 6. The exhibition area could be increased because sponsors would only be exhibiting twice a year. The format used at the recent Brighton meeting was very successful. Both members and exhibiting companies seemed to prefer this more professional approach. 7. for exchange of ideas on information technology and mental illness. Members of the College who are interested are invited to contact Professor McCelland. Recommendations The increase in sponsorship would enable the Programmes and Meetings Committee to invite eminent speakers from overseas. Members are invited to submit any comments to the Registrar, Professor Ann Gath. Approved bv Council January 1992 245 245 The College The College was produced by the Honorary Secretary, Professor Chris Thompson, in 1987,in which a revision of the Quarterly meetings format was suggested, although no further action was taken at that time. Recommendations https://doi.org/10.1192/pb.16.4.245 Published online by Cambridge University Press Information Technology working group An Information Technology working group has been established by the Research Committee of the Royal College of Psychiatrists and is chaired by Professor McClelland of Queen's University Belfast. It is hoped that the group will act as focal point https://doi.org/10.1192/pb.16.4.245 Published online by Cambridge University Press
https://openalex.org/W2888197802	https://www.matec-conferences.org/10.1051/matecconf/201819305071/pdf	English	null	Conceptual framework for managing the competitiveness of the entrepreneurial structures	MATEC web of conferences	2,018	cc-by	4,105	© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Conceptual framework for managing the competitiveness of the entrepreneurial structures Vl di i K nin1 A R m antse a1and Ol Tar tko1 Vladimir Kunin1, Anna Rumyantseva1and Olga Tarutko1 1Private Educational Institution “St. Petersburg University of Management Technologies and Economics”, 44, Lermontovsky Prospekt, St. Petersburg, Russia Vladimir Kunin1, Anna Rumyantseva1and Olga Tarutko1 1Private Educational Institution “St. Petersburg University of Management Technologies and Economics”, 44, Lermontovsky Prospekt, St. Petersburg, Russia Abstract. The paper is devoted to the development of a methodological apparatus for ensuring competitiveness in the formulation of methods and algorithms for assessing competitiveness and its provision in conditions of economic instability. In particular, a method for assessing competitiveness, taking into account both the strategic positioning and operational effectiveness of entrepreneurial structures and their innovative potential, is proposed; the paper also describes the methodology for providing competitiveness and the algorithm for its managing. The findings allow developing specific recommendations for ensuring competitiveness and identifying the reserves for its improvement. Introduction In the market economy conditions the issues of business structures efficiency and performance become more important. Competitiveness as an economic category is inseparably linked with the process of ensuring high performance results. Competitiveness of business structures is a relative indicator that characterizes the existence of differences between goods, works and services of various business structures, as well as between business structures themselves. Despite a rather large number of studies in the field of competitiveness in general and the competitiveness of business structures in particular, the “competitiveness” concept definition lacks the unity of views. In this connection, it is necessary to pay attention to approaches to “competitiveness” concept definition, as well as to disclose the “concept” category content. There is a large number of the “concept” idea interpretations in the literature. Most authors define this category as a way of interpreting, understanding; a system of views or a leading idea. In this paper, the concept is understood as a system of views, laws, principles, approaches, judgments; a leading idea, a certain way of understanding the essence of an idea, category, phenomenon, etc. [1, p.74] The lack of uniformity in determining the “competitiveness” idea is inseparably linked with the economic content of this category. The study of competitiveness and its content is MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 Corresponding author: post_graduate@mail.ru Methodology and theory Conducting analytical procedures for the competitiveness factors of the business structure is necessary in order to identify any opportunities and reserves for ensuring competitiveness. The analysis of competitiveness factors influences the further competitive strategy evolvement that will be aimed at maintaining and developing new and existing competitive advantages. Evaluation of an entrepreneurial structure ability to compete successfully is carried out by comparing the analyzed factors of the studied business structure with the criteria or factors of other business structures competitiveness. The globalization and integration processes inherent in a market economy, as well as an increase in the degree of the Russian economy openness, strengthen the influence of generally accepted standards in the world on Russian business structures. Modern management conditions in the construction sector are characterized by an increase in prices for building materials, energy, transport, etc., and inadequate supply of resources to customers of construction products. These circumstances directly affect the choice of strategy and tactics of the construction sector business structures. The process of ensuring competitiveness is a set of actions aimed at establishing, forming, maintaining and preventing a decline in a given level of competitiveness at all stages of the competitiveness subject functioning. In addition to factors affecting the competitiveness of business structures and areas of competitiveness, an important component in the providing system is an objective instrumentarium for assessing the business structures competitiveness level. The number of indicators and parameters involved in ensuring the entrepreneurial structures competitiveness is quite large and, therefore, it is extremely difficult to take them all into account when making an appraisal. Given this, it is proposed to use in the methodology for assessing the entrepreneurial structures competitiveness only certain indicators that are classified as the most important ones that will act as indicators of the business structure competitivenes s level. As it is shown in [11], the most simple and, at the same time, sufficiently detailed method of assessing the entrepreneurial structures competitiveness is the dynamic method. This method is an effective assessment tool, based on the need to determine the coefficients of operational efficiency and strategic positioning [12, p.205]. Compiling a list of indicators that makes it possible to determine the business structures competitiveness level requires a number of adjustments to the dynamic method of assessing competitiveness. * Corresponding author: post_graduate@mail.ru MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 reflected in the writings of such authors as M. I. Gelvanovsky [2, p.14], V. E. Khrutsky, I. V. Korneeva [3], M. Porter [4], D. S. Voronov [5], P. S. Zavyalov [6, p.8], N. I. Komkov, A. V. Lazarev [7, p.4], M. Meskon, A. Albert, F. Khedouri [8], A. N. Asaul, Sh. M. Mamedov, E. I. Rybnov, N. V. Chepachenko [9, p.23] and others. reflected in the writings of such authors as M. I. Gelvanovsky [2, p.14], V. E. Khrutsky, I. V. Korneeva [3], M. Porter [4], D. S. Voronov [5], P. S. Zavyalov [6, p.8], N. I. Komkov, A. V. Lazarev [7, p.4], M. Meskon, A. Albert, F. Khedouri [8], A. N. Asaul, Sh. M. Mamedov, E. I. Rybnov, N. V. Chepachenko [9, p.23] and others. reflected in the writings of such authors as M. I. Gelvanovsky [2, p.14], V. E. Khrutsky, I. V. Korneeva [3], M. Porter [4], D. S. Voronov [5], P. S. Zavyalov [6, p.8], N. I. Komkov, A. V. Lazarev [7, p.4], M. Meskon, A. Albert, F. Khedouri [8], A. N. Asaul, Sh. M. Mamedov, E. I. Rybnov, N. V. Chepachenko [9, p.23] and others. Summarizing the definitions given in the above-mentioned works and taking into account the signs of competitiveness emphasized by the authors, in this article the competitiveness of entrepreneurial structures is understood to mean the economic category characterizing the actual and potential superiority of the business structure in matters of functioning in a dynamic competitive environment, attracting consumers and meeting their needs, ability to produce and sell a competitive product (constantly increasing its quality), intensifying competitive advantages and expanding the market share achieved without compromising the financial state [10, p.303]. Methodology and theory This circumstance is caused by the fact that in modern managing conditions it is necessary for business structures to have more and more perfect tools for assessing and ensuring competitiveness [11]. A well-known concept of the Balanced Scorecard (BSC), proposed by R. Kaplan and D. Norton in the late 80s of the 20th century, is a good basis for constructing an objective system of entrepreneurial structures competitiveness indicators. This system is an instrument of 2 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 strategic and operational management, the purpose of which is to build the relationship between the business structure strategic objectives, business processes and staff activities at all levels of government, as well as to monitor the implementation of the business structure strategy [13, p.361]. strategic and operational management, the purpose of which is to build the relationship between the business structure strategic objectives, business processes and staff activities at all levels of government, as well as to monitor the implementation of the business structure strategy [13, p.361]. The relationship between the profit margin and sales with the projections of the BSC “Finance”, “Internal business processes” and “Clients and the external environment” is detailed in [11]. As stated in [ibid.], the business structure innovative potential is the indicator of the fourth BSC projection “Personnel, innovation and development”. In this regard, we propose an approach that, along with the factors considered in the dynamic method, takes into account the entrepreneurial structures innovative potential. The following indicators are proposed as indicators of innovative potential: – The intellectual property security coefficient, defined as the share of intangible assets in the total amount of non-current assets; it characterizes the presence of intellectual property in the business structure and rights to it, in the form of trademarks, licenses, certificates, patents that determine effective innovative development. This indicator can be formalized as follows [14, p.12]: FA IС K IС  , (1) (1) FA IС K IС  , where: KIC – intellectual property security coefficient; KIC – intellectual property security coefficient; IC – intellectual property; IC – intellectual property; FA – business structure non-current assets total amount. FA – business structure non-current assets total amount. FA – business structure non-current assets total amount. The formula for calculating the intellectual property security coefficient reflects the degree of equipping the entrepreneurial structure with intellectual capital, in comparison with other basic means of production. KIP – innovative products share index; IP – innovative products volume; Q – business structure sales. Data on the standard values of this indicator vary greatly in the economic literature. In [ibid.] it is noted that the standard value of the innovative products share should exceed the 50% mark. But in [16] it is indicated that the innovative products share in the business structure sales should exceed 30%. In [17] it is stated that the threshold value of this indicator should be at the level of not less than 15%, but it is a matter of the innovative products share on a regional scale. There is no unity of views in determining the threshold value of this indicator in the economic literature. It is necessary to recognize that the amount of innovation costs in the business structure total expenditure, as a rule, needs to be adjusted upwards, as only the introduction of the newest technologies creates conditions for increasing the business structures efficiency. Perspectives of state investment policy in Russia is discussed in [18]. According to the 2016 results, the entrepreneurial structures innovative activity overall level was 8.4% that is 0.9 p.p. lower than in 2015. The values of this indicator have a negative trend for the period from 2012 to 2016. This trend continues in the field of technological innovation, in 2016, 7.3% were engaged in technological innovations. [19, C.6]. Since 2015 the federal statistical observation according to Form No. 4-innovation covers construction organizations. In 2016 financing of technological innovations in construction was carried out entirely at the expense of the entrepreneurial structures own funds [ibid., p.10]. The structure of costs for technological innovation by types of activity in construction differs from similar ones in industrial production, services, and agriculture by the number of expenditure types. There are only two types in the construction organizations, the main of which is the purchase of machinery and equipment (91.3% of all expenses) [ibid., p.13]. Questions of financing of construction with leasing instruments using is discussed in detail in [20] and special aspects of financing technology entrepreneurship is discussed in [21]. According to the data [19, C.13], in 2016 only 2.2% of construction organizations carried out innovative activities, while the share of construction organizations that carried out technological innovation in 2016 was 1.6% [ibid, p.34]. MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 Q – business structure sales. The volume of shipped innovative goods, works and services of innovative-active construction organizations in 2016 was 0.9 mln rubles (in 2015 – 544.5 mln rubles), with the total volume of goods in 2016 – 3159.9 mln rubles (in 2015 – 3599.4 mln rubles) [ibid., p.39]. The above statistics point to a negative trend in the entrepreneurial structures innovative potential, in particular – construction organizations, and make it necessary to take into account the innovative potential in assessing the business structures competitiveness. Methodology and theory As shown in [ibid.], the intellectual property security coefficient may have the following standard values: Low level of innovative activity – the coefficient value is KIC ≤0.05; Average level of innovation activity – the coefficient value is 0.05< KIC ≤0.10; High level of innovative activity – the coefficient value is 0.10< KIC ≤0.15. – The innovative growth coefficient, defined as the share of costs for innovation activity in the total amount of entrepreneurial structure costs. This indicator can be formalized as follows [ibid.]: TC C K I IG  , (2) TC C K I IG  , (2) where: КIG – innovative growth coefficient; КIG – innovative growth coefficient; CI – costs for innovation activity; TC – entrepreneurial structure costs total amount. TC – entrepreneurial structure costs total amount. Standard values of this coefficient in relation to the business structure innovative activity level are as follows: Low level of innovative activity – the coefficient value is 0.35≤КIG≤0.40; Average level of innovation activity – the coefficient value is 0.40< КIG≤0.55; High level of innovative activity – the coefficient value is 0.55<КIG≤0.60 [ibid.]. – The innovative products share index, defined as the specific weight of the innovative products volume in the business structure sales. This indicator can be formalized as follows [15, p.56]: Q IP KIP  , (3) Q IP KIP  , (3) where: KIP – innovative products share index; IP – innovative products volume; 3 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 Results The developed system of indicators allows us to visualize the algorithm for assessing competitiveness. The methodology for calculating operational performance indicators and strategic positioning is discussed in detail in [5], a block diagram of the definition of these integral factors is presented in [22, p.43]. The above coefficients characterizing the business structure innovative potential reflect the growth potential, taking into account the prospects of its innovative development and the share of intangible assets in the business structure general assets that actually represent the level and potential for growth of intellectual capital. Thus, it is proposed to modify the dynamic method of assessing the entrepreneurial structures competitiveness in terms of accounting the innovation potential integral index. The formula for calculating the innovation potential integral index is as follows: p g 5,0 5,0 7,0 5,0 5,0 7,0         IP IG IC K K K N (4) (4) 5,0 5,0 7,0    IG IC N Normalizing the denominator of expression (4), we obtain: 4 4 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 (5) IP IG IC K K K N       29 ,0 29 ,0 42 ,0 In other words, the entrepreneurial structures competitiveness assessment, using a modified dynamic method, is based on the definition of such integrated factors as: strategic positioning, operational efficiency, innovative potential. The modified dynamic method should be considered as the base when assessing the entrepreneurial structures competitiveness. This is due to the fact that it is oriented both to the business structure current state and today’s level of competitiveness, and to calculation the level of its competitiveness in the future that is separated by a certain time lag from the present, owing to the added indicators [23]. In other words, the added indicators allow us to assess the prospects for the entrepreneurial structures competitive development, including the construction sector. For a visual representation of the modified dynamic method for assessing competitiveness, we will present an algorithm for estimating the entrepreneurial structures innovation potential integral factor in the form of a block diagram. A block diagram of the entrepreneurial structures (ES) innovation potential integral factor evaluation is presented in Fig. 1 [ibid.]. Block 3. Innovation potential integral index of covered ES or Block 1.1. Intellectual property security coefficient of covered ES Block 1.2. ES innovative growth coefficient Block 1.3. MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 The following notations are used in Fig. 1: KIC – Intellectual property security coefficient of covered entrepreneurial structure; KSIC – Intellectual property security coefficient of entrepreneurial structure included in the sample; IC – Intellectual property of entrepreneurial structure; ICS – Intellectual property of entrepreneurial structure included in the sample; FA – Non-current assets of entrepreneurial structure; FAS – Non-current assets of entrepreneurial structure included in the sample; KIG – Innovative growth coefficient of entrepreneurial structure; KSIG – Innovative growth coefficient of entrepreneurial structure included in the sample; CI – Costs of entrepreneurial structure for innovation activity; CSI – Costs of entrepreneurial structure included in the sample for innovation activities; TC – Costs of entrepreneurial structure; TCS – Costs of entrepreneurial structure included in the sample; KIP – Innovative products share index of entrepreneurial structure; KSIP – Innovative products share index of entrepreneurial structure included in the sample; IP – Innovative products volume of entrepreneurial structure; IPS – Innovative products volume of entrepreneurial structure included in the sample; Q – Sales of entrepreneurial structure; QS – Sales of entrepreneurial structure included in the sample; N – Innovation potential integral index of entrepreneurial structure; NS – Innovation potential integral index of entrepreneurial structure included in the sample. The following notations are used in Fig. 1: KIC – Intellectual property security coefficient of covered entrepreneurial structure; KSIC – Intellectual property security coefficient of entrepreneurial structure included in the sample; IC – Intellectual property of entrepreneurial structure; ICS – Intellectual property of entrepreneurial structure included in the sample; FA The methodology for ensuring the entrepreneurial structures competitiveness, as well as the degree of internal interaction between its elements, has a strong impact on the competitiveness index. The reaction of the competitiveness management system of an entrepreneurial structure to any changes in the external environment must be immediate, outstripping and suitable to risks and threats of the external entrepreneurial environment; the entrepreneurial structure must be adjusted to increase its dynamism and controllability. The order of managing the entrepreneurial structures competitiveness is determined by the management algorithm. On the basis of all the above, it seems possible to illustrate the algorithm for managing the entrepreneurial structures competitiveness (Fig. 2). At the same time, the management object is an entrepreneurial structure, while competitiveness is the management subject. The purpose of management: Ensuring the business structure competitiveness. Results ES innovative products share index Block 2.3. ES innovative products share index, sample estimation Block 4. Innovation potential integral index of covered ES, sample estimation or Block 2.1. Intellectual property security coefficient of covered ES, sample estimation Block 2.2. ES innovative growth coefficient, sample estimation Block 1.1. Intellectual property security coefficient of covered ES Block 3. Innovation potential integral index of covered ES or Block 1.2. ES innovative growth coefficient Block 1.3. ES innovative products share index Block 1.3. ES innovative products share index Block 2.1. Intellectual property security coefficient of covered ES, sample estimation Block 4. Innovation potential integral index of covered ES, sample estimation Block 2.2. ES innovative growth coefficient, sample estimation p or Block 2.3. ES innovative products share index, sample estimation Fig. 1. A block diagram of the entrepreneurial structures (ES) innovation potential integral factor evaluation 5 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 6 6 https://doi.org/10.1051/matecconf/201819305071 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 Fig.2. Algorithm for managing the entrepreneurial structures competitiveness Di i YES NO NO YES Determination of the evaluation method Research sample preparation Data collection on business structures Assessment of competitiveness Analysis of evaluation results Looking for reserves to improve competitiveness and developing recommendations for improving it Conducting an assessment of competitiveness taking into account the developed activities Events correcting Developed measures adoption Is competitiveness provided? Formulating requirements for research – setting goals, defining tasks Determination of factors and criteria for competitiveness Is competitiveness provided? Recommendations development for further competitiveness improvement Formulating requirements for research – setting goals, defining tasks Analysis of evaluation results Is competitiveness provided? Recommendations development for further competitiveness improvement Looking for reserves to improve competitiveness and developing recommendations for improving it Conducting an assessment of competitiveness taking into account the developed activities Is competitiveness provided? Events correcting Developed measures adoption Fig.2. Algorithm for managing the entrepreneurial structures competitiveness Discussion The desire to increase the entrepreneurial activities efficiency and ensure the entrepreneurial structures sustainable development necessitates a detailed analysis of their competitiveness and the development of measures aimed at ensuring competitiveness in conditions of economic instability. 7 https://doi.org/10.1051/matecconf/201819305071 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 The toolkit for assessing and managing competitiveness presented in this article makes it possible to compare the studied entrepreneurial structure with the actual business structures on the market – competitors, and not with the abstract organization accepted as the standard. A key role in ensuring the business structures competitiveness belongs to integral factors, such as operational efficiency and strategic positioning that in turn, together with the intellectual property security coefficient, innovation growth coefficient and innovation share, characterizing innovation potential, allow us to determine the level of business structures competitiveness. The modified dynamic method, on the one hand, is focused on the current state of the business structure and on its current level of competitiveness; and on the other hand, the added coefficients take into account the competitiveness of the business structure in the future that is separated by a specific time period from the present. Conclusion The issue of improving the strategy aimed at ensuring the entrepreneurial structures competitiveness, both in economic science and in practical activities, is very multifaceted, requires a systematic approach and concentration, in particular, on improving research methods and assessing competitiveness. When assessing the entrepreneurial structures competitiveness, innovation potential should be taken into account as a component that allows to take into account future competitive opportunities associated with the level of innovation in the business structure and with the opportunity in the future to compete with entrepreneurial structures that actively implement innovations in their entrepreneurial activities. The entrepreneurial structures competitiveness management should be in accordance with the methodology proposed in the work, based on the use of accounting for the impact of internal and external factors and the use of feedbacks between management impacts and the results of applying the proposed activities to ensure competitiveness. References 1. R. A. Fatkhutdinov, Contemporary competition, 1 (1), 73-86 (2007) 2. M. I. Gelvanovsky, Our business, 6(8), 10-16 (2011) 3. I. V. Korneeva, Marketing: Textbook and workshop for academic baccalaureate.. (Moscow, Yurayt Publishing House, 2018) 4. E. M. Porter, International competition. Competitive advantages of countries. (Moscow, Alpina Publisher, 2016) 5. D. S. Voronov, Marketing in Russia and abroad, 5, 92-102 (2014) 6. P. S. Zavyalov, Marketing in diagrams, drawings, tables: Textbook. (Moscow, SRC INFRA-M, 2014) 7. N. I. Komkov, A. V. Lazarev, Problems of forecasting, 4, 3-20 (2007) 8. M. Kh. Meskon, M. Albert, F. Khedouri, Fundamentals of Management. 3rd ed. (Transl. from English). (Moscow, Williams Publishing House, 2012) 9. A. N. Asaul, Formation of competitive advantage of business entities in construction. (Petersburg, ANO “IPEV”, 2014) 10. O. A. Tarutko, Analysis and refinement of the category-conceptual apparatus of the entrepreneurial structures competitiveness theory, Development of financial relations in the period of the digital economy formation. Coll. of scient. papers of the 8 MATEC Web of Conferences 193, 05071 (2018) ESCI 2018 https://doi.org/10.1051/matecconf/201819305071 international scientific-practical conference. St. Petersburg, Publishing house of the St. Petersburg University of Management Technologies and Economics , 297-306 (2018) international scientific-practical conference. St. Petersburg, Publishing house of the St. Petersburg University of Management Technologies and Economics , 297-306 (2018) 11. V. A. Kunin, O. A. Tarutko, Problems of Modern Economics, 1 (65), 65-68 (2018) 12. E. A. Tolstikov, Symbol of Science, 11, 204-207 (2016) 13. I. B. Beregovaya, A. A. Morozkin, The system of balanced indicators, Young scientist, 32, 361-364 (2017) 16. P. P. Korsunov, Management of economic systems, 1 (95), 6-16 (2017) 17. Zh. A. Mingaleva, I. I. Platynyuk, Creative economy, 4, 52-58 (2011) 18. E. S. Mityakov, S. N. Mityakov, Comparative analysis of approaches to calculating the generalized index of economic security of Russia, Modern problems of science and education, 3 (2014) 19. A. V. Kalina, I. P. Savelieva, Vestnik of the South Ural State University. Series: Economics and Management, 4, 15-24 (2014) 20. A. Rumyantseva, O. Smeshko, MATEC Web of Conferences, 170, 01083 (2018), DOI: https://doi.org/10.1051/matecconf/201817001083 21. URL: http://www.csrs.ru/archive/stat_2017_inno/innovation_2017.pdf 25. URL: http://rosrid.ru 21. URL: http://www.csrs.ru/archive/stat_2017_inno/innovation_2017.pdf 22. O. Aleksandrova, E. Ivleva, V. Sukhacheva, A. Rumyantseva, E3S Web of Conferences, 33, 03057 (2018) 23. V. Bichurina, A. Rumyantseva, MATEC Web of Conferences, 170, 01084 (2018), https://doi.org/10.1051/matecconf/201817001084 24. V. A. Kunin, O. A. Tarutko, Economics and management, 4 (150), 37-44 (2018) 25 URL: http://rosrid ru 24. V. A. Kunin, O. A. Tarutko, Economics and management, 4 (150), 37-44 (2018) 25. URL: http://rosrid.ru 25. URL: http://rosrid.ru 9
https://openalex.org/W3002072770	https://link.springer.com/content/pdf/10.1057/s41278-020-00148-5.pdf	English	null	Analyzing the relative efficiency of China’s Yangtze River port system	Maritime economics & logistics	2,020	cc-by	10,548	Maritime Economics & Logistics (2020) 22:640–660 https://doi.org/10.1057/s41278-020-00148-5 Maritime Economics & Logistics (2020) 22:640–660 https://doi.org/10.1057/s41278-020-00148-5 ORIGINAL ARTICLE Analyzing the relative efficiency of China’s Yangtze River port system Shilin Ye1,2 · Xinhua Qi1,2 · Yecheng Xu3 Published online: 24 January 2020 © The Author(s) 2020 1 Key Laboratory for Humid Subtropical Eco‑geographical Processes of the Ministry of Education, Fujian Normal University, Fuzhou, People’s Republic of China 2 School of Geographical Sciences, Fujian Normal University, Fuzhou, People’s Republic of China 3 School of Forestry and Wildlife Sciences, Auburn University, Auburn, USA * Xinhua Qi fjqxh@fjnu.edu.cn Keywords River port · Port efficiency · Super-SBM model · Malmquist productivity index · Yangtze River port system 1 Key Laboratory for Humid Subtropical Eco‑geographical Processes of the Ministry of Education, Fujian Normal University, Fuzhou, People’s Republic of China 3 School of Forestry and Wildlife Sciences, Auburn University, Auburn, USA 2 School of Geographical Sciences, Fujian Normal University, Fuzhou, People’s Republic of China 1 Introduction As critical nodes and potential bottlenecks in the international logistical network, ports play a pivotal role in global trade, particularly as hubs, facilitating intermodal transfers at intersections with other modes of transport. Thanks to rapid economic globalization and the development of modern logistics, the function of ports has evolved from a traditional waterway transit node to a complex logistics center, inte- grating transportation, law, information, industry, trade, and other systems (Drewe and Janssen 1998). Currently, ports are deeply embedded in global production, transport, distribution and sales, forming a vital link in the global value and supply chain (Robinson 2002). Ports have become one of the main engines for the promo- tion of regional economic development and an important contributor to a nation’s international competitiveness (Suykens and Van de Voorde 1998; Chin and Tongzon 1998). However, some factors, including global economic integration, improvements in supply chain management methods, the rapid growth of transport infrastructures, and intermodal transportation, have caused increasingly fierce competition among ports worldwide, obliging those ports to provide more value-added logistics services and continuously improve their operational efficiency (Wang and Cullinane 2006; Verhoeven 2010; Da Silva and Rocha 2012). Accordingly, improving port efficiency has become a critical task for local governments, particularly in rapidly develop- ing regions. This increasing competitive pressure on ports as well as the demands of regional economic development have made port efficiency an important topic among scholars since the second half of the 1990s (Wang et al. 2004; González and Trujillo 2008). In recent years, the large economic centers and distant hinterland regions of the Yangtze River economic belt (YZREB) have been more closely connected, with the continuous improvement of comprehensive transportation systems, especially the development of transport along inland waterways, which has promoted the rapid development of the regional economy along the YZREB. Currently, the develop- ments of the Yangtze River’s shipping markets and port systems have received extensive attention from academia, with numerous research studies. These stud- ies principally cover the evolution of the spatial structure of the port systems, the relationship between ports and cities, the optimization of cargo consolidation and distribution systems, etc. (Zhao et al. 2019). However, we still lack sufficient under- standing of the efficiency characteristics of river ports along the Yangtze (Yuen et al. 2013; Barros et al. 2016). Additionally, quantitative analysis of these ports’ efficiency is required. Abstract In spite of the rapid growth of the Yangtze River port system (YRPS) in recent years, knowledge regarding the efficiency characteristics of these ports along the Yangtze River is still limited. This article evaluates the relative efficiency of 22 major ports along the Yangtze River from 2010 to 2016 by using the novel super slacks-based measure (super-SBM) model and the Malmquist productivity index. The YRPS showed a relatively low level of average technical efficiency in 2016, and the tech- nical efficiency values of these ports increased generally from upstream to down- stream. In terms of returns to scale, the ports of Shanghai, Nantong, and Suzhou should pay attention to resource allocation, technological advancement, and insti- tutional innovation in the future. The remaining 19 ports, however, should increase investment in the optimization of production-capacity structures, the upgrading of terminal equipment, the improvement of consolidation and distribution systems, channel regulation, and information sharing among terminals. Our results also show that the production technology, resource utilization, enterprise management, and scale economies of the YRPS improved during 2010–2016, and this significantly enhanced the total factor productivity (TFP). Based on these findings, we propose several development strategies for the YRPS, such as improving the consolidation and distribution systems of ports, encouraging the cross-regional integration of port and shipping enterprises, strengthening the coconstruction and sharing of infrastruc- ture, and optimizing the hub-and-spoke network of the YRPS. Keywords River port · Port efficiency · Super-SBM model · Malmquist productivity index · Yangtze River port system * Xinhua Qi fjqxh@fjnu.edu.cn Vol: Analyzing the relative efficiency of China’s Yangtze River… 641 2 Literature review of port efficiency analysis Port efficiency, which deeply influences port competitiveness and determines whether a port can survive in a fiercely competitive environment, has been broadly investigated. After reviewing the existing literature, we can determine that efficiency mainly focuses on the following two issues.fi fi First, how can port efficiency be measured comprehensively? A number of approaches to measure port efficiency have been adopted in existing literature. In the early days, efficiency measurement was usually based on single or multi- ple indicators, such as single-factor productivity (De Monie 1987), total factor productivity (Kim and Sachish 1986), or multiple regression analysis (Tongzon 1995). These measurements are valuable for describing port efficiency from one or more dimensions and are easy to apply. However, their limitations are also obvious. Port efficiency cannot be easily fully represented by a single indicator, considering that the information delivered by any single indicator is insufficient. On the other hand, the main weakness of multiple indicators lies in the difficulty of judging whether variations in the different indicators improve or reduce port efficiency (Cullinane and Wang 2006a). fi y g Several more reasonable and persuasive approaches have been suggested for a more comprehensive assessment of port efficiency. Among them, data envelop- ment analysis (DEA) (Tongzon 2001; Barros 2003; Cullinane et al. 2005; Yuen et al. 2013; Beuren et al. 2018) and stochastic frontier analysis (SFA) (Liu 1995; Coto-Millan et al. 2000; Cullinane and Song 2003; Pagano et al. 2013; Sere- brisky et al. 2016) have been widely used (Cullinane et al. 2006; Panayides et al. 2009; Merkel and Holmgren 2017); For example, Barros and Athanassiou (2004) applied DEA to estimate the relative efficiency of two Greek and four Portuguese seaports from 1998 to 2000. These authors suggested that scale economies and privatization improve performance. Similarly, Beuren et al. (2018) evaluated and compared the efficiency of the main Brazilian ports by using DEA to assess whether port efficiency is significantly affected by the nature of the cargo that is handled or the management model that is adopted. Their results showed no signif- icant efficiency differences among different management models or types of cargo handled. Pagano et al. (2013) used SFA to compare the effectiveness of privatized Panamanian ports with US ports at varying degrees of privatization; their results estimated the savings and effectiveness gains from privatization.fi f Second, what are the factors that determine port efficiency? Numerous studies have focused on this issue. 1 Introduction In the present work, the efficiency of the Yangtze River port system (YRPS) is measured by using the super slacks-based measure (super-SBM) model (Tone 2002) and Malmquist productivity index (Malmquist 1953). We expect this study to provide a reasonable decision-making basis for port operations and pro- mote the synergetic development of river port systems. The remainder of this manuscript is organized as follows: Section 2 reviews the existing studies on port efficiency. Section 3 presents the characteristics of the Yang- tze River port system. Section 4 details the methodology and describes the data. Section 5 presents the results and discussion, and Sect. 6 describes the conclusions. S. Ye et al. 642 2 Literature review of port efficiency analysis Scholars have attempted to examine how port effi- ciency is associated with variables that are related to management and/or policies (Notteboom et al. 2000; Tongzon and Heng 2005; Cullinane et al. 2005; Yuen et al. 2013; Serebrisky et al. 2016). Most of these studies have concluded that privatization and deregulation in the port sector positively affect efficiency (Culli- nane et al. 2002; Cullinane and Song 2003; Barros and Athanassiou 2004; Pagano et al. 2013); For example, private-sector participation in Asian, European, and American container terminals has increased operational efficiency (Tongzon and Heng 2005). In contrast, other study argued that a clear-cut relationship between Analyzing the relative efficiency of China’s Yangtze River… 643 port ownership or management and efficiency could not be proven (Liu 1995; Notteboom et al. 2000; González and Trujillo 2009). According to an empirical study with a sample of 19 ports in North America and Europe, Valentine and Gray (2002) found that ownership does not significantly affect port efficiency.fii port ownership or management and efficiency could not be proven (Liu 1995; Notteboom et al. 2000; González and Trujillo 2009). According to an empirical study with a sample of 19 ports in North America and Europe, Valentine and Gray (2002) found that ownership does not significantly affect port efficiency.fii iffi Several studies have found that port efficiency increases significantly with port size, driven by economies of scale and agglomeration (Martínez-Budria et al. 1999; Notteboom et al. 2000; Barros 2006; De Oliveira and Cariou 2011; Schøyen and Odeck 2013). Barros and Athanassiou (2004) believed that scale is the main source of port efficiency. In addition, some studies examined the relationship between port efficiency and other factors, such as the geographical location and accessibility of the port (Liu 1995; Caldeirinha and Felicio 2011; Bergantino and Musso 2011), the port’s competitive position (Haralambides et al. 2002; Defilippi and Flor 2008; Yuen et al. 2013; De Oliveira and Cariou 2015), the essential production factors (Dowd and Leschine 1990; Wan et al. 2014), and the economic level of the port hinterland (Tongzon 2002; Cheon 2007).fi The above-cited studies mainly focused on the characteristics of seaport effi- ciency and its influencing factors by using a variety of research methods across different periods or regions. Few studies have addressed the river ports along the Yangtze or in other riverine areas of China and across the world. 1 The mainstream waterway of the Yangtze River is around 2800 km and can be divided into three main reaches: the upper reach from Yichang to Yibin, the middle reach from Wuhan to Yichang, and the lower reach from Shanghai to Wuhan. 2 Literature review of port efficiency analysis In fact, each river port system has its own dynamics, and its spatial and functional development is both location and time specific (Notteboom 2007). This paper focuses exclusively on the nature of port efficiency along the Yangtze, with a specific emphasis on the dynamic characteristics of technical efficiency and total factor productivity by using both the super-SBM model and Malmquist productivity index. 3 Yangtze River port system The Yangtze River originates from the Tanggula Mountains on the Qinghai-Tibet Plateau. The river is 6397 km long,1 the longest river in Asia and the third longest in the world. The drainage area of the river is more than 1.8 million km2, account- ing approximately one-fifth of China’s land mass (Fig. 1). The river flows through three economic zones (eastern, central, and western) of China and plays an increas- ingly important socioeconomic and ecological role in China. Since the beginning of the 21st century, the YZREB has become the largest inland industrial belt and one of the most important manufacturing industry bases in the world (Yu et al. 2015). Meanwhile, as the region with the most developed economy and the highest degree of urban agglomeration in China, the Yangtze River Delta has developed into one of the most important foundations for economic growth. Against this background, the YRPS has transitioned into periods of sustained and rapid development. The ports along the Yangtze River handled over 2.5 billion tons of cargo in 2017, increasing S. Ye et al. 644 Fig. 1 Major ports along the Yangtze River Fig. 1 Major ports along the Yangtze River by more than 8.2% compared with the previous year2 and ranking first among the world’s inland waterway ports. Given its strategic position for China and its criti- cal role in the YZREB, the effective and sustainable development of the YRPS has attracted the interest of policymakers and scholars.i However, the ports along the Yangtze River have faced fiercer competition com- pared with seaports because of their proximity, overlapping hinterlands, and similar shipping conditions within specific areas of the inland waterway. Since the reform of China’s port management system in 2001, all port authorities were devolved from the central government to various local governments, and the latter became the main bodies responsible for the investment, construction, and maintenance of port infra- structure. Subsequently, more port cities along the Yangtze River have joined the spree of port infrastructure construction, creating a serious excess of terminals. At present, more than 3900 cargo berths are present on the mainstream waterway of the Yangtze River, of which 581 are 10,000-ton berths, measuring around 0.7 berths per kilometer of riverbank.3 This situation has caused the disorderly and inefficient uti- lization of riverbank resources and intensified hinterland competition among nearby ports (for instance, Yibin–Luzhou–Chongqing, Chongqing–Wuhan, Wuhu–Ma’anshan, and Nanjing–Suzhou). 2 http://finance.people.com.cn/n1/2018/0105/c1004-29746857.html. 3 http://finance.people.com.cn/n1/2018/0518/c1004-29998355.html. 2 http://finance.people.com.cn/n1/2018/0105/c1004-29746857.html. 3i 3 Yangtze River port system In addition, the development of the Yangtze River ports faces a series of problems: The shipping services sector and related maritime clusters have lagged, consolidation and distribution systems have become insufficient, management Analyzing the relative efficiency of China’s Yangtze River… 645 practices have become inferior, etc. Accordingly, methods to improve the competitive environment and increase the production efficiency of ports have become a priority for the sustainable development of the YRPS. practices have become inferior, etc. Accordingly, methods to improve the competitive environment and increase the production efficiency of ports have become a priority for the sustainable development of the YRPS. 4.1 Super‑SBM model For the SBM model, the efficiency of a DMU (x0, y0) can be measured by the fol- lowing fractional problem: (3) min 휌= 1 −1 m ∑m i=1 s− j xi0 1 + 1 s ∑s i=1 s+ j yi0 (3) (3) S. Ye et al. 646 subject to x0 = X휆+ s− (4) (5) y0 = Y휆−s+ y0 = Y휆−s+ (5) (6) 휆≥0, s−≥0, s+ ≥0, 휆≥0, s−≥0, s+ ≥0, (6) where the value of ρ is the efficiency score at DMU (x0, y0). A DMU (x0, y0) is SBM-efficient if ρ* = 1, which means s−* = s+* = 0.fi fi To further compare and rank SBM-efficient DMUs, Tone (2002) proposed the super-efficiency SBM model, which is derived from the SBM model and the super- efficiency DEA model that was proposed by Andersen and Petersen (1993). The super- efficiency SBM model is described as (7) 𝛿∗= min 𝛿= 1 m ∑m i=1 ̄xi xi0 1 s ∑s r=1 ̄yr xr0 (7) (8) Subject to ̄x ≥ n ∑ j=1,j≠0 𝜆jxj (8) (9) ̄y ≥ n ∑ j=1,j≠0 𝜆jyj (9) (10) ̄x ≥x0, ̄y ≤y0, ̄y ≥0; 𝜆j ≥0, j = 1, … , n. (10) 4.1 Super‑SBM model DEA is a nonparametric technique that is used in operations research and econometrics for multivariate frontier estimation and ranking. This approach can be utilized to calcu- late the relative efficiency levels within a group of decision-making units (DMUs) by comparing the relationship (distance) between a DMU and the efficient frontier (Panay- ides et al. 2009). In the field of port efficiency measurement, DEA has become one of the mainstream technical tools, offering remarkable advantages such as not requiring the adoption of a specific form of frontier production function, nonsubjective weights, and simple data requirements (Barros and Athanassiou 2004; Lai et al. 2012). However, traditional DEA models are radial and angular measurement methods, creating effi- ciency measurements that reflect equiproportional reductions in inputs or outputs onto the best-practice frontier (Farrell 1957). The accuracy of traditional DEA efficiency measurements is affected by the method’s inability to consider input excesses and out- put shortfalls from inefficient DMUs (known as slacks). To overcome the shortcomings of traditional DEA, Tone (2001) proposed a slacks-based measure of efficiency (SBM), which is nonradial and directly handles input or output slacks. The SBM model can be interpreted as profit maximizing, in contrast to traditional DEA models such as the CCR model, which attempt to find the maximum ratio of the virtual output over the virtual input. Consider a production system with n homogeneous DMUs expressed as the input and output matrices X = (xij) ∈ Rm×n and Y = (yij) ∈ Rs×n, respectively. Furthermore, all the inputs and outputs are assumed to be positive, i.e., X > 0 and Y > 0. The production possibility set P is defined as (1) P = {(x, y)\|x ≥X휆, y ≤Y휆, 휆≥0}, (1) where λ is a nonnegative vector in Rn. A certain DMU (x0, y0) can be described as (2) x0 = X휆+ s−, y0Y휆−s+, (2) where λ ≥ 0 and s− and s+ indicate the input and output slack variables, respectively. For the SBM model, the efficiency of a DMU (x0, y0) can be measured by the fol- lowing fractional problem: where λ ≥ 0 and s− and s+ indicate the input and output slack variables, respectively. 4 The 22 sample ports: Luzhou, Chongqing, Yichang, Jingzhou, Yueyang, Wuhan, Huangshi, Jiujiang, Anqing, Chizhou, Tongling, Wuhu, Ma’anshan, Nanjing, Zhenjiang, Taizhou, Yangzhou, Jiangyin, Changzhou, Suzhou, Nantong, and Shanghai. 4.2 Malmquist productivity index Although cross-sectional data enable one to track each producer and its efficiency through a sequence of periods, the results might contain bias arising from situations specific to the period under examination (Kumbhakar and Lovell 2000; Panayides et al. 2009). To overcome this inherent limitation, the Malmquist productivity index (MPI) has been proposed (Malmquist 1953; Caves et al. 1982; Färe et al. 1994) and has become the most widely applied approach for measuring changes in both effi- ciency and productivity over time (Cullinane et al. 2004). The MPI is based on the Shephard distance function (Shephard 1970). Färe et al. (1994) defined distance functions with respect to two different periods and specified the output-based MPI as the geometric mean of two CCD-type MPIs (Caves et al. 1982): (11) M0(xt+1, yt+1, xt, yt) = [ Dt o(xt+1, yt+1) Dt o(xt, yt) × Dt+1 o (xt+1, yt+1) Dt+1 o (xt, yt) ]1∕2 . (11) Analyzing the relative efficiency of China’s Yangtze River… 647 The index can be broken down into two components: The index can be broken down into two components: M0(xt+1, yt+1, xt, yt) = Dt o(xt+1, yt+1) Dt o(xt, yt) × [ Dt o(xt+1, yt+1) Dt+1 o (xt+1, yt+1) × Dt o(xt, yt) Dt+1 o (xt, yt) ]1∕2 , (12) fi (12) [ ] where the ratio outside the brackets measures the changes in technical efficiency (i.e., the change in the distance of the observed production from the maximum potential production) between periods t and t + 1. The geometric mean of the two ratios inside the brackets measures the technological change (TC) between two peri- ods evaluated at xt and xt+1. In addition, if strong disposability of inputs and outputs is assumed (Färe et al. 1994), the technical efficiency change (TEC) can be further decomposed into the pure technical efficiency change (PTEC) and scale efficiency change (SEC), where TEC = PTEC × SEC. The multiplicative decomposition of this index becomes (13) M0(xt+1, yt+1, xt, yt) = Dt+1 o (xt+1, yt+1\|VRS) Dt o(xt, yt\|CRS) × Dt+1 o (xt+1, yt+1\|VRS) Dt o(xt, yt\|VRS) × [ Dt o(xt+1, yt+1\|CRS) Dt+1 o (xt+1, yt+1\|CRS) × Dt o(xt, yt\|CRS) Dt+1 o (xt, yt\|CRS) ]1∕2 , where CRS and VRS represent the constant returns to scale and the variable returns to scale assumptions, respectively. If M > 1, productivity is improved. If M < 1, pro- ductivity is reduced. If M = 1, productivity remains unchanged. 4.3 Data collection One critical issue in port efficiency measurement is sample selection. Although no explicit restrictions exist for the selection of ports, having competitive relationships among the selected ports is preferable in terms of efficiency measurement. With this approach, the ranking of relative efficiencies becomes more valuable and pro- vides more references for the sustainable development of these ports (Panayides et al. 2009). In addition, Ali et al. (1989) and Bowlin (1987) both suggested that the number of DMUs should be at least twice the sum of the input and output vari- ables. To serve the purpose of this study, 22 major ports along the Yangtze River4 were selected as a sample; their locations are depicted in Fig. 1. These ports, which are the primary nodes of the Yangtze River’s shipping network, handle more than 90% of the cargo and container throughput of the Yangtze River Basin. The beaded- string-like distribution of these ports along the Yangtze River and their geographic proximity result in overlapping hinterlands. For a long time, these ports have both S. Ye et al. 648 competed and cooperated with each other in areas such as sources of goods, high- end shipping talent, and shipping cluster by and large. One could reasonably label these ports as a close-knit organic whole. competed and cooperated with each other in areas such as sources of goods, high- end shipping talent, and shipping cluster by and large. One could reasonably label these ports as a close-knit organic whole. While perusing earlier research, it was observed that the input and output vari- ables that were used for measuring port efficiency were quite different. The most frequent input variables often related to the efficient use of land, labor, and capital (Dowd and Leschine 1990), e.g., length of berths, terminal areas, warehouse capac- ity, number of gantry cranes and employees, and quayside water depth (Tongzon 2001; Barros and Athanassiou 2004; Cullinane and Wang 2006b; Wu and Goh 2010; De Oliveira and Cariou 2011; Nguyen et al. 2018). At the same time, cargo through- put, container throughput, and number of ship calls have often been chosen as out- put variables (Valentine and Gray 2001; Itoh 2002; Barros and Athanassiou 2004; Cullinane et al. 2006; Beuren et al. 2018; Nguyen et al. 2018). These studies provide some referencing for the present study. 5 There is a statistical overlap between port cargo throughput and container throughput. Container throughput reflects the total number of import and export containers in a certain port over a period of time, including heavy and empty containers. The cargo throughput refers to the amount of cargo trans- ported in and out of the port area by water, but it does not reflect the number of empty containers han- dled by the port. And this is critical to the port’s production efficiency. Therefore, the simultaneous use of cargo throughput and container throughput as output variables in port efficiency calculations is a com- promise approach under existing data conditions. 4.3 Data collection However, we consider that inputs and out- puts should accurately reflect the actual objectives and processes of port/terminal production, alongside the availability of data on the Yangtze River ports. Therefore, quay-wall length, number of berths, and channel (dredging) depth are used in the present work as inputs, and general cargo throughput and container throughput as outputs.5 Panel data for the 22 major ports in 2010, 2012, 2014, and 2016 were selected, giving a sample of 88 observations. The raw data were mainly obtained from the China Statistical Yearbook, the China Transportation and Communications Year- book, and the China Ports Yearbook, each of which consider several issues. To solve the problem of data unavailability for some ports, data that were collected from the websites or annual reports of sampled ports and terminal operators were also incor- porated into the final dataset. Some important statistics relating to the sample are summarized in Table 1. 5.1 Efficiency of the Yangtze River port system Based on this dataset, the efficiency characteristics of the Yangtze River port system in 2016 were estimated and compared by using the super-SBM DEA model of the MyDEA software package. The efficiency measurements are presented in Table 2. fi These results show that the Yangtze River port system in 2016 had a relatively low average technical efficiency (TE) of 0.642, at a great distance from the production Analyzing the relative efficiency of China’s Yangtze River… 649 ble 1 Summary statistics of the sample ource Data obtained from the China Statistical Yearbook, China Transportation and Communications Yearbook, China Ports Yearbook, etc. Outputs Inputs Cargo throughput (ton) Container throughput (TEU) Quay line length (m) Berth number (unit) Channel dredg- ing depth (m) ean 12,904.6 211.2 22,549.8 267.0 7.6 in. 453.0 0.7 801.7 19.0 2.7 ax. 75,528.9 3713.3 170,970.0 2570.0 15.5 andard deviation 16,040.4 696.6 34,383.8 509.7 3.6 um 1,135,607.2 18,583.7 1,984,378.8 23,498.0 666.1 ble 1 Summary statistics of the sample urce Data obtained from the China Statistical Yearbook, China Transportation and Communications Yearbook, China Ports Yearbook, etc. Outputs Inputs Cargo throughput (ton) Container throughput (TEU) Quay line length (m) Berth number (unit) Channel dredg- ing depth (m) ean 12,904.6 211.2 22,549.8 267.0 7.6 in. 453.0 0.7 801.7 19.0 2.7 ax. 75,528.9 3713.3 170,970.0 2570.0 15.5 andard deviation 16,040.4 696.6 34,383.8 509.7 3.6 m 1,135,607.2 18,583.7 1,984,378.8 23,498.0 666.1 ble 1 Summary statistics of the sample urce Data obtained from the China Statistical Yearbook, China Transportation and Communications Yearbook, China Ports Yearbook, etc. Outputs Inputs Cargo throughput (ton) Container throughput (TEU) Quay line length (m) Berth number (unit) Channel dredg- ing depth (m) ean 12,904.6 211.2 22,549.8 267.0 7.6 in. 453.0 0.7 801.7 19.0 2.7 ax. 75,528.9 3713.3 170,970.0 2570.0 15.5 andard deviation 16,040.4 696.6 34,383.8 509.7 3.6 m 1,135,607.2 18,583.7 1,984,378.8 23,498.0 666.1 S. Ye et al. 650 Table 2 The efficiency of the Yangtze River port system in 2016 IRS represents increasing returns to scale, and DRS represents decreasing returns to scale. TE, PTE, and SE represent the comprehensive technical efficiency, pure technical efficiency, and scale efficiency, respectively. 5.1 Efficiency of the Yangtze River port system Starting with Analyzing the relative efficiency of China’s Yangtze River… 651 SE, Shanghai showed the highest SE (2.714) and was the only port to achieve scale effectiveness, followed by Yangzhou, Nantong, and Changzhou (0.998, 0.993, and 0.923, respectively). These three ports showed relatively high SEs and were very close to the optimal SE state. Moreover, the SEs of the remaining sample ports devi- ated from the optimal state to varying degrees. The standard deviation and mean of the SE were 0.505 and 0.705, respectively, indicating that the SE of the Yangtze River port system demonstrates significant differences, and the actual production scales are quite far from optimal. The scale effect of the Yangtze River port system has not yet been fully realized. Compared with the SE, differences in the PTE were more pronounced: the standard deviation of the PTE was 0.629. Nine ports, includ- ing Suzhou (PTE = 3.478), Jingzhou (PTE = 1.680), Changzhou (PTE = 1.279), and Yueyang (PTE = 1.157), reached the optimal PTE level, comprising 40.9% of the sample ports, which was more than the number of ports with optimal TEs and SEs. The YRPS presented a relatively high average PTE (0.943), indicating that the PTE was very close to the optimal level and was affected by factors such as technol- ogy, operational modes, and enterprise management and decisions. The penultimate column of Table 2 lists the positions of the 22 sample ports in the VRS frontier. The ports with decreasing returns to scale are Shanghai, Nantong, and Suzhou, comprising 13.6% of the sample ports. This result indicates that the development of these three ports, which are located near the Yangtze River Estuary, exceeded the optimal scale. In other words, under the premise of maintaining the current output level, input redundancy should restrict any improvements in the SE of these ports. The remaining ports all show increasing returns to scale, although in actual practice much of their (excess) capacity and resources, particularly in the middle and upper reaches of the Yangtze River, are dysfunctional and obsolete, a structural overcapacity problem caused by blind port competition in the past. There- fore, investing in resources is still an effective method to improve the SE of these ports, from the perspective of input–output ratio. There is currently more capacity than is required to produce the current through- put level. 5.1 Efficiency of the Yangtze River port system Location Port TE PTE SE Returns to scale Rank Upper reach Luzhou 0.219 1.085 0.202 IRS 19 Chongqing 0.370 0.478 0.775 IRS 15 Middle reach Yichang 0.149 1.001 0.149 IRS 21 Jingzhou 0.195 1.680 0.116 IRS 20 Yueyang 0.700 1.157 0.605 IRS 6 Wuhan 0.376 0.668 0.562 IRS 14 Huangshi 0.261 0.655 0.398 IRS 17 Jiujiang 0.459 0.730 0.629 IRS 11 Lower reach Anqing 0.120 0.425 0.283 IRS 22 Chizhou 0.241 0.536 0.449 IRS 18 Tongling 0.634 1.003 0.632 IRS 7 Wuhu 0.457 0.623 0.733 IRS 12 Ma’anshan 0.406 0.584 0.695 IRS 13 Nanjing 0.622 0.719 0.865 IRS 8 Zhenjiang 0.331 0.462 0.716 IRS 16 Taizhou 0.477 0.606 0.787 IRS 10 Yangzhou 1.007 1.009 0.998 IRS 4 Jiangyin 0.522 0.674 0.774 IRS 9 Changzhou 1.181 1.279 0.923 IRS 3 Suzhou 1.795 3.478 0.516 DRS 2 Nantong 0.887 0.893 0.993 DRS 5 Shanghai 2.714 1.000 2.714 DRS 1 SD 0.592 0.629 0.505 Mean 0.642 0.943 0.705 Table 2 The efficiency of the Yangtze River port system in 2016 IRS represents increasing returns to scale, and DRS represents decreasing returns to scale. TE, PTE, and SE represent the comprehensive technical efficiency, pure technical efficiency, and scale efficiency, respectively. frontier. The ports of Shanghai (TE = 2.714), Suzhou (TE = 1.795), Changzhou (TE = 1.181), and Yangzhou (TE = 1.007) were the few ports with TEs greater than one, representing only 3 out of all 22 sample ports. The efficiency scores of other sample ports were significantly lower and did not exceed one, i.e., with plenty of room to improve efficiency through better resource utilization. The standard devia- tion of the YRPS’s TE was 0.592, indicating obvious differences in the TE of these ports; For example, the TE of Shanghai (2.714) was around 23 times that of Anqing (0.120). Furthermore, the spatial distribution of the TEs of ports along the Yang- tze River generally increased from the upper to lower reaches. This result is closely related to the unbalanced development of ports along the Yangtze River over a long period time, caused by great disparities in channel conditions, hinterland economies, consolidation and distribution systems, and government policies.if For the two decompositions of TE, the sample ports showed significant differ- ences in scale efficiency (SE) and pure technical efficiency (PTE). 5.1 Efficiency of the Yangtze River port system However, in fact, much of the production capacity is actually backward or invalid, representing a structural overcapacity problem caused by the blind competi- tion that has been going on for a long time.l Notably, the measurement results of the super-SBM DEA model mainly reflect the relative efficiency characteristics and rankings of different ports in the port sys- tem. The measurement results imply that most of the Yangtze River ports (except for Shanghai, Nantong, and Suzhou) should increase their resource inputs to improve SE. However, this approach seems to be inappropriate from the perspective of the sustainable development of ports. Since the devolution of port management in 2001, the port cities along the Yangtze River have invested blindly in terminal facilities to improve their competitiveness and seize the sources of goods from nearby ports. Currently, a large number of terminals exist along the Yangtze River, but the propor- tion of small, chaotic, scattered, and weak terminals is obviously high. Currently, more than 2700 terminal operators have obtained terminal operation permits along the Yangtze River, of which only about 70 have a cargo throughput of more than 5 million tons (CPHA 2017). At the same time, this situation introduces a series of issues into the YRPS, such as overlapping of port functions, structural surplus of S. Ye et al. 652 Upper reach Middle reach Lower reach Yangtze River Upper reach Middle reach Lower reach Yangtze River cargo handling capacity, extensive utilization of riverbanks, and environmental dam- age. Therefore, investment in resources should focus on expanding the number and scale of terminals and pay more attention to the optimization of production capacity structures, the upgrading of terminal equipment, the improvement of consolidation and distribution systems, channel regulation, and the informatization construction of terminals to maintain the sustainable development of most ports along the Yangtze River. Th 22 i b di id d i h i b d h i 0.0 0.2 0.4 0.6 0.8 1.0 TE PTE SE Efficiency value Upper reach Middle reach Lower reach Yangtze River Fig. 2 Efficiency of three main port groups in 2016 0.0 0.2 0.4 0.6 0.8 1.0 TE PTE SE Efficiency value Fig. 2 Efficiency of three main port groups in 2016 0.0 0.2 0.4 0.6 0.8 1.0 TE PTE SE Efficiency value Fig 2 Efficiency of three main port groups in 2016 Fig. 5.1 Efficiency of the Yangtze River port system Analyzing the relative efficiency of China’s Yangtze River… 653 Table 3 Average values of MPI and its subcategories for the Yangtze River port system in 2010–2016 Period MPI MPI decomposition TC PTEC SEC 2010–2012 1.153 1.105 1.04 1.004 2012–2014 1.083 0.948 0.997 1.146 2014–2016 1.148 1.187 0.997 0.969 Mean 1.128 1.075 1.011 1.037 0.8 1 1.2 1.4 1.6 Luzhou Chongqing Yichang Jingzhou Yueyang Wuhan Huangshi Jiujiang Anqing Chizhou Tongling Wuhu Ma'anshan Nanjing Zhenjiang Taizhou Yangzhou Jiangyin Changzhou Suzhou Nantong Shanghai productivity change MPI TC PTEC SEC Stability Line Fig. 3 Average values of MPI and its decomposition (2010–2016, in order of geographic location along the Yangtze River) Table 3 Average values of MPI and its subcategories for the Yangtze River port system in 2010–2016 Period MPI MPI decomposition TC PTEC SEC 2010–2012 1.153 1.105 1.04 1.004 2012–2014 1.083 0.948 0.997 1.146 2014–2016 1.148 1.187 0.997 0.969 Mean 1.128 1.075 1.011 1.037 0.8 1 1.2 1.4 1.6 Luzhou Chongqing Yichang Jingzhou Yueyang Wuhan Huangshi Jiujiang Anqing Chizhou Tongling Wuhu Ma'anshan Nanjing Zhenjiang Taizhou Yangzhou Jiangyin Changzhou Suzhou Nantong Shanghai productivity change MPI TC PTEC SEC Stability Line Fig. 3 Average values of MPI and its decomposition (2010–2016, in order of geographic location along the Yangtze River) 0.8 1 1.2 1.4 1.6 Luzhou Chongqing Yichang Jingzhou Yueyang Wuhan Huangshi Jiujiang Anqing Chizhou Tongling Wuhu Ma'anshan Nanjing Zhenjiang Taizhou Yangzhou Jiangyin Changzhou Suzhou Nantong Shanghai productivity change MPI TC PTEC SEC Stability Line Fig. 3 Average values of MPI and its decomposition (2010–2016, in order of geographic location along the Yangtze River) productivity change Fig. 3 Average values of MPI and its decomposition (2010–2016, in order of geographic location alon the Yangtze River) 5.1 Efficiency of the Yangtze River port system 2 Efficiency of three main port groups in 2016 cargo handling capacity, extensive utilization of riverbanks, and environmental dam- age. Therefore, investment in resources should focus on expanding the number and scale of terminals and pay more attention to the optimization of production capacity structures, the upgrading of terminal equipment, the improvement of consolidation and distribution systems, channel regulation, and the informatization construction of terminals to maintain the sustainable development of most ports along the Yangtze River. cargo handling capacity, extensive utilization of riverbanks, and environmental dam- age. Therefore, investment in resources should focus on expanding the number and scale of terminals and pay more attention to the optimization of production capacity structures, the upgrading of terminal equipment, the improvement of consolidation and distribution systems, channel regulation, and the informatization construction of terminals to maintain the sustainable development of most ports along the Yangtze River. The 22 river ports can be divided into three main port groups based on their geographic location: the upper-reach port group, the middle-reach port group, and the lower-reach port group. The comparative relationships of the TE, PTE, and SE of each port group are presented in Fig. 2, showing a clear gap in the average TE and its subcategories among each of the port groups. First, the average TE of the upper- and middle-reach port groups was 0.295 and 0.357, respectively, far below that of the lower-reach port group (TE = 0.814) and the average for the entire YRPS (TE = 0.642). Thus, the weakness in the TE of the YRPS mainly lies in the upper- and middle-reach port groups. Second, the three port groups were relatively bal- anced in terms of the average PTE. On average, the middle-reach port group had the highest PTE (0.982), slightly surpassing those of the lower-reach port group (0.949) and the entire YRPS (0.943). This result indicates that the middle-reach port group has certain advantages in aspects such as technology, operational modes, and enter- prise management. Third, the lower-reach port group showed the best performance, with an average SE of 0.863. The average SE of the middle-reach port group was only 0.410, making this group the worst-performing port group and indicating that the scale benefits of this group were very limited. The SE offset the PTE, reducing the average TE of the middle-reach port group. 5.2 Dynamic characteristics of the Yangtze River port system’s efficiency Based on the above analysis, we attempted to deepen our understanding of the char- acteristics of the changes in the efficiency of the YRPS from a dynamic perspec- tive. Hence, the MPI was used to estimate the changes in TFP for the YRPS, from 2010 to 2016. Table 3 presents the calculations of the MPI and its three decompo- sitions, namely PTEC, TC, and SEC, for the periods 2010–2012, 2012–2014, and 2014–2016.i These results show that the sample ports experienced a significant improvement of 12.8% in their MPI between 2010 and 2016. The TC, PTEC, and SEC increased by 7.5%, 1.1%, and 3.7%, respectively. The results also suggest that the production technology, resource utilization, enterprise management, and scale economies in the YRPS improved throughout the study period, significantly improving the total factor productivity. 654 S. Ye et al. Table 4 Correlation coefficient values of MPI and its sources of efficiency change 5% significance level Period MPI-TC MPI-PTEC MPI-SEC 2010–2012 0.091 0.597 0.905 2012–2014 − 0.100 0.285 0.782 2014–2016 0.667 0.362 0.908 Table 4 Correlation coefficient values of MPI and its sources of efficiency change Table 3 also presents the varying changes in the productivity levels of the MPI and its subcategories in the different study periods. The TFP rose consistently throughout the study period. Between 2012 and 2014, TC witnessed a decline in productivity. A slight deterioration in PTEC also occurred after 2012. Finally, a decrease in SEC was also recorded in 2014–2016, meaning that the main sources of TFP increases in the YRPS were different in each period. The 2010–2012 period showed the joint influence of TC, PTEC, and SEC; 2012–2014 showed the influ- ence of SEC; and 2014–2016 showed the influence of TC. Figure 3 depicts the average MPI values and the three decompositions for the 22 sample ports from 2010 to 2016 (the nodes on the horizontal axis are arranged from left to right by their geographic location along the Yangtze River). The differences between sam- ple ports in terms of their MPI and SEC were quite significant, while the differ- ences in TC and PTEC were relatively minor. At the same time, the broken lines in Fig. 3 were located mostly above the stability line (equal to one), signifying that the sample ports performed well in terms of their MPI and its subcategories. 5.2 Dynamic characteristics of the Yangtze River port system’s efficiency Except for Yichang (TC = 0.998), the TC of the other ports were higher than one, indicat- ing that the production technology of the YRPS has generally improved in recent years and has become an important source of port efficiency. The PTEC and SEC of Wuhan, Anqing, Zhenjiang, Taizhou, and Jiangyin were all lower than one. Fur- thermore, four ports were below the stability line, namely Chizhou (PTEC = 0.987) for PTEC and Jingzhou (SEC = 0.897), Wuhu (SEC = 0.968), and Ma’anshan (SEC = 0.977) for SEC, meaning that these ports showed a trend of deteriorating operational management or resource allocation. This finding should be seriously considered by local port management. Finally, apart from Jingzhou (MPI = 0.92), Anqing (MPI = 0.943), Zhenjiang (MPI = 0.956), and Jiangyin (MPI = 0.892), the MPI of the remaining 18 ports was higher than one. From high to low, the productivity growth rates of the sample ports were Jiujiang (MPI = 1.431), Huangshi (MPI = 1.352), Nanjing (MPI = 1.253), Changzhou (MPI = 1.233), Yangzhou (MPI = 1.224), Tongling (MPI = 1.221), Chongqing (MPI = 1.216), Yichang (MPI = 1.206), Luzhou (MPI = 1.185), Yueyang (MPI = 1.179), Nan- tong (MPI = 1.16), Chizhou (MPI = 1.151), Wuhu (MPI = 1.115), Ma’anshan (MPI = 1.09), Wuhan (MPI = 1.071), Suzhou (MPI = 1.07), Shanghai (MPI = 1.07), Taizhou (MPI = 1.04), Zhenjiang (MPI = 0.956), Anqing (MPI = 0.943), Jingzhou (MPI = 0.92), and Jiangyin (MPI = 0.892). Analyzing the relative efficiency of China’s Yangtze River… 655 Analysis of the changes in the correlation coefficient between the MPI and its decompositions in different periods could provide a statistical basis for under- standing the reasons for the changes in TFP. In Table 4, the productivity gains that were achieved from TC only had a statistically meaningful effect (at the 5% sig- nificance level) on the improvement in TFP in 2014–2016. In recent years, under active encouragement from the Ministry of Transport and the port administrative departments along the Yangtze River, port transformation and upgrading strategies6 have achieved certain notable, albeit preliminary, results. Improvements in produc- tion technology played an increasingly significant role in the TFP improvement of the YRPS. The results show that PTEC had a stronger effect on TFP in 2010–2012, which was then followed by periods in which its effect was no longer significant (2012–2014 and 2014–2016). 6 The transformation and upgrading strategies of the Yangtze River ports involve several aspects: (a) promotion of port specialization, intensive development, and the integration of resources such as port terminals, river banks, and routes; (b) strengthening of the division and cooperation among ports, promo- tion of the coordinated development of ports in the upper, middle and lower reaches; encouragement of large-scale port and shipping enterprises to merge; and reorganization or formation of strategic alliances across regions, with capital, technology, and management as the link; and (c) strengthening the conver- gence between port planning, industrial development planning, and urban master planning, and promo- tion of the coordinated development of ports, riverside development zones and logistics parks. 5.2 Dynamic characteristics of the Yangtze River port system’s efficiency Changes in resource allocation, operational manage- ment, and business decisions, represented by PTEC, are believed to be important factors affecting port efficiency. However, we found that the effect of PTEC on the improvement of the overall efficiency of the YRPS greatly declined after 2012, which is not encouraging for the healthy development of river ports. Finally, the correlation coefficients between SEC and TFP were numerically larger, each at a statistical significance level of 5% in the three study periods. In other words, produc- tivity gains from SE were the primary means to improve the TFP of the YRPS. Con- tinuous input (resource) increases appeared to remain a reasonable approach from a statistical perspective. The above findings reveal that the improvement in the overall efficiency of the YRPS heavily depended on the scale benefits from the long-term extensive development of infrastructure, rather than from more strategic improve- ments in PTE. 6 Conclusions The aim of this study is to measure the efficiency of the Yangtze River port sys- tem by using the super-SBM model and Malmquist productivity index. Twenty- two major ports along the Yangtze River were sampled, with the quay-wall length, number of berths, and channel (dredging) depth used as the input variables, and the cargo throughput and container throughput used as the output variables. Several conclusions can be drawn based on this analysis.fi The results of the super-SBM model showed that the overall efficiency of the YRPS was on the low side; only four ports (Shanghai, Suzhou, Changzhou, and Yangzhou) achieved optimal TE. The management and utilization of inputs S. Ye et al. 656 (resources) must improve (Table 2). The TE, SE, and PTE were obviously not equal among ports or port groups. The TE generally increased from the upper to lower reaches. Currently, Shanghai, Nantong, and Suzhou demonstrate decreasing returns to scale; in the future, these ports should transform from relying on resource con- sumption to efficient resource adoption, technological advancement, and institu- tional innovation. The remaining 19 river ports are still in the stage of increasing returns to scale. While expanding, these ports should intensify efforts to optimize production capacity structures, upgrade terminal equipment, improve consolidation and distribution systems, channel regulations, and strengthening information sharing among terminals.f The MPI results showed that the YRPS had differing degrees of improvement in its TFP, PTEC, TC, and SEC from 2010 to 2016 (Table 3). The improvement in TFP was the most obvious, with an increase of 12.8%. However, the PTE of the YRPS showed a continuous downward trend after 2012, which must be considered. At the port level, except for Jingzhou, Anqing, Zhenjiang, and Jiangyin, the TFP of all the remaining 18 ports showed an increasing trend during the study period. TC became an important factor that affected the improvement of TFP after 2014, and the effect of PTEC became insignificant after 2012. At the same time, productivity gains from SEC were the main means to improve the TFP of the YRPS throughout the study period. These results highlight the serious challenges that have impeded the sustainable development of the YRPS in recent years. 7 http://www.ship.sh/news_detail.php?nid=25504. 6 Conclusions These issues have raised considerable concern amongst government authorities at all levels, which are actively promot- ing the transformation, upgrading, and coordinated development of the YRPS by shutting down or relocating small terminals, integrating riverbank line resources, reforming terminal technologies, and setting up provincial-level port groups. How- ever, in spite of the influence of the current administrative and management systems of ports, a performance evaluation system which utilizes economic improvement as its core and the trend of “government entrepreneuralization” (Zhang et al. 2006), excessive competition, and product homogeneity problems still exist among ports along the Yangtze River, and the coordinated development of these ports is gener- ally low. This scenario has greatly reduced the efficiency and overall competitive- ness of the YRPS.ifi Based on the present paper’s findings, the efficiency of the YRPS can be improved through the following approaches: (1) Accelerate the planning and construction of consolidation and distribution systems along the Yangtze River, including upgrading multimodal transport infrastructures, increasing the number of river-crossing tun- nels and bridges, optimizing the locations of dry ports, and improving the informa- tization level of inland navigation. (2) Drawing on the experience of the “Yangtze River Strategy”7 of the Shanghai International Port (Group) Co., Ltd., encourage more large-size port operators, shipping companies, and cargo owners to conduct cross-regional mergers, acquisitions, or informal strategic partnerships of port and shipping enterprises along the river, with capital, technology, and management as Analyzing the relative efficiency of China’s Yangtze River… 657 the links. Strive to extend port services functions and improve the management and capabilities of terminal operators in the upper and middle Yangtze River. (3) Actively promote the integration of ports along the Yangtze River and establish a more stringent riverbank line development. Strengthen the joint construction and sharing of infrastructures (for instance, terminals, navigation waterways, anchor- ages, etc.) and aim at a more intensive utilization of public resources in ports. (4) Optimize the transportation network along the Yangtze River, based on a hub-and- spoke concept, with Shanghai (at the mouth of the Yangtze) as the international hub port; Chongqing (located in the upper reach), Wuhan (located in the middle reach), and Nanjing (located in the lower reach) as regional hub ports; and the remaining river ports as feeder ports. 6 Conclusions The network should offer frequent services in the upper and middle Yangtze River after transshipment in the regional hub ports, utilizing relatively large vessels to and from Shanghai, thereby strengthening the division (as well as the cooperation) between large, medium, and small ports. Such an approach would fully utilize scale effects, improve transport efficiency, and reduce total costs (including transportation and time costs). Overall, we hope that the present study has provided insights into the efficiency characteristics of Yangtze River ports, thus aiding both operational and policy deci- sion-making to improve river port efficiency. However, we did not include labor, land, and information among our input indicators, being limited by the difficulty of port data acquisition, something that may have influenced our results. Enriching data sources, conducting efficiency studies from a terminal-scale perspective, identifying the critical factors influencing port efficiency, and exploring the optimization and upgrading paths of port efficiency may be important directions for future research. Acknowledgements The authors would like to thank the anonymous referees and the Editor-in-Chief of MEL for their careful reading and constructive suggestions. This research is supported by the Natural Sci- ences Foundation of China (grant nos. 41901138 and 41771139) and Program of Humanities and Social Sciences of the Ministry of Education of China (19YJCZH225). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. Ali, I.A., A. Charnes, W.W. Cooper, D. Divine, and J. Stutz. 1989. An application of data envelopment analysis to management of US Army recruitment districts. In Applications of management science, a research annual, ed. R.L. Schultz. New York: JAI Press. Andersen, P., and N.C. Petersen. 1993. A procedure for ranking efficient units in data envelopment analy- sis. Management Science 39 (10): 1261–1264. Andersen, P., and N.C. Petersen. 1993. A procedure for ranking efficient units in data envelopment anal sis. Management Science 39 (10): 1261–1264. Ali, I.A., A. Charnes, W.W. Cooper, D. Divine, and J. Stutz. 1989. An application of data envelopme analysis to management of US Army recruitment districts. In Applications of management scienc a research annual, ed. R.L. Schultz. New York: JAI Press.fi References Ali, I.A., A. Charnes, W.W. Cooper, D. Divine, and J. Stutz. 1989. An application of data envelopment analysis to management of US Army recruitment districts. In Applications of management science, a research annual, ed. R.L. Schultz. New York: JAI Press. Andersen, P., and N.C. Petersen. 1993. A procedure for ranking efficient units in data envelopment analy- sis. Management Science 39 (10): 1261–1264. S. Ye et al. 658 Barros, C.P. 2003. Incentive regulation and efficiency of Portuguese port authorities. Maritime Econom- ics and Logistics 5 (1): 55–69. Barros, C.P. 2006. A benchmark analysis of Italian seaports using data envelopment analysis. Maritime Economics and Logistics 8 (4): 347–365.fi Barros, C.P., and M. Athanassiou. 2004. Efficiency in European seaports with DEA: Evidence from Greece and Portugal. Maritime Economics and Logistics 6 (2): 122–140.fi Barros, C.P., Z. Chen, and P. Wanke. 2016. Efficiency in Chinese seaports: 2002–2012. Maritime Eco- nomics and Logistics 18 (3): 295–316. Bergantino, A.S., and E. Musso. 2011. The role of external factors versus managerial ability in determin- ing seaports’ relative efficiency: an input-by-input analysis through a multi-step approach on a panel of Southern European ports. Maritime Economics and Logistics 13 (2): 121–141. Beuren, M.M., R. Andriotti, G.B.B. Vieira, J.L.D. Ribeiro, and F.J.K. Neto. 2018. On measuring the efficiency of Brazilian ports and their management models. Maritime Economics and Logistics 20 (1): 149–168.fi Bowlin, W.F. 1987. Evaluating the efficiency of US Air Force real-property maintenance activities. Jou nal of the Operational Research Society 38 (2): 127–135.l Caldeirinha, V.R., and J.A. Felicio. 2011. The influence of characterizing factors on port performance, measured by operational, financial and efficiency indicators. MPRA paper, 1 April. http://mpra. ub.uni-muenchen.de/30009/. Accessed 11 Nov 2018. Caves, D.W., L.R. Christensen, and W.E. Diewert. 1982. The economic theory of index numbers and th measurement of input, output, and productivity. Econometrica 50 (6): 1393–1414.fi Cheon, S. 2007. Evaluating impacts of institutional reforms on port efficiency changes Malquimist pr ductivity index for world container ports. Berkeley: University of California. Chin, A., and J. Tongzon. 1998. Maintaining Singapore as a major shipping and air transport hub. In Competitiveness of the Singapore economy: a strategic perspective, ed. T.M. Heng and T.K. Yam, 83–114. Singapore: Singapore University Press, World Scientific.fi ngapore: Singapore University Press, World Scientific i Coto-Millan, P., J. Banos-Pino, and A. Rodriguez-Alvarez. 2000. Economic efficiency in Spanish port some empirical evidence. Maritime Policy and Management 27 (2): 169–174. China Ports and Harbours Association (CPHA). References 2017. Current situation: Port transformation and upgra ing on the road. China Ports 32 (3): 10–13.fi Cullinane, K., and D.W. Song. 2003. A stochastic frontier model of the productive efficiency of Korean container terminals. Applied Economics 35 (3): 251–267. Cullinane, K., and T.F. Wang. 2006a. Data envelopment analysis (DEA) and improving container po efficiency. Research in Transportation Economics 17: 517–566.fi fi Cullinane, K., and T.F. Wang. 2006b. The efficiency of European container ports: a cross-sectional da envelopment analysis. International Journal of Logistics: Research and Applications 9 (1): 19–31. Cullinane, K., D.W. Song, and R. Gray. 2002. A stochastic frontier model of the efficiency of major con- tainer terminals in Asia: assessing the influence of administrative and ownership structures. Trans- portation Research Part A: Policy and Practice 36 (8): 743–762. Cullinane, K., D.W. Song, and T. Wang. 2005. The application of mathematical programming approaches to estimating container port production efficiency. Journal of Productivity Analysis 24 (1): 73–92. fi Cullinane, K., D.W. Song, P. Ji, and T.F. Wang. 2004. An application of DEA windows analysis to tainer port production efficiency. Review of Network Economics 3 (2): 184–206.fi fi Cullinane, K., T.F. Wang, D.W. Song, and P. Ji. 2006. The technical efficiency of container ports: com- paring data envelopment analysis and stochastic frontier analysis. Transportation Research Part A: Policy and Practice 40 (4): 354–374. Da Silva, F.G.F., and C.H. Rocha. 2012. A demand impact study of southern and southeastern ports in Brazil: an indication of port competition. Maritime Economics and Logistics 14 (2): 204–219. De Monie, G. 1987. Measuring and evaluating port performance and productivity. Monographs on port management no. 6. Geneva, Switzerland: United Nations Conference On Trade And Development (UNCTAD). ( ) De Oliveira, G.F., and P. Cariou. 2011. A DEA study of the efficiency of 122 iron ore and coal ports and of 15/17 countries in 2005. Maritime Policy and Management 38 (7): 727–743.fi y g De Oliveira, G.F., and P. Cariou. 2015. The impact of competition on container port (in) efficiency. Tran h A l d 78 124 133 De Oliveira, G.F., and P. Cariou. 2015. The impact of competition on container port (in) efficiency. Trans- portation Research Part A: Policy and Practice 78: 124–133. De Oliveira, G.F., and P. Cariou. 2015. References The impact of competition o portation Research Part A: Policy and Practice 78: 124–133.i , , p p portation Research Part A: Policy and Practice 78: 124–133.i Defilippi, E., and L. Flor. 2008. Regulation in a context of limited competition: a port case. Transporta- tion Research Part A: Policy and Practice 42 (5): 762–773. esearch Part A: Policy and Practice 42 (5): 762–773. Analyzing the relative efficiency of China’s Yangtze River… 659 Dowd, T.J., and T.M. Leschine. 1990. Container terminal productivity: a perspective. Maritime Policy and Management 17 (2): 107–112. Drewe, P., and B. Janssen. 1998. What port for the future? From ‘Mainports’ to ports as nodes of logistic networks. In Accessibility, trade and locational behaviour, ed. A. Reggiani, 241–264. Aldershot: Ashgate. Färe, R., S. Grosskopf, M. Norris, and Z.Y. Zhang. 1994. Productivity growth, technical progress, an efficiency change in industrialized countries. The American Economic Review 84 (1): 66–83.fi fi Farrell, M.J. 1957. The measurement of productive efficiency. Journal of the Royal Statistical Society 12 (3): 253–290.fi González, M.M., and L. Trujillo. 2008. Reforms and infrastructure efficiency in Spain’s container ports. Transportation Research Part A: Policy and Practice 42 (1): 243–257.fi p y ( ) González, M.M., and L. Trujillo. 2009. Efficiency measurement in the port industry: a survey of the empirical evidence. Journal of Transport Economics and Policy 43 (2): 157–192.i Haralambides, H.E., P. Cariou, and M. Benacchio. 2002. Costs, benefits and pricing of dedicated con- tainer terminals. International Journal of Maritime Economics 4 (1): 21–34.fi Itoh, H. 2002. Efficiency changes at major container ports in Japan: a window application of data envel- opment analysis. Review of Urban and Regional Development Studies 14 (2): 133–152. Kumbhakar, S.C., and C.A.K. Lovell. 2000. Stochastic frontier analysis. Cambridge: Cambridge Univer- sity Press. Lai, P.L., A. Potter, and M. Beynon. 2012. The development of benchmarking techniques in airport pe formance evaluation research. Transportation Journal 51 (3): 305–337. Liu, Z. 1995. The comparative performance of public and private enterprises: the case of British port Journal of Transport Economics and Policy 29 (3): 263–274.f Malmquist, S. 1953. Index numbers and indifference surfaces. Trabajos de Estadística 4 (2): 209–242.fi f Martínez-Budria, E., R. Diaz-Armas, M. Navarro-Ibanez, and T. Ravelo-Mesa. 1999. A study of the effi- ciency of Spanish port authorities using data envelopment analysis. International Journal of Trans- port Economics 26 (2): 237–253.fi p Merkel, A., and J. Holmgren. 2017. References Dredging the depths of knowledge: efficiency analysis in the mari- time port sector. Transport Policy 60: 63–74. Nguyen, H.O., H.S. Nghiem, and Y.T. Chang. 2018. A regional perspective of port performance using metafrontier analysis: the case study of Vietnamese ports. Maritime Economics and Logistics 20 (1): 112–130. Notteboom, T. 2007. Container river services and gateway ports: Similarities between the Yangtze River and the Rhine River. Asia Pacific Viewpoint 48 (3): 330–343.fi i Notteboom, T., C. Coeck, and J. Van Den Broeck. 2000. Measuring and explaining the relative efficiency of container terminals by means of Bayesian stochastic frontier models. International Journal of Maritime Economics 2 (2): 83–106.fi Pagano, A.M., G.W. Wang, O.V. Sánchez, and R. Ungo. 2013. Impact of privatization on port efficiency and effectiveness: results from Panama and US ports. Maritime Policy and Management 40 (2): 100–115. Panayides, P.M., C.N. Maxoulis, T.F. Wang, and K.Y.A. Ng. 2009. A critical analysis of DEA applic tions to seaport economic efficiency measurement. Transport Reviews 29 (2): 183–206. fi Robinson, R. 2002. Ports as elements in value-driven chain systems: The new paradigm. Maritime Poli and Management 29 (3): 241–255. Kim, M., and A. Sachish. 1986. The structure of production, technical change and productivity in a port. The Journal of Industrial Economics 35 (2): 209–223.fi Schøyen, H., and J. Odeck. 2013. The technical efficiency of Norwegian container ports: A comparison to some Nordic and UK container ports using Data Envelopment Analysis (DEA). Maritime Econom- ics and Logistics 15 (2): 197–221. Serebrisky, T., J.M. Sarriera, A. Suárez-Alemán, G. Araya, C. Briceño-Garmendía, and J. Schwartz. 2016. Exploring the drivers of port efficiency in Latin America and the Caribbean. Transport Policy 45: 31–45. Shephard, R. 1970. Theory of cost and production functions. Princeton: Princeton University Press. Suykens, F., and E. Van de Voorde. 1998. A quarter a century of port management in Europe: objectives and tools. Maritime Policy and Management 25 (3): 251–261. Shephard, R. 1970. Theory of cost and production functions. Princeton: Princeton University Press. Suykens, F., and E. Van de Voorde. 1998. A quarter a century of port management in Europe: object Suykens, F., and E. Van de Voorde. 1998. A quarter a century of port management in Europe: objectives and tools. Maritime Policy and Management 25 (3): 251–261. y g ( ) Tone, K. 2001. A slacks-based measure of efficiency in data envelopment analysis. European Journal of Operational Research 130 (3): 498–509. S. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Ye et al. 660 Tone, K. 2002. A slacks-based measure of super-efficiency in data envelopment analysis. European Jour- nal of Operational Research 143 (1): 32–41.fi Tongzon, J. 2001. Efficiency measurement of selected Australian and other international ports using data envelopment analysis. Transportation Research Part A: Policy and Practice 35 (2): 107–122. Tongzon, J. 2002. Port choice determinants in a competitive environment. In Annual IAME meeting and conference, Panama, 1–22 November.fi Tongzon, J., and W. Heng. 2005. Port privatization, efficiency and competitiveness: some empirical evi- dence from container ports (terminals). Transportation Research Part A: Policy and Practice 39 (5): 405–424. Tongzon, J.L. 1995. Determinants of port performance and efficiency. Transportation Research Part A: Policy and Practice 29 (3): 245–252.fi Valentine, V.F., and R. Gray. 2001. The measurement of port efficiency using data envelopment analysis. In Proceedings of the 9th world conference on transport research, Seoul, 22–27 July.fi Valentine, V.F., and R. Gray. 2002. An organisational approach to port efficiency. In Proceedings of IAME Panama 2002 conference, Panama, 13–15 November. Verhoeven, P. 2010. A review of port authority functions: towards a renaissance? Maritime Policy and Management 37 (3): 247–270.ffi Wan, Y., A.C.L. Yuen, and A. Zhang. 2014. Effects of hinterland accessibility on US container port effi- ciency. International Journal of Shipping and Transport Logistics 56 (4): 422–440.fi Wang, T.F., and K. Cullinane. 2006. The efficiency of European container terminals and implications f supply chain management. Maritime Economics and Logistics 8 (1): 82–99. Wang, T.F., K.P.B. Cullinane, and D.W. Song. 2004. Industrial concentration in container ports. In Pr ceedings of IAME Izmir 2004 conference, 30 June–2 July.fi Wu, Y.C.J., and M. Goh. 2010. Container port efficiency in emerging and more advanced markets. Trans- portation Research Part E: Logistics and Transportation Review 46 (6): 1030–1042. Yu, X.G., L. Wang, Q.K. Yang, and S.L. Ye. 2015. Background of the Yangtze River economic belt development strategy and geography interpretation of its innovative development. Progress in Geog- raphy 34 (11): 1368–1376. Yuen, A.C.L., A. Zhang, and W. Cheung. 2013. Foreign participation and competition: a way to improve the container port efficiency in China? Transportation Research Part A: Policy and Practice 49: 220–231. Zhang, J.X., J. Yin, and X.L. Luo. 2006. Research of urban spatial development under the circumstances of local government’s entrepreneuralization. Human Geography 21 (4): 1–6. Zhang, J.X., J. Yin, and X.L. Luo. 2006. Research of urban spatial development under the circumstances of local government’s entrepreneuralization. References Human Geography 21 (4): 1–6. Zhao, Y.R., Z.Z. Yang, and H. Haralambides. 2019. Optimizing the transport of export containers along Zhao, Y.R., Z.Z. Yang, and H. Haralambides. 2019. Optimizing the transport of export containers along China’s coronary artery: the Yangtze River. Journal of Transport Geography 77: 11–25. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3008050267	https://europepmc.org/articles/pmc7294224?pdf=render	English	null	Immuno-imaging of ICAM-1 in tumours by SPECT	Nuclear medicine and biology	2,020	cc-by	6,441	a r t i c l e i n f o Purpose: Molecular imaging of cancer cells' reaction to radiation damage can provide a non-invasive measure of tumour response to treatment. The cell surface glycoprotein ICAM-1 (CD54) was identiﬁed as a potential radia- tion response marker. SPECT imaging using an 111In-radiolabelled anti-ICAM-1 antibody was explored. Methods: PSN-1 cells were irradiated (10 Gy), and protein expression changes were investigated using an anti- body array on cell lysates 24 h later. Results were conﬁrmed by western blot, ﬂow cytometry and immunoﬂuo- rescence. We conﬁrmed the afﬁnity of an 111In-labelled anti-ICAM-1 antibody in vitro, and in vivo, in PSN-1- xenograft bearing mice. The xenografts were irradiated (0 or 10 Gy), and [111In]In-anti-ICAM-1 SPECT/CT images were acquired 24, 48 and 72 h after intravenous administration. Article history: Received 28 November 2019 Received in revised form 24 February 2020 Accepted 24 February 2020 Keywords: ICAM-1 Pancreatic cancer SPECT imaging Purpose: Molecular imaging of cancer cells' reaction to radiation damage can provide a non-invasive measure of tumour response to treatment. The cell surface glycoprotein ICAM-1 (CD54) was identiﬁed as a potential radia- tion response marker. SPECT imaging using an 111In-radiolabelled anti-ICAM-1 antibody was explored. Article history: Received 28 November 2019 Received in revised form 24 February 2020 Accepted 24 February 2020 Article history: Received 28 November 2019 Received in revised form 24 February 2020 Accepted 24 February 2020 Keywords: ICAM-1 Pancreatic cancer SPECT imaging Methods: PSN-1 cells were irradiated (10 Gy), and protein expression changes were investigated using an anti- body array on cell lysates 24 h later. Results were conﬁrmed by western blot, ﬂow cytometry and immunoﬂuo- rescence. We conﬁrmed the afﬁnity of an 111In-labelled anti-ICAM-1 antibody in vitro, and in vivo, in PSN-1- xenograft bearing mice. The xenografts were irradiated (0 or 10 Gy), and [111In]In-anti-ICAM-1 SPECT/CT images were acquired 24, 48 and 72 h after intravenous administration. Results: ICAM-1 was identiﬁed as a potential marker of radiation treatment using an antibody array in PSN-1 cell lysates following irradiation, showing a signiﬁcant increase in ICAM-1 signal compared to non-irradiated cells. Western blot and immunohistochemistry conﬁrmed this upregulation, with an up to 20-fold increase in ICAM- 1 signal. Radiolabelled anti-ICAM-1 bound to ICAM-1 expressing cells with good afﬁnity (Kd = 24.0 ± 4.0 nM). [111In]In-anti-ICAM-1 uptake in tumours at 72 h post injection was approximately 3-fold higher than non-speciﬁc isotype-matched [111In]In-mIgG2a control (19.3 ± 2.5%ID/g versus 6.3 ± 2.2%ID/g, P = 0.0002). Immuno-imaging of ICAM-1 in tumours by SPECT Michael Mosley, Julia Baguña Torres, Danny Allen, Bart Cornelissen ⁎ Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom of Great Britain and Northern Ireland Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Ox Northern Ireland a r t i c l e i n f o However, ICAM1 levels, and [111In]In-anti-ICAM-1 uptake in tumours was no different after irradiation (uptake 9.2%ID/g versus 14.8%ID/g). Western blots of the xenograft lysates showed no signiﬁcant differences, conﬁrming these results. Conclusion: Imaging of ICAM-1 is feasible in mouse models of pancreatic cancer. Although ICAM-1 is upregulated post-irradiation in in vitro models of pancreatic cancer, it shows little change in expression in an in vivo mouse xenograft model. © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). journal homepage: www.elsevier.com/locate/nucmedbio journal homepage: www.elsevier.com/locate/nucmedbio ⁎ Corresponding author at: CRUK/MRC Oxford Institute for Radiation Oncology, Depart- ment of Oncology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford OX3 7LJ, United Kingdom of Great Britain and Northern Ireland. E-mail address: bart.cornelissen@oncology.ox.ac.uk (B. Cornelissen). Nuclear Medicine and Biology 84–85 (2020) 73–79 Nuclear Medicine and Biology 84–85 (2020) 73–79 https://doi.org/10.1016/j.nucmedbio.2020.02.014 0969-8051/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2.1. Screening using the human oncology antibody array PSN-1 cells in a T175 ﬂask were irradiated (10 Gy or mock irradiated as a negative control sample; mock-treated samples were treated in an identical fashion to those that were irradiated, but without the radiation equipment turned on), and allowed to recover in a CO2-incubator at 37 °C for 24 h. The cells were washed in PBS, scraped and collected in a centrifuge tube and pelleted at 400 ×g and immediately processed or snap-frozen to −80 °C for storage. The cell pellets were lysed using standard RIPA buffer (with added COMPLETE protease inhibitors [Sigma]), centrifuged at 20,000 ×g to remove debris and stored at −20 °C until further use. The Proteome Proﬁler Human XL Oncology An- tibody Array (#ARY026, Bio-Techne) allows for the measurement of 84 oncology-related proteins in a single sample. Lysates (irradiated and mock-irradiated) were incubated with the Array membranes according to the manufacturer's protocol. Brieﬂy, 1 mg total protein was incubated with the pre-blocked antibody array overnight at 4 °C. Membranes were washed, incubated with the detection antibody cocktail, washed once more, developed and exposed to photographic ﬁlm for up to 10 min. The signal intensities corresponding to the relative abundance of an antibody-antigen complex were quantiﬁed using Matlab software de- veloped in-house [https://www.mathworks.com/matlabcentral/ ﬁleexchange/35128-protein-array-tool]. 2.3. Immunoﬂuorescence microscopy PSN-1 cells were seeded in 8-chamber slides (#IB-80841, Thistle Sci- entiﬁc), and 24 h later were irradiated (10 Gy), or mock-irradiated and allowed to recover for 24 or 48 h, or mock irradiated. The cells were washed and ﬁxed in 4% formaldehyde solution (#252549, Sigma), blocked with 2% BSA for 1 h at room temperature, and stained with anti-ICAM-1 antibody 2213 (Abcam) at 1:100 dilution overnight at 4 °C, followed by Alexa Fluor 488 Goat Anti-Mouse IgG (H & L) antibody (#A11001, Life Technologies) at 1:500 dilution for 1 h at room temper- ature, in blocking buffer, and washes performed with PBS. Immunoﬂu- orescence microscopy was performed on a Leica SP8 confocal microscope system. The level of cellular ﬂuorescence from the Leica ﬂuorescence microscopy images was determined in ImageJ by calculat- ing the corrected total cell ﬂuorescence (CTCF) of each image. This report describes the identiﬁcation of proteins whose expression on pancreatic cancer cell-lines was increased after gamma irradiation, and the selection of one of these molecules, ICAM-1, for further in vitro and in vivo characterization, and imaging using an 111In- labelled anti-ICAM-1 antibody. 2.5. Radiolabelled antibody Anti-ICAM-1 antibody (#2213, Abcam), and an isotype-matched an- tibody (#02-6200, Thermo Fisher Scientiﬁc), were radiolabelled with 111In as previously described [20]. Brieﬂy, 0.3 mg anti-ICAM-1 antibody (#2213, Abcam) was reacted with a 20-fold molar excess of p-SCN-Bn- DTPA in chelex-treated 0.1 M sodium bicarbonate buffer pH 8.6 for 1 h at 37 °C, and the complex puriﬁed on a 1 mL sephadex G50 column, using 0.5 M MES buffer as the eluent. The DTPA-conjugated antibody was then concentrated using an Amicon Ultra 0.5 mL 30 K MWCO ﬁlter unit. DTPA-conjugated antibody (0.1 mg) was radiolabelled with 111In, using 0.5–1.0 MBq per microgram of antibody, for 1 h at room temper- ature. Radiolabelling efﬁciencies of N95% were conﬁrmed by iTLC, and ﬁnal antibody conjugate concentration were measured by Nanodrop spectrophotometry (Thermo Fisher Scientiﬁc). 1. Introduction number of molecular biomarkers that guide clinical decisions following radiation therapy [4,5], especially those imaging extracellular epitopes to facilitate imaging. This therefore highlights the need for alternative imaging biomarkers. Radiotherapy (RT) using ionizing radiation (IR) is an essential com- ponent of treatment for more than half of all newly diagnosed cancer patients, in whom it can irreversibly damage targeted tumour cell DNA to reduce tumour size [1]. Real-time molecular imaging of cellular markers of IR damage would provide a non-invasive measure of tumourigenesis, RT sensitivity and tumour status in response to treat- ment, and would be useful in tailoring RT based on tumour biology in in- dividual patients [2]. Tumour response to treatment varies between patients and anatomical changes may not be measurable using anatom- ical imaging techniques such as CT or MRI, for a signiﬁcant period fol- lowing treatment [3]. This will impact on the timely need to consider changes to treatment in order to improve patient outcome, should the treatment prove non-efﬁcacious. Thus far, there exist only a limited Here, we employed a human pancreatic cancer model, using the pancreatic ductal adenocarcinoma (PDAC) PSN-1 cell line to identify cell-surface markers of radiation damage, in an in vitro screen. Given that there is an urgent clinical need to improve survival rates for pancre- atic cancer patients since these are particularly low in PDAC (a 5-year survival rate of b5% [6]) - in large part because of late diagnosis and the therapy resistance of the disease [7]. Fast decision-making regarding the efﬁcacy of any therapy, including radiotherapy, is therefore para- mount in order to adapt or adjust treatment in a timely fashion [8,9]. PSN-1 cells are not particularly radiosensitive or radioresistant [10]. We report our results after the screening of an antibody array com- paring PSN-1 cell lysates obtained before and after irradiation. This identiﬁed ICAM-1 (Intercellular Adhesion Molecule 1) as being up- regulated in irradiated cells. ICAM-1 is a cell surface glycoprotein, a member of the immunoglobulin superfamily, and is typically expressed on endothelial cells and cells of the immune system [11]. In addition, it M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 74 is reported to be involved in tumour metastasis, cancer progression and is an independent prognostic factor [12]. 2.2. Western blot Western blot was used to conﬁrm antibody array results. PSN-1 cells were irradiated and total cell lysates prepared as described above, on three separate occasions. For this experiment cell recovery times of 2, 24, 48 and 72 h post-irradiation were used, and a separate mock- irradiated control included. Separate 8% Bis-Tris/MOPS PAGE gels (Life Technologies) were loaded with 25 μg of lysate protein per well, and blotted onto PVDF membranes (Invitrogen iBlot-2). The blots were blocked in 5% Bovine Serum Albumin (BSA) at room temperature for 1 h, and incubated with a mouse anti-human ICAM-1 monoclonal anti- body (Abcam, #2213 [MEM-111]) at 1:800 dilution at 4 °C overnight. After washing the membranes with PBS, they were incubated with an 2. Materials and methods The PSN-1 pancreatic adenocarcinoma cell line was obtained from ATCC, validated by STR, and maintained at 37 °C and 5% CO2 in RPMI media (Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS) and L-glutamate (Sigma) up to a maximum passage number of 20 following resuscitation from liquid nitrogen storage. Cells were mon- itored regularly for the absence of mycoplasma. Cells were irradiated using an IBL 637 Cesium-137 γ-ray irradiator to a radiation absorbed dose of 10 Gy (1 Gy/min). 1. Introduction Expression of ICAM-1 is further increased in metastases of the liver [13], where it mediates the forma- tion of a pro-metastatic niche by endothelial cell activation of signalling pathways [14], assisting tumour cell extravasation [15,16], and the re- cruitment of immune cell populations [17]. Additionally, ICAM-1 is overexpressed in triple-negative breast cancer tissue and was previ- ously imaged by near-infrared ﬂuorescence in a mouse xenograft model [18]. Finally, ICAM-1 expression had previously been shown to be upregulated after gamma irradiation of human multiple myeloma cells, potentially leading to an increase in the immunogenicity of tu- mour cells [19]. anti-mouse HRP-conjugated secondary antibody (Bio-Techne, HAF007) at 1:1000 at room temperature for 1 h, and further washes followed. The blots were developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate kit (#34580, Thermo Scientiﬁc), and exposed to a Li-Cor 3600 Blot Scanner. Similar western immunoblot analysis of ICAM-1 expression was performed using total cell lysates pu- riﬁed from PSN-1 xenografts. 2.4. Flow cytometry PSN-1 cells were cultured in ﬂasks to a maximum of 70% conﬂuency, irradiated (10 Gy, or mock-irradiated) and allowed to recover for 24 h. The cells were removed from the ﬂasks using trypsin-free Accutase dis- sociation solution (#A6964, Sigma-Aldrich), blocked in 1% BSA/PBS so- lution for 1 h at room temperature. Increasing amounts of anti-ICAM- 1 antibody 2213 (Abcam) were exposed to 1 million cells in 200 μL ali- quots in blocking buffer for 1 h at room temperature, followed by Alexa Fluor 488 Goat anti-Mouse antibody A11001 at 1:300 dilution in blocking buffer for 1 h at room temperature. Multiple washes in block buffer following centrifugation of the cells at 400 ×g were performed between treatments. Intensity histograms were acquired on a FACSCalibur Flow Cytometer (Becton Dickinson BD), and data analysed using FlowJo software (Becton Dickinson BD). 2.1. Screening using the human oncology antibody array 2.8. Ex vivo autoradiography of xenografts Quantiﬁcation of the biodistribution [111In]In-anti-ICAM-1 and [111In]In-mIgG2a showed signiﬁcantly higher uptake in PSN-1 tumour xenografts of the former compared to the latter, at 72 h post administra- tion (19.9 ± 1.2 versus 6.9 ± 0.5%ID/g; P b 0.0001) (Fig. 3A). Conversely, and surprisingly, no signiﬁcant difference was observed in [111In]In- anti-ICAM-1 uptake in the irradiated tumours compared to mock- treated controls (19.2 ± 1.8 versus 20.7 ± 1.9%ID/g, P N 0.5). Quantiﬁca- tion of SPECT images using VOI analysis conﬁrmed this result (Fig. 3B, C). SPECT image quantiﬁcation by VOI analysis of images acquired 24, 48 and 72 h post administration conﬁrmed these results (20.3 ± 2.9% ID/mL versus 18.3 ± 2.0%ID/mL in mock-irradiated versus irradiated mice at 72 h post injection; P N 0.05). Fig. 3C shows representative MIP images from the SPECT imaging study. Full data of the biodistribution of [111In]In-anti-ICAM-1 and [111In]In-mIgG2a is shown in Fig. 4. After imaging, xenograft tissue from mice was ﬂash-frozen with dry ice and stored at −80 °C overnight. Frozen tissue was sectioned (8 μm) using an OTF5000 cryotome (Bright Instruments Ltd). Tissue sections were thaw-mounted onto Superfrost PLUS glass microscope slides (Menzel-Glaser, Thermo Scientiﬁc) and allowed to dry at room temper- ature. The slides were then exposed to a storage phosphor screen (PerkinElmer, Super Resolution, 12.5 × 25.2 cm) in a standard X-ray cas- sette for 15 h, and the screen imaged using a Cyclone® Plus Storage Phosphor System (PerkinElmer). Autoradiographs were analysed using ImageJ (NIH). After autoradiography, ICAM-1 levels in ex vivo tis- sue were characterized by immunoﬂuorescence. On a separate occasion a second cohort of 7 tumour xenografts were prepared as above that were irradiated or mock-irradiated (10 Gy). Tumour tissue of these an- imals was harvested at 24, 48 and 72 h post-irradiation for western blot analysis of ICAM-1, as described above. Representative sections of xenograft tumours were processed for au- toradiography that conﬁrmed heterogeneous uptake of [111In]In-anti- ICAM-1 in PSN-1 tumours (Fig. 4A). Densitometry conﬁrmed higher up- take of [111In]In-anti-ICAM-1 compared to the [111In]IgG2a negative control conjugate, but little difference between irradiated and non- irradiated xenografts (Fig. 4B). 3. Results 3. Results 3.1. ICAM-1 protein expression is upregulated in PSN-1 cells following irradiation Quantiﬁcation of the antibody array signal allowed comparison of relative protein levels in irradiated (10 Gy) versus mock-irradiated PSN-1 cells. This identiﬁed a number of proteins that exhibited an up to 7-fold increase in expression levels in irradiated cells. Amongst these, the cell surface protein ICAM-1 showed a marked and signiﬁcant increase (Fig. 1A, B). This was conﬁrmed by western blot on an indepen- dent set of cell lysates (Fig. 1C), demonstrating a 4.7 and a 20-fold in- crease in ICAM-1 expression 24 or 48 h after irradiation, respectively. Analysis of cell lysates may overestimate the amount of ICAM-1 expressed at the cell surface. Further investigation by immunoﬂuores- cence microscopy – without permeabilisation of the cell membrane – corroborated this result, with a maximum increase at the 24 h point of 2.7-fold (Fig. 1D). ICAM-1 expression was previously also found in PDAC patient samples (Fig. 1E, adapted from ProteinAtlas.org). One hour later, [111In]In-anti-ICAM-1 or [111In]In-mIgG2a (5 MBq, 5 μg) in sterile PBS (100 μL) was injected intravenously via the lateral tail vein (n = 3 per group). SPECT/CT images were acquired at 24, 48, and 72 h after injection, using a VECTor4CT scanner (MILabs, Utrecht, the Netherlands). Animals were anaesthetised by 4% isoﬂurane gas (0.5 L/min O2) and maintained at 2% and 37 °C throughout the imaging session. The temperature of the animals was maintained at 37 °C, using a custom-built mouse cradle. Image acquisition was performed over 12–17 min using a 1.8 mm pinhole rat collimator (1 frame, 100,000 counts/projection, 35–50 s/bed position). Whole-body CT images were acquired for anatomical reference and attenuation correction (55 kVp, 0.19 mA, 20 ms). Reconstruction of the SPECT images was performed using a γ-ray energy window of 156–190 keV (background weight 2.5), 0.8 mm3 voxels, 2 subsets, and 4 iterations using the manufactur- er's SROSEM reconstruction type. To allow quantiﬁcation of the SPECT data, calibration factors derived from 111In phantoms were used. SPECT images were each registered to CT and then attenuation corrected. Quantiﬁcation of SPECT images using volume-of-interest (VOI) analyses were performed using the PMod software package (Ver- sion 3.807, PMOD Technologies), to calculate the percentage of the injected dose per millilitre per VOI (%ID/mL). 3.2. Anti-ICAM-1 and [111In]In-anti-ICAM-1 antibody characterization 3.2. Anti-ICAM-1 and [111In]In-anti-ICAM-1 antibody characterization 2.6. In vitro saturation binding PSN-1 cells were cultured in 24-well plates at 1 × 104 cells/well and allowed to adhere for 24 h. Cells were then irradiated (10 Gy or mock- irradiated) and allowed to recover for 24 h, and exposed to increasing concentrations of [111In]In-anti-ICAM-1, or [111In]In-mIgG2a control in PBS for 1 h at room temperature. The cells were washed with PBS, lysed in 0.1 M NaOH and counted on a gamma-counter (Perkin Elmer 24802). M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 75 2.7. In vivo SPECT imaging obtained from curve-ﬁts. All data were obtained in at least triplicate and results reported and graphed as mean ± standard deviation. All animal procedures were performed in accordance with the UK Animals (Scientiﬁc Procedures) Act 1986 and with local ethical commit- tee approval. Xenograft tumours were established in the right hind ﬂank of female athymic BALB/c nu/nu mice (Harlan) by subcutaneous injection of PSN-1 cells (2 × 106) in a 100 μL solution of a 1:1 mixture of Matrigel (#356234, Corning) and PBS. When tumours reached a di- ameter of approximately 6 mm or greater, xenografts were irradiated using a Gulmay 320 kV system (2.0 Gy/min) to a radiation absorbed dose of 10 Gy. The radiation set-up allowed irradiation of the right hind quarter, including the tumour and right leg, only. Control animals were mock-irradiated. 3.2. Anti-ICAM-1 and [111In]In-anti-ICAM-1 antibody characterization A commercially available anti-ICAM-1 antibody (#2213, Abcam) was selected to function as a targeting vector for a radiolabelled imaging agent. Evaluation of the antibody by ﬂow cytometry and by saturation binding assay, showed an afﬁnity (Kd) of 1.38 ± 0.39 nM (Fig. 2A). Anti-ICAM-1 antibody bound signiﬁcantly more to irradiated cells than non-irradiated cells (mean ﬂuorescence intensity of 836.9 ± 32.3 versus 454.6 ± 27.2, respectively; P b 0.0001). Radiolabelling with 111In following DTPA conjugation, resulting in [111In]In-anti-ICAM-1 (Fig. 2B), allowed a radioligand saturation bind- ing assay to be performed (Fig. 2C). The afﬁnity Kd of the radiolabelled antibody was calculated as 24.0 ± 4.0 nM. A radiolabelled non- selective control antibody, [111In]In-mIgG2a, bound signiﬁcantly less to PSN-1 cells (P b 0.0001). After the ﬁnal imaging session, mice were euthanized by cervical dislocation, and selected organs, tissues and blood were removed. The amount of radioactivity in each organ was measured using a HiDex gamma counter (Perkin Elmer). Counts per minute were converted into MBq using a calibration curve generated from known standards and the percentage of the injected dose per gram (%ID/g) in each sample was calculated. 3.3. SPECT imaging shows speciﬁc ICAM-1 tumour signal but no difference between untreated and irradiated tumours 3.3. SPECT imaging shows speciﬁc ICAM-1 tumour signal but no difference between untreated and irradiated tumours 2.9. Statistical analyses All statistical analyses and nonlinear regressions were performed using GraphPad Prism v8 (GraphPad Software). One- or two-way ANOVA was used for multiple comparisons, the percentage of the injected dose per gram. F-tests were used to compare best-ﬁt values Western blot analysis of total cell lysates from the xenograft tumours (from a second cohort) showed that ICAM-1 protein expression was far lower in the xenografts when compared to in vitro cell cultures, and M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 Fig. 1. (A) Human oncology antibody array after incubation with mock-treated and irradiated lysates – ICAM-1 signals highlighted. (B) Proteins from antibody array with ≥2-fold increase in expression post-irradiation. (C) A representative western blot showing a maximum increase in signal in PSN-1 cell lysates at the 48 h time-point post-irradiation treatment (IR) when compared to the mock-treated sample. (D) Immunoﬂuorescence microscopy (20×) shows an increase in ICAM-1 signal in PSN-1 cells post-irradiation, with a maximum increase at the 24 h time-point. Scale bar 40 μm. (E) Immunohistochemistry micrograph staining for ICAM-1 in a human PDAC section. (Adapted from ProteinAtlas.org). 76 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 76 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 Fig. 1. (A) Human oncology antibody array after incubation with mock-treated and irradiated lysates – ICAM-1 signals highlighted. (B) Proteins from antibody array with ≥2-fold increase in expression post-irradiation. (C) A representative western blot showing a maximum increase in signal in PSN-1 cell lysates at the 48 h time-point post-irradiation treatment (IR) when compared to the mock-treated sample. (D) Immunoﬂuorescence microscopy (20×) shows an increase in ICAM-1 signal in PSN-1 cells post-irradiation, with a maximum increase at the 24 h time-point. Scale bar 40 μm. (E) Immunohistochemistry micrograph staining for ICAM-1 in a human PDAC section. (Adapted from ProteinAtlas org) conﬁrmed that there was little change in this expression level post- irradiation (Fig. 5). expression in various cells in vitro, such as HUVECs, bone marrow ECs, and HDMECs [26]. The identiﬁcation of ICAM-1 as a potential biomarker of radiation treatment is of particular interest given the involvement of adhesion molecules in the immune response following tumour RT. 2.9. Statistical analyses Irradiation can also induce expression of adhesion molecules on tumour blood and lymphatic vessels, where they play a role in leucocyte migration and extravasation into the tumour [27–29]. Radiation- induced ICAM-1 mediates the transmigration of tumour-promoting CD11b + myeloid cells [30]. Irradiation of a number of human cancer cells has been shown to induce ICAM-1 expression, which enhances activated NK cell-mediated cytotoxicity [31]. 4. Discussion This study sought to identify human tumour cell-surface biomarkers of radiation treatment (RT). Such markers would provide a non- invasive measuring tool, using immuno-PET or SPECT imaging, of tu- mour response to radiation treatment, as an alternative to some of the intranuclearly targeted imaging agents that bind DNA-damage associ- ated nuclear proteins we presented before [5,21]. Pancreatic tumour cells were used as a model system because of the urgent clinical need to improve the particularly poor survival rates for pancreatic cancer pa- tients [22,23]. Here, we provide a proof-of-principle validation of imag- ing of the adhesion molecule ICAM-1, using a radiolabelled antibody for SPECT imaging. Here, we showed that ICAM-1 expression levels in all in vitro assays showed a consistent increase after 10 Gy irradiation (Figs. 1 & 2) – up to a 20-fold increase in western blot analyses. We also showed proof-of- principle that [111In]In-anti-ICAM-1, but not the negative control [111In]In-mIgG2a was taken up in PSN-1 xenografts. Surprisingly, how- ever, we only observed an increased uptake of [111In]In-anti-ICAM-1 in the irradiated tumour on one out of three mice in that group in our in vivo study. Possible explanations for this lack of radiation-induced ef- fect may include: (1) a change in afﬁnity for its target after conjugation and labelling of the ICAM-1 antibody, (2) an increase in ICAM-1 ICAM-1 is a cell surface immunoglobulin glycoprotein, typically expressed on endothelial cells and cells of the immune system [11], and its extracellular location, anchored in the cell membrane, makes it a particularly attractive target for molecular immuno-imaging [24,25]. Radiation doses ranging from 2 to 20 Gy are known to induce ICAM-1 Fig. 2. (A) Calculation of the maximum speciﬁc binding (Bmax) and afﬁnity (Kd) of anti-ICAM-1 antibody using ﬂow cytometry, shows a 1.8-fold increase in Bmax value when comparing mock-treated to irradiated PSN-1 cells. (B) iTLC of 111In-labelled anti-ICAM-1 antibody, showing efﬁcient conjugation of N95%. (C) The binding afﬁnity of the anti-ICAM-1 DTPA/111In radio- conjugate ([111In]In-anti-ICAM-1), at 24 h time-point was determined using an in vitro saturation binding methodology. A negative control [111In]In-mIgG2a was also characterized. Fig. 2. (A) Calculation of the maximum speciﬁc binding (Bmax) and afﬁnity (Kd) of anti-ICAM-1 antibody using ﬂow cytometry, shows a 1.8-fold increase in Bmax value when comparing mock-treated to irradiated PSN-1 cells. (B) iTLC of 111In-labelled anti-ICAM-1 antibody, showing efﬁcient conjugation of N95%. Financial support This research was supported by CRUK through the Oxford Institute for Radiation Oncology (MA, DA, GD, VK, BC). Further support was ob- tained from Pancreatic Cancer Research Fund (BC, JBT). 4. Discussion expression in the irradiated xenografts compared to the mock-treated, overall ICAM-1 levels were lower in the xenograft tumour tissue com- pared to cell lysates from in vitro models (Figs. 1 and 5). expression in the irradiated xenografts compared to the mock-treated, overall ICAM-1 levels were lower in the xenograft tumour tissue com- pared to cell lysates from in vitro models (Figs. 1 and 5). Interference in the ICAM-1 signal obtained by the ICAM-1 monoclo- nal antibody by mouse-speciﬁc expression of the ICAM-1 molecule in vivo (which may be altered by the presence of the xenograft tumour) has not been excluded. However, the antibody used in this study is re- ported to be human-speciﬁc. Furthermore, we [25] and others [33] have previously used murine antibodies for imaging in vivo in mouse without non-speciﬁc background issues. Interference in the ICAM-1 signal obtained by the ICAM-1 monoclo- nal antibody by mouse-speciﬁc expression of the ICAM-1 molecule in vivo (which may be altered by the presence of the xenograft tumour) has not been excluded. However, the antibody used in this study is re- ported to be human-speciﬁc. Furthermore, we [25] and others [33] have previously used murine antibodies for imaging in vivo in mouse without non-speciﬁc background issues. The possibility that conjugation and radio-labelling of the ICAM-1 antibody had an effect on its afﬁnity to its target was investigated. In vitro saturation binding showed that [111In]In-anti-ICAM-1 presented a Kd value of 24.0 ± 4.0 nM (Fig. 2C), although presenting a reduce af- ﬁnity compared to its unmodiﬁed form, still suitable for in vivo imaging. To determine whether the DTPA-conjugation process might explain the difference, we repeated the characterization after decreasing the molar quantity of p-SCN-Bn-DTPA used in the conjugation, but without im- provement in Kd (data not shown). Taken together, our data suggest that the lack of radiation-induced effect is not caused by modiﬁcation of the antibody for radiolabelling. 5. Conclusion We report the identiﬁcation of ICAM-1 as a potential biomarker of radiation treatment of the human pancreatic cancer cell-line PSN-1 in vitro. In vitro assays suggest an increase in ICAM-1 expression follow- ing 10 Gy gamma irradiation. However, although in vivo SPECT imaging conﬁrms the presence of ICAM-1 in PSN-1 cells, and its suitability as a pancreatic cancer cell marker, total and radiation-mediated ICAM-1 ex- pression is reduced in vivo compared to in vitro. There have been previously published examples of differences in speciﬁc gene expression between in vitro and in vivo disease models. A gene expression proﬁling study of xenografts [32] identiﬁed changes in expression levels of numerous genes after growth in mice, in particu- lar a marked decline in ABCB1 transporter levels in HCT-15 colon xeno- graft. Furthermore, differences in expression proﬁles of the adhesion molecules ICAM-1 and VCAM-1, in in vitro versus in vivo models of arte- riovenous malformation disease, after treatment with ionizing irradia- tion, have been previously reported [26], in which already raised levels of ICAM-1 in the in vivo model made changes in expression levels due to irradiation treatment difﬁcult to measure. This possibility was tested in our study, which included a cohort of irradiated xenograft bearing mice where the levels of ICAM-1 expression at various times after irradiation were measured by western blot (Fig. 5). We observed however that, although there were no signiﬁcant changes in ICAM-1 4. Discussion (C) The binding afﬁnity of the anti-ICAM-1 DTPA/111In radio- conjugate ([111In]In-anti-ICAM-1), at 24 h time-point was determined using an in vitro saturation binding methodology. A negative control [111In]In-mIgG2a was also characterized. Fig. 3. (A) Tumour biodistribution quantiﬁcation shows an increase in mean percentage Injected Dose per tumour weight (%ID/g) in mice injected with [111In]In-anti-ICAM-1 compared to [111In]In-mIgG2a. However, there was no signiﬁcant difference in [111In]In-anti-ICAM-1 uptake in the irradiated tumours compared to the mock-treated control. (B) SPECT image VOI quantiﬁcation of each xenograft tumour at each SPECT time-point of 24, 48 and 72 h shows an increase in [111In]In-anti-ICAM-1 compared to [111In]mIgG2a at each of the time-points. There was a small increase in signal at the 24 h time-point in untreated mice compared to irradiated mice. However, there was no difference in [111In]In-anti-ICAM-1 signal in the irradiated tumours compared to the mock-treated control ones in the other 2 time-points. (C) Representative MIP images from the 24 h SPECT imaging study (white dashed circles indicate tumours). 77 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 77 M. Mosley et al. / Nuclear Medicine and B Fig. 3. (A) Tumour biodistribution quantiﬁcation shows an increase in mean percentage Injected Dose per tumour weight (%ID/g) in mice injected with [111In]In-anti-ICAM-1 compared to [111In]In-mIgG2a. However, there was no signiﬁcant difference in [111In]In-anti-ICAM-1 uptake in the irradiated tumours compared to the mock-treated control. (B) SPECT image VOI quantiﬁcation of each xenograft tumour at each SPECT time-point of 24, 48 and 72 h shows an increase in [111In]In-anti-ICAM-1 compared to [111In]mIgG2a at each of the time-points. There was a small increase in signal at the 24 h time-point in untreated mice compared to irradiated mice. However, there was no difference in [111In]In-anti-ICAM-1 signal in the irradiated tumours compared to the mock-treated control ones in the other 2 time-points. (C) Representative MIP images from the 24 h SPECT imaging study (white dashed circles indicate tumours). expression in the untreated PSN-1 cells after growth as a xenograft in inoculated mice, or (3) interference in the in vivo ICAM-1 signal caused by murine ICAM-1 expressed by the host (which may be altered by the presence of the xenograft tumour). References [1] Barton MB, Jacob S, Shaﬁq J, Wong K, Thompson SR, Hanna TP, et al. Estimating the demand for radiotherapy from the evidence: a review of changes from 2003 to 2012. Radiother Oncol 2014;112:140–4. This research was supported by CRUK through the Oxford Institute for Radiation Oncology (MA, DA, BC). Further support was obtained from Pancreatic Cancer Research Fund (BC, JBT). for Radiation Oncology (MA, DA, BC). Further support was obtained from Pancreatic Cancer Research Fund (BC, JBT). Fig. 5. Western blot analysis of total lysates from the xenograft tumours obtained in this study, compared with in vitro prepared PSN-1 cells, both untreated and irradiated. The blot shows that expression of ICAM-1 was generally lower in the PSN-1 xenografts when compared to the cell cultures, and that in the xenografts there was little change in expression post-irradiation when compared to the untreated tumours. [2] Rafat M, Ali R, Graves EE. Imaging radiation response in tumor and normal tissue. Am J Nucl Med Mol Imaging 2015;5:317–32. [3] West CM, Barnett GC. Genetics and genomics of radiotherapy toxicity: towards pre- diction. Genome Med 2011;3:52. [4] Selleck MJ, Senthil M, Wall NR. Making meaningful clinical use of biomarkers. Biomark Insights 2017;12:1177271917715236. [5] Knight JC, Koustoulidou S, Cornelissen B. Imaging the DNA damage response with PET and SPECT. Eur J Nucl Med Mol Imaging 2017;44:1065–78. J g g ; [6] Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605–17. [6] Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605–17. [7] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5–29. [7] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J C [8] Alazraki N. Imaging of pancreatic cancer using ﬂuorine-18 ﬂuorodeoxyglucose pos- itron emission tomography. J Gastrointest Surg 2002;6:136–8. [9] Lee ES, Lee JM. Imaging diagnosis of pancreatic cancer: a state-of-the-art review. World J Gastroenterol 2014;20:7864–77. [10] Fokas E, Prevo R, Pollard JR, Reaper PM, Charlton PA, Cornelissen B, et al. Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pan- creatic tumors to radiation. Cell Death Dis 2012;3:e441. [11] Wai Wong C, Dye DE, Coombe DR. The role of immunoglobulin superfamily cell ad- hesion molecules in cancer metastasis. Int J Cell Biol 2012;2012:340296. [12] Atlas p. https://www.proteinatlas.org/ENSG00000090339-ICAM1/pathology/ pancreatic+cancer; 2019. [13] Khatib AM, Auguste P, Fallavollita L, Wang N, Samani A, Kontogiannea M, et al. Declaration of competing interest The authors disclose no potential conﬂicts of interest. Fig. 4. (A) Autoradiography of a representative xenograft tumour sections conﬁrming speciﬁc uptake of [111In]In-anti-ICAM-1. There is a small increase in uptake of the [111In]anti-ICAM-1 antibody compared to the [111In]In-mIgG2a negative control conjugate, but little difference in uptake of [111In]In-anti-ICAM-1 in xenografts that had been irradiated (IR) compared to mock-treated (MT). (B) Quantiﬁcation of autoradiography image in (A). (C) Biodistribution quantiﬁcation (%ID/g) in mice injected with [111In]In-anti-ICAM-1 compared to [111In]In- mIgG2a, at 72 h post-injection of each IgG molecule. Tumour was either irradiated (IR) or mock treated (MT). 78 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 78 Medicine and Biology 84 85 (2020) 73 79 Fig. 4. (A) Autoradiography of a representative xenograft tumour sections conﬁrming speciﬁc uptake of [111In]In-anti-ICAM-1. There is a small increase in uptake of the [111In]anti-ICAM-1 antibody compared to the [111In]In-mIgG2a negative control conjugate, but little difference in uptake of [111In]In-anti-ICAM-1 in xenografts that had been irradiated (IR) compared to mock-treated (MT). (B) Quantiﬁcation of autoradiography image in (A). (C) Biodistribution quantiﬁcation (%ID/g) in mice injected with [111In]In-anti-ICAM-1 compared to [111In]In- mIgG2a, at 72 h post-injection of each IgG molecule. Tumour was either irradiated (IR) or mock treated (MT). References Char- acterization of the host proinﬂammatory response to tumor cells during the initial stages of liver metastasis. Am J Pathol 2005;167:749–59. Fig. 5. Western blot analysis of total lysates from the xenograft tumours obtained in this study, compared with in vitro prepared PSN-1 cells, both untreated and irradiated. The blot shows that expression of ICAM-1 was generally lower in the PSN-1 xenografts when compared to the cell cultures, and that in the xenografts there was little change in expression post-irradiation when compared to the untreated tumours. [14] Clayton A, Evans RA, Pettit E, Hallett M, Williams JD, Steadman R. Cellular activation through the ligation of intercellular adhesion molecule-1. J Cell Sci 1998;111(Pt 4): 443–53. M. Mosley et al. / Nuclear Medicine and Biology 84–85 (2020) 73–79 79 [15] Rahn JJ, Chow JW, Horne GJ, Mah BK, Emerman JT, Hoffman P, et al. MUC1 mediates transendothelial migration in vitro by ligating endothelial cell ICAM-1. Clin Exp Me- tastasis 2005;22:475–83. [25] Torres JB, Knight JC, Mosley MJ, Kersemans V, Koustoulidou S, Allen D, et al. Imaging of claudin-4 in pancreatic ductal adenocarcinoma using a radiolabelled anti-claudin- 4 monoclonal antibody. Mol Imaging Biol 2018;20:292–9. y g g [26] Raouﬁ-Rad N, McRobb LS, Lee VS, Bervini D, Grace M, Ukath J, et al. In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation. PLoS One 2017;12:e0185393. [16] Ghislin S, Obino D, Middendorp S, Boggetto N, Alcaide-Loridan C, Deshayes F. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro. BMC Cancer 2012;12:455. [27] Walle T, Martinez Monge R, Cerwenka A, Ajona D, Melero I, Lecanda F. Radiation ef- fects on antitumor immune responses: current perspectives and challenges. Ther Adv Med Oncol 2018;10:1758834017742575. [17] Delfortrie S, Pinte S, Mattot V, Samson C, Villain G, Caetano B, et al. Egﬂ7 promotes tumor escape from immunity by repressing endothelial cell activation. Cancer Res 2011;71:7176–86. [18] Zhang Y, Wang M, Liu W, Peng X. Optical imaging of triple-negative breast cancer cells in xenograft athymic mice using an ICAM-1-targeting small-molecule probe. Mol Imaging Biol 2019;21:835–41. [28] Rodriguez-Ruiz ME, Garasa S, Rodriguez I, Solorzano JL, Barbes B, Yanguas A, et al. In- tercellular adhesion molecule-1 and vascular cell adhesion molecule are induced by ionizing radiation on lymphatic endothelium. Int J Radiat Oncol Biol Phys 2017;97: 389–400. References [19] Chiriva-Internati M, Grizzi F, Pinkston J, Morrow KJ, D’Cunha N, Frezza EE, et al. Gamma-radiation upregulates MHC class I/II and ICAM-I molecules in multiple my- eloma cell lines and primary tumors. In Vitro Cell Dev Biol Anim 2006;42:89–95. [29] Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inﬂammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 2007;7:678–89. [20] Mosley M, Knight J, Neesse A, Michl P, Iezzi M, Kersemans V, et al. Claudin-4 SPECT imaging allows detection of aplastic lesions in a mouse model of breast cancer. J Nucl Med 2015;56:745–51. [30] Ni J, Miller M, Stojanovic A, Garbi N, Cerwenka A. Sustained effector function of IL- 12/15/18-preactivated NK cells against established tumors. J Exp Med 2012;209: 2351–65. [21] Cornelissen B, Kersemans V, Darbar S, Thompson J, Shah K, Sleeth K, et al. Imaging DNA damage in vivo using gammaH2AX-targeted immunoconjugates. Cancer Res 2011;71:4539–49. [31] Jeong JU, Uong TNT, Chung WK, Nam TK, Ahn SJ, Song JY, et al. Effect of irradiation- induced intercellular adhesion molecule-1 expression on natural killer cell- mediated cytotoxicity toward human cancer cells. Cytotherapy 2018;20:715–27. [22] Cornelissen B, Knight JC, Mukherjee S, Evangelista L, Xavier C, Caobelli F, et al. Trans- lational molecular imaging in exocrine pancreatic cancer. Eur J Nucl Med Mol Imag- ing 2018;45:2442–55. [32] Hollingshead MG, Stockwin LH, Alcoser SY, Newton DL, Orsburn BC, Bonomi CA, et al. Gene expression proﬁling of 49 human tumor xenografts from in vitro culture through multiple in vivo passages—strategies for data mining in support of thera- peutic studies. BMC Genomics 2014;15:393. [23] Garcea G, Dennison AR, Pattenden CJ, Neal CP, Sutton CD, Berry DP. Survival follow- ing curative resection for pancreatic ductal adenocarcinoma. A systematic review of the literature. JOP 2008;9:99–132. [33] Takashima H, Tsuji AB, Saga T, Yasunaga M, Koga Y, Kuroda JI, et al. Molecular imag- ing using an anti-human tissue factor monoclonal antibody in an orthotopic glioma xenograft model. Sci Rep 2017;7:12341. J [24] Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer 2012;12: 278–87.
https://openalex.org/W4241584932	https://www.dora.lib4ri.ch/wsl/islandora/object/wsl%3A27417/datastream/PDF/Compagno-2021-Brief_communication-%28published_version%29.pdf	English	null	Brief communication: Do 1.0, 1.5, or 2.0 °C matter for the future evolution of Alpine glaciers?	The cryosphere	2,021	cc-by	5,081	Brief communication: Do 1.0, 1.5, or 2.0 ◦C matter for the future evolution of Alpine glaciers? Loris Compagno1,2, Sarah Eggs1,2, Matthias Huss1,2,3, Harry Zekollari4,5,1,2, and Daniel Farinotti1,2 1Laboratory of Hydraulics, Hydrology and Glaciology (VAW), ETH Zurich, Zurich, Switzerland 2Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland 3Department of Geosciences, University of Fribourg, Fribourg, Switzerland 4Department of Geoscience and Remote Sensing, Delft University of Technology, Delft, the Netherlands 5Laboratoire de Glaciologie, Université libre de Bruxelles, Brussels, Belgium Correspondence: Loris Compagno (compagno@vaw.baug.ethz.ch) Received: 26 January 2021 – Discussion started: 10 February 2021 Revised: 4 May 2021 – Accepted: 7 May 2021 – Published: 15 June 2021 Received: 26 January 2021 – Discussion started: 10 February 2021 Revised: 4 May 2021 – Accepted: 7 May 2021 – Published: 15 June 2021 ing of 1.5 ◦C (IPCC, 2018), for example, focused on the ef- fects that would result if the 1.5 ◦C target was to be met and highlighted that, even under such conditions, important and mostly negative changes would occur. A mere 5 years after the Paris Agreement, there is compelling evidence that any reduction in anthropogenic climate change will pay off in the longer term, both from an ecological (IPBES, 2019) and economical (WEF, 2020) perspective. Discussions in prepa- ration for the renewed pledges by signatory parties have, thus, focused on the differences between scenarios close to the original targets. For the upcoming 26th United Nations Climate Change Conference of the Parties, for example, em- phasis is put on separating the impacts occurring under sce- narios of 1.0, 1.5, and 2.0 ◦C of warming above preindus- trial levels. In this brief communication, we focus on the dif- fering impacts that such scenarios would have on glaciers and related water resources in the European Alps. Across the world, glaciers are amongst the most prominent indica- tors of climatic change, providing visual evidence for cli- matic changes occurring over decades. Previous work esti- mated that every kilogram of additionally emitted CO2 would result in a long-term global glacier mass loss of ca. 16 kg (Marzeion et al., 2018) – with important implications for the corresponding landscapes, downstream ecosystems, and wa- ter supplies (IPCC, 2018, 2019). While future projections for the glacier evolution of the European Alps already exist un- der different representative concentration pathways (RCPs) (e.g. Zekollari et al., 2019; Marzeion et al., 2020), targeted information on policy-relevant climate targets, such as the Abstract. Brief communication: Do 1.0, 1.5, or 2.0 ◦C matter for the future evolution of Alpine glaciers? With the Paris Agreement, the urgency of limiting ongoing anthropogenic climate change has been recognised. More recent discussions have focused on the difference of limiting the increase in global average temperatures below 1.0, 1.5, or 2.0 ◦C compared to preindustrial levels. Here, we assess the impacts that such different scenarios would have on both the future evolution of glaciers in the European Alps and the water resources they provide. Our results show that even half-degree differences in global temperature targets have important implications for the changes predicted until 2100, and that – for the most optimistic scenarios – glaciers might start to partially recover, owing to possibly decreasing temperatures after the end of the 21st century. 1 Introduction Climate change is one of the largest challenges that society will face during this century. There is overwhelming con- sensus that limiting the increase of global average temper- atures below a certain threshold is essential if adverse effects are to be avoided. Put forward in 2015 and adopted by 196 parties, the Paris Agreement concurred that such a thresh- old is “well below 2 ◦C above preindustrial levels”, and that efforts should be pursued to “limit the temperature increase to 1.5 ◦C” (UN, 2015). Even under these ambitious climate targets, important environmental changes, such as changes in water availability, migration of species, or glacier loss, are expected to occur. The Special Report on Global Warm- The Cryosphere, 15, 2593–2599, 2021 https://doi.org/10.5194/tc-15-2593-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. The Cryosphere, 15, 2593–2599, 2021 https://doi.org/10.5194/tc-15-2593-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. 2 Data and methods We simulate the evolution of all 3927 glaciers in the Alps independently based on (i) outlines provided by the Ran- dolph Glacier Inventory version 6 (RGI 6.0) (RGI Consor- tium, 2017) and (ii) the consensus ice thickness estimate (Farinotti et al., 2019). Glacier evolution is modelled with the combined mass bal- ance ice ﬂow model GloGEMﬂow (Huss and Hock, 2015; Zekollari et al., 2019), a model that showed robust per- formance in recent model intercomparison projects (e.g. Marzeion et al., 2020). Accumulation is computed based on precipitation and temperature, with a linear transition be- tween solid and liquid precipitation occurring for tempera- tures between 0.5 and 2.5 ◦C. Ablation is calculated with a temperature index model (Hock, 2003), and refreezing is de- termined based on heat conduction. The mass balance mod- ule runs at a monthly resolution, including a parameterisa- tion for day-to-day temperature variability. It is calibrated to reproduce glacier-speciﬁc geodetic volume changes (Zemp et al., 2019) spanning approximatively the period 1959–2013 and covering 57 % of the Alpine glacier area. For more de- tails on the mass balance module and its calibration, refer to Huss and Hock (2015). To evaluate model performance, we compare modelled mass balances with observations provided by the World Glacier Monitoring Service (WGMS, 2020) for 72 glaciers in the European Alps. For glacier-wide annual mass bal- ance, we ﬁnd a bias of 0.07 m water equivalent (w.e. a−1), a root mean square error (RMSE) of 1.07 m w.e. a−1, and a squared correlation coefﬁcient (r2) of 0.29 (see Fig. S1 in the Supplement). Observations aggregated to elevation bands yield a bias of −0.01 m w.e. a−1, a RMSE of 1.47 m w.e. a−1, and a r2 of 0.52. For glacier-wide winter balance, a bias of 0.20 m w.e. a−1, a RMSE of 0.74 m w.e. a−1, and a r2 of 0.26 is found (see Fig. S2). Although correlation coefﬁcients are rather low, the small biases provide conﬁdence in the regional results, which neither indicate overestimation nor underesti- mation. The latter is also conﬁrmed by the comparison of the total glacier volume change in Switzerland (comprising al- most 60 % of the Alpine glacier volume at present) during the period 2000–2020. Our modelled loss is of 24.9 km3, which is very close to the observation-based estimate of 23.6 km3 (Grab et al., 2021). L. Compagno et al.: Why 0.5 ◦C matters 2594 difference between 1.5 and 2.0 ◦C, is not readily available. Here we analyse glacier evolution under low global warm- ing scenarios by re-running the mass balance ice ﬂow model, GloGEMﬂow (Huss and Hock, 2015; Zekollari et al., 2019), with updated climate projections from the sixth phase of the Coupled Model Intercomparison Project (CMIP6). In partic- ular, we focus on Shared Socioeconomic Pathways (SSPs) that would result in global mean temperature changes of +1.0, +1.5, and +2.0 ◦C by 2100 compared to preindustrial conditions (1850–1900) and provide projections for both fu- ture glacier geometries and glacier runoff until the end of the century. In an explorative analysis, we also consider pro- jections until the year 2300, albeit acknowledging that large uncertainties exist when doing so. ﬁrn) and by subtracting refreezing. Throughout the simula- tions, runoff is computed for the area comprised within the RGI 6.0 outlines, implying that, after glacier retreat, runoff contributions from rain and snow melt are still accounted for (in line with Huss and Hock, 2018). ( , ) For the past (1950–2020), GloGEMﬂow is forced with monthly 2 m air temperature and precipitation taken from the ensemble daily gridded observational data set (E-OBS v.21.0) at 0.1◦(11 km) horizontal resolution (Cornes et al., 2018). For the future (2020–2100), we rely on 128 CMIP6 global circulation model (GCM) members. We use direct GCM output since CMIP6 GCM results have not yet been downscaled by regional climate models and since the spa- tial resolution of the climate data was recently shown to only marginally affect model results if suitable model cali- bration at the glacier-speciﬁc scale is performed (Compagno et al., 2021). Consistence between past and future climate is ensured by applying the de-biasing procedure proposed by Huss and Hock (2015). In a nutshell, the procedure uses a set of additive and multiplicative correction factors to adjust both the long-term mean and the long-term variability in the coarse resolution GCMs (100 km) to the level of the high- resolution E-OBS data (see Huss and Hock, 2015, for more details). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. L. Compagno et al.: Why 0.5 ◦C matters 3 Results and discussion Finally, our simulations project the 2080– 2100 August runoff to be 36 ± 15 % (+1.0 ◦C), 44 ± 14 % (+1.5 ◦C), and 55 ± 15 % (+2.0 ◦C) lower on average than it was in 2000–2020. This shift in the magnitude and tim- ing of glacier runoff (i) is similar to what projected in earlier studies (e.g. Huss and Hock, 2018), (ii) is a direct conse- quence of both the reduced glacier volumes (which cause the annual runoff to decrease) and the increased tempera- ture (which causes snowmelt and ice melt to occur earlier in the year), and (iii) will have important consequences for water availability (Huss and Hock, 2018), particularly dur- ing the dry summer months. For the densely populated Euro- pean Alps and the areas downstream, such changes will not only impact ecosystems but also a number of water users, including agriculture or hydropower operators for instance (Beniston et al., 2018). In all three considered warming scenarios, glaciers will be drastically affected. By 2100, glaciers in the European Alps are projected to lose 44 ± 21 % (+1.0 ◦C), 68 ± 12 % (+1.5 ◦C), and 81 ± 8 % (+2.0 ◦C) of their 2020 ice volume (see Fig. 2a). The evolution of the glacier area will be on par (Fig. 2b). By 2100, Alpine glaciers are projected to lose 47 ± 16 % (+1.0 ◦C), 65 ± 10 % (+1.5 ◦C), and 77 ± 8 % (+2.0 ◦C) of their 2020 area (Fig. 2b). Note that, for the +1.0 ◦C temperature target, the projected volume and area losses are only slightly higher than the committed loss, i.e. the loss obtained by assuming the climatic conditions to sta- bilise at the 1988–2017 levels. Indeed, Zekollari et al. (2019) estimated such a committed loss to be of 37 ± 6 % for glacier volume and 35 ± 7 % for the glacier area. In line with the bulk of the literature, the above results all refer to the time horizon until 2100. To gain insights into glacier evolution beyond this horizon, we run GloGEMﬂow with three GCM members that provide climate data until 2300 and that project mean global temperature changes be- low +2.0 ◦C for 2300 (see Fig. S3). 3 Results and discussion The GCMs selected within the three temperature change targets (i.e. +1.0, +1.5, and +2.0 ◦C) show a global mean warming between 1850–1900 and 2071–2100 of +1.00 ± 0.11, +1.49 ± 0.17, and +2.07 ± 0.18 ◦C. For the European Alps, this corresponds to a temperature change of +0.98 ± 0.56, +1.80 ± 0.72, and +2.51 ± 0.73 ◦C, respec- tively (Fig. 1a). During the summer months (June–August; JJA), the temperature increase in the Alps for the two warmer climate targets is even stronger, and is of 0.96 ± 0.88, 2.09 ± 1.24, and 2.81 ± 1.23 ◦C, respectively. These differ- ences agree well with previous assessments (Vautard et al., 2014) and are attributed to stronger warming over land than over the ocean, to enhanced warming at high latitudes com- pared to low latitudes, and to ampliﬁed warming at higher el- evations (Vautard et al., 2014). For a global warming of +1.0, +1.5, and +2 ◦C, global-scale annual precipitation sums for 2071–2100 are projected to increase by 18 ± 8, 29 ± 9, and 42 ± 10 mm a−1, respectively, up from 1141 mm a−1 in the preindustrial baseline (Fig. 1b). For the Alps, the annual pre- cipitation increase is anticipated to be 37 ± 94, 29 ± 135, and 46 ± 145 mm a−1, respectively (the preindustrial baseline is 1210 mm a−1), with slightly higher precipitation changes over the winter season. p Future effects notably include the water availability from presently glacierised areas. Our results indicate that, on a yearly basis, glacier runoff for the period 2080–2100 might decrease by as much as 25 ± 6 % (+1.0 ◦C), 32 ± 8 % (+1.5 ◦C), and 36 ± 10 % (+2.0 ◦C) compared to 2000–2020 levels (Fig. 2c). The decrease follows above-average glacier melt during the last decades and indicates that peak glacier discharge already occurred in the past for many glaciers in the European Alps (consistent with the ﬁndings of, for example, Huss and Hock, 2018). Our model results also show a transition in the timing of the maximum glacier dis- charge during the year; while it historically occurred in Au- gust (2000–2020), it is expected to occur in July (+1.0 and +1.5 ◦C scenarios) or even June–July (+2.0 ◦C) by 2080– 2100 (Fig. 2d). 2 Data and methods Similarly, the modelled rate of glacier area loss (1.4 % per annum for the period 2003–2016) is in very good agreement with the rate of 1.3 % per annum, which was derived by Paul et al. (2020) using Landsat images. For all glaciers with a >1 km length in RGI 6.0 (i.e. 795 glaciers accounting for 95 % of the Alpine glacier vol- ume), we compute ice ﬂow based on the shallow ice approx- imation. The computations follow Zekollari et al. (2019), in- cluding the iterative initialisation procedure used to gener- ate a transient glacier geometry for the RGI inventory year (i.e. 2003 for the majority of Alpine glaciers). For all glaciers with a < 1 km length, glacier evolution is modelled with an elevation-dependent parameterisation that has shown to be in good agreement with higher order ice ﬂow modelling results (Huss et al., 2010). For more information on GloGEMﬂow’s glacier evolution modelling, please refer to Zekollari et al. (2019). Finally, for our main analysis, we only select those GCM members that yield a 21st century global warming of +1.0, +1.5, and +2.0 ◦C compared to the preindustrial levels. The selected members correspond to SSPs that assume low emis- sions, i.e. SSP119, SSP126, and SSP245, and are used to force GloGEMﬂow for the period 2020–2100. This selection is performed by computing the mean global 2 m air temper- ature change over both the land and ocean between the pe- For each glacier, water runoff is calculated at a monthly resolution by summing rain and melt (from ice, snow, and https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 2595 L. Compagno et al.: Why 0.5 ◦C matters might lose as much as 45 ± 12 %, 64 ± 11 %, and 77 ± 6 % of its 2020 volume, respectively. Rhonegletscher (Fig. 3e), one of the most studied and visited glaciers, is projected to lose 55 ± 24 %, 78 ± 11 %, and 91 ± 7 % of its volume in the same time span, while values for other glaciers are found in Table S1 in the Supplement. Our results conﬁrm that global warming can be signiﬁcantly ampliﬁed at the regional scale and that glaciers in the European Alps sensitively respond to even small levels of global atmospheric warming, such as a global air temperature change of +1.0 ◦C. Most impor- tantly, however, is that our results show signiﬁcant differ- ences between the future glacier evolution projected for the three warming scenarios, indicating that any effort to further limit warming will have important effects on glaciers and, thus, the Alpine environment. riod 2071–2100 and preindustrial (1850–1900) levels and by selecting members that are within a given range of tempera- ture increase, namely +1 ± 0.25 ◦C (three model members), +1.5 ± 0.25 ◦C (11 model members), and +2 ± 0.25 ◦C (14 model members). To determine the corresponding warming in the European Alps (see Figs. 1 and S3), we extract all GCM grid cells that are within latitudes of 42.5–49.5◦N, lon- gitudes of 4–16◦E, and at elevations above 500 m. L. Compagno et al.: Why 0.5 ◦C matters Figure 1. Evolution of annual global and Alpine (a) average air temperature and (b) precipitation total compared to the preindustrial (1850– 1900) baseline. Series are smoothed with a 30-year running mean. The coloured bands show the standard deviation of the projection given by individual GCM members. n is the considered number of GCM members. Figure 1. Evolution of annual global and Alpine (a) average air temperature and (b) precipitation total compared to the preindustrial (1850– 1900) baseline. Series are smoothed with a 30-year running mean. The coloured bands show the standard deviation of the projection given by individual GCM members. n is the considered number of GCM members. Figure 2. Modelled evolution of total glacier (a) volume, (b) area, (c) annual runoff, and (d) monthly runoff. The total refers to all glaciers of the European Alps. The time series in panel (c) are smoothed with a 30-year running mean. In all panels, the thick line represents the mean, and the transparent band corresponds to 1 standard deviation of the results obtained by forcing GloGEMﬂow with the selected GCM members. The number of GCM members is given (n) in panel (a). Figure 2. Modelled evolution of total glacier (a) volume, (b) area, (c) annual runoff, and (d) monthly runoff. The total refers to all glaciers of the European Alps. The time series in panel (c) are smoothed with a 30-year running mean. In all panels, the thick line represents the mean, and the transparent band corresponds to 1 standard deviation of the results obtained by forcing GloGEMﬂow with the selected GCM members. The number of GCM members is given (n) in panel (a). three members all come from SSP126 but stem from three different GCMs (see Fig. S3). this result is only based on three GCM members and is, thus, very uncertain, it suggests that considering projections be- yond 2100 might change the current perception of a possi- bly irreversible glacier loss. From the physical point of view, the result that glaciers might regrow after a potential cool- ing of global temperatures is not surprising. 3 Results and discussion Note that one of these GCM members was already considered in the simulations until 2100, whilst the remaining two were not because they show a warming beyond +2.0 ◦C for 2100 (Fig. S3). The Figure 3a–c show the regional distribution of the mod- elled glacier evolution. By 2100, only 1484 ± 324 (+1.0 ◦C), 1033 ± 253 (+1.5 ◦C), and 724 ± 201 (+2.0 ◦C) of today’s roughly 4000 glaciers are anticipated to remain, i.e. to have a volume > 0 by then. In the three scenarios, Grosser Aletschgletscher (Fig. 3d), the largest glacier of the Alps, https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 2596 L. Compagno et al.: Why 0.5 ◦C matters https://doi.org/10.5194/tc-15-2593-2021 L. Compagno et al.: Why 0.5 ◦C matters Still, increas- ing the number of GCM members that consider such longer- term horizons and having different research groups perform- ing similar analyses would help in verifying the robustness of These results show that, owing to slow lowering of air tem- peratures and enhanced precipitation implied by this partic- ular scenario after 2100, slow glacier recovery might happen (Fig. S3). While glacier volume losses of 80 %–85 % are cal- culated for 2100, the experiment projects that Alpine glaciers will regain a part of this volume, reaching a total volume be- tween 28 % and 53 % of the 2020 level by 2300. Although https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 L. Compagno et al.: Why 0.5 ◦C matters 2597 Figure 3. Relative change in glacier volume between 2020 and 2100 for scenarios of (a) +1.0 ◦C, (b) +1.5 ◦C, and (c) +2.0 ◦C of the global temperature increase. Each dot represents one glacier, with the dot’s size being proportional to the glacier area as given by the Randolph Glacier Inventory. The labels “GA” and “Rh” identify the “Grosser Aletschgletscher” and “Rhonegletscher”, respectively. The modelled geometries of these two glaciers are shown in panels (d) and (e) at the inventory date (dashed) for the year 2020 (grey) and for the year 2100 under the three selected scenarios (colours). Figure 3. Relative change in glacier volume between 2020 and 2100 for scenarios of (a) +1.0 ◦C, (b) +1.5 ◦C, and (c) +2.0 ◦C of the global temperature increase. Each dot represents one glacier, with the dot’s size being proportional to the glacier area as given by the Randolph Glacier Inventory. The labels “GA” and “Rh” identify the “Grosser Aletschgletscher” and “Rhonegletscher”, respectively. The modelled geometries of these two glaciers are shown in panels (d) and (e) at the inventory date (dashed) for the year 2020 (grey) and for the year 2100 under the three selected scenarios (colours). this preliminary ﬁnding. We also stress that decisive climate action would be required for steering global temperatures to- wards such an evolution (i.e. SSP126). glacierised surface, with 2080–2100 annual average runoff decreasing by 25 ± 6 % (for a global warming of +1.0 ◦C), 32 ± 8 %, (+1.5 ◦C), and 36 ± 10 % (+2.0 ◦C) compared to 2000–2020 levels. L. Compagno et al.: Why 0.5 ◦C matters Changes in monthly runoff – with a runoff peak projected to occur 1 to 2 months earlier by the end of the century – will be even more pronounced, with August peak runoff reductions of 36 ± 15 %, 44 ± 14 %, and 55 ± 15 %, respectively. For glacierised catchments and the areas down- stream, these changes in water availability can be expected to have important consequences, ranging from the function- ing of ecosystems to the generation of hydroelectricity. Even if our preliminary results based on a strong mitigation sce- nario indicate that slow glacier recovery might happen af- ter the 21st century, we hope that the presented results will contribute in making the point that, when it comes to global climate, every half-degree counts. https://doi.org/10.5194/tc-15-2593-2021 References IPCC: IPCC Special Report on the Ocean and Cryosphere in a Changing Climate, edited by: Pörtner, H.-O., Roberts, D. C., Masson-Delmotte, V., Zhai, P., Tignor, M., Poloczanska, E., Mintenbeck, K., Alegría, A., Nicolai, M., Okem, A., Petzold, J., Rama, B., and Weyer, N. M., 2019. Beniston, M., Farinotti, D., Stoffel, M., Andreassen, L. M., Cop- pola, E., Eckert, N., Fantini, A., Giacona, F., Hauck, C., Huss, M., Huwald, H., Lehning, M., López-Moreno, J.-I., Magnusson, J., Marty, C., Morán-Tejéda, E., Morin, S., Naaim, M., Proven- zale, A., Rabatel, A., Six, D., Stötter, J., Strasser, U., Terzago, S., and Vincent, C.: The European mountain cryosphere: a review of its current state, trends, and future challenges, The Cryosphere, 12, 759–794, https://doi.org/10.5194/tc-12-759-2018, 2018. Marzeion, B., Kaser, G., Maussion, F., and Champollion, N.: Limited inﬂuence of climate change mitigation on short- term glacier mass loss, Nat. Clim. Change, 8, 305–308, https://doi.org/10.1038/s41558-018-0093-1, 2018. Marzeion, B., Hock, R., Anderson, B., Bliss, A., Champollion, N., Fujita, K., Huss, M., Immerzeel, W. W., Kraaijenbrink, P., Malles, J., Maussion, F., Radi´c, V., Rounce, D. R., Sakai, A., Shannon, S., Wal, R., and Zekollari, H.: Partitioning the Uncer- tainty of Ensemble Projections of Global Glacier Mass Change, Earth’s Future, 8, https://doi.org/10.1029/2019EF001470, 2020. Compagno, L., Zekollari, H., Huss, M., and Farinotti, D.: Lim- ited impact of climate forcing products on future glacier evolution in Scandinavia and Iceland, J. Glaciol., 1–17, https://doi.org/10.1017/jog.2021.24, online ﬁrst, 2021. Cornes, R. C., van der Schrier, G., van den Besselaar, E. J. M., and Jones, P. D.: An ensemble version of the E-OBS tempera- ture and precipitation data sets, J. Geophys. Res.-Atmos., 123, 9391–9409, https://doi.org/10.1029/2017JD028200, 2018. Paul, F., Rastner, P., Azzoni, R. S., Diolaiuti, G., Fugazza, D., Le Bris, R., Nemec, J., Rabatel, A., Ramusovic, M., Schwaizer, G., and Smiraglia, C.: Glacier shrinkage in the Alps continues unabated as revealed by a new glacier inven- tory from Sentinel-2, Earth Syst. Sci. Data, 12, 1805–1821, https://doi.org/10.5194/essd-12-1805-2020, 2020. Farinotti, D., Huss, M., Fürst, J. J., Landmann, J., Machguth, H., Maussion, F., and Pandit, A.: A consensus estimate for the ice thickness distribution of all glaciers on Earth, Nat. Geosci., 12, 168–173, https://doi.org/10.1038/s41561-019-0300-3, 2019. RGI Consortium: Randolph Glacier Inventory 6.0, https://doi.org/10.7265/N5-RGI-60, 2017. Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Huss, M., Jouvet, G., Farinotti, D., and Bauder, A.: Fu- ture high-mountain hydrology: a new parameterization of glacier retreat, Hydrol. Earth Syst. Sci., 14, 815–829, https://doi.org/10.5194/hess-14-815-2010, 2010. Acknowledgements. We thank the RGI consortium for the global glacier inventory data. We acknowledge the European Climate As- sessment and Dataset (ECA&D), for the E-OBS data set, and CMIP for the GCM outputs. We also thank the World Glacier Monitor- ing Service (WGMS) for providing mass balance and length change measurements. IPBES: Global assessment report on biodiversity and ecosystem services of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services, edited by: Brondizio, E. S., Settele, J., Díaz, S., and Ngo, H. T., IPBES secre- tariat, Bonn, Germany, available at: https://www.ipcc.ch/srocc/ download/ (last access: 15 January 2021), 2019. IPCC: Global Warming of 1.5◦C, An IPCC Special Report on the impacts of global warming of 1.5◦C above pre-industrial lev- els and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of cli- mate change, sustainable development, and efforts to eradicate poverty, edited by: Masson-Delmotte, V., Zhai, P., Pörtner, H.- O., Roberts, C., Skea, J., Shukla, P. R., Pirani, A., Moufouma- Okia, W., Péan, C., Pidcock, R., Connors, S., Matthews, J. B. R., Chen, Y., Zhou, X., Gomis, M. I., Lonnoy, E., Maycock, T., Tig- nor, M., and Waterﬁeld, T., Cambridge University Press, avail- able at: https://www.ipcc.ch/sr15/download/ (last access: 15 Jan- uary 2021), 2018. Financial support. This research has been supported by the Swiss National Science Foundation (grant no. 184634) and the H2020 Marie Skłodowska-Curie Actions (grant no. 799904). Review statement. This paper was edited by Olivier Gagliardini and reviewed by Nicolas Champollion and one anonymous referee. Review statement. This paper was edited by Olivier Gagliardini and reviewed by Nicolas Champollion and one anonymous referee. 4 Conclusions While there is overwhelming consensus that decisive action has to be taken to limit the unwanted consequences of ongo- ing climatic change (UN, 2015; IPCC, 2018; IPBES, 2019; IPCC, 2019; WEF, 2020), the debate around which temper- ature targets to pursue is all but settled. In this contribution, we quantiﬁed the impact that a global temperature change of +1.0, +1.5, or +2.0 ◦C compared to preindustrial lev- els would have on glaciers of the European Alps. We show that this would correspond to an Alpine warming slightly above global levels and to a moderate increase in precipi- tation. Under such conditions, Alpine glaciers are projected to lose 44 ± 21 %, 68 ± 12 %, and 81 ± 8 % of their 2020 ice volume by 2100, indicating that, even in the most moderate scenario (+1.0 ◦C), about half of the present-day glacier vol- ume will be lost by the end of the century. Preliminary results based on a strong mitigation scenario (SSP126) and running until 2300 indicate that slow recovery might happen after that, emphasising the interest in considering projections that reach beyond the 21st century. The changes in glacier vol- ume will strongly impact the water yield from the presently Code and data availability. Code and data are available upon re- quest. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/tc-15-2593-2021-supplement. https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 L. Compagno et al.: Why 0.5 ◦C matters 2598 modeling. J. Glaciol., 1–19, https://doi.org/10.1017/jog.2021.55, online ﬁrst, 2021. modeling. J. Glaciol., 1–19, https://doi.org/10.1017/jog.2021.55, online ﬁrst, 2021. Author contributions. DF, LC, and SE conceived the study. LC and SE performed the numerical modelling, with support from MH, HZ, and DF. The original code was developed by MH (mass bal- ance component) and HZ (ice ﬂow component). SE retrieved, ho- mogenised and analysed all CMIP6 GCMs, with the support of LC, MH, and DF. LC and DF wrote the paper and produced the ﬁgures, with contributions from all other co-authors. Hock, R.: Temperature index melt modelling in mountain ar- eas, J. Hydrol., 282, 104–115, https://doi.org/10.1016/S0022- 1694(03)00257-9, 2003. Huss, M. and Hock, R.: A new model for global glacier change and sea-level rise, Front. Earth Sci., 3, 54, https://doi.org/10.3389/feart.2015.00054, 2015. Huss, M. and Hock, R.: Global-scale hydrological response to future glacier mass loss, Nat. Clim. Change, 8, 135–140, https://doi.org/10.1038/s41558-017-0049-x, 2018. References Grab, M., Mattea, E., Bauder, A., Huss, M., Rabenstein, L., Hodel, E., Linsbauer A., Langhammer, L., Schmid, L., Church, G., Hell- mann, S., Délèze, K., Schaer, P., Lathion, P., Farinotti, D., and Maurer, H.: Ice thickness distribution of all Swiss glaciers based on extended ground-penetrating radar data and glaciological UN: United nations Paris agreement, United nations framework convention on climate change, Rio de Janeiro, Brazil, and New York, United States, available at: https://unfccc.int/sites/default/ https://doi.org/10.5194/tc-15-2593-2021 https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 2599 https://doi.org/10.5194/tc-15-2593-2021 The Cryosphere, 15, 2593–2599, 2021 L. Compagno et al.: Why 0.5 ◦C matters ﬁles/english_paris_agreement.pdf (last access: 15 January 2021), 2015. Zekollari, H., Huss, M., and Farinotti, D.: Modelling the future evolution of glaciers in the European Alps under the EURO- CORDEX RCM ensemble, The Cryosphere, 13, 1125–1146, https://doi.org/10.5194/tc-13-1125-2019, 2019. ﬁles/english_paris_agreement.pdf (last access: 15 January 2021), 2015. ﬁles/english_paris_agreement.pdf (last access: 15 January 2021), 2015. Vautard, R., Gobiet, A., Sobolowski, S., Kjellström, E., Stege- huis, A., Watkiss, P., Mendlik, T., Landgren, O., Nikulin, G., Teichmann, C., and Jacob, D.: The European climate un- der a 2◦C global warming, Environ. Res. Lett., 9, 034006, https://doi.org/10.1088/1748-9326/9/3/034006, 2014. Zemp, M., Huss, M., Thibert, E., Eckert, N., McNabb, R., Huber, J., Barandun, M., Machguth, H., Nussbaumer, S. U., Gartner- Roer, I., Thomson, L., Paul, F., Maussion, F., Kutuzov, S., and Cogley, J. G.: Global glacier mass changes and their contribu- tions to sea-level rise from 1961 to 2016, Nature, 568, 382–386, https://doi.org/10.1038/s41586-019-1071-0, 2019. WEF: The Global Risks Report 2020, 15th Edn., Tech. rep., World economic forum, Cologny/Geneva, Switzerland, available at: http://www3.weforum.org/docs/WEF_Global_Risk_Report_ 2020.pdf (last access: 15 January 2021), 2020. WGMS: Fluctuations of glaciers database, World Glacier Monitoring Service (WGMS), Zurich, Switzerland, https://doi.org/10.5904/wgms-fog-2020-08, 2020. The Cryosphere, 15, 2593–2599, 2021 https://doi.org/10.5194/tc-15-2593-2021
https://openalex.org/W4378363257	https://egusphere.copernicus.org/preprints/2022/egusphere-2022-842/egusphere-2022-842.pdf	English	null	Comment on egusphere-2022-842	null	2,023	cc-by	22,795	Correspondence: Momme Hell (mhell@brown.edu) Abstract. Sea ice plays an important role in Earth’s energy budget by impacting surface albedo and air-sea ﬂuxes in polar regions. On its margins, sea ice is heavily impacted by waves and we currently do not have routine observations of waves in sea ice and lack understanding of how waves interact with sea ice and how they attenuate. In this paper, we propose methods to separate the two-dimensional (2D) surface wave spectra from sea ice height obser- vations along each ICESat-2 track. A combination of a linear inverse method, called Generalized Fourier Transform (GFT), 5 to estimate the wave spectra along each beam and a Metropolitan Hasting algorithm to estimate the dominant wave’s inci- dent angle was developed. It allows us to estimate the 2D wave signal and its uncertainty from the high-density, unstructured ATL03 ICESat-2 photon retrievals. The GFT is applied to re-binned photon retrials on 25 km segments for all six beams and outperforms a discrete Fourier transform in accuracy while having fewer constraints on the data structure. The Metropolitan Hasting algorithm infers the wave direction from beam pairs every 25 km using coherent crests of the 10 most energetic waves. Both methods together allow a decomposition into a 2D surface wave spectra with the advantage that the residual surface heights can potentially be attributed to other sea ice properties. The described method can routinely decompose ICESat-2 tracks and constrain wave attenuation in sea ice. The Metropolitan Hasting algorithm infers the wave direction from beam pairs every 25 km using coherent crests of the 10 most energetic waves. Both methods together allow a decomposition into a 2D surface wave spectra with the advantage that the residual surface heights can potentially be attributed to other sea ice properties. The described method can routinely decompose ICESat-2 tracks and constrain wave attenuation in sea ice. 10 The Metropolitan Hasting algorithm infers the wave direction from beam pairs every 25 km using coherent crests of the 10 most energetic waves. Both methods together allow a decomposition into a 2D surface wave spectra with the advantage that the residual surface heights can potentially be attributed to other sea ice properties. The described method can routinely decompose ICESat-2 tracks and constrain wave attenuation in sea ice. Directional Surface Wave Spectra And Sea Ice Structure from ICEsat-2 Altimetry Momme C. Hell1 and Christopher Horvat1,2 1Brown University, Providence, RI, USA 2University of Auckland, Auckland, NZ 1Brown University, Providence, RI, USA 2University of Auckland, Auckland, NZ https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction and Problem Description Sea ice covers up to 9% of the world’s oceans, and plays an important role in the energy balance of Earth’s climate. Even 15 though sea ice damps ocean surface waves (Squire, 2007), broad regions along the periphery of the sea-ice-covered ocean are continually under the inﬂuence of surface waves (Rapley, 1984; Horvat et al., 2020; Thomson, 2022; Horvat, 2022). These regions are collectively referred to as the Marginal Ice Zone (MIZ). In the MIZ, waves inﬂuence the thermodynamic and dynamic properties of sea ice, and also impact the coupled exchange between atmosphere and ocean. Currently we do not have a reliable global observations of waves in sea ice, and hence are unable to sufﬁciently understand air-sea exchange and wave 20 1 1 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. propagation in the MIZ. This paper describes how ICESat-2 altimeter observations can be used to record waves spectra in the MIZ, and to infer additional sea ice properties to inform parametrizations of wave attenuation in sea ice. propagation in the MIZ. This paper describes how ICESat-2 altimeter observations can be used to record waves spectra in the MIZ, and to infer additional sea ice properties to inform parametrizations of wave attenuation in sea ice. propagation in the MIZ. This paper describes how ICESat-2 altimeter observations can be used to record waves spectra in the MIZ, and to infer additional sea ice properties to inform parametrizations of wave attenuation in sea ice. Models of wave propagation in sea ice typically evolve the ocean surface wave spectrum, ˜Sh(k) (meter2 k−1, k is the wavenumber), which is attenuated when it comes into contact with sea ice. There has been signiﬁcant debate over the functional form and dependencies of this attenuation (Squire, 2018; Thomson et al., 2021). Yet as it controls how deep waves reach into 25 sea ice, it is vital for modeling MIZ variability and coupled feedbacks in the polar seas. Constraining ice-induced wave attenuation is challenging because wave observations in ice are difﬁcult to make at scale. A majority of observations of waves in ice are carried out using ships or arrays of ﬂoating buoys deployed by ships (or by helicopters from ships, see, for example Thomson, 2022, and references therein). While such observations provide high Models of wave propagation in sea ice typically evolve the ocean surface wave spectrum, ˜Sh(k) (meter2 k−1, k is the wavenumber), which is attenuated when it comes into contact with sea ice. There has been signiﬁcant debate over the functional form and dependencies of this attenuation (Squire, 2018; Thomson et al., 2021). Yet as it controls how deep waves reach into sea ice, it is vital for modeling MIZ variability and coupled feedbacks in the polar seas. 25 temporal frequency observations of wave spectra, they only cover a limited geographic domain, and are limited by the sea ice 30 types and conditions at the original buoy locations. Recently satellite remote sensing technologies have shown promise for describing wave spectra in sea ice regions. SAR imagery is capable of observing wave crests as they move into the MIZ, and the two-dimensional wave spectrum can be constructed in good agreement with in-situ-observed spectra if the sea ice is not rough (Stopa et al., 2018; Ardhuin et al., 2017). However, SAR alone cannot observe continuous spectra as they propagate into temporal frequency observations of wave spectra, they only cover a limited geographic domain, and are limited by the sea ice 30 types and conditions at the original buoy locations. Recently satellite remote sensing technologies have shown promise for describing wave spectra in sea ice regions. SAR imagery is capable of observing wave crests as they move into the MIZ, and the two-dimensional wave spectrum can be constructed in good agreement with in-situ-observed spectra if the sea ice is not rough (Stopa et al., 2018; Ardhuin et al., 2017). However, SAR alone cannot observe continuous spectra as they propagate into the sea ice. 35 The ICESat-2 (IS2) altimeter has the potential for greatly increasing the available observations of wave-ice interactions, either alone, or in combination with other remote sensing instruments (Collard et al., 2022). ICESat-2 carries a single mea- surement tool, ATLAS, a six-beam laser oriented in three weak/strong pairs (Fig. 1a, colored lines) offset at a near-uniform three kilometers on the ground, with a weak-strong beam lateral offset of about 90m meters. ATLAS measures the return time the sea ice. 35 The ICESat-2 (IS2) altimeter has the potential for greatly increasing the available observations of wave-ice interactions, either alone, or in combination with other remote sensing instruments (Collard et al., 2022). ICESat-2 carries a single mea- surement tool, ATLAS, a six-beam laser oriented in three weak/strong pairs (Fig. 1a, colored lines) offset at a near-uniform three kilometers on the ground, with a weak-strong beam lateral offset of about 90m meters. ATLAS measures the return time of individual photons to infer the height of the ice/ocean surface. Typical along-track photon spacings can be centimeters or 40 smaller, and so IS-2 is capable of directly sampling ocean surface waves, particularly over reﬂective sea ice. Recent studies have examined waves in sea ice using IS-2, basing their results on a higher-order sea ice height product derived from photon retrievals (known as ATL07). Horvat et al. (2020) identiﬁed the capability of IS-2 to retrieve ocean waves by examining a storm in the Barents Sea in 2019, and used a simple threshold to establish where and when waves were observed of individual photons to infer the height of the ice/ocean surface. Typical along-track photon spacings can be centimeters or 40 smaller, and so IS-2 is capable of directly sampling ocean surface waves, particularly over reﬂective sea ice. Recent studies have examined waves in sea ice using IS-2, basing their results on a higher-order sea ice height product derived from photon retrievals (known as ATL07). Horvat et al. (2020) identiﬁed the capability of IS-2 to retrieve ocean waves by examining a storm in the Barents Sea in 2019, and used a simple threshold to establish where and when waves were observed in the sea ice to produce global maps of the MIZ. In Collard et al. (2022), IS-2 retrievals during this Barents Sea storm were 45 shown to compare well with model and SAR-based observation data. Brouwer et al. (2021) selected a series of Southern Hemisphere IS-2 retrievals, analyzing wave attenuation using direct spectral transform methods. Both found that areas affected by waves were common in both hemispheres, with repeated measurements of waves hundreds of kilometers into the sea ice zone, particularly in the Southern Hemisphere. in the sea ice to produce global maps of the MIZ. In Collard et al. (2022), IS-2 retrievals during this Barents Sea storm were 45 shown to compare well with model and SAR-based observation data. Brouwer et al. (2021) selected a series of Southern Hemisphere IS-2 retrievals, analyzing wave attenuation using direct spectral transform methods. Both found that areas affected by waves were common in both hemispheres, with repeated measurements of waves hundreds of kilometers into the sea ice zone, particularly in the Southern Hemisphere. Three challenges limit the direct comparison of IS-2 derived wave spectra to observations and models. First, waves propagate 50 at an angle θ relative to the along-track direction of the satellite (Fig. 1b), and observable wave lengths λ are aliased by an unknown factor cos−1 θ (Rapley, 1984; Horvat et al., 2016; Yu et al., 2021). Second, observed surface height variability is a convolution of the dynamic ocean topography, sea ice topography, surface waves, and noise. The surface wave signal can than only successfully reconstructed if these other signals are on a different scale, like the ocean topography, or not periodic, like the sea ice topography and noise Third the fractured nature of sea ice inﬂuence of clouds and changing surface albedo cause 55 Three challenges limit the direct comparison of IS-2 derived wave spectra to observations and models. First, waves propagate 50 at an angle θ relative to the along-track direction of the satellite (Fig. 1b), and observable wave lengths λ are aliased by an unknown factor cos−1 θ (Rapley, 1984; Horvat et al., 2016; Yu et al., 2021). Second, observed surface height variability is a convolution of the dynamic ocean topography, sea ice topography, surface waves, and noise. The surface wave signal can than only successfully reconstructed if these other signals are on a different scale, like the ocean topography, or not periodic, like the sea ice topography and noise. Third, the fractured nature of sea ice, inﬂuence of clouds, and changing surface albedo cause 55 Three challenges limit the direct comparison of IS-2 derived wave spectra to observations and models. First, waves propagate 50 at an angle θ relative to the along-track direction of the satellite (Fig. 1b), and observable wave lengths λ are aliased by an unknown factor cos−1 θ (Rapley, 1984; Horvat et al., 2016; Yu et al., 2021). Second, observed surface height variability is a convolution of the dynamic ocean topography, sea ice topography, surface waves, and noise. The surface wave signal can than only successfully reconstructed if these other signals are on a different scale, like the ocean topography, or not periodic, like 50 the sea ice topography and noise. Third, the fractured nature of sea ice, inﬂuence of clouds, and changing surface albedo cause 55 2 2 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. nominal track a) b) incident wave direction incident wave direction λ’ λ Θ Θ observable wave incident angle wave crests wave direction xi xi+1 xi+2 xi+3 across-track distance yi nominal track along-track distance yi 20 m d ≈ 90 m Figure 1. Illustration of ICESat-2 (IS2) beams intersecting the Marginal Ice Zone (MIZ) under the presence of waves. a) The three beam pairs are shown is red/orange, dark/light green, or dark/light blue lines. The vertical black line is the nominal IS2 reference ground track. The incident waves come from the top-right along the black arrow with wave crests (solid) and valleys (dashed). b) Details view of an ideal monochromatic wave observation by IS2 in sea ice. The IS2 beam pair (light/dark green lines) observes an incident wave (along the black arrow) of wavelength λ with an angle θ as λ′. With the beam-pair distance d one can calculate θ from the phase lag of the incident wave crests. The along-/across-track coordinate system is referenced to the nominal track, while each data point is the weighted mean a 20-meter stencil (see text). nominal track a) b) incident wave direction incident wave direction along track distance yi nominal track a) b) incident wave direction incident wave direction λ’ λ Θ Θ observ wave incide angle wave crests di xi xi+1 xi+2 xi+3 across-track distance yi nominal track along-track distance yi 20 m d ≈ 90 m b) λ’ λ Θ Θ observable wave incident angle wave crests wave direction xi xi+1 xi+2 xi+3 across-track distance yi nominal track along-track distance yi 20 m d ≈ 90 m b) Figure 1. Illustration of ICESat-2 (IS2) beams intersecting the Marginal Ice Zone (MIZ) under the presence of waves. a) The three beam pairs are shown is red/orange, dark/light green, or dark/light blue lines. The vertical black line is the nominal IS2 reference ground track. The incident waves come from the top-right along the black arrow with wave crests (solid) and valleys (dashed). b) Details view of an ideal monochromatic wave observation by IS2 in sea ice. The IS2 beam pair (light/dark green lines) observes an incident wave (along the black arrow) of wavelength λ with an angle θ as λ′. With the beam-pair distance d one can calculate θ from the phase lag of the incident wave crests. The along-/across-track coordinate system is referenced to the nominal track, while each data point is the weighted mean a 20-meter stencil (see text). gaps or irregularities in IS-2 photon retrieval rates, creating an high-density but irregular observation. The method must be applicable to irregular data without generating spurious sources of variance, i.e. artiﬁcial wave energy. All of the above factors complicate direct assessments of spectra and their attenuation in sea ice. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. suppl. Table for granule names), providing two-dimensional wave spectra derived from along-track data in section 3.2, and the decomposition of photon variance in section 4. In section 5 we address how the angle correction and variance decomposition can be used to both (a) developed improved models of attenuation of waves in sea ice, and (b) improve estimates of sea-ice freeboard. 70 suppl. Table for granule names), providing two-dimensional wave spectra derived from along-track data in section 3.2, and the decomposition of photon variance in section 4. In section 5 we address how the angle correction and variance decomposition can be used to both (a) developed improved models of attenuation of waves in sea ice, and (b) improve estimates of sea-ice freeboard. 70 70 2 Along-track Wave Spectra from IS-2 The primary aim of this analysis is to assess surface height variability in the MIZ. Hence we want to use the highest data resolution we can handle, though we are agnostic about the classiﬁcation of photon returns. That is the L2-level product ATL03 from Neumann et al. (2021). For comparison, we show the photon cloud data from ATL03 and the surface heights and type classiﬁcations from the higher level ATL07/10 product in ﬁgure 2 as dark blue, light blue, or orange dots (Kwok et al., 75 2021). By requiring 150 consecutive photons to identify a sea ice segment, the ATL07 product accounts for most of the height variability from the ATL03 product. Yet it misses retrievals in the MIZ (supl. Fig. 2, white and gray area) and within the sea ice (Fig. 2). For better resolution, the following analysis is based on the photon cloud data from ATL03. Here we develop angle-corrected, two-dimensional (2D) wave spectra in sea ice, using photon Here we develop angle-corrected, two-dimensional (2D) wave spectra in sea ice, using photon height data from ICESat-2. We partition surface height variability into waves and sea-ice or noise-related components, which permits a direct assessment 60 of wave energy attenuation and sea-ice properties for evaluation of numerical attenuation schemes. We show this partitioning allows for signiﬁcantly improved sea ice height estimates in the MIZ, which may allow for an expansion of existing higher-level IS-2 products to broader ice-covered regions. We partition surface height variability into waves and sea-ice or noise-related components, which permits a direct assessment 60 of wave energy attenuation and sea-ice properties for evaluation of numerical attenuation schemes. We show this partitioning allows for signiﬁcantly improved sea ice height estimates in the MIZ, which may allow for an expansion of existing higher-level IS-2 products to broader ice-covered regions. We describe the pre-processing of IS-2 along-track photon heights in section 2.1 and develop a (linear) harmonic ﬁtting procedure applied individual IS-2 beams in section 2.2. Subsequently we develop a multi-beam, Monte-Carlo method for bias- 65 correcting along-track wavelengths in section 3. We then demonstrate this method on three example cases (Track 1 to 3, see 3 3 2.1 Data Pre-Processing Linearly inverting photon data requires exact along and across-track information about photon positions. Along-track photon 80 positions are ﬁrst re-referenced to the most equatorward position on the nominal ATLAS ground track. The most equatorward position is evaluated from the ATL07 (Kwok et al., 2021) product and set to the beginning of 1st 100 km section that has at least 0.02 photons per 100km. All tracks are then followed in a poleward direction, until the variance of any of the 6 beams exceeds a factor of 10 the variance of the ﬁrst 15% on the equatorward side of the track (supl. Fig. 2, dashed black lines). Linearly inverting photon data requires exact along and across-track information about photon positions. Along-track photon 80 positions are ﬁrst re-referenced to the most equatorward position on the nominal ATLAS ground track. The most equatorward position is evaluated from the ATL07 (Kwok et al., 2021) product and set to the beginning of 1st 100 km section that has at least 0.02 photons per 100km. All tracks are then followed in a poleward direction, until the variance of any of the 6 beams exceeds a factor of 10 the variance of the ﬁrst 15% on the equatorward side of the track (supl. Fig. 2, dashed black lines). This avoids including impacts from coastal or land ice around the Antarctic coast. The redeﬁned along-track direction x and 85 an across-track direction y are then used as coordinate system throughout the analysis (Fig. 1b, supl. Fig. 1 and 2). After removing the cumulative surface height correction (dem_h taken from the ATL07/10 dataset, Kwok et al., 2021), we bin photon measurements into 20-meter stencils that overlap by 50%. This yields a 10-meter along-track resolution (Fig. 2 green line). Note this differs from the procedure used to form sea ice surface heights in the ATL07 product, which averages This avoids including impacts from coastal or land ice around the Antarctic coast. The redeﬁned along-track direction x and 85 an across-track direction y are then used as coordinate system throughout the analysis (Fig. 1b, supl. Fig. 1 and 2). After removing the cumulative surface height correction (dem_h taken from the ATL07/10 dataset, Kwok et al., 2021), we bin photon measurements into 20-meter stencils that overlap by 50%. This yields a 10-meter along-track resolution (Fig. 2 green line). https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Fig. S2). The same 20-meter stencil also provides an uncertainty estimate σh(x) (proportional to Fig. 2 blue area) representing the varying photon density. This uncertainty is used to deﬁne the data prior (sec. A1) for the harmonic inversion in section 2.2. 00 100 Figure 2. ATL03 photon cloud and ATL07 surface heights for example Track 1. Individual photons are shown in black and the 20-meter weighted average as a green line. The ATL07 Photon heights are are shown as light blue, dark blue, or black dots, with the color corresponds to their type category provided in ATL07. Identiﬁed sea ice leads are shown as red segments on the gray line. The surface slope based on the weighted photon average is shown as a blue line with -1.25 offset, and its uncertainty estimate as blue shading with a -1 offset. The panel b) is an inset of the gray area in panel a). Figure 2. ATL03 photon cloud and ATL07 surface heights for example Track 1. Individual photons are shown in black and the 20-meter weighted average as a green line. The ATL07 Photon heights are are shown as light blue, dark blue, or black dots, with the color corresponds to their type category provided in ATL07. Identiﬁed sea ice leads are shown as red segments on the gray line. The surface slope based on the weighted photon average is shown as a blue line with -1.25 offset, and its uncertainty estimate as blue shading with a -1 offset. The panel b) is an inset of the gray area in panel a). The re-sampled surface height data is used to calculate a series of along-track surface slopes (Fig. 2 thin blue line) by taking the along-track derivative and applying a spike-removing algorithm. Using the along-track surface slope data focuses the timeseries analysis on local photon-height changes, rather then the magnitude of the total surface height ﬁeld. The spike- removal reduces peaks in the slope data, which can be from sea-ice height changes or especially those resulting from ice- ocean transitions. Because the slope ﬁeld approximates the derivative of the height ﬁeld, the spectrum of slopes ˆSc(k) is ˜ ˜ ˜ The re-sampled surface height data is used to calculate a series of along-track surface slopes (Fig. 2 thin blue line) by taking the along-track derivative and applying a spike-removing algorithm. Using the along-track surface slope data focuses the timeseries analysis on local photon-height changes, rather then the magnitude of the total surface height ﬁeld. The spike- removal reduces peaks in the slope data, which can be from sea-ice height changes or especially those resulting from ice- ocean transitions. Because the slope ﬁeld approximates the derivative of the height ﬁeld, the spectrum of slopes ˆSc(k) is 105 readily connected to the spectrum of surface heights ˜Sc(k), as ˜Sh(k) = k ˜Sc(k). The surface height ﬁeld can therefore be directly reconstructed from the slope spectrum, as we show below. The generalized Fourier transform (sec. 2.2) and directional estimates (sec. 3.1) are then applied on 25-km long segments of these surface slopes, with uncertainty estimates (Fig. 2). The 25-km segments also overlap by 50%, providing an along-track spectral estimate every 12.5 km. The re-sampled surface height data is used to calculate a series of along-track surface slopes (Fig. 2 thin blue line) by taking the along-track derivative and applying a spike-removing algorithm. Using the along-track surface slope data focuses the timeseries analysis on local photon-height changes, rather then the magnitude of the total surface height ﬁeld. The spike- removal reduces peaks in the slope data, which can be from sea-ice height changes or especially those resulting from ice- ocean transitions. Because the slope ﬁeld approximates the derivative of the height ﬁeld, the spectrum of slopes ˆSc(k) is 105 readily connected to the spectrum of surface heights ˜Sc(k), as ˜Sh(k) = k ˜Sc(k). The surface height ﬁeld can therefore be directly reconstructed from the slope spectrum, as we show below. The generalized Fourier transform (sec. 2.2) and directional estimates (sec. 3.1) are then applied on 25-km long segments of these surface slopes, with uncertainty estimates (Fig. 2). The 25-km segments also overlap by 50%, providing an along-track spectral estimate every 12.5 km. The re-sampled surface height data is used to calculate a series of along-track surface slopes (Fig. 2 thin blue line) by taking the along-track derivative and applying a spike-removing algorithm. Using the along-track surface slope data focuses the timeseries analysis on local photon-height changes, rather then the magnitude of the total surface height ﬁeld. The spike- removal reduces peaks in the slope data, which can be from sea-ice height changes or especially those resulting from ice- ocean transitions. 2.1 Data Pre-Processing Note this differs from the procedure used to form sea ice surface heights in the ATL07 product, which averages height data for each set of 150 photons along-track. ATL07 has a constant photon count, with the trade-off of irregularly spaced 90 segments of varying length in the MIZ, while our approach provides more regularly-spaced data with the trade-off of having a variable photon count in each stencil and potentially including retrievals of water near sea ice. Stencils with fewer than ﬁve photons are excluded, which also leads to data gaps corresponding to no or very low photon retrievals due to sea-ice leads, open water, clouds, or other noise (suppl. Fig. S2). These data gaps also lead to an irregularity in the data, but here each stencil mean represents the same area and hence better capture wave phase. 95 mean represents the same area and hence better capture wave phase. 95 The photon height in each 20-meter stencil hc(x) is calculated as the mean of the photons weighted by their inverse distance to the stencil center, using a Gaussian weighting function with standard deviation of 10m. We tested other data reduction methods, like using the median or mode of the stencil, ﬁnding results insensitive to the choice of the binning method (suppl. The photon height in each 20-meter stencil hc(x) is calculated as the mean of the photons weighted by their inverse distance to the stencil center, using a Gaussian weighting function with standard deviation of 10m. We tested other data reduction methods, like using the median or mode of the stencil, ﬁnding results insensitive to the choice of the binning method (suppl. 4 4 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2.2 Generalized Fourier Transform (GFT) 110 The data handling needed for DFT can erode the signal substantially, especially in the MIZ (Fig. 3 b,c gray and green lines). The GFT method outlined below works on any grid, incorporates data uncertainty, and does not require periodicity. The GFT can be customized to the frequencies of interest with the additional beneﬁt of providing a standard error in real and wavenumber space. 125 The GFT method outlined below works on any grid, incorporates data uncertainty, and does not require periodicity. The GFT can be customized to the frequencies of interest with the additional beneﬁt of providing a standard error in real and wavenumber space. 125 Because the slope ﬁeld approximates the derivative of the height ﬁeld, the spectrum of slopes ˆSc(k) is 105 readily connected to the spectrum of surface heights ˜Sc(k), as ˜Sh(k) = k ˜Sc(k). The surface height ﬁeld can therefore be directly reconstructed from the slope spectrum, as we show below. The generalized Fourier transform (sec. 2.2) and directional estimates (sec. 3.1) are then applied on 25-km long segments of these surface slopes, with uncertainty estimates (Fig. 2). The 25-km segments also overlap by 50%, providing an along-track spectral estimate every 12.5 km. 105 5 2.2 Generalized Fourier Transform (GFT) 110 We estimate along-track wave spectra using a Generalized Fourier Transform (GFT). The GFT is an harmonic ﬁt of sin- and cos-pair coefﬁcients, which together determine amplitude and phase at each wavenumber. The model complexity is deﬁned by the number of resolved wave numbers and its success depends on prior (assumed) knowledge about the data’s uncertainty and model structure. 115 We use a GFT to overcome several disadvantages arising when implementing a standard Discrete Fourier Transform (DFT). 115 While the DFT is a fast variance-conserving algorithm it requires periodic, continuous and equally-spaced data. The DFT implies that frequency bands are harmonics over a domain or segment length L, which is an arbitrary limitation on the re- solved frequencies. To make segments periodic, often tapering or windowing is applied to the segmented data. In addition to the data’s non-periodicity the common presence of data gaps in IS-2 retrievals requires extrapolation or gap-ﬁlling to create We use a GFT to overcome several disadvantages arising when implementing a standard Discrete Fourier Transform (DFT). 115 While the DFT is a fast variance-conserving algorithm it requires periodic, continuous and equally-spaced data. The DFT implies that frequency bands are harmonics over a domain or segment length L, which is an arbitrary limitation on the re- solved frequencies. To make segments periodic, often tapering or windowing is applied to the segmented data. In addition to the data’s non-periodicity the common presence of data gaps in IS-2 retrievals requires extrapolation or gap-ﬁlling to create continuous equally spaced data suitable for a DFT. These both lead to commonly known problems of the DFT, like energy 120 leaks/compensation in spectral space. The data handling needed for DFT can erode the signal substantially, especially in the MIZ (Fig. 3 b,c gray and green lines). The GFT method outlined below works on any grid, incorporates data uncertainty, and does not require periodicity. The GFT can be customized to the frequencies of interest with the additional beneﬁt of providing a standard error in real and wavenumber space. 125 continuous equally spaced data suitable for a DFT. These both lead to commonly known problems of the DFT, like energy 120 leaks/compensation in spectral space. The data handling needed for DFT can erode the signal substantially, especially in the MIZ (Fig. 3 b,c gray and green lines). leaks/compensation in spectral space. 2.3 Harmonic wave inversion Using Bayes’ theorem (Kachelein et al., 2022), or, alternatively the matrix inversion lemma (Wunsch, 1996), given the data \|b, the most likely estimate of the model parameters ˆp is found as, To ﬁnd the most probable b given a set of model parameters p, we need to estimate the autocovariance matrices of the residual 140 R = rrT , i.e. the error of the data, and the autocovariance matrix of the model P = ppT , where (⟨·⟩) is the expected value. Then the most probable estimate of the data b can be found by estimating the maximum of the posterior probability distribution P(p\|b). Using Bayes’ theorem (Kachelein et al., 2022), or, alternatively the matrix inversion lemma (Wunsch, 1996), given the data \|b, the most likely estimate of the model parameters ˆp is found as, ˆp = HT RH + P−1−1 HT R−1b. 145 ˆp = HT RH + P−1−1 HT R−1b. (6) 145 Once the model parameters ˆp are estimated, the model can be expressed in real space using ˆb (Fig. 3a green lines), or as a power spectrum ˆ˜SGF T (Fig. 3b,c green lines, see suppl. material S1 for derivation). Note that ˆ˜SGF T is substantially different from that of the DFT of the same data (Fig. 3b,c gray and green lines respectively). The frequent gaps in the data, as well as the DFT’s requirement for the data to be period creates artiﬁcial power in the swell’s wavenumber range that lead to miss leading (6) ˆp = HT RH + P−1−1 HT R−1b. (6) 145 Once the model parameters ˆp are estimated, the model can be expressed in real space using ˆb (Fig. 3a green lines), or as a power spectrum ˆ˜SGF T (Fig. 3b,c green lines, see suppl. material S1 for derivation). Note that ˆ˜SGF T is substantially different from that of the DFT of the same data (Fig. 3b,c gray and green lines respectively). The frequent gaps in the data, as well as the DFT’s requirement for the data to be period creates artiﬁcial power in the swell’s wavenumber range that lead to miss leading Once the model parameters ˆp are estimated, the model can be expressed in real space using ˆb (Fig. 3a green lines), or as a power spectrum ˆ˜SGF T (Fig. 3b,c green lines, see suppl. material S1 for derivation). 2.3 Harmonic wave inversion We follow Wunsch (1996), Menke (2018), and Kachelein et al. (2022), using generalized least squares to derive spectral amplitudes in each 25-km segment Xi (Fig. 4). Slope data in each section Xi is a series of unevenly spaced mean-zero data points and is expressed as a column vector b = ∂xz of length Ni where z are the height data. These data are then modeled as the sum of sinusoids having wave numbers in the range of swell and wind waves 130 (1) b = H p + r, where H is an Ni ×2M regressor matrix of basis functions, p is the model parameter vector of length 2M, and r is the vector of the residual timeseries of length Ni. The columns of H are sines and cosines of prescribed wave numbers, km = 2π/λm for wavelengths λm indexed by m = 1,2,..M, i.e.: b = M X m=1 am cos(kmx) + bm sin(kmx) + r, 135 (2) (3) 6 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. with, (4) (5) (4) (5) (5) To ﬁnd the most probable b given a set of model parameters p, we need to estimate the autocovariance matrices of the residual 140 R = rrT , i.e. the error of the data, and the autocovariance matrix of the model P = ppT , where (⟨·⟩) is the expected value. Then the most probable estimate of the data b can be found by estimating the maximum of the posterior probability distribution P(p\|b). Using Bayes’ theorem (Kachelein et al., 2022), or, alternatively the matrix inversion lemma (Wunsch, 1996), given the data \|b, the most likely estimate of the model parameters ˆp is found as, To ﬁnd the most probable b given a set of model parameters p, we need to estimate the autocovariance matrices of the residual 140 R = rrT , i.e. the error of the data, and the autocovariance matrix of the model P = ppT , where (⟨·⟩) is the expected value. Then the most probable estimate of the data b can be found by estimating the maximum of the posterior probability distribution P(p\|b). 2.3.1 Choice of model resolution and degrees of freedom 160 The highest wavenumber is chosen as k′ m = 0.11 rad m−1, a typical period of wind waves of about six seconds. Using evenly spaced wavenumbers with dk = 1.25 × 10−4 this results in M = 869 wavenumbers. 175 dk = 1.25 × 10−4 this results in M = 869 wavenumbers. 175 2.3.1 Choice of model resolution and degrees of freedom 160 The quality of the GFT model depends on the degrees of freedom, the model prior p, and data priors r (eq. 7). While the number of model parameters, 2M, remains ﬁxed throughout the analysis, the number of data points in segment i, Ni, is variable, and may be controlled by the segment length L, 25 km in our case, with a 12.5km overlap (Fig. 4). The number of degrees of freedom is then 2M −Ni and depends on the number of data points in each 25-km segment. A segment with no data gaps and a 10-meter resolution (sec. 2.1) contains Ni = 2500 data points. With 2M = 1738 model 165 parameters, this is then an over determined problem. However, frequent data gaps reduce the number of datapoint per segment, which may result in an underdetermined problem, with more model parameters then data (2M > Ni). The result is then a larger residual r and larger uncertainty estimate ˆperr (eq. 8 and eq. 9). Even in cases where eq. (1) is underdetermined, we are conﬁdent in our wave spectrum estimation within given error because P contains prior knowledge about the shape of the solution, i.e. the shape of typical surface wave spectra (sec. 2.4). 170 A segment with no data gaps and a 10-meter resolution (sec. 2.1) contains Ni = 2500 data points. With 2M = 1738 model 165 parameters, this is then an over determined problem. However, frequent data gaps reduce the number of datapoint per segment, which may result in an underdetermined problem, with more model parameters then data (2M > Ni). The result is then a larger residual r and larger uncertainty estimate ˆperr (eq. 8 and eq. 9). Even in cases where eq. (1) is underdetermined, we are conﬁdent in our wave spectrum estimation within given error because P contains prior knowledge about the shape of the solution, i.e. the shape of typical surface wave spectra (sec. 2.4). 170 The choice of segment length also determines the smallest resolvable wavenumber. For example, a segment length of 25-km resolves a wave with a 20s period at an incident angle of ±75◦about 10 times. We set the lowest resolved wavenumber to k′ 1 = 2.5×10−3 rad m−1 which corresponds to a maximum wavelength of 2500 meters (sec. 3.1). https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2.3 Harmonic wave inversion Note that ˆ˜SGF T is substantially different from that of the DFT of the same data (Fig. 3b,c gray and green lines respectively). The frequent gaps in the data, as well as the DFT’s requirement for the data to be period creates artiﬁcial power in the swell’s wavenumber range that lead to miss leading results 150 results. 150 To estimate the model error, the posterior autocovariance of the difference between estimated and true model parameters is deﬁned as the inverse Hessian, To estimate the model error, the posterior autocovariance of the difference between estimated and true model parameters is deﬁned as the inverse Hessian, Hess−1 = (p −ˆp)(p −ˆp)T = HT RH + P−1−1 , (7) (7) In practice this is calculated from the so called “kernel" matrix H, and the (assumed) Gaussian distributed data and model priors R and P. The inverse Hessian is then be used to estimate the error of the model parameters 155 ˆperr = tr(Hess−1), (8) ˆperr = tr(Hess−1), ˆperr = tr(Hess−1), (8) (Fig. 3d, shown in the same units as the power spectra) and the error of the ﬁt to the modeled data (Fig. 3d, shown in the same units as the power spectra) and the error of the ﬁt to the modeled data, ˆberr = (H2 Hess−1)j, (9) (not shown) where j is a unit vector of length 2M. (not shown) where j is a unit vector of length 2M. (not shown) where j is a unit vector of length 2M. 7 7 2.4 Iterative inversion along each beam The GFT solution ˆb depends on prior assumptions about the wave spectrum: the model prior P. Since the GFT is iteratively applied along each beam, results from a previous (closer to the open ocean) segment inform the subsequent segment as illus- trated in Figure 4. Here we describe how a successive application of the GFT along the IS-2 beam can leads to an efﬁcient solution assuming that the wave’s spectral shape only slowly varies between the segments. 180 trated in Figure 4. Here we describe how a successive application of the GFT along the IS 2 beam can leads to an efﬁcient solution assuming that the wave’s spectral shape only slowly varies between the segments. 180 The initial guess of the model prior Pinit, taken at the most ocean-ward edge of the data is a Pierson-Moskowitz (PM) spectral slope function (based on Pierson and Moskowitz, 1964), to describe the general shape of a narrow banded surface wave ﬁeld. The initial PM form is ﬁt to a DFT of the data, and any locations with missing data are deﬁned with a slope of zero. This gap-ﬁlling creates artiﬁcial ringing in the DFT, but is sufﬁcient to estimate the peak wave number and energy of solution assuming that the wave’s spectral shape only slowly varies between the segments. 180 The initial guess of the model prior Pinit, taken at the most ocean-ward edge of the data is a Pierson-Moskowitz (PM) spectral slope function (based on Pierson and Moskowitz, 1964), to describe the general shape of a narrow banded surface wave ﬁeld. The initial PM form is ﬁt to a DFT of the data, and any locations with missing data are deﬁned with a slope of zero. This gap-ﬁlling creates artiﬁcial ringing in the DFT, but is sufﬁcient to estimate the peak wave number and energy of the spectrum. The PM-spectrum has, in its simplest form only two free parameters, the peak frequency and spectral amplitude, 185 which are ﬁt to the DFT power spectra via an objective function that is regularized by the observed spectral peak of the smoothed data (Appendix A, Hell et al., 2019). The initial inversion of the most equatorward segment X0 is performed using P0 = Pinit in eq. (6), leading to model parameters ˆp0. For this ﬁrst segment, a second inversion is applied on the same data, using an updated prior that is a smoothed the spectrum. 2.4 Iterative inversion along each beam CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 0.1 0.0 0.1 relative slope (m/m) a) Data and Model -12 -6 0 6 12 segment distance (km) @ X=38 km 0.1 0.0 0.1 relative slope (m/m) 0.1 0.0 0.1 relative slope (m/m) a) Data and Model -12 -6 0 6 12 segment distance (km) @ X=38 km 0.1 0.0 0.1 relative slope (m/m) 0.02 0.06 0.1 0 2 4 1e 5 d) Spectral Error gt2l gt2r -3 -1 1 3 Normalized Error 0.0 0.2 0.4 e) PDF of Residual r 0.02 0.06 0.1 0 1 10 2 (m/m)2/k b) Beam gt2l Spectral Estimate 0.02 0.06 0.1 wavenumber k (2 m 1) 0 1 10 2 (m/m)2/k c) Beam gt2r Spectral Estimate GFT Spec FFT Spec Figure 3. Example model and spectral estimate for a 25-km segment in the MIZ for Track 2 (granule 08070210, see suppl. Table). (a) Data (gray), model (dark and light green), and predicted modeled (orange) photon heights for two neighboring beams gt2l (dark green) and gt2r (light green). The data is offset by 0.5. (b and c) Corresponding power spectral estimates using GFT (green) and standard DFT (gray). The thick green lines are smoothed GFT estimates with a rolling 10-wavenumber mean. (d) Power spectral error for both beams as function of wave number. e) Residual PDF of r for both beams respectively. relative slope (m/m) segment distance (km) @ X=38 km 0.02 0.06 0.1 0 2 4 1e 5 d) Spectral Error gt2l gt2r -3 -1 1 3 Normalized Error 0.0 0.2 0.4 e) PDF of Residual r 0.02 0.06 0.1 0 1 10 2 (m/m)2/k b) Beam gt2l Spectral Estimate 0.02 0.06 0.1 wavenumber k (2 m 1) 0 1 10 2 (m/m)2/k c) Beam gt2r Spectral Estimate GFT Spec FFT Spec Figure 3. Example model and spectral estimate for a 25-km segment in the MIZ for Track 2 (granule 08070210, see suppl. Table). (a) Data (gray), model (dark and light green), and predicted modeled (orange) photon heights for two neighboring beams gt2l (dark green) and gt2r (light green). The data is offset by 0.5. (b and c) Corresponding power spectral estimates using GFT (green) and standard DFT (gray). The thick green lines are smoothed GFT estimates with a rolling 10-wavenumber mean. (d) Power spectral error for both beams as function of wave number. 2.4 Iterative inversion along each beam The PM-spectrum has, in its simplest form only two free parameters, the peak frequency and spectral amplitude, 185 which are ﬁt to the DFT power spectra via an objective function that is regularized by the observed spectral peak of the smoothed data (Appendix A, Hell et al., 2019). the spectrum. The PM-spectrum has, in its simplest form only two free parameters, the peak frequency and spectral amplitude, 185 which are ﬁt to the DFT power spectra via an objective function that is regularized by the observed spectral peak of the smoothed data (Appendix A, Hell et al., 2019). The initial inversion of the most equatorward segment X0 is performed using P0 = Pinit in eq. (6), leading to model parameters ˆp0. For this ﬁrst segment, a second inversion is applied on the same data, using an updated prior that is a smoothed The initial inversion of the most equatorward segment X0 is performed using P0 = Pinit in eq. (6), leading to model parameters ˆp0. For this ﬁrst segment, a second inversion is applied on the same data, using an updated prior that is a smoothed version of ˆp0 (Fig. 4, left). The smoothing uses a Lanzos running smoother in wavenumber with a stencil-width of ±0.19 190 2πm−1, or 150 data points. Inversions of the successive segments X1,X2,X3,... are then performed once, with the prior Pi version of ˆp0 (Fig. 4, left). The smoothing uses a Lanzos running smoother in wavenumber with a stencil-width of ±0.19 190 2πm−1, or 150 data points. Inversions of the successive segments X1,X2,X3,... are then performed once, with the prior Pi version of ˆp0 (Fig. 4, left). The smoothing uses a Lanzos running smoother in wavenumber with a stencil-width of ±0.19 190 2πm−1, or 150 data points. Inversions of the successive segments X1,X2,X3,... are then performed once, with the prior Pi version of ˆp0 (Fig. 4, left). The smoothing uses a Lanzos running smoother in wavenumber with a stencil-width of ±0.19 190 2πm−1, or 150 data points. Inversions of the successive segments X1,X2,X3,... are then performed once, with the prior Pi 8 0.1 0.0 0.1 relative slope (m/m) a) Data and Model -12 -6 0 6 12 segment distance (km) @ X=38 km 0.1 0.0 0.1 relative slope (m/m) 2 4 1e 5 d) Spectral Error gt2l gt2r 1 2 (m/m)2/k b) Beam gt2l Spectral Estimate https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. 2.5 Tracking of wave energy through Sea Ice The GFT is applied to each 25 km-segment with more then 250 data points, leading to wave spectral estimates along each beam. 195 In Fig. 5(a-f) we show wavenumber spectra for each segment and for each of beams of Track 3 in the Southern Hemisphere on May 2, 2019. The per-segment cross-beam mean (Fig. 5h) and mean spectral error (Fig. 5i) are derived by weighting each segment by its photon density before taking the mean. We deﬁne this weighted mean and error as our best estimate of the along track spectral evolution of wave energy. The example track shows an attenuating swell signal starting at X = 0 km and a second wave-energy maximum with shorter 200 wavelength (k′ ≈100m) at about X = 150 km in both, the spectrogram of the weighted mean as well as in individual beams (Fig. 5a to f, and h). The corresponding error per wave-number and position indicates larger error were we see wave signals (Fig. 5 i) which are likely just related to larger amplitudes in the data. The estimated wave numbers are the observed along-track wave numbers k′, which are different than the true wave number The example track shows an attenuating swell signal starting at X = 0 km and a second wave-energy maximum with shorter 200 wavelength (k′ ≈100m) at about X = 150 km in both, the spectrogram of the weighted mean as well as in individual beams (Fig. 5a to f, and h). The corresponding error per wave-number and position indicates larger error were we see wave signals (Fig. 5 i) which are likely just related to larger amplitudes in the data. The estimated wave numbers are the observed along-track wave numbers k′, which are different than the true wave number length \|k\| along the incident wave vector k (see Fig. 1). To estimate a wavenumber spectrum along the dominant propagation 205 direction rather then the direction it is observed, we outline a method to correct this bias in the following section. length \|k\| along the incident wave vector k (see Fig. 1). To estimate a wavenumber spectrum along the dominant propagation 205 direction rather then the direction it is observed, we outline a method to correct this bias in the following section. 2.4 Iterative inversion along each beam Schematic of the harmonic inversion algorithm along an ICESat-2 beam, shown over the surface slope ﬁeld of a typical beam in the MIZ. The DFT in each successive segment Xi has a prior Pi derived from the previous segment centered around Xi−1. For those segments with no prior information available, the prior Pinit is generated via a Discrete Fourier Transform (DFT) over the segments centered at Xi. 2.4 Iterative inversion along each beam e) Residual PDF of r for both beams respectively. 0.02 0.06 0.1 0 2 4 1e 5 d) Spectral Error gt2l gt2r e) PDF of Residual r 0.02 0.06 0.1 0 1 10 2 (m/m)2/k b) Beam gt2l Spectral Estimate c) Beam gt2r Spectral Estimate b) Beam gt2l Spectral Estimate b) Beam gt2l Spectral Estimate c) Beam gt2r Spectral Estimate -3 -1 1 3 Normalized Error 0.0 0.2 0.4 0.02 0.06 0.1 wavenumber k (2 m 1) 0 1 10 2 (m/m)2/k GFT Spec FFT Spec Figure 3. Example model and spectral estimate for a 25-km segment in the MIZ for Track 2 (granule 08070210, see suppl. Table). (a) Data (gray), model (dark and light green), and predicted modeled (orange) photon heights for two neighboring beams gt2l (dark green) and gt2r (light green). The data is offset by 0.5. (b and c) Corresponding power spectral estimates using GFT (green) and standard DFT (gray). The thick green lines are smoothed GFT estimates with a rolling 10-wavenumber mean. (d) Power spectral error for both beams as function of wave number. e) Residual PDF of r for both beams respectively. a smoothed version of ˆpi−1. If missing data does not allow for a successful inversion, the algorithm is re-initiated as done to obtain P0 and ˆp0. a smoothed version of ˆpi−1. If missing data does not allow for a successful inversion, the algorithm is re-initiated as done to obtain P0 and ˆp0. 9 9 X1 GFT{1} 1 X3 GFT{3} 3 5 ... init X0 GFT(0) 2 4 X2 GFT(2) X4 GFT(4) DFT(0) 137.5 km 150 km 162.5 km 175 km 187.5 km Along track distance (km) Figure 4. Schematic of the harmonic inversion algorithm along an ICESat-2 beam, shown over the surface slope ﬁeld of a typical beam in the MIZ. The DFT in each successive segment Xi has a prior Pi derived from the previous segment centered around Xi−1. For those segments with no prior information available, the prior Pinit is generated via a Discrete Fourier Transform (DFT) over the segments centered at Xi. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. X 175 km Figure 4. 3.1 Metropolitan estimates of the incident angle The observed wave spectra are distorted by a misalignment between the wave’s incident angle and the beam’s direction (Fig. 1b). While the ICESat-2 track orientations are well known, the surface waves can originate from any direction, and 210 the angle between these two directions is θ. If θ is known, the observed wave number k′, or wavelength λ′ along the beam can be corrected using k = k′cos−1(θ). The same geometrical distortion will also affect estimates of the attenuation rate between Xi-positions along the track (Fig. 1b) because the wave energy attenuates along their dominant propagation direction and not along the direction they are observed by the satellite. 10 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 102 103 Wave length (m) a) gt1r 102 103 b) gt2r 102 103 c) gt3r 102 103 Wave length (m) d) gt1l 102 103 e) gt2l 102 103 f) gt3l g) Photons density (m 1) h) B i ht d i) M Generalized Fourier Transform Slope Spectral Power SH 2019-05-02 granule: 05160312 Generalized Fourier Transform Slope Spectral Power SH 2019-05-02 granule: 05160312 102 103 Wave length (m) a) gt1r 102 103 b) gt2r 102 103 c) gt3r 102 103 Wave length (m) d) gt1l 102 103 e) gt2l 102 103 f) gt3l 50 100 150 200 Distance from the ice edge (km) 10 20 30 g) Photons density (m 1) mean photons density 50 100 150 200 Distance from the ice edge (km) 102 103 h) Beam weighted mean 30 20 Power((m/m)2/k) 50 100 150 200 Distance from the ice edge (km) 102 103 i) Mean error 70 65 60 55 50 Power((m/m)2/k) Generalized Fourier Transform Slope Spectral Power SH 2019-05-02 granule: 05160312 Figure 5. Slope spectra for the six beams (a to f) of example Track 3 (granule 05160312, see suppl. Table) resulting from the GFT inversion (sec. 2.4) for granule 05160312. The photon density per segment (g) is used to calculate the weighted mean spectrum (h) and error (i) for each track segment. 3.1 Metropolitan estimates of the incident angle 103 50 100 150 200 Distance from the ice edge (km) 102 103 i) Mean error h) Beam weighted mean 00 m) 1) ty 50 100 150 200 Distance from the ice edge (km) 102 103 h) Beam weighted mean 50 100 150 200 Distance from the ice edge (km) 102 103 i) Mean error i) Mean error 50 100 150 200 Distance from the ice edge (km) 10 20 30 mean photons density Distance from the ice edge (km) Figure 5. Slope spectra for the six beams (a to f) of example Track 3 (granule 05160312, see suppl. Table) resulting from the GFT inversion (sec. 2.4) for granule 05160312. The photon density per segment (g) is used to calculate the weighted mean spectrum (h) and error (i) for each track segment. To estimate θ from the photon data we make use of the spatial coherence between weak-strong beam pairs. That means wave 215 crests observed in one beam may also be observed in the other. Yet using the coherence has several limitations that have to be taken into account when designing an optimization method: 11 11 Despite these limitations, we describe in the following how the incidence angle θ can still be retrieved 235 With IS-2, the incident wave energy along k is observed along the beam direction for wavenumbers k′ = \|k\|cos−1 θ, or as a distorted wavenumber because of the above described distortion. In the case of IS-2 beam pairs, we know neither θ nor the bandwidth of the incident spectrum (Longuet-Higgins, 1984) and both factors limit the angle inversion based on beam pairs a distorted wavenumber because of the above described distortion. In the case of IS-2 beam pairs, we know neither θ nor the bandwidth of the incident spectrum (Longuet-Higgins, 1984) and both factors limit the angle inversion based on beam pairs alone (suppl. Fig. S8). Despite these limitations, we describe in the following how the incidence angle θ can still be retrieved 235 within these limitations. As explained in section 2.2, the surface wave ﬁeld can be interpreted as the superposition of monochromatic plain waves. For a narrow-banded swell spectrum the majority of the wave energy is contained in a few wave numbers and hence a superposition of the most energetic monochromatic waves explains the majority of the surface slope variability. In the following we optimize the incident angle and phase of the most energetic monochromatic waves numbers using a Metropolis-Hastings algorithm 240 (Foreman-Mackey et al., 2013). We accumulate the marginal distributions of possible incident angles across the dominant wave numbers and beam pairs, which results in a best guess of possible incident angle. This approach leads to directional wave information similar to the maximum entropy method used in wave buoys (Lygre and Krogstad, 1986). We focus on the 25 most-energetic wavenumbers of each beam pair and segment Xi based on the GFT result (sec. 2.2). To We focus on the 25 most-energetic wavenumbers of each beam pair and segment Xi based on the GFT result (sec. 2.2). To identify these wavenumbers, the beam-pairs mean wave power is smoothed using a three-wavenumber running mean to select 245 possible wave-numbers within the distorted narrow-banded spectrum (similar to the thick green lines in Fig. 3 b,c). – The coherence between beam pairs can only usefully be calculated for not too oblique angles ( about ±XX◦, Suppl. Fig. S3, and Yu et al., 2021) and high enough photon densities in both beams. – The coherence between beam pairs can only usefully be calculated for not too oblique angles ( about ±XX◦, Suppl. Fig. S3, and Yu et al., 2021) and high enough photon densities in both beams. – Across-beam coherence relies on an accurate measure of the distance the beams. Uncertainty in the intra-beam distance d can further obscured angle estimates based on coherence (Fig. 1b). Since d varies along the track (suppl. Fig. S1), using the nominal distance rather then the observed value will bias angle estimates. – Across-beam coherence relies on an accurate measure of the distance the beams. Uncertainty in the intra-beam distance d can further obscured angle estimates based on coherence (Fig. 1b). Since d varies along the track (suppl. Fig. S1), using the nominal distance rather then the observed value will bias angle estimates. 220 – The complex generation and propagation history of waves (Kitaigorodskii, 1962; Villas Bôas and Young, 2020; Marechal and Ardhuin, 2021; Hell et al., 2021a) leads to a dynamic distortion in the incident angle. While a monochromatic plane wave would be coherent across beam pairs, estimating its direction is limited by the periodicity of the waves and obser- 225 vational noise. In reality, the incident swell wave energy at any given time is contained in several wavenumbers although concentrated in a narrow-banded 2D-spectrum (Longuet-Higgins and Deacon, 1957). Hence, the bandwidth of the 2D swell ﬁeld limits the coherence between strong/weak beam pairs. While a narrower 2D-spectrum results in coherent wave crests in the across wave-direction, a broader spectrum spans more random-phase waves, which diminishes the coherence between wave crests (suppl. Fig. 8). Less wave-crest coherence in broader spectra limits estimates of the 230 incident angle when the surface is only observed along two beams. With IS-2, the incident wave energy along k is observed along the beam direction for wavenumbers k′ = \|k\|cos−1 θ, or as a distorted wavenumber because of the above described distortion. In the case of IS-2 beam pairs, we know neither θ nor the bandwidth of the incident spectrum (Longuet-Higgins, 1984) and both factors limit the angle inversion based on beam pairs alone (suppl. Fig. S8). This procedure is repeated for each 265 selected wavenumber kn and for each (available) beam pair per segment Xi (Fig. S9 a to c). The best incident angle PDF θP DF (X,k,beampair) can then be derived using the weighted average across wavenumber, beam-pair, or both. The resulting beam- and wavenumber-average PDF of Track 3 at Xi = 87, for example, shows more than one maximum (Fig. 7b). (Here the individual PDFs are weighted by the mean power of the respective wavenumber and the number of data points per segment pair). This more complex marginal distribution comes from different maxima in PDFs of different 270 wavenumbers. If one trusts the angle estimate of a single wavenumber, this can be interpret as a wave ﬁeld with waves from multiple directions. The alternative – likely better – perspective is that the marginal PDF of each wavenumber is not a robust estimate the incident angle, and hence the PDF in ﬁgure 7b is the result of the uncertainty of the method, as explained in the next section. data points per segment pair). This more complex marginal distribution comes from different maxima in PDFs of different 270 wavenumbers. If one trusts the angle estimate of a single wavenumber, this can be interpret as a wave ﬁeld with waves from multiple directions. The alternative – likely better – perspective is that the marginal PDF of each wavenumber is not a robust estimate the incident angle, and hence the PDF in ﬁgure 7b is the result of the uncertainty of the method, as explained in the next section. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. in the local reference system of the segment centered around Xi and y = 0 such that the local along-track coordinate is in the local reference system of the segment centered around Xi and y = 0 such that the local along-track coordinate is η = x −Xi and the across-track coordinate is ν = y with the observable across-track wavenumber l′ = k′ tan(θ), and the 250 phase φ. y g i y g η = x −Xi and the across-track coordinate is ν = y with the observable across-track wavenumber l′ = k′ tan(θ), and the 250 phase φ. η = x −Xi and the across-track coordinate is ν = y with the observable across-track wavenumber l′ = k′ tan(θ), and the 250 phase φ. The monochromatic model is than used to deﬁne the objective function Φn for each wavenumb Φn = \|\|b −ˆhn\|\|2 + βθ Pθ,n(θ), (11) Φn = \|\|b −ˆhn\|\|2 + βθ Pθ,n(θ), where b is the normalized slope data of the beam pair, βθ is a hyper-parameter which controls the regularization Pθ,n of the incidence angle θ for the n-th wavenumber, and Pθ,n(θ,k) is a prior estimate that we describe in sec. 3.1.2. 55 where b is the normalized slope data of the beam pair, βθ is a hyper-parameter which control where b is the normalized slope data of the beam pair, βθ is a hyper-parameter which controls the regularization Pθ,n of the i id l θ f h h b d P (θ k) i i i h d ib i 3 1 2 where b is the normalized slope data of the beam pair, βθ is a hyper-parameter which controls the regularization Pθ,n of the incidence angle θ for the n-th wavenumber, and Pθ,n(θ,k) is a prior estimate that we describe in sec. 3.1.2. 255 The log-probability of the objective function eq. (11) is sampled for each beam pair, selected wavenumber, and along-track position Xi. To derive independent estimates of the incident angle for each n-th wavenumber we use an Metropolis-Hastings Scheme (Marcov-Chain Monte Carlo, MCMC, Foreman-Mackey et al., 2013) by ﬁrst initializing equally-spaced samples of the objective function over the domain θ = [−0.47π,0.47π] and φ = [0,2π) and advancing the ensemble of samples (ensemble of walkers) using MCMC. The MCMC method will quickly cluster walkers in the areas of low-cost, or small objective function 260 (Fig. 6b, black dots). A high density of walker positions is than interpreted as a high likelihood of an incident angle and phase for the chosen wavenumber (Fig. 6b, black dots). We derive a sample of the joint phase and angle distribution by advancing the walkers 300 iterations, and only the last 270 iterations for each walker are used to established the joint histogram D(θ,φ). The joint histogram D is then marginalized of walkers) using MCMC. The MCMC method will quickly cluster walkers in the areas of low-cost, or small objective function 260 (Fig. 6b, black dots). A high density of walker positions is than interpreted as a high likelihood of an incident angle and phase for the chosen wavenumber (Fig. 6b, black dots). We derive a sample of the joint phase and angle distribution by advancing the walkers 300 iterations, and only the last 270 iterations for each walker are used to established the joint histogram D(θ,φ). The joint histogram D is then marginalized We derive a sample of the joint phase and angle distribution by advancing the walkers 300 iterations, and only the last 270 iterations for each walker are used to established the joint histogram D(θ,φ). The joint histogram D is then marginalized for the incident angle θ and normalized to a probability distribution function (Fig. 6c). This procedure is repeated for each 265 selected wavenumber kn and for each (available) beam pair per segment Xi (Fig. S9 a to c). The best incident angle PDF θP DF (X,k,beampair) can then be derived using the weighted average across wavenumber, beam-pair, or both. for the incident angle θ and normalized to a probability distribution function (Fig. 6c). This procedure is repeated for each 265 selected wavenumber kn and for each (available) beam pair per segment Xi (Fig. S9 a to c). The best incident angle PDF θP DF (X,k,beampair) can then be derived using the weighted average across wavenumber, beam-pair, or both. The resulting beam- and wavenumber-average PDF of Track 3 at Xi = 87, for example, shows more than one maximum (Fig. 7b). (Here the individual PDFs are weighted by the mean power of the respective wavenumber and the number of for the incident angle θ and normalized to a probability distribution function (Fig. 6c). For each of these n = 25 observed wavenumbers k′ n, we deﬁne a monochromatic model, identify these wavenumbers, the beam-pairs mean wave power is smoothed using a three-wavenumber running mean to select 245 possible wave-numbers within the distorted narrow-banded spectrum (similar to the thick green lines in Fig. 3 b,c). For each of these n = 25 observed wavenumbers k′ n, we deﬁne a monochromatic model, (10) ˆhn(η,ν \|k′,θ,φ) = cos(k′η + l′ν + φ), ˆhn(η,ν \|k′,θ,φ) = cos(k′η + l′ν + φ), 12 12 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. When combining the systematic under-sampled PDFs they are still statistically robust. Each realization of the joint θ-φ distribution requires 6750 function evaluations for 270 iterations per walker. The walkers auto-correlation is about 20 to 30 iterations, which implies that each joint distribution maybe not be well established (the effective degrees of freedom per walker are about 9 to 14). Hence the marginalization of each joint distribution may misrepresent the angle uncertainty (i.e. a too wide distribution of the walker’s PDF). To reduce uncertainty, we take a super-sample of marginal PDFs, by averaging across 285 wavenumbers and/or the three beam pairs. The super-sampling results in a statistically robust result with 5 × 105 function evaluations per segment Xi, that is about effective 3.5 −5.5 × 103 degrees of freedom per segment. Longer Markov Chains, i.e. more iterations, may result in a better sampling of the individual ﬁt, but may not affect the overall result since they sample from generally smooth objective functions in a low signal-to-noise regime. However, in cases where a directional estimate per wavenumber or beam group is needed, the MCMC iteration length can be adjusted. 290 wavenumber or beam group is needed, the MCMC iteration length can be adjusted. 290 3.1.2 Constraining direction estimates with other data products The choice of βθ = 2 leads to the desired result in breaking the directional ambiguity while not fully determining the incident angle distribution (Fig. 7a). Other values of βθ are tested, but higher values tend to over ﬁt to the prior, and lower values do not break the ambiguity. 305 This method is limited to angles of about ±85◦. Oblique incidence angle can not be captured by this methods. In addition, the model has a 180◦ambiguity such that samples in the +95◦to −95◦arc, that are waves coming from higher latitudes rather than from the equator, would be equally possible. However given the geography of the Antarctic coast for tracks considered here, these are less plausible. but higher values tend to over ﬁt to the prior, and lower values do not break the ambiguity. 305 This method is limited to angles of about ±85◦. Oblique incidence angle can not be captured by this methods. In addition, the model has a 180◦ambiguity such that samples in the +95◦to −95◦arc, that are waves coming from higher latitudes rather than from the equator, would be equally possible. However given the geography of the Antarctic coast for tracks considered here, these are less plausible. but higher values tend to over ﬁt to the prior, and lower values do not break the ambiguity. 305 This method is limited to angles of about ±85◦. Oblique incidence angle can not be captured by this methods. In addition, the model has a 180◦ambiguity such that samples in the +95◦to −95◦arc, that are waves coming from higher latitudes rather than from the equator, would be equally possible. However given the geography of the Antarctic coast for tracks considered here, these are less plausible. 3.1.2 Constraining direction estimates with other data products Sampling of the objective function eq. (11) as described in sec. 3.1 results in a joint distribution of most likely incident angles and phases. These joint distributions may have multiple equally likely incident angles (Fig. 6d blue area) due to the limited across-track observations and distortion. To break the symmetry in the marginalized PDF of incident angles (Fig. 6e) we and phases. These joint distributions may have multiple equally likely incident angles (Fig. 6d blue area) due to the limited across-track observations and distortion. To break the symmetry in the marginalized PDF of incident angles (Fig. 6e) we deﬁne a prior Pθ(θ,k) in the objective function using ridge-regression (Appendix B). The effect of the prior on the joint- and 295 marginalized distribution is shown by comparing Figure 6 b and c with d and e. Here we inform the prior with WW3 global hindcast wave-partitions (Tolman, 2009, using the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) hindcast)). WW3 must be treated with caution due wind-observational biases in the Southern Ocean (Belmonte Rivas and Stoffelen, 2019; Hell et al., 2020). This wave hindcast is currently the only readily available dataset for this global purpose, and priors from observational datasets would improve the quality of this data and the overall wave inversion 300 deﬁne a prior Pθ(θ,k) in the objective function using ridge-regression (Appendix B). The effect of the prior on the joint- and 295 marginalized distribution is shown by comparing Figure 6 b and c with d and e. Here we inform the prior with WW3 global hindcast wave-partitions (Tolman, 2009, using the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) hindcast)). WW3 must be treated with caution due wind-observational biases in the Southern Ocean (Belmonte Rivas and Stoffelen, 2019; Hell et al., 2020). This wave hindcast is currently the only readily available dataset for this global purpose, and priors from observational datasets would improve the quality of this data and the overall wave inversion. 300 The level of certainty in the WW3 prior is expressed in the hyper parameter βθ and the performance of the MCMC sampling is sensitive to its value (eq. 11). Since a validation of the WW3 prior is limited, we set βθ = 2. Its effect on the objective function can be seen by comparing the shading in ﬁgure 6 b and d. 3.1.1 Robustness of the Marginal PDFs 275 The described limitations in retrieving the incident angle (sec. 3.1) indicate a low signal-to-noise regime and demand a careful evaluation of the method for sampling the objective function Φn. While larger samples may ensure convergence of the dis- tribution estimate, a large sampler of each wavenumber, beam pair, and segment may not be necessary, or computationally affordable. We decide for a systematic under-sampling of each realization of the marginal PDFs θ(Xi,k,beampair) and, in a second step, make a super-sample from the marginal PDFs across beam pairs and wavenumber if needed. 280 second step, make a super-sample from the marginal PDFs across beam pairs and wavenumber if needed. 280 13 3.2 Two-dimensional spectra in along-track data 310 With the spectral (sec. 2.2) and angle (sec. 3.1) estimates, we now can describe waves observed along-track in terms of their two-dimensional wavenumber spectra (Fig. 8). The estimated wavenumber amplitudes ˆb (eq. 2) are corrected by cos−1 (θ) using the most likely incident angle (sec. 3.1, Fig. 7b) resulting in the corrected wavenumber spectrogram (Fig. 8a). We use 14 -2 0 2 Slope (m/m) a) Beam Pair -1 0 1 km -2 0 2 0 Angle of Incidence b) Sample Visualization best fit Prior c) Marginal 0 2 Wave Phase 0 Angle of Incidence d) Sample Visualization without prior 0 10 20 Density e) Marginal 3 0 3 Anomalous Cost Incident Angle Sampling model wavelength=168.4m Figure 6. MCMC angle estimate using a monochromatic wave model with and without a prior Pθ. (a) Data of the beam pair gt1l and gt1r (light and dark green) and model (black, eq. 10) for a segment of Track 3 (granule 05160312, centered at Xi = 62.5 km. (b). The objective function with prior Pθ is sampled using a brute-force method (shading) and MCMC (black dots and lines). The prior angle θ0 and prior uncertainty σθ for this wavelength (168.4 meter) are shown as thick orange line and shading. The best ﬁt using a dual-annealing method is shown is shown as red dot (Tsallis and Stariolo, 1996). (c) Marginal PDF of the incident angle θ from MCMC sampling. (d) and (e) same as (b) and (c) but without the prior Pθ. -2 0 2 Slope (m/m) a) Beam Pair -1 0 1 km -2 0 2 0 Angle of Incidence b) Sample Visualization best fit Prior c) Marginal 0 2 Wave Phase 0 Angle of Incidence d) Sample Visualization without prior 0 10 20 Density e) Marginal 3 0 3 Anomalous Cost Incident Angle Sampling model wavelength=168.4m -2 0 2 Slope (m/m) a) Beam Pair -1 0 1 km -2 0 2 0 Angle of Incidence b) Sample Visualization best fit Prior c) Marginal 3 0 3 Anomalous Cost Incident Angle Sampling model wavelength=168.4m Angle of Incidence Angle of Incidence 0 2 Wave Phase 0 Angle of Incidence d) Sample Visualization without prior 0 10 20 Density e) Marginal d) Sample Visualization without prior Figure 6. MCMC angle estimate using a monochromatic wave model with and without a prior Pθ. wavenumber k (sec. 3.1.2, Fig. 8b). 315 The corrected power spectrum and directional distribution (Fig. 8 a,b) can be expressed as directional surface wave spectra every 12.5 km in the MIZ, similar to conventional surface wave buoys (Fig. 8 c to e). This permits tracking the attenuation of wave energy per frequency in MIZ. In the case of Track 3 (granule 05160312) for example, we see a migration of the peak wavelength from about 275 meters to about 300 meters within 12.5 km (Fig. 8 d,e) as shorter waves tend to attenuate faster. The corrected power spectrum and directional distribution (Fig. 8 a,b) can be expressed as directional surface wave spectra every 12.5 km in the MIZ, similar to conventional surface wave buoys (Fig. 8 c to e). This permits tracking the attenuation of wave energy per frequency in MIZ. In the case of Track 3 (granule 05160312) for example, we see a migration of the peak wavelength from about 275 meters to about 300 meters within 12.5 km (Fig. 8 d,e) as shorter waves tend to attenuate faster. Future work will systematically use this analysis to understand attenuation in the MIZ. 320 Future work will systematically use this analysis to understand attenuation in the MIZ. 320 3.2 Two-dimensional spectra in along-track data 310 3.1.2, Fig. 8b). 315 3.2 Two-dimensional spectra in along-track data 310 (a) Data of the beam pair gt1l and gt1r (light and dark green) and model (black, eq. 10) for a segment of Track 3 (granule 05160312, centered at Xi = 62.5 km. (b). The objective function with prior Pθ is sampled using a brute-force method (shading) and MCMC (black dots and lines). The prior angle θ0 and prior uncertainty σθ for this wavelength (168.4 meter) are shown as thick orange line and shading. The best ﬁt using a dual-annealing method is shown is shown as red dot (Tsallis and Stariolo, 1996). (c) Marginal PDF of the incident angle θ from MCMC sampling. (d) and (e) same as (b) and (c) but without the prior Pθ. 15 0.29 0.4 0.51 0.63 0.74 Wavenumber Example Marginal PDF SH 2019-05-02 granule: 05160312 at Xi=87 km a) mean marginal PDFs per wavenumber - /2 - /4 0 /4 /2 Angle 0 1 2 3 Density b) Final weighted marginal PDF Figure 7. Inﬂuence of the WW3 prior on the marginal PDFs of Track 3 (granule 05160312) at Xi = 87 km. (a) Mean marginal PDFs for all beam-pairs as a function of wavenumber. The WW3 spectral partitions are shown as black dots and the interpolated prior θ0,i and its spread σθ,i (eq. B1) are shown as orange line and orange shading. (b) The smoothed weighted mean across the pairs of the most likely incident angle is shown as black thick line and the WW3 priors for all wave numbers used as orange lines. Figure 7. Inﬂuence of the WW3 prior on the marginal PDFs of Track 3 (granule 05160312) at Xi = 87 km. (a) Mean marginal PDFs for all beam-pairs as a function of wavenumber. The WW3 spectral partitions are shown as black dots and the interpolated prior θ0,i and its spread σθ,i (eq. B1) are shown as orange line and orange shading. (b) The smoothed weighted mean across the pairs of the most likely incident angle is shown as black thick line and the WW3 priors for all wave numbers used as orange lines. the most likely angle along the track, although the above analysis can provide angle distributions for each segment Xi and wavenumber k (sec. 3.1.2, Fig. 8b). 15 the most likely angle along the track, although the above analysis can provide angle distributions for each segment Xi and wavenumber k (sec. 3.1.2, Fig. 8b). 315 wavenumber k (sec. 4 Decomposition of along-track data to waves and surface roughness The estimate of the wave signal from sec. 2.3 can be used to decompose wave and ice surface variability. Each photon retrieval is a super-position of ocean waves and sea ice variability like surface roughness, ﬂoe-size, and free board height. A decomposition of surface variability between waves and ice can rely on the coherence of across-beam wave statistics, as well as their common 16 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 50 100 150 100 200 300 400 500 600 corrected wavelength (m) corrected peak observed peak c) d) e) a) Slope Power Spectra (m/m)2 k 1 for SH 2019-05-02 granule: 05160312 -70.0 -60.0 -50.0 -40.0 -30.0 Power 50 100 150 X (km) - /4 0 /4 Angle b) Direction PDFs 0.0 0.5 1.0 1.5 Density 0 30 60 -60 -30 250m 300m c) 0 30 60 -60 -30 250m 300m d) 0 30 60 -60 -30 250m 300m e) Figure 8. Final estimate of the power (a), directional (b), and joint (c,d,e) surface slope spectra every 12.5 km along an ICESat-2 beam (example Track 3). (a) The angle-corrected spectrogram as a function of estimated wavelength λ and distance from the ice-edge X. The power spectral wavelength where corrected by about a factor of three due to a peak incident angle of about 66◦(orange line in b). The observed and corrected peak wavelengths λ′ and λ are shown as dashed and solid black line. (b) Mean directional PDF between ±85◦every 12.5 km. (c) to (e) Rolling-mean smoothed directional wave spectra at the positions indicated at the respective green lines in (a) and (b). 0 a) Slope Power Spectra (m/m)2 k 1 for SH 2019-05-02 granule: 05160312 50 100 150 100 200 300 400 500 600 corrected wavelength (m) corrected peak observed peak c) d) e) a) Slope Power Spectra (m/m)2 k 1 for SH 2019-05-02 granule: 05160312 -70.0 -60.0 -50.0 -40.0 -30.0 Power b) Direction PDFs 150 50 0 30 60 -60 -30 250m 300m c) 0 30 60 -60 -30 250m 300m e) 0 Figure 8. Final estimate of the power (a), directional (b), and joint (c,d,e) surface slope spectra every 12.5 km along an ICESat-2 beam (example Track 3). (a) The angle-corrected spectrogram as a function of estimated wavelength λ and distance from the ice-edge X. 4 Decomposition of along-track data to waves and surface roughness The power spectral wavelength where corrected by about a factor of three due to a peak incident angle of about 66◦(orange line in b). The observed and corrected peak wavelengths λ′ and λ are shown as dashed and solid black line. (b) Mean directional PDF between ±85◦every 12.5 km. (c) to (e) Rolling-mean smoothed directional wave spectra at the positions indicated at the respective green lines in (a) and (b). noise level in wavenumber space. For the purpose of decomposing the data we deﬁne similar signal-to-noise levels across 325 beams in each 25 km segment (Fig. 5 a to f). The results of section 2.3 are used to delineate ATL03 photon heights between wave and sea ice surface variability. We con- struct a binned wave height ﬁeld along-track from the GFT-derived surface slope spectrum, by ﬁltering out high-wavenumber components that likely do not correspond to swell waves. This low-pass ﬁlter is deﬁned by a cutoff-wavenumber kc, which noise level in wavenumber space. For the purpose of decomposing the data we deﬁne similar signal-to-noise levels across 325 beams in each 25 km segment (Fig. 5 a to f). The results of section 2.3 are used to delineate ATL03 photon heights between wave and sea ice surface variability. We con- struct a binned wave height ﬁeld along-track from the GFT-derived surface slope spectrum, by ﬁltering out high-wavenumber components that likely do not correspond to swell waves. This low-pass ﬁlter is deﬁned by a cutoff-wavenumber kc, which noise level in wavenumber space. For the purpose of decomposing the data we deﬁne similar signal-to-noise levels across 25 beams in each 25 km segment (Fig. 5 a to f). g ( g ) The results of section 2.3 are used to delineate ATL03 photon heights between wave and sea ice surface variability. We con- struct a binned wave height ﬁeld along-track from the GFT-derived surface slope spectrum, by ﬁltering out high-wavenumber components that likely do not correspond to swell waves. This low-pass ﬁlter is deﬁned by a cutoff-wavenumber kc, which is the ﬁrst wavenumber where the power spectrum changes slope. A change in the power-spectral slope in log-log scaling 330 from the expected slope of surface wave spectra (k−5/2 or similar, Toba, 1973) to horizontal indicated a change in the signal- to-noise regime in the data (Fig. 9). Decomposing heights into wave and sea ice components allows us to estimate the fraction of the total height variance that is Decomposing heights into wave and sea ice components allows us to estimate the fraction of the total height variance that is neither due to waves nor photon variance on scales shorter than the 10-meter stencil. As shown in ﬁgure 10e, the majority of 345 the total variance is due to the photon variance around its 20-meter stencil mean for scales smaller then the stencil (Fig. 10d red line and black dots, Kwok et al., 2021). In this particular track wave variance than comprises between another 20% to 50% of total photon height variance. The remaining variance about, about 5% to 20%, due to neither waves nor the photon cloud, is from differences in the observed and modeled wave heights, ˆz and z. We assume this is to sea-ice-related variability. The neither due to waves nor photon variance on scales shorter than the 10-meter stencil. As shown in ﬁgure 10e, the majority of 345 the total variance is due to the photon variance around its 20-meter stencil mean for scales smaller then the stencil (Fig. 10d red line and black dots, Kwok et al., 2021). In this particular track wave variance than comprises between another 20% to 50% of total photon height variance. The remaining variance about, about 5% to 20%, due to neither waves nor the photon cloud, is from differences in the observed and modeled wave heights, ˆz and z. We assume this is to sea-ice-related variability. The distribution of the residual statistics is, by model design, approximately Gaussian (Fig. 3e) and hence non-wave signals with 350 non-linear imprint could contaminate the wave estimate and decomposition. In general, this decomposition removes waves as the dominant source of variance on scales larger than 20 meters, allowing for additional analysis of the residual signal, and more consistent surface height signals in wave-affected and low-sea ice regions. 4 Decomposition of along-track data to waves and surface roughness Hence, horizontal slopes at high wavenumber indicates Gaussian (white) noise, while steep wavenumber-slope are the result of wave-wave interaction (Kitaigorodskii, 1962; Hasselmann et al., 1973). The critical 17 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. wavenumber kc between both regimes is found using piecewise regression on the weighted cross-beam log-log power spectrum (Fig. 9, Pilgrim, 2021). 35 wavenumber kc between both regimes is found using piecewise regression on the weighted cross-beam log-log power spectrum (Fig. 9, Pilgrim, 2021). 35 For illustrative purposes we deﬁne a low-pass ﬁlter by setting kc as the cut-off wavenumber. This low-pass ﬁlter potentially creates artiﬁcial ringing in real space and for better results this should be replaced by a more complex ﬁlter design. Here, wavenumbers higher than kc are excluded from the wave height model of all beams (Fig. 10a gray and blue lines and gray area in inlet) by truncating the wavenumber space of the slope model p. From this truncated slope model we can directly construct a coefﬁcient matrix for the wave-height model ˆz for each individual beam by integrating in wavenumber space. The coefﬁcient matrix of the wave-height model ˆz is ˆd = [−b1,−b2,...,−bc,a1,...ac,]T , ˆd = [−b1,−b2,...,−bc,a1,...ac,]T , where bc and ac are the model amplitudes corresponding the the cutoff frequency kc (note the integration of the trig-formula changes order and sign of the indices). The reconstructed wave-height model ˆz can then be directly calculated from the original regressor matrix, ˆz = H ˆd kc −1, ˆz = H ˆd kc −1, (12) with kc = [k1,k2,...,kc,k1,...,kc]T as shown in ﬁgure 10b blue line. The residual between the height model ˆz and the observed 340 smoothed photon heights z, zfree = z −ˆz, is an estimate of the freeboard height absent the inﬂuence of waves (Fig. 10d). The residual zfree has similar data density to the original ATL03 photon retrievals but may reveal secondary, non-wavelike structures in the photon heights as shown in ﬁgure 10d. We provide additional examples in suppl. Fig. S6 and S7. with kc = [k1,k2,...,kc,k1,...,kc]T as shown in ﬁgure 10b blue line. The residual between the height model ˆz and the observed 340 smoothed photon heights z, zfree = z −ˆz, is an estimate of the freeboard height absent the inﬂuence of waves (Fig. 10d). The residual zfree has similar data density to the original ATL03 photon retrievals but may reveal secondary, non-wavelike structures in the photon heights as shown in ﬁgure 10d. We provide additional examples in suppl. Fig. S6 and S7. 5 Discussion ICESat-2 photon data frequently shows wave-like signals in sea ice and these substantially impacts the marginal ice zone. In 355 this paper, we show, for the ﬁrst time, how paired laser-altimeter observations can be converted into directional surface wave spectra. We describe a two-part algorithm that efﬁciently decomposes the IS2 photon retrievals into a surface wave signal ICESat-2 photon data frequently shows wave-like signals in sea ice and these substantially impacts the marginal ice zone. In 355 this paper, we show, for the ﬁrst time, how paired laser-altimeter observations can be converted into directional surface wave spectra. We describe a two-part algorithm that efﬁciently decomposes the IS2 photon retrievals into a surface wave signal 18 10 2 10 1 100 Power (m2/k) a) gt1r b) gt2r c) gt3r 10 2 10 1 wavenumber k 10 3 10 1 101 Power (m2/k) d) gt1l 10 2 10 1 wavenumber k e) gt2l 10 2 10 1 wavenumber k f) gt3l Cut-off Frequency for Displacement Spectral SH 2019-05-02 granule: 05160312 Figure 9. Sections of derived displacement power spectra ˆ˜Sh for Track 3 (Fig. 8) for each beam (a to f). The black lines show the position of the estimated cut-off wavenumber kc based on piece-wise regression on the weighted mean of all beams. Darker colored lines show sections closer to the sea ice edge and lighter colors show sections further into the sea ice. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 10 2 10 1 100 Power (m2/k) a) gt1r b) gt2r c) gt3r 10 2 10 1 wavenumber k 10 3 10 1 101 Power (m2/k) d) gt1l 10 2 10 1 wavenumber k e) gt2l 10 2 10 1 wavenumber k f) gt3l Cut-off Frequency for Displacement Spectral SH 2019-05-02 granule: 05160312 Figure 9. Sections of derived displacement power spectra ˆ˜Sh for Track 3 (Fig. 8) for each beam (a to f). The black lines show the position of the estimated cut-off wavenumber kc based on piece-wise regression on the weighted mean of all beams. Darker colored lines show sections closer to the sea ice edge and lighter colors show sections further into the sea ice. 5 Discussion 10 2 10 1 100 Power (m2/k) a) gt1r b) gt2r c) gt3r 10 2 10 1 wavenumber k 10 3 10 1 101 Power (m2/k) d) gt1l 10 2 10 1 wavenumber k e) gt2l 10 2 10 1 wavenumber k f) gt3l Cut-off Frequency for Displacement Spectral SH 2019-05-02 granule: 05160312 b) t2 ) t3 Cut-off Frequency for Displacement Spectral SH 2019-05-02 granule: 05160312 b) t2 ) t3 Cut-off Frequency for Displacement Spectral SH 2019-05-02 granule: 05160312 Figure 9. Sections of derived displacement power spectra ˆ˜Sh for Track 3 (Fig. 8) for each beam (a to f). The black lines show the position of the estimated cut-off wavenumber kc based on piece-wise regression on the weighted mean of all beams. Darker colored lines show sections closer to the sea ice edge and lighter colors show sections further into the sea ice. as well as variability due to sea ice. The 1st part of the algorithm is based on a linear inversion method to ﬁt wavenumber coefﬁcients to the ATL03 data (sec. 2.2 to 2.4), and the 2nd part uses a non-linear inversion method that optimizes for most- likely wave incident angles (sec. 3.1). 0 as well as variability due to sea ice. The 1st part of the algorithm is based on a linear inversion method to ﬁt wavenumber coefﬁcients to the ATL03 data (sec. 2.2 to 2.4), and the 2nd part uses a non-linear inversion method that optimizes for most- likely wave incident angles (sec. 3.1). 360 The combined method than provides a highly-resolved 2D-surface wave spectra every 12.5 km along each IS-2 track (Fig. 8a,b) as well as an improved surface height estimate when the wave signal is removed (Fig. 10). The surface wave estimate relies on the one hand on the redundancy across beams to optimize the signal-to-noise ratio in wavenumber space and on the other hand on the difference across beams for the angle inversion. The iterative solution proposed here leads to an likely wave incident angles (sec. 3.1). 360 The combined method than provides a highly-resolved 2D-surface wave spectra every 12.5 km along each IS-2 track (Fig. 8a,b) as well as an improved surface height estimate when the wave signal is removed (Fig. 10). 5 Discussion The surface wave estimate relies on the one hand on the redundancy across beams to optimize the signal-to-noise ratio in wavenumber space and on the other hand on the difference across beams for the angle inversion. The iterative solution proposed here leads to an i t t ti f th IS 2 t k t k f t di i l t i l di ti t h i bl (Fi 8 365 interpretation of the IS-2 track as streak of two-dimensional wave spectra, including error estimates on each variable (Fig. 8c 365 to e). We identify the range of wavenumbers that contain wave energy in each segment by establishing a dynamic noise level (sec. 4). Removing the wave energy as a dominant source of variance reveals additional structure in the ATL03 photon cloud data that is not as readily present in the ATL07, or other higher level products (Fig. 10d, or suppl. Fig. S6 and S7), either interpretation of the IS-2 track as streak of two-dimensional wave spectra, including error estimates on each variable (Fig. 8c 365 to e). We identify the range of wavenumbers that contain wave energy in each segment by establishing a dynamic noise level (sec. 4). Removing the wave energy as a dominant source of variance reveals additional structure in the ATL03 photon cloud data that is not as readily present in the ATL07, or other higher level products (Fig. 10d, or suppl. Fig. S6 and S7), either to e). We identify the range of wavenumbers that contain wave energy in each segment by establishing a dynamic noise level (sec. 4). Removing the wave energy as a dominant source of variance reveals additional structure in the ATL03 photon cloud data that is not as readily present in the ATL07, or other higher level products (Fig. 10d, or suppl. Fig. S6 and S7), either because it is obscured by wave signals or the data is not present. A removal of the wave signal may have substantial beneﬁts 370 for classifying photon data for ATL07 products and above. Not removing the wave signal likely leads to aliasing effect of the a es into the freeboard height (compare panel b and d in ﬁg re 10) because it is obscured by wave signals or the data is not present. 5 Discussion A removal of the wave signal may have substantial beneﬁts 370 for classifying photon data for ATL07 products and above. Not removing the wave signal likely leads to aliasing effect of the waves into the freeboard height (compare panel b and d in ﬁgure 10). Our quantiﬁcation of wave energy allows for an improved understanding of photon variance. While in three example tracks have a substantial amount of variance in photon height on scales smaller then 20-meters (Fig. 11a,c,e gray area), the variance on scales longer than 20-meters is clearly dominated by the effect of waves (Fig. 11a,c,e blue area). Especially in the MIZ, 375 Our quantiﬁcation of wave energy allows for an improved understanding of photon variance. While in three example tracks have a substantial amount of variance in photon height on scales smaller then 20-meters (Fig. 11a,c,e gray area), the variance on scales longer than 20 meters is clearly dominated by the effect of waves (Fig 11a c e blue area) Especially in the MIZ 375 have a substantial amount of variance in photon height on scales smaller then 20-meters (Fig. 11a,c,e gray area), the variance on scales longer than 20-meters is clearly dominated by the effect of waves (Fig. 11a,c,e blue area). Especially in the MIZ, 375 19 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 10. Variance decomposition of the photon-cloud data (ATL03) based on the GFT of example Track 2. (a) The observed mean surface height slope b is shown in gray and the truncated model ˆb in blue. The inset shows the corresponding smoothed spectral amplitudes and cut-off wavenumber kc as a black line. (b) The observed photon cloud is shown as black dots, re-binned data z as a red line (sec. 2.1), and the reconstructed surface heights as a thin blue line ˆz (eq. 12). (c) Corresponding ATL07 surface heights product shown as in ﬁgure 2. (d) Figure 10. Variance decomposition of the photon-cloud data (ATL03) based on the GFT of example Track 2. (a) The observed mean surface height slope b is shown in gray and the truncated model ˆb in blue. The inset shows the corresponding smoothed spectral amplitudes and cut-off wavenumber kc as a black line. (b) The observed photon cloud is shown as black dots, re-binned data z as a red line (sec. 5 Discussion 2.1), and the reconstructed surface heights as a thin blue line ˆz (eq. 12). (c) Corresponding ATL07 surface heights product shown as in ﬁgure 2. (d) Residual photon heights (black) and binned heights (red) using ˆz−z. (e) Variance fraction every kilometer with the fraction due to the photon ˆ Figure 10. Variance decomposition of the photon-cloud data (ATL03) based on the GFT of example Track 2. (a) The observed mean surface height slope b is shown in gray and the truncated model ˆb in blue. The inset shows the corresponding smoothed spectral amplitudes and cut-off wavenumber kc as a black line. (b) The observed photon cloud is shown as black dots, re-binned data z as a red line (sec. 2.1), and the reconstructed surface heights as a thin blue line ˆz (eq. 12). (c) Corresponding ATL07 surface heights product shown as in ﬁgure 2. (d) Residual photon heights (black) and binned heights (red) using ˆz−z. (e) Variance fraction every kilometer with the fraction due to the photon cloud (gray), the truncated wave model ˆz (blue), variance of wavenumber > kc (green), and the residual of the model r (red, eq. 1). only a small fraction of this re-binned variance is due to non-wavy features of the surface (Fig. 11a and e gray-blue area at 0 - 50 km from the ice edge). This wave-induced variance in photon cloud of ATL03 can, under certain conditions, also be captured by ATL07. However, we suggest that the ATL07 algorithm also can potentially capture an aliased wave signal, or can fail to provide a sufﬁcient sea- 20 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. a) b) c) d) e) f) Figure 11. Variance decomposition of ATL03 photon cloud data and comparison with ATL07 data. (a) Across-beam averaged variances for Track 3 (granule 05160312, see suppl. Table). The photon cloud variance is shown in black (same as Fig. 10b black dots), the variance of the 20-meter stencils is shown in light blue (same as Fig. 10b red line), and the variance of the low-pass ﬁltered wave model in dark blue with a black outline (same as Fig. 10b blue line). (b) Variance of ATL07 based on the provided segment heights. Gray hatched areas indicate no data in ATL07 as for this track. Black line is repeated from panel (a) for comparison. 5 Discussion 11d), ATL07 does provide data at some but not all locations along-track, even though there is a high photon density throughout the track. In the MIZ, ATL07 variance can exceed the variance estimates of waves, indicating a potential aliasing of wave-induced ice height product at all. While in the case of Track 3 (Fig. 11a,b), waves clearly affect the observation, ATL07 does not provide 380 data in this region (see also suppl. Fig. S2) and so our inversion method using ATL03 allows for improved MIZ freeboard data. In other cases, like Track 2 (Fig. 11d), ATL07 does provide data at some but not all locations along-track, even though there is a high photon density throughout the track. In the MIZ, ATL07 variance can exceed the variance estimates of waves, indicating a potential aliasing of wave-induced ice height product at all. While in the case of Track 3 (Fig. 11a,b), waves clearly affect the observation, ATL07 does not provide 380 data in this region (see also suppl. Fig. S2) and so our inversion method using ATL03 allows for improved MIZ freeboard data. In other cases, like Track 2 (Fig. 11d), ATL07 does provide data at some but not all locations along-track, even though there is a high photon density throughout the track. In the MIZ, ATL07 variance can exceed the variance estimates of waves, indicating a potential aliasing of wave-induced signal to other scales (Fig. 11d). This aliasing can be due to the binning of data based on photon counts which result in varying 385 bin length. Varying bins-length potentially sub-samples the wave’s energy at scales on, or around the Nyquist frequency of the dominant waves. Since both the wavelength and photon density highly vary, it is generally unknown whether or not the photo- count based measure samples these wavelengths correct and does not negatively affect freeboard retrievals or wave energy estimates in the MIZ . signal to other scales (Fig. 11d). This aliasing can be due to the binning of data based on photon counts which result in varying 385 bin length. Varying bins-length potentially sub-samples the wave’s energy at scales on, or around the Nyquist frequency of the dominant waves. 5 Discussion (c and d) Same as (a) and (b) but for Track 2 (granule 08070210). (e and f) Same as (a) and (b) but for Track 4 (granule 05180312) a) b) c) d) e) f) f) Figure 11. Variance decomposition of ATL03 photon cloud data and comparison with ATL07 data. (a) A Figure 11. Variance decomposition of ATL03 photon cloud data and comparison with ATL07 data. (a) Across-beam averaged variances for Track 3 (granule 05160312, see suppl. Table). The photon cloud variance is shown in black (same as Fig. 10b black dots), the variance of the 20-meter stencils is shown in light blue (same as Fig. 10b red line), and the variance of the low-pass ﬁltered wave model in dark blue with a black outline (same as Fig. 10b blue line). (b) Variance of ATL07 based on the provided segment heights. Gray hatched areas indicate no data in ATL07 as for this track. Black line is repeated from panel (a) for comparison. (c and d) Same as (a) and (b) but for Track 2 (granule 08070210). (e and f) Same as (a) and (b) but for Track 4 (granule 05180312) Figure 11. Variance decomposition of ATL03 photon cloud data and comparison with ATL07 data. (a) Across-beam averaged variances for Track 3 (granule 05160312, see suppl. Table). The photon cloud variance is shown in black (same as Fig. 10b black dots), the variance of the 20-meter stencils is shown in light blue (same as Fig. 10b red line), and the variance of the low-pass ﬁltered wave model in dark blue with a black outline (same as Fig. 10b blue line). (b) Variance of ATL07 based on the provided segment heights. Gray hatched areas indicate no data in ATL07 as for this track. Black line is repeated from panel (a) for comparison. (c and d) Same as (a) and (b) but for Track 2 (granule 08070210). (e and f) Same as (a) and (b) but for Track 4 (granule 05180312) ice height product at all. While in the case of Track 3 (Fig. 11a,b), waves clearly affect the observation, ATL07 does not provide 380 data in this region (see also suppl. Fig. S2) and so our inversion method using ATL03 allows for improved MIZ freeboard data. In other cases, like Track 2 (Fig. 5 Discussion We choose to approach this low signal-to-noise problem with a super-sample of marginal distributions derived from independent MCMC samples of monocromatic plane waves. The unweighted mean of this method across all wavenumbers is similar to the lag-cross correlation of the beam pair. However, by focusing on a limited set of energy-containing wavenumbers, the signal-to-noise-ratio improves above a lagged cross-correlation approach. Limiting the beams is limited by the geometry (Fig. 1b), a difﬁcult-to-estimate properties of the wave ﬁeld (i.e. its "groupiness", suppl. Fig. 415 S7), as well as observational noise (sec. 3.1). We choose to approach this low signal-to-noise problem with a super-sample of marginal distributions derived from independent MCMC samples of monocromatic plane waves. The unweighted mean of this method across all wavenumbers is similar to the lag-cross correlation of the beam pair. However, by focusing on a limited set of energy-containing wavenumbers, the signal-to-noise-ratio improves above a lagged cross-correlation approach. Limiting the sample to the most energetic waves and using a prior is what enables an inversion of the approximate wave angle (Fig. 7d). 420 Alternatively, ensemble-based approaches could search for a common direction across wavenumbers simultaneously, but the presented MCMC method has an advantage in that it can sample multiple minima in the objective function. Multiple minima, and therefore multiple potential incidence angles, are a plausible result for two-dimensional wave ﬁelds when observed only along segments (Fig. 6). sample to the most energetic waves and using a prior is what enables an inversion of the approximate wave angle (Fig. 7d). 420 Alternatively, ensemble-based approaches could search for a common direction across wavenumbers simultaneously, but the presented MCMC method has an advantage in that it can sample multiple minima in the objective function. Multiple minima, and therefore multiple potential incidence angles, are a plausible result for two-dimensional wave ﬁelds when observed only along segments (Fig. 6). sample to the most energetic waves and using a prior is what enables an inversion of the approximate wave angle (Fig. 7d). 420 Alternatively, ensemble-based approaches could search for a common direction across wavenumbers simultaneously, but the presented MCMC method has an advantage in that it can sample multiple minima in the objective function. Multiple minima, and therefore multiple potential incidence angles, are a plausible result for two-dimensional wave ﬁelds when observed only along segments (Fig. 6). The low signal-to-noise of the angle inversion requires regularization (sec. 3.1). 5 Discussion Since both the wavelength and photon density highly vary, it is generally unknown whether or not the photo- count based measure samples these wavelengths correct and does not negatively affect freeboard retrievals or wave energy estimates in the MIZ . The provided algorithm has particular use when applied at scale. We expect 10 −15% of the IS-2 tracks in polar regions 390 to be dominated by waves (Brouwer et al., 2021). That implies roughly 3 −4.5 × 103 cases of diverse wave observation in the MIZ (as of June 2022) to sample the large space of wave-ice interaction, which is likely enough to better constrain sea- ice wave interactions. Because IS-2 can also record freeboard heights, ﬂoe sizes, and sea ice types, this analysis can provide complimentary sea ice information to better constrain dynamics in the MIZ. In particular this analysis will be used to 21 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. a) provide data for case studies of wave-ice interaction in the MIZ, 395 b) statistically constrain wave attenuation in sea ice (Fig. 5), and c) leverage the wave-removed residual signal to improve ice classiﬁcation (Fig. 10 d). c) leverage the wave-removed residual signal to improve ice classiﬁcation (Fig. 10 d). We chose a generalized Fourier transform (GFT) method instead of more common methods like DFT, or Lomb-Scargle (LS, sec. 2.2, Lomb, 1976; Scargle, 1982; Wunsch, 1996; Kachelein et al., 2022). In contrast to the DFT and LS, the GFT is variance conserving method that can be applied to unstructured data and does not require periodicity over an (arbitrary) window length. 400 The GFT is customize-able to the wavenumbers of interest and additionally provides uncertainty bounds on all parameters. In turn, it comes with the requirement to apriori know what spectral resolution is needed. Given Atlas’s high resolution photon cloud data, we chose to resolve the plausible wavenumber range for surface waves on a resolution about twice the one of the DFT. Other, more targeted, narrow-banded wavenumber-spaces are possible, but here we choose wavenumber ranges that are the most general for surface waves. A higher resolutions especially at long wavenumber allows us to provide new insights in 405 how narrow-banded the surface wave ﬁeld is; a parameter that is related to the surface’s curvature and likely important for wave-induced sea-ice breakup (Meylan and Squire, 1994). 5 Discussion A major advantage of the GFT is that it can be extended to inversions of the wave ﬁeld for each IS2 track by coupling neighboring or overlapping segments, similar to Kalman inversion methods. We illustrate this by simply iterative updating the the most general for surface waves. A higher resolutions especially at long wavenumber allows us to provide new insights in 405 how narrow-banded the surface wave ﬁeld is; a parameter that is related to the surface’s curvature and likely important for wave-induced sea-ice breakup (Meylan and Squire, 1994). the most general for surface waves. A higher resolutions especially at long wavenumber allows us to provide new insights in 405 how narrow-banded the surface wave ﬁeld is; a parameter that is related to the surface’s curvature and likely important for wave-induced sea-ice breakup (Meylan and Squire, 1994). A major advantage of the GFT is that it can be extended to inversions of the wave ﬁeld for each IS2 track by coupling neighboring or overlapping segments, similar to Kalman inversion methods. We illustrate this by simply iterative updating the data segments and models priors (sec. 2.4, Fig. 4). This coupling of inversions along segments advances the task on hand to 410 solving the coupled inversions consistently along each beam, rather then independently for each segment. The same idea can be extended by coupling the model prior P across beams. This coupled approach ensures smoothly varying wave statistics along and across-track, with the amount of smoothness tuned through the amplitude of P in (eq. 6). The inversion for the wave’s incident angle is based on the cross-beam coherence (sec. 3). The coherence between two data segments and models priors (sec. 2.4, Fig. 4). This coupling of inversions along segments advances the task on hand to 410 solving the coupled inversions consistently along each beam, rather then independently for each segment. The same idea can be extended by coupling the model prior P across beams. This coupled approach ensures smoothly varying wave statistics along and across-track, with the amount of smoothness tuned through the amplitude of P in (eq. 6). The inversion for the wave’s incident angle is based on the cross-beam coherence (sec. 3). The coherence between two g ( ) beams is limited by the geometry (Fig. 1b), a difﬁcult-to-estimate properties of the wave ﬁeld (i.e. its "groupiness", suppl. Fig. 415 S7), as well as observational noise (sec. 3.1). Such an inversion could combine data from IS-2, SAR, and CFOSat to help constrain the surface wave ﬁled in the MIZ or the open ocean (Collard While the here developed methods are customized for ICESat-2 photon retrievals, this approach is applicable to any unstruc- tured quasi-instantaneous observation of a two-dimensional wave ﬁeld. In the case of IS-2, cross-track information is limited, but other future remote-sensing methods may have complimentary information about the surface wave ﬁeld. Such an inversion could combine data from IS-2, SAR, and CFOSat to help constrain the surface wave ﬁled in the MIZ or the open ocean (Collard et al., 2022). 440 Code and data availability. The algorithms are available through Hell (2022a, DOI: 10.5281/zenodo.6908645) and data are available through Hell (2022b, DOI: 10.5281/zenodo.6928350). The IS-2 ATL03 data (Neumann et al., 2021) and ATL07/10 data (Kwok et al., 2021) is available through NSIDC (https://nsidc.org/data/icesat-2/data-sets), or OpenAltimetry (https://openaltimetry.org/data/icesat2/, Khalsa et al., 2020). The wave model data are available through the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) project: ftp.ifremer.fr/ifremer/cersat/products/gridded/wavewatch3/hindcast/. The analysis uses and modiﬁes the icesat2 toolkit (https://read-icesat- 445 2.readthedocs.io/) Code and data availability. The algorithms are available through Hell (2022a, DOI: 10.5281/zenodo.6908645) and data are available through Hell (2022b, DOI: 10.5281/zenodo.6928350). The IS-2 ATL03 data (Neumann et al., 2021) and ATL07/10 data (Kwok et al., 2021) is available through NSIDC (https://nsidc.org/data/icesat-2/data-sets), or OpenAltimetry (https://openaltimetry.org/data/icesat2/, Khalsa et al., 2020). The wave model data are available through the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) project: ftp.ifremer.fr/ifremer/cersat/products/gridded/wavewatch3/hindcast/. The analysis uses and modiﬁes the icesat2 toolkit (https://read-icesat- 445 2.readthedocs.io/) 5 Discussion Since directional wave observation co- 425 located with IS2 tracks are sparse and not readily available, we relied on Wave Watch III (WW3) hindcast models as a prior (IOWAGA Tolman, 2009). The wave hindcasts may perform sufﬁciently well in the Northern Hemisphere but are known to have limitations in the Southern Ocean MIZ, potentially due to wind biases (Belmonte Rivas and Stoffelen, 2019; Hell et al., 22 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2020, 2021b). The lack of certainty in WW3’s peak direction and frequency is expressed the value of the hyper parameter βθ (eq. 11). A value of βθ = 2 leads to the desired behavior of breaking the symmetry (compare shading in ﬁgure 6 b and d) but 430 not imposing the optimization result through the prior (Fig. 7d blue and orange lines). 2020, 2021b). The lack of certainty in WW3’s peak direction and frequency is expressed the value of the hyper parameter βθ (eq. 11). A value of βθ = 2 leads to the desired behavior of breaking the symmetry (compare shading in ﬁgure 6 b and d) but 430 not imposing the optimization result through the prior (Fig. 7d blue and orange lines). The angle inversion is limited to the geometry of the observation and the nature of the surface wave statistics (sec. 3.1). Waves coming from steep angels compared to the IS-2 track cannot be resolved, which limits successful 2D-wave ﬁeld estimates to cases where we have an educated guess of an incident angle that is not too steep. This problem may be overcome by using better, observationally-based priors, or enriching our WW3 priors with data from other sources. 435 The angle inversion is limited to the geometry of the observation and the nature of the surface wave statistics (sec. 3.1). Waves coming from steep angels compared to the IS-2 track cannot be resolved, which limits successful 2D-wave ﬁeld estimates to cases where we have an educated guess of an incident angle that is not too steep. This problem may be overcome by using better, observationally-based priors, or enriching our WW3 priors with data from other sources. 435 better, observationally-based priors, or enriching our WW3 priors with data from other sources. 435 While the here developed methods are customized for ICESat-2 photon retrievals, this approach is applicable to any unstruc- tured quasi-instantaneous observation of a two-dimensional wave ﬁeld. In the case of IS-2, cross-track information is limited, but other future remote-sensing methods may have complimentary information about the surface wave ﬁeld. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. as shown in Figure 3e. At locations with no data this results in a model decay to zero (Fig. 3 orange lines) on scales similar to the data’s auto correlation. A2 Model Prior P 455 As described in section 2.4, the GFT’s prior is initially derived from a PM-spectrum that is ﬁtted to a DFT of the segment data (Fig. A1a gray and black line). The initial prior Pinit is then deﬁned as the peak-normalized PM-spectrum multiplied by the data variance σ(b)2 plus a 10% noise ﬂoor (Fig. A1a green line). The power of the resulting GFT model coefﬁcients ˆp is then shown as red line in ﬁgure A1a. For a segment i with a successfully inversion in the previous segment i−1 we use a smoothed power spectrum based on ˆpi−1 to derive Pi (ﬁg. A1b green line, sec. 2.4). 460 Figure A1. Two GFT ﬁt examples at Xi = 50 km (left) and Xi = 62.5 km (right) with the DFT of the data in gray, the ﬁtted PM-model in black, the rescaled PM-model as prior Pinit (multiplied by a factor of 10), and the ﬁnal GFT inversion expressed as power spectra in red. The number of 2M model parameters and number of N data points are given in the title. Figure A1. Two GFT ﬁt examples at Xi = 50 km (left) and Xi = 62.5 km (right) with the DFT of the data in gray, the ﬁtted PM-model in black, the rescaled PM-model as prior Pinit (multiplied by a factor of 10), and the ﬁnal GFT inversion expressed as power spectra in red. The number of 2M model parameters and number of N data points are given in the title. A1 Data Prior R We deﬁne the data prior R based on the surface slope uncertainty, as We deﬁne the data prior R based on the surface slope uncertainty, as R = βR σ(b)2 σh ∆x R = βR σ(b)2 σh ∆x where the stencil width is ∆x = 20 meters, σ(b) is the standard deviation of the data b within th where the stencil width is ∆x = 20 meters, σ(b) is the standard deviation of the data b within the segment, σh is the vector of standard deviation of each data point (each stencil, sec. 2.1), and βR is a tuning parameter that determines the ratio of the 50 model and data priors in eq. 6 (Fig. 2 blue lines). The standard deviation, or error, of the data is divided by the stencil size to get an error in units of surface slope and R is ampliﬁed by the variance of the data to scale it against the model prior. where the stencil width is ∆x = 20 meters, σ(b) is the standard deviation of the data b within the segment, σh is the vector of standard deviation of each data point (each stencil, sec. 2.1), and βR is a tuning parameter that determines the ratio of the 450 model and data priors in eq. 6 (Fig. 2 blue lines). The standard deviation, or error, of the data is divided by the stencil size to get an error in units of surface slope and R is ampliﬁed by the variance of the data to scale it against the model prior. To avoid over-ﬁtting the ratio of the model prior P and data prior R has to be adjusted. For this we try different values of βR and set it to 102 such that the distribution of the residual r is approximately Gaussian and that \|\|r\|\| = \|\|b −H ˆp\|\| ≈1, \|\|r\|\| = \|\|b −H ˆp\|\| ≈1, 23 Appendix B: Wave-watch III Prior The prior in eq. 11 uses an incident angle θ0(k) with an uncertainty σθ(k), deﬁning the prior Pθ(θ,k) = θ0(k) −θ σθ(k) 2 . (B1) Pθ(θ,k) = θ0(k) −θ σθ(k) 2 . (B1) Both variables in the prior have to be taken from other data sources than IS-2 and here they Both variables in the prior have to be taken from other data sources than IS-2 and here they are derived from WW3 global hindcast wave-partitions (Tolman, 2009, using the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) 65 Both variables in the prior have to be taken from other data sources than IS-2 and here they are derived from WW3 global hindcast wave-partitions (Tolman, 2009, using the Integrated Ocean Waves for Geophysical and other Applications (IOWAGA) 465 hindcast)) and depend on wavenumber. The WW3 hindcast data is selected in a box around the most equatorward photons in ICESat-2 (suppl. Fig. S4 and. sec. 2.1). In cases where this box is within the sea-ice mask of WW3 it is moved equatorward along the IS-2 track until at least 2/3 of the box’s grid cells are not covered by the WW3 sea-ice mask. Within each box, the mean of the peak period, peak direction, and directional spread is calculated for each of the ﬁve WW3 wavenumber partitions (Fig. 7a black dots). These partitions are interpolated and smoothed to the ki wave numbers of interest 470 24 Author contributions. MH developed and programmed the proposed algorithm. MH and CH collaboratively wrote the paper. Competing interests. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. to provide a best guess of the wavenumber-dependent peak direction and spread (Fig. 7a orange line and shading). Note that it is hard to validate the WW3 partitions in the Southern Ocean due to a lack of contemporaneous in-situ observations. The a lack of certainty in the wave-hindcast in combination with (any) smoothing procedure can lead to biases in the directional prior. Hence, we do not expect direct alignment of the WW3 wave directions and those observed in ICESat-2 observations. The WW3 incident angle is here solely used to reduce ambiguities in the objective function (Fig. 6 b and d and Figure 7) and 475 can give a preferred incident wave angle, rather than a certain estimate of the dominant wave angle (Fig. 7). Author contributions. References J., Richter, K., Sell, W., and Walden, H.: Measurements of Wind-Wave Growth and Swell Decay during the Joint North Sea Wave Project (JONSWAP), Ergänzungsheft 8-12, 1973. Foreman-Mackey, D., Hogg, D. W., Lang, D., and Goodman, J.: Emcee: The MCMC Hammer, Publ. Astron. Soc. Pac., 125, 306–312, 495 https://doi.org/10.1086/670067, 2013. Foreman-Mackey, D., Hogg, D. W., Lang, D., and Goodman, J.: Emcee: The MCMC Hammer, Publ. Astron. Soc. Pac., 125, 306–312, 495 https://doi.org/10.1086/670067, 2013. Hasselmann, K., Barnett, T. P., Bouws, E., Carlson, H., Cartwright, D. E., Enke, K., Ewing, J. A., Gienapp, H., Hasselmann, D. E., Kruseman, Hasselmann, K., Barnett, T. P., Bouws, E., Carlson, H., Cartwright, D. E., Enke, K., Ewing, J. A., Gienapp, H., Hasselmann, D. E., Kruseman, P., Meerburg, A., Müller, P., Olbers, D. J., Richter, K., Sell, W., and Walden, H.: Measurements of Wind-Wave Growth and Swell Decay during the Joint North Sea Wave Project (JONSWAP), Ergänzungsheft 8-12, 1973. Hell, M.: Code for Directional Surface Wave Spectra And Sea Ice Structure from ICEsat-2 Altimetry without Data, Zenodo, 500 https://doi.org/10.5281/zenodo.6908645, 2022a. Hell, M.: Data for Directional Surface Wave Spectra And Sea Ice Structure from ICEsat-2 Altimetry, https://doi.org/10.5281/zenodo.6928350, 2022b. Hell, M. C., Cornuelle, B. D., Gille, S. T., Miller, A. J., and Bromirski, P. D.: Identifying Ocean Swell Generation Events from Ross Ice Shelf Hell, M. C., Cornuelle, B. D., Gille, S. T., Miller, A. J., and Bromirski, P. D.: Identifying Ocean Swell Generation Events from Ross Ice Shelf Seismic Data, J. Atmos. Oceanic Technol., 36, 2171–2189, https://doi.org/10.1175/JTECH-D-19-0093.1, 2019. 505 Hell, M. C., Gille, S. T., Cornuelle, B. D., Miller, A. J., Bromirski, P. D., and Crawford, A. D.: Estimating Southern Ocean Storm Positions With Seismic Observations, J. Geophys. Res. Oceans, 125, e2019JC015 898, https://doi.org/10.1029/2019JC015898, 2020. Hell, M. C., Ayet, A., and Chapron, B.: Swell Generation Under Extra-Tropical Storms, J. Geophys. Res. Oceans, 126, e2021JC017 637, https://doi.org/10.1029/2021JC017637, 2021a. Hell, M. C., Cornuelle, B. D., Gille, S. T., and Lutsko, N. J.: Time-Varying Empirical Probability Densities of Southern Ocean Surface Winds: 510 Linking the Leading Mode to SAM and Quantifying Wind Product Differences, J. Clim., 34, 5497–5522, https://doi.org/10.1175/JCLI-D- 20-0629.1, 2021b. Hell, M. C., Cornuelle, B. D., Gille, S. T., and Lutsko, N. J.: Time-Varying Empirical Probability Densities of Southern Ocean Surface Winds: 510 Linking the Leading Mode to SAM and Quantifying Wind Product Differences, J. Clim., 34, 5497–5522, https://doi.org/10.1175/JCLI-D- 20-0629.1, 2021b. References Ardhuin, F., Stopa, J., Chapron, B., Collard, F., Smith, M., Thomson, J., Doble, M., Blomquist, B., Persson, O., Collins, C. O., and Wadhams, 485 P.: Measuring ocean waves in sea ice using SAR imagery: A quasi-deterministic approach evaluated with Sentinel-1 and in situ data, Remote Sensing of Environment, 189, 211–222, https://doi.org/10.1016/j.rse.2016.11.024, 2017. Belmonte Rivas, M. and Stoffelen, A.: Characterizing ERA-Interim and ERA5 Surface Wind Biases Using ASCAT, Ocean Sci., 15, 831–852, https://doi.org/10.5194/os-15-831-2019, 2019. Ardhuin, F., Stopa, J., Chapron, B., Collard, F., Smith, M., Thomson, J., Doble, M., Blomquist, B., Persson, O., Collins, C. O., and Wadhams, 485 P.: Measuring ocean waves in sea ice using SAR imagery: A quasi-deterministic approach evaluated with Sentinel-1 and in situ data, Remote Sensing of Environment, 189, 211–222, https://doi.org/10.1016/j.rse.2016.11.024, 2017. Belmonte Rivas, M. and Stoffelen, A.: Characterizing ERA-Interim and ERA5 Surface Wind Biases Using ASCAT, Ocean Sci., 15, 831–852, https://doi.org/10.5194/os-15-831-2019, 2019. Brouwer, J., Fraser, A. D., Murphy, D. J., Wongpan, P., Alberello, A., Kohout, A., Horvat, C., Wotherspoon, S., Massom, R. A., Cartwright, J., 490 and Williams, G. D.: Altimetric observation of wave attenuation through the Antarctic marginal ice zone using ICESat-2, The Cryosphere, 367, https://doi.org/10.5194/tc-2021-367, 2021. Collard, F., Marié, L., Nouguier, F., Kleinherenbrink, M., Ehlers, F., and Ardhuin, F.: Wind-Wave Attenuation in Arctic Sea Ice: A Discussion of Remote Sensing Capabilities, J. Geophys. Res. Oceans, 127, e2022JC018 654, https://doi.org/10.1029/2022JC018654, 2022. Brouwer, J., Fraser, A. D., Murphy, D. J., Wongpan, P., Alberello, A., Kohout, A., Horvat, C., Wotherspoon, S., Massom, R. A., Cartwright, J., 490 and Williams, G. D.: Altimetric observation of wave attenuation through the Antarctic marginal ice zone using ICESat-2, The Cryosphere, 367, https://doi.org/10.5194/tc-2021-367, 2021. Collard, F., Marié, L., Nouguier, F., Kleinherenbrink, M., Ehlers, F., and Ardhuin, F.: Wind-Wave Attenuation Collard, F., Marié, L., Nouguier, F., Kleinherenbrink, M., Ehlers, F., and Ardhuin, F.: Wind-Wave Attenuation in Arctic Sea Ice: A Discussion of Remote Sensing Capabilities, J. Geophys. Res. Oceans, 127, e2022JC018 654, https://doi.org/10.1029/2022JC018654, 2022. of Remote Sensing Capabilities, J. Geophys. Res. Oceans, 127, e2022JC018 654, https://doi.org/10.1029/2 Foreman-Mackey, D., Hogg, D. W., Lang, D., and Goodman, J.: Emcee: The MCMC Hammer, Publ. Astron. Soc. Pac., 125, 306–312, 495 https://doi.org/10.1086/670067, 2013. Hasselmann, K., Barnett, T. P., Bouws, E., Carlson, H., Cartwright, D. E., Enke, K., Ewing, J. A., Gienapp, H., Hasselmann, D. E., Kruseman, P., Meerburg, A., Müller, P., Olbers, D. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Competing interests. We have no no competing interests. We have no no competing interests. 80 480 Acknowledgements. We thank Bruce Cornuelle and Baylor-Fox Kemper for discussing depths and caveats of the proposed algorithms. C.H. and M.H. were supported by Schmidt Futures—a philanthropic initiative that seeks to improve societal outcomes through the development of emerging science and technologies. 25 R.: The Statistical Analysis of a Random, Moving Surface, Philos. Trans. R. Soc. Lond. Ser. Math. Phys. Sci., 249, 321–387, https://doi.org/10.1098/rsta.1957.0002, 1957. Lygre, A. and Krogstad, H. E.: Maximum Entropy Estimation of the Directional Distribution in Ocean Wave Spectra, J. Phys. Oceanogr., 16, 2052–2060, https://doi.org/10.1175/1520-0485(1986)016<2052:MEEOTD>2.0.CO;2, 1986. 535 2052–2060, https://doi.org/10.1175/1520-0485(1986)016<2052:MEEOTD>2.0.CO;2, 1986. 535 Marechal, G. and Ardhuin, F.: Surface Currents and Signiﬁcant Wave Height Gradients: Matching Numerical Models and High-Resolution Altimeter Wave Heights in the Agulhas Current Region, J. Geophys. Res. Oceans, 126, e2020JC016 564, https://doi.org/10.1029/2020JC016564, 2021. Menke, W.: Geophysical Data Analysis: Discrete Inverse Theory, Academic press, 2018. Marechal, G. and Ardhuin, F.: Surface Currents and Signiﬁcant Wave Height Gradients: Matching Numerical Models and High-Resolution Altimeter Wave Heights in the Agulhas Current Region, J. Geophys. Res. Oceans, 126, e2020JC016 564, https://doi.org/10.1029/2020JC016564, 2021. Menke, W.: Geophysical Data Analysis: Discrete Inverse Theory, Academic press, 2018. Meylan, M. and Squire, V. A.: The Response of Ice Floes to Ocean Waves, J. Geophys. Res. Oceans, 99, 891–900, 540 https://doi.org/10.1029/93JC02695, 1994. Neumann, T. A., Brenner, D., Hancock, D., Robbins, J., Saba, K., Harbeck, K., Gibbons, A., Lee, J., Luthcke, S. B., and Rebold, T.: Meylan, M. and Squire, V. A.: The Response of Ice Floes to Ocean Waves, J. Geophys. Res. Oceans, 99, 891–900, 540 https://doi.org/10.1029/93JC02695, 1994. Neumann, T. A., Brenner, D., Hancock, D., Robbins, J., Saba, K., Harbeck, K., Gibbons, A., Lee, J., Luthcke, S. B., and Rebold, T.: ATLAS/ICESat-2 L2A Global Geolocated Photon Data, Version 5. [ATL03, 2022-04-04], https://doi.org/10.5067/ATLAS/ATL03.005, 2021. Meylan, M. and Squire, V. A.: The Response of Ice Floes to Ocean Waves, J. Geophys. Res. Oceans, 99, 891–900, 540 https://doi.org/10.1029/93JC02695, 1994. https://doi.org/10.1029/93JC02695, 1994. Neumann, T. A., Brenner, D., Hancock, D., Robbins, J., Saba, K., Harbeck, K., Gibbons, A., Lee, J., Luthcke, S. B., and Rebold, T.: ATLAS/ICESat-2 L2A Global Geolocated Photon Data, Version 5. [ATL03, 2022-04-04], https://doi.org/10.5067/ATLAS/ATL03.005, 2021. Pierson, W. J. and Moskowitz, L.: A Proposed Spectral Form for Fully Developed Wind Seas Based on the Similarity Theory of S. A. 545 Kitaigorodskii, J. Geophys. Res., 69, 5181–5190, https://doi.org/10.1029/JZ069i024p05181, 1964. Pierson, W. J. and Moskowitz, L.: A Proposed Spectral Form for Fully Developed Wind Seas Based on the Similarity Theory of S. A. 545 Kitaigorodskii, J. Geophys. Res., 69, 5181–5190, https://doi.org/10.1029/JZ069i024p05181, 1964. Pilgrim, C.: Piecewise-Regression (Aka Segmented Regression) in Python, J. Open Source Softw., 6, 3859, https://doi.org/10.21105/joss.03859, 2021. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Khalsa, S. J. S., Borsa, A., Nandigam, V., Phan, M., Lin, K., Crosby, C., Fricker, H., Baru, C., and Lopez, L.: OpenAltimetry - Rapid Analysis and Visualization of Spaceborne Altimeter Data, Earth Sci Inform, https://doi.org/10.1007/s12145-020-00520-2, 2020. Khalsa, S. J. S., Borsa, A., Nandigam, V., Phan, M., Lin, K., Crosby, C., Fricker, H., Baru, C., and Lopez, L.: OpenAltimetry - Rapid Analysis and Visualization of Spaceborne Altimeter Data, Earth Sci Inform, https://doi.org/10.1007/s12145-020-00520-2, 2020. Kitaigorodskii, S. A.: Applications of the Theory of Similarity to the Analysis of Wind-Generated Wave Motion as a Stochastic Process, Izv Geophys Ser Acad Sci USSR, 1, 105–117, 1962. and Visualization of Spaceborne Altimeter Data, Earth Sci Inform, https://doi.org/10.1007/s12145-020-00520-2, 2020. Kitaigorodskii, S. A.: Applications of the Theory of Similarity to the Analysis of Wind-Generated Wave Motion as a Stochastic Process, Izv Geophys Ser Acad Sci USSR, 1, 105–117, 1962. Kwok, R., Petty, A., Cunningham, G., Markus, T., Hancock, D., Morison, J. H., Palm, S. P., Farrell, S. L., Ivanoff, A., Wimert, J., and ICESat- 525 2 Science Team: ATLAS/ICESat-2 L3A Sea Ice Height, Version 3 [ATL07/10, 2022-04-04], https://doi.org/10.5067/ATLAS/ATL07.003, 2021. Kwok, R., Petty, A., Cunningham, G., Markus, T., Hancock, D., Morison, J. H., Palm, S. P., Farrell, S. L., Ivanoff, A., Wimert, J., and ICESat- 525 2 Science Team: ATLAS/ICESat-2 L3A Sea Ice Height, Version 3 [ATL07/10, 2022-04-04], https://doi.org/10.5067/ATLAS/ATL07.003, 2021. Lomb, N. R.: Least-Squares Frequency Analysis of Unequally Spaced Data, Astrophys Space Sci, 39, 447–462, https://doi.org/10.1007/BF00648343, 1976. 2021. Lomb, N. R.: Least-Squares Frequency Analysis of Unequally Spaced Data, Astrophys Space Sci, 39, 447–462, https://doi.org/10.1007/BF00648343, 1976. Lomb, N. R.: Least-Squares Frequency Analysis of Unequally Spaced Data, Astrophys Space Sci, 39, 447–462, https://doi.org/10.1007/BF00648343, 1976. Longuet-Higgins, M. S.: Statistical Properties of Wave Groups in a Random Sea State, Philos. Trans. R. Soc. Lond. Ser. Math. Phys. Sci., 530 312, 219–250, https://doi.org/10.1098/rsta.1984.0061, 1984. Longuet-Higgins, M. S.: Statistical Properties of Wave Groups in a Random Sea State, Philos. Trans. R. Soc. Lond. Ser. Math. Phys. Sci., 530 312, 219–250, https://doi.org/10.1098/rsta.1984.0061, 1984. Longuet-Higgins, M. S. and Deacon, G. E. R.: The Statistical Analysis of a Random, Moving Surface, Philos. Trans. R. Soc. Lond. Ser. Math. Phys. Sci., 249, 321–387, https://doi.org/10.1098/rsta.1957.0002, 1957. 312, 219 250, https://doi.org/10.1098/rsta.1984.0061, 1984. Longuet-Higgins, M. S. and Deacon, G. E. References Horvat, C.: Floes, the Marginal Ice Zone, and Coupled Wave-Sea-Ice Feedbacks, Phil Trans Roy Soc. A., https://doi org/10 1098/rsta 2021 0252 2022 20-0629.1, 2021b. Horvat, C.: Floes, the Marginal Ice Zone, and Coupled Wave-Sea-Ice Feedbacks, Phil Trans Roy Soc. A., https://doi.org/10.1098/rsta.2021.0252, 2022. Horvat, C., Tziperman, E., and Campin, J.-M.: Interaction of sea ice ﬂoe size, ocean eddies, and sea ice melting, Geophysical Research 515 Letters, 43, 8083–8090, https://doi.org/10.1002/2016GL069742, 2016. Horvat, C., Tziperman, E., and Campin, J. M.: Interaction of sea ice ﬂoe size, ocean eddies, and sea ice melting, Geophysical Research 515 Letters, 43, 8083–8090, https://doi.org/10.1002/2016GL069742, 2016. Horvat, C., Blanchard-Wrigglesworth, E., and Petty, A.: Observing waves in sea ice with ICESat-2, Geophysical Research Letters, https://doi.org/10.1029/2020GL087629, 2020. Horvat, C., Blanchard-Wrigglesworth, E., and Petty, A.: Observing waves in sea ice with ICESat-2, Geophysical Research Letters, https://doi.org/10.1029/2020GL087629, 2020. Kachelein, L., Cornuelle, B. D., Gille, S. T., and Mazloff, M. R.: Harmonic Analysis of Non-Phase-Locked Tides with Red Noise Using the Red_tide Package, J. Atmospheric Ocean. Technol., -1, https://doi.org/10.1175/JTECH-D-21-0034.1, 2022. 520 , , , , , , , y g Red_tide Package, J. Atmospheric Ocean. Technol., -1, https://doi.org/10.1175/JTECH-D-21-0034.1, 2022. 520 26 Rapley C G : First Observations of the Interaction of Ocean Swell with Sea Ice Using Satellite Radar Altimeter Data Nature 307 150–152 Kitaigorodskii, J. Geophys. Res., 69, 5181–5190, https://doi.org/10.1029/JZ069i024p05181, 1964. Pilgrim, C.: Piecewise-Regression (Aka Segmented Regression) in Python, J. Open Source Softw., 6, 3859, https://doi.org/10.21105/joss.03859, 2021. Kitaigorodskii, J. Geophys. Res., 69, 5181–5190, https://doi.org/10.1029/JZ069i024p05181, 1964. Pilgrim, C.: Piecewise-Regression (Aka Segmented Regression) in Python, J. Open Source Softw., 6, 3859, https://doi.org/10.21105/joss.03859, 2021. Rapley, C. G.: First Observations of the Interaction of Ocean Swell with Sea Ice Using Satellite Radar Altimeter Data, Nature, 307, 150–152, Pilgrim, C.: Piecewise-Regression (Aka Segmented Regression) in Python, J. Open Source Softw., 6, 3859, https://doi.org/10.21105/joss.03859, 2021. p g j , Rapley, C. G.: First Observations of the Interaction of Ocean Swell with Sea Ice Using Satellite Radar Altimeter Data, Nature, 307, 150–152, h //d i /10 1038/30 150 0 1984 550 Rapley, C. G.: First Observations of the Interaction of Ocean Swell with Sea Ice Using Satellite Radar Altimeter Data, Nature, 307, 150–152, https://doi.org/10.1038/307150a0, 1984. 550 https://doi.org/10.1038/307150a0, 1984. 550 Scargle, J. D.: Studies in Astronomical Time Series Analysis. II. Statistical Aspects of Spectral Analysis of Unevenly Spaced Data., Astro- phys. J., 263, 835–853, https://doi.org/10.1086/160554, 1982. Squire, V. A.: Of ocean waves and sea-ice revisited, Cold Regions Science and Technology, 49, 110–133, https://doi.org/10.1016/j.coldregions.2007.04.007, 2007. Scargle, J. D.: Studies in Astronomical Time Series Analysis. II. Statistical Aspects of Spectral Analysis of Unevenly Spaced Data., Astro- phys. J., 263, 835–853, https://doi.org/10.1086/160554, 1982. phys. J., 263, 835–853, https://doi.org/10.1086/160554, 1982. Squire, V. A.: Of ocean waves and sea-ice revisited, Cold Regions Science and Technology, 49, 110–133, https://doi.org/10.1016/j.coldregions.2007.04.007, 2007. Squire, V. A.: A Fresh Look at How Ocean Waves and Sea Ice Interact, Phil. Trans. R. Soc. A., 376, 20170 342, 555 https://doi.org/10.1098/rsta.2017.0342, 2018. Squire, V. A.: A Fresh Look at How Ocean Waves and Sea Ice Interact, Phil. Trans. R. Soc. A., 376, 20170 342, 555 https://doi.org/10.1098/rsta.2017.0342, 2018. Stopa, J. E., Sutherland, P., and Ardhuin, F.: Strong and highly variable push of ocean waves on Southern Ocean sea ice, Proceedings of the National Academy of Sciences, 115, 5861–5865, https://doi.org/10.1073/pnas.1802011115, 2018. Stopa, J. E., Sutherland, P., and Ardhuin, F.: Strong and highly variable push of ocean waves on Southern Ocean sea ice, Proceedings of the National Academy of Sciences, 115, 5861–5865, https://doi.org/10.1073/pnas.1802011115, 2018. 27 https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-842 Preprint. Discussion started: 12 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Thomson, J.: Wave propagation in the Marginal Ice Zone: connections and feedbacks within the air-ice-ocean system, Phil Trans R Soc A, 2022. Thomson, J.: Wave propagation in the Marginal Ice Zone: connections and feedbacks within the air ice ocean system, Phil Trans R Soc A, 2022. 560 Thomson, J., Hošeková, L., Meylan, M. H., Kohout, A. L., and Kumar, N.: Spurious Rollover of Wave Attenuation Rates in Sea Ice Caused by Noise in Field Measurements, J. Geophys. Res. Oceans, 126, https://doi.org/10.1029/2020JC016606, 2021. Toba, Y.: Local Balance in the Air-Sea Boundary Processes, Journal of the Oceanographical Society of Japan, 29, 209–220, https://doi.org/10.1007/BF02108528, 1973. 560 Thomson, J., Hošeková, L., Meylan, M. H., Kohout, A. L., and Kumar, N.: Spurious Rollover of Wave Attenuation Rates in Sea Ice Caused by Noise in Field Measurements, J. Geophys. Res. Oceans, 126, https://doi.org/10.1029/2020JC016606, 2021. Toba, Y.: Local Balance in the Air-Sea Boundary Processes, Journal of the Oceanographical Society of Japan, 29, 209–220, https://doi.org/10.1007/BF02108528, 1973. Tolman, H. L.: User Manual and System Documentation of WAVEWATCH III TM Version 3.14, Tech. Note MMAB Contrib., 276, 220, 65 2009. Tolman, H. L.: User Manual and System Documentation of WAVEWATCH III TM Version 3.14, Tech. Note MMAB Contrib., 276, 220, 565 2009. Tsallis, C. and Stariolo, D. A.: Generalized Simulated Annealing, Physica A: Statistical Mechanics and its Applications, 233, 395–406, Tolman, H. L.: User Manual and System Documentation of WAVEWATCH III TM Version 3.14, Tech. Note MMAB Contrib., 276, 220, 565 2009. Tsallis, C. and Stariolo, D. A.: Generalized Simulated Annealing, Physica A: Statistical Mechanics and its Applications, 233, 395–406, https://doi.org/10.1016/S0378-4371(96)00271-3, 1996. Villas Bôas A B and Young W R : Directional Diffusion of Surface Gravity Wave Action by Ocean Macroturbulence J Fluid Mech 890 Tsallis, C. and Stariolo, D. A.: Generalized Simulated Annealing, Physica A: Statistical Mechanics and its Applications, 233, 395–406, https://doi.org/10.1016/S0378-4371(96)00271-3, 1996. Villas Bôas, A. B. and Young, W. R.: Directional Diffusion of Surface Gravity Wave Action by Ocean Macroturbulence, J. Fluid Mech., 890, https://doi.org/10.1017/jfm.2020.116, 2020. 570 https://doi.org/10.1017/jfm.2020.116, 2020. 570 Wunsch, C.: The Ocean Circulation Inverse Problem, Cambridge University Press, Cambridge, https://doi.org/10.1017/CBO9780511629570, 1996. Yu, Y., Sandwell, D. T., Gille, S. T., and Villas Bôas, A. B.: Assessment of ICESat-2 for the Recovery of Ocean Topography, Geophysical Journal International, 226, 456–467, https://doi.org/10.1093/gji/ggab084, 2021. Wunsch, C.: The Ocean Circulation Inverse Problem, Cambridge University Press, Cambridge, https://doi.org/10.1017/CBO9780511629570, 1996. Yu, Y., Sandwell, D. T., Gille, S. T., and Villas Bôas, A. B.: Assessment of ICESat-2 for the Recovery of Ocean Topography, Geophysical Journal International, 226, 456–467, https://doi.org/10.1093/gji/ggab084, 2021. 28
https://openalex.org/W2967048811	https://zenodo.org/records/3478342/files/Hlabangana%20Chem%20Eng%201.pdf	English	null	Effect of media shape on particle breakage in a batch ball mill: Lessons learnt from Population Balance Model and Attainable Region technique	Procedia manufacturing	2,019	cc-by	4,572	Available online at www.sciencedirect.com ScienceDirect Available online at www sciencedirect com S i Di t Available online at www.sciencedirect.com ScienceDirect Available online at www sciencedirect com S i Di t The batch ______ Abstract Keywords: Attainable region (AR), Population balance model, Media shape, Op Peer-review under responsibility of the organizing committee of SMPM 2019. Keywords: Attainable region (AR), Population balance model, Media shape, Optimisation, © 2019 The Authors. Published by Elsevier B.V. Effect of media shape on particle breakage in a batch ball mill: Lessons learnt from Population Balance Model and Attainable Region technique S Khumalo a N Hlabangana a* G Danha b E Muzenda c (SMPM 2019) Effect of media shape on particle breakage in a batch ball mill: Lessons learnt from Population Balance Model and Attainable Region Effect of media shape on particle breakage in a batch ball mill: Lessons learnt from Population Balance Model and Attainable Region technique S. Khumalo , N. Hlabangana , G. Danha , E Muzenda ngineering, Faculty of Engineering and Technology, National University of Science and Technology technique S. Khumalo a, N. Hlabangana a, G. Danha b, E Muzenda c a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cDepartment of Civil & Chemical Engineering, College of Science, Engineering & Technology, University of South Africa, 28 Pioneer Avenue, 1724 Florida Park, Roodepoort, South Africa. __________________________________________________________________________________________ q S. Khumalo a, N. Hlabangana a, G. Danha b, E Muzenda c a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cD t t f Ci il & Ch i l E i i C ll f S i E i i & T h l U i it f S th Af i 28 Pi A 1724 Fl id S. Khumalo , N. Hlabangana , G. Danha , E Muzenda a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cDepartment of Civil & Chemical Engineering, College of Science, Engineering & Technology, University of South Africa, 28 Pioneer Avenue, 1724 Florida Park, Roodepoort, South Africa. 2351-9789 © 2019 The Authors. Published by Elsevier B.V. Peer-review under responsibility of the organizing committee of SMPM 2019. 10.1016/j.promfg.2019.07.003 The batch ______ Abstract g g q g q g g of quartz ore in a laboratory batch ball mill. Using the same milling conditions and grinding equipment, test results were evaluated through the breakage distributions of different size fractions. Results prove that mixing different grinding media increases volume of grinding zones and that translates to an improvement in grinding kinetics and amount of required PSD. The tools are valuable and practical ways of evaluating the breakage of materials on the size fractional base and results of which can be used in the mathematical modelling and optimising of ball milling unit operations. Abstract The batch grinding equation and the Attainable region technique tools were used in evaluating data obtained from the breakage of quartz ore in a laboratory batch ball mill. Using the same milling conditions and grinding equipment, test results were evaluated through the breakage distributions of different size fractions. Results prove that mixing different grinding media increases volume of grinding zones and that translates to an improvement in grinding kinetics and amount of required PSD. The tools are valuable and practical ways of evaluating the breakage of materials on the size fractional base and results of which can The batch grinding equation and the Attainable region technique tools were used in evaluating data obtained from the breakage of quartz ore in a laboratory batch ball mill. Using the same milling conditions and grinding equipment, test results were evaluated through the breakage distributions of different size fractions. Results prove that mixing different grinding media increases volume of grinding zones and that translates to an improvement in grinding kinetics and amount of required PSD. The tools are valuable and practical ways of evaluating the breakage of materials on the size fractional base and results of which can be used in the mathematical modelling and optimising of ball milling unit operations. © 2019 The Authors. Published by Elsevier B.V. Keywords: Attainable region (AR), Population ba p y be used in the mathematical modelling and optim y Peer-review under responsibility of the organizing committee of SMPM 2019. © 2019 The Authors. Published by Elsevier B.V. g p g g p Keywords: Attainable region (AR), Population balance model, Media shape, Op y Peer-review under responsibility of the organizing committee of SMPM 2019. © 2019 The Authors. Published by Elsevier B.V. * Corresponding author: +263776514098 1.1. The population balance model * ScienceDirect Procedia Manufacturing 00 (2019) 000 ScienceDirect Sc e ce ect Procedia Manufacturing 00 (2019) 000 www.elsevier.com/locate/procedia 2nd International Conference on Sustainable Materials Processing and Manufacturing (SMPM 2019) 2nd International Conference on Sustainable Materials Processing and Manufacturing 2nd International Conference on Sustainable Materials Processing and Manufacturing (SMPM 2019) a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cDepartment of Civil & Chemical Engineering, College of Science, Engineering & Technology, University of South Africa, 28 Pioneer Avenue, 1724 Florida Park, Roodepoort, South Africa. __________________________________________________________________________________________ q S. Khumalo a, N. Hlabangana a, G. Danha b, E Muzenda c a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cDepartment of Civil & Chemical Engineering College of Science Engineering & Technology University of South Africa 28 Pioneer Avenue 1724 Florida S. Khumalo , N. Hlabangana , G. Danha , E Muzenda a.Department of Chemical Engineering, Faculty of Engineering and Technology, National University of Science and Technology, Private Bag A.C 939 Ascot, Bulawayo, Zimbabwe bDepartment of Chemical, Materials and Metallurgical Engineering, Faculty of Engineering and Technology, Botswana International University of Science and Technology, Plot 10071 Boseja Ward, Private Bag 16 Palapye, Botswana. cDepartment of Civil & Chemical Engineering, College of Science, Engineering & Technology, University of South Africa, 28 Pioneer Avenue, 1724 Florida Park, Roodepoort, South Africa. 1. Introduction 1 I t d ti Comminution is a vital unit process that is performed in the processing of mineral ores. The ore is slowly reduced in size in tumbling mills to produce material of the preferred size class. This is done to expose, or to free, the valuable mineral from the gangue material before the product is sent to downstream recovery processes. The unit process is energy intensive and any potential savings is of paramount importance as it inspires the efforts for optimisation of size reduction processes. A lot of literature exists that has been dedicated to describe the milling unit operation and the Attainable region technique (AR). _____________________________________________________________________________________________________ 1. Introduction Comminution is a vital unit process that is performed in the processing of mineral ores. The ore is slowly reduced in size in tumbling mills to produce material of the preferred size class. This is done to expose, or to free, the valuable mineral from the gangue material before the product is sent to downstream recovery processes The unit process is energy intensive and any 1. Introduction Comminution is a vital unit process that is performed in the processing of mineral ores. The ore is slowly reduced in size in tumbling mills to produce material of the preferred size class. This is done to expose, or to free, the valuable mineral from the gangue material before the product is sent to downstream recovery processes. The unit process is energy intensive and any potential savings is of paramount importance as it inspires the efforts for optimisation of size reduction processes. p p p p hat has been dedicated to describe the milling unit operation and the Attainable region technique (AR). e material of the preferred size class. This is done to expose, or to free, the valuable mineral from the he product is sent to downstream recovery processes The unit process is energy intensive and any p y p p gy y amount importance as it inspires the efforts for optimisation of size reduction processes. Some key aspects of population balance model and AR will be considered. gangue material before the product is sent to downstream recovery proce potential savings is of paramount importance as it inspires the efforts for op A lot of literature exists that has been dedicated to describe the milling unit o 1.1. Email: nkosikhona.hlabangana@nust.ac.zw Corresponding author: +263776514098 1.3 Media shape Apart from being relatively wasteful of energy, tumbling mills are also inefficient with regard to mineral liberation because of the indiscriminate nature of the grinding force. Accordingly, researchers have made efforts to study the effects of media in an attempt to improve the grinding process [16-19] and have come up with interesting results. Studies on media efficiency have concentrated on comparing their effects on the breakage distribution function and the selection function ignoring other parameters defining mill performance such as downstream processes. The aim of this investigation intends to add value to the understanding of the breakage of the materials as well as to better comprehend optimum conditions required for the downstream processing of mineral ores. Two different tools are applied to the batch grinding data to understand kinetics and maximise the liberation of value material in downstream recovery processes. 1.2. Attainable Region technique The AR is a graphical technique that is equipment-independent and has been applied in the area of comminution to describe the size reduction processes [4]. A complete AR analysis involves a description of the set of all possible outputs for a system [5]. There exist some similarities between the size reduction process and chemical reaction engineering and this suggests that comminution processes can be studied using the AR approach with a mill being considered as basically a reactor system where large feed particles are transformed into smaller product particles. The work already done by different researchers described the construction and application of AR method to optimize size reduction processes done under different operating conditions with different objectives [6-15]. 1. Introduction 1 I t d ti The population balance model Some key aspects of population balan 1 1 Th l i b l d l 1.1. The population balance model Some key aspects of population balan * Corresponding author: +263776514098 1.1. The population balance model * * Corresponding author: +263776514098 1.1. The population balance model * Email: nkosikhona.hlabangana@nust.ac.zw * Corresponding author: +263776514098 76 S. Khumalo et al. / Procedia Manufacturing 35 (2019) 75–79 A number of researchers have used the model [1-3]. It is also known as the batch grinding equation and is made up of the specific rate of breakage (Si), the primary distribution breakage (bij) functions and a residence time distribution function. For a simulation of a ball mill to be successful, operation using laboratory scale results in a relationship of these functions and design of mill and operating variables should be well defined. The tool is centered on first order kinetics and the two functions give a size-mass balance equation for fully mixed batch milling operations. The modelling aspect allows us to construct a simulation model which can easily predict the particle size distribution in terms of the design of mill and the conditions of operation Grinding experiments were done in a laboratory batch mill with diameter 0.282m, length 0.302m and milling conditions in Table 1. During all the tests, mill feeds were constant, (1941g). The sample to be milled was loaded in the mill with the media of appropriate size and weight. The feed material was then ground batch wise using media charges for selected periods (3, 5. 15, 30, 60 and 90 minutes) and a full product size distribution done on the product after each time interval. S. Khumalo et al. / Procedia Manufacturing 35 (2019) 75–79 77 Table 1: Milling conditions for experiments on the ore Mill speed Operational speed (Φ) 75% of critical Media Dimensions Balls 30 mm Cubes 32mm Eclipsoids 40mm* 40mm Mix Ball and cubes (B-C1) 50% B 50% C Mix Ball and cubes (B-C2) 75% B 25% C Mix Ball and Eclipsoids (B-E) 50% B 50% E Material Feed size µm 2360-1700 For the use of the AR, the particle size distribution is usually divided into three or more size classes. For easier use of the tool we selected three sizes classes. This allows us to select the feed material, the intermediate and fine size class. The three size classes were chosen as: Size class 1: Material between 2360μm and 1700μm, Size class 2: Between 150μm and 75μm, Size class 3: Everything less than 75μm. 2. Materials and Methods A quartz ore obtained from a local mine in Johannesburg South Africa was used in the experimental work. A mono sized feed fraction of between 2360 and 1700μm was prepared and used for all the tests. Spheres, cubes and eclipsoids made of cast iron (Figure 1) were prepared and used as grinding media. Figure 1: Photographs of cubes, spheres and eclipsoids and mixture of grinding media shapes used for the batch tests [19] Figure 1: Photographs of cubes, spheres and eclipsoids and mixture of grinding media shapes used for the batch tests [19] Grinding experiments were done in a laboratory batch mill with diameter 0.282m, length 0.302m and milling conditions in Table 1. During all the tests, mill feeds were constant, (1941g). The sample to be milled was loaded in the mill with the media of appropriate size and weight. The feed material was then ground batch wise using media charges for selected periods (3, 5. 15, 30, 60 and 90 minutes) and a full product size distribution done on the product after each time interval. 77 S. Khumalo et al. / Procedia Manufacturing 35 (2019) 75–79 3. Results and Discussion Figure 2 shows a variation of specific rate of breakage and the particle size for all grinding policies. Results show that mixtures offer higher rates of breakage in abnormal region. It is also observed that on adding spheres to cubes, a noticeable increase in breakage rates compared to cubes alone is attained. The authors suggested that further investigations must be done to determine the proportion of cubes in the mixture. Instead of using spheres it is possible to obtain optimal ball to cube mix ratio and use it for size reduction process. On evaluating the selection function for all selected size classes it was noted that breakage function to be normalisable. The rate of conversion of feed to required product is a function of milling conditions and ore properties (i.e. selection function and breakage function). They further established that spheres are most effective and the cubes being the less efficient. Eclipsoids were also effective as the ball especially performing better than balls for coarser material. Figure 2: Variation of the specific rate of breakage with particle size for different grinding media and their mixtures [19] Figure 2: Variation of the specific rate of breakage with particle size for different grinding media and their mixtures [19] Figure 2 shows that the breakage rates for all media shapes are highest at the initial periods of milling but as time progresses and material in the mill gets depleted, cubes seem to be failing to produce a finer size class. This could be due to the reduced probability of particle capture between the cube to cube impacts. Figure 3(a) also reveals that use of cubes alone as a grinding Figure 2 shows that the breakage rates for all media shapes are highest at the initial periods of milling but as time progresses and material in the mill gets depleted, cubes seem to be failing to produce a finer size class. This could be due to the reduced probability of particle capture between the cube to cube impacts. Figure 3(a) also reveals that use of cubes alone as a grinding S. Khumalo et al. / Procedia Manufacturing 35 (2019) 75–79 78 media will not achieve the objective of higher grinding rates and finer material production. Different combinations of grinding media shapes improve the production of finer material. 3. Results and Discussion This implies that if media cost and availability are limiting factors, then it is possible to select and mix what you have and still obtain relatively high values of the required size class material. Figure 4: Attainable region plots for different media and mixing them (a) M2 vs. M1 (b) M3 vs.M1 Figure 4 (a) shows the A.R plot of the variation of mass fraction of material in the intermediate size class versus that in the feed size class obtained by milling with different shapes of grinding media. Similarly, the area bounded by each curve and the x axis is the wanted space termed the A.R and the turning point on each curve at the peak signifies an optimum solution for the maximum production of material in the intermediate size class. Figure 4(b) shows an A.R plot of the variation of mass fraction of material in the fines size class with that in the feed size class for milling done with different media shapes. The concurs with previous A.R plots in that the cubes produce less material in the finer size class while the balls and eclipsoids produce the maximum amount of fines. There are some size reduction industries where fines are a required product e.g the cement production industry. Hence the A.R plot can also be useful in such industries in formulating grinding media policies that give result to optimum production of material in the fines size class. Figure 4: Attainable region plots for different media and mixing them (a) M2 vs. M1 (b) M3 vs.M1 Figure 4: Attainable region plots for different media and mixing them (a) M2 vs. M1 (b) M3 vs.M1 Figure 4 (a) shows the A.R plot of the variation of mass fraction of material in the intermediate size class versus that in the feed size class obtained by milling with different shapes of grinding media. Similarly, the area bounded by each curve and the x axis is the wanted space termed the A.R and the turning point on each curve at the peak signifies an optimum solution for the maximum production of material in the intermediate size class. Figure 4(b) shows an A.R plot of the variation of mass fraction of material in the fines size class with that in the feed size class for milling done with different media shapes. 3. Results and Discussion 0 20 40 60 80 100 0 20 40 60 80 100 Grinding time (mins)(a) Grinding time (mins)(b) Figure 3: Mass fraction of silica material obtained versus grinding time (a) M1 vs. time (b) M2 vs. time 0 0.2 0.4 0.6 0.8 1 CUBES ECLIPSOIDS BALLS %B-50%C 50 50 %B-50%E 75 %B-25%C 0 20 40 60 80 100 Grinding time (mins)(b) 0 20 40 60 80 100 Grinding time (mins)(a) 0 0.2 0.4 0.6 0.8 1 CUBES ECLIPSOIDS BALLS %B-50%C 50 50 %B-50%E 75 %B-25%C Figure 3: Mass fraction of silica material obtained versus grinding time (a) M1 vs. time (b) M2 vs. time Figure 3(b) shows an A.R plot of the mass fraction of intermediate size class material against grinding time for different grinding media shapes. The region bounded by the curves and the x-axis is the wanted or attainable region within which lies solutions of the optimization question. The turning point of the plots give result to the solution of the optimization problem of the maximum amount of the intermediate size class material that can be obtained within the limits of the experimental conditions employed. The A.R plot shows that use of 100% balls or 100% eclipsoids will produce optimum amounts of the required size class material. The A.R method also shows that grinding media mixtures (50%B- 50%E and 75%B-25%C) also give relatively high amounts of the required material. This implies that if media cost and availability are limiting factors, then it is possible to select and mix what you have and still obtain relatively high values of the required size class material. Figure 3(b) shows an A.R plot of the mass fraction of intermediate size class material against grinding time for different grinding media shapes. The region bounded by the curves and the x-axis is the wanted or attainable region within which lies solutions of the optimization question. The turning point of the plots give result to the solution of the optimization problem of the maximum amount of the intermediate size class material that can be obtained within the limits of the experimental conditions employed. The A.R plot shows that use of 100% balls or 100% eclipsoids will produce optimum amounts of the required size class material. The A.R method also shows that grinding media mixtures (50%B- 50%E and 75%B-25%C) also give relatively high amounts of the required material. 4. Conclusion Milling tests were done using similar grinding conditions in order to compare the breakage behaviour of quartz in a batch ball mill using spheres, eclipsoids, cubes, and different mixtures of grinding media. The grinding media mass was kept constant and results showed that each media shape policy offers unique surface area and contact mechanisms during the size reduction process and this was attributed to differences in their geometry. The Attainable Region results show that the spheres and eclipsoids managed to offer high values of the required size class whilst cubes due to their geometry offered a courser product compared to what was required. Using same grinding conditions as researchers [19] who demonstrate that using population balance model, it is possible to improve particle breakage by mixing different grinding media the AR shows that mixing can be done to get optimum amounts of material in the desired size class. The A.R results also make evident that each media policy can be applicable in its own unique industry to give optimum amounts of the required product. Acknowledgement The authors appreciate the National University of Science and Technology, Department of Chemical Engineering where the paper was put together. 3. Results and Discussion The concurs with previous A.R plots in that the cubes produce less material in the finer size class while the balls and eclipsoids produce the maximum amount of fines. There are some size reduction industries where fines are a required product e.g the cement production industry. Hence the A.R plot can also be useful in such industries in formulating grinding media policies that give result to optimum production of material in the fines size class. S. Khumalo et al. / Procedia Manufacturing 35 (2019) 75–79 79 References [1] L. G. Austin, R. R. Klimpel, P. T. Luckie, 1984, Process engineering of size reduction: Ball milling. New York, USA: ME, AIME. [2] K. Shoji, L. G. Austin, F. Smalia, K. Brame, and P. T. Luckie. Further studies of ball and powder filling effects in ball milling. Powder Technology, 1982, 31: pp. 121-26. 3] V. Deniz and T. Onur, Investigation of the breakage kinetic of pumice samples as dependent on powder filling in a ball m Mineral Processing. 2002, 67: pp. 71–78. ine al ocessing. 00 , 67: pp. 7 78. [4] N. Khumalo, D. Glasser, D. Hildebrandt, B. Hausberger, and S. Kauchali. 2006. The application of the attainable region analysis to comminution. Chemical Engineering Science 61: pp 5969–5980. D. Hildebrandt, B. Hausberger, and S. Kauchali. 2006. The application of the attainable region analysis to comminution. Chemical pp 5969–5980. 4] N. Khumalo, D. Glasser, D. Hildebrandt, B. Hausberger, and S. Kauchali. 2006. The application of the attainable region analy Engineering Science 61: pp 5969–5980. [5] Khumalo N, Glasser D, Hildebrandt D, Hausberger B, Kauchali S. An experimental validation of a specified energy-based approach for comminution. Chem. Eng. Sci. 2007; 62: 2765–2776. [6] F.M. Katubilwa, M.H. Moys, D. Glasser, D. Hildebrandt, An attainable region analysis of the effect of ball size on milling. Powder Technology, 2011, 210 (1): pp. 36–46. [7] N. Hlabangana, G. Danha, D. Hildebrandt, D. Glasser, Use of the attainable region approach to determine major trends and optimize particle breakage in a laboratory mill, Powder Technology, 2016, 291: pp. 414–419. a, M.M. Bwalya, D. Hildebrandt, D. Glasser, Application of the attainable region method to determine optimal conditions for wder Technology. 2017; 317: 400-407. [8] N. Hlabangana, G. Danha, M.M. Bwalya, D. Hildebrandt, D. Glasser, Application of the attainable region method to milling and leaching, Powder Technology. 2017; 317: 400-407. [9] N. Khumalo, D. Glasser, D. Hildebrandt, B. Hausberger, S. Kauchali. The application of the attainable region analysis to comminution. Chem. Eng. Sci. 2006; 61: 5969–5980. [10] F.M. Katubilwa, M.H. Moys, D. Glasser, D. Hildebrandt, An attainable region analysis of the effect of ball size on milling. Powder Technology, 2011, 210 (1): pp. 36–46. [11] F.K. Mulenga, M.M. Bwalya. Application of the attainable region technique to the analysis of a full-scale mill in open circuit. Journal of the Southern African Institute of Mining and Metallurgy. 2015; 115 (8): 729 – 740. a, N.G. Mguni, M. S. Madiba, C. References Bhondayi, Determining an optimal interstitial filling condition: An Attainable Region approach, 8; 327: pp. 9-16. 12] N. Hlabangana, G. Danha, N.G. Mguni, M. S. Madiba, C. Bhondayi, Determining an optimal interstitial filling condition: An Powder Technology, 2018; 327: pp. 9-16. [13] N. Hlabangana, G. Danha, E. Muzenda. Effect of ball and feed particle size distribution on the milling efficiency of a ball mill: An attainable region approach, South African Journal [1] of Chemical Engineering, 2018, 25, pp.79-84 [14] G. Danha, D. Legodi, N. Hlabangana, C. Bhondayi, D. Hildebrandt, A fundamental investigation on the breakage of a bed of silica sand particles: An attainable region approach, 2016; 301: pp. 1208-1212. [15] Kelsall D F, Stewart PSB, Weller K R. Continuous grinding in a small wet ball mill Part 5. A study of the influence of media shape. Powder Technology.1973; 8:77-83. ternative to balls as grinding media. World Mining.1983; 36: (10) 59. [16] U. Cloos. Cylpebs: An alternative to balls as grinding media. World Mining.1983; 36: (10) 59. [17] H. Ipek. The effects of grinding media shape on breakage rate, Minerals Engineering. 2006; 19 (1): 91–93. [17] H. Ipek. The effects of grinding media shape on breakage rate, Minerals Engineering. 2006; 19 (1): 91–93. Kong, B.L. Zhang. The effects of grinding media shapes on the grinding kinetics of cement clinker in ball mill. Powder Tech 425. 18] H.Y. Qian, Q.G. Kong, B.L. Zhang. The effects of grinding media shapes on the grinding kinetics of cement clinker in b 2013; 235: 422–425. [19] K.P. Simba, M.H. Moys. Effects of mixtures of grinding media of different shapes on milling kinetics. Minerals Engineering. 2014; 61: 40–46.
https://openalex.org/W4312124415	https://www.aanda.org/10.1051/0004-6361/202244667/pdf	English	null	A new method for age-dating the formation of bars in disc galaxies	Astronomy & astrophysics	2,023	cc-by	19,026	ABSTRACT The epoch in which galactic discs settle is a major benchmark for testing models of galaxy formation and evolution but remains largely unknown. Once discs settle and become suﬃciently self-gravitating, stellar bars are able to form; therefore, determining the ages of bars can shed light on the epoch of disc settling, and on the onset of secular evolution. Nevertheless, timing when the bar formed has proven challenging. In this work we present a new methodology for obtaining the bar age, using the star formation history of nuclear discs. Nuclear discs are rotation-supported structures, built by gas pushed to the centre via bar-induced torques, and their formation is thus coincident with bar formation. In particular, we used integral ﬁeld spectroscopic data from the TIMER survey to disentangle the star formation history of the nuclear disc from that of the underlying main disc, which enables us to more accurately determine when the nuclear disc formed. We demonstrate the methodology on the galaxy NGC 1433 – which we ﬁnd to host an old bar that is 7.5+1.6 −1.1(sys)+0.2 −0.5(stat) Gyr old – and describe a number of tests carried out on both the observational data and numerical simulations. In addition, we present evidence that the nuclear disc of NGC 1433 grows in accordance with an inside-out formation scenario. This methodology is applicable to high-resolution integral ﬁeld spectroscopic data of barred galaxies with nuclear discs, making it ideally suited for the TIMER survey sample. In the future we will thus be able to determine the bar age for a large sample of galaxies, shedding light on the epoch of disc settling and bar formation. Key words. galaxies: bulges – galaxies: evolution – galaxies: formation – galaxies: kinematics and dynamics – galaxies: stellar content – galaxies: structure A new method for age-dating the formation of bars in disc galaxies The TIMER view on NGC1433’s old bar and the inside-out growth of its nuclear disc 55, 80803 München, Germany 8 8 Departamento de Física de la Tierra y Astrofísica, Universidad Complutense de Madrid, 28040 Madrid, Spain 9 I i d Fí i d P í l d l C (IPARCOS) U i id d C l d M d id 28040 M d id 8 Departamento de Física de la Tierra y Astrofísica, Universidad Complutense 9 uto de Física de Partículas y del Cosmos (IPARCOS), Univ 9 Instituto de Física de Partículas y del Cosmos (IPARCOS), Universidad Complutense de Madrid, 28040 Madrid 10 Department of Astrophysics, University of Vienna, Türkenschanzstrasße 17, 1180 Wien, Austria 11 10 Department of Astrophysics, University of Vienna, Türkenschanzstrasße 17, 1180 Wien, Austria 11 11 Universidade de São Paulo, Instituto de Astronomia, Geofísica e Ciências Atmosféricas, Rua do Matão 1226, 05508-090 São Paulo, SP, Brazil 2 European Space Agency, European Space Research and Technology Centre, Keplerlaan 1, 2200 AG Noordwijk, Th 12 European Space Agency, European Space Research and Technology Centre, Keplerlaan 1, 2200 AG Noordwijk, The Netherlands 13 D f A d A h i S i K k N i l U i i D 41566 R bli f K 12 European Space Agency, European Space Research and Technology Centre, Keplerlaan 1, 2200 AG Noordwijk, The Netherlands 13 Department of Astronomy and Atmospheric Sciences, Kyungpook National University, Daegu 41566, Republic of Korea 14 Astrophysics Research Institute, Liverpool John Moores University, IC2 Liverpool Science Park, 146 Brownlow Hill, L3 5RF Liverpool UK European Space Agency, European Space Research and Technology Centre, Keplerlaan 1, 2200 AG Noordwijk, The Netherlands 13 Department of Astronomy and Atmospheric Sciences, Kyungpook National University, Daegu 41566, Republic of Korea 14 Astrophysics Research Institute, Liverpool John Moores University, IC2 Liverpool Science Park, 146 Brownlow Hill, L3 5RF Liverpool, UK 13 Department of Astronomy and Atmospheric Sciences, Kyungpook National University, Daegu 41566, Republic of Korea 14 Astrophysics Research Institute, Liverpool John Moores University, IC2 Liverpool Science Park, 146 Brownlow Hill, L3 5RF Liverpool, UK p 15 Max Planck Institute for Astronomy, Königstuhl 17 69117, Germany 16 y g y 16 Institute of Cosmology and Gravitation, University of Portsmouth, Burnaby Road, Portsmouth PO1 3FX, UK 16 Institute of Cosmology and Gravitation, University of Portsmouth, Burnaby Road, Po 17 Institute of Cosmology and Gravitation, University of Portsmouth, Burnaby Road, Portsmouth PO1 3FX, UK 17 Observatorio Astronómico Nacional, C/Alfonso XII 3, Madrid 28014, Spain gy y y 17 Observatorio Astronómico Nacional, C/Alfonso XII 3, Madrid 28014, Spain Received 2 August 2022 / Accepted 14 November 2022 Received 2 August 2022 / Accepted 14 November 2022 Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics A&A 671, A8 (2023) https://doi.org/10.1051/0004-6361/202244667 c⃝The Authors 2023 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe to Open model. Subscribe to A&A to support open access publication. A8, page 1 of 17 A new method for age-dating the formation of bars in disc galaxies The TIMER view on NGC1433’s old bar and the inside-out growth of its nuclear disc Camila de Sá-Freitas1 , Francesca Fragkoudi1,2,3, Dimitri A. Gadotti1,4, Jesús Falcón-Barroso5,6, Adrian Bittner1,7, Patricia Sánchez-Blázquez8,9, Glenn van de Ven10, Rebekka Bieri3, Lodovico Coccato1, Paula Coelho11, Katja Fahrion12, Geraldo Gonçalves11, Taehyun Kim13, Adriana de Lorenzo-Cáceres5,6, Marie Martig14, Ignacio Martín-Navarro5,6, Jairo Mendez-Abreu5,6, Justus Neumann15,16, and Miguel Querejeta17 Camila de Sá-Freitas1 , Francesca Fragkoudi1,2,3, Dimitri A. Gadotti1,4, Jesús Falcón-Barroso5,6, Adrian Bittner1,7, Patricia Sánchez-Blázquez8,9, Glenn van de Ven10, Rebekka Bieri3, Lodovico Coccato1, Paula Coelho11, Katja Fahrion12, Geraldo Gonçalves11, Taehyun Kim13, Adriana de Lorenzo-Cáceres5,6, Marie Martig14, Ignacio Martín-Navarro5,6, Jairo Mendez-Abreu5,6, Justus Neumann15,16, and Miguel Querejeta17 1 European Southern Observatory, Karl-Schwarzschild-Str. 2, 85748 Garching bei Muenchen, Germany e-mail: camila.desafreitas@eso.org 2 1 European Southern Observatory, Karl-Schwarzschild-Str. 2, 85748 Garching bei Muenchen, Germany e-mail: camila.desafreitas@eso.org 2 Institute for Computational Cosmology, Department of Physics, Durham University, South Road, Durham DH1 3LE, UK 3 2 Institute for Computational Cosmology, Department of Physics, Durham University, South Road, Durham DH1 3LE, UK 3 Max-Planck-Institut für Astrophysik, Karl-Schwarzschild-Str. 1, 85748 Garching, Germany 4 C f E l i A D f Ph i D h U i i S h R d D h DH1 3LE UK p y g y 4 Centre for Extragalactic Astronomy, Department of Physics, Durham University, South Road, Durham DH1 3LE, U 5 Instituto de Astrofísica de Canarias, Calle Vía Láctea s/n, 38205 La Laguna, Tenerife, Spain 6 Departamento de Astrofísica Uni ersidad de La Lag na 38200 La Lag na Tenerife Spain 5 Instituto de Astrofísica de Canarias, Calle Vía Láctea s/n, 38205 La Laguna, Tenerife, Spain 7 Vyoma GmbH, Karl-Theodor-Str. 55, 80803 München, Germany 8 7 Vyoma GmbH, Karl-Theodor-Str. 1. Introduction Bars are common structures in the local Universe: many studies have found that between ∼30% and 70% of disc galaxies host bars (e.g., Eskridge et al. 2000; Menéndez-Delmestre et al. 2007; Barazza et al. 2008; Sheth et al. 2008; Aguerri et al. 2009; Nair & Abraham 2010; Buta et al. 2015; Erwin 2018). There- fore, understanding the time of their formation and how they aﬀect their host galaxies is key to understanding the late-stage evolution of galaxies themselves. monly referred to as pseudo-bulges or disc-like bulges. Simu- lations and theoretical studies ﬁnd that, after the bar forms, it only takes ∼108 yr to form the ND (e.g., Athanassoula 1992a,b; Emsellem et al. 2015; Seo et al. 2019; Baba & Kawata 2020). Thus, one can use the time of the formation of the ND – obtained through galactic archaeology – to time the age of the bar (Gadotti et al. 2015). The study by Gadotti et al. (2015) was oﬀered as a proof of concept, using NGC 4371, to demonstrate the feasibility of timing bar formation with archaeological evi- dence from the ND itself. However, the ND in NGC 4371 has no star formation and is mainly dominated by old stars, which makes it a special case for this type of analysis. More generally, with more complex SFHs, estimating stellar population proper- ties in the central region of disc galaxies is not a trivial task, since the observed light carries tangled information of the ND with the underlying main disc (MD) – and possibly other structural com- ponents that were already present when the ND formed. There- fore, in order to reliably detect the oldest stars in the ND, we need a way to disentangle the light of the ND from that of the underlying MD. g By investigating how galaxies evolve dynamically at dif- ferent redshifts, studies ﬁnd that a large fraction of galaxies at high redshift are rotationally supported (e.g., Shapiro et al. 2008; Schreiber et al. 2009; Epinat et al. 2012; Wisnioski et al. 2015; Rizzo et al. 2020; Lelli et al. 2021), with fractions vary- ing from 70−90% at z ∼1 and 47−74% at z ∼2. Using the James Webb Spatial Telescope. Early Release Observations of the galaxy cluster SMACS 0723, Ferreira et al. (2022) show that the fraction of disc galaxies in the early Universe is still an open question. 1. Introduction They ﬁnd that disc galaxies dominate the mor- phology at z ∼1.5 by more than a factor of ∼10 higher com- pared with previous results from the Hubble Space Telescope. Nevertheless, these rotationally supported discs at higher red- shifts often present higher velocity dispersions, suggesting they are turbulent, unsettled, and thick (e.g., Elmegreen & Elmegreen 2006; Cresci et al. 2009; Newman et al. 2013; although see also Rizzo et al. 2020). Since analytical and numerical work indicates that the bar can only form once the disc is dynamically set- tled, the moment of bar formation marks this transition epoch, during which the discs are at least partially dynamically cold (e.g., Kraljic et al. 2012 and references therein). Exactly how and when the switch from external to internal processes as the driver of galaxy evolution happens is still not clear, but it is associated with the settling of galactic discs. Therefore, timing the epoch of bar formation is a major step forwards in piecing together the diﬀerent phases of galaxy evolution. This is precisely the goal of this work, in which we present a new methodology for disentangling the light of the ND from other structures in the central region of the galaxy. This in turn enables us to derive the bar formation epoch. With the disen- tangled light, we derive independent SFHs for diﬀerent stellar structures in order to time the moment the ND formed and, therefore, when the bar formed. We selected NGC 1433, one of the galaxies in the Time Inference with MUSE in Extragalactic Rings (TIMER) survey (Gadotti et al. 2019), as a pilot galaxy to present this methodology, which will in the future be applied to all TIMER galaxies. This paper is organised as follows. In Sect. 2 we describe the data and explain the reason we selected NGC 1433 for this study. In Sect. 3 we describe the methodology that we developed to dis- entangle the SFH of the ND from that of the underlying MD, and how this can be used to obtain the bar age, as well as the tests of the methodology carried out using hydrodynamic simulations. In Sect. 4 we present our results for the age of the bar in NGC 1433 and the detailed buildup of its ND. In Sect. 5 we discuss the implications of our results in the context of galaxy evolution and the formation of NDs. 1. Introduction We summarise and conclude in Sect. 6. Additional tests of the methodology, including on two control galaxies, are presented in the appendix. Diﬀerent studies have attempted to time when the bar formed for a handful of galaxies using diﬀerent approaches. Gadotti et al. (2015) analysed stellar populations in the nuclear disc (ND) of NGC 4371 using high quality Multi Unit Spectroscopic Explorer (MUSE) data, and found a bar age of about 10 Gyrs. Pérez et al. (2017) used the formation of the boxy/peanut-shaped bulge, which originated from instabilities of the bar, to estimate the age of the bar in NGC 6032 as 10 Gyrs. de Lorenzo-Cáceres et al. (2019) analysed the star for- mation histories (SFHs) of nuclear structures, including inner bars, and ﬁnd that the inner bars in NGC 1291 and NGC 5850 must have formed at least 6.5 Gyrs and 4.5 Gyrs ago, respec- tively. These studies provide observational evidence that bars can be long-lived, which is in agreement with cosmological simula- tions that ﬁnd bars that formed between redshifts 1 and 2 and sur- vive down to z = 0 (e.g., Kraljic et al. 2012; Rosas-Guevara et al. 2020; Fragkoudi et al. 2020, 2021). 1. Introduction Law et al. 2009; Dekel et al. 2009; Oser et al. 2010). As the Uni- verse expands and interactions are less frequent, internal pro- cesses begin to play an important role in the evolution of galaxies (Kormendy & Kennicutt 2004). Constraining the processes that drive galaxy evolution in dif- ferent cosmic epochs is still a work in progress. At higher redshifts, external processes, such as mergers, galaxy inter- actions, and gas inﬂows, have an important impact on how galaxies evolve (e.g., Schreiber et al. 2006; Genzel et al. 2008; Among the most important internal drivers of the evo- lution of disc galaxies are stellar bars, which eﬃciently redistribute angular momentum, as well as both stars and C. de Sá-Freitas et al.: A&A 671, A8 (2023) gas (e.g., Lynden-Bell & Kalnajs 1972; Combes & Gerin 1985; Athanassoula 2003; Munoz-Tunón et al. 2004; Sheth et al. 2005; Romero-Gómez et al. 2007; Di Matteo et al. 2013; Halle et al. 2015; Fragkoudi et al. 2016, 2017). Bars have been linked to a global quenching of star formation in galaxies (e.g., Masters et al. 2012; Schawinski et al. 2014; Haywood et al. 2016; Géron et al. 2021) and are also responsible for inducing bursts of star formation in central regions (e.g., Ishizuki et al. 1990; Ellison et al. 2011; Coelho & Gadotti 2011). Bars are common structures in the local Universe: many studies have found that between ∼30% and 70% of disc galaxies host bars (e.g., Eskridge et al. 2000; Menéndez-Delmestre et al. 2007; Barazza et al. 2008; Sheth et al. 2008; Aguerri et al. 2009; Nair & Abraham 2010; Buta et al. 2015; Erwin 2018). There- fore, understanding the time of their formation and how they aﬀect their host galaxies is key to understanding the late-stage evolution of galaxies themselves. gas (e.g., Lynden-Bell & Kalnajs 1972; Combes & Gerin 1985; Athanassoula 2003; Munoz-Tunón et al. 2004; Sheth et al. 2005; Romero-Gómez et al. 2007; Di Matteo et al. 2013; Halle et al. 2015; Fragkoudi et al. 2016, 2017). Bars have been linked to a global quenching of star formation in galaxies (e.g., Masters et al. 2012; Schawinski et al. 2014; Haywood et al. 2016; Géron et al. 2021) and are also responsible for inducing bursts of star formation in central regions (e.g., Ishizuki et al. 1990; Ellison et al. 2011; Coelho & Gadotti 2011). A8, page 2 of 17 3.1. Building the underlying population contribution and disentangling the nuclear disc light Recent studies deriving radial proﬁles of stellar ages found that galaxies hosting NDs display a drop in average ages towards the centre. This drop in average ages is coincident with the radius of the peak in stellar v/σ (e.g., Falcón-Barroso et al. 2002; Bittner et al. 2020), which implies that these galaxies have a central stellar structure that is younger than the main under- lying population. This agrees with the scenario whereby NDs are structures formed by a relatively late gas inﬂow induced by stellar bars (e.g., Gadotti et al. 2015, 2019; Bittner et al. 2020). Hereafter, we consider the ‘underlying main disc’ to be every stellar population that was present before the formation of the ND. Since the observed light carries combined information of the younger ND and other underlying central structures, the ages derived for the central region of the galaxy can only be consid- ered as an upper limit to the mean stellar age of the ND. Secondly, we aim to mask spaxels dominated by active galactic nucleus (AGN) emission, based on the Baldwin, Phillips & Tervelich (BPT) diagram classiﬁcation (Baldwin et al. 1981), with AON ≥20. We used the emission lines Hα, Hβ, [OIII], and [NII] extracted by DAP, as described above, to build the BPT diagram in Fig. 4. In this section we describe a new strategy to build the under- lying MD and disentangle the light of it from that of the ND. In summary, this methodology consists of deriving a spectrum that represents the main underlying disc (hereafter referred to as the ‘representative spectrum’), using it to build a representa- tive MD data cube, and later subtract it from the observed data. We make two hypothesis: (1) the MD extend all the way to the centre, and its surface brightness proﬁle follows an exponential law and (2) the stellar populations of the MD do not change sig- niﬁcantly in the inner regions and, therefore, we can assume is similar to the spectrum extracted from an aperture surrounding the nuclear ring. The result from this subtraction is expected to be the isolated ND. A step-by-step outline and an illustration of our methodology are shown in Fig. 2. In the third step, we normalise the ﬂux in each spaxel of the representative ring to account for the radial ﬂux increase in the MD towards the centre. 3. Methodology In this section we describe our methodology step-by-step, as illustrated in Fig. 2. We describe how we create the underlying stellar population contribution (Sect. 3.1) and how we isolate the SFH of the ND (Sect. 3.2). In Sect. 3.3 we describe the details in our data analysis and our criterion for obtaining the time of bar formation. In Sect. 3.4 we test our methodology by applying it to a simulated barred galaxy. y g The ﬁrst step is to deﬁne the representative region from which we will extract the representative spectrum. This region is chosen as a ring surrounding the ND – from now on referred to as the ‘representative ring’. The representative ring is expected to be as close as possible to the ND without being contami- nated by its light. For galaxies hosting a ND, such as NGC 1433, we take into consideration the stellar v/σ radial proﬁle (Fig. 3). We deﬁne the inner radius of the representative ring as the ﬁrst minimum point outside the ND. That is the radius outside of which we expect the light from the MD to start dominating and, as expected, it is also the radius with the oldest mean age (see Fig. 3). Nevertheless, we tested diﬀerent positions for the repre- sentative ring to assess the systematic error linked to this deci- sion (see Appendix B.1). In addition, the representative ring has a width of 2′′, so one does not expect strong age and ﬂux gra- dients inside of it. Nonetheless, we performed tests with widths between 1′′ and 4′′ with little variation in the outcome. 2. Sample and data description In order to present the methodology we have developed – which separates the light of NDs from the underlying population – we select NGC 1433 for this pilot study, since it has a ND with similar properties to most NDs in the TIMER sample (e.g., Gadotti et al. 2020; Bittner et al. 2020 – which will be future tar- gets of study). Also, we ensure that our methodology does not produce artiﬁcial bar ages for unbarred galaxies by applying it to two control bar-less galaxies: NGC 1380 and NGC 1084 (see Appendix A). Buta et al. (2015) classiﬁed NGC 1433 morphologically as strongly barred with a nuclear ring/lens and a nuclear bar. In addition, the galaxy is at a distance of 10 Mpc, has an inclina- tion of 34◦, and stellar mass of 2 × 1010 M⊙(see references in Gadotti et al. 2019). Following Gadotti et al. (2020), the radius of the ND is deﬁned as the peak in the v/σ radial proﬁle, which is 440 pc (see Fig. 3). We used data from the TIMER project: the observations were carried out using the MUSE instrument at the European Southern Observatory Very Large Telescope One of the immediate eﬀects that follow bar formation – and the ensuing onset of tangential forces in the disc – is the gas inﬂow to the central parts of the galaxy. This gas inﬂow builds the rotation-supported structures known as NDs, which are com- A8, page 2 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) the data analysis pipeline (DAP) from the PHANGS-MUSE sur- vey (Emsellem et al. 2022) for the original data cube, deriving kinematic and line emission properties. DAP is a module-based pipeline based on the Galaxy IFU Spectroscopy Tool (GIST; Bittner et al. 2019) able to extract properties from data cubes such as kinematic information, emission lines ﬂuxes and more. For the emission lines ﬁtting, DAP uses pPXF (Cappellari 2012), considering emission lines as extra gaussian elements added to the stellar continuum. We measured the kinematic maps for Voronoi-binned (Cappellari & Copin 2003) data with signal to noise of 100 and emission lines spaxel-by-spaxel, between the wavelengths 4800 and 7000 Å, employing the E-MILES simple stellar population (SSP) model library (Vazdekis et al. 2016) to remove the stellar continuum. From the results, we have kine- matic properties such as stellar velocity and velocity dispersion and emission line ﬂuxes. 2. Sample and data description Applying the kinematic information, we shift all spectra to have a ﬁnal velocity of v = 0 km s−1, accounting for both the galaxy recession velocity and internal kinematics. In addition, we also convolved the original data to ensure that every spectra has σ = σmax, where the latter is the highest velocity dispersion in the central region of the origi- nal data cube. For NGC 1433, σmax is 122 km s−1. We point out that we veriﬁed that the convolved data and the original data result in the same stellar population, so such a procedure does not aﬀect our results. Our aim in doing so is to ensure we do not create artiﬁcial wings or artiﬁcial emission/absorption lines when subtracting the spectra (see below). Once the original data were shifted to v = 0 km s−1 and σ = σmax km s−1, we built our underlying MD. (ESO-VLT), in Period 97, from 2016 March to October, in Wide Field Mode. Considering the point spread function full width at half maximum, the spatial resolution of these observations is about 50 pc. Further details on the observations and data reduc- tion can be found in Gadotti et al. (2019). (ESO-VLT), in Period 97, from 2016 March to October, in Wide Field Mode. Considering the point spread function full width at half maximum, the spatial resolution of these observations is about 50 pc. Further details on the observations and data reduc- tion can be found in Gadotti et al. (2019). ( ) In Fig. 1 we display colour composites of our target along with maps of stellar kinematics and population properties: stel- lar velocity and velocity dispersion, the higher-order moments of the line of sight velocity distribution h3 and h4, stellar age, metallicity, and α enhancement, which were produced apply- ing the GIST pipeline (Bittner et al. 2019) to the TIMER MUSE data (more details on the following section). One can see that NGC 1433 has clear signatures of a ND, that is, a rapidly rotat- ing structure detached from the MD and coincident with a drop in velocity dispersion and mean ages. In addition, it also shows an anti-correlation between velocity and h3, and an increase in h4, as expected for NDs. These results are all in agreement with the ﬁndings in Gadotti et al. (2020) and Bittner et al. (2020), respectively. 3.1. Building the underlying population contribution and disentangling the nuclear disc light We normalised each spectra to r = 0 pc, assuming that the disc light proﬁle follows the exponential function I(r) = I0 × e−r/h, (1) I(r) = I0 × e−r/h, (1) where h is the disc scale-length, r is the distance of a given spaxel from the centre (corrected for inclination eﬀects), and I0 is the ﬂux at r = 0 pc. The value of h taken for NGC 1433 comes from Salo et al. (2015) and is 3100 pc. Considering we have I(r) for each spaxel in the representative ring, we divided Before building the MD, we treated the original data cube, taking the kinematic properties into consideration. We ﬁrst used A8, page 3 of 17 A8, page 3 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) ata and derived maps: The top two images are the colour composites of NGC 1433 from the Carnegie-Irvin 2011) and TIMER (top right; Gadotti et al. 2019). Below are maps that show (from left to right and top to b ulation properties derived from the TIMER data using GIST: stellar velocity velocity dispersion h3 h4 age m ig. 1. NGC1433 data and derived maps: The top two images are the colour composites of NGC 1433 from the Carnegie-Irvine Galaxy Survey op left; Ho et al. 2011) and TIMER (top right; Gadotti et al. 2019). Below are maps that show (from left to right and top to bottom) the stellar inematics and population properties derived from the TIMER data using GIST: stellar velocity, velocity dispersion, h3, h4, age, metallicity, and α nhancement. The ND radius is shown with a solid black contour and displays a faster rotation, a drop in velocity dispersion, an anti-correlation etween velocity and h3, and an increase in h4. In addition, the same region corresponds to a drop in mean age and α enhancement and an increase n metallicity. It is clear that NGC 1433 hosts a younger ND with more rotational support in the central region than the underlying population, in greement with Gadotti et al. (2020) and Bittner et al. (2020). Fig. 1. NGC1433 data and derived maps: The top two images are the colour composites of NGC 1433 from the Carnegie-Irvine Galaxy Survey (top left; Ho et al. 2011) and TIMER (top right; Gadotti et al. 2019). 3.1. Building the underlying population contribution and disentangling the nuclear disc light In light of this, we tested the implications of assuming an exponen- tial proﬁle in Appendix B.2, by applying the same methodology to a ﬂat light proﬁle MD. We show that the choice of a proﬁle for the main underlying population does not aﬀect the results noticeably. Next, we subtracted the reconstructed MD from the original data. As mentioned, in order to prevent the creation of artiﬁcial wings from the subtraction, we also used the original data con- volved and shifted to v = 0 km s−1 and σ = σmax km s−1. With that, we can disentangle our original data into the MD and the ND data cubes, as exempliﬁed in Fig. 5. Lastly, we collapse each data cube into a single mean spectra, deriving SFHs for each one. To assure that the collapsed spectra is the correct descrip- tion of the central region for the three data cubes, we masked contributions from the AGN in the centre. Using the BPT clas- siﬁcation (with AON ≥20) together with the light radial proﬁle (Fig. 4, right), we delimited a central region to be masked of around 140 pc (15 spaxels), since it is dominated by AGN emis- sion and can contaminate the total ﬂux of the ND. Nuclear Disc radius Representative ring Fig. 3. v/σ radial proﬁle, displayed in dashed red contours (values in the left-axis), and light-weighted average age, in solid blue contours (values in the right axis) for NGC 1433. We display the median values together with the ﬁrst and third quartiles. The vertical dotted black line marks the ND radius and the hatched area the representative ring. Note that the representative ring is placed in the ﬁrst v/σ minimum outside the ND, which matches the oldest mean age. Fig. 3. v/σ radial proﬁle, displayed in dashed red contours (values in the left-axis), and light-weighted average age, in solid blue contours (values in the right axis) for NGC 1433. We display the median values together with the ﬁrst and third quartiles. The vertical dotted black line marks the ND radius and the hatched area the representative ring. Note that the representative ring is placed in the ﬁrst v/σ minimum outside the ND, which matches the oldest mean age. Due to the high quality of the TIMER data (Gadotti et al. 2019), the decision to collapse the data cube is not motivated by the increase of signal-to-noise ratio. 3.1. Building the underlying population contribution and disentangling the nuclear disc light Nuclear Disc (ND) MUSE Original Main Disc (MD) Representative ring Representative ring close to the nuclear disc, with an inner radius in (v/σ)min Construction of main disc with exponential light profile Spaxel-by-spaxel kinematic corrections, with final velocity of and velocity dispersion of v = 0 σ = σmax Individual star formation histories Collapse of data-cubes into single spectra Representative ring Individual star formation histories Fig. 2. Illustration of the methodology described in Sect. 3 for a galaxy that hosts a ND. In the top left we highlight the ﬁeld of view from MUSE in the centre of the galaxy and the position of the representative region just outside the ND. The position of the representative ring is chosen taking the v/σ radial proﬁle into consideration. From it we derive the representative spectrum used to build the MD. In the bottom left we display the output from the light disentangling: the original data cube (red), the ND data cube (blue), and the representative MD data cube (green). All three data cubes have spaxels corrected to v = 0 km s−1 and σ = σmax km s−1. Lastly, we collapse each data cube into a mean spectrum (as illustrated) and derive SFHs for each one. The steps in our methodology are described to the right. 0 500 1000 1500 R [pc] 0.0 0.5 1.0 1.5 2.0 V/ NGC1433 Nuclear Disc radius Representative ring 4 6 8 Age [Gyrs] Fig. 3. v/σ radial proﬁle, displayed in dashed red contours (values in the left-axis), and light-weighted average age, in solid blue contours (values in the right axis) for NGC 1433. We display the median values together with the ﬁrst and third quartiles. The vertical dotted black line marks the ND radius and the hatched area the representative ring. Note that the representative ring is placed in the ﬁrst v/σ minimum outside the ND, which matches the oldest mean age. we extrapolated the representative spectrum back to a range of radii, again taking an exponential light proﬁle into account for the MD. Although the MD is often described with an expo- nential light proﬁle, recent studies show that discs may not fol- low an exponential light proﬁle all the way to the centre (e.g., Zhu et al. 2018; Breda et al. 2020; Papaderos et al. 2022). 3.1. Building the underlying population contribution and disentangling the nuclear disc light Below are maps that show (from left to right and top to bottom) the stellar kinematics and population properties derived from the TIMER data using GIST: stellar velocity, velocity dispersion, h3, h4, age, metallicity, and α enhancement. The ND radius is shown with a solid black contour and displays a faster rotation, a drop in velocity dispersion, an anti-correlation between velocity and h3, and an increase in h4. In addition, the same region corresponds to a drop in mean age and α enhancement and an increase in metallicity. It is clear that NGC 1433 hosts a younger ND with more rotational support in the central region than the underlying population, in agreement with Gadotti et al. (2020) and Bittner et al. (2020). A8, page 4 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Nuclear Disc (ND) MUSE Original Main Disc (MD) Representative ring close to the nuclear disc, with an inner radius in (v/σ)min Construction of main disc with exponential light profile Spaxel-by-spaxel kinematic corrections, with final velocity of and velocity dispersion of v = 0 σ = σmax Individual star formation histories Collapse of data-cubes into single spectra Representative ring Fig. 2. Illustration of the methodology described in Sect. 3 for a galaxy that hosts a ND. In the top left we highlight the ﬁeld of view from MUSE in the centre of the galaxy and the position of the representative region just outside the ND. The position of the representative ring is chosen taking the v/σ radial proﬁle into consideration. From it we derive the representative spectrum used to build the MD. In the bottom left we display the output from the light disentangling: the original data cube (red), the ND data cube (blue), and the representative MD data cube (green). All three data cubes have spaxels corrected to v = 0 km s−1 and σ = σmax km s−1. Lastly, we collapse each data cube into a mean spectrum (as illustrated) and derive SFHs for each one. The steps in our methodology are described to the right. 1 http://research.iac.es/proyecto/miles/pages/ predicted-masses-and-photometric-observables- based-on-photometric-libraries.php 3.2. Deriving star formation histories and mass assembly Foreachcollapsedspectrum–MUSEoriginal,MD,andcleanND data – we ran the GIST pipeline (Bittner et al. 2019) to derive stel- lar population properties and SFHs. To guarantee consistency in our analysis with previous TIMER work, we used the same GIST conﬁguration as in Bittner et al. (2020). Firstly, GIST employs an un-regularised run of pPXF (Cappellari & Emsellem 2004; Cappellari 2017) to derive stellar kinematic properties. We also include a low-order multiplicative Legendre polynomial in the ﬁt to account for small diﬀerences between the shape of the con- tinuum templates and the observed spectra. Next, GIST employs 3.1. Building the underlying population contribution and disentangling the nuclear disc light pyGandALF (see Bittner et al. 2019) to model emission lines as Gaussians, simultaneously ﬁtting the stellar continuum while the stellar kinematics remains ﬁxed from the previous step. With this, we obtain the emission-subtracted spectra. Lastly, GIST per- forms a regularised pPXF run in the emission-subtracted spectra, inordertoﬁtacombinationofstellarpopulationsandderive mean properties. Since metallicity and stellar velocity dispersion can both be responsible for absorption line broadening, causing pos- sible degeneracies (e.g., Sanchez-Blazquez et al. 2011), we keep the stellar kinematics ﬁxed from previous steps. In addition, to account for extinction and continuum mismatch eﬀects, we apply an eighth-order multiplicative Legendre polynomial in the ﬁt. For the last step, we employ the MILES library (Vazdekis et al. 2015), light-weighted, with [M/Fe] between −1 and +0.4, ages in the range 0.03–14 Gyr, and [α/Fe] enhancements of +0.0 and +0.4. Lastly, we use the regularisation error value of 0.15 derived for TIMER data by Bittner et al. (2020). As described in Cappellari (2017), the regularisation of the SFHs allows one to derive the smoothest and still physically meaningful result. To assess how much our ﬁnal bar age relies on the regularisation error, we tested diﬀerent values in Appendix B.4. For further details on the data analyses, we refer the reader to previous TIMER papers (e.g., Bittner et al. 2020). pyGandALF (see Bittner et al. 2019) to model emission lines as Gaussians, simultaneously ﬁtting the stellar continuum while the stellar kinematics remains ﬁxed from the previous step. With this, we obtain the emission-subtracted spectra. Lastly, GIST per- forms a regularised pPXF run in the emission-subtracted spectra, inordertoﬁtacombinationofstellarpopulationsandderive mean properties. Since metallicity and stellar velocity dispersion can both be responsible for absorption line broadening, causing pos- sible degeneracies (e.g., Sanchez-Blazquez et al. 2011), we keep the stellar kinematics ﬁxed from previous steps. In addition, to account for extinction and continuum mismatch eﬀects, we apply an eighth-order multiplicative Legendre polynomial in the ﬁt. For the last step, we employ the MILES library (Vazdekis et al. 2015), light-weighted, with [M/Fe] between −1 and +0.4, ages in the range 0.03–14 Gyr, and [α/Fe] enhancements of +0.0 and +0.4. Lastly, we use the regularisation error value of 0.15 derived for TIMER data by Bittner et al. (2020). As described in Cappellari (2017), the regularisation of the SFHs allows one to derive the smoothest and still physically meaningful result. NGC1433 -8 -4 0 4 8 [arcsec] MUSE Original -8 -4 0 4 8 [arcsec] -8 -4 0 4 8 [arcsec] Main Disc (MD) -8 -4 0 4 8 [arcsec] Nuclear Disc (ND) 0 6E+05 1E+06 2E+06 2E+06 3E+06 0 2E+04 4E+04 6E+04 8E+04 1E+05 0 6E+05 1E+06 2E+06 2E+06 3E+06 Flux [erg/s/cm2] NGC1433 -8 -4 0 4 8 [arcsec] -8 -4 0 4 8 [arcsec] Main Disc (MD) -8 -4 0 4 8 [arcsec] Nuclear Disc (ND) 0 2E+04 4E+04 6E+04 8E+04 1E+05 0 6E+05 1E+06 2E+06 2E+06 3E+06 Flux [erg/s/cm2] Fig. 5. Illustration of diﬀerent data products derived from the method- Fig. 5. Illustration of diﬀerent data products derived from the method- ology described in Sect. 3. We display the sum of the original MUSE ﬂuxes between 4800 and 5800 Å (top row), and the derived representa- tive MD (bottom row, left) and the ND data (bottom row, right), which is the result of subtracting the representative MD from the original data cube. For the representative MD, it is possible to notice the exponen- tial increase in ﬂux towards the centre. All data cubes were masked for AGN contributions with AON ≥20. pPXF also estimates diﬀerent weights for SSPs with diﬀerent ages, allowing us to build a non-parametric SFH. Each weight represents the fraction of the light formed in the diﬀerent SSPs. In order to convert the light-weighted SFHs to mass-weighted SFHs, we consider the distance to the galaxy to derive the intrin- sic luminosity. We then use the mass-to-light ratios1 predicted from the BaSTI isochrones (Pietrinferni et al. 2004, 2006, 2009, 2013) to convert luminosity into stellar mass. The mass-to-light ratios assume a Kroupa revised initial mass function (Kroupa 2001), and the MILES template library (Vazdekis et al. 2015) with [α/Fe] enhancements of +0.0 and +0.4. These mass-to-light ratios depend on the age, metallicity and [α/Fe] enhancement that best describe the observed data, and account for both stellar and remnants masses. non-collapsed data cube and the bar age is not strongly aﬀected (see Appendix B.3). 3.1. Building the underlying population contribution and disentangling the nuclear disc light Instead, we collapse the data cube to save computational time and guarantee low statistical errors. Nevertheless, we applied the same methodology for the it by e−r/h, extrapolating the observed ﬂux to the centre of the galaxy. We derived the representative spectrum as the mean ﬂux per wavelength of all spectra from the representative ring with v = 0 km s−1, σ = σmax km s−1 and the corresponding ﬂux at r = 0 pc. Finally, to re-construct a data cube of the MD, A8, page 5 of 17 A8, page 5 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Fig. 4. NGC1433 BPT classiﬁcation: Left: BPT diagram (Baldwin et al. 1981) classiﬁcation of each spaxel for the ND in NGC 1433. The BPT classiﬁcation is continued in the middle and right panels, which display the physical position of the diﬀerent spaxels and the light radial proﬁle, respectively. We also display the radius of the inner mask dominated by AGN contributions (dotted contours), the radius of the ND (solid contours), and the representative ring (dashed contours). Fig. 4. NGC1433 BPT classiﬁcation: Left: BPT diagram (Baldwin et al. 1981) classiﬁcation of each spaxel for the ND in NGC 1433. The BPT classiﬁcation is continued in the middle and right panels, which display the physical position of the diﬀerent spaxels and the light radial proﬁle, respectively. We also display the radius of the inner mask dominated by AGN contributions (dotted contours), the radius of the ND (solid contours), and the representative ring (dashed contours). -8 -4 0 4 8 [arcsec] MUSE Original -8 -4 0 4 8 [arcsec] -8 -4 0 4 8 [arcsec] Main Disc (MD) -8 -4 0 4 8 [arcsec] Nuclear Disc (ND) 0 6E+05 1E+06 2E+06 2E+06 3E+06 0 2E+04 4E+04 6E+04 8E+04 1E+05 0 6E+05 1E+06 2E+06 2E+06 3E+06 Flux [erg/s/cm2] NGC1433 Fig. 5. Illustration of diﬀerent data products derived from the method- ology described in Sect. 3. We display the sum of the original MUSE ﬂuxes between 4800 and 5800 Å (top row), and the derived representa- tive MD (bottom row, left) and the ND data (bottom row, right), which is the result of subtracting the representative MD from the original data cube. For the representative MD, it is possible to notice the exponen- tial increase in ﬂux towards the centre. All data cubes were masked for AGN contributions with AON ≥20. A8, page 6 of 17 3.1. Building the underlying population contribution and disentangling the nuclear disc light To assess how much our ﬁnal bar age relies on the regularisation error, we tested diﬀerent values in Appendix B.4. For further details on the data analyses, we refer the reader to previous TIMER papers (e.g., Bittner et al. 2020). 3.3. Age-dating bar formation c: age map of all the stars in the simulated galaxy. The grey box shows the inset that is represented in panel d. d: zoomed-in view around the ND region. The inner and outer dashed lines denote the region used to obtain the SFH of the ‘representative disc region’. The solid black line denotes the radius within which the SFH of the ND is estimated. The scatter points indicate the locations of the pixels used for deriving the SFHs in panel e. The points are colour-coded by the mean age in the pixel. e: the top panel shows the SFHs of the original ND region (solid red), of the representative SFH of the MD (dot-dashed green), and of the ND with the representative SFH subtracted (dashed blue). The bottom panel shows the ratio of the subtracted SFH to the representative SFH. The vertical orange line indicates the time of bar formation in the simulation (3 Gyr), and the vertical dashed line indicates the time at which ND/MD is above 1 (3.3 Gyr). the evolution of barred galaxies; Fragkoudi & Bieri, in prep.). The simulation has two collisionless components (a stellar disc and a dark matter halo) and a collisional component (gaseous disc), which is able to form stars that subsequently return mass, energy, and metals to the interstellar medium via supernova feed- back. We refer the reader to Appendix C for technical details of the simulation. original SFH characterised by an older star formation event fol- lowed by a later burst. Once we are able to disentangle the MD and the ND, we can expect the former’s SFH to map the oldest star formation event and the latter’s to map the youngest burst. p y g If we could perfectly disentangle the light of the ND from the underlying MD, one could simply use the ﬁrst peak in the ND SFH as the formation time of the ND and, consequently, the bar. However, due to gradients in the stellar population properties, the region around the ND that we use to obtain the SFH of the MD might not be identical to the real SFH of the main underlying disc within the ND region. Thus, it might not fully remove the contamination of the MD from the ND light. With that in mind, and by testing our methodology on N-body+hydrodynamic sim- ulations (see Sect. 2 In practice, this is done by sampling from Gaussian distributions at each radius, with a decreasing mean value for the age. 3.3. Age-dating bar formation Considering the scenario in which the ND is formed from a late gas inﬂow due to the bar, in an ideal case, one could expect the A8, page 6 of 17 a C. de Sá-Freitas et al.: A&A 671, A8 (2023) c d a b c d e Fig. 6. Testing the methodology using hydrodynamic simulations. a: face-on projection of the gas temperature. The dust lanes on the leading edge of the bar and the gaseous ND are clearly visible as low temperature regions. b: kinematic map showing the velocity of stars in the y direction with stellar isodensity contours over plotted, which outline the shape of the bar (note the diﬀerent scale compared to panel a). A highly rotating stellar component in the central kiloparsec (i.e. the ND) is clearly evident in the kinematic map. c: age map of all the stars in the simulated galaxy. The grey box shows the inset that is represented in panel d. d: zoomed-in view around the ND region. The inner and outer dashed lines denote the region used to obtain the SFH of the ‘representative disc region’. The solid black line denotes the radius within which the SFH of the ND is estimated. The scatter points indicate the locations of the pixels used for deriving the SFHs in panel e. The points are colour-coded by the mean age in the pixel. e: the top panel shows the SFHs of the original ND region (solid red), of the representative SFH of the MD (dot-dashed green), and of the ND with the representative SFH subtracted (dashed blue). The bottom panel shows the ratio of the subtracted SFH to the representative SFH. The vertical orange line indicates the time of bar formation in the simulation (3 Gyr), and the vertical dashed line indicates the time at which ND/MD is above 1 (3.3 Gyr). e e b d Fig. 6. Testing the methodology using hydrodynamic simulations. a: face-on projection of the gas temperature. The dust lanes on the leading edge of the bar and the gaseous ND are clearly visible as low temperature regions. b: kinematic map showing the velocity of stars in the y direction with stellar isodensity contours over plotted, which outline the shape of the bar (note the diﬀerent scale compared to panel a). A highly rotating stellar component in the central kiloparsec (i.e. the ND) is clearly evident in the kinematic map. 3.3. Age-dating bar formation 3.4), we employed a criterion to time the bar formation epoch as the moment when the ND dominates the star formation, as a signature of the bar bringing gas towards the cen- tre. That corresponds to the ﬁrst time in which the ratio between the star formation in the ND and that in the MD, ND/MD, rises above 1, with a positive slope towards younger ages. In order to verify whether this is a reliable criterion, we test our method- ology using an N-body+hydrodynamic simulation of a barred galaxy below. The simulation is evolved for a total of 3.3 Gyr. The axisym- metric stellar disc that is in place at the start of the simulation (which we refer to as the ‘old stellar component’) rapidly forms a bar after ∼0.3 Gyr (we deﬁne the bar as being fully formed when the m = 2 Fourier mode of the surface density, A2 > 0.3). During and after the formation of the bar, gas piles up at the lead- ing edges of the bar, where it shocks, loses angular momentum, and is funnelled to the centre, where it forms a dense gaseous ND (see panel (a) of Fig. 6), which proceeds to form stars. These new stars – formed out of gas pushed to the centre by the bar – form a highly rotating stellar ND (see panel (b) of Fig. 6), whose size is set by the bar orbits in the inner regions (see Athanassoula 1992b), similar to those observed in the local barred galaxies (e.g., Gadotti et al. 2020). We highlight that, while the new stars formed out of the gas have self-consistent ages – according to when they are formed in the simulation, the old stellar component can have any SFH we assign to it. In order to model the age gradient often found in galaxies, we assigned ages to the old stellar component at the initial snapshot such that a negative age gradient was obtained2 (i.e. with older stars in the centre and younger stars at the edge of 3.4. Testing the method using hydrodynamic simulations The lines are the SFHs derived from the data, while the shaded regions are results from 100 Monte Carlo runs. In the bottom panel we display ND/MD in black, and highlighted with an orange circle is the age where ND/MD rises above one for the ﬁrst time, together with the statistical uncertainty: +0.2 −0.5(stat) Gyr. that best ﬁts the observed spectra and can be understood as a ‘look-back time’ (i.e. t = 0 is the present time). We measure the bar formation epoch of NGC 1433 to occur 7.5+1.6 −1.1(sys)+0.2 −0.5(stat) Gyrs ago, corresponding to a redshift of z ≈1. In order to quantify the statistical error of the method- ology, we perform 100 Monte Carlo runs for each of the col- lapsed spectra (original data, ND data and representative spectra) to derive variations on the bar age. We use the noise information to sample a distribution of ﬂuxes for each wavelength, creating 100 artiﬁcial spectra. We then run pPXF on each of these spec- tra, to obtain the diﬀerent SFHs and the subsequent bar ages. This is shown in Fig. 7 as the shaded area of each SFH. From it, we derive a statistical uncertainty corresponding to +0.2 −0.5(stat) Gyr. The statistical error is subdominant, since, by collapsing the data cubes into single spectra, we achieve signal-to-noise values over 2000. To further quantify uncertainties in the derived bar age, which can be introduced due to various aspects of the methodol- ogy, we perform multiple tests with diﬀerent conﬁgurations (see Appendix B): diﬀerent locations for the representative ring, dif- ferent light proﬁles to describe the increase in density of the main underlying population towards the centre, and diﬀerent regular- isation errors for the pPXF run that results in the derivation of the SFHs. From these tests, we ﬁnd that there is a systematic uncertainty in our measurements of the bar age of the order of +1.6 −1.1(sys) Gyr, which we quote in addition to our statistical errors. p p y g g As can be seen from panel (e) of Fig. 6, this method allows us to recover the time at which the bar formed (which in the simulation occurs at tlookback ∼3 Gyr), which is marked with the vertical solid orange line, while the ratio of the ND over the representative MD SFH (ND/MD) gives a bar age of tlookback = 3.3 Gyr. 3.4. Testing the method using hydrodynamic simulations In order to test the robustness of the methodology developed here and some of the assumptions employed, we used an N-body and hydrodynamic simulation of an isolated Milky-Way- like disc galaxy, which forms a bar and a ND self-consistently (the simulation is part of a suite of models developed to study A8, page 7 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) the disc). This old underlying population, together with the new stars formed out of the gas in the simulation, give rise to the age map of the galaxy at the end of the simulation shown in panel (c) of Fig. 6. 0 5 10 15 103 M NGC1433 - 440 pc MUSE Original Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 4 10 1 102 ND / MD bar Age: 7.5 Gyrs 0 5 10 15 103 M NGC1433 - 440 pc MUSE Original Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 4 10 1 102 ND / MD bar Age: 7.5 Gyrs Fig. 7. Mass-weighted SFHs for NGC 1433 from collapsed spectra of the MUSE original data, ND data, and representative MD data, as illus- trated in Fig. 2. The y-axis corresponds to the mass formed, in solar masses, for each age bin and the x-axis to the age in Gyr, that is, the look-back time. The original data are shown with a solid red line, the subtracted data with a dashed blue line, and the representative spectrum with the dot-dashed green line. The lines are the SFHs derived from the data, while the shaded regions are results from 100 Monte Carlo runs. In the bottom panel we display ND/MD in black, and highlighted with an orange circle is the age where ND/MD rises above one for the ﬁrst time, together with the statistical uncertainty: +0.2 −0.5(stat) Gyr. g We can now extract the SFH for a given ‘pixel’ in the ND region – as we do in the observations – which will contain stars born from gas pushed to the centre by the bar, as well as old stars that were present before the bar formed. We also extract the SFH for pixels in the region just outside the ND, which give us the representative SFH of the underlying main stellar disc (MD). 3.4. Testing the method using hydrodynamic simulations Therefore, as in the methodology used for the observations, we can extract the SFH of both the ND region and the MD. We then subtract the SFH of the MD from the SFH in the ND region, in order to obtain the SFH of the ‘clean’ ND itself. These SFHs are shown in panel (e) of Fig. 6, with solid red for the original total SFH within the ND region, dot-dashed green for the SFH of the main underlying disc, and dashed blue lines for the clean ND SFH. This can be compared to the ‘true’ SFHs of the ND and MD, which are shown in the left panel of Fig. C.1. p g We ﬁnd that due to the age gradient in the underlying disc as well as the gradient in the light proﬁle, there can be a contami- nation of the ‘old component’ in the subtracted ND SFH, which cannot be fully removed by subtracting the underlying MD (see Fig. C.1, where we show how assuming a negative age gradient versus a ﬂat age gradient aﬀects the methodology). The extent to which there is ‘contamination’ by the oldest stars in the ND will depend on both how steep the age gradient is, and on the location of the ring used to obtain the SFH of the MD (see also Fig. C.2). This indicates that we cannot simply use the oldest peak in the subtracted SFH (ND) in order to obtain the time at which the ND formed, but we rather should use the comparison between the subtracted ND and the representative SFH of the MD (i.e. ND/MD). In practice, this is the time when the SFH of the ND increases above that of the MD (i.e. when ND/MD rises above 1), with a positive slope towards younger ages. Fig. 7. Mass-weighted SFHs for NGC 1433 from collapsed spectra of the MUSE original data, ND data, and representative MD data, as illus- trated in Fig. 2. The y-axis corresponds to the mass formed, in solar masses, for each age bin and the x-axis to the age in Gyr, that is, the look-back time. The original data are shown with a solid red line, the subtracted data with a dashed blue line, and the representative spectrum with the dot-dashed green line. 3.4. Testing the method using hydrodynamic simulations Therefore, we ﬁnd that, even with the contamination of older ages in the subtracted spectrum, we can recover the time of bar formation, as the ﬁrst time at which ND/MD rises above one, with an accuracy that will depend on the width of the age bins in the SFHs. The main limitation in obtaining the bar age using this methodology therefore stems from uncertainties in deriving stellar ages – and therefore the SFH – which are typically of the order of ∼1 Gyr (see e.g., Bittner et al. 2020). 5.1. The old bar in NGC 1433 where Σ0 is the mass density at the centre and h is the disc scale-length. Using the mass of the underlying MD within the ND radius, we measure the mass density as 512 M⊙pc−2. This value gives the extrapolated mass for the entire galaxy of 2.74 × 1010 M⊙, which is consistent with the total stellar mass of 2×1010 M⊙derived by Muñoz-Mateos et al. (2015), considering the uncertainties involved. This indicates that our measurements for the total stellar mass of the ND and the underlying main pop- ulation are reliable. In addition, this also shows that the method- ology described above to disentangle the light of the nuclear and underlying discs is trustworthy. Interestingly, our mass estimates indicate that the ND dominates the stellar mass budget in the central region, with the ND being ∼40% more massive than the underlying MD in the same region. y p As the bar evolves, the shape of its light proﬁle may change. Kim et al. (2015) suggested that the bar Sérsic index (nbar) evolves from an exponential (nbar ≈1−2) to a ﬂat (nbar ≈0.2) proﬁle (but see also Anderson et al. 2022). They also measured nbar for 144 local galaxies from the S4G sample and found that the oldest bars have a nbar index close to 0.2. In addition, sim- ulations demonstrate that, as the bar grows older, it becomes more elongated and stronger (e.g., Athanassoula et al. 2013), which can be measured by the bar length, Rbar, and the A2 index. The latter is the second component of the Fourier light/mass distribution in the galaxy and is associated with the bar non- axisymmetry. Lastly, the bar-to-total (Bar/T) luminosity ratio also increases as the bar grows longer and more massive, and the bar morphology evolves to a more rectangular or boxy shape (e.g., Kim et al. 2015). The bar morphology is measured by the index c, where c = 2.0 represents a perfect ellipse and c ≥2.7 a strongly boxy bar (see Gadotti 2011). In summary, an old bar is expected to have a ﬂat light proﬁle (nbar ≤0.7), high strength (A2 ≥0.4), a strongly boxy shape (c ≥2.7), relatively large bar-to-total ratio (Bar/T ≥0.09) and bar length normalised by the disc scale-length Rbar/h ≥1.5. 4. Results After following the methodology to disentangle SFHs described in Sect. 3, we present our results for our pilot study galaxy, NGC 1433. We remind the reader that the chosen criterion to time bar formation epoch is the ﬁrst moment when the ratio ND/MD increases above 1 with positive slope towards younger ages. 1.1 From the SFHs, we can derive estimates of the total stel- lar mass in the isolated ND and the underlying MD within the ND radius, by summing the mass formed through time fol- lowing each curve. However, if NGC 1433 has signiﬁcant age gradients within the central kiloparsec, the very oldest popu- lation in the underlying disc may still be partly present in the isolated ND, as discussed above. Therefore, these would be, Figure 7 shows our main results: the mass of stars formed with diﬀerent age bins of the stellar templates – analogous to SFHs – for the MUSE original (solid-red line and contour), the MD (dot dashed green line) and the ND (dashed blue line and contour) for NGC 1433, together with the ratio ND/MD in the bottom panel. The age is related to the SSP template combination A8, page 8 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) 0 5 10 Age [Gyrs] 0.00 0.05 0.10 0.15 fraction of mass Original data 0 5 10 Age [Gyrs] Nuclear disc data 0 82 164 246 328 411 r [pc] Fig. 8. SFHs for NGC 1433 in diﬀerent galactocentric radii for the original data (left) and the ND data (right). In the y-axis we display the fraction of mass formed. In the upper part of each panel we use arrows to display the mean age for each SFH, following the same colour coding with respect to the distance to the centre. For both data cubes, it is clear that the farther from the centre, the younger the SFH is (with the exception of the last curve), meaning that the fraction of younger stars increases. This is shown even more strikingly with the subtracted data cube: it is clear that the outskirts of the ND are in fact younger, in accordance with the inside-out growth scenario of Bittner et al. (2019). 0 5 10 Age [Gyrs] Nuclear disc data 0 82 164 246 328 411 r [pc] 0 5 10 Age [Gyrs] 0.00 0.05 0.10 0.15 fraction of mass Original data Fig. 8. 5.1. The old bar in NGC 1433 Apart from A2, these val- ues are the median values of the corresponding distributions In addition, we analyse the SFH in diﬀerent radial bins inside the ND region for the original data (left panel) and the ND cleaned data (right panel; Fig. 8). At the top of each panel we dis- play the mean age for each radius, colour-coded according to dis- tance from the centre. As one can see for the cleaned ND results, the SFH and the mean ages gradually get younger at larger radii, in agreement with the inside-out growth scenario (Bittner et al. 2020). We discuss the implications of these ﬁndings further in Sect. 5.2. 4. Results SFHs for NGC 1433 in diﬀerent galactocentric radii for the original data (left) and the ND data (right). In the y-axis we display the fraction of mass formed. In the upper part of each panel we use arrows to display the mean age for each SFH, following the same colour coding with respect to the distance to the centre. For both data cubes, it is clear that the farther from the centre, the younger the SFH is (with the exception of the last curve), meaning that the fraction of younger stars increases. This is shown even more strikingly with the subtracted data cube: it is clear that the outskirts of the ND are in fact younger, in accordance with the inside-out growth scenario of Bittner et al. (2019). 5.1. The old bar in NGC 1433 respectively, an upper limit to the mass of the ND, and a lower limit to the mass of the underlying population within the ND region. For NGC 1433, we measure the underlying MD mass within the ND radius as 2.95 × 108 M⊙and the ‘cleaned’ ND mass as 4.05 × 108 M⊙. To explore whether these values are in agreement with the literature, we extrapolate the underlying MD mass to obtain the total mass of the galaxy assuming an expo- nential function, following Eq. (2): Applying the methodology described in Sect. 3 and considering our chosen criterion, we ﬁnd a bar age of 7.5+1.6 −1.1(sys)+0.2 −0.5(stat) Gyr for NGC 1433, which hosts a ND with a radius of 440 pc (e.g., Gadotti et al. 2019; Bittner et al. 2020). This corresponds to a red- shift z ≈1 and is consistent with other observational studies, such as Simmons et al. (2014), who also ﬁnd massive galaxies with bars at z ≈2. In addition, Gadotti et al. (2015) and Pérez et al. (2017)alsoinferredsimilarbaragesforotherlocalgalaxies,using independent methodologies. Furthermore, this is comparable to predictions from cosmological simulations, which also ﬁnd bars that form between redshifts 1 −2 and that survive down to z = 0 (e.g., Kraljic et al. 2012; Fragkoudi et al. 2020). Our results indi- cate that NGC 1433 hosts an old and long-lived bar, which is in accordance with several observed characteristics of this galaxy and our current understanding of bar formation and bar-driven evolutionary processes. Mh = 2π Z ∞ 0 Σ(r)rdr = 2πΣ0h2, (2) (2) where Σ0 is the mass density at the centre and h is the disc scale-length. Using the mass of the underlying MD within the ND radius, we measure the mass density as 512 M⊙pc−2. This value gives the extrapolated mass for the entire galaxy of 2.74 × 1010 M⊙, which is consistent with the total stellar mass of 2×1010 M⊙derived by Muñoz-Mateos et al. (2015), considering the uncertainties involved. This indicates that our measurements for the total stellar mass of the ND and the underlying main pop- ulation are reliable. In addition, this also shows that the method- ology described above to disentangle the light of the nuclear and underlying discs is trustworthy. Interestingly, our mass estimates indicate that the ND dominates the stellar mass budget in the central region, with the ND being ∼40% more massive than the underlying MD in the same region. 5. Discussion In this section we discuss the results from applying our method- ology to the galaxy NGC 1433 and what they reveal about the formation and evolution of bars and NDs. A8, page 9 of 17 A8, page 9 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) as found in Gadotti (2011) for a sample of about 300 massive barred galaxies. sistent with the inside-out growth picture for NDs. This high- lights how inner structures such as NDs might be assembled in a self-similar way to the corresponding larger-scale structure of the MD. NGC 1433 has nbar = 0.3, A2 = 0.56, a deprojected, nor- malised bar length of 1.2, a boxy shape with c = 2.9, and lastly, a Bar/T = 0.08 (see Kim et al. 2014; Díaz-García et al. 2016). Most of these characteristics are consistent with the expecta- tion of an old bar, also in accordance with our bar age esti- mate of 7.5+1.6 −1.1(sys)+0.2 −0.5(stat) Gyr. Lastly, NGC 1433 has similar characteristics as NGC 4371, which was studied by Gadotti et al. (2015) also using the archaeological evidence present on its ND. The authors investigated the oldest stars in the ND to estimate a bar age of ≈10 Gyr. In addition, NGC 4371 has nbar = 0.2, A2 = 0.62, deprojected normalised bar length of 1.3, c = 2.7, and Bar/T = 0.08 (see Gadotti et al. 2015 and Díaz-García et al. 2016). Both the age and the characteristics of NGC 1433 and NGC 4371 are very consistent with each other and with the sce- nario whereby they are amongst the ﬁrst galaxies to form bars. Finally, we note that Fig. 8 shows a prominent peak in the SFHs at all radii at young ages (∼2.5 Gyr), which is also evident in Fig. 7: there is a late burst of star formation, which occurs at younger ages than the ﬁrst burst associated with the formation of the ND at ∼7.5 Gyr. This implies an event that leads to a renewed inﬂow of gas at late times, which gives rise to such a burst of star formation. 6. Summary and concluding remarks In this study we have developed and presented a new method for dating the bar formation epoch of observed disc galaxies. We summarise this work as follows: – We present a new methodology that allows us to disentan- gle the light from NDs, which are formed by the bar, from the underlying MDs of galaxies, using high-resolution inte- gral ﬁeld spectroscopic data from MUSE on the VLT. This allows us to ﬁnd the time at which the ND formed, by iso- lating the moment when its star formation starts to dominate over the star formation in the underlying population. As NDs are formed due to bar-driven inﬂow – which is concurrent with the formation time of the bar – this allows us to deter- mine the age of the bar. – We performed a number of tests of our methodology, both on observed data and on a hydrodynamic simulation of a barred galaxy (which self-consistently forms a ND), in order to val- idate the robustness of the methodology. – As a pilot study, we applied our methodology to the barred galaxy NGC 1433 from the TIMER survey (Gadotti et al. 2019) and ﬁnd a bar age of 7.5+1.6 −1.1(sys)+0.2 −0.5(stat) Gyr. This implies that NGC 1433 has an old bar that formed around z ∼1. This aligns with a number of observational character- istics of the galaxy, such as its mass, bar strength, and light proﬁle (see Sect. 5.1 for a more detailed discussion on this). Our results are consistent with other studies in the literature that ﬁnd old bars (e.g., Simmons et al. 2014; Gadotti et al. 2015), as well as with results from cosmological simulations (e.g., Kraljic et al. 2012; Fragkoudi et al. 2020) that imply that bars can be old, long-lived structures. 5. Discussion Various mechanisms could give rise to such a late gas inﬂow event through the bar, such as mechanisms that remove angular momentum from gas, for example an interaction or ﬂyby, or – interestingly for this galaxy – this late inﬂow could be related to the buckling of the bar and the formation of the boxy/peanut bulge (e.g., Pérez et al. 2017). The conditions to form a bar are connected with the MD properties, and depend on the galaxy assembling enough mass and settling in more ordered motion. With this in mind, one can expect that the more massive galaxies will reach the necessary conditions ﬁrst, following a downsizing picture (e.g., Sheth et al. 2012). Despite that, another plausible possibility is the one where early interactions and/or minor mergers, which happened after the disc has settled, could have triggered the bar formation (e.g., Noguchi 1987; Gerin et al. 1989; Miwa & Noguchi 1998; Peschken & Łokas 2019; Łokas 2021). Gadotti et al. (2015) argued how this could be possible for NGC 4371, which is mem- ber of the Virgo cluster. Similarly, NGC 1433 is part of the Dorado group (Maia et al. 1989). Following the pre-processing picture, galaxies enter clusters with already some level of pro- cessing due to earlier interactions (Haines et al. 2015) while in groups, which could also be responsible for early bar forma- tion ∼7.5 Gyrs ago. Méndez-Abreu et al. (2010, 2012) found that galaxies in the Coma and Virgo clusters that host bars are mainly massive (109 ≤M/M⊙≤1011). 5.2. The inside-out scenario for the growth of nuclear discs Bittner et al. (2020) showed that in the TIMER sample, the derived age proﬁles of NDs follow a negative gradient, with the outer parts of the ND being younger. This is in accordance with an inside-out growth scenario, in which one can have a grow- ing gaseous ND that forms stars, and/or star formation concen- trated in rings of increasing size, on the external borders of the ND, as shown by Hα maps (Bittner et al. 2020). In this scenario, the gas funnelled by the bar towards the centre piles up near the inner Lindblad resonance of the bar. As the bar grows longer, the radius of the inner Lindblad resonance increases, which leads to the buildup of a ND in an inside-out fashion. – By examining the SFH of the ND of NGC 1433 at diﬀerent radii, we ﬁnd that the disc grows inside out, with younger stars forming at progressively larger radii and the youngest stars forming at the edge of the ND (i.e. at the location of the nuclear ring). This is in agreement with an inside- out growth scenario for NDs (see Bittner et al. 2020, and Sect. 5.2). In order to test this scenario, we analyse the SFHs of the subtracted ND in diﬀerent radial bins, as shown in Fig. 8, and derive the mean ages at each radius (shown as arrows on the top part of the panels). For the original data, the inside-out evidence is subtle, with little change in the mean ages in each radial bin. Nonetheless, with the subtracted data, the inside-out growth of the disc becomes strikingly evident, since in the outer part of the ND the older stellar populations are almost completely absent from the clean ND. This is reﬂected in the mean age at diﬀer- ent radii for the subtracted ND, in which there is a clear gradient towards younger ages at larger radii. This result is also testament that the methodology we develop to subtract the underlying disc component is robust. It shows that the ND of NGC 1433 is con- We will apply the methodology presented here to the full TIMER sample of barred galaxies, which will provide, for the ﬁrst time, robust age determinations for bars in a sizeable sample of disc galaxies. 5.2. The inside-out scenario for the growth of nuclear discs This will enable us to compare the age of bars with var- ious galaxy properties, such as the total mass, the bar length, and the pattern speed, enabling us to place important constraints on the evolution of dynamical properties of disc galaxies with time, the epoch of disc settling, and the eﬀects of bar-driven evolution. A8, page 10 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Acknowledgements. We thank the anonymous referee for the constructive report. This work was supported by STFC grant number ST/T000244/1. Based on observations collected at the European Southern Observatory under ESO pro- grammes 296.B-5054, 097.B-0640 and 099.B-0242. Raw and reduced data are available at the ESO Science Archive Facility. A.d.L.C. acknowledges ﬁnancial support from the Spanish Ministry of Science and Innovation (MICINN) through the Spanish State Research Agency, under Severo Ochoa Centres of Excel- lence Programme 2020-2023 (CEX2019-000920-S). K.F. acknowledges support through the ESA research fellowship program. P.C. acknowledges support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2021/08813- 7) and Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq, 310555/2021-3). G.G. acknowledges support from Coordenação de Aperfeiçoa- mento de Pessoal de Nível Superior (CAPES). T.K. was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grants (No. 2019R1I1A3A02062242) funded by the Ministry of Edu- cation, grants (No. 2022R1A4A3031306) funded by the Korean government (MSIT), and grants (WISET 2021-541) funded by the Korea Foundation for Women In Science, Engineering and Technology. J.M.A. acknowledges the sup- port of the Viera y Clavijo Senior program funded by ACIISI and ULL. P.S.B. acknowledges ﬁnancial support from the Spanish Ministry of Science, Inno- vation and Universities (MCIUN) under grant number PID2019-107427GB- C31. G.v.d.V. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation pro- gramme under grant agreement No 724857 (Consolidator Grant ArcheoDyn). J.N. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 694343) and the Science and Technology Facilities for support through the Consolidated Grant Cosmology and Astrophysics at Portsmouth, ST/S000550/1. Gadotti, D. A., Bittner, A., Falcón-Barroso, J., et al. 2020, A&A, 643, A14 Gao, H., Ho, L. C., Barth, A. J., & Li, Z.-Y. 2019, ApJ, 244, 34 Genzel, R., Burkert, A., Bouché, N., et al. 2900 Kormendy, J., & Kennicutt, R. C. Jr. 2004, ARA&A, 42, 603 Kraljic, K., Bournaud, F., & Martig, M. 2012, ApJ, 757, 60 Kroupa, P. 2001, MNRAS, 322, 231 p Law, D. R., Steidel, C. C., Erb, D. K., et al. 2009, ApJ, 697, 2057 L lli F Di T d E M F li F l 2021 S i 371 71 Law, D. R., Steidel, C. C., Erb, D. K., et al. 2009, ApJ, 697, 2057 Lelli, F., Di Teodoro, E. M., Fraternali, F., et al. 2021, Science, 371, 713 Łokas, E. L. 2021, A&A, 647, A143 Lynden-Bell, D., & Kalnajs, A. 1972, MNRAS, 157, 1 Lynden-Bell, D., & Kalnajs, A. 1972, MNRAS, 157, 1 Lynden-Bell, D., & Kalnajs, A. 1972, MNRAS, 157, 1 Maia, M., Da Costa, L., & Latham, D. W. 1989, ApJS, 69, 809 Maia, M., Da Costa, L., & Latham, D. W. 1989, ApJS, 69, 809 Masters, K. L., Nichol, R. C., Haynes, M. P., et al. 2012, MNRAS, 424, 2180 McDermid, R. M., Alatalo, K., Blitz, L., et al. 2015, MNRAS, 448, 3484 ez-Abreu, J., Sánchez-Janssen, R., & Aguerri, J. 2010, ApJ, 711, L Méndez-Abreu, J., Sánchez-Janssen, R., Aguerri, J., Corsini, E., & Zarattini, S. 2012, ApJ, 761, L6 References Menéndez-Delmestre, K., Sheth, K., Schinnerer, E., Jarrett, T. H., & Scoville, N. Z. 2007, ApJ, 657, 790 Miwa, T., & Noguchi, M. 1998, ApJ, 499, 149 Aguerri, J., Méndez-Abreu, J., & Corsini, E. 2009, A&A, 495, 491 Aguerri, J., Méndez-Abreu, J., & Corsini, E. 2009, A&A, 495, 491 Muñoz-Mateos, J. C., Sheth, K., Regan, M., et al. 2015, ApJS, 21 Anderson, S. R., Debattista, V. P., Erwin, P., et al. 2022, MNRAS, 513, 1642 Athanassoula, E. 1992a, MNRAS, 259, 328 Anderson, S. R., Debattista, V. P., Erwin, P., et al. Athanassoula, E. 1992a, MNRAS, 259, 328 Athanassoula, E. 1992a, MNRAS, 259, 328 Munoz-Tunón, C., Caon, N., & Aguerri, J. A. L. 2004 Munoz-Tunón, C., Caon, N., & Aguerri, J. A. L. 2004, AJ, 127, 58 , , , , Athanassoula, E. 1992b, MNRAS, 259, 345 Athanassoula, E. 1992b, MNRAS, 259, 345 Nair, P. B., & Abraham, R. G. 2010, ApJ, 714, L260 Nair, P. B., & Abraham, R. G. 2010, ApJ, 714, L260 , , , , p , , Navarro, J. F., Frenk, C. S., & White, S. D. M. 1997, ApJ, 490, 493 Athanassoula, L. 2003, Galaxies and Chaos (Springer), 313 Navarro, J. F., Frenk, C. S., & White, S. D. M. 1997, ApJ, 4 Navarro, J. F., Frenk, C. S., & White, S. D. M. 1997, ApJ, 490, 493 Newman, S. F., Genzel, R., Schreiber, N. M. F., et al. 2013, ApJ, 767, 104 Noguchi M 1987 MNRAS 228 635 Newman, S. F., Genzel, R., Schreiber, N. M. F., et al. 2013, ApJ, 767, 104 Athanassoula, E., Machado, R. E., & Rodionov, S. 2013, MNRAS, 429, 1949 Athanassoula, E., Machado, R. E., & Rodionov, S. 2013, M Noguchi, M. 1987, MNRAS, 228, 635 Noguchi, M. 1987, MNRAS, 228, 635 Baba, J., & Kawata, D. 2020, MNRAS, 492, 4500 Baba, J., & Kawata, D. 2020, MNRAS, 492, 4500 Baldwin, J. A., Phillips, M. M., & Terlevich, R. 1981, PASP, 93, 5 Oser, L., Ostriker, J. P., Naab, T., Johansson, P. H., & Burkert, A. 2010, ApJ, 725, 2312 Barazza, F. D., Jogee, S., & Marinova, I. 2008, ApJ, 675, 1194 Papaderos, P., Breda, I., Humphrey, A., et al. 2022, A&A, 658, A74 Bittner, A., Falcón-Barroso, J., Nedelchev, B., et al. 2019, A&A, 628, A117 Bittner, A., Sánchez-Blázquez, P., Gadotti, D. A., et al. 2020, A&A, 643, A65 Pérez, I., Martínez-Valpuesta, I., Ruiz-Lara, T., et al. References 2017, MNRAS, 470, L122 Breda, I., Papaderos, P., & Gomes, J.-M. 2020, A&A, 640, A20 Perret, V. 2016, Astrophysics Source Code Library [record ascl:1607.002] p Buta, R. J., Sheth, K., Athanassoula, E., et al. 2015, ApJS, 217, 32 R. J., Sheth, K., Athanassoula, E., et al. 2015, ApJS, 217, 32 Cappellari, M. 2012, Astrophysics Source Code Library [record ascl:1210.002] Perret, V., Renaud, F., Epinat, B., et al. 2014, A&A, 562, A1 pp p y Cappellari, M. 2017, MNRAS, 466, 798 Peschken, N., & Łokas, E. L. 2019, MNRAS, 483, 2721 Pietrinferni, A., Cassisi, S., Salaris, M., & Castelli, F. 2004, ApJ, 612, 168 Cappellari, M., & Copin, Y. 2003, MNRAS, 342, 345 Cappellari, M., & Emsellem, E. 2004, PASP, 116, 138 Pietrinferni, A., Cassisi, S., Salaris, M., & Castelli, F. 2006, ApJ, 642, 797 Coelho, P., & Gadotti, D. A. 2011, ApJ, 743, L13 Pietrinferni, A., Cassisi, S., Salaris, M., Percival, S., & Ferguson, J. W. 2009, ApJ, 697, 275 Combes, F., & Gerin, M. 1985, A&A, 150, 327 Pietrinferni, A., Cassisi, S., Salaris, M., & Hidalgo, S. 2013, A&A, 558, A46 Cresci, G., Hicks, E., Genzel, R., et al. 2009, ApJ, 697, 115 Rizzo, F., Vegetti, S., Powell, D., et al. 2020, Nature, 584, 201 Dekel, A., Sari, R., & Ceverino, D. 2009, ApJ, 7 de Lorenzo-Cáceres, A., Sánchez-Blázquez, P., Méndez-Abreu, J., et al. 2019, MNRAS 484 5296 de Lorenzo-Cáceres, A., Sánchez-Blázquez, P., Méndez-Abreu, J., et al. 2019, MNRAS, 484, 5296 Romero-Gómez, M., Athanassoula, E., Masdemont, J. J., & García-Gómez, C. 2007, A&A, 472, 63 p Erwin, P. 2018, MNRAS, 474, 5372 p Erwin, P. 2018, MNRAS, 474, 5372 Shapiro, K. L., Genzel, R., Schreiber, N. M. F., et al. 2008 Eskridge, P. B., Frogel, J. A., Pogge, R. W., et al. 2000, AJ, 119, 536 Sheth, K., Vogel, S. N., Regan, M. W., Thornley, M. D., & Teuben, P. J. 2005, ApJ, 632, 217 g , , g , , gg , , , , , Falcón-Barroso, J., Peletier, R. F., & Balcells, M. 2002, MNRAS, 335, 74 p Sheth, K., Elmegreen, D. M., Elmegreen, B. G., et al. 2008, ApJ, 675, 1141 Sheth, K., Elmegreen, D. M., Elmegreen, B. G., et al. 2008, ApJ, 675, 1141 Sheth K Melbourne J Elmegreen D M et al 2012 ApJ 758 136 Ferreira, L., Adams, N., Conselice, C. J., et al. 2022, ApJ, 938, L2 Sheth, K., Elmegreen, D. M., Elmegreen, B. G., et al. 2008, ApJ, 675, 1141 Sheth, K., Melbourne, J., Elmegreen, D. M., et al. 2012, ApJ, 758, 136 Fragkoudi, F., Athanassoula, E., & Bosma, A. 2016, MNRAS, 462, L41 Sheth, K., Melbourne, J., Elmegreen, D. M., et al. 2012, ApJ, 758, 136 Sheth, K., Melbourne, J., Elmegreen, D. M., et al. 2012, ApJ, 758, 136 Simmons, B. D., Melvin, T., Lintott, C., et al. 2014, MNRAS, 445, 3466 T i R 2002 A&A 385 337 g Fragkoudi, F., Di Matteo, P., Haywood, M., et al. 2017, A&A, 606, A47 Simmons, B. D., Melvin, T., Lintott, C., et al. 2014, MNRAS, 445, 3466 Simmons, B. D., Melvin, T., Lintott, C., et al. 2014, MNRAS, 445, 3466 Teyssier, R. 2002, A&A, 385, 337 Fragkoudi, F., Grand, R. J. J., Pakmor, R., et al. 2020, MNRAS, 494, 5936 Teyssier, R. 2002, A&A, 385, 337 Vazdekis, A., Coelho, P., Cassisi, S., et al. 2015, MNRAS, 449, 1177 Fragkoudi, F., Grand, R. J. J., Pakmor, R., et al. 2021, A&A, 650, L16 Gadotti, D. A. 2011, MNRAS, 415, 3308 g , , , , , , Gadotti, D. A. 2011, MNRAS, 415, 3308 Gadotti, D. A. 2011, MNRAS, 415, 3308 Vazdekis, A., Koleva, M., Ricciardelli, E., Röck, B., & Falcón-Barroso, J. 2016, MNRAS, 463, 3409 Gadotti, D. A., Seidel, M. K., Sánchez-Blázquez, P., et al. 2015, A&A, 584, A90 Wisnioski, E., Schreiber, N. F., Wuyts, S., et al. 2015, ApJ, 799, 209 Zhu, L., van de Ven, G., Méndez-Abreu, J., & Obreja, A. 2018, MNRAS, 479, 945 Gadotti, D. 5.2. The inside-out scenario for the growth of nuclear discs 2008, ApJ, 687, 59 Gerin, M., Combes, F., & Athanassoula, E. 1989, Dyn. Astrophys. Discs, 219 Gerin, M., Combes, F., & Athanassoula, E. 1989, Dyn. Astrophys. Discs, 219, 111 111 Géron, T., Smethurst, R. J., Lintott, C., et al. 2021, MNRAS, 507, 4389 Haines, C., Pereira, M., Smith, G. P., et al. 2015, ApJ, 806, 101 Halle, A., Di Matteo, P., Haywood, M., & Combes, F. 2015, A&A, 578, A58 Haywood, M., Lehnert, M., Di Matteo, P., et al. 2016, A&A, 589, A66 H L C Li Z Y B th A J S i M S & P C Y 2011 A JS 197 Ho, L. C., Li, Z.-Y., Barth, A. J., Seigar, M. S., & Peng, C. Y. 2011, ApJS, 197, 21 21 Ishizuki, S., Kawabe, R., Ishiguro, M., et al. 1990, Nature, 344, 224 Kim T Gadotti D A Sheth K et al 2014 ApJ 782 64 21 Ishizuki, S., Kawabe, R., Ishiguro, M., et al. 1990, Nature, 344, 224 Kim, T., Gadotti, D. A., Sheth, K., et al. 2014, ApJ, 782, 64 Kim, T., Sheth, K., Gadotti, D. A., et al. 2015, ApJ, 799, 99 Kimm, T., & Cen, R. 2014, ApJ, 788, 121 Ishizuki, S., Kawabe, R., Ishiguro, M., et al. 1990, Nature, 344, 224 Ki T G d i D A Sh h K l 2014 A J 782 64 Kim, T., Gadotti, D. A., Sheth, K., et al. 2014, ApJ, 782, 64 Kim, T., Gadotti, D. A., Sheth, K., et al. 2014, ApJ, 782, 64 Kim, T., Sheth, K., Gadotti, D. A., et al. 2015, ApJ, 799, 99 Kimm, T., & Cen, R. 2014, ApJ, 788, 121 Kim, T., Sheth, K., Gadotti, D. A., et al. 2015, ApJ, 799, 99 Kim, T., Sheth, K., Gadotti, D. A., et al. 2015, ApJ, 799, 99 Kimm, T., & Cen, R. 2014, ApJ, 788, 121 Kimm, T., & Cen, R. 2014, ApJ, 788, 121 Kimm, T., Cen, R., Devriendt, J., Dubois, Y., & Slyz, A. 2015, MNRAS, 451, 2900 Kimm, T., Cen, R., Devriendt, J., Dubois, Y Kimm, T., Cen, R., Devriendt, J., Dubois, Y., & Slyz, A. 2015, MNRAS, 451, 2900 MNRAS, 484, 5296 Díaz-García, S., Salo, H., Laurikainen, E., & Herrera-Endoqui, M. 2016, A&A, 587, A160 Rosas-Guevara, Y., Bonoli, S., Dotti, M., et al. 2020, MNRAS, 491, 25 Salo, H., Laurikainen, E., Laine, J., et al. 2015, ApJS, 219, 4 Di Matteo, P., Haywood, M., Combes, F., Semelin, B., & Snaith, O. N. 2013, A&A, 553, A102 Sanchez-Blazquez, P., Ocvirk, P., Gibson, B. K., Pérez, I., & Peletier, R. F. 2011, MNRAS, 415, 709 Ellison, S. L., Nair, P., Patton, D. R., et al. 2011, MNRAS, 416, 2182 Sarzi, M., Iodice, E., Coccato, L., et al. 2018, A&A, 616, A121 Elmegreen, B. G., & Elmegreen, D. M. 2006, ApJ, 650, 644 Schawinski, K., Urry, C. M., Simmons, B. D., et al. 2014, MNRAS, 440, 8 g g p Emsellem, E., Renaud, F., Bournaud, F., et al. 2015, MNRAS, 446, 2468 y Schreiber, N. F., Genzel, R., Lehnert, M., et al. 2006, ApJ, 645, 1062 Emsellem, E., Schinnerer, E., Santoro, F., et al. 2022, A&A, 659, A191 Schreiber, N. F., Genzel, R., Bouché, N., et al. 2009, ApJ, 706, 1364 p Seo, W.-Y., Kim, W.-T., Kwak, S., et al. 2019, ApJ, 872, 5 Epinat, B., Tasca, L., Amram, P., et al. 2012, A&A, 539, A92 Seo, W.-Y., Kim, W.-T., Kwak, S., et al. 2019, ApJ, 872, 5 p Shapiro, K. L., Genzel, R., Schreiber, N. M. F., et al. 2008, ApJ, 682, 231 B.1. The location of the representative spectrum Ideally, the representative ring should be located immediately after the end of the ND so that it is not contaminated by light from it and represents, as closely as possible, the underlying population in the region where the ND dominates. However, the end of the ND is not trivial to pinpoint, as this structure may gradually fade into the MD. If the region is too close to the ND, it may be aﬀected by its young star formation, but if it is too far, it may not map the underlying MD old star forma- tion. Our methodology used the physical justiﬁcation based on kinematic maps to select the representative region. Nevertheless, to constrain how much the position of the representative ring could aﬀect our ﬁnal result, we produce a number of tests with the representative ring at diﬀerent positions closer to the cen- tre than the position we employed. The results are summarised in Fig. B.1. We note that the mask used that the mask used for the representative ring of Fig. B.1 is slightly diﬀerent than the one used in the main result. For this case, we masked all spaxels with AGN contribution, with no threshold for AON (as oppo- site of AON ≥20 used for the main result). With this, the bar age is slightly older, 8 Gyrs (Fig. B.1, last row, center). With this test we aim to assess the relative systematic error due to the location of the representative ring, therefore this does not aﬀect the conclusions. We further note that the change in bar age due to the diﬀerent mask is well within the systematic error uncertainties. Fig. A.1. Same as Fig. 7 but for NGC 1380 (top) and NGC 1084 (bot- tom). It is clear that for both galaxies neither the representative MD nor the ‘cleaned’ data show important deviations in their SFHs. In order to assess whether our methodology creates spurious results for galaxies that do not host a ND, we applied the same methodology for two control galaxies: NGC 1380 and NGC 1084. In order to assess whether our methodology creates spurious results for galaxies that do not host a ND, we applied the same methodology for two control galaxies: NGC 1380 and NGC 1084. Considering NGC 1380, Gao et al. (2019) describe the galaxy as an inclined system with a classical bulge and no clear presence of a bar. p Erwin, P. 2018, MNRAS, 474, 5372 A., Sánchez-Blázquez, P., Falcón-Barroso, J., et al. 2019, MNRAS, 482, 506 A8, page 11 of 17 A8, page 11 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) archive, PI: Carollo, C. M., programme ID 099.B-0242, using MUSE in Wide Field Mode. Appendix A: Control galaxies: NGC 1380 and NGC 1084 2 8 16 105 M NGC1380 Original data Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 2 100 102 ND / MD 0 20 40 60 103 M NGC1084 Original data Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 2 100 102 ND / MD Fig. A.1. Same as Fig. 7 but for NGC 1380 (top) and NGC 1084 (bot- tom). It is clear that for both galaxies neither the representative MD nor the ‘cleaned’ data show important deviations in their SFHs. Figure A.1 shows the outcome of these tests. It is clear that the subtracted spectra show SFHs similar to those in the original spectra. Therefore, our methodology does not artiﬁcially pro- duce diﬀerences in the SFHs of the regions where the nuclear and main underlying discs dominate, which would wrongly be attributed to the formation of the bar. 2 8 16 105 M NGC1380 Original data Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 10 2 100 102 ND / MD 105 M Appendix B: Further tests of the methodology In order to test our methodology and assess the eﬀects of sys- tematic errors, we tested diﬀerent conﬁgurations to assess the possible range of bar ages thus deduced. Our tests include varying the position of the representative ring, using diﬀer- ent light proﬁles to build the underlying MD, not collaps- ing the data cubes, but rather deriving a mean SFH from the SFHs corresponding to each individual spaxel in the data cubes, and lastly, changing the pPXF regularisation error parameter. Age [Gyrs] 0 20 40 60 103 M NGC1084 Original data Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 2 100 102 ND / MD p Considering all the possible diﬀerent conﬁgurations, we esti- mate the ﬁnal systematic errors as 7.5+1.6 −1.1(sys) Gyr. In the follow- ing we describe each of the tests individually. B.1. The location of the representative spectrum The galaxy is at a distance of 21.2 Mpc with inclination of 47◦(see references in Sarzi et al. 2018 and Gao et al. 2019). We consider the eﬀective radius of the bulge to be 1080 pc (12.1 arcsec) from the photometric decomposition performed in Gao et al. (2019) in lieu of the ND radius. The data for NGC 1380 comes from the ESO archive, PI: Sarzi, M., pro- gramme ID 296.B-5054, using MUSE in Wide Field Mode. Fur- ther details about the galaxy and the observations can be found in Sarzi et al. (2018). With the exception of the most central representative ring, for underlying MDs following an exponential light proﬁle, the derived bar ages are constrained to the range 7–9 Gyr. 3 See http://leda.univ-lyon1.fr/ledacat.cgi B.2. Different main disc proﬁles: Flat versus exponential As a second control galaxy, NGC 1084 is a bar-less galaxy (Gao et al. 2019) with inclination3 of 49.9◦that also does not host a ND. Gao et al. (2019) also describe its morphology with a broken inner disc and a bulge with eﬀective radius of ≈150 pc (4.3 arcsec). The data for NGC 1084 comes from the ESO In order to build the underlying MD, one has to assume a light proﬁle that describes it. Although galaxy discs are usu- ally assumed as having exponential light proﬁles, recent stud- ies demonstrate that some cases may follow a ﬂat light pro- ﬁle (e.g. Zhu et al. 2018; Breda et al. 2020; Papaderos et al. 2022). In order to assess how much such a decision may A8, page 12 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Exponential main disc Flat main disc Results for diﬀerent conﬁgurations in the methodology presented in Sect. 3, varying the position of the representative ring (row b ssumed light proﬁle of the main underlying disc. To be continued on the next page. Exponential main disc Flat main disc Fig. B.1. Results for diﬀerent conﬁgurations in the methodology presented in Sect. 3, varying the position of the representative ring (row by row) and the assumed light proﬁle of the main underlying disc. To be continued on the next page. A8, page 13 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Exponential main disc Flat main disc Fig. B.2. Results for diﬀerent conﬁgurations in the methodology presented in Sect. 3, varying the position of the representative ring (row by row) and the assumed light proﬁle of the main underlying disc. In the ﬁrst column, we display the stellar velocity dispersion map, indicating the representative ring position with the dashed lines (the solid line corresponds to the peak in v/σ at the outskirts of the ND). In the middle and left columns we display results for exponential and ﬂat light proﬁles, respectively. One can see that the diﬀerent conﬁgurations result in bar ages in the range 7.0–9.5 Gyr, with the exception of the representative ring closest to the centre. The middle panel of the bottom row corresponds to the standard conﬁguration of our methodology. We note that the mask used that the mask used for the representative ring is slightly diﬀerent than the one used for the main result. B.2. Different main disc proﬁles: Flat versus exponential With this, the bar age is slightly older, 8 Gyrs. Exponential main disc Flat main disc Fig. B.2. Results for diﬀerent conﬁgurations in the methodology presented in Sect. 3, varying the position of the representative ring (row by row) and the assumed light proﬁle of the main underlying disc. In the ﬁrst column, we display the stellar velocity dispersion map, indicating the representative ring position with the dashed lines (the solid line corresponds to the peak in v/σ at the outskirts of the ND). In the middle and left columns we display results for exponential and ﬂat light proﬁles, respectively. One can see that the diﬀerent conﬁgurations result in bar ages in the range 7.0–9.5 Gyr, with the exception of the representative ring closest to the centre. The middle panel of the bottom row corresponds to the standard conﬁguration of our methodology. We note that the mask used that the mask used for the representative ring is slightly diﬀerent than the one used for the main result. With this, the bar age is slightly older, 8 Gyrs. 0 5 10 15 103 M NGC1433 - 440 pc MUSE Original Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 4 10 1 102 ND. / MD. Bar Age: 8.50 Gyrs 0 5 10 15 103 M NGC1433 - 440 pc MUSE Original Nuclear Disc (ND) Main Disc (MD) 0 5 10 14 Age [Gyrs] 10 4 10 1 102 ND. / MD. Bar Age: 8.50 Gyrs Fig. B.3. Same as Fig. 7, but for the test with spaxel-by-spaxel SFHs instead of a single collapsed spectrum produced for each data cube. The presented SFHs are mean SFHs calculated over all spaxels. aﬀect our ﬁnal result, we compared exponential and ﬂat pro- ﬁles as extreme possible cases. The results are summarised in Fig. B.1. NGC1433 - 440 pc For each representative ring position, one sees that the ﬁnal bar age derived using a ﬂat proﬁle is 0.5-1.0 Gyr older than that derived with an exponential proﬁle (with the exception of the most central representative ring). 103 M Considering the light proﬁle choice together with the posi- tion of the representative ring, the derived bar ages vary between 7.0-9.5 Gyr. B.4. pPXF regularisation As described in Cappellari (2017), regularisation allows one to have the smoothest SFH solution, without aﬀecting the physi- cal reality of it. In our methodology, we used the regul_err value of 0.15 derived by Bittner et al. (2020) for the TIMER sample following the procedure developed by McDermid et al. (2015). Nevertheless, in order to assess how much our ﬁnal result depends on it, we produce tests with values of regul_err in the range 0.1-5.0. The results are summarised in Fig. B.4. Since regul = 1/regul_err, the larger the regularisation error, the less regularised the solution is. The simulation used in Sect. 3.3 has both a collisionless (stellar disc and dark matter halo) and collisional (gaseous disc) compo- nent and is part of a suite of isolated disc simulations developed to study the evolution of barred galaxies (these will be presented in detail in Fragkoudi & Bieri, in prep.). Here we describe the main properties of the simulation that are relevant for this study. The simulation is run using the adaptive mesh reﬁnement (AMR) code RAMSES (Teyssier 2002). The AMR grid is reﬁned using a quasi-Lagrangian strategy, and has seven reﬁnement levels, with the maximum resolution reached in the simulation being 10 pc. The initial conditions of the model are created with the Markov chain Monte Carlo code DICE (Perret et al. 2014; Perret 2016). The total mass of the system is Mtot = 2 × 1012M⊙, with 98.5%, 1.425% and 0.075% of this distributed in the dark matter, stel- lar and gaseous components, respectively. The dark matter and stellar components have particles with masses 3.7 × 104M⊙and 4.3 × 105M⊙, respectively. The dark matter halo has a Navarro- Frenk-White proﬁle (Navarro et al. 1997) with a scale-length of 3 kpc, while the stars (gas) are modelled as an exponential disc, with a scale-length of 3 kpc (4 kpc) and a scale-height of 150 pc (50 pc). Gas in the simulation cools via atomic and metal- dependant cooling processes. As one can see, by changing the regularisation error parame- ter our bar age has variations of ±0.5 Gyr. Considering the vari- ations due to the representative ring position and the MD light proﬁle, this leaves us with systematic errors of +1.6 −1.1 Gyr, after summing in quadrature the maximum variations we ﬁnd with the four tests on systematic eﬀects. B.3. Spaxel-by-spaxel analysis The original conﬁguration uses a regulari- sation error of 0.15. The systematic eﬀect of employing diﬀerent regularisations is constrained to 0.5 Gyr. B.3. Spaxel-by-spaxel analysis Considering the elevated signal-to-noise ratio in the TIMER data (Gadotti et al. 2019), it would be possible to run the same methodology spaxel-by-spaxel, deriving spatially resolved SFHs, and then obtaining a mean SFH for each data cube. This would be in contrast to our regular methodology, in which the individual spectra in the data cubes are collapsed into a single spectrum, to then proceed to the derivation of the SFH. The disadvantages of doing so spaxel by spaxel are that the statis- tical uncertainties for each SFH would be larger and the com- putational time would increase signiﬁcantly. Since our goal is to apply the same methodology for the whole TIMER sample, it is too detrimental to require substantial computational time for the analysis of an individual galaxy. Nevertheless, to ver- ify that the results do not diﬀer signiﬁcantly with the diﬀerent approaches, we applied the alternative methodology using our data for NGC 1433. For the spatially resolved products, we con- sidered the mean SFHs to measure the bar age. The result is Fig. B.3. Same as Fig. 7, but for the test with spaxel-by-spaxel SFHs instead of a single collapsed spectrum produced for each data cube. The presented SFHs are mean SFHs calculated over all spaxels. shown in Fig. B.3 and, as one can see, the derived bar age is not substantially diﬀerent; in fact, the diﬀerence is within typ- ical uncertainties in the derivation of stellar ages. With that in mind, we decided to keep our regular methodology employing collapsed spectra to ensure smaller statistical errors and lower computational requirements. A8, page 14 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Regularization error of 0.10 Regularization error of 0.15 Regularization error of 0.30 Regularization error of 0.45 Regularization error of 1.00 Regularization error of 5.00 Fig. B.4. Same as Fig. 7 but for diﬀerent values of the regularisation error parameter applied by pPXF. The original conﬁguration uses a regulari- sation error of 0.15. The systematic eﬀect of employing diﬀerent regularisations is constrained to 0.5 Gyr. Regularization error of 0.15 Regularization error of 0.10 Regularization error of 0.30 Regularization error of 0.15 Regularization error of 5.00 Regularization error of 1.00 Regularization error of 0.45 Regularization error of 5.00 Regularization error of 0.45 Fig. B.4. Same as Fig. 7 but for diﬀerent values of the regularisation error parameter applied by pPXF. B.4. pPXF regularisation Finally, the regularisation error of 1.00 leads to a SFH that looks similar to that of the simulation presented in Fig. 6 (panel e), which could lead to the incorrect interpretation that the ﬁrst peak (∼8 Gyrs) is a contamination of the MD and the second peak (∼5 Gyrs) is the real bar formation. We point out however that, even though they might look similar at a ﬁrst glance, one should expect that contamination from the MD would be from the oldest stars, as showed from the diﬀerent tests for the simu- lated galaxies (Appendix C.1). Since the oldest peak of the ND is younger than the one from the MD, we do not expect this to be a sign contamination. In addition, this feature is not a well converged feature (as it is not evident for regularisation errors of 0.45 and 5) and is particular to a singular regularisation error. As noted in the main body of the paper, the peak at younger ages could arise through a number of mechanisms that induce gas inﬂow, such as the formation of the boxy/peanut bulge (e.g. Pérez et al. 2017) as well as through an interaction or ﬂyby that removes angular momentum from gas in the disc, causing a burst in gas inﬂow to the central regions through the bar. Star formation is modelled as a Schmidt law, (C.1) ˙ρ⋆= ϵ⋆ρgas/tﬀ, ˙ρ⋆= ϵ⋆ρgas/tﬀ, where ˙ρ⋆ is the local star formation rate, and tﬀ = p3π/(32Gρgas) is the free-fall time. Star formation is triggered when the gaseous density ρgas is larger than 1 cm−3 and the tem- perature is less than 100 K, with an eﬃciency of ϵ⋆= 1%. Core- collapse supernova feedback is implemented by assuming that a fraction of the stellar population, ηSN = 0.2, will explode as supernovae. The explosion itself is modelled using the mechan- ical feedback implementation presented in Kimm & Cen (2014) where ˙ρ⋆ is the local star formation rate, and tﬀ = p3π/(32Gρgas) is the free-fall time. Star formation is triggered when the gaseous density ρgas is larger than 1 cm−3 and the tem- perature is less than 100 K, with an eﬃciency of ϵ⋆= 1%. Core- collapse supernova feedback is implemented by assuming that a fraction of the stellar population, ηSN = 0.2, will explode as supernovae. The explosion itself is modelled using the mechan- ical feedback implementation presented in Kimm & Cen (2014) where ˙ρ⋆ is the local star formation rate, and tﬀ = p3π/(32Gρgas) is the free-fall time. Star formation is triggered A8, page 15 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Negative gradient Flat Gradient 1 2 3 M? [106MØ] True ND True MD 0 2 4 6 8 10 12 Age [Gyr] 10°1 100 101 102 ND/MD Fig. C.1. Testing the bar age criterion and how the age gradient in the underlying MD aﬀects our methodology for a simulated galaxy. In the left panel we show the ‘true’ SFH of the ND and MD. The true SFH of the ND is deﬁned as the SFH of all stars formed from the gas pushed to the centre after the bar forms within the ND radius, rND. The true SFH of the MD is deﬁned as the SFH of all the ‘old’ stars in the disc that were present before the bar and ND formed in the disc, within the same radius (i.e. rND). As one can see, in the bottom panel, the bar age criterion – the ﬁrst moment at which ND/MD is above 1 with a positive slope towards younger ages (the vertical grey dashed line) – successfully times the moment the bar is formed (given by the vertical orange line). By comparing this and Fig. ˙ρ⋆= ϵ⋆ρgas/tﬀ, 6 we see that the methodology we present in this paper – which involves using a representative region around the ND to model the MD – is able to recover the SFH of the ND and, therefore, the bar age. In the middle and right panels we explore how the age gradient of the old underlying MD aﬀects our methodology: in the middle panels we show a case where there is a negative age gradient applied to the underlying MD, while in the right panels we show the eﬀects of a ﬂat age gradient on the obtained results. The top panels show the average age gradient as a function of radius, and the bottom panels show how this aﬀects the SFHs of the original ND region (red), of the representative SFH of the underlying MD (green), and of the cleaned ND (blue), and the lower panels show the ND/MD ratio. Flat Gradient 1 2 3 M? [106MØ] True ND True MD 0 2 4 6 8 10 12 Age [Gyr] 10°1 100 101 102 ND/MD Fig. C.1. Testing the bar age criterion and how the age gradient in the underlying MD aﬀects our methodology for a simulated galaxy. In the left panel we show the ‘true’ SFH of the ND and MD. The true SFH of the ND is deﬁned as the SFH of all stars formed from the gas pushed to the centre after the bar forms within the ND radius, rND. The true SFH of the MD is deﬁned as the SFH of all the ‘old’ stars in the disc that were present before the bar and ND formed in the disc, within the same radius (i.e. rND). As one can see, in the bottom panel, the bar age criterion – the ﬁrst moment at which ND/MD is above 1 with a positive slope towards younger ages (the vertical grey dashed line) – successfully times the moment the bar is formed (given by the vertical orange line). By comparing this and Fig. 6 we see that the methodology we present in this paper – which involves using a representative region around the ND to model the MD – is able to recover the SFH of the ND and, therefore, the bar age. ˙ρ⋆= ϵ⋆ρgas/tﬀ, In the middle and right panels we explore how the age gradient of the old underlying MD aﬀects our methodology: in the middle panels we show a case where there is a negative age gradient applied to the underlying MD, while in the right panels we show the eﬀects of a ﬂat age gradient on the obtained results. The top panels show the average age gradient as a function of radius, and the bottom panels show how this aﬀects the SFHs of the original ND region (red), of the representative SFH of the underlying MD (green), and of the cleaned ND (blue), and the lower panels show the ND/MD ratio. within the radius corresponding to the ND (rND). The true SFH of the MD is deﬁned as the SFH of all the ‘old’ stars that were present before the bar and ND formed in the MD, within the same radius rND. As we can see, the bar age is well constrained by the moment when ND/MD is above 1 with positive slope towards younger ages for the ﬁrst time. We can compare true SFHs to the recovered SFHs from our methodology in Fig. 6, where we see that the SFH of the ND is well recovered, apart from at the oldest ages. and Kimm et al. (2015). In this implementation, the supernova explosion is injected into the surrounding interstellar medium according to the phase of the explosion (energy conserving or momentum conserving); in other words, it is injected as momen- tum or thermal energy, depending on whether the cooling radius is resolved. More details on the feedback implementation can be found in (Kimm & Cen 2014; Kimm et al. 2015). C.1. Testing the methodology with hydrodynamic simulations g In addition, we test how the underlying age gradient of the MD in the galaxy can aﬀect the determination of the bar age. In the right panel of Fig. C.1, we show a case where there is no age gradient in the underlying MD of the galaxy. By subtracting the main underlying disc (as obtained from the representative ring around the ND) from the original data, we obtain a peak in ND/MD that corresponds to the time of bar formation, while before this peak, ND/MD is consistently below < 1. When a negative age gradient is added to the old disc in the simulation (middle panel), we ﬁnd similar results, with the diﬀerence that, at old ages, ND/MD rises again above one. This happens because there are old stars that are present in the ND region, which are not present in the ‘representative’ MD region which is at larger radii. Therefore, due to the age gradient, there is a lack of old stars in the ring, which leads to a rise in ND/MD for the oldest In this section we describe some of the tests carried out on the simulations that allowed us to assess the eﬀects of some of the assumptions in our methodology. In particular, we tested how an assumed underlying age gradient of the old disc, as well as how the location of the ring used to obtain the representative spectrum, aﬀected the results. In the left panel of Fig. C.1, we show what are the true SFHs of the ND and the underlying MD in the simulations. Then, we show that we can accurately reproduce the time the bar formed using these two values and our chosen criterion for the time of bar formation (i.e. the ﬁrst moment when the ratio of ND/MD increases above 1 with positive slope towards younger ages; see Sect. 3). We deﬁne the true SFH of the ND as the SFH of all stars formed from the gas pushed to the centre after the bar forms A8, page 16 of 17 C. de Sá-Freitas et al.: A&A 671, A8 (2023) Fig. C.2. Testing the eﬀects of ring location on the methodology for a simulated galaxy. C.1. Testing the methodology with hydrodynamic simulations From left to right we show cases where the ring used to reconstruct the representative SFH of the underlying MD is placed at larger radii, in a case where the model has a negative age gradient in the underlying MD. The top rows show the locations of the rings with respect to the ND region, and the bottom panels show the SFHs. We see that when the ring is placed at larger radii, the representative SFH of the MD does not fully match the ND region at the oldest ages. Fig. C.2. Testing the eﬀects of ring location on the methodology for a simulated galaxy. From left to right we show cases where the ring used to reconstruct the representative SFH of the underlying MD is placed at larger radii, in a case where the model has a negative age gradient in the underlying MD. The top rows show the locations of the rings with respect to the ND region, and the bottom panels show the SFHs. We see that when the ring is placed at larger radii, the representative SFH of the MD does not fully match the ND region at the oldest ages. panels of Fig. C.2). Therefore, the representative ring should not be placed at too large radii from the ND, as the farther away it is placed, the more diﬀerent the stellar populations will be from those of the underlying MD within the ND region. As described in Sect. 3, we therefore select the radius just outside the ND, where the average age is oldest (see Fig. 3). This ensures we select a region outside the inﬂuence of the ND itself, but without going to larger radii where the stellar population properties of the representative region will be signiﬁcantly diﬀerent from those of the underlying MD in the ND region. populations. This eﬀect will be larger, the larger the age gradient (which will vary from galaxy to galaxy). Due to this, for our criterion of determining the bar age, we explicitly require that ND/MD be above 1 with a positive slope towards younger ages (rather than simply ND/MD > 1, as this could occur at old ages due to a strong age gradient in the inner galaxy). g g g g y In Fig. C.1. Testing the methodology with hydrodynamic simulations C.2 we test how the location of the ring aﬀects our methodology, in a case where there is a negative age gradient (if there is no age gradient, the location of the ring does not have a signiﬁcant eﬀect on the methodology, as long as the SFH of the MD is appropriately rescaled to take into account the higher surface density in the centre of the galaxy, due to its exponential proﬁle). When the ring is placed at larger radii, the representative SFH will be lacking more of the old stars that are present at smaller radii, which can be seen by the increase in ND/MD > 1 for old ages, as we move to larger radii (i.e. the middle and right We also note that we applied our methodology to other sim- ulations with the same initial conditions, but with modiﬁcations in the star formation and stellar feedback prescriptions, and we were able to recover the bar age reliably also with these changes (i.e. the methodology does not depend on the details of the star formation and stellar feedback prescriptions in the simulation). A8, page 17 of 17
https://openalex.org/W3002295425	https://findresearcher.sdu.dk/ws/files/168570301/Ferreira2020_Article_AlternativeLengtheningOfTelome.pdf	English	null	Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation	Journal of neuro-oncology	2,020	cc-by	9,744	Citation for pulished version (APA): Ferreira, M. S. V., Sørensen, M. D., Pusch, S., Beier, D., Bouillon, A.-S., Kristensen, B. W., Brümmendorf, T. H., Beier, C. P., & Beier, F. (2020). Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation. Journal of Neuro-Oncology, 147(1), 1-14. https://doi.org/10.1007/s11060-020-03394-y Download date: 24. Oct. 2024 University of Southern Denmark Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation Ferreira, Monica Sofia Ventura; Sørensen, Mia Dahl; Pusch, Stefan; Beier, Dagmar; Bouillon, Anne-Sophie; Kristensen, Bjarne Winther; Brümmendorf, Tim Henrik; Beier, Christoph Patrick; Beier, Fabian Citation for pulished version (APA): Ferreira, M. S. V., Sørensen, M. D., Pusch, S., Beier, D., Bouillon, A.-S., Kristensen, B. W., Brümmendorf, T. H., Beier, C. P., & Beier, F. (2020). Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation. Journal of Neuro-Oncology, 147(1), 1-14. https://doi.org/10.1007/s11060-020-03394-y Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. 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Please direct all enquiries to puresupport@bib.sdu.dk Journal of Neuro-Oncology (2020) 147:1–14 https://doi.org/10.1007/s11060-020-03394-y LABORATORY INVESTIGATION 4 Department of Neuropathology, University of Heidelberg, Heidelberg, Germany 5 Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany 6 German Cancer Consortium (DKTK), Heidelberg, Germany 7 Department of Neurology, University Hospital Odense, Sdr. Boulevard 29, 5000 Odense, Denmark Abstract IDH1R132H downregulates ATRX expression in vitro resulting in ALT, which may contribute to the strong association of IDH1R132H mutations, ATRX loss, and ALT. words Isocitrate dehydrogenase · D2HG · Telomerase · Telomere length · Q-FISH · TERT promote Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation Monica Sofia Ventura Ferreira1 · Mia Dahl Sørensen2,3 · Stefan Pusch4,5,6 · Dagmar Beier3,7 · Anne‑Sophie Bouillon1 · Bjarne Winther Kristensen2,3 · Tim Henrik Brümmendorf1 · Christoph Patrick Beier3,7 · Fabian Beier1 Monica Sofia Ventura Ferreira1 · Mia Dahl Sørensen2,3 · Stefan Pusch4,5,6 · Dagmar Beier3,7 · Anne‑Sophie Bouillon Bjarne Winther Kristensen2,3 · Tim Henrik Brümmendorf1 · Christoph Patrick Beier3,7 · Fabian Beier1 Received: 24 October 2019 / Accepted: 7 January 2020 / Published online: 20 January 2020 © The Author(s) 2020 Abstract Purpose Isocitrate dehydrogenase 1 (IDH1) mutations are associated with improved survival in gliomas. Depending on the IDH1 status, TERT promoter mutations affect prognosis. IDH1 mutations are associated with alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations and alternative lengthening of telomeres (ALT), suggesting an interaction between IDH1 and telomeres. However, little is known how IDH1 mutations affect telomere maintenance.i Purpose Isocitrate dehydrogenase 1 (IDH1) mutations are associated with improved survival in gliomas. Depending on the IDH1 status, TERT promoter mutations affect prognosis. IDH1 mutations are associated with alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations and alternative lengthening of telomeres (ALT), suggesting an interaction between IDH1 and telomeres. However, little is known how IDH1 mutations affect telomere maintenance. Methods We analyzed cell-specific telomere length (CS-TL) on a single cell level in 46 astrocytoma samples (WHO II-IV) by modified immune-quantitative fluorescence in situ hybridization, using endothelial cells as internal reference. In the same samples, we determined IDH1/TERT promoter mutation status and ATRX expression. The interaction of IDH1R132H mutation and CS-TL was studied in vitro using an IDH1R132H doxycycline-inducible glioma cell line system. Results Virtually all ALTpositive astrocytomas had normal TERT promoter and lacked ATRX expression. Further, all ALTpositive samples had IDH1R132H mutations, resulting in a significantly longer CS-TL of IDH1R132H gliomas, when compared to their wildtype counterparts. Conversely, TERT promotor mutations were associated with IDHwildtype, ATRX expression, lack of ALT and short CS-TL. ALT, TERT promoter mutations, and CS-TL remained without prognostic significance, when correcting for IDH1 status. In vitro, overexpression of IDHR132H in the glioma cell line LN319 resulted in downregulation of ATRX and rapid TERT-independent telomere lengthening consistent with ALT. Conclusion ALT is the major telomere maintenance mechanism in IDHR132H mutated astrocytomas, while TERT promoter mutations were associated with IDHwildtype glioma. IDH1R132H downregulates ATRX expression in vitro resulting in ALT, which may contribute to the strong association of IDH1R132H mutations, ATRX loss, and ALT. f Methods We analyzed cell-specific telomere length (CS-TL) on a single cell level in 46 astrocytoma samples (WHO II-IV) by modified immune-quantitative fluorescence in situ hybridization, using endothelial cells as internal reference. In the same samples, we determined IDH1/TERT promoter mutation status and ATRX expression. The interaction of IDH1R132H mutation d CS TL di d i i i IDH1R132H d li i d ibl li ll li Conclusion ALT is the major telomere maintenance mechanism in IDHR132H mutated astrocytomas, while TERT promoter mutations were associated with IDHwildtype glioma. * Fabian Beier fbeier@ukaachen.de Immunohistochemistry Formaldehyde-fixed paraffin embedded (FFPE) sections of three µm from pre-surgery tissue biopsies were used for this study. FFPE sections were stained as described previously using primary antibodies against IDH1R132H (H09, 1:100, Dianova, Germany) [30] and ATRX [29] (HPA001906, 1:100, Atlas Antibodies, Sweden) epitopes. TERT promoter mutations (TERTpmut) are common in glioma and found in 80% of all primary GBM [14–16], repre- senting one possible TMM. TERTpmut disrupt the tight tran- scriptional suppression of TERT in somatic cells resulting in increased TERT expression and telomerase activity in vitro in glioma [17]. A mechanism of homologous recombination [18] to main- tain TL, known as “alternative lengthening of telomeres” (ALT), was also identified in gliomas. About 20–63% of adult low-grade glioma and 11% of adult GBM use ALT as an additional mechanism for telomere maintenance [19, 20]. Dysfunction of the α-thalassemia/mental retardation syndrome X-linked (ATRX)/death-associated protein 6 (DAXX) com- plex is known to result in ALT along with more widespread genomic destabilization [21–23]. ATRX and DAXX are cen- tral components of a chromatin-remodeling complex required for the incorporation of H3.3 histone proteins into the telo- meric regions of chromosomes [24, 25]. 75% of grade II–III astrocytomas and secondary GBM harbor ATRX abberations [26–28] linking IDH1 mutations with ATRX and ALT. DNA extraction, polymerase chain reaction and mutational analysis by sequencing Mutations in the TERT promoter region were identified by PCR and Sanger sequencing as described previously [31]. The detailed protocol can be found in the Supplementary Materials and methods. Patients Tissue samples from 46 astrocytoma patients were included for the study. The Regional Committee on Health Research Ethics approved the study for Southern Denmark (S2DO90080) and Danish Data Protection Agency (file number: 2009-41-3070) and all patients provide informed consent. The use of tissue was not prohibited by any patient according to the Danish Tissue Application Register. All methods were performed in accordance with the relevant guidelines and regulations. All patients underwent pri- mary surgery between 1991 and 2005 at the Department of Neurosurgery, Odense University Hospital, Denmark. All cases were independently reviewed and reclassified by M.D.S and B.W.K. (senior neuropathologist) according to the 2016 WHO guidelines [2] as described in [29]. Clinical data were extracted from the respective electronic patient journal. Clinical and neuropathological characteristics of the astrocytoma patients are shown in Table 1. Telomeres determine the proliferative capacity of mam- malian cells. Telomere length (TL) shortens with each cell division until cell proliferation is arrested once the maximal number of cell divisions is reached (“Hayflick limit”). In case of further replication, cells undergo chromosomal instability and induction of apoptosis. Expression of human telomerase reverse transcriptase (TERT) allows for the stabilization and elongation of telomeres. Telomere maintenance mechanisms (TMM) are necessary for immortality of cancer cells by over- coming genetic instability associated with critical telomere shortening [13]. Introduction Along this line, determination of isocitrate dehydrogenase (IDH) 1 mutation status became mandatory in the updated 2016 WHO classification [3]. IDH1 mutations are driver mutations of low-grade gliomas and found in 80% [4] but are not detectable in primary glioblastoma (GBM) [2, 5]. The most common IDH1 mutations in glioma (> 95%) result in an amino acid substitution at arginine 132 (R132), which resides in the enzyme’s active site [4]. Despite the abundance of evidence in support of a major pathophysiological and prog- nostic role of IDH1 mutations [6–8], the precise mechanism of how IDH1 mutations modulate malignancy is still not com- pletely understood. While IDH1 has a major role in the citric acid cycle, R132H mutation (IDHR132H) results in a gain of function; IDH1R132H catalyzes conversion of alpha-ketoglutar- ate into the oncometabolite D-2-hydroxyglutarate (D2HG) [9]. D2HG inhibits dioxygenases that depend on alpha-ketoglutar- ate, like Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and histone-lysine demethylases [10]; this results in increased CpG island methylation [11] and a stable reshaping of the epigenome (CpG island methylation phenotype, CIMP) changing transcriptional programs and altering the differentia- tion state [12]. Despite the strong associations observed, few data is available how the different pathways interact on telomere maintenance. The aim of this study was to investigate the interplay among IDH1, TL, ATRX and TERT/ALT using a newly developed distinctive methodology that allowed deter- mination of glioma cell-specific telomere length (CS-TL) on a single cell level. Introduction Christoph Patrick Beier and Fabian Beier have contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11060-020-03394-y) contains supplementary material, which is available to authorized users. * Fabian Beier fbeier@ukaachen.de 1 Department of Haematology, Oncology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany 2 Department of Pathology, University Hospital Odense, Sdr. Boulevard 29, 5000 Odense, Denmark 3 Department of Clinical Research, University of Southern Denmark, Sdr. Boulevard 29, 5000 Odense, Denmark Christoph Patrick Beier and Fabian Beier have contributed equally to this work. Christoph Patrick Beier and Fabian Beier have contributed equally to this work. Gliomas are the most common primary brain tumors in adults and represent a histologically defined entity with a high molecular heterogeneity determining prognosis and response Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11060-020-03394-y) contains supplementary material, which is available to authorized users. * Fabian Beier fbeier@ukaachen.de 1 Department of Haematology, Oncology, Medical Faculty, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany 2 Department of Pathology, University Hospital Odense, Sdr. Boulevard 29, 5000 Odense, Denmark 3 Department of Clinical Research, University of Southern Denmark, Sdr. Boulevard 29, 5000 Odense, Denmark :(0123 1 3456789) 3 Journal of Neuro-Oncology (2020) 147:1–14 2 to therapy [1, 2]. Along this line, determination of isocitrate dehydrogenase (IDH) 1 mutation status became mandatory in the updated 2016 WHO classification [3]. IDH1 mutations are driver mutations of low-grade gliomas and found in 80% [4] but are not detectable in primary glioblastoma (GBM) [2, 5]. The most common IDH1 mutations in glioma (> 95%) result in an amino acid substitution at arginine 132 (R132), which resides in the enzyme’s active site [4]. Despite the abundance of evidence in support of a major pathophysiological and prog- nostic role of IDH1 mutations [6–8], the precise mechanism of how IDH1 mutations modulate malignancy is still not com- pletely understood. While IDH1 has a major role in the citric acid cycle, R132H mutation (IDHR132H) results in a gain of function; IDH1R132H catalyzes conversion of alpha-ketoglutar- ate into the oncometabolite D-2-hydroxyglutarate (D2HG) [9]. D2HG inhibits dioxygenases that depend on alpha-ketoglutar- ate, like Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and histone-lysine demethylases [10]; this results in increased CpG island methylation [11] and a stable reshaping of the epigenome (CpG island methylation phenotype, CIMP) changing transcriptional programs and altering the differentia- tion state [12]. to therapy [1, 2]. Cell culture, proliferation and clonogenicity For the cell culture experiments the doxycycline-inducible GBM cell line LN319 expressing IDH1 wildtype (IDH1WT) and IDH1R132H was used. Cells were cultured in Dulbecco’s 3 Journal of Neuro-Oncology (2020) 147:1–14 3 Table 1 Patient characteristics and pathology Diffuse astrocytoma Anaplastic astrocytoma Glioblastoma All Patients (n) 24 9 13 46 Age Mean 45.2 47.6 65.3 51.4 Range 2.6–78.5 29.5–71.0 49.4–77.5 2.6–78.5 Sex (n) Male 14 (58.3%) 3 (33.3%) 10 (76.9%) 27 (58.7%) Female 10 (42.7%) 6 (66.7%) 3 (23.1%) 19 (41.3%) Status (n) Alive 3 (12.5%) 0 (0%) 0 (0%) 3 (6.5%) Dead 21 (87.5%) 9 (100%) 13 (100%) 43 (93.5%) Overall survival (months) Median 76.9 24.0 7.5 32.6 Range 3.4–280.0 4.7–48.8 1.5–34.2 1.5–280.0 IDH1 status (n) Mutated 18 (75.0%) 4 (44.4%) 1 (7.7%) 23 (50.0%) Wildtype 6 (25.0%) 5 (55.6%) 12 (92.3%) 23 (50.0%) ATRX status (n) Loss 20 (83.3%) 4 (44.4%) 4 (30.8%) 28 (60.9%) Retained 4 (16.7%) 5 (55.6%) 9 (69.2%) 18 (39.1) 1P/19Q status (n) CO-DEL 0 0 0 0 1P-DEL 0 1 (11.1%) 1 (7.7%) 2 (4.3%) 19-DEL 0 0 0 0 NON-DEL 5 (20.8%) 2 (22.2%) 2 (15.3%) 9 (19.6%) Not determined 19 (79.2%) 6 (66.7%) 10 (76.9%) 35 (76.0%) hTERT promoter mutation Mutated 6 (25.0%) 3 (33.3%) 9 (69.2%) 18 (39.1%) C228T 5 (83.3%) 2 (66.7%) 8 (88.9%) 15 (83.3%) C250T 1 (16.7%) 1 (33.3%) 1 (11.1%) 3 (16.7%) None 17 (75.0%) 5 (55.6%) 4 (30.8%) 26 (56.5%) Not determined 1 (4.1%) 1 (11.1%) 0 2 (4.3%) ALT status Negative 12 (50%) 5 (55.6%) 12 (92.3%) 29 (63.0%) Positive 12 (50%) 4 (44.4%) 1 (7.7%) 17 (37.0%) KI67 proliferation rate Mean (%) 3.3 9.7 10.2 6.7 Range (%) 0–30 3–20 2–35 0–35 CS-TL Median 11.8 13.6 3.5 11.0 Range − 1.1 to 36.7 − 3.9 to 31.5 − 6.0 to 25.2 − 6.0 to 36.7 Modified Eagle Medium (DMEM) (Gibco, Germany) sup- plemented with 10% tetracycline-free fetal calf serum (FCS) (Clontech, USA) and standard antibiotics (Gibco, Germany). 1 µM doxycycline was used (Sigma Aldrich, Germany) to induce expression. Cell proliferation was assessed using the CellTiter-Blue Assay (Promega, Germany) as described pre- viously [32] using the FLUOstarOPTIMA (BMG Labtech, Germany) fluorometer. Modified Eagle Medium (DMEM) (Gibco, Germany) sup- plemented with 10% tetracycline-free fetal calf serum (FCS) (Clontech, USA) and standard antibiotics (Gibco, Germany). 1 µM doxycycline was used (Sigma Aldrich, Germany) to induce expression. (D)‑2‑hydroxyglutarate (DGH2) assay The DGH2 assay used was based on an enzymatic assay as previously described [9]. The detailed protocol can be found in the Supplementary Materials and methods. Quantitative fluorescence in situ hybridization (Q‑FISH) TL analysis was done by a modified protocol of immuno- quantitative fluorescence in situ hybridization (Q-FISH) as previously described [31–35]. FFPE sections of the cohort were deparaffinized and rehydrated before antigen retrieval in 10 mM citrate buffer (pH 6.0). Slides were permeabilized with 0.2% Triton X-100 and blocked for 30 min in serum- free buffer (Rotiblock 1:10, Roth, Germany). Actin fibers were first stained with primary antibody mouse anti-human alfa-SMA (1:200, DAKO, Germany) and a goat anti-mouse Alexa Fuor 633 (1:100, Thermo Fisher, Germany) as sec- ondary antibody. Next, cells were post-fixed in formalin for 30 s and dehydrated with increasing ethanol series before telomere staining. For cells in culture, cells were recovered from culture, fixed in methanol:acetic acid (3:1), cytospin, air dried and dehydrated with ethanol before telomeres were stained. Telomere staining consisted in providing a hybridization mixture containing the Cy3-(C3TA2) peptide nucleic acid (PNA) probe (Panagene, South Korea) to the slides for 3 min at 85 °C for DNA denaturation. Slides were then hybridized for 2 h at room temperature in a humidified chamber. Next, slides were washed with a formamide-based buffer, DAPI stained, and mounted with Vectashield antifade mounting medium (Vector Labs, USA). Fluorescence was acquired with the high-resolution laser-scanning microscope LSM710 (Zeiss, Germany). H&E stained sections were ana- lyzed in parallel for all cases to identify tumor areas. Fluo- rescent image capture was done with ×63 optical magnifica- tion and ×1.2 zoom. A multi-tracking mode of 0.5 μm-steps was used to acquire images of DAPI, Cy3 and Alexa Fluor ALT assessment All astrocytoma cases were assessed for the presence of ALT phenotype using telomere Q-FISH staining. ALT positivity was identified by large, ultrabright, clumpy, intranuclear foci of telomere FISH signals, as previously described [20, 21]. A tumor was defined as ALT-positive, when the following two criteria were fullfilled: (1) the presence of ultra-bright intranuclear foci of telomere FISH signals (ALT-associated telomeric foci), with integrated total signal intensities for individual foci being > 10-fold the mean signal intensities per cell of all telomeric signals from endothelial cells within the same case and (2) the number of cells with ALT-associ- ated telomeric foci being 1% or more of the total number of tumor cells assessed per case [20, 21]. Cell culture, proliferation and clonogenicity Cell proliferation was assessed using the CellTiter-Blue Assay (Promega, Germany) as described pre- viously [32] using the FLUOstarOPTIMA (BMG Labtech, Germany) fluorometer. For colony-forming unit assays, 2500 cells/well were seeded in a 6-well format for 10 days before colonies were fixed and stained with Cristal Violet (Sigma, Germany). For agar assays, 8000 cells/well were seeded in a 6-well format and incubated for 3 weeks before cells were stained with Cristal Violet. Images were acquired with a Cool Snap™ HQ2 digital camera (Photometrics, USA) on an Axiophot 2 microscope (Carl Zeiss, Germany). Quantification was done 1 3 Journal of Neuro-Oncology (2020) 147:1–14 4 633 stainings. Maximum projection of five single consecu- tive steps of 1.2 µm each was done for TL quantification using Definiens software (Definiens, Germany). Nuclei and telomeres were detected based on the respective DAPI and Cy3 intensity. Alfa-SMA was used to identify endothelial cells that were used as an internal control to correct for TL inter-individual variability [32–38]. A mean number of 150 tumor cells and 100 endothelial cells were assessed per case. To determine the tumor cell-specific telomere length (CS- TL), the difference (Δ telomere length) between the TL of astrocytoma cells and the TL of endothelial cells was calcu- lated and designated in arbitrary units (a.u.) of fluorescence. using ImageJ software (open source). Results are means of three repeated experiments. ATRX and TERT Immunofluorescence and western blotting Immunofluorescence for ATRX and TERT followed previ- ously published protocols [32, 35, 37]. Western blot was car- ried out according to previous published standard protocols. The detailed protocol can be found in the Supplementary Materials and methods. RNA extraction, cDNA synthesis and mRNA expression Determination of TERT and ATRX mRNA expression was carried out as described presviously[32]. Gene expression is expressed in fold change according to the 2−ΔΔct method. Additional information can be found in the Supplementary Materials and methods. Statistical analysis The data was collected via Microsoft Excel 2007 and ana- lyzed using Stata version 15 (StataCorp LP, USA) or Graph- Pad Prism 5.0 (GraphPad Software Inc, USA). ANOVA with Bonferroni correction was used for comparison of more than two groups. Student’s unpaired t test was used to compare differences between two groups. Fisher’s exact test was used to analyze categorical data. Overall survival was defined from the day of initial surgery until death or date of censor- ing (July 1st, 2018). Survival data were analysed and we using Kaplan–Meier and the multivariate Cox regression 1 3 Journal of Neuro-Oncology (2020) 147:1–14 5 ig. 1 Glioma cell-specific telomere length in IDH1WT and DH1R132H glioma. a Overview of biomarkers status in glioma atients (n = 46). b Telomere length in glioma vs endothelial cells. c Telomere length (in arbitrary units, a.u.) in IDH1WT and IDH1R132H lioma samples; d Telomere length (in a.u.) in IDH1WT and DH1R132H glioma stratified by WHO tumor grade (DA diffuse astro- cytoma, AA anaplastic astrocytoma, GBM gliobastoma multiform); e Kaplan–Meier survival curves (in months) of patients with CS-TL below (CS-TLshort) or above median; f Kaplan–Meier survival curves (in months) of patients with CS-TL below (CS-TLshort) or above median stratified based on IDH1 mutational status Fig. 1 Glioma cell-specific telomere length in IDH1WT and IDH1R132H glioma. a Overview of biomarkers status in glioma patients (n = 46). b Telomere length in glioma vs endothelial cells. c Telomere length (in arbitrary units, a.u.) in IDH1WT and IDH1R132H glioma samples; d Telomere length (in a.u.) in IDH1WT and IDH1R132H glioma stratified by WHO tumor grade (DA diffuse astro- cytoma, AA anaplastic astrocytoma, GBM gliobastoma multiform); e Kaplan–Meier survival curves (in months) of patients with CS-TL below (CS-TLshort) or above median; f Kaplan–Meier survival curves (in months) of patients with CS-TL below (CS-TLshort) or above median stratified based on IDH1 mutational status 1 3 Journal of Neuro-Oncology (2020) 147:1–14 6 found significant differences of CS-TL beween IDH1WT/ TERTpWT and IDH1WT/TERTpmut or IDH1R132H/TERTpWT and IDH1R132H/TERTpmut tumors (Fig. 2c). model to adjust for age, WHO grade, and IDH1 status. Sur- vival curves were compared using the log-rank test. found significant differences of CS-TL beween IDH1WT/ TERTpWT and IDH1WT/TERTpmut or IDH1R132H/TERTpWT and IDH1R132H/TERTpmut tumors (Fig. 2c).i TERTp status was significantly associated with reduced survival (HR 2.05; 95% CI 1.10–3.86; p = 0.022) in the entire cohort (Fig. 2d). In vitro overexpression of mutant IDHR132H in a GBM cell line To understand the role of IDH1R132H mutations in regulating CS-TL in glioma, we used a doxycycline-inducible GBM cell line (LN319) expressing IDH1WT and IDH1R132H. The cell line was previously described and the presence of the IDH1R132H mutation was confirmed by sequencing [39]. IDHR132H expression induced by doxycycline significantly stimulated D2HG synthesis (p = 0.01; Fig. 4a), and com- promised proliferation and clonogenicity (Supplementary Figure S3A-B). In this in vitro model, induced IDH1R132H overexpression also resulted in a significant increase in TL after nine population doublings (Fig. 4b; Supplementary Figure S3C) and a significant downregulation of ATRX at transcriptional level (Fig. 4c). Telomere FISH detected a significant increase in ALTpos cells in IDH1R132H cells (Fig. 4d, left panels) and loss of ATRX protein expression ALT, ATRX mutations and telomere length Using telomere FISH, we studied the presence of ALT (ALTpos) on a single cell level in our cohort (Fig. 3a). 37% of the patients had ALTpos tumors (Table 1) of which 100% also had an IDH1R132H mutation (Fig. 3b). Further, loss of ATRX expression (Fig. 3c) and TERTpWT (Fig. 3d) were associated with the presence of ALT. Both, presence of ALT and loss of ATRX expression were significantly associated with longer telomeres (p < 0.001, Fig. 3e, f) and improved survival (HR 0.42; 95% CI 0.22–0.81; p = 0.007 and HR 0.46; 95% CI 0.4–0.85; p = 0.012, respectively; Fig. 3g, h). However, this association disappeared when adjusting for age, WHO grade and IDH1 status i a multi-variate analysis (ALT status: HR 0.62; 95% CI 0.23–1.72; p = 0.36; ATRX status: HR 1.12; 95% CI 0.43–2.93; p = 0.82). Statistical analysis However, in patients with an IDH1R132H tumor, TERTp status did not significantly associate with survival (Fig. 2e), and multivariate analysis unveiled that TERTp status was not an independent prognostic factor when adjusting for age, WHO grade, and IDH1 status (HR 1.06; 95% CI 0.50–2.24; p = 0.87). Association of IDH1R132H mutations and telomere length The incidence of IDH1R132H mutations in our cohort was 50% (Fig. 1a, Table 1). Single cell-based telomere analysis using modified immuno-Q-FISH (Supplementary Figure S1A, B) revealed significantly longer TL in astrocytoma as compared to endothelial cells (p < 0.0001) and a substan- tial inter-individual variability (Fig. 1b). To overcome this issue, we used the endothelial cells as internal control, which allowed correction of the TL of astrocytoma cells for age and for inter-individual variability (i.e. CS-TL). The CS-TL was significantly longer in IDH1R132H-mutated tumors as compared to IDH1WT tumors (Fig. 1c) irrespective of tumor grade (Fig. 1d), while there were no significant differences among the different tumor types (p = 0.19, Supplementary Figure S1C). IDH1R132H mutation was significantly asso- ciated with improved patient survival (HR 0.28; 95% CI 0.14–0.50; p < 0.001, Supplementary Figure S1C) with simi- lar tendency (HR 0.47; 95% CI 0.21–1.08; p = 0.076) when adjusting for age and WHO grade. CS-TL did not signifi- cantly correlate with prognosis (HR 0.98; p = 0.95) (Fig. 1e). However, when dichotomizing patients based on IDH1 status and CS-TL, patients with IDH1R132-mutated tumors and long CS-TL had significantly poorer survival than patients with IDH1R132-mutated tumors and short CS-TL (HR 1.80; 95% CI 1.03–3.16; p = 0.030, Fig. 1F). No significant prognostic value was found for patients with IDHWT tumors (HR 0.98; 95% CI 0.59–1.65; p = 0.95, Fig. 1f). hTERT promoter mutations, IDH1 mutations, and telomere length [42], our data supports the evidence that TERTpmut are associated with shorter TL in gliomas. Fig. 3 ATRX, alternative lengthening and survival. a Representa- tive images showing Q-FISH stained gliomas with (ALTpos) and without ALT (ALTneg, magnification: 756x). b Number of IDH1WT and IDH1R132 patients stratified according to ALT status. c Num- ber of ATRXWT and ATRXmut patients stratified according to ALT status. d Number of TERTpWT and TERTpmut patients stratified according to ALT status. e Glioma-specific telomere length (CS-TL in arbitrary units, a.u.) in ALTneg and ALTpos tumors. f Glioma-spe- cific telomere length (CS-TL in arbitrary units, a.u.) in tumors with retained (ATRXret) ATRX and in tumors with ATRX loss (ALTloss). g Kaplan–Meier survival curves (in months) of glioma patients accord- ing to ALT status. h Kaplan–Meier survival curves (in months) of glioma patients according to ATRX mutational status ◂ Fig. 3 ATRX, alternative lengthening and survival. a Representa- tive images showing Q-FISH stained gliomas with (ALTpos) and without ALT (ALTneg, magnification: 756x). b Number of IDH1WT and IDH1R132 patients stratified according to ALT status. c Num- ber of ATRXWT and ATRXmut patients stratified according to ALT status. d Number of TERTpWT and TERTpmut patients stratified according to ALT status. e Glioma-specific telomere length (CS-TL in arbitrary units, a.u.) in ALTneg and ALTpos tumors. f Glioma-spe- cific telomere length (CS-TL in arbitrary units, a.u.) in tumors with retained (ATRXret) ATRX and in tumors with ATRX loss (ALTloss). g Kaplan–Meier survival curves (in months) of glioma patients accord- ing to ALT status. h Kaplan–Meier survival curves (in months) of glioma patients according to ATRX mutational status ◂ Here we showed that the IDH1R132H mutation is directly associated with a lack of ATRX expression and conse- quently, ALT as described previously [46]. In our sample, all tumors with ALT bear IDH1R132H mutations and lost ATRX expression. Together with the inverse association of ALT with TERTpmut, our data suggests that ALT is the major TMM in IDH1R132H astrocytoma. Conversely, TERTp mutations appear to be the crucial TMM in IDHWT astrocytoma. Immunofluorescence for TERT protein expression with DAPI counterstaining (Fig. 4d, right panels) illustrated the TERT-independent increase of TL in IDHR132H cells. Our data suggests a dichotomy of mechanisms in astro- cytoma depending on the presence of IDH1 mutations (Fig. 5). Discussion Maintenance mechanisms of telomeres are promising thera- peutic targets and small molecule telomerase inhibitors are currently tested in clinical trials and filed for approval for the treatment of myeloproliferative syndromes [40, 41]. Exact understanding of telomere biology in glioma is therefore of high translational importance, given the completely diverse prognostic impact of TERTp mutations according to the IDH1 mutation status. Here we provide evidence suggest- ing that glioma subgroups with and without IDH1 mutations use different TMM and describe a new pathway linking the IDH1R132H mutations to ALT. We also showed that overexpression of IDH1R132H in gli- oma cells in vitro result in a phenotype that fully mimicked all phenomena observed in the patient samples. Overex- pression of IDH1R132H in the glioma cells resulted in D2HG production, decreased proliferation in vitro, loss of ATRX expression in vitro and ALT. Reduced ATRX expression due to IDH1R132H overexpression suggests that IDH1R132H alone is sufficient to diminish ATRX expression and thereby induce ALT. However, the importance and functional rel- evance of this mechanism needs to be further confirmed in vivo. Further, it remains to be clarified, why IDHR132H mutated glioma cells favor ATRX mutations, although there is a second, alternative pathway to suppress ATRX. One major limitation of published studies on telomere biology in solid cancer was missing availability of tech- niques that allow the determination of TL on a single cell level. In most studies performed so far [42–44], TL in tumor samples was actually derived from a mixture of vessels, immune cells and normal brain or tumor cells. This impaired significantly the validity and robustness of such studies. Fur- thermore, these studies rarely controlled for inter-individual (mostly genetic) variability of TL or the presence of ALT. In our study, we developed a modified immuno-Q-FISH technique for the determination of the TL in glioma cells at a single cell level, thus allowing to overcome some of these limitations not only by measuring tumor cells individu- ally but also by using endogenous endothelial cells as non- malignant controls. Using this technique, we could show an increased CS-TL in IDH1R132H mutated as compared to IDH1WT tumors. An obvious mechanism linking IDH1R132H phenotype to the loss of ATRX in human glioma may be the existence of a typical hypermethylation/CpG island methylation of the ATRX gene. Our data partially supports the results found by Ohba et al. hTERT promoter mutations, IDH1 mutations, and telomere length The incidence of TERTpmut was 43% of which 84% harbored a C228T point mutation and 16% had a C250T point muta- tion (Supplementary Figure S2A) (Table 1). The presence of TERTpmut was associated with IDH1WT (p = 0.007) (Fig. 2a). TERTpmut tumors had slightly shorter CS-TL than TERTpWT tumors (p = 0.05) (Fig. 2b). When analyzing CS-TL according to IDH1 and TERTpmut status, we con- firmed the increased CS-TL of IDH1R132 tumors but did not 1 3 ournal of Neuro-Oncology (2020) 147:1–14 Fig. 4d) middle panels). Induced IDHR132H overexpres- ion neither altered the TERTpmut status after short or long-term culture (Supplementary Figure S3D) nor signi cantly increased TERT mRNA expression level (Fig. 4 g. 2 TERT promotor, glioma cell-specific telomere length and DH status. a Distribution of IDH1WT and IDH1R132H among the ERTpmut and TERTpWT patients (p = 0.001, X2-test). b Glioma- pecific telomere length (CS-TL, in arbitrary units, a.u.) in TERTpWT and TERTpmut tumors. c CS-TL (in a.u.) of glioma patients acco ing to IDH1 and TERTpmut status. d Kaplan–Meier survival curves TERTpWT and TERTpmut patients. e Kaplan–Meier survival curves TERTpWT and TERTpmut patients stratified after IDH1 status 7 Journal of Neuro-Oncology (2020) 147:1–14 Fi 2 TERT t li ll ifi t l l th d d TERT mut t CS TL (i ) f li ti t d Fig. 2 TERT promotor, glioma cell-specific telomere length and IDH status. a Distribution of IDH1WT and IDH1R132H among the TERTpmut and TERTpWT patients (p = 0.001, X2-test). b Glioma- specific telomere length (CS-TL, in arbitrary units, a.u.) in TERTpWT and TERTpmut tumors. c CS-TL (in a.u.) of glioma patients accord- ing to IDH1 and TERTpmut status. d Kaplan–Meier survival curves of TERTpWT and TERTpmut patients. e Kaplan–Meier survival curves of TERTpWT and TERTpmut patients stratified after IDH1 status (Fig. 4d) middle panels). Induced IDHR132H overexpres- sion neither altered the TERTpmut status after short or (Fig. 4d) middle panels). Induced IDHR132H overexpres- sion neither altered the TERTpmut status after short or long-term culture (Supplementary Figure S3D) nor signifi- cantly increased TERT mRNA expression level (Fig. 4e). 1 3 Journal of Neuro-Oncology (2020) 147:1–14 8 8 Journal of Neuro-Oncology (2020) 147:1 1 3 Journal of Neuro-Oncology (2020) 147:1–14 9 association of ALT and TERTp mutations in the multivari- ate analysis have to be interpreted with caution. In line with Heidenreich et al. hTERT promoter mutations, IDH1 mutations, and telomere length In one tumor with TERTp mutations and ALT, the co-existance of two distinct TMM in IDH1R132H cells of the same tumor (ALT and telomerase-dependent mechanisms) may exist, similar to the mosaic hypothesis previously sug- gested for other tumor types, e.g. sarcomas [47]. The pro- posed dichotomy indicates that treatment strategies targeting telomere maintenance, e.g. treatment with telomerase-inhib- itors or ALT targeted treatment such e.g. PARP inhibitors or ATRX directed drugs [48, 49], must be personalized to the patient according to TMM. It is likely that the use of telomerase inhibitors can be ineffective or even detrimen- tal in treating patients with IDHR132H gliomas. Thus, the determination of ALT could be usefull as predictive marker to identify patients not responding to telomerase inhibitors. Discussion [50], who showed that IDH1 mutations do not select for or induce ATRX mutations or TERTpmut. How- ever, we found no significant reactivation of TERT. Concerning the role of ARTX mutations in gliomas [21, 23, 26–28], we confirmed the association with survival[45] and with ALT[21] as well as the association among ALT, ATRX expression loss, and IDH1R132H. However, the sam- ples size of our cohort does not allow sound conclusions the prognostic relevance of the different TMM and the lacking i In conclusion, we found that ALT is the major TMM in IDH1R132H astrocytomas and that IDH1R132H mutations can directly suppress ATRX expression resulting in ALT. 1 3 Journal of Neuro-Oncology (2020) 147:1–14 10 Fig. 4 Telomerelength after induction of IDH1R132H expression. a Production of the metabolite (inM) in the glioma cell line LN319 after the doxycycline-induced overexpression of IDH1R132H and control conditions. D2HG was measured in the medium supernatant after three population doublings in culture. b Telomere length in the doxycycline induced IDH1WT and IDH1R132H cell lines, after nine population doublings in culture (IDH1WT = 33.76 ± 1.54 a.u, n = 48 vs. IDH1R132H = 47.3 ± 1.93 a.u, n = 29). c Fold change of ATRX mRNA expression after doxycycline treatment and nine popula- tion doublings as compared to untreated controls. d Representative images of Q-FISH stained doxycycline induced IDH1WT (upper row) and IDH1R132H cell lines (lower row). All images correspond to cell culture after 14 population doublings. The presence of ALT (left panel: telomere FISH (red), DAPI counterstaining), ATRX protein expression (middle panel: ATRX immunofluorescence (green), DAPI counterstaining), and TERT expression (right panel: TERT immuno- fluorescence (green), DAPI counterstaining) is given (magnification 756x). e TERT expression levels (fold change) at the mRNA level of doxycycline-induced IDH1WT and IDH1R132H cell lines after nine population doublings in culture images of Q-FISH stained doxycycline induced IDH1WT (upper row) and IDH1R132H cell lines (lower row). All images correspond to cell culture after 14 population doublings. The presence of ALT (left panel: telomere FISH (red), DAPI counterstaining), ATRX protein expression (middle panel: ATRX immunofluorescence (green), DAPI counterstaining), and TERT expression (right panel: TERT immuno- fluorescence (green), DAPI counterstaining) is given (magnification 756x). e TERT expression levels (fold change) at the mRNA level of doxycycline-induced IDH1WT and IDH1R132H cell lines after nine population doublings in culture Fig. 4 Telomerelength after induction of IDH1R132H expression. images of Q-FISH stained doxycycline induced IDH1WT (upper row) and IDH1R132H cell lines (lower row). All images correspond to cell culture after 14 population doublings. The presence of ALT (left panel: telomere FISH (red), DAPI counterstaining), ATRX protein expression (middle panel: ATRX immunofluorescence (green), DAPI counterstaining), and TERT expression (right panel: TERT immuno- fluorescence (green), DAPI counterstaining) is given (magnification 756x). e TERT expression levels (fold change) at the mRNA level of doxycycline-induced IDH1WT and IDH1R132H cell lines after nine population doublings in culture Discussion a Production of the metabolite (inM) in the glioma cell line LN319 after the doxycycline-induced overexpression of IDH1R132H and control conditions. D2HG was measured in the medium supernatant after three population doublings in culture. b Telomere length in the doxycycline induced IDH1WT and IDH1R132H cell lines, after nine population doublings in culture (IDH1WT = 33.76 ± 1.54 a.u, n = 48 vs. IDH1R132H = 47.3 ± 1.93 a.u, n = 29). c Fold change of ATRX mRNA expression after doxycycline treatment and nine popula- tion doublings as compared to untreated controls. d Representative 1 3 11 Journal of Neuro-Oncology (2020) 147:1–14 Fig. 5 Overview of the two mechanisms of telomere maintenance. a Acquisition of IDH1R132H mutation leads to reduced ATRX expression and ALT. b Acquisition of TERTpmut mutation results in increased telomerase expression and telomere elongation via telomerase are handled by the DKFZ Technology Transfer department. The other authors have nothing to disclose. Acknowledgements Open Access funding provided by Projekt DEAL. We would like to thank Andreas von Deimling for his excellent com- ments on the manuscript. Further, we are thankful to Anne Abels for the excellent technical assistance. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Author contributions MSVF: Performed the experiments analyzed, interpreted the data and wrote the manuscript. MDS: Performed the IHC experiments, analyzed and interpreted the data, and revised the manuscript. SP: Provided and helped performing the 2HG and the LN319 IDHR132H assays and revised the manuscript. DB: Provided clinical data and interpreted the data. ASB: performed experiments and analyzed the data. BWK: Provided patient samples, clinical data and interpreted the data. References 1. Huse JT, Phillips HS, Brennan CW (2011) Molecular subclas- sification of diffuse gliomas: seeing order in the chaos. Glia 59:1190–1199. https://doi.org/10.1002/glia.21165 Discussion THB provided fincancial support, analyzed and interpreted the data and wrote the manuscript; FB, CPB con- ceived and planned the study design, interpreted the data, and wrote the manuscript. Funding This work was supported by a Grant START grant (RWTH Aachen University) to M.S.V.F. and Region of Southern Denmark Grant to C.P.B. (17/18517). Fig. 5 Overview of the two mechanisms of telomere maintenance. a Acquisition of IDH1R132H mutation leads to reduced ATRX expression and ALT. b Acquisition of TERTpmut mutation results in increased telomerase expression and telomere elongation via telomerase Compliance with ethical standards Cancer Genome Atlas Research N, Brat DJ, Verhaak RG, Aldape KD, Yung WK, Salama SR, Cooper LA, Rheinbay E, Miller CR, Vitucci M, Morozova O, Robertson AG, Noushmehr H, Laird PW, Cherniack AD, Akbani R, Huse JT, Ciriello G, Poisson LM, Barnholtz-Sloan JS, Berger MS, Brennan C, Colen RR, Colman H, Flanders AE, Giannini C, Grifford M, Iavarone A, Jain R, Joseph I, Kim J, Kasaian K, Mikkelsen T, Murray BA, O’Neill BP, Pachter L, Parsons DW, Sougnez C, Sulman EP, Vandenberg SR, Van Meir EG, von Deimling A, Zhang H, Crain D, Lau K, Mallery D, Morris S, Paulauskis J, Penny R, Shelton T, Sherman M, Yena P, Black A, Bowen J, Dicostanzo K, Gastier-Foster J, Leraas KM, Lichtenberg TM, Pierson CR, Ramirez NC, Taylor C, Weaver S, Wise L, Zmuda E, Davidsen T, Demchok JA, Eley G, Ferguson ML, Hutter CM, Mills Shaw KR, Ozenberger BA, Sheth M, Sofia HJ, Tarnuzzer R, Wang Z, Yang L, Zenklusen JC, Ayala B, Baboud J, Chudamani S, Jensen MA, Liu J, Pihl T, Raman R, Wan Y, Wu Y, Ally A, Auman JT, Balasundaram M, Balu S, Baylin SB, Beroukhim R, Bootwalla MS, Bowlby R, Bristow CA, Brooks D, Butterfield Y, Carlsen R, Carter S, Chin L, Chu A, Chuah E, Cibulskis K, Clarke A, Coetzee SG, Dhalla N, Fennell T, Fisher S, Gabriel S, Getz G, Gibbs R, Guin R, Hadji- panayis A, Hayes DN, Hinoue T, Hoadley K, Holt RA, Hoyle AP, Jefferys SR, Jones S, Jones CD, Kucherlapati R, Lai PH, Lander E, Lee S, Lichtenstein L, Ma Y, Maglinte DT, Mahadeshwar HS, Marra MA, Mayo M, Meng S, Meyerson ML, Mieczkowski PA, Moore RA, Mose LE, Mungall AJ, Pantazi A, Parfenov M, Park PJ, Parker JS, Perou CM, Protopopov A, Ren X, Roach J, Sabedot TS, Schein J, Schumacher SE, Seidman JG, Seth S, Shen H, Simons JV, Sipahimalani P, Soloway MG, Song X, Sun H, Tabak B, Tam A, Tan D, Tang J, Thiessen N, Triche T Jr, Van Den Berg DJ, Veluvolu U, Waring S, Weisenberger DJ, Wilkerson MD, Wong T, Wu J, Xi L, Xu AW, Yang L, Zack TI, Zhang J, Aksoy BA, Arachchi H, Benz C, Bernard B, Carlin D, Cho J, DiCara D, Frazer S, Fuller GN, Gao J, Gehlenborg N, Haussler D, Heiman DI, Iype L, Jacobsen A, Ju Z, Katzman S, Kim H, Knijnenburg T, Kreisberg RB, Lawrence MS, Lee W, Leinonen K, Lin P, Ling S, Liu W, Liu Y, Liu Y, Lu Y, Mills G, Ng S, Noble MS, Paull E, Rao A, Reynolds S, Saksena G, Sanborn Z, Sander C, Schultz N, Senbabaoglu Y, Shen R, Shmulevich I, Sinha R, Stuart J, Sumer SO, Sun Y, Tasman N, Taylor BS, Voet D, Weinhold N, Weinstein JN, Yang D, Yoshihara K, Zheng S, Zhang W, Zou L, Abel T, Sadeghi S, Cohen ML, Eschbacher J, Hattab EM, Raghunathan A, Schniederjan MJ, Aziz D, Barnett G, Barrett W, Bigner DD, Boice L, Brewer C, Calatozzolo C, Campos B, Carlotti CG Jr, Chan TA, Cuppini L, Curley E, Cuzzubbo S, Devine K, DiMeco F, Duell R, Elder JB, Fehrenbach A, Finocchiaro G, Friedman W, Fulop J, Gardner J, Hermes B, Herold-Mende C, Jungk C, Kendler A, Lehman NL, Lipp E, Liu O, Mandt R, McGraw M, McLen- don R, McPherson C, Neder L, Nguyen P, Noss A, Nunziata R, Ostrom QT, Palmer C, Perin A, Pollo B, Potapov A, Potapova O, Rathmell WK, Rotin D, Scarpace L, Schilero C, Senecal K, Shimmel K, Shurkhay V, Sifri S, Singh R, Sloan AE, Smolenski K, Staugaitis SM, Steele R, Thorne L, Tirapelli DP, Unterberg A, Vallurupalli M, Wang Y, Warnick R, Williams F, Wolinsky Y, Bell 7. Compliance with ethical standards Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, Jewell CM, Johnson ZR, Irvine DJ, Guarente L, Kelleher JK, Vander Heiden MG, Iliopoulos O, Stephanopoulos G (2011) Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481:380–384. https://doi.org/10.1038/ nature10602 8. Nobusawa S, Watanabe T, Kleihues P, Ohgaki H (2009) IDH1 mutations as molecular signature and predictive factor of sec- ondary glioblastomas. Clin Cancer Res 15:6002–6007. https:// doi.org/10.1158/1078-0432.CCR-09-0715 9. Pusch S, Schweizer L, Beck AC, Lehmler JM, Weissert S, Balss J, Miller AK, von Deimling A (2014) D-2-hydroxyglutarate producing neo-enzymatic activity inversely correlates with frequency of the type of isocitrate dehydrogenase 1 mutations found in glioma. Acta Neuropathol Commun 2:19. https://doi. org/10.1186/2051-5960-2-19 10. Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, Ito S, Yang C, Wang P, Xiao MT, Liu LX, Jiang WQ, Liu J, Zhang JY, Wang B, Frye S, Zhang Y, Xu YH, Lei QY, Guan KL, Zhao SM, Xiong Y (2011) Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Can- cer Cell 19:17–30. https://doi.org/10.1016/j.ccr.2010.12.014 11. Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Berman BP, Pan F, Pelloski CE, Sulman EP, Bhat KP, Ver- haak RG, Hoadley KA, Hayes DN, Perou CM, Schmidt HK, Ding L, Wilson RK, Van Den Berg D, Shen H, Bengtsson H, Neuvial P, Cope LM, Buckley J, Herman JG, Baylin SB, Laird PW, Aldape K (2010) Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 17:510–522. https://doi.org/10.1016/j.ccr.2010.03.017 12. Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, Campos C, Fabius AW, Lu C, Ward PS, Thompson CB, Kauf- man A, Guryanova O, Levine R, Heguy A, Viale A, Morris LG, Huse JT, Mellinghoff IK, Chan TA (2012) IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483:479–483. https://doi.org/10.1038/nature10866 13. Blackburn EH (2005) Telomeres and telomerase: their mecha- nisms of action and the effects of altering their functions. FEBS Lett 579:859–862 14. Yuan P, Cao JL, Abuduwufuer A, Wang LM, Yuan XS, Lv W, Hu J (2016) Clinical characteristics and prognostic signifi- cance of TERT promoter mutations in cancer: a cohort study and a meta-analysis. PLoS ONE 11:e0146803. https://doi. org/10.1371/journal.pone.0146803 15. Compliance with ethical standards p g g 2. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella- Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW (2016) The 2016 World Health Organization Clas- sification of Tumors of the Central Nervous System: a summary. 2. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella- Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW (2016) The 2016 World Health Organization Clas- sification of Tumors of the Central Nervous System: a summary. 2. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella- Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW (2016) The 2016 World Health Organization Clas- sification of Tumors of the Central Nervous System: a summary. Conflict of interest Stefan Pusch is patent holder of a patient on the 2HG assay used in this manuscript. All terms concerning this patent 1 3 12 Journal of Neuro-Oncology (2020) 147:1–14 S, Rosenberg M, Stewart C, Huang F, Grimsby JL, Radenbaugh AJ, Zhang J (2015) Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N Engl J Med 372:2481–2498. https://doi.org/10.1056/NEJMoa1402121 Acta Neuropathol 131:803–820. https://doi.org/10.1007/s0040 1-016-1545-1 S, Rosenberg M, Stewart C, Huang F, Grimsby JL, Radenbaugh AJ, Zhang J (2015) Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N Engl J Med 372:2481–2498. https://doi.org/10.1056/NEJMoa1402121 3. Eckel-Passow JE, Lachance DH, Molinaro AM, Walsh KM, Decker PA, Sicotte H, Pekmezci M, Rice T, Kosel ML, Smirnov IV, Sarkar G, Caron AA, Kollmeyer TM, Praska CE, Chada AR, Halder C, Hansen HM, McCoy LS, Bracci PM, Marshall R, Zheng S, Reis GF, Pico AR, O’Neill BP, Buckner JC, Giannini C, Huse JT, Perry A, Tihan T, Berger MS, Chang SM, Prados MD, Wie- mels J, Wiencke JK, Wrensch MR, Jenkins RB (2015) Glioma groups based on 1p/19q, IDH, and TERT promoter mutations in tumors. N Engl J Med 372:2499–2508. https://doi.org/10.1056/ NEJMoa1407279 5. 5. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD (2009) IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765–773. https://doi.org/10.1056/ NEJMoa0808710 6. Dunn GP, Andronesi OC, Cahill DP (2013) From genomics to the clinic: biological and translational insights of mutant IDH1/2 in glioma. Neurosurg Focus 34:E2. https://doi.org/10.3171/2012.12. FOCUS12355 4. Compliance with ethical standards Kannan K, Inagaki A, Silber J, Gorovets D, Zhang J, Kastenhuber ER, Heguy A, Petrini JH, Chan TA, Huse JT (2012) Whole-exome sequencing identifies ATRX mutation as a key molecular determi- nant in lower-grade glioma. Oncotarget 3:1194–1203. https://doi. org/10.18632/oncotarget.689 p g g 19. Langford LA, Piatyszek MA, Xu R, Schold SC Jr, Shay JW (1995) Telomerase activity in human brain tumours. Lancet 346:1267–1268 20. Heaphy CM, Subhawong AP, Hong SM, Goggins MG, Mont- gomery EA, Gabrielson E, Netto GJ, Epstein JI, Lotan TL, Westra WH, Shih Ie M, Iacobuzio-Donahue CA, Maitra A, Li QK, Eberhart CG, Taube JM, Rakheja D, Kurman RJ, Wu TC, Roden RB, Argani P, De Marzo AM, Terracciano L, Torbenson M, Meeker AK (2011) Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. Am J Pathol 179:1608–1615. https://doi.org/10.1016/j. ajpath.2011.06.018 28. Liu XY, Gerges N, Korshunov A, Sabha N, Khuong-Quang DA, Fontebasso AM, Fleming A, Hadjadj D, Schwartzentruber J, Majew- ski J, Dong Z, Siegel P, Albrecht S, Croul S, Jones DT, Kool M, Tonjes M, Reifenberger G, Faury D, Zadeh G, Pfister S, Jabado N (2012) Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 muta- tions. Acta Neuropathol 124:615–625. https://doi.org/10.1007/s0040 1-012-1031-3 29. Sorensen MD, Dahlrot RH, Boldt HB, Hansen S, Kristensen BW (2018) Tumour-associated microglia/macrophages predict poor prognosis in high-grade gliomas and correlate with an aggressive tumour subtype. Neuropathol Appl Neurobiol 44:185–206. https:// doi.org/10.1111/nan.12428 21. Heaphy CM, de Wilde RF, Jiao Y, Klein AP, Edil BH, Shi C, Bettegowda C, Rodriguez FJ, Eberhart CG, Hebbar S, Offer- haus GJ, McLendon R, Rasheed BA, He Y, Yan H, Bigner DD, Oba-Shinjo SM, Marie SK, Riggins GJ, Kinzler KW, Vogelstein B, Hruban RH, Maitra A, Papadopoulos N, Meeker AK (2011) Altered telomeres in tumors with ATRX and DAXX mutations. Science 333:425. https://doi.org/10.1126/science.1207313 30. Dahlrot RH, Kristensen BW, Hjelmborg J, Herrstedt J, Hansen S (2013) A population-based study of high-grade gliomas and mutated isocitrate dehydrogenase 1. Int J Clin Exp Pathol 6:31–40 22. Compliance with ethical standards Ceccarelli M, Barthel FP, Malta TM, Sabedot TS, Salama SR, Murray BA, Morozova O, Newton Y, Radenbaugh A, Pagnotta SM, Anjum S, Wang J, Manyam G, Zoppoli P, Ling S, Rao AA, Grifford M, Cherniack AD, Zhang H, Poisson L, Carlotti CG Jr, Tirapelli DP, Rao A, Mikkelsen T, Lau CC, Yung WK, Rabadan R, Huse J, Brat DJ, Lehman NL, Barnholtz-Sloan JS, Zheng S, Hess K, Rao G, Meyerson M, Beroukhim R, Cooper L, Akbani R, Wrensch M, Haussler D, Aldape KD, Laird PW, 1 3 3 Journal of Neuro-Oncology (2020) 147:1–14 13 Gutmann DH, Network TR, Noushmehr H, Iavarone A, Ver- haak RG (2016) Molecular profiling reveals biologically dis- crete subsets and pathways of progression in diffuse glioma. Cell 164:550–563. https://doi.org/10.1016/j.cell.2015.12.028 localization at specific genomic regions. Cell 140:678–691. https ://doi.org/10.1016/j.cell.2010.01.003 Gutmann DH, Network TR, Noushmehr H, Iavarone A, Ver- haak RG (2016) Molecular profiling reveals biologically dis- crete subsets and pathways of progression in diffuse glioma. Cell 164:550–563. https://doi.org/10.1016/j.cell.2015.12.028 25. Lewis PW, Elsaesser SJ, Noh KM, Stadler SC, Allis CD (2010) Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telom- eres. Proc Natl Acad Sci USA 107:14075–14080. https://doi. org/10.1073/pnas.1008850107 16. Arita H, Narita Y, Fukushima S, Tateishi K, Matsushita Y, Yoshida A, Miyakita Y, Ohno M, Collins VP, Kawahara N, Shibui S, Ichimura K (2013) Upregulating mutations in the TERT promoter commonly occur in adult malignant glio- mas and are strongly associated with total 1p19q loss. Acta Neuropathol 126:267–276. https://doi.org/10.1007/s0040 1-013-1141-6 g p 26. Jiao Y, Killela PJ, Reitman ZJ, Rasheed AB, Heaphy CM, de Wilde RF, Rodriguez FJ, Rosemberg S, Oba-Shinjo SM, Nagahashi Marie SK, Bettegowda C, Agrawal N, Lipp E, Pirozzi C, Lopez G, He Y, Friedman H, Friedman AH, Riggins GJ, Holdhoff M, Burger P, McLendon R, Bigner DD, Vogelstein B, Meeker AK, Kinzler KW, Papadopoulos N, Diaz LA, Yan H (2012) Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas. Oncotarget 3:709–722. https://doi.org/10.18632/oncotarget .588 17. Hiraga S, Ohnishi T, Izumoto S, Miyahara E, Kanemura Y, Mat- sumura H, Arita N (1998) Telomerase activity and alterations in telomere length in human brain tumors. Cancer Res 58:2117–2125 18. Cesare AJ, Reddel RR (2010) Alternative lengthening of tel- omeres: models, mechanisms and implications. Nat Rev Genet 11:319–330. https://doi.org/10.1038/nrg2763 27. Compliance with ethical standards Lovejoy CA, Li W, Reisenweber S, Thongthip S, Bruno J, de Lange T, De S, Petrini JH, Sung PA, Jasin M, Rosenbluh J, Zwang Y, Weir BA, Hatton C, Ivanova E, Macconaill L, Hanna M, Hahn WC, Lue NF, Reddel RR, Jiao Y, Kinzler K, Vogelstein B, Papadopoulos N, Meeker AK, Consortium ALTSC (2012) Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway. PLoS Genet 8:e1002772. https://doi.org/10.1371/journ al.pgen.1002772 31. Ferreira MSV, Crysandt M, Braunschweig T, Jost E, Voss B, Bouil- lon AS, Knuechel R, Brummendorf TH, Beier F (2018) Presence of TERT promoter mutations is a secondary event and associates with elongated telomere length in myxoid liposarcomas. Int J Mol Sci. https://doi.org/10.3390/ijms19020608 32. Ventura Ferreira MS, Bienert M, Muller K, Rath B, Goecke T, Oplander C, Braunschweig T, Mela P, Brummendorf TH, Beier F, Neuss S (2018) Comprehensive characterization of chorionic villi- derived mesenchymal stromal cells from human placenta. Stem Cell Res Ther 9:28. https://doi.org/10.1186/s13287-017-0757-1 pg 23. Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K, Sturm D, Fontebasso AM, Quang DA, Tonjes M, Hovestadt V, Albrecht S, Kool M, Nantel A, Konermann C, Lin- droth A, Jager N, Rausch T, Ryzhova M, Korbel JO, Hielscher T, Hauser P, Garami M, Klekner A, Bognar L, Ebinger M, Schuh- mann MU, Scheurlen W, Pekrun A, Fruhwald MC, Roggendorf W, Kramm C, Durken M, Atkinson J, Lepage P, Montpetit A, Zakrze- wska M, Zakrzewski K, Liberski PP, Dong Z, Siegel P, Kulozik AE, Zapatka M, Guha A, Malkin D, Felsberg J, Reifenberger G, von Deimling A, Ichimura K, Collins VP, Witt H, Milde T, Witt O, Zhang C, Castelo-Branco P, Lichter P, Faury D, Tabori U, Plass C, Majewski J, Pfister SM, Jabado N (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482:226–231. https://doi.org/10.1038/natur e10833 33. Hummel S, Ventura Ferreira MS, Heudobler D, Huber E, Fahren- kamp D, Gremse F, Schmid K, Muller-Newen G, Ziegler P, Jost E, Blasco MA, Brummendorf TH, Holler E, Beier F (2015) Telomere shortening in enterocytes of patients with uncontrolled acute intes- tinal graft-versus-host disease. Blood 126:2518–2521. https://doi. org/10.1182/blood-2015-03-633289 34. Compliance with ethical standards Ziegler S, Schettgen T, Beier F, Wilop S, Quinete N, Esser A, Masouleh BK, Ferreira MS, Vankann L, Uciechowski P, Rink L, Kraus T, Brummendorf TH, Ziegler P (2017) Accelerated telomere shortening in peripheral blood lymphocytes after occupational poly- chlorinated biphenyls exposure. Arch Toxicol 91:289–300. https:// doi.org/10.1007/s00204-016-1725-8 35. Beier F, Foronda M, Martinez P, Blasco MA (2012) Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita. Blood 120:2990–3000 24. Goldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Dewell S, Law M, Guo X, Li X, Wen D, Chapgier A, DeKelver RC, Miller JC, Lee YL, Boydston EA, Holmes MC, Gregory PD, Greally JM, Rafii S, Yang C, Scambler PJ, Gar- rick D, Gibbons RJ, Higgs DR, Cristea IM, Urnov FD, Zheng D, Allis CD (2010) Distinct factors control histone variant H3.3 36. Beier F, Balabanov S, Buckley T, Dietz K, Hartmann U, Rojewski M, Kanz L, Schrezenmeier H, Brummendorf TH (2005) Accelerated telomere shortening in glycosylphosphatidylinositol (GPI)-negative 1 Journal of Neuro-Oncology (2020) 147:1–14 14 A, Giannini C, Tihan T, Berger MS, Wiemels JL, Bracci PM, Eckel- Passow JE, Lachance DH, Clarke J, Taylor JW, Luks T, Wiencke JK, Jenkins RB, Wrensch MR (2017) Adult infiltrating gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT. Acta Neuropathol 133:1001–1016. https://doi. org/10.1007/s00401-017-1690-1 compared with GPI-positive granulocytes from patients with par- oxysmal nocturnal hemoglobinuria (PNH) detected by proaerolysin flow-FISH. Blood 106:531–533 A, Giannini C, Tihan T, Berger MS, Wiemels JL, Bracci PM, Eckel- Passow JE, Lachance DH, Clarke J, Taylor JW, Luks T, Wiencke JK, Jenkins RB, Wrensch MR (2017) Adult infiltrating gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT. Acta Neuropathol 133:1001–1016. https://doi. org/10.1007/s00401-017-1690-1 l 37. Beier F, Martinez P, Blasco MA (2015) Chronic replicative stress induced by CCl4 in TRF1 knockout mice recapitulates the origin of large liver cell changes. J Hepatol 63:446–455 46. Mukherjee J, Johannessen T-CA, Ohba S, Chow TT, Jones LE, Pan- dita A, Pieper RO (2018) Mutant IDH1 cooperates with ATRX loss to drive the alternative lengthening of telomere (ALT) phenotype in glioma. Cancer Res. https://doi.org/10.1158/0008-5472.can-17-2269 38. Compliance with ethical standards Beier F, Masouleh BK, Buesche G, Ventura Ferreira MS, Schneider RK, Ziegler P, Wilop S, Vankann L, Gattermann N, Platzbecker U, Giagounidis A, Gotze KS, Nolte F, Hofmann WK, Haase D, Kreipe H, Panse J, Blasco MA, Germing U, Brummendorf TH (2015) Tel- omere dynamics in patients with del (5q) MDS before and under treatment with lenalidomide. Leuk Res. https://doi.org/10.1016/j. leukres.2015.09.003 47. Gocha AR, Nuovo G, Iwenofu OH, Groden J (2013) Human sarco- mas are mosaic for telomerase-dependent and telomerase-independ- ent telomere maintenance mechanisms: implications for telomere- based therapies. Am J Pathol 182:41–48. https://doi.org/10.1016/j. ajpath.2012.10.001 39. Birner P, Pusch S, Christov C, Mihaylova S, Toumangelova-Uzeir K, Natchev S, Schoppmann SF, Tchorbanov A, Streubel B, Tuettenberg J, Guentchev M (2014) Mutant IDH1 inhibits PI3K/Akt signaling in human glioma. Cancer 120:2440–2447. https://doi.org/10.1002/ cncr.28732 48. Kovatcheva M, Liao W, Klein ME, Robine N, Geiger H, Crago AM, Dickson MA, Tap WD, Singer S, Koff A (2017) ATRX is a regulator of therapy induced senescence in human cells. Nat Commun 8:386. https://doi.org/10.1038/s41467-017-00540-5 40. Tefferi A, Lasho TL, Begna KH, Patnaik MM, Zblewski DL, Finke CM, Laborde RR, Wassie E, Schimek L, Hanson CA, Gangat N, Wang X, Pardanani A (2015) A pilot study of the telomerase inhibi- tor imetelstat for myelofibrosis. N Engl J Med 373:908–919 49. Koschmann C, Calinescu AA, Nunez FJ, Mackay A, Fazal-Salom J, Thomas D, Mendez F, Kamran N, Dzaman M, Mulpuri L, Kra- sinkiewicz J, Doherty R, Lemons R, Brosnan-Cashman JA, Li Y, Roh S, Zhao L, Appelman H, Ferguson D, Gorbunova V, Meeker A, Jones C, Lowenstein PR, Castro MG (2016) ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma. Sci Transl Med 8:328ra328. https://doi.org/10.1126/scitr anslmed.aac8228 i 41. Baerlocher GM, Oppliger Leibundgut E, Ottmann OG, Spitzer G, Odenike O, McDevitt MA, Roth A, Daskalakis M, Burington B, Stu- art M, Snyder DS (2015) Telomerase inhibitor imetelstat in patients with essential thrombocythemia. N Engl J Med 373:920–928 42. Heidenreich B, Rachakonda PS, Hosen I, Volz F, Hemminki K, Weyerbrock A, Kumar R (2015) TERT promoter mutations and telomere length in adult malignant gliomas and recurrences. Onco- target 6:10617–10633. https://doi.org/10.18632/oncotarget.3329 50. Compliance with ethical standards Ohba S, Mukherjee J, Johannessen TC, Mancini A, Chow TT, Wood M, Jones L, Mazor T, Marshall RE, Viswanath P, Walsh KM, Perry A, Bell RJ, Phillips JJ, Costello JF, Ronen SM, Pieper RO (2016) Mutant IDH1 expression drives TERT promoter reactivation as part of the cellular transformation process. Cancer Res 76:6680–6689. https://doi.org/10.1158/0008-5472.CAN-16-0696 43. Gao K, Li G, Qu Y, Wang M, Cui B, Ji M, Shi B, Hou P (2016) TERT promoter mutations and long telomere length predict poor survival and radiotherapy resistance in gliomas. Oncotarget 7:8712– 8725. https://doi.org/10.18632/oncotarget.6007 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p g g 44. Wang S, Chen Y, Qu F, He S, Huang X, Jiang H, Jin T, Wan S, Xing J (2014) Association between leukocyte telomere length and glioma risk: a case-control study. Neuro-Oncol 16:505–512. https ://doi.org/10.1093/neuonc/not240 g 45. Pekmezci M, Rice T, Molinaro AM, Walsh KM, Decker PA, Hansen H, Sicotte H, Kollmeyer TM, McCoy LS, Sarkar G, Perry 1 3 3
https://openalex.org/W2524469494	https://hull-repository.worktribe.com/preview/443855/2016-10-03%20Watson.pdf	English	null	Getting practice into evidence	Obzornik zdravstvene nege	2,016	cc-by	1,969	Leading article/Uvodnik Getting practice into evidence S prakso podprti dokazi Leading article/Uvodnik Roger Watson Make sure you read the title correctly as it is more common to see titles that examine 'getting evidence into practice'. I am not just playing with words here; I think we have had at least two decades of calls to close the gap between evidence and practice which assume that the evidence exists and, if it exists, it must be implemented (Watson, 2015). However, 'evidence' is rarely unequivocal and, where the benefits may be small, it is not easy—and probably not worth—convincing those who hold health service budgets to make changes which may or may not be more expensive.h staff and their allied health and medical colleagues see the problem, they will not know that it is being fixed. Unless these frontline staff are asked what problems they face, and they rarely are in my experience, then nobody will know what their problems are. But how does this relate to my title 'Getting practice into evidence'? By getting practice into evidence I mean that the evidence presented to frontline healthcare workers should address the problems they face. To achieve that they must be given the opportunity to express the problems they face in their clinical work and the evidence - where it exists - should be sought to help them address those problems. In that way, the evidence will be based on practice issues and, thereby, on practice and is more likely to be owned, adopted and implemented. Where the evidence does not exist - and this is remarkably common - then this provides the opportunity for research projects. These are likely to be small scale and not definitive but they will be the first steps in the direction of solving recognised problems and contribute to the body of knowledge and, ultimately, to the evidence base. The problem with evidence is that its generation is usually policy driven, either at national level or at whatever organisational level health services are administered across the globe (Watson, 2002). A problem becomes apparent, a public scandal over health care arises, such as at the Mid-Staffordshire Hospital in England (Francis, 2013) or, more commonly, something is costing too much and the search for solutions begins. The assumption is that something can be done about it, driven by the belief that something must be done about it. Obzornik zdravstvene nege, 50(3), pp. 180–182. Obzornik zdravstvene nege, 50(3), pp. 180–182. Professor Roger Watson, PhD, RN, FAAN; Faculty of Health and Social Care, University of Hull, HU6 7RX UK, Hull, U Correspondence e-mail/Kontaktni e-naslov: R.Watson@hull.ac.uk Professor Roger Watson, PhD, RN, FAAN; Faculty of Health and Social Care, University of Hull, HU6 7RX UK, Hull, UK Correspondence e-mail/Kontaktni e-naslov: R.Watson@hull.ac.uk Received/Prejeto: 11. 7. 2016 Accepted/Sprejeto: 25. 8. 2016 Leading article/Uvodnik Getting practice into evidence S prakso podprti dokazi This is the classic 'top down' approach and it is expedited, usually, by evidence synthesis. If it is by research, such an agenda is set that the academics who take up these projects have little leeway to exercise their imaginations and, actually, make a discovery. In a sense, the answer is 'begged' and if it is not found then the project is deemed to have failed; the very antithesis of science which ensues through curiosity and inquiry.h y Over the past few decades, small scale research seems to have been eschewed in favour of large scale, multi-million euro, multidisciplinary and large team collaborative research. This is clearly an effective strategy for problems which can be addressed this way and the RN4CAST project is an obvious and very successful example (Aiken, et al., 2014). However, while staff-to-patient ratios are an issue for frontline staff, I doubt many are vexed about the proportion of graduate nurses in the profession and, in terms of staff to patient ratios, frontline staff have no control over this. Small scale local research projects are ideal for solving local problems and they should not be dismissed, as so often they are. They can lead to greater things and it is impossible to predict where they will lead. The problem with the 'top down' approach to evidence implementation is that evidence-driven changes are usually being implemented by people who did not know they had the problem in the first place. If they do not see the sense in the change, then, unless coerced into doing so by micro-management, they will subvert the change anyway; such is the fate of so many good policy driven intentions. Unless frontline nursing http://dx.doi.org/10.14528/snr.2016.50.3.105 Watson, R., 2016. / Obzornik zdravstvene nege, 50(3), pp. 180–182. 181 However, getting practice into evidence and back again into practice does not happen by accident. Presently, we provide undergraduate nurses and allied health students with training in research and evidence- based practice. This, of course, is essential but it is only part of the solution. At the clinical level there needs to be a commitment and a strategy to ensuring that evidence and practice are linked. Strategy means budgets and personnel and a visible commitment to the cause. Therefore, how many hospitals have a research or evidence-based practice unit - actually concerned with doing research and not, as in so many cases, simply regulating it? Leading article/Uvodnik Getting practice into evidence S prakso podprti dokazi How many hospitals have a group related to research and evidence-based practice that includes frontline clinical staff? prostora za oblikovanje novih idej in dejanska znanstvena odkritja. Če rezultati ne ustrezajo željam in potrebam, projekt ne bo uspešen, kar predstavlja pravo nasprotje znanosti, ki se razvija na temelju vedoželjnosti in raziskovanja. j j Problem pristopa z »vrha navzdol« pri z dokazi podprti praksi je predvsem to, da spremembe, ki jih narekujejo dokazi, izvajajo posamezniki, ki se niti ne zavedajo, da problem obstaja. Če se izvajalci ne zavedajo pomena sprememb, jih ne bodo podpirali in jih bodo izvajali le pod pritiskom neposrednih vodij. Tako pogosto propadejo številne dobre namere zdravstvene politike. Če se v vodstvih zdravstvene nege in povezanih zdravstvenih disciplin problema ne bodo zavedali, tudi ne bodo vedeli, ali je problem rešen. Če vodilnih delavcev ne bomo povprašali, s kakšnimi problemi se soočajo, in to se po mojih izkušnjah le redko zgodi, nihče ne bo vedel, kakšni so ti problemi. In kako se to povezuje z naslovom prispevka »S prakso podprti dokazi«? f I will not end this editorial with a solution, rather I will point you to one excellent example where solutions are being sought which is the Evidence-based Council at Sheffield Teaching Hospitals National Health Service Trust in England (http://tinyurl.com/z39o2p2). Here an identifiable and funded group of committed individuals ask what the problems are, help staff to find solutions and then help them to publish those solutions. They also hold annual celebrations of achievement. I consider this an exemplary demonstration of how to bring practice and evidence closer together and if you want to know more, then information is easy to obtain on Google. Zagovarjam stališče, da bi morali dokazi iz prakse, predstavljeni vodilnim zdravstvenim delavcem, naslavljati probleme, s katerimi se le-ti soočajo. Zdravstveni delavci bodo učinkovito opravljali svoje delo le, če bodo imeli možnost izraziti probleme, ki jih v klinični praksi zaznavajo. Potrebno je poiskati dokaze (kjer le-ti obstajajo), ki bi omogočili reševanje problemov. Tako bi dokazi temeljili na problemih iz klinične prakse, torej na praksi, kar je lažje privzeti, sprejeti in izvajati. Kjer dokazi ne obstajajo, kar se dogaja izjemno pogosto, se ponujajo možnosti za raziskovanje. Taki raziskovalni projekti bi bili verjetno manjšega obsega in nedokončni, predstavljali pa bi prvi korak in usmeritev pri reševanju prepoznanih problemov ter prispevali nova spoznanja in končno tudi širitev baze dokazov. Slovenian translation/Prevod v slovenščino Ste prav prebrali naslov? Pogosteje se namreč srečujemo s prispevki, ki preučujejo »z dokazi podprto prakso« V naslovu se ne poigravam z besedami – že vsaj dve desetletji se pojavljajo zahteve, da se izpolni vrzel med dokazi in klinično prakso, ki predpostavlja, da dokazi obstajajo, in če obstajajo, jih je potrebno v praksi uporabiti (Watson, 2015). Vendar pa so »dokazi« le redko nedvoumni, in kadar so koristi majhne, ni enostavno oziroma ni vredno prepričevati plačnikov zdravstveni storitev, da uvajajo spremembe, ki so (ali morda tudi ne) povezane z večjimi stroški. V zadnjih desetletjih se manjše raziskave umikajo večjim multidisciplinarnim raziskovalnim projektom, ki so podprti z večmilijonskimi sredstvi in kjer sodelujejo številni znanstveniki različnih disciplin. To je seveda uspešna strategija za reševanje določenih problemov; kot odličen primer lahko izpostavimo projekt RN4CAST (Aiken, et al., 2014). Čeprav se vodilni zdravstveni delavci zavedajo problema številčnega razmerja med zdravstvenimi delavci in pacienti, pa je manj izražena skrb glede števila diplomiranih medicinskih sester med izvajalci zdravstvene nege oz. števila pacientov na posamezno diplomirano medicinsko sestro, na kar imajo vodilne medicinske sestre le neznaten vpliv. Z manjšimi raziskovalnimi projekti lahko uspešno rešujemo lokalne probleme, zato jih ni smiselno opuščati, čeprav se danes to žal pogosto dogaja. Rezultati takih raziskovanj so lahko osnova za pomembne spremembe in težko je v celoti predvideti njihovo vrednost. p j Problem dokazov je predvsem to, da njihovo oblikovanje vodi zdravstvena politika na nacionalni ravni ali na ravni organizacij, ki opravljajo zdravstvene storitve v različnih delih sveta (Watson, 2002). Iskanje ustreznih rešitev se lahko prične šele, ko je problem na področju zdravstva v javnosti prepoznan in morda doseže sramotne razsežnosti, kot kaže primer bolnišnice Mid-Staffordshire (Mid-Staffordshire Hospital) v Angliji (Francis, 2013), ali pa največkrat zato, ker je trenutno reševanje problema povezano z velikimi stroški. Predpostavlja se, da je rešitev možno najti zato, ker jo je potrebno najti. To je klasičen pristop »z vrha navzdol«, ki se običajno pospeši s sintezo dokazov. Če to sintezo izvedejo znanstveniki – raziskovalci, imajo pri tem le malo manevrskega Zavedati se moramo, da vključevanje prakse v iskanje dokazov in le-teh nazaj v klinično prakso ni samodejen proces. Danes se medicinske sestre in Watson, R., 2016. / Obzornik zdravstvene nege, 50(3), pp. 180–182. 182 dosežke tudi obeležijo. Gre za odličen zgled zbliževanja prakse in dokazov, še več o njem si lahko preberete na spletu. dosežke tudi obeležijo. Slovenian translation/Prevod v slovenščino Gre za odličen zgled zbliževanja prakse in dokazov, še več o njem si lahko preberete na spletu. drugi zdravstveni delavci že v času dodiplomskega izobraževanja usposabljajo za raziskovalno delo in z dokazi podprto prakso. To so seveda temelji, vendar le del rešitve. V klinični praksi so potrebni predanost zagotavljanju povezovanja dokazov s prakso in temu ustrezne strategije. Strategije vključujejo financiranje in zagotavljanje kompetentnih zdravstvenih strokovnjakov ter izraženo predanost doseganju teh ciljev. Torej, koliko bolnišnic ima svoje enote, ki se ukvarjajo z raziskovanjem in z dokazi podprto prakso? Koliko bolnišnic dejansko opravlja raziskovalno delo in ga ne le usmerja, kar se v večini primerov dogaja? Koliko bolnišnic v to delo vključuje vodilne medicinske sestre? Cite as/Citirajte kot: Watson, R., 2016. Getting practice into evidence. Obzornik zdravstvene nege, 50(3), pp. 180–182. http://dx.doi.org/10.14528/snr.2016.50.3.105 / j Watson, R., 2016. Getting practice into evidence. Obzornik zdravstvene nege, 50(3), pp. 180–182. http://dx.doi.org/10.14528/snr.2016.50.3.105 Literature/Literatura Aiken, L.H., Sloane, D., Bruyneel, L., Van den Heede, K., Griffiths, P. & Busse, R. 2014. Nurse staffing and education and hospital mortality in nine European countries: a retrospective observational study. Lancet, 383(9931), pp. 1824–1830. Francis, R., 2013. Report of the Mid Staffordshire NHS Foundation Trust Public Inquiry: executive summary. London: The Stationery Office. V zaključku svojega prispevka namesto rešitev navajam odličen zgled iskanja rešitev. V okviru »Evidence-based Council at Sheffield Teaching Hospitals National Health Service Trust« v Angliji (http://tinyurl.com/z39o2p2) deluje skupina neodvisnih, priznanih in predanih strokovnjakov, ki na osnovi ugotovljenih problemov zaposlenim rešitve pomagajo iskati in jih tudi objavljati, vsako leto svoje Watson, R., 2002. Faking an argument: prejudice-based views and the illusion of scholarship. Nurse Education Today, 22(4), pp. 273–274. http://dx.doi.org/10.1054/nedt.2002.0788 PMid:12030746 http://dx.doi.org/10.1054/nedt.2002.0788 PMid:12030746 Watson, R., 2105. Who cares about evidence? Working Papers in the Health Sciences, 1(14), p. 1. etting practice into evidence. Obzornik zdravstvene nege, 50(3), pp. 180–182. http://dx.doi.org/10.14528/snr.2016.50.3.105
https://openalex.org/W4360612976	https://pure.ulster.ac.uk/ws/files/118365380/fpsyg-14-1129912.pdf	English	null	Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study	Frontiers in psychology	2,023	cc-by	16,728	General rights Th i ht General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of th output without obtaining the permission of the author(s). If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study Farrell, D., Moran, J., Zat, Z., Miller, P. W., Knibbs, L., Papanikolopoulos, P., Prattos, T., McGowan, I., McLaughlin, D., Barron, I., Mattheß, C., & Kiernan, M. D. (2023). Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study. Frontiers in Psychology, 14, 2-19. Article 1129912. Advance online publication. https://doi.org/10.3389/fpsyg.2023.1129912 Link to publication record in Ulster University Research Portal Link to publication record in Ulster University Research Portal Publication Status: Published online: 23/03/2023 DOI: 10.3389/fpsyg.2023.1129912 Document Version Publisher's PDF, also known as Version of record Document Licence: CC BY Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study Farrell, D., Moran, J., Zat, Z., Miller, P. W., Knibbs, L., Papanikolopoulos, P., Prattos, T., McGowan, I., McLaughlin, D., Barron, I., Mattheß, C., & Kiernan, M. D. (2023). Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study. Frontiers in Psychology, 14, 2-19. Article 1129912. Advance online publication. https://doi.org/10.3389/fpsyg.2023.1129912 Take down policy Th R h P t Take down policy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 TYPE Original Research PUBLISHED 23 March 2023 DOI 10.3389/fpsyg.2023.1129912 Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study OPEN ACCESS EDITED BY Francisco Sampaio, Escola Superior de Enfermagem do Porto, Portugal Derek Farrell 1,2, Johnny Moran 1, Zeynep Zat 1, Paul W. Miller 3, Lorraine Knibbs 1, Penny Papanikolopoulos 1, Tessa Prattos 1, Iain McGowan 2, Derek McLaughlin 2, Ian Barron 4, Cordula Mattheß 1 and Matthew D. Kiernan 5 1 Department for Violence Prevention, Trauma and Criminology (VPTC), School of Psychology, University of Worcester, Worcester, United Kingdom, 2 School of Nursing and Midwifery, Queen’s University, Belfast, Northern Ireland, United Kingdom, 3 School of Nursing, Magee Campus, Ulster University, Northern Ireland, United Kingdom, 4 Centre for International Education, College of Education, University of Massachusetts, Amherst, MA, United States, 5 Northern Hub for Veteran and Military Families’ Research, Northumbria University, Newcastle upon Tyne, United Kingdom Farrell D, Moran J, Zat Z, Miller PW, Knibbs L, Papanikolopoulos P, Prattos T, McGowan I, McLaughlin D, Barron I, Mattheß C and Kiernan MD (2023) Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study. Front. Psychol. 14:1129912. doi: 10 3389/fpsyg 2023 1129912 Objective: Frontline mental health, emergency, law enforcement, and social workers have faced unprecedented psychological distress in responding to the COVID-19 pandemic. The purpose of the RCT (Randomized Controls Trial) study was to investigate the effectiveness of a Group EMDR (Eye Movement Desensitization and Reprocessing) therapy (Group Traumatic Episode Protocol— GTEP) in the treatment of Post-Traumatic Stress Disorder (PTSD) and Moral Injury. The treatment focus is an early intervention, group trauma treatment, delivered remotely as video-conference psychotherapy (VCP). This early intervention used an intensive treatment delivery of 4x2h sessions over 1-week. Additionally, the group EMDR intervention utilized therapist rotation in treatment delivery. Group early intervention eye movement desensitization and reprocessing therapy as a video-conference psychotherapy with frontline/emergency workers in response to the COVID-19 pandemic in the treatment of post-traumatic stress disorder and moral injury—An RCT study OPEN ACCESS EDITED BY Francisco Sampaio, Escola Superior de Enfermagem do Porto, Portugal REVIEWED BY Selvira Draganovic, International University of Sarajevo, Bosnia and Herzegovina Wilis Srisayekti, Padjadjaran University, Indonesia Yvette Hendrix, Onze Lieve Vrouwe Gasthuis (OLVG), Netherlands Malindi Van Der Mheen, Amsterdam University Medical Center, Netherlands CORRESPONDENCE Derek Farrell d.farrell@worc.ac.uk SPECIALTY SECTION This article was submitted to Psychology for Clinical Settings, a section of the journal Frontiers in Psychology RECEIVED 22 December 2022 ACCEPTED 22 February 2023 PUBLISHED 23 March 2023 Introduction Sritharan et al., 2020; Beames et al., 2021; Billings et al., 2021; Do and Frank, 2021; Norman et al., 2021; Carmassi et al., 2022; Feingold et al., 2022; Ghahramani et al., 2022; Smith et al., 2022). COVID-19 risk factors on mental health depend on the individual and the context. These included the risk heightened stress and anxiety, limited resources and protection, exposure to the virus and fear of exposing family, partners, and relatives, long shift patterns, sleep disruption and deprivation, burnout, and broader impact on work/life balance (Johnson et al., 2020; Raudenská et al., 2020; Sasangohar et al., 2020; Shreffler et al., 2020; de Kock et al., 2021; Lasalvia et al., 2021). Additionally, pre-existing anxiety/depression, exposure to previous adverse life experiences, and dissociative symptoms may contribute to PTSD in frontline health and emergency workers (Greenberg et al., 2020; Williamson et al., 2020; Billings et al., 2021; Greene et al., 2021; Miguel-Puga et al., 2021). Frontline mental health, emergency, law enforcement, and social workers have faced unprecedented psychological distress in responding to the COVID-19 pandemic, with around 22% meeting the criteria for Post-Traumatic Stress Disorder [PTSD]–a mental health condition caused by a traumatic experience (Wild et al., 2016, 2022; Johnson et al., 2020; Greene et al., 2021; Nagarajan et al., 2022; Watson, 2022; Yunitri et al., 2022). Continued psychological distress potentially may result in adverse outcomes including substance misuse, suicide risk, burnout, compassion fatigue, and secondary traumatization (Arpacioglu et al., 2021; Hooper et al., 2021). Although the impact of psychological harm may have delayed onset, implementing effective early intervention measures that seek to mitigate the potential development of PTSD is essential (Haugen et al., 2012; Hooper et al., 2021; Brunet et al., 2022). Early intervention in the aftermath of psychological trauma is defined as a treatment administered at the earliest possible time, sometimes within hours of the traumatic event, but is considered any intervention within the first 3 months after exposure (Dyregrov and Regel, 2012) consider four important principles related to early trauma interventions: immediacy, proximity, expectancy, and flexibility. Acknowledging the COVID-19 pandemic has sharply increased the demand on frontline emergency workers; nonetheless, organizations require evidence-based information about available psychological programs to address the mental health needs of staff (Jecker et al., 2020; Rasheed et al., 2020; Ruck et al. (2013) stipulate that early trauma interventions are designed to neither prevent, nor treat, PTSD. COPYRIGHT © 2023 Farrell, Moran, Zat, Miller, Knibbs, Papanikolopoulos, Prattos, McGowan, McLaughlin, Barron, Mattheß and Kiernan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Methods: The study’s design comprised a delayed (1-month) treatment intervention (control) versus an active group. Measurements included the International Trauma Questionnaire (ITQ), Generalized Anxiety Disorder Assessment (GAD-7), Patient Health Questionnaire (PHQ-9), Moral Injury Events Scale (MIES), and a Quality-of- Life psychometric (EQ-5D), tested at T0, T1: pre—treatment, T2: post-treatment, T3: 1-month follow-up (FU), T4: 3-month FU, and T5: 6-month FU. The Adverse Childhood Experiences – International version (ACEs), Benevolent Childhood Experience (BCEs) was ascertained at pre-treatment only. N = 85 completed the study. Results: Results highlight a significant treatment effect within both active and control groups. Post Hoc comparisons of the ITQ demonstrated a significant 01 Frontiers in Psychology frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 difference between T1 pre (mean 36.8, SD 14.8) and T2 post (21.2, 15.1) (t11.58) = 15.68, p < 0.001). Further changes were also seen related to co-morbid factors. Post Hoc comparisons of the GAD-7 demonstrated significant difference between T1 pre (11.2, 4.91) and T2 post (6.49, 4.73) (t = 6.22) = 4.41, p < 0.001; with significant difference also with the PHQ-9 between T1 pre (11.7, 5.68) and T2 post (6.64, 5.79) (t = 6.30) = 3.95, p < 0.001, d = 0.71. The treatment effect occurred irrespective of either ACEs/BCEs during childhood. However, regarding Moral Injury, the MIES demonstrated no treatment effect between T1 pre and T5 6-month FU. The study’s findings discuss the impact of Group EMDR therapy delivered remotely as video-conference psychotherapy (VCP) and the benefits of including a therapist/rotation model as a means of treatment delivery. However, despite promising results suggesting a large treatment effect in the treatment of trauma and adverse memories, including co-morbid symptoms, research results yielded no treatment effect in frontline/emergency workers in addressing moral injury related to the COVID-19 pandemic. Conclusion: The NICE (2018) guidance on PTSD highlighted the paucity of EMDR therapy research used as an early intervention. Clinical Trial Registration: Clinicaltrials.gov, ISRCTN16933691. Clinical Trial Registration: Clinicaltrials.gov, ISRCTN16933691. KEYWORDS EMDR early intervention, group treatment, COVID-19, emergency and frontline workers, therapist rotation, posttraumatic stress disorder, moral injury Frontiers in Psychology COPYRIGHT The primary rationale for this study was to address this critical issue. In summary, treatment results for group EMDR, delivered virtually, intensively, using therapist rotation are tentatively promising, however, the moral dimensions of trauma need consideration for future research, intervention development, and potential for further scalability. The data contributes to the emerging literature on early trauma interventions. Clinical Trial Registration: Clinicaltrials.gov, ISRCTN16933691. Clinical Trial Registration: Clinicaltrials.gov, ISRCTN16933691. frontiersin.org Introduction In addition to Acute Stress Disorder [ASD] (Brunet et al., 2022) and PTSD, another critical issue to return to, is moral injury. In reviewing the academic literature outlined in Figure 1, moral injury results from an act, or failure to act, that creates an ethical transgression, either by self or witnessed, which damages one’s conscience or moral compass (Litz et al., 2009) refers to the impact of moral injury as emotional, psychological, social, behavioral, and spiritual. However, there is insufficient explanation of moral injury within the current understanding of PTSD and its subsequent treatment (Barnes et al., 2019; Griffin et al., 2019; Koenig et al., 2019, 2020). Treating core PTSD symptoms does not address moral trauma—the same is true vice-versa (Farnsworth, 2019; Borges et al., 2020). A further definition of moral injury provided by Brock and Lettini (2012) describes it as a ‘wound of the soul’ when existing core moral foundations cannot be justified, sufficiently processed, and integrated into a reliable identity and meaning system sustains relationships and human flourishing. A study carried out with military mental health nurses by Jamieson et al. (2020a,b) relates moral injury to frontline workers. They describe it as experiencing existential, psychological, social, emotional, and spiritual/ religious damage arising from a violation or betrayal (by omission or commission) of the core moral framework and manifesting through feelings of shame, guilt, stigma, and self-condemning, or self-sabotaging behaviors. The core aspects of moral injury pertaining to the COVID-19 pandemic are highlighted in Figure 1 (Koenig et al., 2019; Borges et al., 2020; Williamson et al., 2020; Amsalem et al., 2021; Cartolovni et al., 2021). p A humanitarian crisis emerges when an event threatens a population’s health, safety, and well-being. COVID-19 creates a familiar narrative frequently witnessed in humanitarian emergencies where demand for mental health provision outstrips the supply available (Carriere, 2014; Dunkley, 2018; Eichfeld et al., 2019; Mattheß et al., 2019, 2020; Farrell et al., 2020; Pupat et al., 2022). As Bryant (2022) highlighted earlier, interventions need adaptation to meet the needs of as many as possible. One such adaptation within EMDR therapy is Group Interventions. Kaptan et al. Introduction Early organization interventions are useful in facilitating mutual support, identifying those that may require additional assistance, improving social cohesion, reducing harmful responses, and improving occupational functioning (Creamer et al., 2012; Dyregrov and Regel, 2012; Richins et al., 2020). Bryant (2021, 2022) and Magruder et al. (2017) consider that despite the continued debate surrounding the optimal strategies used in the immediate period following trauma exposure, the specific objective should always be preventing the onset of PTSD and other co-morbidities. 02 frontiersin.org Farrell et al. Farrell et al. 10.3389/fpsyg.2023.1129912 Group Protocol with Children. Results suggested that Group EMDR protocols might effectively improve a wide range of mental health- related outcomes, including post-traumatic stress disorder (PTSD), depression and anxiety, compared with pre-treatment and control groups. However, Kaptan et al. (2021) concluded that the included studies are limited to methodological challenges with a high risk of bias. Group Protocol with Children. Results suggested that Group EMDR protocols might effectively improve a wide range of mental health- related outcomes, including post-traumatic stress disorder (PTSD), depression and anxiety, compared with pre-treatment and control groups. However, Kaptan et al. (2021) concluded that the included studies are limited to methodological challenges with a high risk of bias. The dynamic nature of PTSD in the immediate aftermath of trauma presents both opportunities and challenges concerning early trauma interventions (Price et al., 2018). Furthermore, the malleability of symptoms post-adversity exposure suggests that early interventions can alter the course of trauma sequelae, provided those interventions are both flexible and adaptive (Asmussen et al., 2019; Roberts et al., 2019; Hooper et al., 2021; Bryant, 2022). Therefore, the primary endeavor of any early trauma intervention response should be: The Group Trauma Episode Protocol [GTEP], developed by Shapiro and Laub (2008), is an evidence-based EMDR intervention used in the treatment of recent natural and human disasters (Acarturk et al., 2016; Roberts, 2018; Womersley and Arikut-Treece, 2019; Kaptan et al., 2021; Pink et al., 2022). An essential aspect of GTEP is that the client does not disclose any details regarding their trauma experiences. Within EMDR therapy this is known as ‘Blind 2 therapist (Farrell et al., 2020, 2022). There are distinct advantages to this. The rationale surrounding non-disclosure may potentially involve trauma memories that invoke shame, survivor guilt, moral injury, or fear of recrimination and stigma. Introduction Dimensions surrounding moral injury are pertinent as these either relates to inner core values, or external such as betrayal or breaches of trust. 1. Reduce trauma stress reactions. 2. Provide authentic support. 3. Enhance coping. 4. Empower resilience. 5. Minimize the risk of burnout and vicarious traumatization. 6. Diminish the risk of developing other mental health and psychological difficulties with the potential to impact psychological well-being. Roberts et al. (2019) published a systematic review of 61 studies of early psychological interventions. Their review supported trauma- focused cognitive behavioral therapy (CBT-TF), cognitive therapy without exposure, and eye movement desensitization and reprocessing (EMDR) for individuals reporting traumatic stress symptoms. They concluded that the research was more robust for CBT-TF. However, NICE guidance on PTSD (2018) highlights the lack of research supporting using EMDR as an early trauma intervention. For frontline and emergency workers, stigma remains a substantial barrier to seeking psychological support (Clement et al., 2015; Gronholm et al., 2021). Diop et al. (2022), in a study investigating 723 frontline workers, determined that 44% expressed concerns about being stigmatized and excluded from serving those affected by COVID-19, highlighting that stigma can be both internal and external. A study carried out in India with frontline healthcare workers in direct management of COVID-19 patients (Sachdeva et al., 2021) declared that 75% experienced self-stigma – primarily in the form of guilt in potentially exposing their families to the coronavirus. EMDR therapy is an empirically supported treatment for psychological trauma, endorsed by the World Health Organisation (2013), United Nations High Commission for Refugees (2022) and the International Society for Traumatic Stress Studies (2019). It is a psychological treatment for pathogenic (trauma) memories and their associated stress symptoms using a model of pathogenesis and change known as adaptive information processing (Hase et al., 2017; Hase, 2021; Farrell et al., 2022; Laliotis and Shapiro, 2022; Wippich et al., 2023). Shapiro (2017) considers EMDR to desensitize disturbing memories and stimulates the reprocessing of associated thoughts, feelings, and sensations towards adaptive resolution. Although a recent systematic review concluded EMDR is effective for first responders, they concurred with NICE (2018) in further indicating the quality of studies as weak or medium, highlighting several gaps and unanswered questions within the academic literature including high risk of bias, limited availability of data including safety and harm- related information (Oosterbaan et al., 2019; Bryant, 2021, 2022; Kaptan et al., 2021; Morris et al., 2022). frontiersin.org Frontiers in Psychology Introduction (2021) undertook a review of 22 studies using Group EMDR Interventions of which 13 studies examined the EMDR Integrative Group Treatment Protocol (IGTP), four studies the EMDR Group Traumatic Episode Protocol (G-TEP), four studies of EMDR Integrative Group Treatment Protocol for Ongoing Traumatic Stress and one study considered the EMDR Implementing an early trauma intervention during COVID-19, and in government-enforced lockdown, presented several logistical 03 Farrell et al. 10.3389/fpsyg.2023.1129912 FIGURE 1 Key aspects of moral injury and COVID-19. Key aspects of moral injury and COVID-19. challenges. Firstly, the public health management of COVID-19 involved social distancing to reduce risk of infection. One of the management strategies utilized to address this required a shift towards video-conferencing psychotherapy [VCP] (Crowe et al., 2020). As Healthcare providers closed their doors to face-to-face intervention, video-conferencing treatment platforms became, for many, the only viable option (Turgoose et al., 2018; Watts et al., 2020; Wind et al., 2020; Farrell et al., 2022). To date, several research studies indicate that psychological treatment, delivered remotely though video-conference platforms, are both safe and feasible. Furthermore, that they offer greater flexibility, and improve equity of access (Appleton et al., 2021; Broadbear et al., 2021; Cantone et al., 2021; Cataldo et al., 2021; Dharwadkar et al., 2021; Oudshoorn et al., 2021; Puspitasari et al., 2021; Shklarski et al., 2021a,b; Vera San Juan et al., 2021; Milosevic et al., 2022). Smith et al. (2022), however, considers that although the evidence-base is promising further interrogation and research is needed to further explore the ethical and moral dimensions of video- conference psychotherapy. methodology, larger sample sizes with participants who meet clear diagnostic criteria and including necessary follow-up data is essential. Addressing these salient issues was the primary driver in the inception of this COVID-19 randomized-control clinical trial. Mindful of the issues raised so far, this clinical trial wished to explore the following: early trauma intervention, use of video-conference psychotherapy, group treatment, Blind-2- therapist, intensive treatment, and therapist rotation – with a primary treatment target group Emergency Workers on the frontline of the COVID-19 pandemic who demonstrated trauma sequelae. The broad research objectives for the study were: 1. Is Early Intervention EMDR Video Group Therapy (VGTEP) a safe, and efficient treatment intervention for Frontline/ Emergency/Keyworkers, who experience psychological trauma in response to COVID-19 with regards to trauma sequalae and co-morbid symptoms. 2. Frontiers in Psychology Introduction Is Early Intervention EMDR Video Group Therapy (VGTEP) a relevant and effective treatment intervention for Frontline/ Emergency/Keyworkers, who experience psychological trauma in response to COVID-19 with regards to both recruitment and retention to the study? According to Herman and Kallivayalil (2018), outcome research supports group interventions in the treatment of PTSD; however, they conclude that there is limited evidence to support the superiority of one group model over another. The research evidence exploring group trauma treatment for PTSD using VCP is limited, especially regarding COVID-19. One group trauma treatment approach is Eye Movement Desensitization and Reprocessing (EMDR). Most current research demonstrates its effectiveness with Post-Traumatic Stress Disorder [PTSD] and Complex PTSD. However, EMDR therapy has been used extensively as an early intervention as part of trauma capacity building throughout the world (Farrell et al., 2011, 2013, 2020; Eichfeld et al., 2019; Mattheß et al., 2019, 2020; Covers et al., 2021; Tarquinio et al., 2021). Table 1 highlights the advantages and disadvantages of using EMDR therapy as an early intervention using virtual platforms. Disadvantages Disadvantages o Reliant on effective communication and handover between team members o Potential divergence in confidence and ability in running treatment sessions o Requires strong team adhesion, team support and robust clinical supervision o Requires ‘buy in’ from clients and active client engagement o Client’s may consider the intervention as not bespoke to their needs o Focus of the intervention does not address individual issues outside the remit of the study o Difficult to research and evaluate the dyadic relationship The RCT generates six hypotheses to determine that the Virtual EMDR Group Intervention was safe, relevant, effective, and efficient: Interest in participation into the study was such that recruitment, although from the Island of Ireland, also included international participants from United States, Canada, Australia, New Zealand, Greece, and Turkey. The treatment intervention used for the clinical trial was the EMDR Group Traumatic Episode Protocol – version 9 (Shapiro, 2019–unpublished) used as a video-conference psychotherapy (VGTEP). Hypothesis 1: There is no difference between active and control (delayed) groups regarding trauma sequelae—measured with the International Trauma Questionnaire (ITQ) (Cloitre et al., 2018) when comparing T1, T2, T3, T4, and T5. A Randomized Control Trial (RCT) Delayed Start Design used for the clinical study compared an active treatment intervention versus a control group (delayed treatment – 1 month) using the same intervention. The clinical trial conducted from July 2020 – March 2022 during the primary lockdown periods. The RCT consisted of two cohorts: Hypothesis 2: There is no difference between active and control (delayed) groups regarding co-morbid sequelae – measured with the Patient Health Questionnaire (PHQ-9) (Kroenke et al., 2001) and the Generalized Anxiety Disorder (GAD-7) (Williams, 2014) over the T1-T5 time periods. o Active Cohort 1: EMDR therapy VGTEP Treatment (4 sessions of approximately 2 h duration which equated to 8 h total treatment intervention) implemented as an intensive intervention for 1 week. Hypothesis 3: Treating the trauma sequelae will reduce the level of moral injury as demonstrated with the Moral Injury Events Scale (MIES) ((Nash et al., 2013), demonstrated between T1 and T5, for both active and control groups. o Control Cohort 2: 4 weeks Delayed intervention of EMDR therapy VGTEP. Treatment involved an intensive intervention within 1 week. Hypothesis 4: Using Trauma-Focused Therapist rotation will demonstrate no change in clinical diagnosis between T1 and T5, for both active and control groups. Design The University of Worcester (United Kingdom) granted ethical approval for the study [CBPS19200030]. The research study strictly adhered to the approval granted. Additionally, the study adhered to the British Psychological Society (BPS) Guidelines on ‘Conducting Research with Human Participants during COVID-19 (2020). The study registered as a clinical trial ID ISRCTN16933691 (2020) and hosted by the Trauma Response Network (TRN) – Ireland, an NGO organization offering early trauma interventions. Research participants recruited via a social networking and advertising strategy coordinated by TRN Ireland, which involved media outlets and radio stations in Ireland, Northern Ireland, and Great Britain. Replicating an innovative approach used in the treatment of PTSD (van Minnen et al., 2018) utilized therapist rotation in delivering the treatment intervention. In this contemporary approach Trauma Therapists rotate between patients during the treatment intervention. As Table 2 accentuates, there are several distinct advantages and disadvantages to using therapist rotation (Krampe and Ehrenreich, 2012; van Minnen et al., 2018): As Kaptan et al. (2021) emphasized, more trauma-focused clinical trials, especially early intervention, incorporating robust 04 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 TABLE 1 Advantages and disadvantages of early intervention EMDR therapy using visual platforms. Frontiers in Psychology Design Advantages Disadvantages o Brief intervention for the purpose of trauma symptom reduction o System of triage/ risk assessment in environments of limited resources o Provides trauma informed psychoeducation o Implementation of trauma informed stress regulation strategies o Distinct trauma confrontation intervention o Empowers individual and community resilience o Potential for implementation and delivery on consecutive days (intensive treatment) o Health economic benefits o Potential for ‘tasking-shifting’ to allied/ non-mental health workers o Enables specific teaching and learning in a focused trauma treatment intervention o Greater flexibility, logistical, linguistic, and cultural adaptability o Promotes re-invention, resilience, and post-traumatic growth o Access to technology o Unreliable technolog o Concerns about priv o Often dependent on o Difficulty to respond o Not appropriate for o Concerns about risk o Restricts forms of no o Ethical and legal co different geographic Advantages Disadvantages o Brief intervention for the purpose of trauma symptom reduction o System of triage/ risk assessment in environments of limited resources o Provides trauma informed psychoeducation o Implementation of trauma informed stress regulation strategies o Distinct trauma confrontation intervention o Empowers individual and community resilience o Potential for implementation and delivery on consecutive days (intensive treatment) o Health economic benefits o Potential for ‘tasking-shifting’ to allied/ non-mental health workers o Enables specific teaching and learning in a focused trauma treatment intervention o Greater flexibility, logistical, linguistic, and cultural adaptability o Promotes re-invention, resilience, and post-traumatic growth o Access to technology and software o Unreliable technology and functioning o Concerns about privacy and confidentiality o Often dependent on access to Wi-Fi and power supply o Difficulty to respond in crisis situations o Not appropriate for serious mental health problems and concerns o Concerns about risk o Restricts forms of non-verbal communication o Ethical and legal concerns of carrying out treatment with clients from different geographical areas/ regions TABLE 2 Advantages and disadvantages of therapist rotation model in EMDR therapy. TABLE 2 Advantages and disadvantages of therapist rotation model in EMDR therapy. Design Advantages Disadvantages o Reducing interpersonal dependency on individual therapists o Reducing trauma therapists’ anxiety regarding trauma confrontation (exposure) o Improves ‘patient-focused’ intervention o Increased confidence in carrying out trauma-focused protocolized interventions o Increases trauma treatment fidelity o Greater cogency in overall patient treatment plan o Improving confidence in directly challenging trauma avoidance behaviors o Reduces the risk of vicarious traumatization of trauma therapists o Reliant on effective communication and handover between team members o Potential divergence in confidence and ability in running treatment sessions o Requires strong team adhesion, team support and robust clinical supervision o Requires ‘buy in’ from clients and active client engagement o Client’s may consider the intervention as not bespoke to their needs o Focus of the intervention does not address individual issues outside the remit of the stu o Difficult to research and evaluate the dyadic relationship TABLE 2 Advantages and disadvantages of therapist rotation model in EMDR therapy. Advantages Disadvantages o Reducing interpersonal dependency on individual therapists o Reducing trauma therapists’ anxiety regarding trauma confrontation (exposure) o Improves ‘patient-focused’ intervention o Increased confidence in carrying out trauma-focused protocolized interventions o Increases trauma treatment fidelity o Greater cogency in overall patient treatment plan o Improving confidence in directly challenging trauma avoidance behaviors o Reduces the risk of vicarious traumatization of trauma therapists o Reliant on effective communication and handover between team members o Potential divergence in confidence and ability in running treatment sessions o Requires strong team adhesion, team support and robust clinical supervision o Requires ‘buy in’ from clients and active client engagement o Client’s may consider the intervention as not bespoke to their needs o Focus of the intervention does not address individual issues outside the remit of the study o Difficult to research and evaluate the dyadic relationship TABLE 2 Advantages and disadvantages of therapist rotation model in EMDR therapy. frontiersin.org Design Advantages Disadvantages o Reducing interpersonal dependency on individual therapists o Reducing trauma therapists’ anxiety regarding trauma confrontation (exposure) o Improves ‘patient-focused’ intervention o Increased confidence in carrying out trauma-focused protocolized interventions o Increases trauma treatment fidelity o Greater cogency in overall patient treatment plan o Improving confidence in directly challenging trauma avoidance behaviors o Reduces the risk of vicarious traumatization of trauma therapists o Reliant on effective commu o Potential divergence in con o Requires strong team adhe o Requires ‘buy in’ from clien o Client’s may consider the i o Focus of the intervention d o Difficult to research and ev TABLE 2 Advantages and disadvantages of therapist rotation model in EMDR therapy. Advantages Disadvantag o Reducing interpersonal dependency on individual therapists o Reducing trauma therapists’ anxiety regarding trauma confrontation (exposure) o Improves ‘patient-focused’ intervention o Increased confidence in carrying out trauma-focused protocolized interventions o Increases trauma treatment fidelity o Greater cogency in overall patient treatment plan o Improving confidence in directly challenging trauma avoidance behaviors o Reduces the risk of vicarious traumatization of trauma therapists o Reliant on effecti o Potential diverge o Requires strong t o Requires ‘buy in’ o Client’s may con o Focus of the inte o Difficult to resea Disadvantages o Reliant on effective communication and handover between team members o Potential divergence in confidence and ability in running treatment sessions o Requires strong team adhesion, team support and robust clinical supervision o Requires ‘buy in’ from clients and active client engagement o Client’s may consider the intervention as not bespoke to their needs o Focus of the intervention does not address individual issues outside the remit of the study o Difficult to research and evaluate the dyadic relationship The clinical trial used the following measures: The clinical trial used the following measures: o Adults (18 years and above). o Front Line Health, Emergency and Social Care Workers experiencing Psychological Distress and Trauma in response to frontline working addressing the COVID-19 virus. o International Trauma Questionnaire (ITQ) – an 18 question, self- reporting measure focused on both PTSD and Complex PTSD, consistent with ICD-11 (Hyland et al., 2017; Cloitre et al., 2018; Redican et al., 2021). o Currently in active employment. o Symptoms indicative of psychological distress and impact of psychological well-being and functioning measured using the Impact of Events Scale Revised Score of 24 and above. o Generalized Anxiety Disorder Assessment (GAD-7) which objectifies and assesses the degree of anxiety severity (Williams, 2014). Exclusion criteria: o Patient Health Questionnaire (PHQ-9) which objectifies and assesses the degree of depression severity (Kroenke et al., 2001). o Non-trauma exposure. o Non-trauma exposure. o Subjective Unit of Disturbance/Distress (SUD) – a scale of 0 to 10, measuring the subjective intensity of disturbance or distress currently experienced by an individual (Wolpe, 1969). o Impact of Events Scale Revised score of 23 or below. o Undergoing current treatment (physical and/or psychological). d f h o Undergoing current treatment (physical and/or psychological). o Present evidence of psychosis. o Moral Injury Event Scale (MIES) – 9-point self-reporting scale exploring perceived transgressions, betrayals, or violations of an individual’s moral code (Nash et al., 2013). o Present evidence of psychosis. As mentioned earlier as this was a Republic of Ireland Research initiative, research participants were recruited either from the Island of Ireland or internationally. A recruitment initiative was carried out utilizing media organizations within the Republic of Ireland. Recruitment to participate in the clinical trial used self-selection access through the Trauma Response Network (TRN) Ireland website through a secure platform. Each participant was also assessed by TRN Ireland administrative staff, who provided further information about the study outlining what participation involved. Those wishing to participate were provided with a detailed, research participant information sheet and research consent form. o Adverse Childhood Experience International Questionnaire (ACE-IQ) – tested at Pre point only with the intention to measure exposure to adverse childhood experiences (< 18 years old). Disadvantages The study incorporated six data points including T0 (control group), T1 (pre), T2 (post), T3 (1-month FU), T4 (3-month FU), and T5 (6-month FU) (Marcus et al., 2004; Ostacoli et al., 2018). All included research clients provided written consent before enrolment into the clinical trial. Hypothesis 5: A negative correlation exists, within the target population, between Adverse Childhood Experiences (ACEs) and Benevolent Childhood Experiences (BCEs) (Narayan et al., 2018) 05 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 regarding evidence supporting resilience and posttraumatic growth. regarding evidence supporting resilience and posttraumatic growth. Mirabilis Health Institute, Belfast, Northern Ireland, provided medical supervision for the clinical trial and was responsible for clinical risk assessment and triage. Hypothesis 6: There is no difference between active and control groups regarding quality of life using the EQ-5D – generic measure of an individual’s health status when comparing between T1 and T5. In determining the sample size1 was used setting α = 0.05, β = 0.2. A power calculation indicated an initial sample size estimation of N = 72 with N = 36 in each arm of the study. This figure allowed for a 10% drop-out rate. Randomization done through sequence generation using a computer algorithm allocating each research participant a unique client number (UCN) to ensure anonymity to the clinical team. Figure 2 highlights N = 192 clients assessed for eligibility and N = 97 individuals excluded from the study as their IES-R scores were < 24, leaving a total of N = 95 subject to randomization. Participants The clients used for this clinical trial were Frontline Health and Social Care workers or Emergency/First Responders directly working on the frontline of the COVID-19 pandemic utilizing the following inclusion/exclusion criteria: 1 http://www.openepi.com/Menu/OE_Menu.htm Frontiers in Psychology Measures Inclusion criteria: Treatment intervention EMDR Blind 2 therapist protocol. Clinical Leads and Emotional Protection workers were also randomly allocated to the VGTEP treatment intervention ensuring therapist rotation by the TRN Administrative team. Once research participants met the study’s inclusion criteria the TRN administrative team randomly assigned participants using a random allocation software package to either the active group Immediate Treatment Group) or a 1-month Delayed Treatment Group. Each group intervention involved a 1-week block of intensive treatment consisting of four, two hours sessions of Group EMDR therapy, delivered as a remote treatment intervention. The platform used for the VGTEP sessions was ZOOM Enterprise version 5.9.1. The TRN Ireland administrative team emailed a ZOOM link for each of the treatment sessions – evenings of Monday, Wednesday, and Thursday, and Saturday morning. At the commencement of each treatment session research participants were instructed to only use the unique client number as a means of identification, and not their name. Clinical members of the research teams had no means of identifying any of the research participants. Participants were required to attend all four treatment sessions. Each session lasted approximately 2 h. Each group consisted of six participants who remained as a group for the duration of the treatment intervention. In addition, the VGTEP session lead, and the emotional protection worker (EPW), were also randomly allocated to the treatment sessions. As per the research protocol, session leads and EPWs introduced themselves at the start of each VGTEP treatment session, and importantly, however, the research participants revealed no information about themselves. Additionally, it was emphasized to the research participants that the VGTEP intervention required no disclosure of anything about the trauma memory – consistent with the The EMDR Group Treatment Intervention delivered as video- conference psychotherapy (VGTEP) consisted of seven steps: 1. Trauma regulation exercise, 2. Trauma target selection, 3. Anchor to a past resource, 4. Identification of a future belief, 5. Trauma confrontation of the target trauma memory, 6. Enhancement of resilience and post-trauma growth, and 7. Closure grounding exercise. Dual attention, and bilateral stimulation, a distinct element of EMDR therapy, are used in steps 2 and 5. Step 5 addresses three of the significant points of disturbance relating to the target trauma memory. Trauma processing within the confrontation stage of VGTEP involves 27 occasions of focus on the trauma target material. The clinical trial used the following measures: o Benevolent Childhood Experiences Score (BCEs) (Narayan et al., 2017) – tested at Pre point only: this is a measure of exposure to benevolent factors that occurred in childhood that may impact on resilience and post-traumatic growth (< 18 years old). o EQ-5D – generic measure of an individual’s health status. The EQ-5D is a preference based HRQL across five dimensions; mobility, self-care, usual activities, pain/discomfort, and anxiety/ depression. The Impact of Events Scale–Revised (IES-R; Weiss and Marmar, 1997) was the primary screening tool used for the purpose of recruitment only. The IES-R has been tested for factor structure, internal consistency, concurrent validity, and discriminative validity by Beck et al. (2008). An IES-R score of 24 was used as the threshold for entering the study. The IES-R is an internationally recognized psychometric tool to assist in a diagnosis of Post-Traumatic Stress Disorder. The IES-R consists of 22 questions and provides four scores: total, intrusion, avoidance, and hyperarousal. The scoring is a maximum of 88. Asukai et al. (2002) stipulate that an IES-R score of 24–32 indicates clinical concern of either partial PTSD or at least some of its symptoms. Additionally (Creamer et al., 2003) advises that scores between 33 and 38 represent a cut-off score for a probable diagnosis of PTSD. A score of 39 and above is enough to suppress the immune system’s functioning (Izutsu et al., 2004, 2008). Measure evaluated after each VGTEP session: SUD. Measure evaluated after each VGTEP session: SUD. Measures evaluated at T1 only: ACE-IQ and BCEs. Measures evaluated at T0, T1, T2, T3, T4, and T5: ITQ, GAD-7, PHQ-9, EQ-5D. Measures evaluated at T0, T1, T2, T3, T4, and T5: ITQ, GAD-7, PHQ-9, EQ-5D. Measures evaluated at T1 and T5 only: MIES. All psychometrics were sent out electronically to all research participants via the Trauma Response Network Ireland Administrators and was collected independent to the research team. 06 frontiersin.org frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 FIGURE 2 Flow of participants through the research clinical trial. FIGURE 2 Flow of participants through the research clinical trial. Frontiers in Psychology frontiersin.org Data analysis Metric points conducted at T0 (control), and then T1 (pre-treatment), T2 (post—treatment), T3 (1-mth FU), T4 (3-mth FU), and T5 (6-mth FU). Table 3 highlights N = 85 completed the study up to, and including, T5. Data Analysis used the Jamovi (version 2.3.21.0) statistical software package. Descriptive data analyzed used either independent sample t-tests or fischer exact test. For further descriptive statistics, mean ±, standard deviation (±SD) for numerical variables and percentage (%) for categorical data. Tests for normality and homogeneity of variance determined no violations, therefore repeated measures RM-ANOVA analyzed each variable at T0, T1, T2, T3, T4, and T5 time points. The rationale for utilizing RM-ANOVAS is that they have been frequently used in various RCT studies testing EMDR therapy (de Roos et al., 2011; Jarero et al., 2018; Osorio, 2018; Ostacoli et al., 2018; Yurtsever et al., 2018). Skewness and kurtosis ensured normality of the ACEs and BCEs scores, as well as Pearson r to determine correlation coefficients. The p value of <0.05 considered significant. Using RM-ANOVA models Eta squared η2 is used as an indication of effect size (Olejnik and Algina, 2003). In testing Hypothesis 1: There is no difference between active and control (delayed) groups regarding trauma sequelae—measured with the International Trauma Questionnaire (ITQ) when comparing T1, T2, T3, T4, and T5. As mentioned earlier results revealed no statistical change between T0 and T1 for the control group, therefore, Figure 3 highlights the descriptive results for the complete cohort N = 85 for periods T1, T2, T3, T4, and T5. A repeated measures RM-ANOVA conducted to compare the impact of the Virtual GTEP intervention on trauma symptoms using the International Trauma Questionnaire (ITQ) evaluated at T0 (control group), T1 – pre, T2 – post, T3–1-mth FU, T4–3-mth FU, and T5–6-mth FU. For the control (delayed) group there was no statistical difference between T0 and T1. Results Regarding treatment fidelity and integrity a treatment protocol manual was created for the clinical trial, based on the Elan Shapiro GTEP Training Manual – version 9 (unpublished manual). Each treatment session was video recorded and was independently fidelity checked by an international expert in the Group Traumatic Episode Protocol (GTEP). Additionally, all psychometrics were acquired independently to the research team, with the entire data set only handed over to the team for data analysis once all the 6-month follow-up data had been obtained. Within the research trial, 64 VGTEP treatment sessions were carried out during two periods of government lockdown during the COVID-19 pandemic. This equated to approximately 128 h of treatment intervention, including both active (N = 46) and control (N = 39) groups. Per the therapist rotation model, N = 11 EMDR therapists carried out the 64 treatment interventions, and N = 26 Emotional Protection Team members were utilized. VGTEP Therapists, Emotional Protection Workers, and research participants were blinded in the intervention. Treatment intervention Each VGTEP session was digitally recorded and subjected to treatment fidelity checks carried out by an independent EMDR Europe Accredited Consultant/Clinical Supervisor. When necessary, the Emotional Protection worker utilized the breakout room within ZOOM to manage any participant if they needed I:I support or guidance. Before the commencement of the trial, training was provided for both the VGTEP clinical leads and Emotional Protection workers, this ensured familiarity with the clinical trial, treatment fidelity, triage and risk assessment procedures, welfare checks, team support, and clinical and research supervision. Mirabilis Health Institute, Belfast, Northern Ireland, provided onward referral and Consultant Medical Supervision for the clinical trial. 07 frontiersin.org Farrell et al. Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 Data analysis Results, highlighted in Figure 3, demonstrated a significant difference in the Characteristics Active: group EMDR (n = 46) Control: delayed group EMDR (n = 39) Total sample (n = 85) p value Age, mean (SD) 46.4 (9.78) 45.5 (11.9) 45.99 (10.72) 0.3401 Gender no 0.7922 - Male 9 9 18 - Female 37 30 67 Location 0.3842 - All Ireland 22 23 45 - International 24 16 40 Occupation category 0.7872 - Frontline Healthcare 35 33 68 - First Responders/ Emergency Workers 10 7 17 Trauma Onset 0.0142 * - A: less that 6-months 2 10 12 - B: 6–12 months 41 27 68 - C: 1 year + 3 2 5 ACE scores mean, (SD) 2.78 (2.46) 3.36 (2.49) 3.05 (2.47) 0.8561 BCE scores mean (SD) 8.09 (2.14) 8.00 (1.84) 8.05 (1.99) 0.4211 1Independent samples t-test. 2Fischer exact test. sig p < 0.05. 08 Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 (delayed) treatment interventions. In determining the overall effect size of the treatment intervention regarding the ITQ, as a repeated measures ANOVA used Lakens (2013) cites Cohen (1992) in providing a benchmark for η2 as an indication of effect size: η2 = 0.01 (small), η2 = 0.06 (medium), and η2 = 0.14 (large). Results of the ITQ treatment effect size calculated at η2 = 0.420 indicating a large treatment effect. reduction of the ITQ scores following the treatment intervention over time F(4–332) = 106.84, p < 0.001. Post Hoc comparisons of the ITQ demonstrated a significant difference between T1 pre (mean 36.8, SD 14.8) and T2 post (21.2, 15.1) (t11.58) = 15.68, p < 0.001). This was also seen between T1 pre and T3 1-mth FU (16.8; 14.5) (t13.77) = 20.06, p < 0.001; T1 pre and T4 3-mth FU (14.9, 15.7) (t14.02) = 21.98, p < 0.001; and T1 pre and T5 6-mth FU (12.9, 14.7) (t15.11) = 24.12, p < 0.001. Finally, there was a significant difference between T2 post and T4 3-mth FU d = 0.41, and T5 6-mth FU d. Figure 4 highlights the T1 and T5 scores, including outliers in the T5 dataset. f During the VGTEP session there are stages where the participants subjective unit of disturbance is assessed. Data analysis This subjective measure ascertained in steps 1, 2, 5, and 6 of the VGTEP protocol, however, per the research protocol SUD measures obtained pre and post step 1, and at points step 2 and Concerning the ITQ, post hoc comparison determined no statistically significant difference between the active and the controlled Frontiers in Psychology 09 frontiersin.org y gif p p p , p p FIGURE 3 International trauma questionnaire (ITQ) T1–T5 active and control group (N=85) @ p < 0,001. T1, pre; T2, post; T3, 1-mth FU; T4, 3-mth FU; T5, 6-MTH FU. FIGURE 4 International trauma questionnaire (ITQ) T1–T5 active and control (delayed) groups (N=85) @ p < 0.001. FIGURE 3 International trauma questionnaire (ITQ) T1–T5 active and control group (N=85) @ p < 0,001. T1, pre; T2, post; T3, 1-mth FU; T4, 3-mth FU; T5, 6-MTH FU. FIGURE 3 International trauma questionnaire (ITQ) T1–T5 active and control group (N=85) @ p < 0,001. T1, pre; T2, post; T3, 1-mth FU; T4, 3-mth FU; T5, 6-MTH FU FIGURE 3 International trauma questionnaire (ITQ) T1–T5 active and control group (N=85) @ p < 0,001. T1, pre; T2, post; T3, 1-mth FU; T4, 3-m FU. FIGURE 4 International trauma questionnaire (ITQ) T1–T5 active and control (delayed) groups (N=85) @ p < 0.001. FIGURE 4 International trauma questionnaire (ITQ) T1–T5 active and control (delayed) groups (N=85) @ p < 0.001. FIGURE 4 International trauma questionnaire (ITQ) T1–T5 active and control (delayed) groups (N=85) @ p < 0.001. 09 Frontiers in Psychology frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 piece @ p < 0.001, with an effect size calculated at η2 = 0.189, and trauma confrontation η2 = 0.295. piece @ p < 0.001, with an effect size calculated at η2 = 0.189, and trauma confrontation η2 = 0.295. step 6. The rationale for this is to consider changes in subjective distress during both the trauma regulation (step 1) and trauma confrontation (steps 2–6) parts of the VGTEP protocol. Figure 5 highlights the alterations in the SUD during the trauma confrontation stage of the N = 340 participant episodes of VGTEP. The SUD treatment effect size calculated at η2 = 0.295, p < 0.001, indicating a large treatment effect. In summary, when reviewing hypothesis 1 the null is supported – no difference was observed between the active and control groups indicating that the VGTEP treatment intervention was effective for both groups. Data analysis f Testing Hypothesis 2: There is no difference between active and control groups regarding co-morbid sequelae – measured with the GAD-7 and the PHQ-9 at T1, T2, T3, T4, and T5. Table 4 demonstrates alterations in the SUDs phenomenology (mean, SD, median) during the entire VGTEP session including both trauma regulation and confrontation stages. Results suggest that the Trauma Regulation element has a distinct treatment effect overall (η2 = 0.189, p < 0.001), and therefore is of clinical relevance and benefit. Further repeated measures conducted to compare the impact of the trauma intervention on both the Generalized Anxiety Disorder Assessment (GAD-7) and the Patient Health Questionnaire (PHQ-9), evaluated at T0 (control group), T1 – pre, T2 – post, T3–1-mth FU, T4–3-mth FU, and T5–6-mth FU. Results determined no statistical difference between the two treatment group interventions for either the GAD-7 or the PHQ-9. Figure 5 plots the alterations in SUD for both trauma regulation (Step 1 pre and post) and confrontation (Step 2 and 6). Results also highlight the range scores in that within each VGTEP session SUD scores decreased, increased, and flatlined, however, the general trend was a statistically significant (p < 0.001) decrease (Figures 6, 7). For the GAD-7 results demonstrated a significant reduction in both groups over time F(5–190) = 42.3, p < 0.001. There was no difference between T0 and T1. However, Post Hoc comparisons of the GAD-7 demonstrated significant difference between T1 pre (11.2, 4.91) and T2 post (6.49, 4.73) (t = 6.22) = 4.41, p < 0.001; T1 and T3 1-mth FU (5.42, 4.75) (t = 8.67) = 5.78, p < 0.001; T1 and T4 3-mth (5.12, 4.64) (t = 7.78) = 7.78, p < 0.001; T1 and T5 6-mth FU i For the VGTEP SUD scores for the individual (N = 340) sessions results demonstrated a significant reduction in both groups over time for both trauma regulation (Step 1) and trauma confrontation (Step 2 and 6) with no statistical difference between the active and control (delayed) groups. The overall effect size for the trauma regulation FIGURE 5 Combined ITQ T1 pre and T5 6-mth FU scores active and control (delayed) groups (N=85) @ p < 0.001. TABLE 4 Descriptive data measuring Subjective unit of disturbance during VGTEP session (N = 340). TABLE 4 Descriptive data measuring Subjective unit of disturbance during VGTEP session (N = 340). Data analysis Treatment group VGTEP step 1 pre VGTEP step 1 post VGTEP step 2 (pre) VGTEP step 6 (post) Mean (SD) Active (N = 46) 5.06 (2.14) 2.87 (2.23) 7.40 (1.98) 4.25 (2.64) Control (N = 39) 5.05 (2.34) 3.03 (2.00) 6.98 (1.92) 4.42 (2.18) Median Active (N = 46) 5 3 8 4 Control (N = 39) 5 3 7 4 10 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 FIGURE 6 GAD-7 psychometric scores for active and control groups (total N=85). FIGURE 7 PHQ-9 psychometric scores for active and control groups (total N=85). FIGURE 6 GAD-7 psychometric scores for active and control groups (total N=85). FIGURE 6 GAD-7 psychometric scores for active and control groups (total N=85). FIGURE 6 GAD-7 psychometric scores for active and control groups (total N=85). FIGURE 6 GAD-7 psychometric scores for active and control groups (total N=85). RE 6 -7 psychometric scores for active and control groups (total N=85). FIGURE 7 PHQ-9 psychometric scores for active and control groups (total N=85). FIGURE 7 PHQ-9 psychometric scores for active and control groups (total N=85). Regarding hypothesis 2 the null hypothesis is accepted – no difference was observed between the active and control groups demonstrating that the VGTEP intervention was influential in the treatment of co-morbid features of anxiety and depression. (4.73, 4.27) (t = 8.93) = 6.72, p < 0.001. In determining the overall effect size of the treatment intervention regarding the GAD-7, the overall effect size calculated as η2 = 0.202 indicating a large treatment effect. f For the Patient Health Questionnaire (PHQ-9) results also demonstrated a significant reduction in both groups over time F(5–190) = 43.3, p < 0.001. Furthermore, like the ITQ and GAD-7, there was no statistical difference between T0 and T1. However, post hoc comparison of the PHQ-9 demonstrated significant differences between T1 pre (11.7, 5.68) and T2 post (6.64, 5.79) (t = 6.30) = 3.95, p < 0.001, d = 0.71; T1 and T3 1-mth FU (5.86, 5.77) (t = 6.22) = 4.95, p < 0.001, d = 0.82; T1 and T4 3-mth FU (5.33, 5.24) (t = 6.39) = 5.49, p < 0.001; and T1 and T5 6-mth FU (4.54, 4.94) (t = 8.89) = 7.15, p < 0.001. In determining the overall effect size of the treatment intervention regarding the GAD-7, the overall effect size calculated as η2 = 0.136 which is on the threshold of a large treatment effect. Frontiers in Psychology frontiersin.org Data analysis Hypothesis 3: Treating the trauma sequelae will reduce the level of moral injury in active and control groups. The Moral Injury Events Scale (MIES) was selected for this study as it has been adapted and applied within civilian research, has the advantage of measuring both events and symptoms, and, as a psychometric, is brief and straightforward in wording (Nash et al., 2013; Koenig et al., 2019). Although the MIES has a three-factor structure: moral violations either perpetrated or witnessed by the individual, and betrayal experiences results at this stage only focused on total scores. Further analysis will be presented in a future paper. Figure 8 demonstrate no treatment effect between T1 pre and T5 11 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 FIGURE 8 Moral injury events scale (MIES): T1 Pre and T5 6-mth FU (N=85).. FIGURE 8 Moral injury events scale (MIES): T1 Pre and T5 6-mth FU (N=85).. Moral injury events scale (MIES): T1 Pre and T5 6-mth FU (N=85).. TABLE 5 Changes in clinical diagnosis following VGTEP intervention using the ITQ at T1 and T5 (N = 85). 6-month FU, measured by the MIES, of the VGTEP intervention in both active and control groups. TABLE 5 Changes in clinical diagnosis following VGTEP intervention using the ITQ at T1 and T5 (N = 85). Initial diagnosis @ T1 pre-treatment No. Final diagnosis @ T5 6-mth FU No. o Sub-clinical PTSD1 24 Sub-clinical PTSD 23 PTSD 1 Complex PTSD 0 o PTSD 28 Sub-clinical PTSD 28 PTSD 0 Complex PTSD 0 o Complex PTSD 33 Sub-clinical PTSD 24 PTSD 2 Complex PTSD 7 1Sub-clinical IES-R score of 24-32 (clinical concern). Regarding hypothesis 3, the results do not support this hypothesis. Although the VGTEP intervention effectively treated trauma, anxiety, and depression symptoms, results demonstrate no discernible treatment effect on moral injury. Hypothesis 4: Using Trauma-Focused Therapist rotation will demonstrate no change in clinical diagnosis between T1 and T5, for both active and control groups. Regarding the diagnoses of PTSD and Complex PTSD, using the ITQ as a diagnostic indicator, Table 5 highlights the impact of the VGTEP treatment intervention at post-treatment (N = 85). At T1 N = 24 had sub-clinical PTSD, N = 28 PTSD, and N = 33 Complex PTSD. Frontiers in Psychology frontiersin.org Data analysis TABLE 6 Correlation matrix between ACEs and BCEs. Total ACE Score Total BCE score Total ACE Score Pearson’s r — p value — Total BCE score Pearson’s r −0.312* — p value 0.004 — p < 0.05, p < 0.01. TABLE 6 Correlation matrix between ACEs and BCEs. TABLE 6 Correlation matrix between ACEs and BCEs. At T5 6-month FU, every research participant contacted to provide brief feedback about their lived experience of undergoing the VGTEP intervention. A further research paper will explore this in more detail. However, Figure 10 highlights the primary response which clustered around 13 themes. The overwhelming feedback appeared to suggest the VGTEP intervention to have been effective, helpful, and timely. Frontiers in Psychology Data analysis Of the research participants with sub-clinical PTSD at the start of the treatment intervention results indicated that 95.83% remained sub-clinical at T5 6-month FU, with 4.17% meeting the criterion for PTSD. Those meeting the criterion for Complex PTSD results suggest a 72.73% improvement to sub-clinical, however, 21.21% showed limited improvement from the VGTEP treatment intervention. 1Sub-clinical IES-R score of 24-32 (clinical concern). significant difference between T1 pre (65.02, 17.99) and T5 (79.19, 14.84) t(−6.99) = −14.3, p < 0.001. In determining the overall effect size of the treatment intervention regarding the EQ-5D, calculated as η2 = 0.159 indicating a large treatment effect. There was not statistical difference between each of the groups. A more in-depth review of the health economic data will follow in a later paper. Despite using a therapist rotation model within the clinical trial results highlight significant alterations in diagnosis at T5 6-mth FU. The VGTEP interventions had marked efficacy with both the sub-clinical and the PTSD research participants. In the sub-category, only one research participant was referred for additional help and support. With a dropout rate of 10.53% from the clinical trial, and 11.76% finding the treatment intervention insufficient, the overall recovery rate from the VGTEP intervention is estimated at 77.71%. Results confirm that hypothesis 4 is not supported – a favorable recovery rate was achieved using a therapist rotation model. Hypothesis 6: A correlation exists, within the target population, between Adverse Childhood Experiences (ACEs) and Benevolent Childhood Experiences (BCEs) regarding evidence supporting resilience and posttraumatic growth. Hypothesis 5: There is no difference between active and control groups regarding quality of life using the EQ-5D – a generic measure of an individual’s health status and quality of life. A Pearson correlation coefficient was computed to assess the linear relationship between Adverse Childhood Experiences (ACEs) and Benevolent Childhood Experiences (BCEs). Results revealed a moderate, negative correlation between the two r = −0.312, *p = <0.01 as highlighted in Table 6. Bivariate analysis of either ACEs or BCEs revelated no other linear relationships with the ITQ, GAD-7, PHQ-9, MIES, or EQ-5D. Results indicate that the VGTEP treatment Figure 9 highlights that for the EQ-5D results demonstrated a significant reduction in both groups over time F(1–83) = 48.8, p < 0.001. Post Hoc comparisons of the EQ-5D demonstrated a 12 Farrell et al. 10.3389/fpsyg.2023.1129912 FIGURE 9 Improvements in EQ-5D between T1 and T5 active and control groups. frontiersin.org FIGURE 10 Primary themes related to the qualitative feedback received from research participants post T5 (6-mth FU/end of treatment). Although results are favorable regarding the ITQ, GAD-7, PHQ-9, SUD, and EQ-5D, demonstrating that the VGTEP intervention appeared relevant to the research population under investigation, results also suggest that the intervention was well tolerated, potentially safe, and effective as a treatment intervention. Intriguingly, there was no demonstrable difference between active and control (delayed) groups. Despite promising results, caution also needs to be exercised. The absence of another trauma-focused treatment intervention as a realistic comparator suggests that more research is required to ascertain a more reliable efficiency indicator. For example, future early intervention should consider comparing VGTEP with another Trauma-focused CBT Group Intervention. The GTEP protocol involves 27 episodes of trauma confrontation and requires further research and investigation, particularly into its distinctive parts – which are essential, which not? Results from this study demonstrate an overall treatment effect; however, further dismantling studies need to explore whether there might be more effective or efficient means of achieving similar or better results. Results from Table 5 are intriguing. Within the first category 28% were sub-clinical PTSD, and post-treatment 96% remained in this category. Could this potentially suggest that early intervention Group EMDR prevented the onset of PTSD? Further research is needed to explore this, however, even though results from this study are promising caution against over-reach is important. Of further interest relate to the PTSD and Complex PTSD data which suggest the favorability of the Group EMDR intervention. Again, these results are cautiously promising, however, further research is needed. Overall, the dropout rate for the study was just below 11%. There are two ways of looking at this; firstly, this level is particularly favorable to the context in which the research study operated – during a global pandemic. A contrary perspective relates to the tolerability of any psychological treatment intervention. There will always be dropouts related to factors unrelated to the study intervention – this was certainly the case for some but by no means all. However, following up on those that did drop out and relate to the treatment intervention highlighted that for some, it was too intense, too powerful, and too overwhelming, bearing in mind everything else going on in people’s lives at the time. Roberts et al. (2019) conclude little evidence that most multiple- session trauma treatment interventions have little impact on primary outcome measures. However, our study’s results do not concur with this perspective. FIGURE 10 Primary themes related to the qualitative feedback received from research participants post T5 (6-mth FU/end of treatment). non-disclosure of any trauma material. This aspect has advantages and disadvantages. Advantages include a greater willingness to work on traumatic material without the need for disclosure. This maximizes both power and control for the research participants. If trauma memories involve shame, fear of retribution, or a shattering of assumptive networks then there are clear benefits to non-disclosure. Disadvantages include a difficulty in demonstrating cause and effect. Although research participants were invited to work with recent material there was no means to accurately monitor this. This both a strength and limitation to this study. That said, previous research demonstrates qqclear benefits from working with non-disclosed trauma material (Farrell et al., 2020, 2022). Non-disclosure of trauma material does have another distinct advantage as non-disclosure of trauma material potentially reduces the risk of vicarious traumatization of therapists. However, more research is needed to explore this aspect further to ascertain if there is veracity in this argument. FIGURE 10 Primary themes related to the qualitative feedback received from research participants post T5 (6-mth FU/end of treatment). FIGURE 10 Primary themes related to the qualitative feedback received from research participants post T5 (6-mth FU/end of treatment). The headline results from this RCT suggest that EMDR therapy, delivered remotely through a virtual platform was effective in the treatment of both PTSD and complex PTSD. This is an important finding considering the critical elements of group treatment, remote intervention, intensive delivery, and therapist rotation. When demand outstrips supply regarding access to trauma interventions research supporting the benefits of group treatment suggest significant resource benefits. Further research is needed to explore the potential benefits of this in relation to task-shifting. The study’s results shed interesting light on the therapist rotation model. Instinctively, therapist rotation seems counterintuitive, considering the therapist relationship critical to outcome. Results from this study run counter to this narrative. Why is therapist rotation effective? There are several reasons to consider. The VGTEP intervention is highly structured – there are distinct benefits from this, reassurance, familiarity, containment, etc., however, the intervention is relatively passive. This argument may provide a rationale for why VGTEP was effective for PTSD/C-PTSD, but not Moral Injury (MI), is that MI requires more active engagement. This would require further investigation and study, and if proven correct, would necessitate further adaptation in EMDR group interventions. However, based upon this data set, results do not support its efficacy as a treatment for Moral Injury. Discussion Undertaking any large-scale, early intervention clinical trial is always difficult and present significant logistical challenges. Doing so during periods of Government-Imposed lockdown was also immensely demanding. The rationale for the study was to carry out a multi-component research project which focused on an important population at the frontline of the COVID-19 response. The realities of the COVID-19 pandemic, and carrying out clinical research, meant that everyone involved – the research participants, research team, and clinicians were all exposed to the ‘lived experience’ realities of COVID-19. Therefore, a reasonable conclusion is that the universality level for this whole study was extraordinarily high. As highlighted within the literature review, defining early intervention is not straight forward. Considering the ongoing nature of the pandemic for the entire duration of this RCT there is an argument cogent argument for this qualifying as an early intervention study. TABLE 7 Qualitative factors which impacted upon T2–T5 psychometric data. TABLE 7 Qualitative factors which impacted upon T2–T5 psychometric data. Theme Number and % Referred for follow-up care after T2 9 (10.58%) Struggled with continued post-COVID symptoms/ complications (long COVID) @ T5 5 (5.89%) Additional post-trauma experiences not COVID-19 related post T2 4 (4.71%) intervention was effective regardless of a research participants ACE or BCE scores. Table 7 highlights qualitative data related to the research participants which focused on the drop-outs from the study. Each participant was followed-up and offered further support and intervention. As their withdrawal impacted upon the T2 – T5 psychometric data their data removed from the overall data set. Although a 10.58% drop-out rate is low it is also reasonable to assume that not every client, once they have direct experience of working in this way, will find it useful. An important reality of the current time was that lack of availability of trauma-informed early intervention for frontline emergency workers paradoxically assisted recruitment for our study. The consequence of this resulted in most research participants highly motivated, and appreciative to being included. Despite the eventual recruitment, a larger sample would have further enhanced the study’s findings. However, as this was an unfunded study, it was essential to make the limited resources we had stretch as far as possible. 13 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 non-disclosure of any trauma material. This aspect has advantages and disadvantages. Advantages include a greater willingness to work on traumatic material without the need for disclosure. Frontiers in Psychology frontiersin.org Discussion This maximizes both power and control for the research participants. If trauma memories involve shame, fear of retribution, or a shattering of assumptive networks then there are clear benefits to non-disclosure. Disadvantages include a difficulty in demonstrating cause and effect. Although research participants were invited to work with recent material there was no means to accurately monitor this. This both a strength and limitation to this study. That said, previous research demonstrates qqclear benefits from working with non-disclosed trauma material (Farrell et al., 2020, 2022). Non-disclosure of trauma material does have another distinct advantage as non-disclosure of trauma material potentially reduces the risk of vicarious traumatization of therapists. However, more research is needed to explore this aspect further to ascertain if there is veracity in this argument.h Conclusion Another critical point of note relates to Adverse and Benevolent Childhood Experiences. Results from this study parallel an earlier study (Farrell et al., 2022) demonstrating a trauma treatment effect irrespective of either individual or collective ACEs or BCEs. Although results show a moderate, negative correlation between ACEs and BCEs, this certainly needs further research and exploration. The evidence from this study suggests that adverse memories process in EMDR therapy regardless of the form the adversity takes. The way benevolent factors potentially mitigate does require further investigation, particularly utilizing the theoretical framework of Adaptive Information Processing – the model underpinning EMDR therapy. The COVID-19 pandemic has presented significant challenges individually, collectively, and geopolitically. Ascertaining treatment interventions that can be delivered remotely safely, relevant to trauma populations, that are both effective and efficient, is paramount. In addition, the advantage of treatment given through video-conference platforms potentially increases availability and access. Critical to this relates to technical components, including availability, access, and functionality in making such treatment interventions viable on a fundamental level of scalability.hfi We recognize that follow periods reported within this RCT study are short (Dumoulin et al., 2018), however we took the pragmatic decision to use three-and six-month timeframes for follow up given the relative urgency to provide online psychological support to frontline workers during the pandemic (Drissi et al., 2021). A recent systemic review (Wilson et al., 2018) suggests follow up periods of between 4 weeks and 6 months is not uncommon in EMDR intervention research. For example, in what appears to be the only trial of online EMDR (Lenferink et al., 2020), Spence et al., 2013 used a 3 month follow up period when examine the use of internet delivered EMDR. We are therefore content that the design chosen for the study is in line with other EMDR related randomized controlled trials, however, acknowledge that the longer-term maintenance of benefits from the intervention may not be fully understood. This research suggests the potential efficacy of the Group EMDR intervention; incorporating a therapist rotation model adds a contemporary facet that seems counterintuitive to the existing academic literature. In addressing the global burden of psychological trauma and the necessity for task-shifting, this study offers interesting findings to this debate. The primary objective and rationale for this study were Frontline and Emergency colleagues working on the frontline of COVID-19 traumatized by their endeavors. Data availability statement A further, distinct limitation of the study relates to the issue of moral injury. Further research is needed to adapt EMDR group interventions more specifically more sufficiently for this important issue. Secondly, with the benefit of hindsight, incorporating more qualitative data surrounding the participants experience would have been useful. Presently, there is no absolute clarity surrounding agents of change other than from the psychometric data. Thirdly, it would have been useful to ascertain better insight into the target memories worked on by the research participants. This was always going to be challenging based the critical aspect of non-disclosure. A further aspect related to this would have been to determine if research participants may indeed have been willing to disclose their trauma targets. Fourthly, would be to consider if more detailed debriefing sessions after the VGTEP intervention may have further enhanced the study’s findings. Fifthly, as the research participants were ‘self- selecting’ this leads to the possibility of bias and ambiguity. By not capturing or clarifying motivation for being part of the study there is a fair degree of uncertainty about this aspect. Furthermore, a distinct limitation was not being able to ascertain which trauma targets were moral injury, or not. A final limitation relates to the sub-clinical PTSD participants. The data set suggests that many from this group remained ‘sub-clinical’ post-intervention, however, there is not means to determine cause and effect, that the intervention prevented the onset of PTSD. To ascertain this with more clarity further research is needed. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found in the article/supplementary material. Frontiers in Psychology FIGURE 10 Primary themes related to the qualitative feedback received from research participants post T5 (6-mth FU/end of treatment). There is, nonetheless, an important narrative to consider at this juncture – results from this study suggest a significant treatment effect for PTSD but not for moral injury. Why might this be the case? This is an intriguing question. A potential consideration relates to the ‘unknowingness’ of the trauma memory clients use to target and reprocess. The EMDR GTEP and VGTEP interventions are ostensibly passive treatments. Within conventional EMDR therapy, moral dimensions are often addressed more actively with the utilization of Cognitive Interweaves, which are much more bespoke to the client’s lived experiences. However, more research is needed to explore this further, but results suggest a potential need for modifying the existing group EMDR protocols to address moral dimensions of trauma more effectively. There are four other factors to consider. Firstly, treatment was delivered as video-conference psychotherapy. Overall results suggest that this was generally well tolerated. Although there were occasional technical glitches, fortunately, these were relatively infrequent. Indeed, the ZOOM platform performed robustly for much of the study—Secondly, the intense delivery of the treatment within 1 week. Again, results suggest that this was well tolerated, although admittedly, it does require a significant commitment from research participants. Further research needs to explore the potential health economic benefits of delivering trauma treatment in this manner. Thirdly, the VGTEP intervention involves 14 Farrell et al. Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 Author contributions DF conceived the study, was the chief investigator for the entire project, the principal data analyst, and the primary author of this research manuscript. JM was responsible for project management. MK was responsible for secondary data analysis, senior leadership, and support to the project. LK, PM, IM, DM, TP, PP, CM, and IB were vital research team members and treatment interventions. ZZ was responsible for treatment fidelity. DF, LK, JM, PM, IM, DM, TP, and PP were all clinical leads regarding the VGTEP treatment intervention. All authors contributed to the article and approved the submitted version. A contemporary debate within the EMDR therapy literature relates to the value of trauma regulation. The data presented in Figure 5 suggests treatment benefits for trauma regulation and trauma confrontation phases with VGTEP; however, the effect size is more significant for trauma confrontation. Nevertheless, results suggest the clear advantage of utilizing trauma regulation strategies within the VGTEP intervention, even if this provides a grounding, presentness, or even a sense of community-building. Further dismantling studies need to explore this aspect in more detail. Conclusion This study contributes to the emerging knowledge base that Group EMDR therapy has something significant to contribute. Ethics statement The studies involving human participants were reviewed and approved by University of Worcester. The patients/participants provided their written informed consent to participate in this study. frontiersin.org References comparing regions with increasing pandemic incidence. J. Affect. Disord. 298, 209–216. doi: 10.1016/j.jad.2021.10.128 Acarturk, C., Konuk, E., Cetinkaya, M., Senay, I., Sijbrandij, M., Gulen, B., et al. (2016). The efficacy of eye movement desensitization and reprocessing for post- traumatic stress disorder and depression among Syrian refugees: results of a randomized controlled trial. Psychol. Med. 46, 2583–2593. doi: 10.1017/S0033291716001070 comparing regions with increasing pandemic incidence. J. Affect. Disord. 298, 209–216. doi: 10.1016/j.jad.2021.10.128 Acarturk, C., Konuk, E., Cetinkaya, M., Senay, I., Sijbrandij, M., Gulen, B., et al. (2016). The efficacy of eye movement desensitization and reprocessing for post- traumatic stress disorder and depression among Syrian refugees: results of a randomized controlled trial. Psychol. Med. 46, 2583–2593. doi: 10.1017/S0033291716001070 Carriere, R. C. (2014). Scaling up what works: using EMDR to help confront the World’s burden of traumatic stress. J. EMDR Pract. Res. 8, 187–195. doi: 10.1891/1933-3196.8.4.187 Amsalem, D., Lazarov, A., Markowitz, J. C., Naiman, A., Smith, T. E., Dixon, L. B., et al. (2021). Psychiatric symptoms and moral injury among US healthcare workers in the COVID-19 era. BMC Psychiatry 21:546. doi: 10.1186/s12888-021-03565-9 Amsalem, D., Lazarov, A., Markowitz, J. C., Naiman, A., Smith, T. E., Dixon, L. B., et al. (2021). Psychiatric symptoms and moral injury among US healthcare workers in the COVID-19 era. BMC Psychiatry 21:546. doi: 10.1186/s12888-021-03565-9 Čartolovni, A., Stolt, M., Scott, P. A., and Suhonen, R. (2021). Moral injury in healthcare professionals: A scoping review and discussion. Nurs. Ethics 28, 590–602. doi: 10.1177/0969733020966776 Appleton, R., Williams, J., Needle, J. J., Schlief, M., Jordan, H., Sheridan Rains, L., et al. (2021). Implementation, adoption, and perceptions of Telemental health during the COVID-19 pandemic: systematic review. J. Med. Internet Res. 23:e31746. doi: 10.2196/31746 Cataldo, F., Chang, S., Mendoza, A., and Buchanan, G. (2021). A perspective on client- psychologist relationships in videoconferencing psychotherapy: literature review. JMIR Mental Health 8:e19004. doi: 10.2196/19004 Arpacioglu, S., Gurler, M., and Cakiroglu, S. (2021). Secondary traumatization outcomes and associated factors among the health care workers exposed to the COVID-19. Int. J. Soc. Psychiatry 67, 84–89. doi: 10.1177/0020764020940742 Clement, S., Schauman, O., Graham, T., Maggioni, F., Evans-Lacko, S., Bezborodovs, N., et al. (2015). What is the impact of mental health-related stigma on help-seeking? A systematic review of quantitative and qualitative studies. Psychol. Med. 45, 11–27. doi: 10.1017/S0033291714000129 Asmussen, K., McBride, T., and Waddell, S. (2019). The potential of early intervention for preventing and reducing ACE-related trauma. Soc. Policy Soc. 18, 425–434. References Beacon Press. de Kock, J. H., Latham, H. A., Leslie, S. J., Grindle, M., Munoz, S.-A., Ellis, L., et al. (2021). A rapid review of the impact of COVID-19 on the mental health of healthcare workers: implications for supporting psychological well-being. BMC Public Health 21:104. doi: 10.1186/s12889-020-10070-3 Borges, L. M., Barnes, S. M., Farnsworth, J. K., Bahraini, N. H., and Brenner, L. A. (2020). A commentary on moral injury among health care providers during the COVID-19 pandemic. Psychol. Trauma Theory Res. Pract. Policy 12, S138–S140. doi: 10.1037/tra0000698 de Roos, C., Greenwald, R., den Hollander-Gijsman, M., Noorthoorn, E., van Buuren, S., and de Jongh, A. (2011). A randomized comparison of cognitive behavioral therapy (CBT) and eye movement desensitization and reprocessing (EMDR) in disaster- exposed children. Eur. J. Psychotraumatol. 2:5694. doi: 10.3402/ejpt.v2i0.5694 Broadbear, J. H., Heidari, P., Dharwadkar, N. P., Cheney, L., and Rao, S. (2021). Telehealth psychotherapy for severe personality disorder during COVID-19: experience of Australian clinicians. Global J. Health Sci. 13:61. doi: 10.5539/gjhs.v13n12p61 Brunet, A., Rivest-Beauregard, M., Lonergan, M., Cipolletta, S., Rasmussen, A., Meng, X., et al. (2022). PTSD is not the emblematic disorder of the COVID-19 pandemic; adjustment disorder is. BMC Psychiatry 22:300. doi: 10.1186/ s12888-022-03903-5 Dharwadkar, N. P., Broadbear, J. H., Heidari, P., Cheney, L., and Rao, S. (2021). Psychotherapy via Telehealth during the COVID-19 pandemic in australia–experience of clients with a diagnosis of borderline personality disorder. Global J. Health Sci. 14:29. doi: 10.5539/gjhs.v14n1p29 Bryant, R. A. (2021). A critical review of mechanisms of adaptation to trauma: implications for early interventions for posttraumatic stress disorder. Clin. Psychol. Rev. 85:101981. doi: 10.1016/j.cpr.2021.101981 Diop, N., Anderson, I., Gwezera, B., Fihn, J., Gohar, F., Morgos, D., et al. (2022). Mental health and psychosocial support concerns among frontline workers within the eastern and southern Africa COVID-19 response. Pan African Med. J. 41:9032. doi: 10.11604/pamj.supp.2022.41.2.29032 Bryant, R. A. (2022). “Early intervention after trauma” in Evidence based treatments for trauma-related psychological disorders (Springrm, Cham: Springer International Publishing), 135–159. doi: 10.1007/978-3-030-97802-0_7 Do, D. P., and Frank, R. (2021). U.S. frontline workers and COVID-19 inequities. Prev. Med. 153:106833. doi: 10.1016/j.ypmed.2021.106833 Cantone, D., Guerriera, C., and ARCHITRAVO, M., (2021). A sample of Italian psychotherapists express their perception and opinions of online psychotherapy during the covid-19 pandemic. Riv. Psichiatr. 56, 98–204. doi: 10.1708/3654.36347 Drissi, N., Ouhbi, S., Marques, G., de la Torre Díez, I., Ghogho, M., and Janati Idrissi, M. A. (2021). Publisher’s note Finally, we thank EMDR All Ireland Board—President Gus Murray for the vital support of the research project. Finally, we thank EMDR All Ireland Board—President Gus Murray for the vital support of the research project. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Acknowledgments We thank Trauma Response Ireland and Sonjia Maher for her invaluable contribution and administrative support. Additionally, we would like to thank Clare Smith, Adam Horner, Gus Murray, Bev Taylor, Carol Norris, Imelda Buckley, Yvonne Bayo, and the many colleagues who assisted this study as the Emotional Protection team. 15 frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References doi: 10.1017/S1474746419000071 Cloitre, M., Shevlin, M., Brewin, C. R., Bisson, J. I., Roberts, N. P., Maercker, A., et al. (2018). The international trauma questionnaire: development of a self-report measure of ICD-11 PTSD and complex PTSD. Acta Psychiatr. Scand. 138, 536–546. doi: 10.1111/ acps.12956 Asukai, N., Kato, H., Kawamura, N., Kim, Y., Yamamoto, K., Kishimoto, J., et al. (2002). RELIABILIGY and validity of the JAPANESE-language version of the impact of event scale-REVISED (IES-r-j): four studies of different traumatic events. J. Nerv. Ment. Dis. 190, 175–182. doi: 10.1097/00005053-200203000-00006 Cohen, J. (1992). Statistical Power Analysis. Curr. Direct. Psychol. Sci. 1, 98–101. doi: 10.1111/1467-8721.ep10768783 Barnes, H. A., Hurley, R. A., and Taber, K. H. (2019). Moral injury and PTSD: often co-occurring yet mechanistically different. J. Neuropsychiatry Clin. Neurosci. 31, A4– A103. doi: 10.1176/appi.neuropsych.19020036 Covers, M. L. V., de Jongh, A., Huntjens, R. J. C., de Roos, C., van den Hout, M., and Bicanic, I. A. E. (2021). Early intervention with eye movement desensitization and reprocessing (EMDR) therapy to reduce the severity of post-traumatic stress symptoms in recent rape victims: a randomized controlled trial. Eur. J. Psychotraumatol. 12:3188. doi: 10.1080/20008198.2021.1943188 Beames, J. R., Christensen, H., and Werner-Seidler, A. (2021). School teachers: the forgotten frontline workers of Covid-19. Australas. Psychiatry 29, 420–422. doi: 10.1177/10398562211006145 Creamer, M., Bell, R., and Failla, S. (2003). Psychometric properties of the impact of event scale—Revised. Behav. Res. Ther. 41, 1489–1496. doi: 10.1016/j. brat.2003.07.010 Beck, J. G., Grant, D. M., Read, J. P., Clapp, J. D., Coffey, S. F., Miller, L. M., et al. (2008). The impact of event scale-revised: psychometric properties in a sample of motor vehicle accident survivors. J. Anx. Disorders 22, 187–198. Creamer, M. C., Varker, T., Bisson, J., Darte, K., Greenberg, N., Lau, W., et al. (2012). Guidelines for peer support in high-risk organizations: an international consensus study using the delphi method. J. Trauma. Stress. 25, 134–141. doi: 10.1002/jts.21685 Billings, J., Ching, B. C. F., Gkofa, V., Greene, T., and Bloomfield, M. (2021). Experiences of frontline healthcare workers and their views about support during COVID-19 and previous pandemics: a systematic review and qualitative meta-synthesis. BMC Health Serv. Res. 21:923. doi: 10.1186/s12913-021-06917-z Crowe, M., Inder, M., Farmar, R., and Carlyle, D. (2020). Delivering psychotherapy by video conference in the time of COVID-19: some considerations. J. Psychiat. Mental Health Nurs. 28, 751–752. doi: 10.1111/jpm.12659 Brock, R. N., and Lettini, G. (2012). Soul repair: Recovering from moral injury after war. References A systematic literature review on e-mental health solutions to assist health care workers during COVID-19. Telemed. E-Health 27, 594–602. doi: 10.1089/ tmj.2020.0287 Carmassi, C., Dell’Oste, V., Bui, E., Foghi, C., Bertelloni, C. A., Atti, A. R., et al. (2022). The interplay between acute post-traumatic stress, depressive and anxiety symptoms on healthcare workers functioning during the COVID-19 emergency: A multicenter study Dumoulin, C., Cacciari, L. P., and Hay-Smith, E. J. C. (2018). Pelvic floor muscle training versus no treatment, or inactive control treatments, for urinary incontinence in 16 Frontiers in Psychology frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 women. Cochrane Database Syst. Rev. 2018:CD005654. doi: 10.1002/14651858. CD005654.pub4 women. Cochrane Database Syst. Rev. 2018:CD005654. doi: 10.1002/14651858. CD005654.pub4 Jamieson, N., Usher, K., Maple, M., and Ratnarajah, D. (2020b). Invisible wounds and suicide: moral injury and veteran mental health. Int. J. Ment. Health Nurs. 29, 105–109. doi: 10.1111/inm.12704 Dunkley, F. (2018). Psychosocial support for humanitarian aid workers: a roadmap of trauma and critical incident care. London UK: Routledge. Jarero, I., Givaudan, M., and Osorio, A. (2018). Randomized controlled trial on the provision of the EMDR integrative group treatment protocol adapted for ongoing traumatic stress to female patients with cancer-related posttraumatic stress disorder symptoms. J. EMDR Pract. Res. 12, 94–104. doi: 10.1891/1933-3196.12.3.94 Dyregrov, A., and Regel, S. (2012). Early interventions following exposure to traumatic events: implications for practice from recent research. J. Loss Trauma 17, 271–291. doi: 10.1080/15325024.2011.616832 Jecker, N. S., Wightman, A. G., and Diekema, D. S. (2020). Prioritizing frontline workers during the COVID-19 pandemic. Am. J. Bioeth. 20, 128–132. doi: 10.1080/15265161.2020.1764140 Eichfeld, C., Farrell, D., Mattheß, M., Bumke, P., Sodemann, U., Ean, N., et al. (2019). Trauma stabilization as a sole treatment intervention for post-traumatic stress disorder in Southeast Asia. Psychiatry Q. 90, 63–88. doi: 10.1007/s11126-018-9598-z Johnson, S. U., Ebrahimi, O., and v., & Hoffart, A., (2020). PTSD symptoms among health workers and public service providers during the COVID-19 outbreak. PLoS One 15:e0241032. doi: 10.1371/journal.pone.0241032 Farnsworth, J. K. (2019). Is and ought: descriptive and prescriptive cognitions in military-related moral injury. J. Trauma. Stress. 32, 373–381. doi: 10.1002/jts.22356 Farrell, D., Fadeeva, A., Zat, Z., Knibbs, L., Miller, P., Barron, I., et al. (2022). A stage 1 pilot cohort exploring the use of EMDR therapy as a videoconference psychotherapy during COVID-19 with frontline mental health workers: A proof-of-concept study Utilizing a virtual blind 2 therapist protocol. Front. Psychol. 13:1855. References Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs. Front. Psychol. 4:863. doi: 10.3389/ fpsyg.2013.00863 Greenberg, N., Docherty, M., Gnanapragasam, S., and Wessely, S. (2020). Managing mental health challenges faced by healthcare workers during covid-19 pandemic. BMJ 368:m1211. doi: 10.1136/bmj.m1211 Laliotis, D., and Shapiro, F. (2022). “EMDR therapy for trauma-related disorders” in Evidence based treatments for trauma-related psychological disorders. eds. U. Schnyder and M. Cloitre (Springer, Cham: Springer International Publishing), 227–254. Greene, T., Harju-Seppänen, J., Adeniji, M., Steel, C., Grey, N., Brewin, C. R., et al. (2021). Predictors and rates of PTSD, depression and anxiety in UK frontline health and social care workers during COVID-19. Eur. J. Psychotraumatol. 12:2781. doi: 10.1080/20008198.2021.1882781 Lasalvia, A., Amaddeo, F., Porru, S., Carta, A., Tardivo, S., Bovo, C., et al. (2021). Levels of burn-out among healthcare workers during the COVID-19 pandemic and their associated factors: a cross-sectional study in a tertiary hospital of a highly burdened area of north-East Italy. BMJ Open 11:e045127. doi: 10.1136/ bmjopen-2020-045127 Griffin, B. J., Purcell, N., Burkman, K., Litz, B. T., Bryan, C. J., Schmitz, M., et al. (2019). Moral injury: an integrative review. J. Trauma. Stress. 32, 350–362. doi: 10.1002/ jts.22362 Lenferink, L. I. M., Meyerbröker, K., and Boelen, P. A. (2020). PTSD treatment in times of COVID-19: A systematic review of the effects of online EMDR. Psychiatry Res. 293:113438. doi: 10.1016/j.psychres.2020.113438 Gronholm, P. C., Nosé, M., van Brakel, W. H., Eaton, J., Ebenso, B., Fiekert, K., et al. (2021). Reducing stigma and discrimination associated with COVID-19: early-stage pandemic rapid review and practical recommendations. Epidemiol. Psychiatr. Sci. 30:e15. doi: 10.1017/S2045796021000056 Litz, B. T., Stein, N., Delaney, E., Lebowitz, L., Nash, W. P., Silva, C., et al. (2009). Moral injury and moral repair in war veterans: A preliminary model and intervention strategy. Clin. Psychol. Rev. 29, 695–706. doi: 10.1016/j.cpr.2009.07.003 Hase, M. (2021). The structure of EMDR therapy: A guide for the therapist. Front. Psychol. 12:753. doi: 10.3389/fpsyg.2021.660753 Magruder, K. M., McLaughlin, K. A., and Elmore Borbon, D. L. (2017). Trauma is a public health issue. Eur. J. Psychotraumatol. 8:1375338. doi: 10.1080/20008198.2017.1375338 Hase, M., Balmaceda, U. M., Ostacoli, L., Liebermann, P., and Hofmann, A. (2017). The AIP model of EMDR therapy and pathogenic memories. Front. Psychol. 8:1578. doi: 10.3389/fpsyg.2017.01578 Marcus, S., Marquis, P., and Sakai, C. (2004). Three-and 6-month follow-up of EMDR treatment of PTSD in an HMO setting. Int. J. Stress. Manag. References doi: 10.3389/ fpsyg.2022.901855 Vera San Juan, N., Shah, P., Schlief, M., Appleton, R., Nyikavaranda, P., Birken, M., et al. (2021). Service user experiences and views regarding telemental health during the COVID-19 pandemic: A co-produced framework analysis. PLoS One 16:e0257270. doi: 10.1371/journal.pone.0257270 Kaptan, S. K., Dursun, B. O., Knowles, M., Husain, N., and Varese, F. (2021). Group eye movement desensitization and reprocessing interventions in adults and children: A systematic review of randomized and nonrandomized trials. Clin. Psychol. Psychother. 28, 784–806. doi: 10.1002/cpp.2549 Farrell, D. P., Keenan, P. S., Ali, M. W., Bilal, S., Tareen, S. M., Keenan, L., et al. (2011). Training Pakistani mental health workers in EMDR in the aftermath of the 2005 earthquake in northern Pakistan. Couns. Psychol. Q. 24, 127–137. doi: 10.1080/09515070.2011.589599 Koenig, H. G., Youssef, N. A., Ames, D., Teng, E. J., and Hill, T. D. (2020). Examining the overlap between moral injury and PTSD in US veterans and active-duty military. J. Nerv. Ment. Dis. 208, 7–12. doi: 10.1097/NMD.0000000000001077 Farrell, D., Keenan, P., Knibbs, L., and Hicks, C. (2013). A Q-methodology evaluation of an EMDR europe HAP facilitators training in Pakistan. J. EMDR Pract. Res. 7, 174–185. doi: 10.1891/1933-3196.7.4.174 Koenig, H. G., Youssef, N. A., and Pearce, M. (2019). Assessment of moral injury in veterans and active-duty military personnel with PTSD: A review. Front. Psych. 10:443. doi: 10.3389/fpsyt.2019.00443 Farrell, D., Kiernan, M. D., de Jongh, A., Miller, P. W., Bumke, P., Ahmad, S., et al. (2020). Treating implicit trauma: a quasi-experimental study comparing the EMDR therapy standard protocol with a “blind 2 Therapist” version within a trauma capacity building project in northern Iraq. J. Int. Human. Act. 5:3. doi: 10.1186/ s41018-020-00070-8 Krampe, H., and Ehrenreich, H. (2012). Therapeutic alliance and multiple psychotherapy in the context of therapist rotation: experiences with OLITA. Neurol. Psychiatry Brain Res. 18, 137–152. doi: 10.1016/j.npbr.2012.05.003 Feingold, J. H., Hurtado, A., Feder, A., Peccoralo, L., Southwick, S. M., Ripp, J., et al. (2022). Posttraumatic growth among health care workers on the frontlines of the COVID-19 pandemic. J. Affect. Disord. 296, 35–40. doi: 10.1016/j.jad.2021.09.032 Kroenke, K., Spitzer, R. L., and Williams, J. B. W. (2001). The PHQ-9. J. Gen. Intern. Med. 16, 606–613. doi: 10.1046/j.1525-1497.2001.016009606.x Ghahramani, S., Kasraei, H., Hayati, R., Tabrizi, R., and Marzaleh, M. A. (2022). Health care workers’ mental health in the face of COVID-19: a systematic review and meta-analysis. Int. J. Psychiatry Clin. Pract. 23, 1–10. doi: 10.1080/13651501.2022.2101927 Lakens, D. (2013). References 11, 195–208. doi: 10.1037/1072-5245.11.3.195 Haugen, P. T., Evces, M., and Weiss, D. S. (2012). Treating posttraumatic stress disorder in first responders: A systematic review. Clin. Psychol. Rev. 32, 370–380. doi: 10.1016/j.cpr.2012.04.001 Mattheß, C., Farrell, D., Mattheß, M., Bumke, P., Sodemann, U., and Mattheß, H. (2019). The therapeutic value of trauma stabilization in the treatment of post-traumatic stress disorder – A southeast Asian study. Asian J. Psychiatr. 41, 45–49. doi: 10.1016/j. ajp.2018.09.010 Herman, J. L., and Kallivayalil, D. (2018). Group trauma treatment in early recovery. Guilford Publications. New York, United States. Mattheß, C., Farrell, D., Mattheß, M., Bumke, P., Sodemann, U., and Mattheß, H. (2020). Trauma stabilization as an effective treatment for children with post-traumatic stress problems in South-East Asia. Int. J. Ment. Health Nurs. 29, 725–735. doi: 10.1111/ inm.12707 Hooper, J. J., Saulsman, L., Hall, T., and Waters, F. (2021). Addressing the psychological impact of COVID-19 on healthcare workers: learning from a systematic review of early interventions for frontline responders. BMJ Open 11:e044134. doi: 10.1136/ bmjopen-2020-044134 Miguel-Puga, J. A., Cooper-Bribiesca, D., Avelar-Garnica, F. J., Sanchez-Hurtado, L. A., Colin-Martínez, T., Espinosa-Poblano, E., et al. (2021). Burnout, depersonalization, and anxiety contribute to post-traumatic stress in frontline health workers at COVID-19 patient care, a follow-up study. Brain Behav. 11:e02007. doi: 10.1002/brb3.2007 Hyland, P., Shevlin, M., Brewin, C. R., Cloitre, M., Downes, A. J., Jumbe, S., et al. (2017). Validation of post-traumatic stress disorder (PTSD) and complex PTSD using the international trauma questionnaire. Acta Psychiatr. Scand. 136, 313–322. doi: 10.1111/acps.12771 Izutsu, T., Shibuya, M., Tsutsumi, A., Konishi, T., and Kawamura, N. (2008). The relationship between past traumatic experience and sickness absence. Int. J. Soc. Psychiatry 54, 83–89. doi: 10.1177/0020764007083874 Milosevic, I., Cameron, D. H., Milanovic, M., McCabe, R. E., and Rowa, K. (2022). Face-to-face versus video teleconference group cognitive Behavioral therapy for anxiety and related disorders: A preliminary comparison. Can. J. Psychiatry 67, 391–402. doi: 10.1177/07067437211027319 Izutsu, T., Tsutsumi, A., Asukai, N., Kurita, H., and Kawamura, N. (2004). Relationship between a traumatic life event and an alteration in stress response. Stress. Health 20, 65–73. doi: 10.1002/smi.997 Morris, H., Hatzikiriakidis, K., Savaglio, M., Dwyer, J., Lewis, C., Miller, R., et al. (2022). Eye movement desensitization and reprocessing for the treatment and early intervention of trauma among first responders: A systematic review. J. Trauma. Stress. 35, 778–790. doi: 10.1002/jts.22792 Jamieson, N., Maple, M., Ratnarajah, D., and Usher, K. (2020a). Military moral injury: A concept analysis. Int. References J. Ment. Health Nurs. 29, 1049–1066. doi: 10.1111/inm.12792 17 Frontiers in Psychology frontiersin.org Farrell et al. Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 Nagarajan, R., Krishnamoorthy, Y., Basavarachar, V., and Dakshinamoorthy, R. (2022). Prevalence of post-traumatic stress disorder among survivors of severe COVID-19 infections: A systematic review and meta-analysis. J. Affect. Disord. 299, 52–59. doi: 10.1016/j.jad.2021.11.040 Sachdeva, A., Nandini, H., Kumar, V., Chawla, R. K., and Chopra, K. (2021). From stress to stigma – mental health considerations of health care workers involved in COVID19 management. Indian J. Tuberc. 69, 590–595. doi: 10.1016/j.ijtb.2021.09.007 Sasangohar, F., Jones, S. L., Masud, F. N., Vahidy, F. S., and Kash, B. A. (2020). Provider burnout and fatigue during the COVID-19 pandemic: lessons learned from a high-volume intensive care unit. Anesth. Analg. 131, 106–111. doi: 10.1213/ANE.0000000000004866 Narayan, A. J., Ippen, C. G., Harris, W. W., and Lieberman, A. F. (2017). Assessing angels in the nursery: A pilot study of childhood memories of benevolent caregiving as protective influences. Infant Ment. Health J. 38, 461–474. doi: 10.1002/imhj.21653 Shapiro, F. (2017). Eye movement desensitization and reprocessing (EMDR) therapy: Basic principles, protocols, and procedures. Guilford Publications. Narayan, A. J., Rivera, L. M., Bernstein, R. E., Harris, W. W., and Lieberman, A. F. (2018). Positive childhood experiences predict less psychopathology and stress in pregnant women with childhood adversity: A pilot study of the benevolent childhood experiences (BCEs) scale. Child Abuse Negl. 78, 19–30. doi: 10.1016/j.chiabu.2017.09.022 Shapiro, E., and Laub, B. (2008). Early EMDR intervention (EEI): A summary, a theoretical model, and the recent traumatic episode protocol (R-TEP). J. EMDR Pract. Res. 2, 79–96. doi: 10.1891/1933-3196.2.2.79 Nash, W. P., Marino Carper, T. L., Mills, M. A., Au, T., Goldsmith, A., and Litz, B. T. (2013). Psychometric evaluation of the moral injury events scale. Mil. Med. 178, 646–652. doi: 10.7205/MILMED-D-13-00017 Shklarski, L., Abrams, A., and Bakst, E. (2021a). Navigating changes in the physical and psychological spaces of psychotherapists during Covid-19: when home becomes the office. Pract. Innov. 6, 55–66. doi: 10.1037/pri0000138 Norman, S. B., Feingold, J. H., Kaye-Kauderer, H., Kaplan, C. A., Hurtado, A., Kachadourian, L., et al. (2021). Moral distress in frontline healthcare workers in the initial epicenter of the COVID-19 pandemic in the United States: relationship to PTSD symptoms, burnout, and psychosocial functioning. Depress. Anxiety 38, 1007–1017. doi: 10.1002/da.23205 Shklarski, L., Abrams, A., and Bakst, E. (2021b). Will we ever again conduct in-person psychotherapy sessions? References Factors associated with the decision to provide in-person therapy in the age of COVID-19. J. Contemp. Psychother. 51, 265–272. doi: 10.1007/ s10879-021-09492-w Shreffler, J., Huecker, M., and Petrey, J. (2020). The impact of COVID-19 on healthcare worker wellness: A scoping review. Western. J. Emerg. Med. 21, 1059–1066. doi: 10.5811/ westjem.2020.7.48684 Olejnik, S., and Algina, J. (2003). Generalized eta and omega squared statistics: measures of effect size for some common research designs. Psychol. Methods 8, 434–447. doi: 10.1037/1082-989X.8.4.434 Smith, K., Moller, N., Cooper, M., Gabriel, L., Roddy, J., and Sheehy, R. (2022). Video counselling and psychotherapy: A critical commentary on the evidence base. Couns. Psychother. Res. 22, 92–97. doi: 10.1002/capr.12436 Oosterbaan, V., Covers, M. L. V., Bicanic, I. A. E., Huntjens, R. J. C., and de Jongh, A. (2019). Do early interventions prevent PTSD? A systematic review and meta-analysis of the safety and efficacy of early interventions after sexual assault. Eur. J. Psychotraumatol. 10:2932. doi: 10.1080/20008198.2019.1682932 Spence, J., Titov, N., Johnston, L., Dear, B. F., Wootton, B., Terides, M., et al. (2013). Internet-delivered eye movement desensitization and reprocessing (iEMDR): an open trial. F1000Research 2:79. doi: 10.12688/f1000research.2-79.v2 Osorio, A. (2018). Randomized controlled trial on the EMDR integrative group treatment protocol for ongoing traumatic stress with adolescents and young adults patients with cancer. Am. J. Appl. Psychol. 7:50. doi: 10.11648/j.ajap.20180704.11 Sritharan, J., Jegathesan, T., Vimaleswaran, D., and Sritharan, A. (2020). Mental health concerns of frontline workers during the COVID-19 pandemic: A scoping review. Global J. Health Sci. 12:89. doi: 10.5539/gjhs.v12n11p89 Ostacoli, L., Carletto, S., Cavallo, M., Baldomir-Gago, P., di Lorenzo, G., Fernandez, I., et al. (2018). Comparison of eye movement desensitization reprocessing and cognitive behavioral therapy as adjunctive treatments for recurrent depression: the European depression EMDR network (EDEN) randomized controlled trial. Front. Psychol. 9:74. doi: 10.3389/fpsyg.2018.00074 Tarquinio, C., Brennstuhl, M.-J., Rydberg, J. A., Bassan, F., Peter, L., Tarquinio, C. L., et al. (2021). EMDR in telemental health counseling for healthcare workers caring for COVID-19 patients: A pilot study. Issues Ment. Health Nurs. 42, 3–14. doi: 10.1080/01612840.2020.1818014 Oudshoorn, C. E. M., Frielink, N., Riper, H., and Embregts, P. J. C. M. (2021). Experiences of therapists conducting psychological assessments and video conferencing therapy sessions with people with mild intellectual disabilities during the COVID-19 pandemic. Int. J. Dev. Disab. 10, 1–9. doi: 10.1080/20473869.2021.1967078 Turgoose, D., Ashwick, R., and Murphy, D. (2018). Systematic review of lessons learned from delivering tele-therapy to veterans with post-traumatic stress disorder. J. References Telemed. Telecare 24, 575–585. doi: 10.1177/1357633X17730443 Pink, J., Ghomi, M., Smart, T., and Richardson, T. (2022). Effects of EMDR group traumatic episode protocol on burnout within IAPT health care professionals: A feasibility and acceptability study. J. EMDR Pract. Res. 16, 215–227. doi: 10.1891/ EMDR-2022-0029 van Minnen, A., and Hendriks, L., Kleine, R. de, Hendriks, G.-J., Verhagen, M., and de Jongh, A. (2018). Therapist rotation: a novel approach for implementation of trauma- focused treatment in post-traumatic stress disorder. Eur. J. Psychotraumatol., 9,:1492836. doi:10.1080/20008198.2018.1492836 Watson, P. (2022). Stress, PTSD, and COVID-19: the utility of disaster mental health interventions during the COVID-19 pandemic. Curr. Treat. Opt. Psychiatry 9, 14–40. doi: 10.1007/s40501-021-00253-z Price, M., van Stolk-Cooke, K., Brier, Z. M. F., and Legrand, A. C. (2018). mHealth solutions for early interventions after trauma: improvements and considerations for assessment and intervention throughout the acute post-trauma period. MHealth 4:22. doi: 10.21037/mhealth.2018.06.03 Watts, S., Marchand, A., Bouchard, S., Gosselin, P., Langlois, F., Belleville, G., et al. (2020). Telepsychotherapy for generalized anxiety disorder: impact on the working alliance. J. Psychother. Integr. 30, 208–225. doi: 10.1037/int0000223 Pupat, A., Dewailly, A., Guidot, F., Duagani, Y., Kawesa, E., Carriere, R., et al. (2022). Global initiative for stress and trauma treatment—traumatic stress relief training for allied and Para-professionals to treat traumatic stress in underserved populations: A case study. Europ. J. Trauma Dissociation 6:100229. doi: 10.1016/j.ejtd.2021.100229 Weiss, D. S., and Marmar, C. R. (1997). “The impact of event scale—revised,’’ in Assessing psychological trauma and post traumatic stress disorder: a handbook for practitioners. eds. P. Wilson and T. Keane (New York: Guilford Press), 399–411. Puspitasari, A. J., Heredia, D., Coombes, B. J., Geske, J. R., Gentry, M. T., Moore, W. R., et al. (2021). Feasibility and initial outcomes of a group-based teletherapy psychiatric day program for adults with serious mental illness: open, nonrandomized trial in the context of COVID-19. JMIR Mental Health 8:e25542. doi: 10.2196/25542 Wild, J., McKinnon, A., Wilkins, A., and Browne, H. (2022). Post-traumatic stress disorder and major depression among frontline healthcare staff working during the COVID-19 pandemic. Br. J. Clin. Psychol. 61, 859–866. doi: 10.1111/bjc.12340 Rasheed, J., Jamil, A., Hameed, A. A., Aftab, U., Aftab, J., Shah, S. A., et al. (2020). A survey on artificial intelligence approaches in supporting frontline workers and decision makers for the COVID-19 pandemic. Chaos, Solitons Fractals 141:110337. doi: 10.1016/j. chaos.2020.110337 Wild, J., Warnock-Parkes, E., Grey, N., Stott, R., Wiedemann, M., Canvin, L., et al. (2016). Womersley, G., and Arikut-Treece, Y. (2019). Collective trauma among displaced populations in northern Iraq: A case study evaluating the therapeutic interventions of the free Yezidi foundation. Intervention 17:18. doi: 10.4103/INTV. INTV_56_18 Yunitri, N., Chu, H., Kang, X. L., Jen, H.-J., Pien, L.-C., Tsai, H.-T., et al. (2022). Global prevalence and associated risk factors of posttraumatic stress disorder during COVID-19 pandemic: A meta-analysis. Int. J. Nurs. Stud. 126:104136. doi: 10.1016/j. ijnurstu.2021.104136 Yurtsever, A., Konuk, E., Akyüz, T., Zat, Z., Tükel, F., Çetinkaya, M., et al. (2018). An eye movement desensitization and reprocessing (EMDR) group intervention for Syrian refugees with post-traumatic stress symptoms: results of a randomized controlled trial. Front. Psychol. 9:493. doi: 10.3389/fpsyg.2018.00493 Yurtsever, A., Konuk, E., Akyüz, T., Zat, Z., Tükel, F., Çetinkaya, M., et al. (2018). An eye movement desensitization and reprocessing (EMDR) group intervention for Syrian refugees with post-traumatic stress symptoms: results of a randomized controlled trial. Front. Psychol. 9:493. doi: 10.3389/fpsyg.2018.00493 pandemic: A meta-analysis. Int. J. Nurs. Stud. 126:104136. doi: 10.1016/j. ijnurstu.2021.104136 pandemic: A meta-analysis. Int. J. Nurs. Stud. 126:104136. doi: 10.1016/j. ijnurstu.2021.104136 Womersley, G., and Arikut-Treece, Y. (2019). Collective trauma among displaced populations in northern Iraq: A case study evaluating the therapeutic interventions of the free Yezidi foundation. Intervention 17:18. doi: 10.4103/INTV. INTV_56_18 Yunitri, N., Chu, H., Kang, X. L., Jen, H.-J., Pien, L.-C., Tsai, H.-T., et al. (2022). Global prevalence and associated risk factors of posttraumatic stress disorder during COVID-19 References Internet-delivered cognitive therapy for PTSD: a development pilot series. Eur. J. Psychotraumatol. 7:1019. doi: 10.3402/ejpt.v7.31019 Williams, N. (2014). The GAD-7 questionnaire. Occup. Med. 64:224. doi: 10.1093/ occmed/kqt161 Raudenská, J., Steinerová, V., Javůrková, A., Urits, I., Kaye, A. D., Viswanath, O., et al. (2020). Occupational burnout syndrome and post-traumatic stress among healthcare professionals during the novel coronavirus disease 2019 (COVID-19) pandemic. Best Pract. Res. Clin. Anaesthesiol. 34, 553–560. doi: 10.1016/j.bpa.2020.07.008 Williamson, V., Murphy, D., and Greenberg, N. (2020). COVID-19 and experiences of moral injury in front-line key workers. Occup. Med. 70, 317–319. doi: 10.1093/ occmed/kqaa052 Redican, E., Nolan, E., Hyland, P., Cloitre, M., McBride, O., Karatzias, T., et al. (2021). A systematic literature review of factor analytic and mixture models of ICD-11 PTSD and CPTSD using the international trauma questionnaire. J. Anxiety Disord. 79:102381. doi: 10.1016/j.janxdis.2021.102381 Wilson, G., Farrell, D., Barron, I., Hutchins, J., Whybrow, D., and Kiernan, M. D. (2018). The use of eye-movement desensitization reprocessing (EMDR) therapy in treating post-traumatic stress disorder-A systematic narrative review. Front. Psychol. 9:923. doi: 10.3389/fpsyg.2018.00923 Richins, M. T., Gauntlett, L., Tehrani, N., Hesketh, I., Weston, D., Carter, H., et al. (2020). Early post-trauma interventions in organizations: A scoping review. Front. Psychol. 11:1176. doi: 10.3389/fpsyg.2020.01176 Wind, T. R., Rijkeboer, M., Andersson, G., and Riper, H. (2020). The COVID-19 pandemic: the ‘black swan’ for mental health care and a turning point for e-health. Internet Interv. 20:100317. doi: 10.1016/j.invent.2020.100317 Roberts, A. K. P. (2018). The effects of the EMDR group traumatic episode protocol with cancer survivors. J. EMDR Pract. Res. 12, 105–117. doi: 10.1891/1933-3196.12.3.105 Wippich, A., Howatson, G., Allen-Baker, G., Farrell, D., Kiernan, M., and Scott-Bell, A. (2023). Eye movement desensitization reprocessing as a treatment for PTSD in conflict-affected areas. Psychol. Trauma Theory Res. Pract. Policy. 1–7. doi: 10.1037/tra0001430 Roberts, N. P., Kitchiner, N. J., Kenardy, J., Lewis, C. E., and Bisson, J. I. (2019). Early psychological intervention following recent trauma: A systematic review and meta- analysis. Eur. J. Psychotraumatol. 10:1695486. doi: 10.1080/20008198.2019.1695486 Wolpe., (1969). The practice of behavior therapy. New York: Pergamon Press. Pergamon Press. Ruck, S., Bowes, N., and Tehrani, N. (2013). Evaluating trauma debriefing within the UK prison service. J. Forensic Pract. 15, 281–290. doi: 10.1108/JFP-09-2012-0018 18 Frontiers in Psychology frontiersin.org Farrell et al. 10.3389/fpsyg.2023.1129912 10.3389/fpsyg.2023.1129912 19 frontiersin.org Frontiers in Psychology Frontiers in Psychology
https://openalex.org/W4210756944	https://www.jmir.org/2020/8/e21265/PDF	English	null	Effect of an E-Learning Module on Personal Protective Equipment Proficiency Among Prehospital Personnel: Web-Based Randomized Controlled Trial (Preprint)	null	2,020	cc-by	9,455	Corresponding Author: Corresponding Author: Laurent Suppan, MD Division of Emergency Medicine Department of Anesthesiology, Clinical Pharmacology, Intensive Care and Emergency Medicine University of Geneva Hospitals and Faculty of Medicine Rue Gabrielle-Perret-Gentil 4 Geneva, 1211 Switzerland Phone: 41 789151845 Email: laurent.suppan@hcuge.ch Corresponding Author: Laurent Suppan, MD Division of Emergency Medicine Department of Anesthesiology, Clinical Pharmac University of Geneva Hospitals and Faculty of M Rue Gabrielle-Perret-Gentil 4 Geneva, 1211 Switzerland Phone: 41 789151845 Email: laurent.suppan@hcuge.ch Corresponding Author: Laurent Suppan, MD Division of Emergency Medicine Department of Anesthesiology, Clinical Pharmacology, Intensive Care and Emergency Medicine University of Geneva Hospitals and Faculty of Medicine Rue Gabrielle-Perret-Gentil 4 Geneva, 1211 Switzerland Phone: 41 789151845 Email: laurent.suppan@hcuge.ch Original Paper Original Paper Effect of an E-Learning Module on Personal Protective Equipment Proficiency Among Prehospital Personnel: Web-Based Randomized Controlled Trial Laurent Suppan1, MD; Mohamed Abbas2, MD; Loric Stuby3, CAS; Philippe Cottet1, MD; Robert Larribau1, MD; Eric Golay1, MAS; Anne Iten2, MD; Stephan Harbarth2, MD, MSc; Birgit Gartner1, MD; Mélanie Suppan4, MD 1Division of Emergency Medicine, Department of Anesthesiology, Clinical Pharmacology, Intensive Care and Emergency Medicine, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland 2Infection Control Program and WHO Collaborating Centre on Patient Safety, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland 3 J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 1 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al KEYWORDS personal protective equipment; COVID-19; electronic learning; prehospital; randomized controlled trial; protection; equipment; safety; gamified; online learning; communication personal protective equipment; COVID-19; electronic learning; prehospital; randomized controlled trial; protection; equipment; safety; gamified; online learning; communication of learners nor the repetitive use of equipment for demonstration as could be the case for live simulation [11,12]. of learners nor the repetitive use of equipment for demonstration as could be the case for live simulation [11,12]. Goal of This Investigation Adequate use of personal protective equipment (PPE) is of paramount importance to ensure health care workers’ safety and to avoid shortages of such equipment in the context of the coronavirus disease (COVID-19) pandemic [1,2]. Protection guidelines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have rapidly evolved following publication of new evidence regarding its mode of transmission, making prompt adaptation of the guidelines both frequent and necessary [3-5]. Prehospital personnel is particularly at risk of contamination as they usually work in a challenging environment and have to stay next to their patients in the narrow space of the ambulance for extended periods of time. This risk is further increased when high-risk procedures such as endotracheal intubation have to be performed [6-8]. The purpose of this study was to evaluate whether a specifically designed gamified e-learning module [13] could improve the rate of adequate PPE choice by prehospital personnel in the context of the COVID-19 pandemic. Our hypothesis was that knowledge of PPE guidelines would be inconsistent between prehospital personnel, and that an e-learning module may increase and standardize knowledge regarding the use of PPE. This could help limit both underuse and overuse of such equipment. JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Conclusions: Among prehospital personnel with an already relatively high knowledge of and experience with PPE use, both web-based study paths increased the rate of adequate choice of PPE. There was no major added value of the gamified e-learning module apart from preserving participants' confidence in their ability to correctly use PPE. Abstract Background: To avoid misuse of personal protective equipment (PPE), ensure health care workers’ safety, and avoid shortages, effective communication of up-to-date infection control guidelines is essential. As prehospital teams are particularly at risk of contamination given their challenging work environment, a specific gamified electronic learning (e-learning) module targeting this audience might provide significant advantages as it requires neither the presence of learners nor the repetitive use of equipment for demonstration. Objective: The aim of this study was to evaluate whether a gamified e-learning module could improve the rate of adequate PPE choice by prehospital personnel in the context of the coronavirus disease (COVID-19) pandemic. Methods: This was an individual-level randomized, controlled, quadruple-blind (investigators, participants, outcome assessors, and data analysts) closed web-based trial. All emergency prehospital personnel working in Geneva, Switzerland, were eligible for inclusion, and were invited to participate by email in April 2020. Participants were informed that the study aim was to assess their knowledge regarding PPE, and that they would be presented with both the guidelines and the e-learning module, though they were unaware that there were two different study paths. All participants first answered a preintervention quiz designed to establish their profile and baseline knowledge. The control group then accessed the guidelines before answering a second set of questions, and were then granted access to the e-learning module. The e-learning group was shown the e-learning module right after the guidelines and before answering the second set of questions. Results: Of the 291 randomized participants, 176 (60.5%) completed the trial. There was no significant difference in baseline knowledge between groups. Though the baseline proportion of adequate PPE choice was high (75%, IQR 50%-75%), participants’ description of the donning sequence was in most cases incorrect. After either intervention, adequate choice of PPE increased significantly in both groups (P<.001). Though the median of the difference in the proportion of correct answers was slightly higher in the e-learning group (17%, IQR 8%-33% versus 8%, IQR 8%-33%), the difference was not statistically significant (P=.27). Confidence in the ability to use PPE was maintained in the e-learning group (P=.27) but significantly decreased in the control group (P=.04). J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 1 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ Online Platform Before performing a 1:1 randomization, stratification was achieved according to professional status (EMTs, paramedics, and emergency physicians). Email addresses were then sorted according to alphabetical order, and an investigator (MS) who did not have access to the email addresses database was given the number of participants by category and performed the randomization using a computer-generated table. The randomization key was then combined with the list of email addresses and entered in the mailing component by the only investigator who had access to the system (L Suppan). As the list of email addresses and allocations were solely present in an encrypted database, all other investigators were blinded as to group allocation. An online platform [20] developed under the Joomla! 3.9 content management system (Open Source Matters) was created specifically for the purpose of this study. A mailing management component (AcyMailing 5.10, Acyba), a survey component (Community Surveys Pro 5.4.0, CoreJoomla), and a form builder component used to issue completion certificates (BreezingForms Pro 1.9.0, Crosstec) were installed on the website. L Suppan was the only author with access to the platform’s administration console. No maintenance or update was planned on the server, platform, or content during the study period. Setting The study took place in April 2020 in Geneva, Switzerland; the detailed organization of local prehospital emergency services has already been described elsewhere [19]. There are five levels of increasing expertise working in the prehospital field. Ambulance drivers all have a basic life support certificate but are never called upon to deal with emergency situations. Emergency medical technicians (EMTs) are certified after 1 year of training, and can either transport stable patients on their own, or team up with a paramedic to deal with more difficult prehospital situations. The highest level of nonphysician care is provided by paramedics, who complete a 3-year curriculum. Whenever a potentially life-threatening emergency is identified by emergency dispatchers, paramedics are sent in teams of two. A medical reinforcement by way of a light vehicle, Service Mobile d’Urgence et de Réanimation (SMUR), staffed by a paramedic and an emergency physician can either be dispatched at the same time as the ambulance or be called upon by paramedics if specialized medical care is required on site. Emergency physicians are either senior residents or fellows working in one of the following departments: emergency medicine, anesthesiology, or general internal medicine. They can be supervised, either by call or on-site, by a senior specialist emergency physician, which represents the highest level of prehospital care in this setting. Consistency of specific “free-text” questions, such as age, was checked by means of regular expression (“regex”) rules. All answers were automatically checked for completeness by the system before participants were allowed to proceed to the next page. Custom validation messages were displayed to inform users who had not answered a question. Participants were not allowed to correct or review their answers once a page had been completed. Subjects and Inclusion and Exclusion Criteria Chief ambulance officers of all services were asked to provide one of the investigators (L Suppan) with a list of all the professional email addresses of their EMTs, paramedics, and emergency physicians. All the email addresses received from these officers were included. Email addresses of ambulance drivers were excluded as these drivers usually only deal with interhospital transfers and almost never don PPE. Senior specialist emergency physicians were also excluded, as they are few in number and are usually involved in the writing of the guidelines or in the creation of the learning material; in addition, some of them are authors of this study. Finally, the email addresses of the paramedics who participated in the creation of this study or the learning material were also excluded. Apart from the physician-staffed prehospital medical service, there are seven different ambulance companies in Geneva, two of which belong to public organizations while the remaining five are privately owned. As each company has its own continuous education structure as well as its own equipment, medical devices, and protocols, one cannot expect all paramedics to share the same knowledge level regarding all aspects of prehospital emergencies. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al The regional ethics committee issued a “Declaration of no objection” (Req-2020-00374) as the population studied was not considered vulnerable according to the Swiss federal law on human research [17]. As the purpose of the study was to examine the effect of two different study paths only on providers’ knowledge and attitude toward PPE, registration of the trial was not performed as it is not deemed necessary by the International Committee of Medical Journal Editors [18]. sequences. Within the module, trigger mechanisms are used to check that the user had accessed and completed all required steps before being allowed to proceed to the following section. The e-learning package is available on the study website where it can be viewed and downloaded freely. The comprehensive prehospital COVID-19 guidelines from the Geneva University Hospitals, version 1.11, was also used in this study [21]. Two quizzes were created by BG and L Suppan: a preintervention quiz designed to establish the participants’ baseline knowledge regarding PPE, their use and indication, and a postintervention quiz to assess whether these parameters had changed. Both quizzes contained 10 closed questions, either multiple choice or multiple answer. Questions designed to assess PPE choice were preceded by short clinical scenarios. Each quiz was displayed over 5 pages. The number of questions was limited to reduce attrition. These quizzes were tested and validated by all coinvestigators. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 3 (page number not for citation purposes) Study Design This was an individual-level, stratified, randomized, controlled, quadruple-blind (investigators, participants, outcome assessors, and data analysts) closed web-based trial (Figure 1) designed following the CONSORT-EHEALTH (Consolidated Standards of Reporting Trials of Electronic and Mobile HEalth Applications and onLine TeleHealth) guidelines [14,15] and incorporating relevant elements from the CHERRIES (Checklist for Reporting Results of Internet E-Surveys) statement [16]. To avoid misuse of PPE, effective communication of the corresponding guidelines to frontline health care workers is necessary [9]. However, continuous education has been massively disrupted due to the cancellation of continuous education sessions to limit disease transmission [10]. In this challenging context, electronic learning (e-learning) might provide significant advantages as it requires neither the presence Figure 1. Study design. Figure 1. Study design. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 2 http://www.jmir.org/2020/8/e21265/ (page number not for citation purposes) O J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 2 (page number not for citation purposes) J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 2 http://www.jmir.org/2020/8/e21265/ http://www.jmir.org/2020/8/e21265/ http://www.jmir.org/2020/8/e21265/ Study Sequence As guidelines differ from region to region, we decided to only include prehospital staff working in the Geneva emergency medical system, thereby using a convenience sample rather than performing a sample size calculation. After clicking on the survey link, a welcome screen containing detailed information about the study was displayed. This welcome screen was identical for both groups and, similarly to the email messages, did not convey any information regarding the study sequence to ensure the participants were adequately blinded. Data Collection Data was electronically recorded and securely stored in an encrypted MariaDB database (Version 5.5.5; MariaDB Foundation). At the end of the study, all data was extracted to CSV format by the only investigator who had access (L Suppan). No personally identifiable data (including name, date of birth, email, or IP address) was ever asked for or recorded. To improve the response rate, the chief ambulance and medical officers of all companies were asked to encourage their paramedics, EMTs, and emergency physicians to participate in the study. Participation reminders were also sent to all prehospital personnel along with a daily COVID-19 information newsletter. Outcomes The main outcome was defined as the difference in the proportion of correct choice of PPE before and after the course, assessed by means of short clinical scenarios. Apart from the survey link, the emails contained information regarding the study length and objectives as well as a short data protection statement. Participants were informed that they would be presented with the most recent version of the prehospital COVID-19 guidelines as well as with an e-learning module, though the order in which these materials would be shown was not explained. They were informed that, by clicking on the survey link, they consented to participate in the study and were provided with the names and electronic addresses of five investigators (BG, EG, L Stuby, L Suppan, and PC), whom they could contact at any time. As collected data were irreversibly anonymized, it was impossible for users to ask for their own answers to be deleted once the survey had been completed. Secondary outcomes were stratification of the main outcome by profession and by personal history of COVID-19 (whether or not the participant had been infected), accuracy of donning and doffing sequences reconstitutions, differences in the rates of overuse and underuse of PPE, confidence in one’s ability to use PPE, perceived usefulness of the course, and satisfaction regarding the course. The latter three outcomes were assessed by means of a 5-point Likert scale. Data Curation and Availability The extracted data were imported into Stata (StataCorp LLC). Variables were renamed to facilitate their understanding by the blinded data analysts. All data that could have enabled data analysts to identify group allocation were removed, and data fields were sorted accordingly. Incomplete questionnaires were excluded at this stage. The control and e-learning groups were renamed using city names (Moscow and Nairobi), and all relevant data were exported by L Suppan to a Stata .dta file and sent to L Stuby and MA for analysis. Participation was not mandatory. No monetary incentive or prize was offered to the survey participants. As the e-learning module was akin to a continuous education session, participants were informed before beginning the survey that they would be able to print a continuous education certificate upon completion. As the certificate component was independent from both the mailing and the survey components, participants were ensured they could generate the certificate without their identity being disclosed. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Adaptive questioning was used in this section to avoid displaying irrelevant questions. Participants were then asked a series of general questions related to SARS-CoV-2 and the COVID-19 pandemic. Adaptive questioning was used in this section to avoid displaying irrelevant questions. Participants were then asked a series of general questions related to SARS-CoV-2 and the COVID-19 pandemic. Enrolment and Consent Individual emails that were identical for all participants except for the unique links that pointed to one of the two study paths were sent on April 13, 2020 (Multimedia Appendices 1 and 2). These unique links were automatically created by the survey component but were not recorded in the mailing component other than in generic form (there was no way the individual tokens could be linked to the email addresses). The webmaster could therefore only know whether the email had been opened and the survey started, but was prevented from reconciling the email address with the survey answers, thereby guaranteeing user anonymization. Using unique links served two purposes: they allowed the participants to resume the course in case they were interrupted and also avoided double entries. Given the current circumstances, and as the vast majority of prehospital professionals had had their holidays cancelled and were therefore at work, participants were only given 12 days to complete the study, with email reminders sent on days 3, 7, and 10. The control group was then shown the prehospital COVID-19 guidelines. They were then asked to answer a second set of questions before being prompted to evaluate the learning path up to this point. Only then could they access the e-learning module and download their certificate. The e-learning group followed the same path at first, but accessed the e-learning module immediately after being shown the guidelines. This group then completed the same second set of questions, was asked to evaluate the learning path (which in this case included the e-learning module), and was finally allowed to download the completion certificate. http://www.jmir.org/2020/8/e21265/ Study Material A previously described gamified e-learning module created under Storyline 3 (Articulate Global) was used in this study [13]. The module contains 19 sections and embeds 7 video http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 4 (page number not for citation purposes) Outcomes Assessment Though most outcomes were electronically recorded and their interpretation therefore was generally independent of subjective human evaluation, comments had to be assessed by outcome After clicking on the start button, participants were asked a series of questions designed to gather demographic-related data. http://www.jmir.org/2020/8/e21265/ J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 4 (page number not for citation purposes) J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 4 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al homogeneity tests. Binomial paired data were assessed using the McNemar test. assessors. Two assessors (L Stuby and PC), blinded as to group allocation, were asked to independently assess all comments. The nature of comments were to be rated as “positive,” “negative,” or “neutral” regarding the study, and as to whether they challenged Infection Prevention and Control (IPC) guidelines (binary, yes versus no). Disagreements were solved by sending the unclear comments to a third outcome assessor (BG), who was blinded to the previous assessments. assessors. Two assessors (L Stuby and PC), blinded as to group allocation, were asked to independently assess all comments. The nature of comments were to be rated as “positive,” “negative,” or “neutral” regarding the study, and as to whether they challenged Infection Prevention and Control (IPC) guidelines (binary, yes versus no). Disagreements were solved by sending the unclear comments to a third outcome assessor (BG), who was blinded to the previous assessments. Stratification was defined a priori based on expertise level (EMTs, paramedics, or physicians) and COVID-19 status (negative; confirmed, quarantined; confirmed, cured or unknown). Two post hoc sensitivity analyses were conducted. Statistical Analysis Statistical analysis was performed using Stata 15. Continuous independent outcomes were assessed using the Student t test or the Mann-Whitney rank-sum test depending on normality. Categorical outcomes were assessed using Fisher exact test. A two-sided P<.05 was considered significant. Normality of distribution was first assessed graphically. In case of doubt, the Shapiro-Wilk test was applied. Data Availability The original data has been deposited to Mendeley Data [22]. Figure 2. Study flowchart. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 5 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ Characteristics of Study Subjects Of the 291 randomized participants, 176 (60.5%) completed the trial (Figure 2). No change was made to the web platform after the first batch of emails was sent. There was no significant downtime (the server was available more than 99% of the time throughout the study period). Just three participants were unable to complete the trial owing to technical problems, the exact nature of which could not be determined: two were not able to access the e-learning module, and one could not access the guideline (in PDF format). The study path was completed in one session by 82.7% of the participants (143/173). Continuous paired data were assessed using either the paired Student t test or the Wilcoxon matched-pairs signed-rank test depending on normality. The sign test for matched pair was used if symmetry could not be proven. Categorical paired data were analyzed using asymptotic symmetry and marginal Figure 2. Study flowchart. Figure 2. Study flowchart. The blinded data analysts (L Stuby and MA) excluded two surveys from the e-learning group as the participants’ professions did not match the target population (one “ambulance driver” and one “other”). They also excluded one survey from the control group because of incoherent answers (Multimedia Appendix 3). Participant characteristics are described in Table 1. the control group because of incoherent answers (Multimedia Appendix 3). Participant characteristics are described in Table 1. The blinded data analysts (L Stuby and MA) excluded two surveys from the e-learning group as the participants’ professions did not match the target population (one “ambulance driver” and one “other”). They also excluded one survey from J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 5 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Table 1. Participant characteristicsa. Characteristics of Study Subjects E-learning (n=88) Control (n=85) Characteristics Profession, n (%) 10 (11.4) 5 (5.9) Student paramedic 12 (13.6) 11 (12.9) Emergency medical technician 60 (68.2) 61 (71.8) Paramedic 6 (6.8) 8 (9.4) Physician 28 (31.8) 32 (37.6) Gender, female, n (%) 34 (28-40) 35 (30-42) Age (years), median (Q1-Q3) 7 (2-12) 9 (3-15) Professional experience (years), median (Q1-Q3) Prior infection prevention and control course, n (%) 70 (79.6) 73 (85.1) No/does not remember 18 (20.4) 12 (14.1) Yes 6 (6.8) 7 (8.3) Coronavirus disease status, positive, n (%) 84 (95.4) 79 (92.9) Local guideline seen, yes, n (%) 5 (2-8) 5 (3-10) Last time guideline seen (days), median (Q1-Q3) 32 (36.4) 28 (32.9) Specific coronavirus disease course followed, yes, n (%) E-learning (n=88) Control (n=85) Specific coronavirus disease course followed, yes, n (%) aTotals may not equal to 100% due to rounding. Adequate choice of PPE was significantly increased in both groups after the intervention (P<.001; Figure 3). Though the median of the difference was higher in the e-learning group (17%, IQR 8%-33% versus 8%, IQR 8%-33%), it did not reach statistical significance (P=.27). This difference was similar regardless of stratification by profession or history of COVID-19 (Table 2). No participant was able to describe the correct doffing sequence, which was assessed postintervention. The doffing sequence originally displayed in the survey was left unchanged by 8% of the participants (14/173, 7 per group). Adequate choice of PPE was significantly increased in both groups after the intervention (P<.001; Figure 3). Though the median of the difference was higher in the e-learning group (17%, IQR 8%-33% versus 8%, IQR 8%-33%), it did not reach statistical significance (P=.27). This difference was similar regardless of stratification by profession or history of COVID-19 (Table 2). No participant was able to describe the correct doffing sequence, which was assessed postintervention. The doffing sequence originally displayed in the survey was left unchanged by 8% of the participants (14/173, 7 per group). J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 6 (page number not for citation purposes) J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 7 (page number not for citation purposes) Main Results There was no significant difference in baseline knowledge between groups. Though the baseline proportion of adequate PPE choice was high (75%, IQR 50%-75%), description of the donning sequence (assessed preintervention) was in most cases incorrect, as only 7 (4%) of the participants were able to reconstitute it accurately, with a similar proportion between groups. The donning sequence initially displayed in the survey was left unchanged by 7% of the participants (12/173, 6 per group). http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Figure 3. Change in proportion (%) of adequate choice of personal protective equipment. Figure 3. Change in proportion (%) of adequate choice of personal protective equipment. Figure 3. Change in proportion (%) of adequate choice of personal protective equipment. Table 2. Adequate choice of personal protective equipment (main outcome). P value E-learning (n=88) Control (n=85) Outcomes .27 17 (8 to 33) 8 (8 to 33) Main outcome: difference in percentage of correct answers, median (Q1-Q3) Main outcome by profession (%), median (Q1-Q3) .15 17 (8 to 33) 8 (0 to 25) Paramedic .49 29 (25 to 33) 25 (8 to 33) Paramedic student .88 8 (–8 to 33) 25 (8 to 33) Emergency medical technician .30 13 (–8 to 42) 25 (13 to 38) Physician Main outcome by coronavirus disease status (%), median (Q1-Q3) .20 25 (8 to 33) 8 (8 to 33) Negative for coronavirus disease .37 0 (–8 to 17) 8 (–8 to 33) Positive for coronavirus disease Confidence in the ability of using PPE was identical before and after the course in the e-learning group (P=.27), but was significantly lower after the course in the control group (P=.04). While most participants found the course useful (68.5%, 95% CI 61.5%-75.3%), the proportion of participants finding the course “very useful” was significantly higher (P=.01) in the e-learning group (33.0%, 95% CI 23.2%-42.8% versus 11.8%, 95% CI 4.9%-18.7%). Participants were generally satisfied regarding the course (60.0%, 95% CI 49.6%-70.4% for the e-learning group versus 62.5%, 95% CI 52.4%-72.6%; P=.28; Figure 4). Table 2. Adequate choice of personal protective equipment (main outcome). Paramedic Positive for coronavirus disease course “very useful” was significantly higher (P=.01) in the e-learning group (33.0%, 95% CI 23.2%-42.8% versus 11.8%, 95% CI 4.9%-18.7%). Participants were generally satisfied regarding the course (60.0%, 95% CI 49.6%-70.4% for the e-learning group versus 62.5%, 95% CI 52.4%-72.6%; P=.28; Figure 4). Main Results course “very useful” was significantly higher (P=.01) in the e-learning group (33.0%, 95% CI 23.2%-42.8% versus 11.8%, Confidence in the ability of using PPE was identical before and after the course in the e-learning group (P=.27), but was significantly lower after the course in the control group (P=.04). While most participants found the course useful (68.5%, 95% CI 61.5%-75.3%), the proportion of participants finding the J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 7 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Figure 4. Participant satisfaction. Figure 4. Participant satisfaction. The proportion of positive comments was similar between groups (14%, 95% CI 7%-21% in the e-learning group versus 19%, 95% CI 11%-27%; P=.16). Participants who expressed disagreements with IPC recommendations were also evenly divided (5.7%, 95% CI 3.2%-8.2% in the e-learning group versus 4.7%, 95% CI 0.2%-9.2%; P>0.99). The proportion of positive comments was similar between groups (14%, 95% CI 7%-21% in the e-learning group versus 19%, 95% CI 11%-27%; P=.16). Participants who expressed disagreements with IPC recommendations were also evenly divided (5.7%, 95% CI 3.2%-8.2% in the e-learning group versus 4.7%, 95% CI 0.2%-9.2%; P>0.99). guidelines recently. Most participants were therefore aware of the local recommendations, and the anxiety generated by the COVID-19 pandemic probably acted as a catalyst regarding their interest in such guidelines [23]. Another factor that might partly explain the lack of a significant difference is the sample size, which limited the power to detect small differences. Nevertheless, as PPE guidelines vary not only between countries, but also between Swiss cantons, it might have been inadequate to draw conclusions from a pooled group of paramedics working in different cantons with different guidelines [24]. Moreover, though increasing the sample size would increase the chances of finding a significant difference, too small an increase would not be clinically relevant regardless of the P value obtained [25]. Lack of significance might therefore also be consecutive to lack of effect of the e-learning module regarding knowledge acquisition in this particular population. Two post hoc sensitivity analyses were performed. The first was achieved by excluding all participants who answered they were either “very unsatisfied” or “unsatisfied” with the course. Main Results The second was performed by excluding participants whose compliance regarding COVID-19–specific IPC guidelines could be doubted; this included those who did not answer “systematic wearing of a double pair of gloves” to the question “Which of these measures is NOT one of the infection prevention measures?” (40 [45%] in the e-learning group versus 35 [41%]). These analyses did not demonstrate a significant change regarding the main outcome (P=.89 and P=.29, respectively). Though necessary to assess participants’ knowledge and attitudes regarding PPE prior to the interventions, the first set of questions, along with the study title, might have acted as a primer and focused the participants’ attention on the specific contents that would be tested postintervention [26]. This effect might have further dampened the potential impact of the e-learning module. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 8 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ Conclusions Among prehospital personnel with an already relatively high knowledge and experience regarding PPE use, both web-based study paths increased the rate of adequate choice of PPE. There was no major added value of the gamified e-learning module apart from preserving participants' confidence in their ability to correctly use PPE. Perspectives The larger impact such a web-based study might have had, regardless of the effect of a specific intervention such as the gamified e-learning module, should be assessed, as a possible change in PPE consumption or infection rate among prehospital providers could ensue. Though incorrect answers regarding the donning sequence are easily understandable as they might primarily result from a lack of knowledge, an inability to correctly rebuild the doffing sequence is questioning. No less than four different broad categories of causes could be involved: ineffectiveness of the teaching material, inadequate sequence, flawed means of assessing the participants’knowledge, or disagreement with the procedure outlined in the guidelines and in the e-learning module. As this result was unforeseen, the method used in this study was unfortunately ill-suited to the evaluation of the underlying causes. Nevertheless, with less than 10% of participants having left the initially displayed sequences unchanged, a technical flaw can reasonably be ruled out. The potential impact of this gamified e-learning module on less experienced and less primed participants should also be evaluated to confirm (or refute) the theories outlined in this discussion. The gamified e-learning module can thus be freely downloaded from the study website in both a web (HTML5 with Flash fallback) and SCORM (Shareable Content Object Reference Model) format. Acknowledgments The authors would like to thank Mr Michel Hofer for his support. Main Results In this study, the proportion of those making an adequate choice regarding PPE increased significantly after prehospital personnel followed either web-based study path. Though the median of the increase was twice that of the e-learning group, this difference was not statistically significant. The relatively low level of satisfaction displayed by the participants should also be taken into account. Though e-learning modules and serious games usually increase participant satisfaction when compared to more traditional methods [27,28], such an effect could not be found in this study. Disagreement with IPC guidelines might, at least in part, explain why some participants were dissatisfied [5,29,30]. Indeed, the vast majority of paramedics working in Geneva have had some prior training regarding the use of PPE in situations other than the COVID-19 Failure to reach significance might be explained by many different factors. The high baseline proportion of adequate choice of PPE in both groups may have dampened any relative impact of the intervention as there was little room left for improvement. Although this result might seem counterintuitive given the high rate of participants who responded that they did not attend (or did not recall attending) an IPC course prior to this study, more than 90% declared they had consulted the local http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX XSL•FO RenderX Suppan et al JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al pandemic. Preparations for high-risk transfers during the 2014 Ebola epidemic [31] and practical exercises in the context of simulated major incidents involving the presence of hazardous materials [32] have presented paramedics with many different PPE guidelines and protocols over the last few years. As the latter situations require more stringent donning and doffing procedures than those described in the COVID-19 context, some paramedics seem to feel that IPC recommendations do not go far enough. There is however a delicate balance between underprotection and overuse of scarcely available resources that must be maintained [33,34]. Moreover, it can hardly be expected to have all paramedics don maximal PPE for all interventions. This would not only considerably increase intervention time, but also make delicate interventions more difficult given the bulk of the PPE and lead to more difficult communications. It could also increase fatigue as such equipment has been shown to be quite uncomfortable [35]. Secondary Outcomes Despite its limitations, this study also has some strengths, among which the quadruple-blind design and the relatively high response rate should be acknowledged. Moreover, as all answers were electronically recorded, there was no risk of an outcome assessment bias. Finally, as neither the control nor the e-learning path requires the physical presence of either participants or instructors, the framework used in this study could serve as the building ground for courses in the context of an epidemic or a pandemic such as the current COVID-19 situation. Though confidence in the ability to use PPE was maintained in the e-learning group, it significantly decreased after reviewing the guidelines in the control group. Participants who initially felt confident in their knowledge might have felt it was challenged after being asked specific questions [36,37]. The e-learning module probably helped restore their altered confidence, as interactive presentations, as well as gamification, have been shown to increase this feeling [38,39]. Nevertheless, most participants were unable, regardless of their assigned intervention, to reconstruct either the donning (assessed preintervention) or the doffing (assessed postintervention) sequences. This potential limitation has already been highlighted in the paper describing the development of the gamified e-learning module used in this study [13], though cut scenes were embedded to provide direct demonstration [40,41]. Limitations Apart from the abovementioned limitations, the ever-increasing knowledge regarding SARS-CoV-2 and COVID-19 might render both the guideline and the gamified e-learning module used in this study obsolete. However, current technological tools might mitigate this effect as they allow for a quick adaptation, even of highly interactive content [42]. Another limitation is the relatively small number of questions asked pre- and postintervention. Keeping the total number of questions and the time required to complete either study path relatively low was necessary to limit attrition [16,43]. This strategy was altogether successful as dropouts amounted to less than 15% in either group, a rather lower proportion than generally reported [44]. Attrition was further limited thanks to the use of unique identifiers, which allowed as many as 30 participants (17.3%) to complete their assigned study path in more than one session. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 9 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al Editorial Notice This randomized study was not prospectively registered. As the purpose of the study was to examine the effect of two different study paths only on providers’ knowledge and attitude toward PPE, registration of the trial was not performed as it is not deemed necessary by the International Committee of Medical Journal Editors. The editor is therefore following the guidelines suggested by the ICMJE and not mandating prospective registration of this randomized trial. However, readers are advised to carefully assess the validity of any potential explicit or implicit claims related to primary outcomes or effectiveness, as retrospective registration does not prevent authors from changing their outcome measures retrospectively. Multimedia Appendix 1 First email sent to participants belonging to the control group. [PDF File (Adobe PDF File), 45 KB-Multimedia Appendix 1] Multimedia Appendix 2 First email sent to participants belonging to the e-learning group. [PDF File (Adobe PDF File), 45 KB-Multimedia Appendix 2] Multimedia Appendix 3 Details of the answer set excluded by the blinded data analysts. [PDF File (Adobe PDF File), 126 KB-Multimedia Appendix 3] References Occupational exposure to infection risk and use of personal protective equipment by emergency medical personnel in the Republic of Korea. Am J Infect Control 2016 Jun 01;44(6):647-651 [FREE Full text] [doi: 10.1016/j.ajic.2015.12.022] [Medline: 26874405] 9. Oh HS, Uhm D. Occupational exposure to infection risk and use of personal protective equipment by emergency medical personnel in the Republic of Korea. Am J Infect Control 2016 Jun 01;44(6):647-651 [FREE Full text] [doi: 10.1016/j.ajic.2015.12.022] [Medline: 26874405] j j 10. Rose S. Medical Student Education in the Time of COVID-19. JAMA 2020 Mar 31;323(21):2131. [doi: 10.1001/jama.2020.5227] [Medline: 32232420] 10. Rose S. Medical Student Education in the Time of COVID-19. JAMA 2020 Mar 31;323(21):2131. [doi: 10.1001/jama.2020.5227] [Medline: 32232420] j 11. Ahmed H, Allaf M, Elghazaly H. COVID-19 and medical education. Lancet Infect Dis 2020 Jul;20(7):777-778 [FREE Full text] [doi: 10.1016/S1473-3099(20)30226-7] [Medline: 32213335] 11. Ahmed H, Allaf M, Elghazaly H. COVID-19 and medical education. Lancet Infect Dis 2020 Jul;20(7):777-778 [FREE Full text] [doi: 10.1016/S1473-3099(20)30226-7] [Medline: 32213335] 11. Ahmed H, Allaf M, Elghazaly H. COVID-19 and medical education. text] [doi: 10.1016/S1473-3099(20)30226-7] [Medline: 32213335] 12. Wittich C, Agrawal A, Cook D, Halvorsen A, Mandrekar J, Chaudhry S, et al. E-learning in graduate medical education: survey of residency program directors. BMC Med Educ 2017 Jul 11;17(1):114 [FREE Full text] [doi: 10.1186/s12909-017-0953-9] [Medline: 28697744] 12. Wittich C, Agrawal A, Cook D, Halvorsen A, Mandrekar J, Chaudhry S, et al. E-learning in graduate medical education: survey of residency program directors. BMC Med Educ 2017 Jul 11;17(1):114 [FREE Full text] [doi: 10.1186/s12909-017-0953-9] [Medline: 28697744] 13. Suppan M, Gartner B, Golay E, Stuby L, White M, Cottet P, et al. Teaching Adequate Prehospital Use of Personal Protective Equipment During the COVID-19 Pandemic: Development of a Gamified e-Learning Module. JMIR Serious Games 2020 Jun 12;8(2):e20173 [FREE Full text] [doi: 10.2196/20173] [Medline: 32516115] 13. Suppan M, Gartner B, Golay E, Stuby L, White M, Cottet P, et al. Teaching Adequate Prehospital Use of Personal Protective Equipment During the COVID-19 Pandemic: Development of a Gamified e-Learning Module. JMIR Serious Games 2020 Jun 12;8(2):e20173 [FREE Full text] [doi: 10.2196/20173] [Medline: 32516115] 14. Eysenbach G, CONSORT-EHEALTH Group. CONSORT-EHEALTH: improving and standardizing evaluation reports of Web-based and mobile health interventions. J Med Internet Res 2011 Dec 31;13(4):e126 [FREE Full text] [doi: 10.2196/jmir.1923] [Medline: 22209829] 14. Eysenbach G, CONSORT-EHEALTH Group. CONSORT-EHEALTH: improving and standardizing evaluation reports of Web-based and mobile health interventions. Conflicts of Interest Most of the authors participated in the development of the gamified e-learning module tested in this trial. Nevertheless, as this module is freely available, the authors deny any financial conflict of interest. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 9 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH References Teaching Adequate Prehospital Use of Personal Protective Equipment During the COVID-19 Pandemic: Development of a Gamified e-Learning Module. JMIR Serious Games 2020 Jun 12;8(2):e20173 [FREE Full text] [doi: 10.2196/20173] [Medline: 32516115] 14. Eysenbach G, CONSORT-EHEALTH Group. CONSORT-EHEALTH: improving and standardizing evaluation reports of Web-based and mobile health interventions. J Med Internet Res 2011 Dec 31;13(4):e126 [FREE Full text] [doi: 10.2196/jmir.1923] [Medline: 22209829] 2. Ranney ML, Griffeth V, Jha AK. Critical Supply Shortages - The Need for Ventilators and Personal Protective Equipment during the Covid-19 Pandemic. N Engl J Med 2020 Apr 30;382(18):e41. [doi: 10.1056/NEJMp2006141] [Medline: 32212516] 3 S Ch i i i l d i f i i l d h i l i f l i 3. Wax RS, Christian MD. Practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-nCoV) patients. Can J Anaesth 2020 May;67(5):568-576 [FREE Full text] [doi: 10.1007/s12630-020-01591-x] [Medline: 32052373] 4. Guan L, Zhou L, Zhang J, Peng W, Chen R. More awareness is needed for severe acute respiratory syndrome coronavirus 2019 transmission through exhaled air during non-invasive respiratory support: experience from China. Eur Respir J 2020 Mar 20;55(3):2000352 [FREE Full text] [doi: 10.1183/13993003.00352-2020] [Medline: 32198275] 5. Rhee C, Baker MA, Klompas M. The COVID-19 infection control arms race. Infect Control Hosp Epidemiol 2020 May 08:1-3 [FREE Full text] [doi: 10.1017/ice.2020.211] [Medline: 32381140] 6. Teter J, Millin MG, Bissell R. Hand hygiene in emergency medic [doi: 10.3109/10903127.2014.967427] [Medline: 25415186] 6. Teter J, Millin MG, Bissell R. Hand hygiene in emergency medical services. Prehosp Emerg Care 2015 6. Teter J, Millin MG, Bissell R. Hand hygiene in emergency medical services. Prehosp Emerg Care 2015 Nov 21;19(2):313-319. [doi: 10.3109/10903127.2014.967427] [Medline: 25415186] 6. Teter J, Millin MG, Bissell R. Hand hygiene in emergency medical serv [doi: 10.3109/10903127.2014.967427] [Medline: 25415186] 7. Valdez MK, Sexton JD, Lutz EA, Reynolds KA. Spread of infectious microbes during emergency m J Infect Control 2015 Jun;43(6):606-611 [FREE Full text] [doi: 10.1016/j.ajic.2015.02.025] [Medl Valdez MK, Sexton JD, Lutz EA, Reynolds KA. Spread of infectious microbes during emergency med J Infect Control 2015 Jun;43(6):606 611 [FREE Full text] [doi: 10 1016/j ajic 2015 02 025] [Medline: 8. Lockhart SL, Duggan LV, Wax RS, Saad S, Grocott HP. Personal protective equipment (PPE) for both anesthesiologists and other airway managers: principles and practice during the COVID-19 pandemic. Can J Anaesth 2020 Apr 23:1005-1015 [FREE Full text] [doi: 10.1007/s12630-020-01673-w] [Medline: 32329014] 9. Oh HS, Uhm D. http://www.jmir.org/2020/8/e21265/ References References 1. Livingston E, Desai A, Berkwits M. Sourcing Personal Protective Equipment During the COVID-19 Pandemic. JAMA 2020 Mar 28:E1-E3. [doi: 10.1001/jama.2020.5317] [Medline: 32221579] 2. Ranney ML, Griffeth V, Jha AK. Critical Supply Shortages - The Need for Ventilators and Personal Protective Equipment during the Covid-19 Pandemic. N Engl J Med 2020 Apr 30;382(18):e41. [doi: 10.1056/NEJMp2006141] [Medline: 32212516] 3. Wax RS, Christian MD. Practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-nCoV) patients. Can J Anaesth 2020 May;67(5):568-576 [FREE Full text] [doi: 10.1007/s12630-020-01591-x] [Medline: 32052373] 4. Guan L, Zhou L, Zhang J, Peng W, Chen R. More awareness is needed for severe acute respiratory syndrome coronavirus 2019 transmission through exhaled air during non-invasive respiratory support: experience from China. Eur Respir J 2020 Mar 20;55(3):2000352 [FREE Full text] [doi: 10.1183/13993003.00352-2020] [Medline: 32198275] 5. Rhee C, Baker MA, Klompas M. The COVID-19 infection control arms race. Infect Control Hosp Epidemiol 2020 May 08:1-3 [FREE Full text] [doi: 10.1017/ice.2020.211] [Medline: 32381140] 6. Teter J, Millin MG, Bissell R. Hand hygiene in emergency medical services. Prehosp Emerg Care 2015 Nov 21;19(2):313-319. [doi: 10.3109/10903127.2014.967427] [Medline: 25415186] 7. Valdez MK, Sexton JD, Lutz EA, Reynolds KA. Spread of infectious microbes during emergency medical response. Am J Infect Control 2015 Jun;43(6):606-611 [FREE Full text] [doi: 10.1016/j.ajic.2015.02.025] [Medline: 26042849] 8. Lockhart SL, Duggan LV, Wax RS, Saad S, Grocott HP. Personal protective equipment (PPE) for both anesthesiologists and other airway managers: principles and practice during the COVID-19 pandemic. Can J Anaesth 2020 Apr 23:1005-1015 [FREE Full text] [doi: 10.1007/s12630-020-01673-w] [Medline: 32329014] 9. Oh HS, Uhm D. Occupational exposure to infection risk and use of personal protective equipment by emergency medical personnel in the Republic of Korea. Am J Infect Control 2016 Jun 01;44(6):647-651 [FREE Full text] [doi: 10.1016/j.ajic.2015.12.022] [Medline: 26874405] 10. Rose S. Medical Student Education in the Time of COVID-19. JAMA 2020 Mar 31;323(21):2131. [doi: 10.1001/jama.2020.5227] [Medline: 32232420] 11. Ahmed H, Allaf M, Elghazaly H. COVID-19 and medical education. Lancet Infect Dis 2020 Jul;20(7):777-778 [FREE Full text] [doi: 10.1016/S1473-3099(20)30226-7] [Medline: 32213335] 12. Wittich C, Agrawal A, Cook D, Halvorsen A, Mandrekar J, Chaudhry S, et al. E-learning in graduate medical education: survey of residency program directors. BMC Med Educ 2017 Jul 11;17(1):114 [FREE Full text] [doi: 10.1186/s12909-017-0953-9] [Medline: 28697744] 13. Suppan M, Gartner B, Golay E, Stuby L, White M, Cottet P, et al. References hug-ge.ch/sites/smur/files/guideline_covid19_1_11c.pdf [accessed 2020-04-22] g p 21. Brigade Sanitaire Cantonale. Coronavirus de la maladie COVID-19: prise en charge préhospitalière. URL: https://smur. hug-ge.ch/sites/smur/files/guideline_covid19_1_11c.pdf [accessed 2020-04-22] g g g p 22. Suppan M, Stuby L, Abbas M, Gartner B, Suppan L. COVID-19 - Prehospital PPE Dataset. Mendeley Data. 2020. URL: https://data.mendeley.com/datasets/ffh678bv42/1 [accessed 2020-08-17] 22. Suppan M, Stuby L, Abbas M, Gartner B, Suppan L. COVID-19 - Prehospital PPE Dataset. Mendeley Data. 2020. URL: https://data.mendeley.com/datasets/ffh678bv42/1 [accessed 2020-08-17] p y 23. Pappa S, Ntella V, Giannakas T, Giannakoulis VG, Papoutsi E, Katsaounou P. Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: A systematic review and meta-analysis. Brain Behav Immun 2020 Aug;88:901-907 [FREE Full text] [doi: 10.1016/j.bbi.2020.05.026] [Medline: 32437915] g [ ] [ j ] [ ] 24. Glass GV. Primary, Secondary, and Meta-Analysis of Research. Educational Researcher 1976 Nov 1;5(10):3-8. [doi: 10.3102/0013189X005010003] 25. Ranganathan P, Pramesh C, Buyse M. Common pitfalls in statistical analysis: Clinical versus statistical significance. Perspect Clin Res 2015;6(3):169-170 [FREE Full text] [doi: 10.4103/2229-3485.159943] [Medline: 26229754] 26. Latimier A, Riegert A, Peyre H, Ly ST, Casati R, Ramus F. Does pre-testing promote better rete NPJ Sci Learn 2019 Sep 24;4(1):15. [doi: 10.1038/s41539-019-0053-1] [Medline: 31583117] 26. Latimier A, Riegert A, Peyre H, Ly ST, Casati R, Ramus F. Does pre testing promote better retentio NPJ Sci Learn 2019 Sep 24;4(1):15. [doi: 10.1038/s41539-019-0053-1] [Medline: 31583117] NPJ Sci Learn 2019 Sep 24;4(1):15. [doi: 10.1038/s41539-019-0053-1] [Medline: 31583117] 27. Rouleau G, Gagnon M, Côté J, Payne-Gagnon J, Hudson E, Dubois C, et al. Effects of E-Learning in a Continuing Education Context on Nursing Care: Systematic Review of Systematic Qualitative, Quantitative, and Mixed-Studies Reviews. J Med Internet Res 2019 Oct 02;21(10):e15118 [FREE Full text] [doi: 10.2196/15118] [Medline: 31579016] Context on Nursing Care: Systematic Review of Systematic Qualitative, Quantitative, and Mixed-Studies Reviews. J Med Internet Res 2019 Oct 02;21(10):e15118 [FREE Full text] [doi: 10.2196/15118] [Medline: 31579016] 28. Lozano-Lozano M, Fernández-Lao C, Cantarero-Villanueva I, Noguerol I, Álvarez-Salvago F, Cruz-Fernández M, et al. A Blended Learning System to Improve Motivation, Mood State, and Satisfaction in Undergraduate Students: Randomized Controlled Trial. J Med Internet Res 2020 May 22;22(5):e17101 [FREE Full text] [doi: 10.2196/17101] [Medline: 32441655] y 29. Godlee F. Protect our healthcare workers. BMJ 2020 Apr 02:m1324. [doi: 10.1136/bmj.m1324] 30. Houghton C, Meskell P, Delaney H, Smalle M, Glenton C, Booth A, et al. References Barriers and facilitators to healthcare workers' adherence with infection prevention and control (IPC) guidelines for respiratory infectious diseases: a rapid qualitative evidence synthesis. Cochrane Database Syst Rev 2020 Apr 21;4:CD013582. [doi: 10.1002/14651858.CD013582] [Medline: 32315451] 31. Schibler M, Vetter P, Cherpillod P, Petty T, Cordey S, Vieille G, et al. Clinical features and viral kinetics in a rapidly cured patient with Ebola virus disease: a case report. The Lancet Infectious Diseases 2015 Sep;15(9):1034-1040. [doi: 10.1016/s1473-3099(15)00229-7] 32. Lettre d'information ERNS 19. Confédération suisse. 2019. URL: https://www.vbs.admin.ch/content/vbs-internet/fr/ verschiedene-themen-des-vbs/die-schweizer-sicherheitspolitik/die-sicherheitsverbundsuebung--svu19-/_jcr_content/ contentPar/tabs/items/documents/tabPar/downloadlist/downloadItems/30_1567670674944.download/ Lettre%20information%20ERNS%2019%20-%20Aout%202019.pdf [accessed 2020-08-18] verschiedene-themen-des-vbs/die-schweizer-sicherheitspolitik/die-sicherheitsverbundsuebung--svu19-/_jcr_cont contentPar/tabs/items/documents/tabPar/downloadlist/downloadItems/30_1567670674944.download/ Lettre%20information%20ERNS%2019%20-%20Aout%202019.pdf [accessed 2020-08-18] p g j contentPar/tabs/items/documents/tabPar/downloadlist/downloadItems/30_1567670674944.download/ Lettre%20information%20ERNS%2019%20-%20Aout%202019.pdf [accessed 2020-08-18] 33. Rowan NJ, Laffey JG. Challenges and solutions for addressing critical shortage of supply chain for personal and protective equipment (PPE) arising from Coronavirus disease (COVID19) pandemic - Case study from the Republic of Ireland. Sci Total Environ 2020 Jul 10;725:138532 [FREE Full text] [doi: 10.1016/j.scitotenv.2020.138532] [Medline: 32304970] 34. Delgado D, Wyss Quintana F, Perez G, Sosa Liprandi A, Ponte-Negretti C, Mendoza I, et al. Personal Safety during the COVID-19 Pandemic: Realities and Perspectives of Healthcare Workers in Latin America. Int J Environ Res Public Health 2020 Apr 18;17(8):2798 [FREE Full text] [doi: 10.3390/ijerph17082798] [Medline: 32325718] 34. Delgado D, Wyss Quintana F, Perez G, Sosa Liprandi A, Ponte-Negretti C, Mendoza I, et al. Personal Safety during the COVID-19 Pandemic: Realities and Perspectives of Healthcare Workers in Latin America. Int J Environ Res Public Health 2020 Apr 18;17(8):2798 [FREE Full text] [doi: 10.3390/ijerph17082798] [Medline: 32325718] p j p 35. Kang J, O'Donnell JM, Colaianne B, Bircher N, Ren D, Smith K. Use of personal protective equipment among health care personnel: Results of clinical observations and simulations. Am J Infect Control 2017 Jan 01;45(1):17-23. [doi: 10.1016/j.ajic.2016.08.011] [Medline: 28065328] 35. Kang J, O'Donnell JM, Colaianne B, Bircher N, Ren D, Smith K. Use of personal protective equipment among health care personnel: Results of clinical observations and simulations. Am J Infect Control 2017 Jan 01;45(1):17-23. [doi: 10.1016/j.ajic.2016.08.011] [Medline: 28065328] j j 36. Serra M, DeMarree K. Unskilled and unaware in the classroom: College students' desired grades predict their biased grade predictions. Mem Cognit 2016 Oct;44(7):1127-1137. [doi: 10.3758/s13421-016-0624-9] [Medline: 27270923] 36. Serra M, DeMarree K. Unskilled and unaware in the classroom: College students' desired grades predict their biased grade predictions. Mem Cognit 2016 Oct;44(7):1127-1137. [doi: 10.3758/s13421-016-0624-9] [Medline: 27270923] p g ( ) [ ] [ ] 37. References J Med Internet Res 2011 Dec 31;13(4):e126 [FREE Full text] [doi: 10.2196/jmir.1923] [Medline: 22209829] J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 10 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al 15. de Kraker MEA, Abbas M, Huttner B, Harbarth S. Good epidemiological practice: a narrative review of appropriate scientific methods to evaluate the impact of antimicrobial stewardship interventions. Clin Microbiol Infect 2017 Nov;23(11):819-825 [FREE Full text] [doi: 10.1016/j.cmi.2017.05.019] [Medline: 28571767] 15. de Kraker MEA, Abbas M, Huttner B, Harbarth S. Good epidemiological practice: a narrative review of appropriate scientific methods to evaluate the impact of antimicrobial stewardship interventions. Clin Microbiol Infect 2017 Nov;23(11):819-825 [FREE Full text] [doi: 10.1016/j.cmi.2017.05.019] [Medline: 28571767] j 16. Eysenbach G. Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). J Med Internet Res 2004 Sep 29;6(3):e34 [FREE Full text] [doi: 10.2196/jmir.6.3.e34] [Medline: 15471760] 17. CC 810.30 Federal Act of 30 September 2011 on Research involving Human Beings (Human Research Act, HRA). URL: 16. Eysenbach G. Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). J Med Internet Res 2004 Sep 29;6(3):e34 [FREE Full text] [doi: 10.2196/jmir.6.3.e34] [Medline: 15471760] 16. Eysenbach G. Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). J Med Internet Res 2004 Sep 29;6(3):e34 [FREE Full text] [doi: 10.2196/jmir.6.3.e34] [Medline: 15471760] 17 CC 810 30 Federal Act of 30 September 2011 on Research involving Human Beings (Human Research Act HRA) URL: 18. Clinical Trials Registration. International Committee of Medical Journal Editors. URL: http://www.icmje.org/about-icmje/ faqs/clinical-trials-registration/ [accessed 2020-06-10] 19. Chan M, Fehlmann CA, Pasquier M, Suppan L, Savoldelli GL. Endotracheal Intubation Success Rate in an Urban, Supervised, Resident-Staffed Emergency Mobile System: An 11-Year Retrospective Cohort Study. J Clin Med 2020 Jan 16;9(1):238 [FREE Full text] [doi: 10.3390/jcm9010238] [Medline: 31963162] 19. Chan M, Fehlmann CA, Pasquier M, Suppan L, Savoldelli GL. Endotracheal Intubation Success Rate in an Urban, Supervised, Resident-Staffed Emergency Mobile System: An 11-Year Retrospective Cohort Study. J Clin Med 2020 Jan 16;9(1):238 [FREE Full text] [doi: 10.3390/jcm9010238] [Medline: 31963162] j Gamified E-Learning Module. URL: https://coronavirus.anesth.ch [accessed 2020-08-18] j 20. Gamified E-Learning Module. URL: https://coronavirus.anesth.ch [accessed 2020-08-18] g p [ ] 21. Brigade Sanitaire Cantonale. Coronavirus de la maladie COVID-19: prise en charge préhospitalière. URL: https://smur. JOURNAL OF MEDICAL INTERNET RESEARCH Suppan et al 38. Tan AJQ, Lee CCS, Lin PY, Cooper S, Lau LST, Chua WL, et al. Designing and evaluating the effectiveness of a serious game for safe administration of blood transfusion: A randomized controlled trial. Nurse Educ Today 2017 Aug;55:38-44. [doi: 10.1016/j.nedt.2017.04.027] [Medline: 28521248] j 39. Dankbaar MEW, Richters O, Kalkman CJ, Prins G, Ten Cate OTJ, van Merrienboer JJG, et al. Comparative effectiveness of a serious game and an e-module to support patient safety knowledge and awareness. BMC Med Educ 2017 Feb 02;17(1):30 [FREE Full text] [doi: 10.1186/s12909-016-0836-5] [Medline: 28148296] 40. Koka A, Suppan L, Cottet P, Carrera E, Stuby L, Suppan M. Teaching the National Institutes of Health Stroke Scale to Paramedics (E-Learning vs Video): Randomized Controlled Trial. J Med Internet Res 2020 Jun 09;22(6):e18358 [FREE Full text] [doi: 10.2196/18358] [Medline: 32299792] 40. Koka A, Suppan L, Cottet P, Carrera E, Stuby L, Suppan M. Teaching the National Institutes of Health Stroke Scale to Paramedics (E-Learning vs Video): Randomized Controlled Trial. J Med Internet Res 2020 Jun 09;22(6):e18358 [FREE Full text] [doi: 10.2196/18358] [Medline: 32299792] 41. Brame CJ. Effective Educational Videos: Principles and Guidelines for Maximizing Student Learning from Video Content. CBE Life Sci Educ 2016;15(4):1-6 [FREE Full text] [doi: 10.1187/cbe.16-03-0125] [Medline: 27789532] 41. Brame CJ. Effective Educational Videos: Principles and Guidelines for Maximizing Student Learning from Video Content. CBE Life Sci Educ 2016;15(4):1-6 [FREE Full text] [doi: 10.1187/cbe.16-03-0125] [Medline: 27789532] 42. Kwag K, González-Lorenzo M, Banzi R, Bonovas S, Moja L. Providing Doctors With High-Quality Information: An Updated Evaluation of Web-Based Point-of-Care Information Summaries. J Med Internet Res 2016 Jan 19;18(1):e15 [FREE Full text] [doi: 10.2196/jmir.5234] [Medline: 26786976] j 43. Eysenbach G. The law of attrition. J Med Internet Res 2005;7(1):e11 [FREE Full text] [doi: 10.2196/jmir.7.1.e11] [Medline: 15829473] 44. Hochheimer CJ, Sabo RT, Krist AH, Day T, Cyrus J, Woolf SH. Methods for Evaluating Respondent Attrition in Web-Based Surveys. J Med Internet Res 2016 Nov 22;18(11):e301 [FREE Full text] [doi: 10.2196/jmir.6342] [Medline: 27876687] References Osterhage JL, Usher EL, Douin TA, Bailey WM. Opportunities for Self-Evaluation Increase Student Calibration in an Introductory Biology Course. CBE Life Sci Educ 2019 Jun;18(2):ar16 [FREE Full text] [doi: 10.1187/cbe.18-10-0202] [Medline: 31025916] 37. Osterhage JL, Usher EL, Douin TA, Bailey WM. Opportunities for Self-Evaluation Increase Student Calibration in an Introductory Biology Course. CBE Life Sci Educ 2019 Jun;18(2):ar16 [FREE Full text] [doi: 10.1187/cbe.18-10-0202] [Medline: 31025916] J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 11 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 12 (page number not for citation purposes) Abbreviations CHERRIES: Checklist for Reporting Results of Internet E-Surveys CONSORT-EHEALTH: Consolidated Standards of Reporting Trials of Electronic and Mobile HEalth Applications and onLine TeleHealth COVID-19: coronavirus disease E-learning: electronic learning EMT: emergency medical technician IPC: infection prevention and control PPE: personal protective equipment SARS-CoV-2: severe acute respiratory syndrome coronavirus 2 SCORM: Shareable Content Object Reference Model SMUR: Service Mobile d’Urgences et de Réanimation CHERRIES: Checklist for Reporting Results of Internet E-Surveys CONSORT-EHEALTH: Consolidated Standards of Reporting Trials of Electronic and Mobile HEalth Applications and onLine TeleHealth COVID-19: coronavirus disease E-learning: electronic learning EMT: emergency medical technician IPC: infection prevention and control PPE: personal protective equipment SARS-CoV-2: severe acute respiratory syndrome coronavirus 2 SCORM: Shareable Content Object Reference Model SMUR: Service Mobile d’Urgences et de Réanimation Edited by G Eysenbach; submitted 13.06.20; peer-reviewed by C Miranda, H Schröder; comments to author 31.07.20; revised version received 02.08.20; accepted 03.08.20; published 21.08.20 Please cite as: Suppan L, Abbas M, Stuby L, Cottet P, Larribau R, Golay E, Iten A, Harbarth S, Gartner B, Suppan M Effect of an E-Learning Module on Personal Protective Equipment Proficiency Among Prehospital Personnel: Web-Based Randomized Controlled Trial J Med Internet Res 2020;22(8):e21265 URL: http://www.jmir.org/2020/8/e21265/ doi: 10.2196/21265 PMID: 32747329 ©Laurent Suppan, Mohamed Abbas, Loric Stuby, Philippe Cottet, Robert Larribau, Eric Golay, Anne Iten, Stephan Harbarth, Birgit Gartner, Mélanie Suppan. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 21.08.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. J Med Internet Res 2020 \| vol. 22 \| iss. 8 \| e21265 \| p. 12 (page number not for citation purposes) http://www.jmir.org/2020/8/e21265/ http://www.jmir.org/2020/8/e21265/ XSL•FO RenderX
https://openalex.org/W4385641870	https://www.e3s-conferences.org/10.1051/e3sconf/202340105027/pdf	English	null	Modeling of the process of mechano activation of filler particles in polymer composites	E3S web of conferences	2,023	cc-by	4,492	Umida Ziyamukhamedova1,2, Hatice Evlen1,2, Jasurbek Nafasov1, Zilola Jalolova3, and Elbek Turgunaliyev1,3, and Erkin Rakhmatov4 Umida Ziyamukhamedova1,2, Hatice Evlen1,2, Jasurbek Nafasov1, Zilola Jalolova3, an Elbek Turgunaliyev1,3, and Erkin Rakhmatov4 1Tashkent State Transport University, Tashkent, Uzbekistan 2Karabuk University, Turkey 3Andijan machine building institute, Andijan, Uzbekistan 4University of Economics and Education, Uzbekistan Abstract. The article presents the results of the study of the filler particles activation mechanism in the production of composite polymeric materials modified by mechanical activation. The studies were conducted in simulated conditions of motion of filler particles taking into account the trajectory of motion during milling in progressive-rotational activation units using analytical expression of velocity distribution of each filler particle in the composite material. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). *Corresponding author: z.umida1973@yandex.ru E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 1 Introduction Materials with improved mechanical and technological properties, both solid metal materials [1-3] and plastic ones based on various polymers [4-6] and elastomers [7-9], used in mechanical engineering cover more and more applications in practice in production conditions. The development of materials science, especially related to composite materials in many cases is based on the methods and conditions of their production, where one of the most common and effective is mechanical and mechanochemical activation of the components [10, 11]. Mechanochemistry is a science that develops on the edge of chemistry and mechanics: it is also closely related to other fields of science and technology, physics, especially solid-state physics, biophysics, physical chemistry, polymer technology, chemistry and technology of inorganic substances, biochemistry, molecular biology, and bionics [10]. Mechanochemistry, by selecting and processing certain facts and regularities taken from all these fields of science, in turn puts forward its own regularities and conclusions that contribute to the development and enrichment of the originating sciences. In the most general definition, mechanochemistry can be called a science that studies the mutual transformations of mechanical and chemical energy occurring in bodies. But the creation of a single, comprehensive representation that comprehensively reflects all aspects of the mechanochemical process must be based on knowledge of the complex structure of the object under study, which is determined by the number of levels of supramolecular organization. The mechanochemical phenomenon includes two main components: mechanochemical, which determines the transformation of mechanical energy into E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 chemical energy, and chemo mechanical, which represents the release of mechanical energy, due to the course of chemical reactions [10]. From the above review, it follows that mechanical activation is one of the main indicators of the effectiveness of the activation process itself, so it is important to study the mechanism of the processes, depending on the activity of the particles of the components involved in this process. chemical energy, and chemo mechanical, which represents the release of mechanical energy, due to the course of chemical reactions [10]. From the above review, it follows that mechanical activation is one of the main indicators of the effectiveness of the activation process itself, so it is important to study the mechanism of the processes, depending on the activity of the particles of the components involved in this process. 1 Introduction In practice, various varieties of installations providing fast and fine grinding of the processed materials are widely used in production conditions [10]. Most often vibromills of different types and designs are used, which differ mainly by technological and design features. In accordance with technological features, they are subdivided according to the type of grinding (dry grinding or wet) and the nature of work (batch or continuous). Structural features include the type of exciter (eccentric or unbalanced), the shape of the mill body (cylindrical, rectangular), the type of its support, etc. A wide choice of mechanical activation units gives an opportunity to choose equipment for modification of composite materials of various purposes, but in our opinion more profound theoretical research of the process is required in order to increase the capabilities of this modification method. In this connection an interest arose to study the mechanism of particle motion during mechanical activation and its influence on the process itself. 2 Objects and methods of research At reception of a composite material, structural appointment the basic purpose of filling is reception of the strengthened polymeric composite material, i.e. a material with the improved complex of physical and mechanical properties. It is achieved both by introduction of fibrous reinforcing fillers and fine-dispersed fillers. Dispersed fillers used to impart special wear properties to the material include minerals, carbon black, graphite, metal powders, etc. Another group of disperse fillers, which are increasingly being used nowadays, are modified materials in the form of disperse particles. Their chemical composition, which can be both simple elementary (AB) and multicomponent (АnBmXp, Аm(BnXc)m-x(DiGf)m-y ) of complex structure with many strong primary and weak secondary, such as hydrogen and Van Derwalas, plays important role in choosing fillers. It should be noted that the above-mentioned set of compositions can mainly fit minerals, which contain metal oxides and their complex structure associated multicomponent. One of the representatives of minerals of complex structure with a multi- component composition is kaolin, in addition, its production marks produced by enrichment (AKF-78, 80, AKS-30, AKT-10), which was used in our studies in obtaining composite polymeric materials. Both thermoplastics and reactoplastic polymers can be used as binders. But in the process of reinforcement of any composite polymers with filling there is a change of physical and mechanical properties mechat their limits in relation to amount and correlation of heterogeneous components, which is the reason of considering the necessity of their plasticization with organic and inorganic components in particular ecological and accessible ones. The world petrochemical industry is one of the largest industries [12-15], which produces olefin products, where as in any production enterprise annually generates more than 109 thousand tons of waste products - pyrolysis condensate, which we used as a carbon-containing plasticizer. To study the process of mechanical activation under laboratory conditions, the installation “Rocker mill” was developed (Figure 1). 2 2 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 Fig. 1. Schematic of a rocker mill: 1 - electric motor; 2 - connection sleeve; 3 - crank; 4 - stone; 5 - beaker; 6 - rocker; 7 - mill body; 8 - grinding bodies. Fig. 1. Schematic of a rocker mill: 1 - electric motor; 2 - connection sleeve; 3 - crank; 4 - stone; 5 - beaker; 6 - rocker; 7 - mill body; 8 - grinding bodies. 2 Objects and methods of research The principle of operation of the unit is as follows: the rotation of the crank causes the vibrations of the beater, and the beater in turn provides torsional and onset motion of the stone, where the mill body, loaded with grinding bodies and grinded material, is attached. The frequency of rotation of the shaft is from 1000 to 3000 rpm, the amplitude of vibration of the mill body is 600 mm. The grinding bodies, receiving frequent impulses from the walls of the mill shell, make complex movements. They bounce, collide and slide on the walls of the mill shell. As a result of friction against the walls of the mill, they begin to rotate. The particles of the ground material are subject to impact, compression and shearing forces of variable magnitude. The impact impulse of a single grinding body in a rocker-type mill is relatively large compared with the impact impulse in a rotating ball mill. 3 Results and their discussion To study the motion of composite polymeric materials, particles of kaolin filler with pyrolysis containing carbon and residual paraffins in an oscillatory-rotational unit occurs along a truncated cone trajectory. Each filler particle participates in structural changes between solid dispersed and liquid polymer components. The filler particles in the process of mechanocativation are activated by the volume of the particles depending on the abrasion trajectory depending on the direction of movement depending on the trajectory of the equipment container where the activation process takes place. In this regard, we set the task of modeling the dynamics of particle motion in the process of mechanoactivation of heterocomposite materials. In the section of truncated cone type, taking into account that the approximation of an arbitrary point of the truncated cone is equal (Figure 2):  tg r r z ) ( 0   (1) (1) But as we know from figure 1, the height of the truncated cone also has an expression of the following form: 3 3 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027  tg r r H ) ( 0 1   (2)  tg r r H ) ( 0 1   (2) (2) where, 1 0  r r . From where we obtain the coordinate for the driving filler particles in hetero-composites: where, 1 0  r r . 3 Results and their discussion From where we obtain the coordinate for the driving filler particles in hetero-composites: H r r r r z 0 1 0    (3) (3) As is known from analytical geometry, the cross-sectional area of a truncated cone  S S S , , 0 is defined by the equations: 2 0 0 r S   ; 2 1 1 r S   and 2 r S    (4) 2 0 0 r S   ; 2 1 1 r S   and 2 r S    (4) Then the second flow rate of milling particles of heterocomposites in the beaker is determined by the equations: Then the second flow rate of milling particles of heterocomposites in the beaker is determined by the equations: H r V S Q 2 1 1 1 1    (5) 0 0 0 V S Q  (6) and    V S Q (7) H r V S Q 2 1 1 1 1    (5) 0 0 0 V S Q  (6) and    V S Q (7) (7) If there is no inflow of particle mass of components from outside, the second flow rate through the cross section will be constant [16] i.e. (1): If there is no inflow of particle mass of components from outside, the second flow rate through the cross section will be constant [16] i.e. (1): const SV Q   (8) (8) In the rotational motion of the center of the section, there is a compression of the flow, there is a change in density - the particles of the components of mechanoactivation. Fig. 2. Scheme of motion of filler particles of composite materials in the oscillatory-rotational machine of mechanical activation Fig. 2. 3 Results and their discussion Scheme of motion of filler particles of composite materials in the oscillatory-rotational machine of mechanical activation E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 ences 401, 05027 (2023) https://doi.org/10.1051/e3sconf/202340105 nferences 401, 05027 (2023) O - 2023 https://doi.org/10.1051/e3sconf/202340 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 CONMECHYDRO - 2023 But from the mass conservation condition of the particle flux [17] we have: const VS Q    (9) (9) where -is the density of the particle at each arbitrary point of the inner part of the beaker area of the mechanically activated unit. where -is the density of the particle at each arbitrary point of the inner part of the beaker area of the mechanically activated unit. From the condition of non-breakable flow of filler particles of the components in obtaining wear-resistant hetero-composites of the assumed continuous area we get the equality:   0  VS d  (10) (10) or in differential form: or in differential form: 0    V dV S dS dp  (11) (11) where p - is the pressure on the particles during the rotational motion of the beaker, and is defined by the following formula: where p - is the pressure on the particles during the rotational motion of the beaker, and is defined by the following formula: n p p        0 0   (12) (12) 0 p and 0  — pressure and density of the particle of wear-resistant hetero-composites , in the position of equilibrium; n - polytropy index; 0 p and 0  — pressure and density of the particle of wear-resistant hetero-composites , in the position of equilibrium; n - polytropy index; In a polytropic process, the flow of a filler particle of composite materials is modeled as an ideal medium. 3 Results and their discussion Then, in the absence of vortexing, we obtain expressions for pressure, density and temperature from the Bernoulli integral as 1 2 1 2 0 0 1           V V p p (13)            2 1 2 0 0 1 V V (14) 1 2 1 2 0 0 1           V V p p (13)            2 1 2 0 0 1 V V (14) and          2 1 2 0 0 1 V V T T (15) where 1 1  n  (16) (13) (14) and and (15) where (16) E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 2 max 2 1 0 V V   (17) 1 1 2 2 1 0    n n a V  (18) (18) (18) Vmax- maximum flow velocity of inseparably bonded particles of hetero-composites. Vmax- maximum flow velocity of inseparably bonded particles of hetero-composites. F li (5) f h h i id h i b k f i l fl d i Vmax- maximum flow velocity of inseparably bonded particles of hetero-composites. From equality (5) for the change inside the activator beaker of particle flux density o i l i i l h Vmax- maximum flow velocity of inseparably bonded particles of hetero-composites. 3 Results and their discussion From equality (5) for the change inside the activator beaker of particle flux density of composite polymeric materials we have: y p y p p From equality (5) for the change inside the activator beaker of particle flux density of composite polymeric materials we have:          0 0 2 1 2 0 0 0 1 1 1          V V (19) (19) The pattern of relative changes in the density of hetero-composite materials particles in a rotating beaker follows from the following: The pattern of relative changes in the density of hetero-composite materials particles in a rotating beaker follows from the following:                0 2 1 2 0 0 1 1 1     V V . (20) From equality (4) we find 1 1 1 V S SV    and given the last equations we get: From equality (4) we find 1 1 1 V S SV    and given the last equations we get:       0 2 1 2 0 0 1 1 1 1         V V V S SV . (21) smallness of       1 2 1 2 0 V V  the friction of the particles of the components of Given the smallness of       1 2 1 2 0 V V  the friction of the particles of the components of Given the smallness of       1 2 1 0 V V  the friction of the particles of the components of composite materials, the last equality can be written in the form: composite materials, the last equality can be written in the form: composite materials, the last equality can be written in the form: composite materials, the last equality can be written in the form:       1 1 2 1 2 0 1 1 V V S S V V . (22) (22) Then we obtain a quadratic equation for the particle velocity distribution of hetero- composites. 3 Results and their discussion As a ratio of particle velocity: 0 1 1 2 1 2 0 2 1 2    V V r r V V   (23) (23) 6 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 From there we get the expression for the flow velocity distribution of wear-resistant hetero-composites:     0 2 1 2 0 2 1 1 2 1 4                  r r r r V V (25) (25) or or 4 1 4 0 2 1 2 1 4 2 r r r r V V      (26) (26) Knowing the incompressibility of the particle flow of the activated filler when using the process of mechanical activation in obtaining composite materials with improved physical and mechanical properties, we can write that the tangential stress of the particle flow 1 0   and the velocity distribution we get: 2 2 1 1 r r V V  (27) 2 2 1 1 r r V V  (27) (27) Thus, the distribution of the velocity of filler particles during mechanical activation to obtain composite materials in the progressively rotating motion of the installation at each point of the medium, provided that there is no gravity is obtained:  z r r r r V V 0 1 2 0 2 1 1 ˆ ˆ ˆ    . (28)  z r r r r V V 0 1 2 0 2 1 1 ˆ ˆ ˆ    . (28) (28) As we mentioned above, one of the most common methods of modification is the mechanochemical method, which is increasingly used in studies of interstructural phenomena at the phase boundary [10, 17-20]. In order to study the effect of mechanoactivation as one of the methods of physical modification of mineral filler, we conducted structural studies of AKT-10 filler together with soot-containing waste after mechanoactivation. 4 Conclusions Analyses made by formula (28) show that the particle velocity distribution of hetero- composites of the inclined wall of the truncated cone inside the beaker decreases, depending on the angle of inclination. Progressive - rotational motion of each particle of hetero-composite materials moves with a variable radius, under the action of gravity and the intensity of the flow. The optimal duration of time for mechanochemical processing in vibro-planetary mechanisms for materials that are planned to be included as fillers in polymer composite materials is 30-35 minutes. Since during this time the dispersed fillers have enough charge to form interstructural bonds, increasing the duration of time can be considered uneconomical. In the process of mechanical activation of fillers, increasing the frequency of rotations of the electric motor shaft from 1000 revolutions per minute does not significantly affect the tensile strength of the obtained material. 3 Results and their discussion When the filler is mechanochemically treated at four different values of the rotation frequency of the vibro-planetary mechanism electrode, n1=750 rev/min, n2=1000 rev/min, n3=1500 rev/min, n4=3000 rev/min, different rotation frequencies values, we can see the graph of the dependence of the tensile strength on the time of mechanoactivation from Figure 3. 7 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 CONMECHYDRO - 2023 Fig. 3. Graph of dependence of extensor stiffness on mechanoactivation time at different values of rotation frequency Fig. 3. Graph of dependence of extensor stiffness on mechanoactivation time at different values of rotation frequency Fig. 3. Graph of dependence of extensor stiffness on mechanoactivation time at different values of rotation frequency As can be seen from the graph, the optimum value of the mechanochemical processing time of dispersed mineral materials, which are included as fillers in polymer composite materials, is 30-35 minutes. For more than this time, the effect of mechanochemically treated materials on the mechanical properties of the obtained polymer composite material has not changed significantly. When the number of rotations of the mechanoactivator electrode reaches 1000 times per minute, the degree of dispersion of processed fillers has a certain value. We can see that the degree of dispersion of the processed material remains almost unchanged at further values of the rotation frequency (Figure 4). a b c d Fig. 4. Gemini 500 (FE-SEM) Scanning Electron Microscope Analysis of Mechanically Activated Fillers at Different Times, ×30.00 К а) t1= 5 min; b) t2= 15 min; с) t3= 30 min; d) t4= 50 min a b b a c d d c Fig. 4. Gemini 500 (FE-SEM) Scanning Electron Microscope Analysis of Mechanically Activated Fillers at Different Times, ×30.00 К а) t1= 5 min; b) t2= 15 min; с) t3= 30 min; d) t4= 50 min 8 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 Microscopic analysis shows that the materials used as fillers form active surfaces during mechanochemical processing in grinders when certain values of time are reached. If the processing process is continued, the increase in the density of the processed filler particles due to the joining and formation of agglomerates leads to a decrease in the interstructural connection of the fillers with the polymer (Figure 4-d). References 1. Otabek Toirov and Nodirjon Tursunov, “Development of production technology of rolling stock cast parts”, E3S Web of Conferences 264, 05013 (2021). https://doi.org/10.1051/e3sconf/202126405013. 1. Otabek Toirov and Nodirjon Tursunov, “Development of production technology of rolling stock cast parts”, E3S Web of Conferences 264, 05013 (2021). https://doi.org/10.1051/e3sconf/202126405013. 2. Otabek Toirov, Nodirjon Tursunov, Shavkat Alimukhamedov, and Lochinbek Kuchkorov, “Improvement of the out-of-furnace steel treatment technology for improving its mechanical properties”, E3S Web of Conferences 365, 05002 (2023). https://doi.org/10.1051/e3sconf/202336505002. 2. Otabek Toirov, Nodirjon Tursunov, Shavkat Alimukhamedov, and Lochinbek Kuchkorov, “Improvement of the out-of-furnace steel treatment technology for improving its mechanical properties”, E3S Web of Conferences 365, 05002 (2023). https://doi.org/10.1051/e3sconf/202336505002. 3. Lochinbek Kuchkorov, Shavkat Alimukhamedov, Nodirjon Tursunov, and Otabek Toirov, “Effect of different additives on the physical and mechanical properties of liquid-glass core mixtures”, E3S Web of Conferences 365, 05009 (2023). https://doi.org/10.1051/e3sconf/202336505009. 3. Lochinbek Kuchkorov, Shavkat Alimukhamedov, Nodirjon Tursunov, and Otabek Toirov, “Effect of different additives on the physical and mechanical properties of liquid-glass core mixtures”, E3S Web of Conferences 365, 05009 (2023). https://doi.org/10.1051/e3sconf/202336505009. 4. Ziyamukhamedova, U., Rakhmatov, E., & Nafasov, J. (2021, April). Optimization of the composition and properties of heterocomposite materials for coatings obtained by the activation-heliotechnological method. In Journal of Physics: Conference Series (Vol. 1889, No. 2, p. 022056). IOP Publishing. 4. Ziyamukhamedova, U., Rakhmatov, E., & Nafasov, J. (2021, April). Optimization of the composition and properties of heterocomposite materials for coatings obtained by the activation-heliotechnological method. In Journal of Physics: Conference Series (Vol. 1889, No. 2, p. 022056). IOP Publishing. 5. Ziyamukhamedova, U., Djumabaev, A., Urinov, B., & Almatayev, T. (2021). Features of structural adaptability of polymer composite coatings. In E3S Web of Conferences (Vol. 264, p. 05011). EDP Sciences. 5. Ziyamukhamedova, U., Djumabaev, A., Urinov, B., & Almatayev, T. (2021). Features of structural adaptability of polymer composite coatings. In E3S Web of Conferences (Vol. 264, p. 05011). EDP Sciences. 6. Ziyamukhamedova, U. A., Almataev, T. O., Dzhumabaev, A. B., & Bakirov, L. Y. (2022, June). Improvement of methods and means of testing non-conventional tribosystems. In AIP Conference Proceedings (Vol. 2432, No. 1, p. 030031). AIP Publishing LLC. 7. Teshabayeva, E., Ibadullayev, A., Chorshanbiyev, U., & Vapayev, M. (2022, June). Modification of composite elastomeric materials for polyfunctional purposes. In AIP Conference Proceedings (Vol. 2432, No. 1, p. 030082). AIP Publishing LLC. 9 E3S Web of Conferences 401, 05027 (2023) CONMECHYDRO - 2023 https://doi.org/10.1051/e3sconf/202340105027 8. References Ibadullaev, A., Teshabayeva, E., Kakharov, B., & Babaev, A. (2022, June). Elastomeric materials based on new ingredients. In AIP Conference Proceedings (Vol. 2432, No. 1, p. 030021). AIP Publishing LLC. 9. Ibadullaev, A., Nigmatova, D., & Teshabaeva, E. (2021, July). Radiation Resistance of Filled Elastomer Compositions. In IOP Conference Series: Earth and Environmental Science (Vol. 808, No. 1, p. 012043). IOP Publishing. 10. U. Ziyamukhamedova, N. Tursunov, G. Miradullaeva, J. Nafasov, “Effects of mechanochemical activation on the morphology of polymer fillers”, Safety issues in transport, Gomel, November 24-25, 2022., p. 29-30. 11. Jean-Louis Do and Tomislav F. Mechanochemistry: A Force of Synthesis. ACS Cent. Sci. 2017, 3, 1, 13–19. 12. Miradullaeva, G., Rakhmatov, E., Bozorov, O., Ziyamukhamedova, U., & Shodiev, B. (2021). Mathematical modeling of rheological properties during structure formation of heterocomposite potting materials and coatings and their application. In International Scientific Conference on Energy, Environmental and Construction Engineering (pp. 346-355). Springer, Cham. 13. Official site of JV Uz-Kor Gas Chemical. - Access mode: https://www.uz- kor.com/index.php/ru/ 14. Gaibullaev, S. A., & Tursunov, B. Zh. (2020). Pyrocondensate is the most important raw material for chemical synthesis. Universum: Engineering Sciences, (6-2(75)), 84- 86. 15. Isakulova, M. Sh. (2022). Technology of processing tar product raw materials. Central Asian Research Journal for Interdisciplinary Studies (CARJIS), 2(3), 260-267. 16. Khudaykulov S.I., Tursunkhojaev P.M., Ravshanov S.S., Normatov A.M. "Modeling of grain particles motion in the chambers of denuding drum. Materials of the international scientific-technical conference "Applied mathematics, mechanics of liquid, gas and plasma" Samarkand. 27-30 June 2007. pp. 121-124. 17. Medvedev, A. S., Korshunov, B. G., & Khavskii, N. N. (1995). Physical intensification methods for mineral raw materials leaching. Mineral Processing and Extractive Metallurgy Review, 15(1-4), 48-49. 18. Sokolova, M. D., Davydova, M. L., & Shadrinov, N. V. (2018). The modification of surface of dispersed fillers for their application in polymer–elastomer composites. Protection of metals and physical chemistry of surfaces, 54, 845-852. 19. Nurmetov, K., Riskulov, A., & Ikromov, A. (2022, August). Physicochemical aspects of polymer composites technology with activated modifiers. In AIP Conference Proceedings (Vol. 2656, No. 1, p. 020011). AIP Publishing LLC. 20. Nurmetov, K., Riskulov, A., Azimov, S., & Kuchkorov, L. (2022, June). Structures of functional elements manufactured using the composite materials. In AIP Conference Proceedings (Vol. 2432, No. 1, p. 030059). AIP Publishing LLC. DOI: 10.1063/5.0089888. 10
https://openalex.org/W2973434654	https://hal-normandie-univ.archives-ouvertes.fr/hal-02346582/document	English	null	VCAM-1 targeted alpha-particle therapy for early brain metastases	Neuro-oncology	2,019	cc-by	10,753	To cite this version: Aurélien Corroyer-Dulmont, Samuel Valable, Nadia Falzone, Anne-Marie Frelin-Labalme, Ole Tietz, et al.. VCAM-1 Targeted Alpha-Particle Therapy for Early Brain Metastases. Neuro-Oncology, 2020, 22 (3), pp.357-368. 10.1093/neuonc/noz169. hal-02346582 VCAM-1 Targeted Alpha-Particle Therapy for Early Brain Metastases Aurélien Corroyer-Dulmont, Samuel Valable, Nadia Falzone, Anne-Marie Frelin-Labalme, Ole Tietz, Jérôme Toutain, Manuel Sarmiento Soto, Didier Divoux, Laurent Chazalviel, Elodie A. Pérès, et al. To cite this version: Aurélien Corroyer-Dulmont, Samuel Valable, Nadia Falzone, Anne-Marie Frelin-Labalme, Ole Tietz, et al.. VCAM-1 Targeted Alpha-Particle Therapy for Early Brain Metastases. Neuro-Oncology, 2020, 22 (3), pp.357-368. 10.1093/neuonc/noz169. hal-02346582 Aurélien Corroyer-Dulmont, Samuel Valable, Nadia Falzone, Anne-Marie Frelin-Labalme, Ole Tietz, Jérôme Toutain, Manuel Sarmiento Soto, Didier Divoux, Laurent Chazalviel, Elodie A. Pérès, et al. Neuro-Oncology 22(3), 357–368, 2020 \| doi:10.1093/neuonc/noz169 \| Advance Access date 20 September 2019 HAL Id: hal-02346582 https://normandie-univ.hal.science/hal-02346582v1 Submitted on 8 Dec 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. VCAM-1 targeted alpha-particle therapy for early brain metastases Aurélien Corroyer-Dulmont, Samuel Valable, Nadia Falzone, Anne-Marie Frelin-Labalme, Ole Tietz, Jérôme Toutain, Manuel Sarmiento Soto, Didier Divoux, Laurent Chazalviel, Elodie A. Pérès, Nicola R. Sibson,# Katherine A. Vallis,# and Myriam Bernaudin# Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Normandie University, UNICAEN, CEA, CNRS, ISTCT/CERVOxy group, GIP CYCERON, Caen, France (A.C-D., S.V., J.T., D.D., L.C., E.A.P., M.B.); Grand National Heavy Ion Accelerator, Caen, France (A-M.F-L.); Cancer Research UK and Medical Research Council, Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK (A.C-D., N.F., O.T., M.S.S., N.R.S., K.A.V.) Normandie University, UNICAEN, CEA, CNRS, ISTCT/CERVOxy group, GIP CYCERON, Caen, France (A.C-D., S.V., J.T., D.D., L.C., E.A.P., M.B.); Grand National Heavy Ion Accelerator, Caen, France (A-M.F-L.); Cancer Research UK and Medical Research Council, Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK (A.C-D., N.F., O.T., M.S.S., N.R.S., K.A.V.) #Authors contributed equally to this work. #Authors contributed equally to this work. Corresponding Author: Dr Aurélien Corroyer-Dulmont, Unité ISTCT, GIP Cyceron, Bd H Becquerel, BP 5229, 14074 Caen Cedex (corroyer@cyceron.fr). Corresponding Author: Dr Aurélien Corroyer-Dulmont, Unité ISTCT, GIP Cyceron, Bd H Becquerel, BP 5229, 14074 Caen Cedex (corroyer@cyceron.fr). Abstract Background. Brain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic chal- lenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the thera- peutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti–VCAM-1 antibody (212Pb-αVCAM-1). Methods. Human breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT. Results. 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statisti- cally significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P < 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P < 0.01). No major toxicity was observed. Conclusions. The present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition. Importance of the Study treatment against tumor cells, while minimizing healthy brain tissue damage owing to the short range of the alpha-particles. As we previously showed that VCAM-1 is expressed in endothelial cells adjacent to early BM in patients, our new targeted alpha-particle therapy has the potential for clinical translation. In addition, we have recently developed a fully human- ized anti-human VCAM-1 antibody to facilitate clinical trials of this strategy. Taken together, our results pre- sented here indicate that radio-immunotherapy with VCAM-1 is an interesting new approach to the treat- ment of BM. Brain metastases remain a significant challenge de- spite the many advances in the treatment of meta- static breast cancer. Current available treatments of BM originating from primary breast cancer are inef- fective in a significant proportion of patients, because of the late stage detection. VCAM-1, in preclinical and clinical settings, has been found to be overexpressed by endothelial cells during the early stages of BM development, representing a promising therapeutic target. We report that labeling anti–VCAM-1 anti- bodies with an alpha-emitting radionuclide, 212Pb (212Pb-αVCAM-1), provides a precisely targeted shown that VCAM-1 can be used to detect BM very early by targeting an MRI-detectable contrast agent to this ad- hesion molecule.6,8 We now propose that this target could be used to direct therapy specifically to the site of brain micrometastases. Targeted radionuclide therapy, in which a radiotherapeutic agent is selectively delivered to tumor cells, is an area of active research.9 The recent introduc- tion of a novel bone-seeking alpha-emitter, 223Ra (Xofigo), for the treatment of metastatic prostate cancer has high- lighted this class of radionuclides as an effective treatment for disseminated disease.10 Alpha emitters used for tar- geted alpha therapy (TAT) emit high energy alpha-particles (between 5 and 8 MeV) with associated linear energy transfer of 50–230 keV/µm to targeted cancer cells over a short range of several cell diameters (40–80 µm). For this reason, TAT has been proposed as a treatment for meta- static tumors.11 The local control of many types of primary cancer has im- proved with oncological advances in recent years, but in some cases, prolongation of survival has been associated with the eventual emergence of brain metastases (BM). Importance of the Study The availability of sensitive methods of brain imaging has also contributed to the increasingly frequent diagnosis of BM.1 Breast cancer, the most common malignancy in women in developed countries, carries an approximately 20% risk of BM, and the incidence is even higher in women with human epidermal growth factor receptor 2–positive disease.2 Even in cases where control of the primary cancer has favorable impact on overall survival (OS), a significant proportion of patients die as a result of BM,3 and the presence of BM is a poor prognostic factor, with an average OS of about 6 months.1 In the case of multiple BM, treatment consists mainly of external radiation therapy and/or, in a minority of cases, surgery. The radiotherapy protocol used depends on multiple parameters, such as KPS, molecular features, and the number and volume of tumors and consists of ei- ther whole-brain radiotherapy (WBRT) or image-guided stereotactic radiosurgery.4 However, despite the limited therapeutic effect (an increase in OS of 2‒4 mo), both radio- therapy protocols may induce cognitive deterioration in the case of multiple BM.5 Increasing the dose of external radio- therapy in an effort to improve tumor control is therefore not currently possible. The second reason for the low OS is that BM tend to present late when tumors are well established. This reflects the fact that during the early micrometastatic stages of development, the blood–brain barrier (BBB) re- mains intact, thus preventing detection of BM with con- ventional passive contrast (gadolinium) enhanced MRI. However, treatment during the earlier stages of develop- ment is likely to confer a much greater therapeutic benefit than in the later highly aggressive stages. The pressing ther- apeutic challenge for BM, therefore, is the need to treat (i) at an early stage when relatively few metastatic tumor cells have invaded the brain parenchyma, and (ii) in a molecularly targeted manner to avoid healthy brain toxicity. In a previous in silico dosimetric modeling study, we found that lead-212 (212Pb), a novel alpha-particle emitting radionuclide proposed for theranostic applications, when combined with an antibody against VCAM-1 would be suitable for the treatment of early BM.12 It was found that during the early micrometastatic stages of a breast cancer BM model, tumor cells grew co-optively around blood vessels with a maximum penetration depth of ≤47.8 μm from VCAM-1 expressing at 21 days after intracardiac injection of human breast carcinoma cells (MDA231BR- GFP). Key Point Key Point © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons org/licenses/by/4 0/) which permits unrestricted reuse distribution and reproduction in any medi p ( p // ativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, vided the original work is properly cited. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 358 358 Autoradiography An image phosphor plate was exposed for 23 h to brain sections (prepared from samples as part of the biodistribution study) and analyzed using a Cyclone Storage Phosphor System (Perkin Elmer). The phosphor plate was calibrated by exposure to different amounts of 212Pb (1 Bq, 2 Bq, 4 Bq, 10 Bq) for 23 h, thus allowing image intensity to be related to the amount of radioactivity in bec- querels. After autoradiography, VCAM-1 immunostaining was performed on the brain sections. Co-registration of autoradiography images and VCAM-1 staining using PMOD software allowed evaluation of 212Pb-αVCAM-1 and 212Pb-IgG uptake in healthy brain tissue and VCAM-1 posi- tive BM at 2, 4, 8, and 24 h post-injection (p.i.). Therapy Study To evaluate the therapeutic potential of 212Pb-αVCAM-1, five groups of mice were used. A control group consisted of an- imals with BM that did not receive treatment. The 212Pb-IgG and 208Pb-αVCAM-1 groups were animals with BM and were included for comparison to 212Pb-αVCAM-1 (208Pb is a nonradioactive isotope of 212Pb). Lastly, 212Pb-αVCAM-1 was compared with WBRT. Immunohistochemistry was performed to evaluate damage to DNA double-strand breaks (DSB; γH2AX) 4 h after treatment. The effect of treat- ment on tumor cell proliferation and vascularization was characterized 3 days after treatment by Ki67 and CD31 immunostaining. Animals were then followed periodically by MRI to evaluate the number and volume of BM and OS. Magnetic Resonance Imaging Importance of the Study Monte Carlo simulation of radiation dose deposi- tion showed that 212Pb-labeled anti–VCAM-1 antibodies would provide a therapeutic dose to tumor cells adjacent to blood vessels, without the need for antibodies to cross the BBB. The aim of the current study, therefore, was to deter- mine whether VCAM-1 targeted 212Pb (212Pb-αVCAM-1) can prevent or delay the development of early BM. To achieve this aim, 3 substudies were performed: (i) a biodistribution study to assess the specificity and the dosimetry of the therapeutic approach; (ii) a therapeutic efficacy study for 212Pb-αVCAM-1 in BM, including com- parison to external beam radiotherapy (EBRT), as WBRT is the current treatment of choice for multiple BM; and (iii) a study to determine systemic and brain toxicity of 212Pb-αVCAM-1. We have previously shown that vascular cell adhesion molecule 1 (VCAM-1) is upregulated on the surface of en- dothelial cells (in the vessel lumen) during seeding of met- astatic cells to the brain and during the micrometastatic stages of development within the brain.6,7 We have also Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Neuro- Oncology where A0 is the maximum activity, λr and λd the rise and decay constants of the activity in the organ, and t the time. The cumulated activity A was then calculated by inte- grating At between 0 and 24 h. Materials and Methods Cell Culture We used the human breast carcinoma cell line that pref- erentially metastasizes to the brain, MDA-231-Br (kindly provided by Dr Patricia S. Steeg of the National Cancer Institute).6,7 Cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich) with 1 g/L of glucose sup- plemented with 2 mM glutamine (Gibco), 10% fetal calf serum (Eurobio), and 1 mg/mL penicillin/streptomycin (Sigma) at 37°C. Biodistribution Study All MRI procedures were performed as previously de- scribed.13 The diffusion parameters were obtained from diffusion-weighted spin-echo planar images (6 diffusion directions, with 6 b values: 200, 500, 750, 1000, 1500, and 2000 s/mm2 and 2 reference images; b≈0s/mm2). Twenty-one days after intracardiac injection of MDA- 231-Br cells, either 212Pb-αVCAM-1 (1 MBq; antibody mass, 27.03 µg; injectate volume, 100 µL) or an equivalent amount of 212Pb–immunoglobulin G (IgG) was administered intra- venously to BM-bearing animals or to control animals that had not received intracardiac MDA-231-Br cells. Animals were euthanized 2, 4, 8, 24, and 72 h after 212Pb-αVCAM-1 injection and 4 h after 212Pb-IgG, and 100 µL of blood was collected. Animals were then perfused with saline so- lution and organs removed to evaluate radioactivity biodistribution. The organs analyzed were brain, liver, kid- neys, lungs, spleen, heart, bone, and muscle. Whole brain cryosections of 20 µm thickness were prepared for auto- radiography. In this study, 4 animals were used for each time point for the 2 radio-immunoconjugates. Activity was measured for the various organs (including brain sections) at different times: 2, 4, 8, and 24 h. These measurements were fitted by Mouse Brain Metastasis Model All animal investigations were performed under the cur- rent European directive (2010/63/EU). This study was undertaken with the permission of the regional committee on animal ethics (C2EA-54) and the French Ministry of Higher Education, Research, and Innovation (project #10083). Naval Medical Research Institute nu/nu mice (25 g, 8 wk old, female; Janvier Labs) were maintained in specific pathogen-free housing and were fed γ-irradiated laboratory food and water ad libitum (ONCOModels). Procedures were performed on mice under general anes- thesia (5% isoflurane for induction, 2% for maintenance in 70%-N2O/30%-O2). Body temperature was monitored and maintained at 37.5 ± 0.5°C throughout the experiments. For the BM model, mice were placed in the supine position and 1.75e105 cells in 100 µL of phosphate buffered saline (PBS)– glutamine 2 mM were injected into the left ventricle of the heart guided by ultrasound imaging (CX50, MSI-FAS). Animals were then followed periodically by MRI (7T-MRI Bruker, Cyceron imaging platform) over a 16-day period to follow BM development. Animals were then assigned to biodistribution, therapy, or toxicity substudies. Survival Study Following treatment, mice were followed in a survival study. Prior to the initiation of the study, we defined 40 days as an arbitrary endpoint for OS outcome based on previous studies17 or when maximum tumor burden was reached. Toxicity Study To evaluate the radiotoxicity of 212Pb-αVCAM-1 on brain microstructure, diffusion MRI was used. FA, MD, AD, and RD were quantified to characterize the white matter organ- ization and are predicted to be impacted by radiotoxicity. Moreover, cellularity was evaluated with ADC and kur- tosis, two standard non-Gaussian diffusion parameters, which would be expected to increase and decrease, re- spectively, in the case of radiotoxicity in normal brain.19 All diffusion parameters were evaluated in whole brain region of interest. Submandibular blood collection was used for platelet and white blood cell counts at 24 h prior and at various times after radio-immunoconjugate injection. Counting was performed manually after standard blood smear hematoxylin and eosin staining. At the end of the study, animals were euthanized and blood was harvested for transaminase assays. Throughout the study, the weight of animals was measured as an indicator of global toxicity. External Beam Radiation Therapy WBRT treatment was performed using an Xrad-225Cx ir- radiator (PXi, Cyceron platform) (225 kV X-rays; 3.3 Gy/ min). A cone beam CT image was acquired for anatom- ical delineation. Treatment planning was performed using SmART-Plan software (Maastro Clinic), with segmentation, targeting, and planning performed using the cone beam CT image. Animals received WBRT to a total dose of 12 Gy delivered in 3 fractions of 4 Gy on 3 consecutive days, using a 10 mm collimator. Clonogenic Assay For X-ray treatment, an Xrad-225Cx irradiator was used (dose rate: 1.96 Gy/min; PXi, Cyceron platform). Tumor cells were plated in 6-well plates (750 cells/well), exposed to X-rays (0, 2, 4, 6, or 8 Gy) 2 h after cell seeding and in- cubated for 12 days. For TAT, the same number of cells was plated, and after 4 h exposed to 212Pb (0, 5, 10, 20, 30, or 50 kBq). Culture medium was replaced by fresh medium and cells were incubated for 12 days to allow colony forma- tion. Colonies were stained with 2% crystal violet (Sigma- Aldrich) diluted in 20% ethanol. Colonies were counted manually. Four biological repeats of clonogenic assays were performed with, for each experiment, 3 wells per ra- diation dose. SF2 (survival fraction at 2Gy) and D50 (dose corresponding for SF = 50%) were obtained from the sur- vival curves and used to compare radiosensitivity between X-rays and TAT. In vitro irradiations were modeled using Monte Carlo simulations performed with GATE.18 Targeted Alpha-Particle Therapy with 212Pb-αVCAM-1 Targeted Alpha-Particle Therapy with 212Pb-αVCAM-1 All reagents were obtained from Sigma-Aldrich, unless otherwise stated. 212Pb(NO3)2 (Orano Med) was conjugated to VCAM-1 antibody (clone: M/K; Merck Millipore) using a protocol based on previous publications.15 Briefly, metal chelator TCMC (Macrocyclics) was conjugated in 15-fold molar excess with VCAM-1 antibody using thiocyanate (SCN) chemistry resulting in a ligand to antibody ratio of 2 as determined by an Arsenzo III spectrophotometric assay. One milligram of TCMC-αVCAM-1 was then incu- bated with 37 MBq of 212Pb(NO3)2 at 37°C in 0.15 M NH4OAc buffer for 30 min. Radiochemical purity was determined to be 93.5% using instant thin layer chromatography (iTLC) in 0.1 M EDTA buffer (pH 8.4). Radio-immunoconjugate was diluted in PBS before injection into animals. The tail vein intravenous injected dose was 1 MBq per mouse, similar to a previous report.15 The above procedure was used for the radioconjugation of 212Pb-TCMC-IgG and 208Pb-TCMC- αVCAM-1. To assess the integrity of 212Pb-TCMC-VCAM-1, plasma was harvested at 2, 4, 8, 24, and 72 hours p.i. and the iTLC method was used to assess the ratio of conju- gated to unconjugated 212Pb. Image Processing and Analyses BM were delineated manually on all adjacent T2-weighted slices. BM volume was calculated by multiplication of the sum of contiguous tumor surface areas by the slice thick- ness. Enumeration of BM was done manually using MR images. Diffusion parameters including fractional anisot- ropy (FA) and apparent diffusion coefficient (ADC) were obtained from maps generated using Paravision software. Mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps were calculated from eigenvalues with MD = (λ1 + λ2 + λ3)/3, AD = λ1, and RD = (λ2 + λ3)/214. Kurtosis was estimated by fitting the signal obtained at all b values according to the non-Gaussian diffusion model as A (t) = A0 · 1 −e−r·t · ed·t A (t) = A0 · 1 −e−r·t · ed·t (1) Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 360 examined at 20x magnification for VCAM-1 and at 40x for Ki67, γH2AX, CD31, PDGFRβ, CD68, and GFAP using a Leica DMi8 microscope. BM were identified by Hoechst 33342 counterstaining and through green fluorescent protein ex- pression of MDA-231-Br cells. Whole brain images were obtained using Metavue software. For quantification of VCAM-1, CD31, γH2AX, and Ki67, three slices per animal were used and vessels, foci, and nuclei were automati- cally counted (ImageJ). Quantitative results of γH2AX and Ki67 staining are expressed as the percentage area of bio- marker expression relative to the total tumor area. Vessel diameter was quantified as previously described.16 follows, assuming signal levels remain high compared with background noise: S(b) = S0.exp[−bADC + (bADC)2K/6]. Kurtosis characterizes the deviation from a mono- exponential decay and is null when water Brownian mo- tion obeys a Gaussian law. As such, K increases with the heterogeneity of the cellular environment. Image analysis was performed with ImageJ software. 212Pb-αVCAM-1 Increases Overall Survival 212Pb-αVCAM-1 Increases Overall Survival A detailed depiction of the biodistribution study, including number of animals per group, is illustrated in Fig. 1A. The stability of the 212Pb-αVCAM-1 complex in blood was evaluated showing that 212Pb-αVCAM-1 remained intact from 2 to 24 h after injection when 92.78 ± 2.19% of radio- activity present was due to 212Pb-TCMC-αVCAM-1. Whole body biodistribution in BM-bearing mice revealed that 212Pb-αVCAM-1 was predominantly present in the blood, liver, kidneys, and spleen up to 24 h p.i. (Fig. 2A). Three days after injection, 212Pb-αVCAM-1 retention was ob- served in only the spleen (0.27 ± 0.037% injected dose per gram tissue). The absorbed dose to all normal organs was <1 Gy, while blood and kidney received the highest ab- sorbed doses of 0.84 and 0.72 Gy, respectively (Table 1). Concerning the brain, the uptake of 212Pb-αVCAM-1 by healthy tissue and at sites of VCAM-1 positive BM was assessed autoradiographically (Fig. 2B). Four hours after injection, no significant 212Pb-αVCAM-1 uptake was ob- served in mouse brain without metastases. Similarly, in animals with VCAM-1 positive BM that received non- specific treatment (212Pb-IgG), there was no signifi- cant uptake in brain. In contrast, 212Pb-αVCAM-1 uptake was significantly greater (P < 0.001) in animals with BM (0.215 ± 0.060 Bq) following 212Pb-αVCAM-1 administra- tion compared with those without BM (0.018 ± 0.014 Bq) and those with BM that received 212Pb-IgG (0.038 ± 0.009 Bq) (Fig. 2C). Kinetic studies (Fig. 2D) clearly revealed that the uptake of 212Pb-αVCAM-1 was significantly greater in BM than in healthy tissues from 2 h to 8 h p.i.; no sig- nificant difference was observed at 24 h p.i. These curves allowed us to calculate the accumulated activity in BM and healthy brain tissues and revealed a 6-fold greater accumulated activity in BM (5.52e108 ± 1.41e108 and 0.92e108 ± 0.89e108 disintegrations per gram of BM and healthy tissue, respectively). To evaluate treatment efficacy, a survival study was con- ducted (Fig. 3E). Animals in the untreated BM group reached the tumor volume limit at 23.8 ± 1.3 days after tumor cell injection. Animals treated with 212Pb-IgG and 208Pb-αVCAM-1 presented similar OS at 24.0 ± 1.0 days and 23.3 ± 1.3 days, respectively (no significant difference com- pared with untreated controls). 212Pb-αVCAM-1 Increases Overall Survival In contrast, WBRT signifi- cantly improved OS to 27.6 ± 2.4 days (P < 0.05 vs control groups), while 212Pb-αVCAM-1 treatment further improved survival to 35.6 ± 2.1 days (P < 0.01 vs BM and BM + WBRT groups). To explore the difference in efficacy of the 2 radiation modalities observed in vivo, in vitro clonogenic assays were performed. The SF2 was 0.6 ± 0.06 and 0.026 ± 0.001 for X-rays and 212Pb, respectively (P < 0.001; Fig. 3F). The D50 was 2.41 ± 0.18 and 0.382 ± 0.04 for X-rays and 212Pb, respectively (P < 0.001). The relative biological effect at 1 and 2 Gy was 4.80 and 23.08, respectively, suggesting a more pronounced radiosensitivity of MDA-231-Br cells to 212Pb than to X-rays. Statistical Analyses (40.4 ± 11.3 at 24 days) of metastases (Fig. 3A, C–D). WBRT reduced both tumor growth and the number of MRI- detectable BM in comparison to the untreated BM group (5.11 ± 1.49 mm3 and 32.4 ± 9.06 for tumor volume and number, respectively, at 24 days; P < 0.01 for both param- eters; Fig. 3A, C–D). Interestingly, 212Pb-αVCAM-1 treat- ment significantly reduced tumor growth and number of BM in comparison to the control group (P < 0.001) and to the WBRT group (3.04 ± 0.60 mm3 and 18.8 ± 1.64 for tumor volume and number, respectively, at 24 days; P < 0.01). Treatment effects were visible as early as 3 days after the beginning of the treatment (Fig. 3B). Data cutoff for BM and BM + WBRT groups in Fig. 3B panels C and D reflects the time at which maximum tumor burden was reached. All data are expressed as mean ± SD. Student's t-test was used to compare radiosensitivity of cells to X-rays and 212Pb, and one-way ANOVA followed by Tukey's post-hoc test was used to compare differences between treatment groups. Two-way ANOVA (group and time effects) followed by Tukey's post-hoc test was used to assess differences in the volume and number of BM between treatment groups. A log-rank test was used to compare survival. Statistical analyses were obtained using JMP (SAS Institute). Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 212Pb-αVCAM-1 Decreased Cell Proliferation and Increased DNA DSBs Staining by γH2AX revealed a significant increase in DNA DSBs in BM + WBRT and BM + 212Pb-αVCAM-1 groups in comparison to the BM (no treatment) group (Fig. 4A and D). Moreover, a significant difference was also observed between BM + WBRT and BM + 212Pb-αVCAM-1 groups (P < 0.01), with a greater number of γH2AX-positive cells in the BM + 212Pb-αVCAM-1 group. Staining for Ki67 demon- strated similar results, with a significant decrease in prolif- eration following WBRT and 212Pb-αVCAM-1 treatments in comparison to the BM group, and a significant difference between the BM + WBRT and BM + 212Pb-αVCAM-1 groups (Fig. 4B and D). 212Pb-αVCAM-1 Reduces the Number and Volume of Brain Metastases Immunohistochemistry Animals were euthanized and perfused with saline so- lution 4 h (for inflammation and DNA DSB staining) or 3 days (for cell proliferation and vasculature staining) after 212Pb-αVCAM-1 treatment. Inflammation, DNA DSBs, tumor cell proliferation, pericytes, microglia, and astro- cytes were evaluated using primary antibodies against VCAM-1 (5 µg/mL, SoutherBiotech), γH2AX (2 µg/mL, Abcam), Ki67 (0.35 µg/mL, Dako), CD31 (5 µg/mL, PB Bioscience), platelet derived growth factor receptor beta (PDGFRβ) (2 µg/mL, Santa-Cruz), CD68 (1 µg/mL, Merck Millipore), and glial fibrillary acidic protein (GFAP) (3 µg/ mL, Dako), respectively. Immunostaining protocols were performed as previously described.13 Tissue sections were Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 361 Neuro- Oncology Neuro- Oncology Statistical Analyses Results Whole Body Biodistribution Reveals High Uptake of 212Pb-αVCAM-1 at Sites of Brain Metastases No Effect of 212Pb-αVCAM-1 on Brain Vascularization and Microenvironment A detailed schedule of the therapy study is illustrated in Fig. 1B. As expected, without treatment, the BM group experienced continuous tumor growth in terms of both volume (9.05 ± 4.34 mm3 at 24 days) and number VCAM-1 is expressed on the luminal surface of en- dothelial cells close to early BM. The potential Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 362 Inoculation of tumour cells D21 D0 2h - 4h - 8h - 24h - 72h post-injection Autopsy (Biodistribution/autoradiography/ immunohistochemistry (αVCAM-1)) Anatomical MRI A Biodistribution study D20 Group 1 (n = 4) No brain metastases 212 Pb-αVCAM-1 Group 2 (n = 4) Brain metastases 212Pb-IgG Group 3 (n = 4) Brain metastases 212Pb-αVCAM-1 D22 D24 Treatments A xicity of 212Pb-αVCAM-1 treatment on brain blood ssels and the brain microenvironment was as- ssed immunohistochemically by CD31, PDGFRβ, CD68, and GFAP immunostaining. As expected, v sels close to the tumor (Fig. 4C) appear tortuous, rupted, and larger in comparison to vessels in hea Inoculation of tumour cells D21 D0 2h - 4h - 8h - 24h - 72h post-injection Autopsy (Biodistribution/autoradiography/ immunohistochemistry (αVCAM-1)) Anatomical MRI Inoculation of tumour cells D16 208/212Pb-X D24 Anatomical MRI D21 3 days post-treatment Autopsy Immunohistochemistry (Ki67-CD31) Anatomical MRI WBRT D18-19-20 D0 4 hours post-treatment Autopsy Immunohistochemistry (γH2AX) Treatments: Anatomical MRI D27 D31 D38 C A Biodistribution study Therapy study D20 Group 1 (n = 4) No brain metastases 212 Pb-αVCAM-1 Group 2 (n = 4) Brain metastases 212Pb-IgG Group 3 (n = 4) Brain metastases 212Pb-αVCAM-1 Group 1 (n = 6) Brain metastases Control Group 2 (n = 3) Brain metastases 212Pb-IgG Group 3 (n = 3) Brain metastases 208Pb-α VCAM-1 Group 4 (n = 8) Brain metastases WBRT Group 5 (n = 8) Brain metastases 212Pb-αVCAM-1 B D22 D24 Treatments Inoculation of tumour cells D21 D0 Plateletand white bloodcell counts Treatments Diffusion MRI Toxicity study D20 Group 1 (n = 5) No brain metastases saline solution Group 2 (n = 5) No brain metastases 212Pb-αVCAM-1 Group 3 (n = 5) Brain metastases 212Pb-IgG Group 4 (n = 5) Brain metastases 212Pb-αVCAM-1 D22 – D24 – D28 – D31 – D35 – D38 – D42 Transaminase assay Diffusion MRI Fig. 1 Experimental paradigm for (A) biodistribution, (B) therapy, and (C) toxicity studies. No Effect of 212Pb-αVCAM-1 on Brain Vascularization and Microenvironment xicity of 212Pb-αVCAM-1 treatment on brain blood ssels and the brain microenvironment was as- ssed immunohistochemically by CD31, PDGFRβ, CD68, and GFAP immunostaining. As expected, ves- sels close to the tumor (Fig. 4C) appear tortuous, dis- rupted, and larger in comparison to vessels in healthy D21 D0 2h - 4h - 8h - 24h - 72h post-injection Autopsy (Biodistribution/autoradiography/ immunohistochemistry (αVCAM-1)) Anatomical MRI Inoculation of tumour cells D16 208/212Pb-X D24 Anatomical MRI D21 3 days post-treatment Autopsy Immunohistochemistry (Ki67-CD31) Anatomical MRI WBRT D18-19-20 D0 4 hours post-treatment Autopsy Immunohistochemistry (γH2AX) Treatments: Anatomical MRI D27 D31 D38 C Biodistribution study Therapy study D20 Group 1 (n = 4) No brain metastases 212 Pb-αVCAM-1 Group 2 (n = 4) Brain metastases 212Pb-IgG Group 3 (n = 4) Brain metastases 212Pb-αVCAM-1 Group 1 (n = 6) Brain metastases Control Group 2 (n = 3) Brain metastases 212Pb-IgG Group 3 (n = 3) Brain metastases 208Pb-α VCAM-1 Group 4 (n = 8) Brain metastases WBRT Group 5 (n = 8) Brain metastases 212Pb-αVCAM-1 B D22 D24 Inoculation of tumour cells D21 D0 Plateletand white bloodcell counts Treatments Diffusion MRI Toxicity study D20 Group 1 (n = 5) No brain metastases saline solution Group 2 (n = 5) No brain metastases 212Pb-αVCAM-1 Group 3 (n = 5) Brain metastases 212Pb-IgG Group 4 (n = 5) Brain metastases 212Pb-αVCAM-1 D22 – D24 – D28 – D31 – D35 – D38 – D42 Transaminase assay Diffusion MRI ig. 1 Experimental paradigm for (A) biodistribution, (B) therapy, and (C) toxicity studies. Biodistribution study Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Zoom represent VCAM-1 staining (red) in this specific healthy or BM region (blue: A 0 5 10 15 20 212Pb (%ID/g) after injection 2 h Pb212-αVCAM -1 4 h Pb212-αVCAM-1 8 h Pb212-αVCAM-1 24 h Pb212-αVCAM-1 72 h Pb212-αVCAM-1 Blood Liver Kidney Lung Spleen Heart Bone Muscle A 0 5 10 15 20 212Pb (%ID/g) after injection 2 h Pb212-αVCAM -1 4 h Pb212-αVCAM-1 8 h Pb212-αVCAM-1 24 h Pb212-αVCAM-1 72 h Pb212-αVCAM-1 Blood Liver Kidney Lung Spleen Heart Bone Muscle A A Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 0 0.1 0.2 0.3 0.4 0 4 8 12 16 2 212Pb-uptake (Bq) Time post-injection (da Brain metas Healthy brai 0 0.1 0.2 0.3 212Pb uptake 4 h post- injection (Bq) BM+212Pb-αVCAM-1 No BM+212Pb-αVCAM-1 0.4 0 Autoradiography 4h post-injection (D21) Activity (Bq) B Merged 0 5 212Pb (% BM+212Pb-IgG T2w MRI (D20) * ## C D * * *** VCAM-1 VCAM-1 VCAM-1 Blood Liver Kidney Lung Spleen Heart Bone Muscle No BM+212Pb- αVCAM-1 BM+212Pb αVCAM BM+212Pb-lgG 2 Biodistribution of 212Pb-αVCAM-1: whole body and brain metastases. (A) Whole body 212Pb-αVCAM-1 uptake biodistributi and 72h post-injection. Mean ± SD, n = 4 for all time points. (B) Top: Representative T2-weighted MRI of brain from animals w am + 212Pb-αVCAM-1), with tumor with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respectively tases appear in hyperintensity in T2-weighted (red arrows). Zoom represent VCAM-1 staining (red) in this specific healthy or BM echst staining). Middle: Representative autoradiography of 212Pb-IgG/αVCAM-1 uptake. Bottom: Merged T2-weighted MRI and oradiography pictures (threshold: mean value of healthy tissue + 1.96SD for P < 0.05). (C) 212Pb-αVCAM-1 brain uptake 4 h pos mals without BM (sham + 212Pb-αVCAM-1) or with BM with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCA ly). Mean ± SD, n = 4 for all groups, P < 0.001 vs sham + 212Pb-αVCAM-1 group and ###P < 0.001 vs BM + 212Pb-IgG group. (D) 0 0.1 0.2 0.3 0.4 0 4 8 12 16 20 24 212Pb-uptake (Bq) Time post-injection (days) Brain metastases Healthy brain tissue 0 0.1 0.2 0.3 212Pb uptake 4 h post- injection (Bq) BM+212Pb-αVCAM-1 No BM+212Pb-αVCAM-1 0.4 0 Autoradiography 4h post-injection (D21) Activity (Bq) B Merged 0 212Pb BM+212Pb-IgG T2w MRI (D20) * ### C D * * * NS VCAM-1 VCAM-1 VCAM-1 Blood Liver Kidney Lung Spleen Heart Bone Muscle No BM+212Pb- αVCAM-1 BM+212Pb- αVCAM-1 BM+212Pb-lgG Fig. Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Autopsy B Anatomical MRI oxicity of 212Pb-αVCAM-1 treatment on brain blood vessels and the brain microenvironment was as- sessed immunohistochemically by CD31, PDGFRβ, CD68, and GFAP immunostaining. As expected, ves- sels close to the tumor (Fig. 4C) appear tortuous, dis rupted, and larger in comparison to vessels in healthy C Group 5 (n = 8) Brain metastases 212Pb-αVCAM-1 Inoculation of tumour cells D21 D0 Plateletand white bloodcell counts Treatments Diffusion MRI Toxicity study D20 Group 1 (n = 5) No brain metastases saline solution Group 2 (n = 5) No brain metastases 212Pb-αVCAM-1 Group 3 (n = 5) Brain metastases 212Pb-IgG Group 4 (n = 5) Brain metastases 212Pb-αVCAM-1 D22 – D24 – D28 – D31 – D35 – D38 – D42 Transaminase assay Diffusion MRI Fig. 1 Experimental paradigm for (A) biodistribution, (B) therapy, and (C) toxicity studies. C Toxicity study toxicity of 212Pb-αVCAM-1 treatment on brain blood vessels and the brain microenvironment was as- sessed immunohistochemically by CD31, PDGFRβ, toxicity of 212Pb-αVCAM-1 treatment on brain blood vessels and the brain microenvironment was as- sessed immunohistochemically by CD31, PDGFRβ, CD68, and GFAP immunostaining. As expected, ves- sels close to the tumor (Fig. 4C) appear tortuous, dis- rupted, and larger in comparison to vessels in healthy Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Neuro- Oncology Neuro Oncology 0 0.1 0.2 0.3 0.4 0 4 8 12 16 20 24 212Pb-uptake (Bq) Time post-injection (days) Brain metastases Healthy brain tissue 0 0.1 0.2 0.3 212Pb uptake 4 h post- injection (Bq) BM+212Pb-αVCAM-1 No BM+212Pb-αVCAM-1 0.4 0 Autoradiography 4h post-injection (D21) Activity (Bq) B A Merged 0 5 10 15 20 212Pb (%ID/g) after injection BM+212Pb-IgG T2w MRI (D20) * ### C D * * *** NS 2 h Pb212-αVCAM -1 4 h Pb212-αVCAM-1 8 h Pb212-αVCAM-1 24 h Pb212-αVCAM-1 72 h Pb212-αVCAM-1 VCAM-1 VCAM-1 VCAM-1 Blood Liver Kidney Lung Spleen Heart Bone Muscle No BM+212Pb- αVCAM-1 BM+212Pb- αVCAM-1 BM+212Pb-lgG Fig. 2 Biodistribution of 212Pb-αVCAM-1: whole body and brain metastases. (A) Whole body 212Pb-αVCAM-1 uptake biodistribution at 2, 4, 8, 24, and 72h post-injection. Mean ± SD, n = 4 for all time points. (B) Top: Representative T2-weighted MRI of brain from animals without tumor (sham + 212Pb-αVCAM-1), with tumor with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respectively). Brain me- tastases appear in hyperintensity in T2-weighted (red arrows). Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 2 Biodistribution of 212Pb-αVCAM-1: whole body and brain metastases. (A) Whole body 212Pb-αVCAM-1 uptake biodistribution at 2, 4, 8, 24, and 72h post-injection. Mean ± SD, n = 4 for all time points. (B) Top: Representative T2-weighted MRI of brain from animals without tumor (sham + 212Pb-αVCAM-1), with tumor with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respectively). Brain me- tastases appear in hyperintensity in T2-weighted (red arrows). Zoom represent VCAM-1 staining (red) in this specific healthy or BM region (blue: Hoechst staining). Middle: Representative autoradiography of 212Pb-IgG/αVCAM-1 uptake. Bottom: Merged T2-weighted MRI and thresholded autoradiography pictures (threshold: mean value of healthy tissue + 1.96SD for P < 0.05). (C) 212Pb-αVCAM-1 brain uptake 4 h post-injection in animals without BM (sham + 212Pb-αVCAM-1) or with BM with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respec- tively). Mean ± SD, n = 4 for all groups, P < 0.001 vs sham + 212Pb-αVCAM-1 group and ###P < 0.001 vs BM + 212Pb-IgG group. (D) Quantitative analyses of 212Pb-αVCAM-1 uptake in BM and healthy brain tissue. Mean ± SD, n = 4 for both groups, P < 0.001 vs sham + 212Pb-αVCAM-1 group. NS = non-significant. BM+212Pb-αVCAM-1 No BM+212Pb-αVCAM-1 0.4 0 Autoradiography 4h post-injection (D21) Activity (Bq) B Merged BM+212Pb-IgG T2w MRI (D20) VCAM-1 VCAM-1 VCAM-1 Bl L Kid L Spl H B Mus 2 Biodistribution of 212Pb-αVCAM-1: whole body and brain metastases. (A) Whole bo and 72h post-injection. Mean ± SD, n = 4 for all time points. (B) Top: Representative T2- am + 212Pb-αVCAM-1), with tumor with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG tases appear in hyperintensity in T2-weighted (red arrows). Zoom represent VCAM-1 stain echst staining). Middle: Representative autoradiography of 212Pb-IgG/αVCAM-1 uptake. oradiography pictures (threshold: mean value of healthy tissue + 1.96SD for P < 0.05). Discussion tissue. However, immunostaining quantification of vessel diameter and surface did not demonstrate an ef- fect of WBRT or 212Pb-αVCAM-1 treatments on vessels in comparison to the BM (no treatment) control group (Fig. 4E). From a preliminary study, we also observed that at 3 days after treatments, pericytes (detected by the PDGFRβ staining) and astrocyte activation (GFAP) do not appear to be modified whatever the treatment (Supplementary Fig. 1A–C). However, for CD68 staining, there appears to be a trend toward an increase in mi- croglial/macrophage activation in the radiotherapy- treated groups compared with the control group (and no obvious difference between the radiotherapy modal- ities), but more investigations would be needed to con- clude on these effects. A significant proportion of patients with controlled primary cancer die as a result of BM.3 Current treatment options for patients with BM from breast cancer remain limited, firstly because EBRT is performed when BM are already estab- lished and, secondly because it may induce a decline in cognitive ability for long-term survivors. Consequently, there is a critical need to develop new therapeutic ap- proaches to target the early stages of BM. In this study, we investigated the therapeutic potential of a targeted molecular radiotherapeutic using 212Pb, an alpha emitter, combined with an antibody against an early BM mo- lecular biomarker (VCAM-1). To reflect the clinical situation as closely as possible, we used a preclinical model of BM using intracardiac injection of human metastatic breast cancer cells mimicking the invasion process observed in humans.20 To the best of our knowledge, this study is the first investigating the potential of TAT for the treatment of early BM. Uptake of 212Pb-αVCAM-1 to the region encompassing tumor cells was combined with a very low healthy brain tissue uptake, yielding a ratio of BM/healthy brain tissue dose deposition of ~6. This result is similar to findings in a clinical study targeting BM with 124I-radretumab for dosimetry purposes, which re- ported a tumor/healthy brain tissue ratio of 4.95.21 This 6-fold increase in accumulated activity in BM compared with healthy brain tissue could provide a better therapeutic outcome than with WBRT, where dose to normal tissue is unavoidable, leading to cognitive decline. The high uptake at sites of BM was combined with a marked increase in radiosensitivity of the human breast cancer cell line used in 212Pb compared with WBRT. Discussion The therapy study revealed better tumor control with 212Pb-αVCAM-1 in comparison to X-ray treatment, which could be ascribed to the higher radiosensitivity of cells to the high linear energy transfer of 212Pb compared with X-rays, leading to a greater number of irreparable DSBs.22 Another study, in a glioblastoma model, using an alpha emitter com- bined with an antibody targeting tumor vessels, 225Ac-E4G10, showed similar improvements in OS to the current study, but with a lower injected amount of radioactivity. The observed differences between these and the current study may reflect the longer half-life of 225Ac (10 days vs 10.6 h for 212Pb) and the BBB permeability observed in glioblastoma that is not ob- served in early stage BM, enabling greater alpha emitter up- take within the tumor area.23 Diffusion Imaging Shows that 212Pb-αVCAM-1 Does Not Affect Brain White Matter and Cellularity Diffusion Imaging Shows that 212Pb-αVCAM-1 Does Not Affect Brain White Matter and Cellularity A detailed schedule of the toxicity study is shown in Fig. 1C. As presented in Supplementary Fig. 2, the integrity of white matter was preserved after 212Pb-αVCAM-1 treatment compared with the control group. No signif- icant differences were observed for diffusion metrics between the 3 animal groups (Supplementary Fig. 2E and F). Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 4, whereas combined treatment allows better tumor control (decrease in tumor volume and number of BM) compared with each treatment alone, it involves significant toxicity (probably as a result of too much accumulated dose), which does not allow increased OS. However, the OS is not worst than that observed with WBRT, as all the animals died about one week after treatment, as observed in the animals receiving WBRT. In a preliminary study, we investigated combining the 2 ra- diotherapy treatments. As shown in Supplementary Fig. 4, whereas combined treatment allows better tumor control (decrease in tumor volume and number of BM) compared with each treatment alone, it involves significant toxicity (probably as a result of too much accumulated dose), which does not allow increased OS. However, the OS is not worst than that observed with WBRT, as all the animals died about one week after treatment, as observed in the animals receiving WBRT. Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 (C 0 0.1 0.2 0.3 212Pb uptake 4 h post- injection (Bq) * ### C D No BM+212Pb- αVCAM-1 BM+212Pb- αVCAM-1 BM+212Pb-lgG BM+212Pb-αVCAM-1 No BM+212Pb-αVCAM-1 0.4 0 Autoradiography 4h post-injection (D21) Activity (Bq) B BM+212Pb-IgG T2w MRI (D20) VCAM-1 VCAM-1 VCAM-1 K Sp M 0 0.1 0.2 0.3 0.4 0 4 8 12 16 20 24 212Pb-uptake (Bq) Time post-injection (days) Brain metastases Healthy brain tissue 0 0.1 0.2 0.3 212Pb uptake 4 h post- injection (Bq) * ### C D * * * NS No BM+212Pb- αVCAM-1 BM+212Pb- αVCAM-1 BM+212Pb-lgG B C 212Pb-uptake (Bq) D 0 0.1 0.2 0.3 0.4 0 4 8 12 16 20 24 212Pb-uptake (Bq) Time post-injection (days) Brain metastases Healthy brain tissue D * * * NS α α B D 212Pb-uptake (Bq) Time post-injection (days) Fig. 2 Biodistribution of 212Pb-αVCAM-1: whole body and brain metastases. (A) Whole body 212Pb-αVCAM-1 uptake biodistribution at 2, 4, 8, 24, and 72h post-injection. Mean ± SD, n = 4 for all time points. (B) Top: Representative T2-weighted MRI of brain from animals without tumor (sham + 212Pb-αVCAM-1), with tumor with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respectively). Brain me- tastases appear in hyperintensity in T2-weighted (red arrows). Zoom represent VCAM-1 staining (red) in this specific healthy or BM region (blue: Hoechst staining). Middle: Representative autoradiography of 212Pb-IgG/αVCAM-1 uptake. Bottom: Merged T2-weighted MRI and thresholded autoradiography pictures (threshold: mean value of healthy tissue + 1.96SD for P < 0.05). (C) 212Pb-αVCAM-1 brain uptake 4 h post-injection in animals without BM (sham + 212Pb-αVCAM-1) or with BM with 212Pb-IgG/αVCAM-1 conjugated (BM + 212Pb-IgG and BM + 212Pb-αVCAM-1, respec- tively). Mean ± SD, n = 4 for all groups, P < 0.001 vs sham + 212Pb-αVCAM-1 group and ###P < 0.001 vs BM + 212Pb-IgG group. (D) Quantitative analyses of 212Pb-αVCAM-1 uptake in BM and healthy brain tissue. Mean ± SD, n = 4 for both groups, P < 0.001 vs sham + 212Pb-αVCAM-1 group. NS = non-significant. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 364 Combination of 212Pb-αVCAM-1 and WBRT Increases Tumor Control but Dose Optimization Is Required In a preliminary study, we investigated combining the 2 ra- diotherapy treatments. As shown in Supplementary Fig. 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity Two weeks after treatment, weight loss was: sham + sa- line solution = 0.85 ± 0.81 g; sham + 212Pb-αVCA M-1 = 3.68 ± 1.27 g, and BM + 212Pb-αVCAM-1 = 6 .20 ± 3.02 g (P < 0.01 vs sham + saline solution group) (Supplementary Fig. 3A). To evaluate 212Pb-αVCAM-1 systemic toxicity, platelets and white blood cells were counted and compared with baseline. Despite a slight de- crease 4 to 15 days after treatment, no significant differ- ences were observed between groups (Supplementary Fig. 3B and C). Concerning liver toxicity, no significant changes were observed in aspartate and alanine trans- aminases (Supplementary Fig. 3D and E). Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Neuro- Oncology Neuro- 0 25 50 75 100 20 25 30 35 40 Overall survival (%) Time (days) C BM+WBRT T2w MRI (D16) T2w MRI (D20) BM T2w MRI (D27) D E T2w MRI (D31) T2w MRI (D38) T2w MRI (D24) $$ * $$$ BM+WBRT BM A 0 1 2 3 4 5 15 20 25 30 35 40 Normalized number of brain metastatic foci Time (days) B BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 BM+208Pb-αVCAM-1 BM+212Pb-lgG BM BM+WBRT 0.01 0.1 1 0 1 2 3 4 5 6 Survival fraction Dose (Gy) * X-rays 212Pb IN VITRO RADIOSENSITIVITY SF2 D50 X-rays 0.60 (0.06) 2.41 (0.18) 212Pb 0.026(0.001)* 0.382 (0.04) * F 0 10 20 30 40 15 20 25 30 35 40 Normalized tumour volume Time (days) BM+212Pb-αVCAM-1 BM+WBRT BM BM+212Pb-αVCAM-1 BM+WBRT BM 3 Treatment effect on number and volumes of BM and OS. (A) Representative T2-weighted MR images for the 3 treatment groups at nning of EBRT (D16-D17-D19) and TAT (D20) treatments at 4 time points after treatments. (B) Three-dimensional representation of BM (r in the brain (gray) in the different groups at D24. (C) Quantitative analyses of tumor volume after treatment normalized to D16. Mean ± S 5 for all groups. P < 0.001 for time effect, P < 0.01 and *P < 0.001 vs BM and $$P < 0.01 vs WBRT group. (D) Quantitative analyses of num M after treatment normalized to D16. Mean ± SD, n = 5 for all groups. P < 0.001 for time effect, P < 0.01, and *P < 0.001 vs BM and $$$P < 0.0 WBRT group. 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity (E) Kaplan–Meier curves of survival, n = 5 for BM, n = 3 for 208Pb-αVCAM-1, n = 3 for 212Pb-IgG, n = 5 for WBRT, and n = 5 b-αVCAM-1 groups. P < 0.05 between WBRT and BM, 208Pb-αVCAM-1 and 212Pb-IgG and P < 0.01 between 212Pb-αVCAM-1 and all the ot ups. (F) Quantitative analyses of in vitro MDA-231-Br radiosensitivity for X-rays and 212Pb. Mean ± SD, n = 4 for X-rays and 212Pb treatmen 0.001 for dose effect, P < 0.001 between 212Pb and X-ray treatment. SF2 = surviving fraction for 2Gy; D50 = dose that gives SF of 50%. C $$ 0 10 20 30 40 15 20 25 30 35 40 Normalized tumour volume Time (days) BM+212Pb-αVCAM-1 BM+WBRT BM C C A Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Time (days) D D * $$$ 0 1 2 3 4 5 15 20 25 30 35 40 Normalized number of brain metastatic foci Time (days) Time (days) BM+212Pb-αVCAM-1 BM+WBRT BM Normalized number of brain metastatic foci mic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 Time (days) 0 25 50 75 100 20 25 30 35 40 Overall survival (%) Time (days) E BM+212Pb-αVCAM-1 BM+208Pb-αVCAM-1 BM+212Pb-lgG BM BM+WBRT E T2w MRI (D38) 0.01 0.1 1 0 1 2 3 4 5 6 Survival fraction Dose (Gy) * X-rays 212Pb F F B B Survival fraction BM+WBRT BM+212Pb-αVCAM-1 BM BM Fig. 3 Treatment effect on number and volumes of BM and OS. (A) Representative T2-weighted MR images for the 3 treatment groups at the beginning of EBRT (D16-D17-D19) and TAT (D20) treatments at 4 time points after treatments. (B) Three-dimensional representation of BM (red) within the brain (gray) in the different groups at D24. (C) Quantitative analyses of tumor volume after treatment normalized to D16. Mean ± SD, n = 5 for all groups. P < 0.001 for time effect, P < 0.01 and *P < 0.001 vs BM and $$P < 0.01 vs WBRT group. (D) Quantitative analyses of number of BM after treatment normalized to D16. Mean ± SD, n = 5 for all groups. P < 0.001 for time effect, P < 0.01, and *P < 0.001 vs BM and $$$P < 0.001 vs WBRT group. 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity (E) Kaplan–Meier curves of survival, n = 5 for BM, n = 3 for 208Pb-αVCAM-1, n = 3 for 212Pb-IgG, n = 5 for WBRT, and n = 5 for 212Pb-αVCAM-1 groups. P < 0.05 between WBRT and BM, 208Pb-αVCAM-1 and 212Pb-IgG and P < 0.01 between 212Pb-αVCAM-1 and all the other groups. (F) Quantitative analyses of in vitro MDA-231-Br radiosensitivity for X-rays and 212Pb. Mean ± SD, n = 4 for X-rays and 212Pb treatments. P < 0.001 for dose effect, P < 0.001 between 212Pb and X-ray treatment. SF2 = surviving fraction for 2Gy; D50 = dose that gives SF of 50%. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 366 With regard to systemic toxicity, although weight significantly decreased after treatment with b-αVCAM-1, no blood or liver toxicity was ob- ved. These findings are consistent with pre- us reports of intravenously administered 212Pb In a preliminary study we observed that 212Pb-αVCAM- had no effect on this BM microenvironment; pericyte and astrocyte activation do not appear to be modifie by either form of radiotherapy. Additional studies ar needed to investigate specifically the effect of both treat A Healthy brain tissue BM+WBRT BM Ki67/Hoechst B Healthy brain tissue BM+WBRT BM γH2AX/Hoechst 15μm 25μm BM BM+WBRT γH2AX staining (% of total brain metastases surface) 0.84 (0.66) 22.82 (4.38)* 46.29 (13.14)**/$$ Ki67 staining (% of total brain metastases surface) 46.42 (8.00) 29.73 (8.79) 9.88 (9.78)**/$ D BM BM+WBRT Vessel diameter (μm) 7.05 (1.31) 8.86 (0.72) 6.99 (1.04) Vessel surface (%) 40.46 (21.26) 29.27 (13.03) 35.04 (17.85) C E BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 CD31/Hoechst 25 m μ g. 4 Immunohistochemical determination of treatment effect. (A) Representative images of γH2AX immunostaining (DNA DSB damage, red) th Hoechst 33342 nuclear counterstaining (blue). (B) Representative images of Ki67 immunostaining (cell proliferation, red) with Hoechst 342 counterstaining (blue). White arrows indicate BM foci. (C) Representative images of CD31 immunostaining (endothelium, red) with Hoechst 342 nuclear counterstaining (blue). (D) Data represent the mean (+SD) per group for the percentage of γH2AX and Ki67 positive cells. P < 0.05, P < 0.01, *P < 0.001 vs BM group, $P < 0.05 and $$P < 0.01 vs WBRT group. (E) Data represent the mean (+SD) per group for vessel diameter and rface analyzed by histology. Keywords alpha-particle therapy \| early brain metastases \| VCAM-1 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity In the clinic, patients with BM are treated mainly with radiotherapy, and a number of preclinical studies have highlighted the impact of external radiotherapy on the tumor microenvironment and inflammatory pro- cesses in particular.26 Lugade and colleagues showed an increase in VCAM-1 on B16 melanoma tumor vessels in vivo following external radiotherapy.27 Similarly, in the present study we found an increase in vascular VCAM-1 expression 24 h and 72 h following WBRT in tumor ves- sels, but not in healthy brain vessels (Supplementary Fig. 5). On the basis of our findings, we propose that external radiotherapy prior to 212Pb-αVCAM-1 adminis- tration could increase the specificity of our therapeutic approach by upregulating VCAM-1 locally at brain me- tastasis sites. This aspect highlights the importance of evaluating the impact of EBRT and targeted radionuclide therapy and the schedule of each treatment.9,28 Further studies are necessary with regard to the combination and the dose escalation of external radiotherapy and 212Pb-αVCAM-1 in a dose escalation manner, in particular with regard to balancing healthy tissue preservation and tumor control. Combining WBRT with 212Pb-αVCAM-1 shows great promise; however, additional studies are required to optimize the dose prescription of the com- bined treatments to reduce the toxicity observed in the present study Supplementary Material Supplementary data are available at Neuro-Oncology online. Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity A A Healthy brain tissue BM+WBRT BM γH2AX/Hoechst 15μm BM+212Pb-αVCAM-1 Downloaded from https://academic.oup.com/neuro-oncology/article/22/3/357/5571872 by guest on 08 December 2020 B B Healthy brain tissue BM+WBRT BM Ki67/Hoechst 25μm BM+212Pb-αVCAM-1 C C C CD31/Hoechst 25 m μ BM BM+WBRT γH2AX staining (% of total brain metastases surface) 0.84 (0.66) 22.82 (4.38) 46.29 (13.14)**/$$ Ki67 staining (% of total brain metastases surface) 46.42 (8.00) 29.73 (8.79) 9.88 (9.78)**/$ D BM BM+WBRT Vessel diameter (μm) 7.05 (1.31) 8.86 (0.72) 6.99 (1.04) Vessel surface (%) 40.46 (21.26) 29.27 (13.03) 35.04 (17.85) E BM+212Pb-αVCAM-1 BM+212Pb-αVCAM-1 mmunohistochemical determination of treatment effect. (A) Representative images of γH2AX immunostaining (DNA DSB dam echst 33342 nuclear counterstaining (blue). (B) Representative images of Ki67 immunostaining (cell proliferation, red) with H D E Fig. 4 Immunohistochemical determination of treatment effect. (A) Representative images of γH2AX immunostaining (DNA DSB damage, red) with Hoechst 33342 nuclear counterstaining (blue). (B) Representative images of Ki67 immunostaining (cell proliferation, red) with Hoechst 33342 counterstaining (blue). White arrows indicate BM foci. (C) Representative images of CD31 immunostaining (endothelium, red) with Hoechst 33342 nuclear counterstaining (blue). (D) Data represent the mean (+SD) per group for the percentage of γH2AX and Ki67 positive cells. P < 0.05, P < 0.01, *P < 0.001 vs BM group, $P < 0.05 and $$P < 0.01 vs WBRT group. (E) Data represent the mean (+SD) per group for vessel diameter and surface analyzed by histology. With regard to systemic toxicity, although weight was significantly decreased after treatment with 212Pb-αVCAM-1, no blood or liver toxicity was ob- served. These findings are consistent with pre- vious reports of intravenously administered 212Pb radio-immunotherapy.24 In a preliminary study we observed that 212Pb-αVCAM-1 had no effect on this BM microenvironment; pericytes and astrocyte activation do not appear to be modified by either form of radiotherapy. Additional studies are needed to investigate specifically the effect of both treat- ments on the BM microenvironment and, in particular, Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Neuro- Oncology on inflammatory processes (eg, microglia/macrophage phenotype). We found no difference in vessel diameter or surface area. Interestingly, it has been shown in a model of glioblastoma with another alpha emitter (225Ac) that tumor vessel permeability and perfusion were altered,25 and we cannot rule this out here. Funding This work was supported by CNRS, CEA, Université de Caen- Normandie, the French Ministère de l'Enseignement Supérieur et de la Recherche, the Conseil Régional-Normandie and the European Union-Fonds Européen de Développement Régional (FEDER), The HABIONOR European project. Co-funding was provided by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine” 2015–2020. FRC, ANR-11-LABEX-0018-01; ANR-10-EQPX1401. M.S.S., N.R.S., K.A.V., O.T., and N.F. were supported by Cancer Research UK (grant number C5255/ A15935), the Medical Research Council (MC_PC_12004), the Engineering and Physical Sciences Research Council (EPSRC) Oxford Centre for Drug Delivery Devices (EP/L024012/1), the CRUK Oxford Centre, and the CRUK/EPSRC Cancer Imaging Centre Oxford (C5255/A16466). In the current study we used the MDA-231-Br cell line, a metastatic human breast carcinoma. However, further preclinical studies are necessary to determine whether the observed effects with 212Pb-αVCAM-1 treatment are conserved in early BM originating not only from breast cancer, but also from primary lung cancer and melanoma, which also have a propensity to spread to the brain. More generally, VCAM-1 has been shown to be highly involved in tumor growth in a number of dif- ferent cancers, including melanoma, leukemia, renal, osteosarcoma, and gastric cancers, as well as down- stream metastatic processes, further increasing the potential scope of 212Pb-αVCAM-1 therapy.29 Finally, the promising results generated using the MDA- 231-Br model should be supplemented by similar studies in other tractable models of BM, especially in a syngeneic model. Conflict of interest statement. The authors declare no con- flict of interest. Authorship statement. Conception and design: ACD, NF, SV, MS, NS, KV, and MB. Method development: ACD, NF, SV, AMF, JT, DD, LC, EP, and OT. Data acquisition: ACD, SV, AMF, JT, DD, LC, and OT. Data analysis and interpretation: All authors. Writing, review, and/or revision of the manuscript: All authors. Study su- pervision: ACD, SV, NF, NS, KV, and MB. Acknowledgment We thank Dr Patricia S. Steeg of the National Cancer Institute for providing the MDA-MD-231Br cell line and Orano Med (France) for providing the 212Pb. In conclusion, we have demonstrated the therapeutic efficacy of a new TAT for early stage treatment in a pre- clinical model of breast cancer BM, with this first proof- in-principle investigation. 212Pb-αVCAM-1 accumulated selectively at sites of BM resulting in a favorable metas- tasis/healthy brain tissue radiation absorbed dose ratio. Moreover, 212Pb-αVCAM-1 uptake was associated with a 212Pb-αVCAM-1 Does Not Induce Major Systemic Toxicity For BM, a change in vessel permeability close to the tumor cells would be of interest in terms of systemic treatment, considering the constitutively low vessel permeability. This possi- bility warrants further investigation in future preclinical studies with more quantitative evaluation of BBB integ- rity using T1-weighted MRI with gadolinium DOTA con- trast. In the clinic, patients with BM are treated mainly with radiotherapy, and a number of preclinical studies have highlighted the impact of external radiotherapy on the tumor microenvironment and inflammatory pro- cesses in particular.26 Lugade and colleagues showed an increase in VCAM-1 on B16 melanoma tumor vessels in vivo following external radiotherapy.27 Similarly, in the present study we found an increase in vascular VCAM-1 expression 24 h and 72 h following WBRT in tumor ves- sels, but not in healthy brain vessels (Supplementary Fig. 5). On the basis of our findings, we propose that external radiotherapy prior to 212Pb-αVCAM-1 adminis- tration could increase the specificity of our therapeutic approach by upregulating VCAM-1 locally at brain me- tastasis sites. This aspect highlights the importance of evaluating the impact of EBRT and targeted radionuclide therapy and the schedule of each treatment.9,28 Further studies are necessary with regard to the combination and the dose escalation of external radiotherapy and 212Pb-αVCAM-1 in a dose escalation manner, in particular with regard to balancing healthy tissue preservation and tumor control. Combining WBRT with 212Pb-αVCAM-1 shows great promise; however, additional studies are required to optimize the dose prescription of the com- bined treatments to reduce the toxicity observed in the present study significant therapeutic effect and no major toxicity. These findings suggest that early BM, which are normally not ac- cessible to systemic treatment owing to the presence of the BBB, could be controlled using this novel therapeutic approach. on inflammatory processes (eg, microglia/macrophage phenotype). We found no difference in vessel diameter or surface area. Interestingly, it has been shown in a model of glioblastoma with another alpha emitter (225Ac) that tumor vessel permeability and perfusion were altered,25 and we cannot rule this out here. For BM, a change in vessel permeability close to the tumor cells would be of interest in terms of systemic treatment, considering the constitutively low vessel permeability. This possi- bility warrants further investigation in future preclinical studies with more quantitative evaluation of BBB integ- rity using T1-weighted MRI with gadolinium DOTA con- trast. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases 368 alpha-radioimmunotherapy of small volume peritoneal carcinomatosis using 212Pb. PLoS One. 2013;8(7):e69613. References 16. Corroyer-Dulmont A, Pérès EA, Petit E, et al. Noninvasive assess- ment of hypoxia with 3-[18F]-fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([18F]-FMISO): a PET study in two experimental models of human glioma. Biol Chem. 2013;394(4):529–539. 1. Ostrom QT, Wright CH, Barnholtz-Sloan JS. Brain metastases: epidemi- ology. Handb Clin Neurol. 2018;149:27–42. 17. Perera M, Ribot EJ, Percy DB, et al. In vivo magnetic resonance imaging for investigating the development and distribution of experimental brain metastases due to breast cancer. Transl Oncol. 2012;5(3):217–225. 2. Cheng X, Hung MC. Breast cancer brain metastases. Cancer Metastasis Rev. 2007;26(3-4):635–643. 3. Robinson AG, Young K, Balchin K, Ashworth A, Owen T. Causes of death and subsequent treatment after initial radical or palliative therapy of stage III non-small cell lung cancer. Curr Oncol. 2015;22(5):333. 18. Jan S, Santin G, Strul D, et al. GATE: a simulation toolkit for PET and SPECT. Phys Med Biol. 2004;49(19):4543–4561. 19. Pérès EA, Etienne O, Grigis A, Boumezbeur F, Boussin FD, Le Bihan D. Longitudinal study of irradiation-induced brain microstructural alter- ations with S-index, a diffusion MRI biomarker, and MR spectroscopy. Int J Radiat Oncol Biol Phys. 2018;102(4):1244–1254. 4. Andrews DW, Scott CB, Sperduto PW, et al. Whole brain radiation therapy with or without stereotactic radiosurgery boost for patients with one to three brain metastases: phase III results of the RTOG 9508 ran- domised trial. Lancet. 2004;363(9422):1665–1672. 20. Daphu I, Sundstrøm T, Horn S, et al. In vivo animal models for studying brain metastasis: value and limitations. Clin Exp Metastasis. 2013;30(5):695–710. 5. Brown PD, Jaeckle K, Ballman KV, et al. Effect of radiosurgery alone vs radiosurgery with whole brain radiation therapy on cognitive function in patients with 1 to 3 brain metastases: a randomized clinical trial. JAMA. 2016;316(4):401–409. 21. Poli GL, Bianchi C, Virotta G, et al. Radretumab radioimmunotherapy in patients with brain metastasis: a 124I-L19SIP dosimetric PET study. Cancer Immunol Res. 2013;1(2):134–143. 6. Serres S, Soto MS, Hamilton A, et al. Molecular MRI enables early and sensitive detection of brain metastases. Proc Natl Acad Sci U S A. 2012;109(17):6674–6679. 22. Asaithamby A, Chen DJ. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation. Mutat Res. 2011;711(1-2):87–99. 7. Soto MS, Serres S, Anthony DC, Sibson NR. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Functional role of endothe- lial adhesion molecules in the early stages of brain metastasis. Neuro Oncol. 2014;16(4):540–551. 23. Behling K, Maguire WF, Di Gialleonardo V, et al. Remodeling the vas- cular microenvironment of glioblastoma with α-particles. J Nucl Med. 2016;57(11):1771–1777. 8. Cheng VWT, Soto MS, Khrapitchev AA, et al. VCAM-1-targeted MRI en- ables detection of brain micrometastases from different primary tumors. Clin Cancer Res. 2019;25(2):533–543. 24. Tan Z, Chen P, Schneider N, et al. Significant systemic therapeutic ef- fects of high-LET immunoradiation by 212Pb-trastuzumab against pros- tatic tumors of androgen-independent human prostate cancer in mice. Int J Oncol. 2012;40(6):1881–1888. 9. Gill MR, Falzone N, Du Y, Vallis KA. Targeted radionuclide therapy in combined-modality regimens. Lancet Oncol. 2017;18(7):e414–e423.i 25. Sattiraju A, Xiong X, Pandya DN, et al. Alpha particle enhanced blood brain/tumor barrier permeabilization in glioblastomas using integrin alpha-v beta-3-targeted liposomes. Mol Cancer Ther. 2017;16(10):2191–2200. 10. Maffioli L, Florimonte L, Costa DC, et al. New radiopharmaceutical agents for the treatment of castration-resistant prostate cancer. Q J Nucl Med Mol Imaging. 2015;59(4):420–438. 11. Ménager J, Gorin JB, Fichou N, et al. [Alpha-radioimmunotherapy: principle and relevance in anti-tumor immunity]. Med Sci (Paris). 2016;32(4):362–369. 26. Arnold KM, Flynn NJ, Raben A, et al. The impact of radiation on the tumor microenvironment: effect of dose and fractionation schedules. Cancer Growth Metastasis. 2018;11:1179064418761639. 12. Falzone N, Ackerman NL, Rosales LF, et al. Dosimetric evaluation of radionuclides for VCAM-1-targeted radionuclide therapy of early brain metastases. Theranostics. 2018;8(1):292–303. 27. Lugade AA, Sorensen EW, Gerber SA, Moran JP, Frelinger JG, Lord EM. Radiation-induced IFN-gamma production within the tumor microenviron- ment influences antitumor immunity. J Immunol. 2008;180(5):3132–3139. 13. Corroyer-Dulmont A, Pérès EA, Gérault AN, et al. Multimodal imaging based on MRI and PET reveals [(18)F]FLT PET as a specific and early in- dicator of treatment efficacy in a preclinical model of recurrent glioblas- toma. Eur J Nucl Med Mol Imaging. 2016;43(4):682–694. 28. Corroyer-Dulmont A, Falzone N, Kersemans V, et al. Improved outcome of 131I-mIBG treatment through combination with external beam ra- diotherapy in the SK-N-SH mouse model of neuroblastoma. Radiother Oncol. 2017;124(3):488–495. 14. Alexander AL, Lee JE, Lazar M, Field AS. Diffusion tensor imaging of the brain. Neurotherapeutics. 2007;4(3):316–329. 29. Schlesinger M, Bendas G. Vascular cell adhesion molecule-1 (VCAM- 1)—an increasing insight into its role in tumorigenicity and metastasis. Int J Cancer. 2015;136(11):2504–2514. 15. Boudousq V, Bobyk L, Busson M, et al. Acknowledgment We thank Dr Patricia S. Steeg of the National Cancer Institute for providing the MDA-MD-231Br cell line and Orano Med (France) for providing the 212Pb. Corroyer-Dulmont et al. Targeted α-particle therapy for brain metastases Comparison between intern- alizing anti-HER2 mAbs and non-internalizing anti-CEA mAbs in
https://openalex.org/W2755224844	https://journal.ugm.ac.id/jcef/article/download/26507/17260	English	null	Hydraulic and Hydrologic Modeling of Steep Channel of Putih River, Magelang District, Central Java Province, Indonesia	Journal of the civil engineering forum	2,017	cc-by-sa	6,581	3.1 Watershed A watershed is an area of land draining into a stream at a given location. One of the watershed morphology that reflects on flow discharge is watershed hydrologic length and slope. Hydrologic length is the length of the main stream from the outlet to the watershed boundary. Hydrologic length reflects on time behavior of discharge. Watershed slope is the total elevation change of the main stream divided by hydrologic length. The watershed slope is an important factor in that affects runoff momentum. The longitudinal profile of the river, which is extracted from topographic maps, can be used to divide stream reaches into slope categorization that reflects profile morphology as listed in Table 1(Rosgen, 1994). Hydraulic and Hydrologic Modeling of Steep Channel of Putih River, Magelang District, Central Java Province, Indonesia Adi Putri Anisa Widowati Master of Engineering in Natural Disaster Management, Universitas Gadjah Mada, INDONESIA adi.putri.a@mail.ugm.ac.id 1 INTRODUCTION The development of Geographic Information System (GIS) has a significant impact on hydrologic and hydraulic modeling. As GIS-based hydrologic and hydraulic data are now widely available, many recent hydrologic and hydraulic models have been developed based on GIS. Lahar flood occur regularly in Putih River, Central Java Province. Since the upstream is located near the peak of active volcano Merapi, Putih River has a huge amount of lahar producing sediment materials on its upstream. Located on the steep upstream, the sediment materials, when saturated by rainfall, have a high probability to produce destructive lahar flood threatening the populated downstream area. This research is aimed to assess the lahar flood in Putih River by applying a whole modeling process ranging from hydrologic modeling to hydraulic modeling. Miyata, et al. (2014) examined the effects of intensive local rainfall on flashflood events using a grid based distributed hydrological model. The hydrology model is able to both evaluate the basin response due to rainfall, in form of flow, and to identify contributions of each sub-basins to the quickly rising stream discharge. ABSTRACT Hydrologic and hydraulic modeling are important to be conducted to examine the watershed response based on a rainfall input, especially over disaster-prone watershed such as Putih River watershed in Magelang, Central Java Province. A GIS-based grid- based distributed rainfall-runoff model was used to simulate the rainfall-runoff transformation. A two-dimensional hydrodynamic flow modeling was then carried out to simulate the flood processes on the stream and floodplain area. A sensitivity analysis was conducted on infiltration rate, Manning’s n value, and rainfall intensity. Infiltration rate, Manning’s n value, and rainfall intensity give considerable effects to the resulted flow hydrographs. The modeling results show that the results of hydrologic-hydraulic modeling is in good agreement with the observed results. Keywords: Hydrologic-hydraulic model, rainfall-runoff model, two-dimensional hydrodynamic model peak discharge conforming to the field observed data, and also show that the model can be used to evaluate the effect of change in land cover and soil on the stream flow due to human activity. Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum 3.4 Hydrologic – Hydraulic Model Precipitation contributes to various storage and flow processes. The precipitation which becomes streamflow may reach the stream by overland flow, subsurface flow, or both (Chow, et al., 1988). Precipitation contributes to various storage and flow processes. The precipitation which becomes streamflow may reach the stream by overland flow, subsurface flow, or both (Chow, et al., 1988). The passage of overland flow into a channel can be viewed as a lateral flow. A discharge of the overland flow, q0 per unit width, passes into the channel, with the length of the channel, Lc, so the discharge in the channel is Q = q0 Lc. To find the depth and velocity at various points along the channel, an iterative solution of Manning’s equation is necessary. Figure 1. The design hyetograph derived by Alternating Block Method (ABM). In Japan, the equation to calculate rainfall intensity for any duration based on daily rainfall depth is as follow (Mori, et al., 2003): 2 / 1 3 / 2 1 S R n u  (2) (2) 3 / 2 24 24 24             t R it (1) (1) where u is flow velocity (m/s), n is Manning’s roughness coefficient, R is hydraulic radius (m), and S is energy line gradient. where it is rainfall intensity for any duration t (mm/hour), R24 is daily rainfall depth (mm), and t is rainfall duration (hour). Infiltration is the process of water penetrating from the ground surface into the soil. Many factors influence the infiltration rate, including the condition of the soil surface and its vegetative cover, the properties of the soil, such as porosity and hydraulic conductivity, and the current moisture content of the soil (Chow, et al., 1988). Infiltration is the process of water penetrating from the ground surface into the soil. Many factors influence the infiltration rate, including the condition of the soil surface and its vegetative cover, the properties of the soil, such as porosity and hydraulic conductivity, and the current moisture content of the soil (Chow, et al., 1988). 2 HYDROLOGY AND HYDRAULIC MODEL Zhang and Savenije (2005) simulated saturation overland flow by relating the variable source area to both the topography and groundwater level. Koren, et al. (2005) evaluated the performance of a grid-based distributed hydrology model when transforming rainfall to stream flow which resulted in runoff volumes that was in good agreement with the observed data. Liu, et al. (2009) modeled runoff volume and peak discharge based on several flood events using grid-based distributed kinematic hydrological model with rainfall data derived from climate radar. The research was able to reproduce 125 Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum Table 1. Categorization of river bed-slope steepness (Rosgen, 1994) Slope Categorization < 4 % (2.3°) Gentle 4% - 10% (2.3°- 5.7°) Steep >10% (5.7°) Very steep 3 2 Design Hyetograph Table 1. Categorization of river bed-slope steepness (Rosgen, 1994) input which is different to another depending on several climatic, topographic, and geological factors. Figure 2. The flow hydrograph at a basin outlet during a rainfall-runoff simulation by a particular observed rainfall event Peak discharge Rainfall Flow Flow rate Time Rainfall Lag time Peak discharge Rainfall Flow Flow rate Ti Lag time Rainfall 3.2 Design Hyetograph Flow rate Hyetograph is a plot of incremental rainfall intensity against time interval. There are several methods to develop hypothetical rainfall distribution, one of which is Alternating Block Method (Chow, et al., 1988). The general shape of design hyetograph by ABM is shown in Figure 1. Time Figure 2. The flow hydrograph at a basin outlet during a rainfall-runoff simulation by a particular observed rainfall event Figure 1. The design hyetograph derived by Alternating Block Method (ABM). Rainfall intensity Rainfall duration Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum B in A in A A q q x q t h         (5) B in A in A A q q x q t h         (5) A A A A h K i q  (6) The scheme of hydrology model in hillslope domain is shown by Figure 2. In each grid, there are 3 layers on which runoff flow, sub-surface flow, and vertical flow between layers are calculated. The relation of a grid with its 8 surrounding grids is defined by flow direction. Flow direction of a grid to adjacent grid is determined by a grid whose elevation level is the lowest among those 8 surrounding grids. (5) t A A A A h K i q  (6) A A A A h K i q  (6) where qin_A is infiltration flow from surface to the layer A, qin_B is infiltration flow from layer A to layer B, iA is hydraulic gradient of flow in layer A, and KA is permeability coefficient of layer A. The calculation of lateral flow discharge and flow depth in layer B uses the equation same as of the layer A (Miyata, et al., 2014). Figure 3. The illustration of hydrologic model scheme on hillslope domain (Miyata, et al., 2014) Furthermore, the calculation of discharge on stream domain, qCH, uses the same kinematic wave model which is being used in flow model in hillslope domain. The composition of stream grid is carried out as a series of cross sections with lateral coming from adjacent right and left grids. The following equation is used to calculate the flow in stream domain: Figure 3. The illustration of hydrologic model scheme on hillslope domain (Miyata, et al., 2014) L r x q t h CH CH        (7) 3 5 CH M CH CH h n i q  (8) The illustration (Figure 3) explains the process of rainfall dropping on a surface of a hillslope grid which infiltrates to the layer A. The saturated flow of layer A then percolates to the layer B. Journal of the Civil Engineering Forum The deeper percolation of flow on layer B to the deeper layer could occur, yet percolation flow to the deeper layer is assumed not flowing back to the slope or stream. (7) (8) where r is rainfall on a stream grid, L is lateral flows from left and right adjacent grid, iCH is hydraulic gradient of stream flow, and nM is Manning’s roughness coefficient. The hydrologic model calculates both runoff flow and lateral flow based on Manning equation and Darcy’s law. The runoff flow calculation takes into account the water balance from rainfall, flow from adjacent upstream grid, and flow to adjacent downstream grid, infiltration flow to the lower layer, and evaporation. The relation between depth of surface runoff flow, hOF, and runoff discharge per unit width, qOF, is represented in the following equations: The hydrologic model calculates both runoff flow and lateral flow based on Manning equation and Darcy’s law. The runoff flow calculation takes into account the water balance from rainfall, flow from adjacent upstream grid, and flow to adjacent downstream grid, infiltration flow to the lower layer, and evaporation. 3.3 Hydrograph A streamflow or discharge hydrograph is a graph or table showing the flow rate as a function of time at a given location on the stream. Figure 2 shows components of a streamflow hydrograph during a storm (Chow, et al., 1988). The rising limb is the limb that has a positive gradient indicating the rising of discharge (point A to B). The recession limb is the limb that has a negative gradient indicating the falling of discharge (point B to C). Point C to D represents baseflow recession. Lag time between peak rainfall and peak discharge indicates the time required for a surface runoff flow to reach an outlet point where the hydrograph is observed. The shape of the hydrograph describes the response of a basin toward a rainfall Hydrologic-hydraulic model is used to model rainfall- runoff transformation. The output of the model is flow hydrograph at a particular basin outlet. The model is a grid-based distributed model and consists of two domains. The first domain is hillslope and the second one is stream. The calculation in hillslope domain includes water balance calculation and flow routing on hillslope, while the calculation in stream domain consists of channel flow routing. 126 Vol. 3 No. 3 (September 2017) b) Rainfall and water level data b) Rainfall and water level data Hourly rainfall data for Putih River watershed is obtained from Yogyakarta Sabo Center. Hourly rainfall data for Putih River watershed is obtained from Yogyakarta Sabo Center. Hourly rainfall data for Putih River watershed is obtained from Yogyakarta Sabo Center. 4.2 Data Availability by y H gh y vM x uM t M                 ) ( ) ( (12) (12) 4.1 Location of Study Several physical parameters for hydrologic-hydraulic simulation such as Manning coefficient and properties of bed material are collected from previous researches, namely Widowati (2015), and Musthofa (2014). The research was carried out on Putih River watershed in Magelang District, Central Java Province. It has upstream end on the peak of Merapi Volcano. It flows downward the Merapi slope to the southwest direction until it intercepts with Blongkeng River on the downstream end. The boundary of Putih River watershed is shown by Figure 4. 3.5 Two-Dimensional Hydrodynamic Model SIMLAR v1.0 The relation between depth of surface runoff flow, hOF, and runoff discharge per unit width, qOF, is represented in the following equations: SIMLAR is developed by Yogyakarta Sabo Centre in cooperation with Universitas Gadjah Mada (Hardjosuwarno, et al., 2012). The hydrodynamic model is used to model flow along a particular stream or channel. The flow which is modeled using SIMLAR is of debris flow type. The debris flow is calculated as a fluid unity of debris flow and suspended sediments. Some equations used in the model are described as follows. r x q t h OF OF       (3) 3 / 5 OF M OF OF h n i q  (4) (3) (4) To calculate the permanent flow velocity at a uniform channel cross section, Manning equation is used. Based on flow velocity from the Manning equation, flow discharge at a particular channel cross section is calculated using the following equation. where r is intensity of effective rainfall (rainfall – infiltration), iOF is the energy line gradient (approached by surface slope gradient), and nM is Manning’s roughness coefficient (Miyata, et al., 2014). uA Q  (9) uA Q  (9) Sub-surface lateral flows occur between layer A and layer B. The equation to calculate lateral discharge per unit width, qA, and the flow depth, hA, in layer A, are: where Q is flow discharge (m3/s), and A is flow area of a channel cross section (m2). where Q is flow discharge (m3/s), and A is flow area of a channel cross section (m2). 127 Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Figure 4. Location of study area in Putih River watershed For a non-permanent and non-uniform flow, the debris equations are developed considering the mass conservation principles and force and momentum conservation principles. a) Topography data Topography data for Putih River is LiDAR DEM of 20 m resolution which is obtained from Yogyakarta Sabo Center Topography data for Putih River is LiDAR DEM of 20 m resolution which is obtained from Yogyakarta Sabo Center Topography data for Putih River is LiDAR DEM of 20 m resolution which is obtained from Yogyakarta Sabo Center where H is depth of flow (m), M is flow discharge per unit width at x direction (m2/s), N is flow discharge per unit width at y direction (m2/s), g is gravity acceleration (m2/s), and τ is shear stress. b) Rainfall and water level data 3.5 Two-Dimensional Hydrodynamic Model SIMLAR v1.0 Mass conservation equation for two-dimensional flow is: 0          y N x M t H (10) (10) Force and momentum equation for two-dimensional flow is: At x direction: At x direction:     bx x H gh y vN x uN t N                 (11) At y direction: by y H gh y vM x uM t M                 ) ( ) ( (12)     bx x H gh y vN x uN t N                 (11) (11) Figure 4. Location of study area in Putih River watershed At y direction: 5.1 Delineation of Putih River watershed 5.1 Delineation of Putih River watershed The result of the watershed delineation gives the total area of Putih River watershed (Figure 5) as 23.011 km2 with a total length of the main river of 21.639 km. The sub-watershed has an area of 8.432 km2. The sub watershed of Putih River has riverbed slope ranging from 3.0° to 7.6°. Figure 6. Rainfall event of December 8th 2010 in sensitivity analysis in Putih River. Figure 5. The delineation of Putih River watershed and sub- watershed. Outlet point of sub- watershed Figure 6. Rainfall event of December 8th 2010 in sensitivity analysis in Putih River. Figure 7. The scheme of stream and watershed used in rainfall-runoff modeling in Putih River. Outlet point of sub- watershed Outlet point of sub- watershed Figure 5. The delineation of Putih River watershed and sub- watershed. 4.3 Research Tools Several basic data processing applications such as Microsoft Word, Microsoft Excel, Notepad++ are used to process the data and conduct the whole simulation. The GIS data preparation is done using ArcMap 10.1, QGIS and GRASSGIS application. The hydrology simulation of transforming rainfall-runoff is carried out using the grid-based distributed hydrology model (Miyata, 2014). The flood hydrodynamic simulation is conducted using 2 dimensional hydrodynamic model SIMLAR (Hardjosuwarno, et al., 2012). 128 Journal of the Civil Engineering Forum Journal of the Civil Engineering Forum Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) 5 RESULTS AND DISCUSSIONS The area of Putih River inflow sub-basin is 8.43 km2. Figure 7 presents the scheme of slope and stream domain of the simulation. Figure 7 presents the scheme of slope and stream domain of the simulation. Figure 6. Rainfall event of December 8th 2010 in sensitivity analysis in Putih River. Figure 7. The scheme of stream and watershed used in rainfall-runoff modeling in Putih River. 5.2 Rainfall – Runoff Modeling The rainfall-runoff modeling is conducted using kinematic distributed model. The final results are flow hydrograph on the outflow point. A sensitivity analysis was conducted on infiltration rate, Manning’s n value, and rainfall intensity. The rainfall event used during the simulation of sensitivity analysis is the rainfall event on December 8th 2010 recorded at Ngepos station (Figure 6). Figure 7. The scheme of stream and watershed used in rainfall-runoff modeling in Putih River. 5.2.1 Sensitivity analysis on infiltration rate In the hydrograph with high infiltration rate (f = 5.0 mm/hour), this additional rising part of hydrograph is not observed because the flow is already being infiltrated into the soil along its path to the stream. Small infiltration rate also gives flow a chance to accumulate faster, thus hydrograph of small infiltration rate rises earlier. Table 2. Peak discharge (Q) and and peak time (tp) of simulated hydrographs in sensitivity analysis of infiltration rate (f) in Putih River. f (mm/hour) Peak 1 Peak 2 Q (m3/s) t (min) Q (m3/s) t (min) 0.6 2.395 85 3.403 185 1.2 1.190 90 2.736 195 2.2 1.813 90 1.812 210 3.6 1.333 95 0.850 250 5.0 0.910 95 0.286 325 Table 3. Peak discharge (Q) and peak time (tp) of simulated hydrographs in sensitivity analysis of infiltration rate (f) in Putih River. n Peak 1 Peak 2 Q (m3/s) tp (min) Q (m3/s) tp (min) 0.3 1.981 90 1.751 145 0.4 1.433 90 1.062 185 0.5 1.183 90 0.676 225 0.6 1.103 95 0.438 275 0.7 0.910 95 0.286 325 0.8 0.816 100 0.186 390 High infiltration rate enables more surface f low to infiltrate into the soil and thus result in low surface runoff discharges. High infiltration rate also diminishes the discharge of flows from far-grids that enter the stream at a later time which is indicated by later peaks in the hydrographs of small infiltration rate. The later rising parts of the hydrographs of small infiltration rate (f = 0.6, 1.2, and 2.2 mm/hour) appear due to the flow from slope-grids located in farther distance from the stream that just enter the stream at a later time. In the hydrograph with high infiltration rate (f = 5.0 mm/hour), this additional rising part of hydrograph is not observed because the flow is already being infiltrated into the soil along its path to the stream. Small infiltration rate also gives flow a chance to accumulate faster, thus hydrograph of small infiltration rate rises earlier. For a given rainfall intensity and infiltration coefficient, smaller Manning’s slope n values results in higher flow velocity than higher n values. High flow velocity also gives effect in reducing the chance of the flow to be infiltrated into the soil. Thus, smaller n values produce greater and faster flow discharge entering the stream than higher n values. 5.2.1 Sensitivity analysis on infiltration rate The sensistivity analysis used a constant slope Manning’s n = 0.7 and a constant channel Manning’s n = 0.03. Figure 8 shows the results of sensitivity analysis on infiltration rate in Putih River upstream watershed. Table 2 provides the values of the resulted peak discharge and peak time. The simulated watershed boundary in Putih River covers Putih River upstream sub-basin with an outlet point located at approximately upstream of PUD-02. Figure 8. Simulated channel flow discharge hydrographs at outlet point using different values of infiltration rate in Putih River. 36,5 0 20 40 60 80 0 1 2 3 4 0 50 100 150 200 250 300 350 400 450 500 550 Rainfall intensity (mm/hr) Discharge (m3/s) Simulation time (minute) Rainfall Dec 8th 2010 f = 0.6 mm/hour f = 1.2 mm/hour f = 2.2 mm/hour f = 3.6 mm/hour f = 5.0 mm/hour Figure 8. Simulated channel flow discharge hydrographs at outlet point using different values of infiltration rate in Putih River. 129 Vol. 3 No. 3 (September 2017) Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum Thus, it was taken the Manning’s n values ranging from 0.3 to 0.8 for sensitivity analysis of Manning’s n value. The result of sensitivity analysis of Manning’s n values is shown by Figure 9. Table 3 provides the values of the resulted peak discharge and peak time. Table 2. Peak discharge (Q) and and peak time (tp) of simulated hydrographs in sensitivity analysis of infiltration rate (f) in Putih River. f (mm/hour) Peak 1 Peak 2 Q (m3/s) t (min) Q (m3/s) t (min) 0.6 2.395 85 3.403 185 1.2 1.190 90 2.736 195 2.2 1.813 90 1.812 210 3.6 1.333 95 0.850 250 5.0 0.910 95 0.286 325 High infiltration rate enables more surface f low to infiltrate into the soil and thus result in low surface runoff discharges. High infiltration rate also diminishes the discharge of flows from far-grids that enter the stream at a later time which is indicated by later peaks in the hydrographs of small infiltration rate. The later rising parts of the hydrographs of small infiltration rate (f = 0.6, 1.2, and 2.2 mm/hour) appear due to the flow from slope-grids located in farther distance from the stream that just enter the stream at a later time. 5.2.1 Sensitivity analysis on infiltration rate In addition, small n value which results in high flow velocity is correlated with a faster draining time. Flow with high velocity is easier to be drained out of slopes than slow flow, thus makes it quicker for the flow both to form the peak discharge and to decline. So, small n value makes the hydrographs sharper: shorter duration to reach the peak discharge, higher peak discharge rate, and steeper recession limb. 5.2.3 Modeling with variation of rainfall intensity 5.2.3 Modeling with variation of rainfall intensity boundary of the modeling is the upstream of PU-C0 Sukowati. The inflow hydrograph (Figure 11) was obtained from rainfall-runoff modeling in Putih River upstream sub-basin of January 9th 2011 rainfall event from Ngepos ARR station, using infiltration rate of f = 5 mm/hour and Manning’s slope n value of n = 0.7. The hydrodynamic modeling of debris flow in Putih River was conducted for 210 minutes. The design hyetograph was derived using a modified Mononobe equation and distributed using Alternating Block Method (ABM) technique. The results of modeling with variation of rainfall intensity in Putih River is shown in Figure 10. Although the infiltration rate and Manning’s slope n value are set constant of f = 5 mm/hour and n = 0.7 respectively, each rainfall produces different shape of hydrograph. Figure 11. Inflow hydrograph for Putih River hydrodynamic modeling. 0 30 60 90 0 3 6 9 0 200 400 600 Rainfall intensity (mm/hour) Discharge (m3/s) Time (minute) Rainfall January 9th 2011 Simulated flow hydrograph The hypothetical rainfall of P2-hours = 10 mm produces a very low hydrograph with maximum peak discharge of 0.7 m3/s at t = 215 minute. The hypothetical rainfall P2-hours = 15 mm produces a terraced hydrograph with a discharge rising at t = 180 min and a peak discharge rate of 3,541 m3/s at t = 285 minute. The hypothetical rainfall P2-hours = 20 mm starts to rise at t = 155 minutes and reaches maximum discharge rate 9,277 m3/s at t = 245 minutes. The hypothetical rainfall P2- hours = 25 mm starts to rise at t = 145 minutes and reaches maximum discharge rate 15.99 m3/s at t = 225 minute. Figure 11. Inflow hydrograph for Putih River hydrodynamic modeling. The result of hydrodynamic lahar modeling is a spread of water and sediment inundation on the simulated river and riverbank. Figure 12 shows the simulated sediment propagation at t = 60, 120, 180, and 210 minutes of simulation time layered over an aerial image of Putih River watershed. It is observed that sediment of debris flow is still concentrated around the inflow point at t = 60 minutes. At t = 120 minutes, the sediment flow reaches PU-RD5. The sediment flow reaches PU-C2 (Gempol), downstream of Magelang public road, at t = 180 minutes. The hydrographs have different shape one another. 5.2.2 Sensitivity analysis on Manning’s slope n values Referring to the Manning’s n value from literature study (Engman, 1986), the surface condition in Putih River is considered ranging from grass to woods. Figure 9. Simulated channel flow discharge hydrographs at outlet point by different Manning’s slope n values in Putih River. 36,5 0 20 40 60 80 0 1 2 3 0 50 100 150 200 250 300 350 400 450 500 550 600 Rainfall intensity (mm/hr) Discharge (m3/s) Simulation time (minute) Rain Dec 8th 2010 n = 0.3 n = 0.4 n = 0.5 n = 0.6 n = 0.7 n = 0.8 Figure 9. Simulated channel flow discharge hydrographs at outlet point by different Manning’s slope n values in Putih River. gure 9. Simulated channel flow discharge hydrographs at outlet point by different Manning’s slope n value 130 Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Figure 10. Comparison of the simulated channel flow hydrographs at outlet point by different hypothetical rainfalls in Putih River. 0 5 10 15 20 0 50 100 150 200 250 300 350 400 450 500 550 Discharge (m3/s) Simulation time (minute) P-2hour = 25 mm P-2hour = 20 mm P-2hour = 15 mm P-2hour = 10 mm Figure 10. Comparison of the simulated channel flow hydrographs at outlet point by different hypothetical rainfalls in Putih River. 5.2.3 Modeling with variation of rainfall intensity The hydrographs of higher rainfall depth rise earlier have higher discharge. The maximum discharge also occurs earlier in the hydrographs of higher rainfall. By the specified rainfall distribution, the maximum discharge occurs after the rain stops. The rainfall intensity affects the flow discharge greatly, and produces hydrographs of rainfall intensity distinctly. 5.3 Hydrodynamic Modeling The hydrodynamic debris flow modeling was carried out on Putih River basin. The inflow point is assigned at the downstream of PUD-02. The downstream 131 Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum Figure 12. Simulated sediment spread of debris flow in Putih River at t = 210 minutes Figure 12. Simulated sediment spread of debris flow in Putih River at t = 210 minutes Figure 13. Sediment inundation near Magelang public road. Based on the simulation results of debris flow modeling until t = 210 minutes, the overtopping of water and sediment to the river side is not visually observed. Meanwhile, the velocity of water flow and sediment flow from simulation result is still too low compared to the field event. From the recorded event, when a lahar was triggered by a rainfall in the upstream of Putih River, the lahar flow reached the downstream near Magelang road within 1 hour (60 minutes). However, the simulation results show that the travel of water and sediment took 2 hour (120 minutes) to arrive at the downstream area near Magelang road. One of the public facility vulnerable toward Puti River debris flow is Magelang public road locate upstream of PU-C2 (Gempol). An image of sedimen inundation near Magelang road is shown by Figur 13, in which two courses of Putih River reach ar described, one is the old stream course before debri flow of Merapi 2010 eruption which is represented b a dashed line, and another one is the new stream course after the debris flow of Merapi 2010 eruptio represented by a continuous line. According to the field report of debris flow occurrence prior to Merapi eruption in Octobe Sabo structure Stream (after 2010) Stream (before 2010) Magelang Public Road Sediment t =210 min Value 2 m -2 m Legend Figure 13. Sediment inundation near Magelang public road. Sabo structure Stream (after 2010) Stream (before 2010) Magelang Public Road Sediment t =210 min Value 2 m -2 m Legend Figure 13. Sediment inundation near Magelang public road. Sabo structure Stream (after 2010) Stream (before 2010) Magelang Public Road Sediment t =210 min Value 2 m -2 m Legend Figure 13. Sediment inundation near Magelang public road. One of the public facility vulnerable toward Putih River debris flow is Magelang public road located upstream of PU-C2 (Gempol). Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) November 2010, the debris flow in Putih River overtopped to and inundated the Magelang road several times, one of which was the debris flow occurrence at January 9th 2011. The new stream course was formed due to the huge and massive force of the debris flow which overtopped the road. However, the simulated results indicated that debris flow based on rainfall of January 9th 2011 remained through the old stream course. Some part of the flow seemed to start entering the new stream course. It may be caused by the lack of DEM resolution which was 20 m. A hydrodynamic modeling of lahar flow using DEM with smaller resolution which more represents river topography should be conducted to have a more accurate result. At CS 1, it is observed that an erosion occurs with the maximum erosion depth of 0.73 m at t = 210 minutes. The riverbed has been lowered due to the erosion. At CS 2, it is observed that a sediment inundation occurs with the maximum sediment inundation depth of 0.20 m at t = 210 minutes. At CS 3, it is observed that a sediment inundation occurs with the maximum sediment inundation depth of 0.49 m at t = 210 minutes. At CS 4, it is observed that a sediment inundation occurs with the maximum sediment inundation depth of 0.94 m at t = 210 minutes. According to the field event record of lahar flow in Putih River at January 19th 2011 (Delson, 2012; SID Jumoyo, 2015), the lahar flow sediment material inundated the Magelang road with the sediment inundation depth ranging from 2 to 3 meters. Compared to the simulation results, the highest simulated sediment inundation depth around Magelang road is 0.94 m at t = 210 minutes. During the event of January 19th 2011 lahar flow, the lahar flow was also reported to overtop the road and flow into the old stream course. The simulated results also show an indication of the water and sediment flow overtopping the road and starting to flow into the old stream course. However, due to the simulation duration was limited to 210 minutes only, the farther propagation of water and sediment flow could not be observed yet. 5.3 Hydrodynamic Modeling An image of sediment inundation near Magelang road is shown by Figure 13, in which two courses of Putih River reach are described, one is the old stream course before debris flow of Merapi 2010 eruption which is represented by a dashed line, and another one is the new stream course after the debris flow of Merapi 2010 eruption represented by a continuous line. One of the public facility vulnerable toward Putih River debris flow is Magelang public road located upstream of PU-C2 (Gempol). An image of sediment inundation near Magelang road is shown by Figure 13, in which two courses of Putih River reach are described, one is the old stream course before debris flow of Merapi 2010 eruption which is represented by a dashed line, and another one is the new stream course after the debris flow of Merapi 2010 eruption represented by a continuous line. Based on the simulation results of debris flow modeling until t = 210 minutes, the overtopping of water and sediment to the river side is not visually observed. Meanwhile, the velocity of water flow and sediment flow from simulation result is still too low compared to the field event. From the recorded event, when a lahar was triggered by a rainfall in the upstream of Putih River, the lahar flow reached the downstream near Magelang road within 1 hour (60 minutes). However, the simulation results show that the travel of water and sediment took 2 hour (120 minutes) to arrive at the downstream area near Magelang road. According to the field report of debris flow occurrence prior to Merapi eruption in October- 132 Journal of the Civil Engineering Forum Journal of the Civil Engineering Forum Vol. 3 No. 3 (September 2017) Flow Simulation SIMLAR Version 1.0.211], Yogyakarta: Sabo Office. 6.2 Recommendations Of all the research results, there are several recommendation for a better future research: Miyata, S., 2014. Manuals of Simulation Program for GIS Based Rainfall-Runoff Model, Kyoto: DPRI Kyoto University. a) The rainfall-runoff model should be applied on basins with different topography and stream structure, such as basins with finer slope and more stream tributaries. Mori, K., Ishii, H., Somatani, A. & Hatakeyama, A., 2003. Hidrologi untuk Pengairan [Manual on Hydrology]. 9th ed. Jakarta: Paradnya Paramita. b) The radar-derived rainfall data should be applied as rainfall input in order to produce a spatially better result. Musthofa, A., 2014. Simulasi Banjir Bandang untuk Peringatan Dini dan Bahaya, Yogyakarta: Gadjah Mada University. c) A hydrodynamic modeling of lahar flow using DEM with smaller resolution should be conducted to have a more accurate result. Rosgen, D. L., 1994. A classification of natural rivers. Catena, 22(2), pp. 169 - 199. d) A longer duration of hydrodynamic modeling of lahar flow should be conducted to observe the longer debris flow propagation over a stream reach. d) A longer duration of hydrodynamic modeling of lahar flow should be conducted to observe the longer debris flow propagation over a stream reach. Shusuke, M., Masaharu, F., Takuji, T. & Tsujimoto, H., 2014. Flash Flood due to Local and Intensive Rainfall in an Alpine Catchment. Nara, International Sabo Association. Several conclusions of this research are: Several conclusions of this research are: a) Infiltration rate affects the magnitude of flow discharge. The higher infiltration rate, the lower the resulted runoff flow discharge. Infiltration rate also affects the shape and peaks hydrograph Koren, V. et al., 2005. Evaluation of a grid-based distributed model over a large area. BrazIAHS Scientific Assembly. b) Manning’s n value affects the flow velocity. The small Manning’s n value results in fast- accumulated flow and high discharge. Liu, J., Feng, J. & Otache, M. Y., 2009. A grid-based kinematic distributed hydrological model for digital river basin. In Advances in Water Resources and Hydraulic Engineering. Heidelberg ed. Berlin: Springer. c) Rainfall intensity dominantly affects the typical hydrograph, yet, the effect of rainfall intensity toward the hydrograph parameter is different from one rainfall intensity and other intensities. Miyata, S., 2014. Manuals of Simulation Program for GIS Based Rainfall-Runoff Model, Kyoto: Kyoto University. 6.1 Conclusions Hardjosuwarno, S. et al., 2012. Panduan Pengoperasian Program Simulasi 2D Banjir Debris Veris 1.0.2011. Yogyakarta: Balai Sabo Yogyakarta. Journal of the Civil Engineering Forum Four cross-sectional stream profiles (CS 1, CS 2, CS 3, and CS 4) at the Putih River reach which intersects Magelang public road are extracted to observe the effect of sediment erosion and inundation near the vulnerable public road. Figure 14 shows the riverbed changes due to sediment erosion and sediment inundation at the specified cross-sectional stream profiles. The cross-sectional stream profiles are extracted at t = 180 minutes and t = 210 minutes only based on the time the sediment flow has reached the Magelang road. 385 386 387 388 10 30 50 70 Elevation (m) m CS 1 initial bed t = 180 minutes t = 210 minutes Figure 14. Riverbed change due to sediment at CS 1, CS 2, CS 3, and CS 4. 385 386 387 388 10 30 50 70 Elevation (m) m CS 1 initial bed t = 180 minutes t = 210 minutes 382 383 384 385 40 60 80 100 120 Elevation (m) m CS 2 initial bed t = 180 minutes t = 210 minutes 384 385 386 387 10 20 30 40 50 60 Elevation (m) m CS 3 initial bed t = 180 minutes t = 210 minutes 383 384 385 386 5 25 45 65 Elevation (m) m CS 4 initial bed t = 180 minutes t = 210 minutes 382 383 384 385 40 60 80 100 120 Elevation (m) m CS 2 initial bed t = 180 minutes t = 210 minutes 383 384 385 386 5 25 45 65 Elevation (m) m CS 4 initial bed t = 180 minutes t = 210 minutes 384 385 386 387 10 20 30 40 50 60 Elevation (m) m CS 3 initial bed t = 180 minutes t = 210 minutes Figure 14. Riverbed change due to sediment at CS 1, CS 2, CS 3, and CS 4. 133 REFERENCES Chow, V. T., Maidment, D. R. & Mays, L. W., 1988. Applied Hydrology. New York: McGraw-Hill, Inc.. SID Jumoyo, 2015. Sistem Informasi Desa (SID) Jumoyo, Kecamatan Salam, Kabupaten Magelang. [Online] Available at: http://jumoyo-magelang.sid.web.id [Accessed June 2017]. Delson, 2012. Kajian Lahar Hujan Kali Putih Setelah Letusan Gunungapi Merapi 2010 [Study on Lahar at Putih River after Merapi Eruption 2010], Yogyakarta: Faculty of Engineering, Universitaws Gadjah Mada. Widowati, A. A. P., 2015. Pengaruh Modifikasi Resolusi Spasial DEM Terhadap Sebaran Sedimen Hasil Simulasi Aliran Lahar Menggunakan SIMLAR v1.0 pada Sungai Kali Putih [Effect of DEM Resolution Modification on Deposited Sediment using SIMLAR v1.0 in Putih River], Yogyakarta: Undergraduate Thesis, Faculty of Engineering, Universitas Gadjah Mada. Engman, E. T., 1986. Roughness coefficients for routing surface runoff. Journal of Irrigation Drainage Engineering, 112(1), pp. 39-53. Hardjosuwarno, S. et al., 2012. Panduan Pengoperasian Program Simulasi 2D Banjir Debris SIMLAR Veris 1.0.2011 [Guideline fo 2Dr Debris 134
https://openalex.org/W2789758434	https://www.nature.com/articles/s41598-018-23130-x.pdf	English	null	Publisher Correction: Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes	Scientific reports	2,018	cc-by	667	1University of Campinas, Institute of Geosciences, Campinas, 13083-970, Brazil. 2University of Concepción, Institute of Applied Economic Geology, Concepción, Casilla, 160-C, Chile. 3University of Campinas, School of Civil Engineering, Architecture and Urban Design, Campinas, 13083-889, Brazil. 4Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, C1428EHA, Argentina. 5CONICET-Universidad de Buenos Aires, Instituto de Astronomía y Física del Espacio (IAFE), Buenos Aires, C1428ZAA, Argentina. 6University of Colorado, Boulder, 80302, USA. 7Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Potsdam, 14473, Germany. 8André Tosello Institute, Campinas, 13087-010, Brazil. Correspondence and requests for materials should be addressed to A.A.N. (email: Alexey.Novoselov@udec.cl) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Publisher Correction: Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes Alexey A. Novoselov1,2, Dailto Silva1, Jerusa Schneider 3, Ximena Celeste Abrevaya4,5, Michael S. Chaffin6, Paloma Serrano7, Margareth Sugano Navarro1, Maria Josiane Conti8 & Carlos Roberto de Souza Filho1 Correction to: Scientific Reports https://doi.org/10.1038/s41598-017-04161-2, published online 21 June 2017h lished: xx xx xxxx OPEN Publisher Correction: Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes x OPEN Published: xx xx xxxx Alexey A. Novoselov1,2, Dailto Silva1, Jerusa Schneider 3, Ximena Celeste Abrevaya4,5, Michael S. Chaffin6, Paloma Serrano7, Margareth Sugano Navarro1, Maria Josiane Conti8 & Carlos Roberto de Souza Filho1 Correction to: Scientific Reports https://doi.org/10.1038/s41598-017-04161-2, published online 21 June 2017 This Article contains an error in Equation 9. = − + ∑ − z z Affinity (z z ) i 1 2 1i 2i 2 should read: z z Affinity (z z ) i 1 2 1i 2i 2 = − = ∑ − = − + ∑ − z z Affinity (z z ) i 1 2 1i 2i 2 should read: z z Affinity (z z ) i 1 2 1i 2i 2 = − = ∑ − Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 1University of Campinas, Institute of Geosciences, Campinas, 13083-970, Brazil. 2University of Concepción, Institute of Applied Economic Geology, Concepción, Casilla, 160-C, Chile. 3University of Campinas, School of Civil Engineering, Architecture and Urban Design, Campinas, 13083-889, Brazil. 4Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, C1428EHA, Argentina. 5CONICET-Universidad de Buenos Aires, Instituto de Astronomía y Física del Espacio (IAFE), Buenos Aires, C1428ZAA, Argentina. 6University of Colorado, Boulder, 80302, USA. 7Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Potsdam, 14473, Germany. 8André Tosello Institute, Campinas, 13087-010, Brazil. Correspondence and requests for materials should be addressed to A.A.N. (email: Alexey.Novoselov@udec.cl) 1University of Campinas, Institute of Geosciences, Campinas, 13083-970, Brazil. 2University of Concepción, Institute of Applied Economic Geology, Concepción, Casilla, 160-C, Chile. 3University of Campinas, School of Civil Engineering, Architecture and Urban Design, Campinas, 13083-889, Brazil. Publisher Correction: Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes x OPEN 4Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, C1428EHA, Argentina. 5CONICET-Universidad de Buenos Aires, Instituto de Astronomía y Física del Espacio (IAFE), Buenos Aires, C1428ZAA, Argentina. 6University of Colorado, Boulder, 80302, USA. 7Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Potsdam, 14473, Germany. 8André Tosello Institute, Campinas, 13087-010, Brazil. Correspondence and requests for materials should be addressed to A.A.N. (email: Alexey.Novoselov@udec.cl) SCIentIfIC REPOrtS \| (2018) 8:4790 \| DOI:10.1038/s41598-018-23130-x SCIentIfIC REPOrtS \| (2018) 8:4790 \| DOI:10.1038/s41598-018-23130-x 1
https://openalex.org/W4286274224	https://zenodo.org/records/6868836/files/Accuracy%20assessment%20of%20marker%20recognition%20using%20ultra%20wide%20angle%20camera.pdf	English	null	Accuracy assessment of marker recognition using ultra wide angle camera	Technology audit and production reserves	2,022	cc-by	4,656	1. Introduction to the no need to use additional equipment, its comfortable ergonomics, wide range of provided features, and variety of tools for software development for this device [3]. Despite all the advantages, Microsoft HoloLens 2 has limitations. Both design flaws of device and drawbacks of AR tech nology itself make the Microsoft HoloLens 2 ineffective to be applicable in certain scenarios. The modern state of augmented reality (AR) technology allows it usage in various fields of human activity, including medicine. AR makes it possible to interact with virtual objects in the real world. In this way, AR technology is more attractive compare to virtual reality when used in applications for medical visualization [1]. Tracking method used in Microsoft HoloLens 2 is inside- out tracking. Only those cameras and sensors that are built into the device itself provide information on the orienta tion of the device in the real world. In order to provide update of spatial relationships of virtual and real objects, such method needs information from the tracking of fidu cial markers attached in the real scene. Fiducial markers provide unambiguous identification of object position and pose in three-dimensional space [4, 5]. The most widely used cases of AR technology in medi cine are the visualization of the patient’s state indicators during examination and the visualization of surgical in strument positions during surgery. For mentioned cases, the output can be provided into medical card helmet, interactive MRI scan, and any other device with AR technology on board. Conventional smart phone, tablet, and even computers with plugged camera can be used as AR devices to superimpose virtual objects onto physical objects in real space. However, the most convenient in case of visualization for medical purposes is usage of special helmets or head mounted displays. They do not take up hands and the imposition of information on the user’s field of view (FoV) allows users to immerse fully in the process of interaction with augmented objects [2, 3]. According to the manufacturer information, the mono scopic camera of Microsoft HoloLens 2 covers 52 degrees [6], while the FoV of user, excluding the rotation of the eyes and peripheral vision, is 114 degrees [7]. In practice, us age of mentioned monoscopic camera results in a small area beyond which interaction with virtual objects are not possible. INFORMATION TECHNOLOGIES INFORMATION TECHNOLOGIES UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» UDC 004.932:616-073.756.8 DOI: 10.15587/2706-5448.2022.259068 Article type «Reports on Research Projects» How to cite Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 ACCURACY ASSESSMENT OF MARKER RECOGNITION USING ULTRA WIDE ANGLE CAMERA Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and failures referred to the distortion occurs are available in less than 0.2 % of all cases. This gives a possibility to increase workspace for interaction with markers compare to the Microsoft HoloLens 2. At the same time, the workspace for interaction could not reach the actual view space of the camera since recognition fails in cases where marker’s body is partially visible in the captured image (i. e., marker position is at the image boundaries). Keywords: augmented reality, marker recognition, ArUco fiducial markers, recognition accuracy, surgical navi gation, ultra-wide angle camera. Received date: 30.04.2022 Accepted date: 11.06.2022 Published date: 16.06.2022 © The Author(s) 2022 This is an open access article under the Creative Commons CC BY license How to cite Alkhimova, S., Davydovych, I. (2022). Accuracy assessment of marker recognition using ultra wide angle camera. Technology Audit and Production Reserves, 3 (2 (65)), 6–10. doi: http://doi.org/10.15587/2706-5448.2022.259068 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 ACCURACY ASSESSMENT OF MARKER RECOGNITION USING ULTRA WIDE ANGLE CAMERA Svitlana Alkhimova, Illia Davydovych Modern devices that support augmented reality technology are widely used in various fields of human activity, including medicine. Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. h d h l d k h k d Modern devices that support augmented reality technology are widely used in various fields of human activity, including medicine. Head mounted displays may provide an attractive alternative to traditional surgery naviga tion systems because allow users to stand at the first point of view and interact with objects in their surroundings naturally. Thus, the object of research in this study is recognition accuracy of fiducial markers in zones where ultra- wide angle camera distort the most. This is motivated by the need to increase user workspace for interaction with markers compare to the workspace provided with such popular augmented reality device as Microsoft HoloLens 2. In this study, the recognition accuracy is evaluated using ArUco square markers with taking into account different marker sizes and their positions in the camera view space. The marker positions include the center of the camera view space as well as such zones where lenses distort the most as top left, top right, bottom left, and bottom right corners. Obtained results show that recognition accuracy is good enough to be applicable for surgical navigation and failures referred to the distortion occurs are available in less than 0.2 % of all cases. This gives a possibility to increase workspace for interaction with markers compare to the Microsoft HoloLens 2. At the same time, the workspace for interaction could not reach the actual view space of the camera since recognition fails in cases where marker’s body is partially visible in the captured image (i. e., marker position is at the image boundaries). Keywords: augmented reality marker recognition ArUco fiducial markers recognition accuracy surgical navi including medicine. 2. Research methodology In this study, binary square fiducial markers were used as visual markers to be recognized. The main advantage of such markers is that camera pose can be obtained with only one marker usage [13]. Moreover, binary square fiducial markers are widely used in medical research and practice, as they are simple, highly reliable, and multifunctional solution [14–16]. Of the factors mentioned above, usability of such aug mented reality-based system as Microsoft HoloLens 2 will benefit from an increased FoV and achieved a good enough level of recognition accuracy of fiducial markers. Ultra-wide angle camera with high resolution can be a solution to increase FoV. Such a camera has a lens whose focal length is shorter than 24 mm in full-frame equivalent FoV. Depending on the shape and location of the lens group, the angle of view can range from 52 to 180 degrees (Fig. 1). ArUco library is an open source popular library for detection of square fiducial markers. ArUco library is an open source popular library for detection of square fiducial markers [17]. ArUco library was chosen as it has high accuracy of recognition on all three axes of the Cartesian plane and faster than similar tools for markers detection. This choice also provides compliance with the hardware capabilities of AR devices (i. e., most of them are not equipped with any sensors other than cameras). At the same time, usage of ultra-wide angle cameras pro vides images that usually suffer from distortions. Distortion of the geometry and proportions of the objects at the image can affect the accuracy of recognition [10, 11]. However, these defects can be corrected. The most common way to get rid of distortions is to calibrate the internal parameters of the camera with the help of calibration boards [12]. The disadvantages are that calibration should be performed in dividually for each camera, requires additional time and availability of appropriate software. The ArUco library includes several dictionaries contain ing sets of tokens with different numbers of bits. Having a smaller bit size helps to identify markers better for cases when marker size in image is too small, but a larger bit size can help to get position in three-dimensional space more accurately [13]. Therefore, the object of research in this study is recogni tion accuracy of fiducial markers in AR-based system ap plicable for surgical navigation. INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES have a lower resolution of the light-sensitive matrix compared to monoscopic camera. This leads to obtaining of a lower level of recognition accuracy of fiducial markers [8, 9]. 1. Introduction Presence of two pairs of stereoscopic cameras on the glasses sides could solve the issue with a small FoV. However, stereoscopic cameras of Microsoft HoloLens 2 The most commercially successful AR device is the Microsoft HoloLens 2 (Microsoft Corporation, USA) due TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 6 ISSN 2664-9969 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES ISSN 2664-9969 For experiments A and B where the same bit size of markers were used, better recognition accuracy (by 5.4 % on average) was observed for markers with larger physical sizes. For experiments B and C where the same physical size of markers were used, better recognition accuracy (by 6.2 % on average) was observed for markers with smaller bit sizes. It can be explained as captured images of markers with different physical sizes have different resolution in case the markers have the same position and at a fixed distance from the camera. This, in its turn, could lead to loss of information on some marker’s details or ele ments of inner binary matrix may not be clear in the captured image. In these experiments, the ultra-wide angle camera was represented by the 8MP ultra-wide angle camera of Xiao mi Redmi Note 8 Pro (Xiaomi Inc., China). The camera properties are: f/2.2 aperture, 13 mm focal length (FoV is 120 degrees), 1/4.0″ diagonal light-sensitive matrix, 1.12 μm pixel size. Distance from the camera position to the markers position was restricted to 70 cm. This value was taken from [18]. It is based on the assumption that interaction with the object to which the markers are applied is constrained by anthropometric and biomechanical limits (i. e., the maximal distance an average individual is able to reach forward from shoulder to fingertip). The analysis of the images with failed recognition showed that most of the failures occurred because of lighting is sues, i. e., due to insufficient illumination of the scene or presence of glare from lighting on the marker surface. The other significant part of failures was referred to the cases where only part of the fiducial marker was visible in the image. As any specific preprocessing for distortion correction was not applied to the images extracted from the video files, few recognition failures were referred to the distortion occurs (i. e., less than 0.2 % of all cases). To find out failed recognition cases due to the distortion presence, correction algorithm was applied to the input images copied beforehand. The OpenCV module was utilized to provide radial and tangential correction. Coefficients of radial and tangential were determined from the calibration data obtained before the experiments. INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES Difference between the initial recognition rate and the rated obtained on the corrected data was considered as a measure of failed cases referred the distortion occurs. In each experiment, video data were collected from scenes with markers placed in the FoV of the camera in five diffe rent positions (i. e., in the center, top left, top right, bottom left, and bottom right corners of the camera view space). The position of the camera was fixed for all experiments. In each experiment, video data were collected from scenes with markers placed in the FoV of the camera in five diffe rent positions (i. e., in the center, top left, top right, bottom left, and bottom right corners of the camera view space). The position of the camera was fixed for all experiments. Since the current study is interested in analyzing a re cognition accuracy of fiducial markers in AR-based system applicable for surgical navigation, interaction with individual components in such system is an important aspect for its operator. Therefore, operator interacted with markers and rotated them in any direction during the video record ing process. Video record lasted about 2 minutes for each marker position. In total, 15 videos were recorded. Since the current study is interested in analyzing a re cognition accuracy of fiducial markers in AR-based system applicable for surgical navigation, interaction with individual components in such system is an important aspect for its operator. Therefore, operator interacted with markers and rotated them in any direction during the video record ing process. Video record lasted about 2 minutes for each marker position. In total, 15 videos were recorded. The marker recognition was implemented using Python programming language (version 3.9.1), OpenCV open-source computer vision library (version 4.5.5), and cv2.aruco mo dule [13]. The program input was images previously extracted from the video files. In case of success recognition, the pro gram saved the coordinates of marker’s corners and center to be used to confirm marker recognition. The processed images also were saved at the program output. Images with failed marker recognition were marked and saved separately. In order for the marker to be successfully recognized, it should be positioned on white-light background or have white border around the marker to enable detection. 2. Research methodology Different physical sizes and number of bits of markers were used in this study to assess their possible impact on the recognition accuracy (Fig. 2). Experiments were conducted with markers that have 4×4 bits and 3×3 cm physical sizes (Experiment A), 4×4 bits and 5×5 cm physi cal sizes (Experiment B), 7×7 bits and 5×5 cm physical sizes (Experiment C). The aim of this research is to analyze a recognition accuracy of fiducial markers in zones where ultra-wide- angle camera distort the most with taking into account different marker sizes. a b Fig. 1. Comparison of captures images by wide angle and ultra-wide angle camera: a – example of an image from monoscopic camera of Microsoft HoloLens 2 (FoV is 52 degrees); b – example of an image from 8MP camera of Xiaomi Redmi Note 8 Pro (FoV is 120 degrees) Fig. 1. Comparison of captures images by wide angle and ultra-wide angle camera: a – example of an image from monoscopic camera of Microsoft HoloLens 2 (FoV is 52 degrees); b – example of an image from 8MP camera of Xiaomi Redmi Note 8 Pro (FoV is 120 degrees) a b c Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker with 4×4 bits and 5×5 cm physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C) Fig. 2. Markers used in this study: a – marker with 4×4 bits and 3×3 cm physical sizes (Experiment A); b – marker wi physical sizes (Experiment B); c – marker with 7×7 bits and 5×5 cm physical sizes (Experiment C TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 7 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES In addition, the white elements of inner binary matrix of the marker should have good contrast respect to the black frame and the marker’s body should be fully visible in the image. Among the mentioned issues related to markers recogni tion failures, the cases caused due to insufficient illumina tion can be solved through applying algorithms of exposure compensation and the cases caused due to distortion occurs can be solved through calibration procedure with further applying of distortion correction algorithms. As for the cases caused due to the image contained only part of the marker, possibility to increase the recognition rate is com plicated by the lack of built-in methods to process markers partially visible in the image. For this reason, the workspace for interaction with markers is smaller than the actual view space provided by the ultra-wide angle camera (i. e., by 15.85 % smaller for markers that have 3×3 cm physi cal sizes and by 25.96 % smaller for markers that have 5×5 cm physical sizes in our experiments). In order for the marker to be successfully recognized, it should be positioned on white-light background or have white border around the marker to enable detection. In addition, the white elements of inner binary matrix of the marker should have good contrast respect to the black frame and the marker’s body should be fully visible in the image. A h i d i l d k i 3. Research results and discussion The recognition accuracy was calculated as the frac tion of the number of images with recognized marker to the total number of images with certain marker position in given experiment. Table 1 lists the recognition accuracy of the markers by experiments. In all three experiments, the used algo rithm successfully recognized most of markers in images captured by ultra-wide angle camera. Table 1 Table 1 Markers recognition accuracy by experiments, % Position Experiment A Experiment B Experiment C center 91.8 93.1 89.4 up right 71.7 74.0 72.0 up left 82.4 89.8 84.6 down right 75.0 89.8 78.1 down left 84.1 85.4 76.9 Markers recognition accuracy by experiments, % TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 References 1. Vassallo, R., Rankin, A., Chen, E. C., Peters, T. M. (2017, March). Hologram stability evaluation for Microsoft HoloLens. Medical Imaging 2017: Image Perception, Observer Performance, and Technology Assessment, 10136, 295–300. doi: http://doi.org/ 10.1117/12.2255831 1. Vassallo, R., Rankin, A., Chen, E. C., Peters, T. M. (2017, March). Hologram stability evaluation for Microsoft HoloLens. Medical Imaging 2017: Image Perception, Observer Performance, and Technology Assessment, 10136, 295–300. doi: http://doi.org/ 10.1117/12.2255831 2. Eckert, M., Volmerg, J. S., Friedrich, C. M. (2019). Augmented Reality in Medicine: Systematic and Bibliographic Review. JMIR mHealth and uHealth, 7 (4), e10967. doi: http://doi.org/ 10.2196/10967 3. Moro, C., Phelps, C., Redmond, P., Stromberga, Z. (2020). HoloLens and mobile augmented reality in medical and health science education: A randomised controlled trial. British Journal of Educational Technology, 52 (2), 680–694. doi: http://doi.org/ 10.1111/bjet.13049 Despite its contributions, this study has some limitations to be considered. First, let’s use data collected under the same lighting conditions. Poor lighting conditions affect the result of the marker recognition. Especially, it happens when final texture or geometry of marker in the image gets corrupted. Second, this study has taken into account only 2D ArUco markers. Usage of 3D cube with an ArUco marker attached to each side of the cube can affect the recognition accuracy as angle between the camera plane and the marker plane does not exceed 45 degrees. Finally, our study did not compare results of marker recognition from different commercial or open-source software, which can be applied for that. Only OpenCV library was used. 4. Olson, E. (2011). AprilTag: A robust and flexible visual fi ducial system. 2011 IEEE international conference on robo tics and automation, 3400–3407. doi: http://doi.org/10.1109/ icra.2011.5979561 5. Romero-Ramirez, F. J., Mu oz-Salinas, R., Medina-Carnicer, R. (2018). Speeded up detection of squared fiducial markers. Image and Vision Computing, 76, 38–47. doi: http://doi.org/10.1016/ j.imavis.2018.05.004 6. Microsoft: Learn about HoloLens 2 features and review technical specs. Available at: https://www.microsoft.com/en-us/hololens/ hardware Last accessed: 20.05.2022 7. Howard, I. P., Rogers, B. J. (1995). Binocular vision and stereopsis. Oxford psychology series No. 29. Oxford University Press, 736. doi: https://doi.org/10.1093/acprof:oso/9780195084764.001.0001 In the future, it is necessary to further examine recogni tion accuracy at the image boundaries and find a way to expand the workspace for interaction with fiducial markers close to the maximum possible. 8. Brand, M., Wulff, L. A., Hamdani, Y., Sch ppstuhl, T. (2020). References Accuracy of Marker Tracking on an Optical See-Through Head Mounted Display. Annals of Scientific Society for Assembly, Handl ing and Industrial Robotics. Vieweg, Berlin, Heidelberg: Springer, 21–31. doi: http://doi.org/10.1007/978-3-662-61755-7_3 Markers recognition accuracy by experiments, % Since usage of ultra-wide angle camera aims to solve difficulties related to limited workspace in AR-based system, distribution of failed recognition cases where the fiducial marker left the workspace (i. e., partially visible in the image) was analyzed separately (Fig. 3). Regardless of the physical sizes and number of bits of markers, the recognition accuracy in the center of image is on average higher than in the corners. The recognition accuracy in the corners relative to the center of image degrades by 13.5 %, 8.35 %, and 11.5 % on average in Experiment A, B, and C, respectively. The results of this analysis revealed that despite the limited workspace, interaction with marker placed in the corners of the camera view space leads to cases where only part of the fiducial marker was visible in the image. On average, the mentioned factor reached 20.47 % of the total number of all unrecognized cases. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 8 8 ISSN 2664-9969 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES INFORMATION TECHNOLOGIES Fig. 3. Distribution of failed recognition cases where only part of the fiducial marker was visible in the image: a – Experiment A; b – Experiment B; c – Experiment C a b c a Fig. 3. Distribution of failed recognition cases where only part of the fiducial marker was visible in the image: a – Experiment A; b – Experiment B; c – Experiment C There was a noticeable increase in the percentage of failed recognition cases where the fiducial marker crossed the right side of workspace and was partially visible in the image. Right-side cases occurred more than 2.15 times often than lift-side cases. It can be explained by biome chanical impact during the interaction with marker, as operator was right-handed person. Since a movement to the left side by the right hand would be farther than reaching to the right side, it was more easer to cross the right side of workspace for the right-handed operator. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 4. Conclusions 9. Thabit, A., Niessen, W. J., Wolvius, E. B., van Walsum, T. (2022). Evaluation of marker tracking using mono and stereo vision in Microsoft HoloLens for surgical navigation. Medical Imaging 2022: Image-Guided Procedures, Robotic Interventions, and Mo deling, 12034, 253–262. doi: http://doi.org/10.1117/12.2607262 In this study, we evaluated recognition accuracy of fiducial markers in zones where ultra-wide angle came ra distort the most with taking into account different marker sizes. 10. Zhao, H., Ying, X., Shi, Y., Tong, X., Wen, J., Zha, H. (2020). RDCFace: radial distortion correction for face recognition. Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 7721–7730. doi: http://doi.org/10.1109/ cvpr42600.2020.00774 It is possible to conclude that the proposed usage of ultra-wide-angle camera is feasible for fiducial markers recognition in AR-based system applicable for surgical navigation. The accuracy that could be achieved in dis tortion zones of ultra-wide angle camera is sufficient in case where marker’s body is fully visible in the captured image. The recognition failures referred to the distortion occurs were less than 0.2 % of all cases. However, issues of failed recognition of makers partially visible in the image reduce the workspace for interaction with fiducial markers compare to the actual view space provided by the ultra-wide angle camera. 11. Miki, D., Abe, S., Chen, S., Demachi, K. (2020). Robust human motion recognition from wide-angle images for video surveillance in nuclear power plants. Mechanical Engineering Journal, 7 (3), 19–00533–19–00533. doi: http://doi.org/10.1299/mej.19-00533 12. Remondino, F., Fraser, C. (2006). Digital camera calibration methods: considerations and comparisons. International Archives of the Photogrammetry, Remote Sensing and Spatial Informa tion Sciences, 36 (5), 266–272. doi: https://doi.org/10.3929/ ethz-b-000158067 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 9 9 Corresponding author TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 INFORMATION AND CONTROL SYSTEMS: INFORMATION TECHNOLOGIES ISSN 2664-9969 Recognition, 47 (6), 2280–2292. doi: http://doi.org/10.1016/ j.patcog.2014.01.005 Recognition, 47 (6), 2280–2292. doi: http://doi.org/10.1016/ j.patcog.2014.01.005 13. OpenCV: Tutorials for contrib modules. Detection of ArUco Markers. Available at: https://docs.opencv.org/4.x/d5/dae/tuto rial_aruco_detection.html Last accessed: 20.05.2022 18. Looker, J., Garvey, T. (2015). Reaching for Holograms: Assess ing the Ergonomics of the Microsoft™ Hololens™ 3D Gesture Known as the «Air Tap». Proceedings from International Design Congress. Gwangju: KSDS, 504–511. 14. Liu, X., Plishker, W., Shekhar, R. (2021). Hybrid electromag netic-ArUco tracking of laparoscopic ultrasound transducer in laparoscopic video. Journal of Medical Imaging, 8 (1). doi: http:// doi.org/10.1117/1.jmi.8.1.015001 15. O dal, P., Heczko, D., Vysock , A., Mlotek, J., Nov k, P., Virgala, I. et. al. (2020). Improved Pose Estimation of Aruco Tags Using a Novel 3D Placement Strategy. Sensors, 20 (17), 4825. doi: http://doi.org/10.3390/s20174825 Svitlana Alkhimova, PhD, Department of Biomedical Cyberne Svitlana Alkhimova, PhD, Department of Biomedical Cyberne tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- *Svitlana Alkhimova, PhD, Department of Biomedical Cyberne tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- 0002-9749-7388, e-mail: alkhimova.svitlana@lll.kpi.ua tics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: http://orcid.org/0000- 0002-9749-7388, e-mail: alkhimova.svitlana@lll.kpi.ua 16. Luzon, J. A., Stimec, B. V., Bakka, A. O., Edwin, B., Ignjatovic, D. (2020). Value of the surgeon’s sightline on hologram registra tion and targeting in mixed reality. International Journal of Computer Assisted Radiology and Surgery, 15 (12), 2027–2039. doi: http://doi.org/10.1007/s11548-020-02263-3 Illia Davydovych, Department of Biomedical Cybernetics, National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Kyiv, Ukraine, ORCID: https://orcid.org/0000-0001- 9987-8267 17. Garrido-Jurado, S., Mu oz-Salinas, R., Madrid-Cuevas, F. J., Mar n-Jim nez, M. J. (2014). Automatic generation and detec tion of highly reliable fiducial markers under occlusion. Pattern TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 3/2(65), 2022 10
https://openalex.org/W3040233034	https://hal.science/hal-03322494/document	English	null	A Unified Theory of Human Judgements and Decision-Making under Uncertainty	Entropy	2,020	cc-by	24,341	To cite this version: Raffaele Pisano, Sandro Sozzo. A unified theory of human judgements and decision-making under uncertainty. Entropy, 2020, 22 (7), 738, 34 p. 10.3390/e22070738. hal-03322494 Distributed under a Creative Commons Attribution 4.0 International License Received: 6 June 2020; Accepted: 1 July 2020; Published: 3 July 2020 Abstract: Growing empirical evidence reveals that traditional set-theoretic structures cannot in general be applied to cognitive phenomena. This has raised several problems, as illustrated, for example, by probability judgement errors and decision-making (DM) errors. We propose here a uniﬁed theoretical perspective which applies the mathematical formalism of quantum theory in Hilbert space to cognitive domains. In this perspective, judgements and decisions are described as intrinsically non-deterministic processes which involve a contextual interaction between a conceptual entity and the cognitive context surrounding it. When a given phenomenon is considered, the quantum-theoretic framework identiﬁes entities, states, contexts, properties and outcome statistics, and applies the mathematical formalism of quantum theory to model the considered phenomenon. We explain how the quantum-theoretic framework works in a variety of judgement and decision situations where systematic and signiﬁcant deviations from classicality occur. Keywords: quantum structures; concept theory; decision theory; cognitive fallacies; disjunction effect; Ellsberg paradox; quantum modelling; historical perspectives Raffaele Pisano 1 and Sandro Sozzo 2,* 1 Institute of Electronics, Microelectronics and Nanotechnology (IEMN), Lille University, Avenue Poincaré CS 60069, CEDEX, 59652 Villeneuve d’Ascq, France; raffaele.pisano@univ-lille.fr 2 School of Business and Centre for Quantum Social and Cognitive Science (IQSCS), University of Leicester, University Road, Leicester LE1 7RH, UK * Correspondence: ss831@le.ac.uk; Tel.: +44-116-294-4692 1 Institute of Electronics, Microelectronics and Nanotechnology (IEMN), Lille University, Avenue Poincaré CS 60069, CEDEX, 59652 Villeneuve d’Ascq, France; raffaele.pisano@univ-lille.fr 2 School of Business and Centre for Quantum Social and Cognitive Science (IQSCS), University of Leicester, University Road, Leicester LE1 7RH, UK * Correspondence: ss831@le ac uk; Tel : +44 116 294 4692 y * Correspondence: ss831@le.ac.uk; Tel.: +44-116-294-4692 HAL Id: hal-03322494 https://hal.science/hal-03322494v1 Submitted on 20 May 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License entropy entropy Article Raffaele Pisano 1 and Sandro Sozzo 2,* (II) for every E ∈A , E′ = S \ E ∈A ; (I) S ∈A ; (III) for every countable family {Ei ∈A }i∈N, ∪i∈NEi ∈A . 1. Introduction Set-theoretic algebraic structures, like Boolean algebras, are the building blocks of classical (Boolean) logic and classical (Kolmogorovian) probability. It is, for example, well known that the elementary connectives of conjunction, disjunction and negation in Boolean logic are represented by the set-theoretic operations of intersection ∩, union ∪and complementation \, respectively. Similarly, conjunctions and disjunctions of two events are represented by the same set-theoretic operations in Kolmogorovian probability theory [1–3]. Set-theoretic structures were originally used in classical physics and later applied to a variety of disciplines, from psychology to economics, ﬁnance, computer science and statistics. This is why one usually refers to them as “classical structures”. In particular, Kolmogorovian probability models have characterized the experimental revolution which occurred in cognitive psychology in the 1970s [4]. In this regard, it is worth to brieﬂy summarize the fundamental deﬁnitions and results of Kolmogorovian probability theory that are needed for the scopes of the present paper. Let S ̸= ∅, let P(S ) be the power set of S , and let A ⊆P(S ). We say that A is a “σ-algebra” if it satisﬁes the following properties: (I) S ∈A ; (II) for every E ∈A , E′ = S \ E ∈A ; (III) for every countable family {Ei ∈A }i∈N, ∪i∈NEi ∈A . www.mdpi.com/journal/entropy Entropy 2020, 22, 738; doi:10.3390/e22070738 www.mdpi.com/journal/entropy 2 of 34 Entropy 2020, 22, 738 Entropy 2020, 22, 738 It straightly follows from the deﬁnition that, in a σ-algebra A , for every countable family {Ei ∈A }i∈N, ∩i∈NEi ∈A . A σ-algebra A is in particular distributive, that is, for every E, F, G ∈A , E ∩(F ∪G) = (E ∩F) ∪(E ∩G) and E ∪(F ∩G) = (E ∪F) ∩(E ∪G). It then follows from distributivity that, for every E, F ∈A , E = (E ∩F) ∪(E ∩F′). Let now S ̸= ∅, let A ⊆P(S ) be a σ-algebra, and let p : E ∈A 7→p(E) ∈[0, 1] be an application over A . We say that p is a “normalized probability measure” if it satisﬁes the following conditions: (1) p(S ) = 1 (normalization); (2) for every countable family {Ei ∈A }i∈N of pairwise disjoint sets, that is, Ei ∩Ej = ∅, i ̸= j, p(∪i∈NEi) = ∑i∈N p(Ei) (additivity). 1. Introduction (2) for every countable family {Ei ∈A }i∈N of pairwise disjoint sets, that is, Ei ∩Ej = ∅, i ̸= j, p(∪i∈NEi) = ∑i∈N p(Ei) (additivity). The sets in A correspond to “events” of the real world. Conditions (1) and (2) were introduced by The sets in A correspond to “events” of the real world. Conditions (1) and (2) were introduced by Kolmogorov, hence, the normalized probability measure p is called “Kolmogorovian probability” [1]. Then, let p : E ∈A 7→p(E) ∈[0, 1] be a Kolmogorovian probability, and let E, F ∈A , with p(E), p(F) ̸= 0. We deﬁne the “conditional probability” p(E\|F) of E given F as Kolmogorov, hence, the normalized probability measure p is called “Kolmogorovian probability” [1]. Then, let p : E ∈A 7→p(E) ∈[0, 1] be a Kolmogorovian probability, and let E, F ∈A , with p(E), p(F) ̸= 0. We deﬁne the “conditional probability” p(E\|F) of E given F as p(E\|F) = p(E ∩F) p(F) . (1) (1) The conditional probability p(F\|E) of F given E is deﬁned in an analogous way, and the “Bayes formula” p(F)p(E\|F) = p(E ∩F) = p(F ∩E) = p(E)p(F\|E) (2) (2) holds. The following laws of Kolmogorovian probability can be ﬁnally derived. For every E, F ∈ p(E) = p(F)p(E\|F) + p(F′)p(E\|F′), (3) (3) which is known as the “law of total probability”, and which is known as the “law of total probability”, and (4) p(E ∩F) ≤p(E), p(F) ≤p(E ∪F), (4) which is known as the “law of monotonicity”. which is known as the “law of monotonicity”. Growing empirical evidence in cognitive psychology reveals that the classical structures above, when applied to complex processes, like judgement and decision-making (DM), lead to predictions that do not agree with empirical data [4,5]. Deviations from classicality in these domains can be roughly divided into two main groups, as follows [6]. (i) Probability judgement errors. Given two events E and F, people judge the conjunction event ‘E and F’ (disjunction event ‘E or F’) as more (less) probable than the event E or/and F separately. This entails a “violation” of the of the law of monotonicity (cfr., Equation (4)). (ii) DM errors. Given two actions f and g, people prefer f over g if they know that an event E occurs, and also if they know that E does not occur, but they prefer g over f if they do not know whether E occurs or not. This entails a “violation” of the law of total probability (cfr., Equation (3)). Over-/under-extension effects in membership judgements on conceptual combinations [7,8] and conjunctive/disjunctive fallacies [9,10] are examples of type-(i) errors. The disjunction effect [11] and empirical violations of expected utility theory (EUT) [12] are instead examples of type-(ii) errors. Over-/under-extension effects in membership judgements on conceptual combinations [7,8] and conjunctive/disjunctive fallacies [9,10] are examples of type-(i) errors. The disjunction effect [11] and empirical violations of expected utility theory (EUT) [12] are instead examples of type-(ii) errors. The existence of situations of type-(i) and type-(ii) entails, in particular, that: The existence of situations of type-(i) and type-(ii) entails, in particular, that: • the most natural set-based models of cognition cannot in general be used to represent human judgements and decisions; the most natural set-based models of cognition cannot in general be used to represent human the most natural set-based models of cognition cannot in general be used to represent human judgements and decisions; • the usual interpretation of human behaviour in terms of classical (Boolean) logic and classical (Kolmogorovian) probability theory is problematical when human judgements and decisions are at stake. 3 of 34 Entropy 2020, 22, 738 The famous “Kahneman-Tversky research programme” considers the above empirical deviations from classicality as genuine fallacies of human reasoning and provides a theoretical framework for them which assumes individual heuristics and judgement biases [5,13], in substantial agreement with Simon’s theory of bounded rationality [14]. which is known as the “law of monotonicity”. This explains why terms like error, effect, fallacy, paradox, contradiction, and so forth, are typically used in these cases. A major limitation of the Kahneman-Tversky research programme is that it works at an intuitive level and has manifold applications, but it does not provide a general theory of human judgements and decisions. This has led various scholars to explore alternative mathematical structures which are more appropriate than classical structures to model these phenomena. On the other side, quantum theory is certainly one of the most successful theoretical achievement that humans have ever produced. Originally elaborated to cope with speciﬁc phenomena where empirical results did not agree with the predictions of classical physics, for example, black-body radiation, photoelectric effect, atomic modelling, and so forth, quantum theory is nowadays applied to any conceivable scale, from the micro-world to living matter. We add that classical physics had to face at the beginning of the 1900s problems of incompatibility between classical structures and their underlying logic and empirical results that are similar to the problems discussed here. For example, from the beginning of the 1900s, the problem of black-body radiation represented the critical root of quantum theory in a period in which already the space in physics had started to be conceived differently from the Euclidean one. Thus, the logical structure of the forthcoming theories changed as well. Regarding the black-body radiation, the main epistemological and foundational problem concerned the way in which the intensity of electromagnetic radiation emitted by a black body (i.e., a perfect absorber, also known as a “cavity radiator”) depended on the frequency of the radiation (i.e., the colour of light) and the temperature of the body. In other words, a mathematical function was looked for which was able to work with both energy and frequencies. This situation was logically the opposite that scientists had faced in the 1800s with the previous theory of light. Other theories such as thermodynamics, analytical theory of heat and electromagnetism were involved in the debate (see, e.g., References [15–20]). In its modern versions, quantum theory is formulated in a mathematical language that uses Hilbert spaces, unit vectors, self-adjoint operators and unitary dynamics, which give rise to speciﬁcally non-classical algebraic and probabilistic structures, like non-Boolean lattices, non-Kolmogorovian probabilities, non-commutative algebras, and so forth. which is known as the “law of monotonicity”. In the SCoP formalism, tests involving judgements and decisions are considered as complex phenomena in which: (a) the conceptual entity under study is prepared by the test in a deﬁned state and each participant is confronted with this uniquely prepared state; (b) an interaction occurs on a cognitive level between the conceptual entity and the participant, which acts as a (measurement) context for the entity; (a) the conceptual entity under study is prepared by the test in a deﬁned state and each participant is confronted with this uniquely prepared state; (b) an interaction occurs on a cognitive level between the conceptual entity and the participant, which acts as a (measurement) context for the entity; (c) the interaction is in general non-deterministic and the state of the conceptual entity is transformed into a new state; (d) the state change makes actual (potential) some properties of the conceptual entity which were potential (actual) in the initial state; (e) when the responses of all participants are collected, a statistics of outcomes is obtained. (a) the conceptual entity under study is prepared by the test in a deﬁned state and each participant is confronted with this uniquely prepared state; is confronted with this uniquely prepared state; (b) an interaction occurs on a cognitive level between the conceptual entity and the participant, which acts as a (measurement) context for the entity; (b) an interaction occurs on a cognitive level between the conceptual entity and the participant, which acts as a (measurement) context for the entity; (c) the interaction is in general non-deterministic and the state of the conceptual entity is transformed into a new state; (d) the state change makes actual (potential) some properties of the conceptual entity which were potential (actual) in the initial state; (e) when the responses of all participants are collected, a statistics of outcomes is obtained. (d) the state change makes actual (potential) some properties of the conceptual entity which were potential (actual) in the initial state; when the responses of all participants are collected, a statistics of outcomes is obtained. e) when the responses of all participants are collected, a statistics of outcomes is obtained. (e) The third step consists in the development of a unified quantum representation. which is known as the “law of monotonicity”. Indeed, whenever a specific cognitive phenomenon is studied and one identifies points (a)–(e) in it, namely, the conceptual entity, its states, properties, the relevant contexts and the statistics of measurement outcomes, then the notions in (a)–(e) are represented in the quantum-theoretic framework in exactly the same way as entities, states, context, properties, probabilities and dynamics are represented in the Hilbert space formalism of quantum theory. Proceeding in this way, one can in principle apply the quantum-theoretic framework to any judgement and decision. The theoretical framework presented here is alternative to the Kahneman-Tversky research programme, because it considers the empirical deviations from classicality in cognition as natural expressions of genuine quantum structures in human reasoning, which overlap with classical reasoning and reveal a separate layer of reasoning, namely, “emergent”, or “conceptual”, reasoning [34]. For the sake of completeness, we summarize the content of this paper in the following. We respectively review in Sections 2 and 3 the type-(i) errors that occur in conceptual conjunctions/disjunctions and conjunctive/disjunctive fallacies. Then, we respectively review in Sections 4 and 5 the type-(ii) errors that occur in the disjunction effect and Ellsberg-type violations of EUT. We also sketch limitations of traditional modelling techniques and explanations for both types of cognitive fallacies. Next, we present in Section 6 the SCoP formalism for conceptual entities, applying the formalism to human probability judgements in Section 6.1 and to human decisions in in Section 6.2. In these sections, we also show how a representation in Hilbert space can be constructed. This quantum-theoretic framework enables successful modelling of membership judgements in conceptual combinations, which is shown is Section 7, the conjunction and disjunction fallacy, which is shown in Section 7.1, the disjunction effect, which is shown in Section 7.2, and Ellsberg-type paradoxes, which is shown in Section 8. Finally, in Section 9, we offer some concluding remarks. which is known as the “law of monotonicity”. In addition, quantum effects, like contextuality, emergence, entanglement, interference, nonlocality, superposition, and indistinguishability, do not admit a classical counterpart (see, e.g., Reference [21]). Because of their generality and ﬂexibility, quantum structures in Hilbert space may provide the mathematical tools to deal with the classically problematical phenomenology above and, indeed, quantum mathematical structures have systematically and successfully been applied to a variety of complex systems in psychology, social science, biology and computer science (see, e.g., References [4,22–29] and references therein). We review and deepen in the present paper the contribution to the “quantum cognition research programme” that we have provided in a decade of collaboration between the Brussels and Leicester research teams (see, e.g., References [30–35] and references therein). More precisely, we put forward a unitary theoretical framework in which both judgements and decisions are described as intrinsically non-deterministic context-dependent processes and provide the foundations for applying quantum theory in Hilbert space as a general theory for these processes. The ensuing models, originally designed to represent speciﬁc phenomena, then ﬁnd their foundation and justiﬁcation in the general quantum-theoretic framework presented here. The foundation of a quantum modelling can then be split into three main steps, as follows. The ﬁrst step consists in the recognition that, in any judgement/decision process, exactly as in a quantum measurement process, a contextual inﬂuence (of a cognitive, rather than physical, type) occurs where one outcome is actualized from a set of possible outcomes. Like in quantum physics, 4 of 34 Entropy 2020, 22, 738 the (measurement) context does not reveal pre-existing properties of the entity but, it rather actualizes properties that were only potential in the initial state in which the entity had been prepared—unless the initial state was chosen to be an eigenstate of the measurement in object. the (measurement) context does not reveal pre-existing properties of the entity but, it rather actualizes properties that were only potential in the initial state in which the entity had been prepared—unless the initial state was chosen to be an eigenstate of the measurement in object. The second step consists in the development of a “State-Context-Property (SCoP) formalism”, originally worked out to provide a realistic and operational foundation of quantum physics [36,37]. 2. Probability Judgement Errors: Over- and Under-Extension Effects The issue of what a concept is and how concepts combine has intrigued and fascinated epistemologists and cognitive scientists for a long time. Leaving aside the dispute between the former and the latter on the ontology of a concept, one however recognises at once the transition needed when one moves from a very abstract concept, as Thing, to more concrete natural concepts, as Fruit, . . . , Apple, ..., to ﬁnally come to an object, as This Apple. Concepts and categorization are the areas in cognition that deal with the ancient philosophical “problem of universals”, that is, whether unique particular objects or events can be treated “equivalently” as members of a class. According to the “classical view of concepts”, which can 5 of 34 Entropy 2020, 22, 738 be traced back to Aristotelian school and has been dominant until the 1950s, all instances of a concept share a common set of necessary and sufﬁcient deﬁning properties. It is however well known that this classical view contrasts with some empirical results related to the way in which people concretely judge and combine concepts. In particular, the following aspects have been identiﬁed in empirical studies on categorization (see, e.g., Reference [38]). (i) Concepts are “vague,” “fuzzy,” or “graded,” notions. For example, people judge an item like Robin as more typical than Stork when typicality is deﬁned with respect to the concept Bird. (i) Concepts are “vague,” “fuzzy,” or “graded,” notions. For example, people judge an item like Robin as more typical than Stork when typicality is deﬁned with respect to the concept Bird. p g , y, g , p , p p j g Robin as more typical than Stork when typicality is deﬁned with respect to the concept Bird. (ii) The meaning of a concept depends on the “context” in which the concept is used. For example, an item like Snake, or Spider would score a low typicality with respect to the concept Pet. But, if typicality is deﬁned with respect to the concept Weird Pet, or Pet of a Weird Person, then Snake, or Spider, would score a high typicality. To account for these features of “vagueness,” “graded typicality,” “context dependence,” several alternative theories of concepts have been proposed since the 1970s. Limiting ourselves to the most celebrated ones, we recall the following theories. 2. Probability Judgement Errors: Over- and Under-Extension Effects • “Prototype theory.” A given concept is associated with a “prototype,” which has a deﬁned set of characteristic, not deﬁning, features. Similarity with the prototype establishes the membership of an item with respect to the concept [39,40]. • “Prototype theory.” A given concept is associated with a “prototype,” which has a deﬁned set of characteristic, not deﬁning, features. Similarity with the prototype establishes the membership of an item with respect to the concept [39,40]. • “Exemplar theory.” A given concept is deﬁned by a salient set of instances of it stored in memory [41,42]. • “Exemplar theory.” A given concept is deﬁned by a salient set of instances of it stored in memory [41,42]. • “Theory theory.” A concept is similar to a “mini-theory,” and concepts stand in relation to one other in the same way as the terms of a scientiﬁc theory [43,44]. Despite mutual differences, traditional theories of concepts share the view that concepts have the form of “ﬁxed mental representations,” whereas context only plays a secondary role in all of them. On the other side, a “combination problem” raises in all traditional concept theories of concepts, namely, how the combination, for example, conjunction, disjunction, negated conjunction, of two given concepts can be consistently represented in terms of the representation of the individual concepts. The combination problem becomes even harder when the combination of more than two concepts is investigated. At ﬁrst glance, one may be tempted to believe that concepts combine by means of set-theoretic rules that are similar to those used to combine propositions in classical (Boolean) logic. In order to incorporate conceptual vagueness into such a set-theoretic perspective, Zadeh proposed to represent concepts by using “fuzzy set logic” [45]. Given a concept A and an item X, a “graded membership” µX(A) ∈[0, 1] exists such that, for any two concepts A and B, the conceptual conjunction ‘A and B’ satisﬁes the “minimum rule of fuzzy set conjunction” µ(A and B) = min h µ(A), µ(B) i , (5) (5) and the conceptual disjunction ‘A or B’ satisﬁes the “maximum rule of fuzzy set disjunction” µ(A or B) = max h µ(A), µ(B) i . (6) (6) However, a wide range of empirical findings reveal that classical set-theoretic structures and, in particular, this fuzzy set representation, are not able to model even simple combinations of two concepts. (1) “Pet-Fish problem”, or “Guppy effect”. 2. Probability Judgement Errors: Over- and Under-Extension Effects People judge an item like Guppy to be a very typical example of the conjunction Pet-Fish, without judging Guppy to be a typical example of either Pet or Fish [46]. (1) “Pet-Fish problem”, or “Guppy effect”. People judge an item like Guppy to be a very typical example of the conjunction Pet-Fish, without judging Guppy to be a typical example of either Pet or Fish [46]. Entropy 2020, 22, 738 6 of 34 (2) “Overextension effects in the conjunction”. For several items, people judge the membership weight of the item with respect to the conjunction to be higher than the membership weight of the item with respect to one or both the component concepts [7]. (2) “Overextension effects in the conjunction”. For several items, people judge the membership weight of the item with respect to the conjunction to be higher than the membership weight of the item with respect to one or both the component concepts [7]. (2) “Overextension effects in the conjunction”. For several items, people judge the membership weight of the item with respect to the conjunction to be higher than the membership weight of the item with respect to one or both the component concepts [7]. (3) “Under-extension effects”. For several items, people judge the membership weight of the item with respect to the disjunction to be lower than the membership weight of the item with respect to one or both the component concepts [8]. (3) “Under-extension effects”. For several items, people judge the membership weight of the item with respect to the disjunction to be lower than the membership weight of the item with respect to one or both the component concepts [8]. (4) “Borderline contradictions”. A signiﬁcant number of people judge the proposition “John is tall and John is not tall” to be true, in particular, for some borderline cases of “John” [47]. (4) “Borderline contradictions”. A signiﬁcant number of people judge the proposition “John is tall and John is not tall” to be true, in particular, for some borderline cases of “John” [47]. In the late 1980s, James Hampton performed a series of cognitive tests on the conjunction and the disjunction of two concepts [7,8]. Let us illustrate an example of these tests in the following. Let us consider, for example, the concepts Fruits, Vegetables, Fruits and Vegetables, Fruits or Vegetables. 2. Probability Judgement Errors: Over- and Under-Extension Effects Let us choose a set number of items, e.g, Apple, Tomato, Broccoli, Olive, Coconut, Almond, Raisin, Acorn, Tomato, Mushrooms, and so forth. A sample of participants are asked to judge membership of a given item with respect to Fruits, Vegetables and, for example, their disjunction Fruits or Vegetables. A 7-point Likert scale {−3, −2, −1, 0, +1, +2, +3} is used as a judgement of membership, where the positive values +1, +2 and +3 indicate that the participant considers the item to be a member of the concept, and the degree of membership increases with the value, thus the value +3 indicates that the participant considers the item to be a “strong member” of the concept. Analogously, negative numbers indicate non-membership, again in increasing order, thus the value −3 indicates that the participant considers the item to be a “strong non-member” of the concept. The value 0 indicates that the participant is unresolved about membership or non-membership of the item. The relative frequencies of positive responses are considered, in the large number limit, as probabilities of membership and are called “membership weights.” Hampton collected membership weights of several items with respect to different pairs of natural concepts and their conjunction or disjunction, ﬁnding signiﬁcant deviations from classical set-theoretic structures and introducing a speciﬁc terminology to classify such deviations. g p gy y Let X be an item and let µ(A), µ(B), µ(A and B) and µ(A or B) be the membership weights of X with respect to the concepts A, B, their conjunction ‘A and B’ and their disjunction ‘A or B’, respectively. We say that X is: “overextended with respect to the conjunction” if µ(A and B) > µ(A) or µ(A and B) > µ(B); • “overextended with respect to the conjunction” if µ(A and B) > µ(A) or µ(A and B) > µ(B); • “underextended with respect to the disjunction” if µ(A or B) < µ(A) or µ(A or B) < µ(B); p j µ( ) µ( ) µ( ) µ( ) • “underextended with respect to the disjunction” if µ(A or B) < µ(A) or µ(A or B) < µ(B); • “double overextended with respect to the conjunction” if µ(A and B) > µ(A) and µ(A and B) > µ(B); • “double underextended with respect to the disjunction” if µ(A or B) < µ(A) and µ(A or B) < µ(B). 2. Probability Judgement Errors: Over- and Under-Extension Effects Overextension and double overextension express a violation of the law of monotonicity of Kolmogorovian probability for the conjunction of two events. Let us consider, for example, the item Razor. Hampton measured membership of Razor with respect to Weapons, Tools and their conjunction Weapons and Tools, ﬁnding µ(A) = 0.63, µ(B) = 0.78 and µ(A and B) = 0.83, respectively. Hence, Razor is double overextended with respect to the conjunction Weapons and Tools [7]. However, the deviation from classical structures in conceptual conjunctions is even deeper. To illustrate this aspect, let us introduce the following deﬁnition. We say that the membership weights µ(A), µ(B) and µ(A and B) of the item X with respect to the concepts A, B and their conjunction ‘A and B’, respectively, correspond to “classical data for the 22, 738 7 of 34 Entropy 2020, 22, 738 7 of 34 conjunction” if a σ-algebra A , a Kolmogorovian probability measure p : A −→[0, 1], and events EA, EB ∈A exist such that: conjunction” if a σ-algebra A , a Kolmogorovian probability measure p : A −→[0, 1], and events EA, EB ∈A exist such that: µ(A) = p(EA), (7) µ(B) = p(EB), (8) µ(A and B) = p(EA ∩EB). (9) (7) (7) (8) (9) (8) (9) One can then prove that µ(A), µ(B) and µ(A and B) are classical data for the conjunction if and only if the following inequalities are simultaneously satisﬁed: µ(A and B) −min h µ(A), µ(B) i ≤ 0, (10) µ(A) + µ(B) −µ(A and B) ≤ 1. (11) (10) (11) Inequality (10) expresses the law of monotonicity for the conjunction, while inequality (11) is called the “Kolmogorovian conjunction factor” [22]. Inequality (10) expresses the law of monotonicity for the conjunction, while inequality (11) is called the “Kolmogorovian conjunction factor” [22]. Similarly, underextension and double underextension express a violation of the law of monotonicity of Kolmogorovian probability for the disjunction of two events. Let us consider, for example, the item Razor. Hampton measured membership of Curry with respect to Spices, Herbs and their disjunction Spices or Herbs, ﬁnding µ(A) = 0.90, µ(B) = 0.40 and µ(A or B) = 0.75, respectively. Hence, Curry is underextended with respect to the conjunction Spices or Herbs [8]. However, other non-classical effects are at play in the disjunction case too. 2. Probability Judgement Errors: Over- and Under-Extension Effects Indeed, we say that the membership weights µ(A), µ(B) and µ(A or B) of the item X with respect to the concepts A, B and their disjunction ‘A or B’, respectively, correspond to “classical data for the disjunction” if a σ-algebra A , a Kolmogorovian probability measure p : A −→[0, 1], and events EA, EB ∈A exist such that: µ(A) = p(EA), (12) µ(B) = p(EB), (13) µ(A or B) = p(EA ∪EB). (14) (12) (12) (13) (14) (13) (14) (14) One can then prove that µ(A), µ(B) and µ(A or B) are classical data for the disjunction if and only if the following inequalities are simultaneously satisﬁed: One can then prove that µ(A), µ(B) and µ(A or B) are classical data for the disjunction if and only if the following inequalities are simultaneously satisﬁed: µ(A or B) −max h µ(A), µ(B) i ≥ 0, (15) µ(A) + µ(B) −µ(A or B) ≥ 0. (16) (15) (16) (15) (15) (15) (16) (16) Inequality (15) expresses the law of monotonicity for the conjunction, while inequality (16) is called the “Kolmogorovian conjunction factor” [22]. Let us consider, for example, the item Olive. Hampton measured membership of Olive with respect to Fruits, Vegetables and their disjunction Fruits or Vegetables, ﬁnding µ(A) = 0.50, µ(B) = 0.10 and µ(A or B) = 0.80, respectively. In this case, inequality (15) is satisﬁed, whereas inequality (16) is violated. Hence, the membership weights Olive correspond to non-classical data. As mentioned in Section 1, over- and under-extension effects in membership judgements are examples of type-(i) errors. Additional examples of these non-classical effects will be considered in the next section. (i) Linda is a bank teller. (i) Linda is a bank teller. (ii) Linda is a bank teller and is active in the feminist movement. The test revealed that overall 85% of the participants judged event (ii) as more probable than event (i). This empirical pattern violates the law of monotonicity of Kolmogorovian probability (see Equation (4), Section 1). Indeed, if we denote by p(EA) the probability that the event “Linda is a bank teller” occurs and by p(EB) the probability that the event “Linda is active in the feminist movement” occurs, then the conjunction event “Linda is a bank teller and is active in the feminist movement” is associated with the probability p(EA ∩EB) in a Kolmogorovian probability framework, and the monotonicity law requires in particular that p(EA ∩EB) ≤p(EA), in contrast to the empirical ﬁndings in Reference [9]. The test was performed in two distinct ways. The test was performed in two distinct ways. • “Joint test”. The same group of participants were asked to judge the likelihood of both the individual event and the conjunction event. • “Joint test”. The same group of participants were asked to judge the likelihood of both the individual event and the conjunction event. • “Separate test”. One group of participants were asked to judge the likelihood of the individual event and another group of participants were asked to judge the likelihood of the conjunction event. • “Separate test”. One group of participants were asked to judge the likelihood of the individual event and another group of participants were asked to judge the likelihood of the conjunction event. A fallacy was identiﬁed in both types of tests. A large empirical literature exists in cognitive psychology which conﬁrms the existence of a conjunction fallacy in the “Linda story problem”. However, this is not the only probability judgement error that has been identiﬁed. A more convincing test was performed by Morier and Borgida in 1984 [10]. People participating in the test were asked to rank the likelihood of the following events: (i) Linda is a feminist; (ii) Linda is a bank teller; (iii) Linda is a feminist and a bank teller; (iv) Linda is a feminist or a bank teller. Let us denote by µ(A), µ(B), µ(A and B), and µ(A or B) the judgement probabilities in points (i), (ii), (iii), and (iv), respectively. 3. Probability Judgement Errors: Conjunctive and Disjunctive Fallacies The identiﬁcation of fallacies in cognition and the role played by psychological insights into social science marked the beginning of new disciplines, for example, behavioural economics, and allowed Daniel Kahneman to receive the Nobel Prize in Economic Science in 2002. 8 of 34 Entropy 2020, 22, 738 Entropy 2020, 22, 738 One of the key results in his research, obtained by Kahneman in 1983, in collaboration with Amos Tversky, was the discovery of the “conjunction fallacy” [9]. A sample of participants underwent a test in which they were presented the following story about an hypothetical woman named “Linda”. “Linda is 31 years old, single, outspoken and very bright. She majored in philosophy. As a student, she was deeply concerned with issues of discrimination and social justice, and also participated in anti-nuclear demonstrations.” “Linda is 31 years old, single, outspoken and very bright. She majored in philosophy. As a student, she was deeply concerned with issues of discrimination and social justice, and also participated in anti-nuclear demonstrations.” The test required participants to indicate which of the following events they judged as more likely i) Linda is a bank teller. (i) Linda is a bank teller. Let us now sketch the most celebrated explanations for conjunctive and disjunctive fallac Let us now sketch the most celebrated explanations for conjunctive and disjunctive fallacies. • Kahneman-Tversky explanation. The heuristics of representativeness is introduced to explain the conjunction fallacy. The event “being a feminist and a bank teller” is judged as more representative than the event “being a bank teller” for Linda’s story [5,9]. This explanation can be criticised, as the heuristics hypothesis works well at an intuitive level, but a general theory of heuristics is missing and different heuristics have to be assumed each time to accommodate different types of fallacies [6,48]. • Misunderstanding explanation. People misunderstand the Linda problem, in the sense that they misinterpret the terms “and” and/or “probability” in the story. This explanation can be criticised, as the fallacy also occurs when the terms “and” and “probability” are not mentioned in the story [48]. • Quantum probability explanation. People judge the sentence “Linda is a feminist and a bank teller” as an ordered sequence, namely, “Linda is a feminist”, then “Linda is a bank teller”. The two questions are incompatible in the standard quantum sense, that is, they produce different statistical distributions when asked in a different order [4,6]. This explanation can be criticised, as new tests seem to conﬁrm that the conjunction fallacy is not directly related to question order effects [49]. Conjunctive and disjunctive fallacies have been identiﬁed in several cognitive tests involving Linda-like stories (see, e.g., References [4,6,48–51]). It should be noted that the original story considered a likely event (L), namely, “Linda is a feminist”, and a unlikely event (U), namely, “Linda is a bank teller”. More recent variants consider the other possible combinations “UU” and “LL”, in both conjunctions and disjunctions. Consider, for example, the following story. “Bill is 34 years old. He is intelligent, but unimaginative, compulsive and generally lifeless. In college, he was strong in mathematics.” People were asked in Reference [50] to rank likelihood of the following events People were asked in Reference [50] to rank likelihood of the following events. (i) Linda is a bank teller. Then, Morier and Borgida found that the mean probability judgements are ordered as follows: (17) µ(A) = 0.83 > µ(A or B) = 0.60 > µ(A and B) = 0.36 > µ(B) = 0.26. (17) Thus, the test revealed the simultaneous presence of a conjunction fallacy and also a “disjunction fallacy”. As mentioned in Section 1, Kahneman and Tversky developed a research programme, assuming heuristics and biases, to account in a descriptive way how people make judgements and take decisions in the presence of uncertainty [5,9]. The main steps of it are summarized in the following. (1) Human judgement and DM rest on a preliminary subjective assessment of the probabilities of uncertain events. Entropy 2020, 22, 738 9 of 34 (2) In these processes, people rely on a limited number of heuristics principles, for example, representativeness, availability, adjustment and anchoring, and so forth. (2) In these processes, people rely on a limited number of heuristics principles, for example, representativeness, availability, adjustment and anchoring, and so forth. (2) In these processes, people rely on a limited number of heuristics principles, for example, representativeness, availability, adjustment and anchoring, and so forth. (3) Judgement heuristics are simple strategies/mental processes that people use to ﬁnd quick solutions to a complex problem by focusing on the most relevant aspects of it. (3) Judgement heuristics are simple strategies/mental processes that people use to ﬁnd quick solutions to a complex problem by focusing on the most relevant aspects of it. (4) These heuristics are generally very useful, but they may also lead to severe and systematic errors, or “biases”, because the ensuing judgements are all based on data of limited validity. (4) These heuristics are generally very useful, but they may also lead to severe and systematic errors, or “biases”, because the ensuing judgements are all based on data of limited validity. (5) Examples of biases are given by the already mentioned over-/under-estimation of probabilities and other fallacies. (5) Examples of biases are given by the already mentioned over-/under-estimation of probabilities and other fallacies. Despite individual differences, the programme in (1)–(5) is substantially compatible with Simon’s theory of bounded rationality, that is, rationality is limited in judgements/decisions by the tractability of the problem, the cognitive limitations of the mind, and the time available to make a choice: people thus seek a satisfactory, rather than an optimal, solution [14]. (i) Linda is a bank teller. (U) Bill is a reporter; (LU) Bill is an accountant and plays jazz for a hobby; (UU) Bill is a reporter and surfs for a hobby; (L) Bill works for the Inland Revenue; (U) Bill plays jazz for a hobby; (LL) Bill is an accountant and works for the Inland Revenue; (U) Bill surfs for a hobby; (L) Bill is an accountant. 4. Decision-Making Errors: The Disjunction Effect We present in the next two sections examples of type-(ii) errors (cfr., Section 1). Let us preliminarily note that both the disjunction effect, which is discussed in this section and Ellsberg paradox, which will be discussed in Section 5, entail a violation of a fundamental axiom of EUT, namely, the “sure-thing principle”. The sure-thing principle was formulated by Savage within his subjective EUT (SEUT) formulation [2]. He was inspired by the following story. We present in the next two sections examples of type-(ii) errors (cfr., Section 1). Let us preliminarily note that both the disjunction effect, which is discussed in this section and Ellsberg paradox, which will be discussed in Section 5, entail a violation of a fundamental axiom of EUT, namely, the “sure-thing principle”. The sure-thing principle was formulated by Savage within his subjective EUT (SEUT) formulation [2] y g p p The sure-thing principle was formulated by Savage within his subjective EUT (SEUT) formulation [2] He was inspired by the following story. “A businessman contemplates buying a certain piece of property. He considers the outcome of the next presidential election relevant. So, [. . . ], he asks whether he would buy if he knew that the Democratic candidate were going to win, and decides that he would. Similarly, he considers whether he would buy if he knew that the Republican candidate were going to win, and again ﬁnds that he would. Seeing that he would buy in either event, he decides that he should buy, even though he does not know which event obtains [...].” “A businessman contemplates buying a certain piece of property. He considers the outcome of the next presidential election relevant. So, [. . . ], he asks whether he would buy if he knew that the Democratic candidate were going to win, and decides that he would. Similarly, he considers whether he would buy if he knew that the Republican candidate were going to win, and again ﬁnds that he would. Seeing that he would buy in either event, he decides that he should buy, even though he does not know which event obtains [...].” Tversky and Shafir performed a test on the sure-thing principle in which they asked a sample of participants to bet on a gamble that can be played twice, that is a “two-stage gamble” [11]. (L) Bill is an accountant. Empirical results clearly suggest that conjunctive and disjunctive fallacies are systematically present and do not depend on likelihood of events. Tables 1 and 2 are illustrative in this direction. 10 of 34 Entropy 2020, 22, 738 Table 1. Test by Fisk and Pidgeon on the conjunction fallacy [50]. Values obtained from the quantum model in Section 7.1 are also reported. Case µ(A) µ(B) µ(A and B) θc \|A⟩ e−iθc\|B⟩ LL 0.84 0.62 0.71 94.65 (0.92, 0, 0.4) (0.27, 0.74, −0.62) LL 0.59 0.85 0.63 111.28 (0.77, 0, 0.64) (0.32, 0.86, −0.39) LU 0.76 0.28 0.37 111.15 (0.87, 0, 0.49) (0.48, 0.23, −0.85) LU 0.85 0.31 0.42 119.82 (0.92, 0, 0.39) (0.35, 0.43, −0.83) UU 0.33 0.11 0.13 118.19 (0.82, 0, 0.57) (−0.23, −0.91, 0.33) Table 2. Test by Fisk on the disjunction fallacy [51]. Values obtained from the quantum in Section 7.1 are also reported. Case µ(A) µ(B) µ(A or B) θd \|A⟩ e−iθd\|B⟩ LL 0.82 0.6 0.78 74.88 (0.91, 0, 0.42) (0.3, 0.72, −0.63) LL 0.85 0.62 0.72 93.60 (0.92, 0, 0.39) (0.26, 0.74, −0.62) LU 0.61 0.2 0.47 79.28 (0.62, 0, 0.78) (−0.56, −0.7, 0.45) LU 0.82 0.31 0.62 81.02 (0.91, 0, 0.42) (0.39, 0.4, −0.83) UU 0.36 0.11 0.26 82.78 (0.8, 0, 0.6) (−0.25, −0.91, 0.33) UU 0.3 0.08 0.23 75.04 (0.84, 0, 0.55) (−0.19, −0.94, 0.28) Table 2. Test by Fisk on the disjunction fallacy [51]. Values obtained from the quantum in Section 7.1 are also reported. Table 2. Test by Fisk on the disjunction fallacy [51]. Values obtained from the quantum in Section 7.1 are also reported. Case µ(A) µ(B) µ(A or B) θd \|A⟩ e−iθd\|B⟩ 4. Decision-Making Errors: The Disjunction Effect Let us consider a gamble that has probability 0.5 of winning $200 and probability 0.5 of losing $100. At each stage of the test, respondents were asked whether they wanted to play the gamble. They were presented the following three situations. (i) Respondents knew they had won the ﬁrst gamble. (ii) Respondents knew they had lost the ﬁrst gamble. (iii) Respondents did not know the outcome of the ﬁrst gamble. According to the sure-thing principle, if respondents decide to play again in both situation (i) and situation (ii), then they should play again also in situation (iii). Tversky and Shaﬁr instead found that: (1) 69% of the respondents who knew they had won the ﬁrst gamble decided to play ag 69% of the respondents who knew they had won the ﬁrst gamble decided to play again. (2) 59% of the respondents who knew they had lost the ﬁrst gamble decided to play aga 59% of the respondents who knew they had lost the ﬁrst gamble decided to play again. 36% of the respondents who did not know the outcome of the ﬁrst gamble decided to play again (3) 36% of the respondents who did not know the outcome of the ﬁrst gamble decided to play The two-stage gamble test violates the sure-thing principle. Moreover, it also violates the law of total probability of Kolmogorovian probability. Indeed, let us denote by p(EP) the probability that the 11 of 34 Entropy 2020, 22, 738 respondent plays again without knowing the outcome of the ﬁrst gamble, by p(EW) the probability that the respondent wins the ﬁrst gamble, by p(EL) the probability that the respondent loses the ﬁrst gamble, by p(EP\|EW) the conditional probability that the respondent plays again knowing they have won the ﬁrst gamble, and by p(EP\|EL) the conditional probability that the respondent plays again knowing they have lost the ﬁrst gamble. Then, one should have p(EP) = p(EW)p(EP\|EW) + p(EL)p(EP\|EL), (18) (18) according to the law of total probability (see Equation 3, Section 1). But, it is easy to show that one cannot ﬁnd Kolmogorovian probabilities p(EW) and p(EL) = 1 −p(EW) such that the data in Reference [11] are reproduced, that is, p(EP) = 0.36, p(EP\|EW) = 0.69 and p(EP\|EL) = 0.59, and the law of total probability is satisﬁed. This deviation from classicality is known as the “disjunction effect”. 4. Decision-Making Errors: The Disjunction Effect A similar result was obtained in another test of the sure-thing principle, the “Hawaii problem” [11], and also the “prisoner’s dilemma” can be formulated as a disjunction effect [52,53]. Tversky and Shaﬁr explained the effect suggesting that people are generally “averse to uncertainty”, that is, they prefer sure over unsure choices. In the two-stage gamble, participants prefer to play again in both cases where they have certainty about the outcome of the ﬁrst gamble, while they refuse to play again when they do not know the outcome of the ﬁrst gamble. Recent two-stage gamble have found an empirical pattern that substantially agrees with Tversky-Shaﬁr’s ﬁndings [52,53] (see Table 3). Table 3. Average data on the two-stage gamble tests by Tversky and Shaﬁr (TS 1992) [11], Kühberger, Kamunska and Perner (KKP 2001) [52], and Lambdin and Burdsal (LB 2007) [53] on the disjunction effect. Values obtained from the quantum in Section 7.2 are also reported. Test µ(A) µ(B) µ(A or B) θd \|A⟩ e−iθd\|B⟩ TS 1992 0.69 0.58 0.37 137.26 (0.73, 0, 0.68) (0.61, 0.45, −0.66) KKP 2001 0.72 0.47 0.48 107.37 (0.85, 0 ,0.53) (0.45, 0.51, −0.73) LB 2007 0.63 0.45 0.41 106.75 (0.79, 0, 0.61) (0.57, 0.36, −0.74) Table 3. Average data on the two-stage gamble tests by Tversky and Shaﬁr (TS 1992) [11], Kühberger, Kamunska and Perner (KKP 2001) [52], and Lambdin and Burdsal (LB 2007) [53] on the disjunction effect. Values obtained from the quantum in Section 7.2 are also reported. Tversky-Shafir’s explanation is that people are generally “averse to uncertainty”, that is, they prefer sure over unsure actions. In the two-stage gamble test, participants specifically prefer to play again in both cases where they have certainty about the outcome of the first gamble, while they refuse to play again when they do not know the outcome of the first gamble. 5. Decision-Making Errors: Ellsberg-Type Paradoxes EUT is the prevalent theory of DM under uncertainty. Because of its intuitive character, mathematical simplicity and predictive success, EUT has been widely applied to a wide range of choice problems in economics, finance, management and also medicine. The main tenet of EUT is that, in situations of uncertainty individual agents (or, decision-makers) choose in such a way to maximize their utility, or degree of satisfaction. In the 1940s, von Neumann and Morgenstern proposed a set of “reasonable” axioms on human preferences which allow to uniquely represent uncertain gambles (or, lotteries) by means of a suitable expected utility (EU) functional [54]. However, this formulation only deals with the uncertainty that can be formalized by known probabilities (“objective uncertainty”, or risk). On the other side, situations frequently occur in social science where uncertainty cannot be formalized by known probabilities (“subjective uncertainty, or “ambiguity”) [55]. The so-called “Bayesian paradigm” minimizes the distinction introducing the notion of “subjective probability”: even when probabilities are not known, people may still form their own beliefs (or, priors), and they would aim to maximize EU with respect to these priors [56]. And, indeed, Savage presented in the 1950s an axiomatic formulation Entropy 2020, 22, 738 12 of 34 of EUT which extends von Neumann and Morgenstern’s to subjective uncertainty (Savage’s SEUT, cfr., Section 4) [2]. Let us provide a simpliﬁed presentation of SEUT. Let S be a (discrete, ﬁnite) set of all physical states of nature, let {E1, . . . , En} be a family of mutually exclusive and exhaustive elementary events, that is, for every i, j = 1, . . . , n, i ̸= j, Ei = {si} ⊆S , Ei ∩Ej = ∅, and ∪n i=1Ei = S . For every i = 1, . . . , n, let p(Ei) be the (subjective) probability that Ei occurs and suppose that p is a Kolmogorovian probability measure. Then, let {x1, . . . , xn} be a family of monetary payoffs (payoffs are special cases of consequences in SEUT). For every i = 1, . . . , n, let u(xi) be the utility value of xi (u is a risk-dependent continuous and strictly increasing utility function in SEUT). Finally, let f = (E1, x1; . . . , En, xn) deﬁne an “act”. 5. Decision-Making Errors: Ellsberg-Type Paradoxes Then, the EU of f with respect to the Kolmogorovian probability measure p is deﬁned as W( f ) = n ∑ i=1 p(Ei)u(xi). (19) (19) Savage proved that, if the set F of all acts satisﬁes a number of “reasonable” axioms, including the sure-thing principle, then, for every f, g ∈F, a single Kolmogorovian probability measure p and a single (up to positive afﬁne transformations) utility function u exist such that f is preferred to g (or, f ≿g) if and only if W( f ) ≥W(g) [2]. Savage’s theorem is compelling from a normative point of view and testable from a descriptive point of view. Regarding the former, if decision-makers are “rational”, in the sense that their decisions reveal axioms satisfying preferences, then they must all behave as if they maximized an EU with respect to a single Kolmogorovian probability measure interpreted as their subjective probability. Regarding the latter, the validity of SEUT and its underlying axioms can be concretely tested. And, indeed, deviations from classicality have occurred in various DM tests. More specifically, Daniel Ellsberg proved in two seminal thought experiments, the “three-color example” and the “two-urn example”, that decision-makers violate the predictions of SEUT, because they prefer in general acts with known probabilities over acts with unknown probabilities, instead of maximizing EU [12]. Let us start by the three-color example. One urn contains 30 red balls and 60 balls that are either yellow or black, the latter in unknown proportion. One ball will be drawn at random from the urn. Then, a person is asked to bet on pairs of the acts f1, f2, f3 and f4 in Table 4. Table 4. Payoff table for the Ellsberg three-color example. Table 4. Payoff table for the Ellsberg three-color example. p(ER) = 1/3 p(EY) + p(EB) = 2/3 Act Red Yellow Black f1 $100 $0 $0 f2 $0 $0 $100 f3 $100 $100 $0 f4 $0 $100 $100 Ellsberg suggested that most individuals will choose f1, revealing the preference f1 ≻f2, and f4, revealing the preference f4 ≻f3. This is intuitively reasonable, as f1 and f4 are unambiguous acts, that is, are associated with events over known probabilities, whereas f2 and f3 are ambiguous acts, that is, are associated with events over unknown probabilities. This attitude of decision-makers to prefer known over unknown probabilities is called “ambiguity aversion” [12]. 5. Decision-Making Errors: Ellsberg-Type Paradoxes Ellsberg preferences above violate the sure-thing principle of SEUT, because the latter predicts consistency of preferences, that is, f1 ≻ f2 if and only if f3 ≻ f4. Indeed, if we denote by p(ER) = 1/3, p(EY) and p(EB) the Kolmogorovian probabilities associated with the events “a red ball is drawn”, “a yellow ball is drawn” and “a black ball is drawn”, respectively, then the EUs are 13 of 34 Entropy 2020, 22, 738 such that f1 ≻f2 if and only if W( f1) > W( f2) if and only if (1/3 −p(EB))(u(100) −u(0)) > 0 if and only if W( f3) > W( f4) if and only if f3 ≻f4. Equivalently, no assignment of Kolmogorovian probabilities p(ER) = 1/3, p(EY) and p(EB) reproduces Ellsberg preferences f1 ≻f2 and f4 ≻f3, with W( f1) > W( f2) and W( f4) > W( f3), respectively, whence the “Ellsberg paradox”. Tests on simple urns, but also on ﬁnancial, management, insurance and medical choices, generally conﬁrm Ellsberg preferences, in most cases indicating ambiguity aversion attitudes (ambiguity seeking attitudes have been identiﬁed in a few cases; for a review of empirical studies, see, for example, References [57,58]). A recent test on the three-color example, performed by ourselves, has found a rate of 0.82 for the preference f1 ≻f2 and a rate of 0.78 for the preference f4 ≻f3. The test thus agrees with empirical patterns found in the literature [59]. Various extensions of SEUT have been elaborated to cope with the Ellsberg paradox and non-neutral attitudes towards ambiguity. These “non-EU models” in general weaken the sure-thing principle and explicitly incorporate ambiguity in their formulations. Major models include Choquet EU, cumulative prospect theory, max-min EU, α-max min EU, variational preferences, robust control, and second order beliefs (for a review of theoretical studies, see, for example, References [58,60] and references therein). The models above accommodate the Ellsberg paradox and have manifold applications in social science. More important, they already depart from the assumption that only Kolmogorovian probabilities can represent subjective priors. However, Mark Machina has recently proved that major non-EU models cannot reproduce his hypothesized preferences in two thought experiments, namely, the “50/51 example” and the “reﬂection example” [61,62]. Some DM tests have conﬁrmed Machina preferences against the predictions of the non-EU models above [59,63]. 5. Decision-Making Errors: Ellsberg-Type Paradoxes The situation becomes even more problematical if one considers the Ellsberg two-urn example, which constitutes the paradigm of real life tests involving managerial and medical decisions. We briefly review the two-urn example and its consequences in the following. Consider two urns, “urn I” with 100 balls that are either red or black in unknown proportion, and “urn II” exactly with 50 red balls and 50 black balls. One ball is to be drawn at random from each urn. Then, free of charge, a person is asked to bet on pairs of the acts f1, f2, f3 and f4 in Table 5. Table 5. Payoff matrix for the Ellsberg two-urn example. Urn I Urn II Acts ER: Red Ball EB: Black Ball ER: Red Ball EB: Black Ball p(ER) ∈[0, 1] p(EB) = 1 −p(ER) p(ER) = 1/2 p(EB) = 1/2 f1 $100 $0 f2 $100 $0 f3 $0 $100 f4 $0 $100 Table 5. Payoff matrix for the Ellsberg two-urn example. In this case, acts f2 and f4 are unambiguous, whereas acts f1 and f3 are ambiguous. Thus, Ellsberg suggested that, while decision-makers will be generally indifferent between f1 and f3 and between f2 and f4, but they will prefer f2 over f1 and f4 over f3, thus revealing preferences f2 ≻f1 and f4 ≻f3, together with an ambiguity aversion attitude. The predictions of SEUT are incompatible with the Ellsberg preferences above. Indeed, if we we denote by p(ER) and p(EB) = 1 −p(EB) the Kolmogorovian probabilities associated with the events “a red ball is drawn from Urn I” and “a black ball is drawn from Urn I”, respectively, then the EUs are such that f2 ≻f1 if and only if W( f2) > W( f1) if and only if (p(ER) −1/2)(u(100) −u(0)) < 0 if and only if W( f3) > W( f4) if and only if f3 ≻f4. Tests on the two-urn example generally conﬁrm Ellsberg preferences, hence an ambiguity aversion attitude of decision-makers. We have tested the two-urn Entropy 2020, 22, 738 14 of 34 example too, ﬁnding a rate of 0.82 for the preference f2 ≻f1 and a rate of 0.84 for the preference f4 ≻f3. The test thus agrees with empirical patterns found in the literature [59]. In each bet, the two-urn example consists in comparing a risky option (Urn II) with an ambiguous option (Urn I). 6. Elaboration of a SCoP Formalism for Cognitive Domains As anticipated in Section 1, the quantum cognition research programme rests on the recognition that complex cognitive phenomena, like judgements and decisions, exhibit peculiar aspects which prevent in general the application of traditional modelling techniques based on classical set-theoretic formalisms. In particular, the following features have arisen from the phenomena studied in Sections 2–5. (i) Judgements and decisions are intrinsically and unavoidably probabilistic processes. Judgements and decisions involve constructive processes which create rather than record. (iii) In these processes, context plays a fundamental role in determining the ﬁnal response among a range of possible alternatives. (iii) In these processes, context plays a fundamental role in determining the ﬁnal response among a range of possible alternatives. g p (iv) Different and mutually exclusive alternatives generally disturb each other. (v) The unrestricted validity classical (Boolean) logic and (Kolmogorovian) probability cannot be assumed in these cases. (v) The unrestricted validity classical (Boolean) logic and (Kolmogorovian) probability cannot be assumed in these cases. These and other considerations have lead several scholars to start using systematically the formalism of quantum theory in Hilbert space, as pure mathematical formalism detached from its physical interpretation, in these classically problematical cognitive phenomena (see, e.g., References [36,37] and references therein). In this section, we aim to present a uniﬁed view on how and why the mathematical formalism of quantum theory is able to provide a general theoretical framework for human cognition. In other words, we aim to provide a foundation of human cognition which justiﬁes the use of quantum theory as a general theory for it. The starting point of our research was the recognition that, in view of points (i)–(v), the systems (or, entities) studied in cognitive domains exhibit deep analogies with quantum entities in the description of what occurs in a laboratory where preparations and measurements are performed. Indeed, an experiment, or test, on a quantum entity is typically performed in a physics laboratory. The quantum entity preliminarily undergoes a preparation procedure designed by the experimenter, at the end of which “the entity is in a deﬁned state”. This state expresses the “physical reality” of the entity, in the sense that, as a consequence of being in that state, the entity has some “actual” properties independently of any measurement that can be performed on it. 5. Decision-Making Errors: Ellsberg-Type Paradoxes This is the typical scenario one ﬁnds in real life tests involving ﬁnancial, managerial and medical decisions (see, e.g., References [64,65]). One observes in these tests a shift from ambiguity averse to ambiguity seeking attitudes, and viceversa, as due to the presence of “hope and fear effects”. In these situations, indeed, a benchmark value is set which determines whether the decision leads to a “gain”, for example, a better than expected return on an investment, or a “loss”, for example, a failure in a medical treatment, and intuition suggests that the level of probability will play a crucial role in the ﬁnal decision: if the probability of a gain (loss) is high, then a fear effect (hope effect) occurs in which people tend to be ambiguity averse (seeking). But, as the probability of a gain (loss) decreases, people tend to be less ambiguity averse (seeking), reaching a crossover point in which they become ambiguity seeking (averse), which indicates a shift from a fear (hope) to a hope (fear) effect [64,65]. Shifts of ambiguity attitudes, as due to hope and fear effects, cannot be explained, either from the point of view of traditional SEUT nor from the point of view of more general non-EU models. The consequence of the empirical ﬁndings summarized in this section is that, according to many authors, a uniﬁed theory of human choices under uncertainty is still missing (see, e.g., Reference [66]). 6. Elaboration of a SCoP Formalism for Cognitive Domains Then, when a measurement is performed on the quantum entity, the macroscopic apparatus operates as a measurement context which interacts, on a physical level, with the entity and changes its state in a way that is generally 15 of 34 15 of 34 Entropy 2020, 22, 738 indeterministic, that is, neither controllable nor predictable. Then, the quantum entity, its states, contexts, properties, and the mutual statistical relations are represented in the Hilbert space formalism. Similarly, a cognitive test, or measurement, is typically performed in a “psychology laboratory”, that is, a spatio-temporal domain where participants give their responses. Suppose, for example, that a judgement/decision test is performed in which a sample of participants have to write down their responses on a paper picking them from a list on a structured questionnaire. The information contained in the questionnaire and the meaning content of the situation that is the object of the decision (literally, what is written in the questionnaire and which has to be judged/decided on) deﬁne a preparation procedure for a “conceptual entity”, at the end of which we can say that “the entity is in a deﬁned state”. Thus, a preparation does take place when a cognitive test is performed and each participant “is confronted with this one and unique state”, independently of any belief the participant has about it, because the state was prepared by the experimenter designing the test, before and independently of any individual participating in the test. This state has a conceptual, rather than a physical, nature, but it is a “state of affairs”, because it expresses the meaning content of the questionnaire that was prepared by the experimenter. As such, it is not a mental state or a state of belief. In addition, this state is independent of any operation that can be performed on the entity, hence it expresses the “conceptual reality” of the entity at a given time. The state of a conceptual entity can change under the effect of the surrounding context, which has in general a cognitive nature and may include the individual participating in the test. Indeed, when the test is performed and a participant is asked to express a judgement or make a decision on the questionnaire, the participant operates as a context which interacts, on a cognitive level, with the entity and changes its state in a way that is generally indeterministic, that is, neither controllable nor predictable. 6. Elaboration of a SCoP Formalism for Cognitive Domains A property a ∈L can be either actual or potential for Ω, depending on its state. (4) µ is the “state-transition probability function”, that is, an application µ : Σ × M × Σ −→[0, 1], such that, for every p, q ∈Σ, e ∈M , µ(q, e, p) is the probability that the state p of Ωis changed to the state q by the context e. (4) µ is the “state-transition probability function”, that is, an application µ : Σ × M × Σ −→[0, 1], such that, for every p, q ∈Σ, e ∈M , µ(q, e, p) is the probability that the state p of Ωis changed to the state q by the context e. (5) ν is the “property-applicability probability function”, that is, an application ν : Σ × L −→[0, 1], such that, for every p, ∈Σ, a ∈L , ν(p, a) is the probability that the property a is actual in the state p of Ω. (5) ν is the “property-applicability probability function”, that is, an application ν : Σ × L −→[0, 1], such that, for every p, ∈Σ, a ∈L , ν(p, a) is the probability that the property a is actual in the state p of Ω. The above realistic-operational description of any conceptual entity involved in a judgement or a decision in the SCoP formalism suggest representing these entities, states, contexts, properties, probabilities and dynamics within the Hilbert space formalism, in the sense that, in the modelling of a given phenomenon in the cognitive domain, once one identifies the conceptual entity, its states, contexts, properties and statistics of responses, one represents them by means of the usual representation in Hilbert space. Before coming to the applications, we stress that this description of a judgement/decision process differs from that of other quantum-based approaches to cognition, where the state corresponds to a “mental state of the individual participating in the test” (see, e.g., References [4,6,25]). While the latter description is frequently used, we believe that it does not completely capture all elements involved in such processes, namely, preparation, contextual interaction, state change. In particular, the notion of “state of a conceptual entity” introduces a new element, not directly related to beliefs and not previously used in cognitive science, at the best of our knowledge. 6. Elaboration of a SCoP Formalism for Cognitive Domains The notion is mainly borrowed from physics, as a test in cognitive science, like an experiment in physics, is a bridge between a preparation and a measurement. We believe that this notion of state should be a constitutive element of any statistical theory. This is why we prefer to adopt the description presented here, as it also adheres more closely to the interpretation of the Hilbert space formalism expounded in modern manuals of quantum theory (see, e.g., Reference [21]). In the following, we will specify the SCoP formalism for probability judgement tests, like those performed on conceptual combinations (Section 6.1), and for DM tests, like those performed on Ellsberg-type situations (Section 6.2), and sketch how a Hilbert space representation of these phenomena can be constructed. 6. Elaboration of a SCoP Formalism for Cognitive Domains In the SCoP formalism, Ωis described by the 5-tuple (Σ, M , L , µ, ν), where: Entropy 2020, 22, 738 16 of 34 (1) Σ is the set of all states of Ω. A state p ∈Σ is the result of a preparation procedure of Ωat the end of which Ωis in the state p. (1) Σ is the set of all states of Ω. A state p ∈Σ is the result of a preparation procedure of Ωat the end of which Ωis in the state p. (2) M is the set of all contexts of Ω. A context e ∈M has typically a cognitive nature and interacts with Ωchanging in general its state. (3) L is the set of all properties of Ω. A property a ∈L can be either actual or potential for Ω, depending on its state. (4) µ is the “state-transition probability function”, that is, an application µ : Σ × M × Σ −→[0, 1], such that, for every p, q ∈Σ, e ∈M , µ(q, e, p) is the probability that the state p of Ωis changed to the state q by the context e. (5) ν is the “property-applicability probability function”, that is, an application ν : Σ × L −→[0, 1], such that, for every p, ∈Σ, a ∈L , ν(p, a) is the probability that the property a is actual in the (1) Σ is the set of all states of Ω. A state p ∈Σ is the result of a preparation procedure of Ωat the end of which Ωis in the state p. (1) Σ is the set of all states of Ω. A state p ∈Σ is the result of a preparation procedure of Ωat the end of which Ωis in the state p. (2) M is the set of all contexts of Ω. A context e ∈M has typically a cognitive nature and interacts with Ωchanging in general its state. (2) M is the set of all contexts of Ω. A context e ∈M has typically a cognitive nature and interacts with Ωchanging in general its state. (3) L is the set of all properties of Ω. A property a ∈L can be either actual or potential for Ω, depending on its state. (3) L is the set of all properties of Ω. 6. Elaboration of a SCoP Formalism for Cognitive Domains More specifically, when the first participant enters the laboratory and fills out the questionnaire, the participant “interacts with this conceptual state presented to her/him in the test”, but prepared, for example, on a paper, by the experimenter, and changes this state. When the second participant enters the laboratory and fills out the questionnaire, the participant again interacts with this independently prepared conceptual state and contextually changes it, and so on – different individuals may determine different changes of state. As a consequence of this “(measurement) context-induced actualization of potential”, a statistics of responses can be collected, which can be interpreted as outcome probabilities in the large number limit. In addition, some properties of the conceptual entity, which were actual (potential) in the initial state in which the entity is originally prepared, will become potential (actual) in the final state, as a consequence of this contextual interaction. Now, research on the foundations of quantum physics and quantum probability has allowed to recover the Hilbert space formalism of quantum theory from “operationally reasonable” axioms, resting on well deﬁned empirical notions, directly connected with the operations that are performed in a physics laboratory. In particular, one line of research, initiated by Jauch [67] and Piron [68] in Geneva, and further developed by the research team in Brussels (see, e.g., References [36,37]), has produced a “realistic” and “operational” foundation of this kind, which was called the “State Context Property” (SCoP) formalism. In the ScoP formalism, any physical entity is described by means of the basic notions of “state,” “context,” “property,” and their mutual statistical relations. If suitable axioms are imposed on the mathematical structure underlying the SCoP formalism, then the Hilbert space structure of quantum theory emerges as a unique mathematical representation, up to isomorphisms (see, e.g., References [36,67,68]). Then, following the analogies above between quantum and conceptual entities, we have elaborated a realistic-operational foundation of conceptual entities, in which any entity of this kind is described in terms of its states, contexts, properties, and statistics of outcomes [69]. We resume and deepen the SCoP description of a conceptual entity in the following. Let Ωbe a conceptual entity, which can be a concept, a combination of concepts, a proposition, or a more complex DM entity. 6.1. Application of the SCoP Formalism to Human Probability Judgements Let us now consider a membership judgement test in which each participant is asked to judge membership of the item X with respect to the concept A. In the SCoP formalism, such test corresponds to a measurement with only two outcomes, or “yes-no measurement”, performed by means of a membership measurement context eM, in which a property aX of the conceptual entity ΩA is tested. Hence, the probability of membership, or membership weight, of the item X with respect to the concept A is formalized by means of a property-applicability probability ν(pA, aX). Let us now come to the representation in Hilbert space and start by a membership judgement test. The conceptual entity ΩA is associated with an abstract Hilbert space H, and the initial state pA of A is represented by a unit vector \|A⟩∈H. The membership measurement context eM is represented by a hermitian operator or, equivalently, by a spectral family {M, 1 −M}, where M is an orthogonal projection operator over H and 1 is the identity operator. Then, the membership weight µ(A) of X with respect to A coincides with the probability that the outcome “yes” is obtained in the interaction of ΩA in the state pA with eM, and is calculated through the Born rule of quantum probability, that is, µ(A) = ∥M\|A⟩∥2. The Born rule also applies to measurements with more than two outcomes. For example, in a typicality judgement test involving the typicality of a family {X1, . . . , Xn} of n distinct items with respect to the concept A, the typicality measurement context eT is represented by the spectral measure {M1, . . . , Mn}, where ∑n i=1 Mi = 1 and MiMj = δijMi, i, j = 1, . . . , n, and the probability µi(A) that the item Xi is judged as a typical example of the concept A is given by the quantum probability µi(A) = ∥Mi\|A⟩∥2, i = 1, . . . , n. An interesting aspect concerns the ﬁnal state of a conceptual entity ΩA after a judgement test. We assume the existence of a non-empty class of cognitive tests that correspond to ideal ﬁrst kind measurements in the standard quantum sense, that is, measurements that satisfy the “Lüders postulate”. Suppose for example that the item Xi, i = 1, . . . 6.1. Application of the SCoP Formalism to Human Probability Judgements We apply in this section the SCoP formalism to the judgements that are expressed when participants are asked about typicality and membership probabilities with respect to concepts and their combinations. This will allow us to construct a general Hilbert space representation of the phenomenology encountered in Section 2. Let A be a concept and let us consider judgement tests in which a sample of participants are asked in a questionnaire to judge typicality or membership of speciﬁc items with respect to the concept A, as in Section 2. We associate A with the conceptual entity ΩA following the prescriptions in Section 6. In particular, the information and meaning content of the questionnaire provides a preparation of ΩA at the end of which ΩA is in a deﬁned state pA. A context e interacts with the conceptual entity ΩA and can change its initial state pA. A relevant context is the one introduced by the judgement test itself. Indeed, when the test starts, an interaction occurs between the conceptual entity ΩA and each participant, in which the state pA of ΩA generally changes, being transformed into another state p. This interaction, which occurs at a cognitive level, as 17 of 34 Entropy 2020, 22, 738 we have seen in Section 6, is speciﬁcally described by a measurement context. For example, when in a typicality judgement each participant is asked to pick the most typical item in the family {X1, ..., Xn} of n distinct items and the participant chooses the item Xi, i = 1, . . . , n, then the initial state pA of ΩA is transformed by the typicality measurement context eT into pXi, as a consequence of the contextual interaction between ΩA in pA and eT. The change of state of ΩA due to a context e may be either “deterministic”, hence in principle predictable under the assumption that both pA and e are perfectly known, or “intrinsically probabilistic”, that is, only the state-transition probability µ(p, e, pA) that the state pA of ΩA changes to the state p is known. Speciﬁcally, in the typicality judgement test above, the typicality of the item Xi with respect to the concept A is formalized by means of a state-transition probability µ(pXi, eT, pA), where eT is the typicality measurement context and pXi is the ﬁnal state of ΩA at the end of the interaction with eT. 6.2. Application of the SCoP Formalism to Human Decisions plication of the SCoP Formalism to Human Decisions 6.2. Application of the SCoP Formalism to Human Decisions We apply in this section the realistic-operational description of a conceptual entity to the DM tests studied in Section 5 and sketch how a Hilbert space representation can be constructed in these cases. Let us consider a decision problem where n mutually exclusive and exhaustive elementary events, E1, . . . , En may occur and the decision-maker has to take a decision between pairs of acts giving different payoffs which depend on the occurrence of E1, . . . , En. If the required decision has to be taken in the form of a written answer on a questionnaire, a story is preliminarily presented to each individual participating in the test. What each individual reads in the questionnaire, interacts with and decides on deﬁnes a preparation of a conceptual entity, which we call “DM entity”, ΩDM. A state p of ΩDM has a conceptual, not physical, nature and incorporates aspects of uncertainty (e.g., ambiguity). A context e does not pertain to ΩDM but can interact with it. The interaction of ΩDM with a context e may determine a change of the state of ΩDM from p to a different state q. The probability of such a state transition is given by the state-transition probability µ(q, e, p), in agreement with the prescriptions in Section 6. We can also complete the SCoP description of ΩDM introducing properties and property-applicability probabilities, which is not relevant to Ellsberg-type situations but may play a role in more complex decision problems. A ﬁrst cognitive context eC relevant to ΩDM is the context associated with the the n mutually exclusive and exhaustive elementary events E1, . . . , En. It corresponds to a measurement that can be performed on ΩDM in a given state p and has n distinct outcomes, or eigenvalues. Each eigenvalue is associated with a ﬁnal state pi of ΩDM, which is an eigenstate of eC, as it satisﬁes the condition µ(pi, eC, pi) = 1. Thus, the context eC is connected with the elementary event Ei by the fact that the (subjective) probability µ(Ei, p) that the event Ei occurs when the conceptual entity ΩDM is in the state p is formalized by means of the state-transition probability µ(pi, eC, p), i = 1, . . . , n. 6.2. Application of the SCoP Formalism to Human Decisions Then, in analogy with SEUT, we can deﬁne an act f = (E1, x1; . . . , En, xn) by associating the monetary payoff xi to the event Ei, i = 1, . . . , n. Let us now come to the realistic-operational description of the DM process and consider a decision between acts f1 and f2 and a decision between acts f3 and f4 in Table 4, Section 5, for the sake of simplicity. Suppose that the meaning content of the questionnaire has prepared the conceptual entity ΩDM in the initial state p0. Whenever an individual starts pondering between f1 and f2, before a decision is taken, the individual’s attitude towards ambiguity, for example, ambiguity aversion, is described as a new cognitive context e12 operating on ΩDM in the initial state p0 and changing p0 to a new state p12. The (subjective) probability that the event Ei occurs in the state p12 is thus µ(Ei, p12) = µ(pi, eC, p12), i = 1, . . . , n. Similarly, whenever the individual starts pondering between f3 and f4, before a decision is taken, the individual’s attitude towards ambiguity, for example, again ambiguity aversion, is described as another cognitive context e34 operating on ΩDM in the initial state p0 and changing p0 to a new state p34. The (subjective) probability that the event Ei occurs in the state p34 is thus µ(Ei, p34) = µ(pi, eC, p34), i = 1, . . . , n. The ambiguity averse final states p34 and p34 are responsible of the “inversion of preferences” which occur in Ellsberg-type situations [70]. Finally, the actual decision between acts f1 and f2 is operationally described as a decision measurement context eD1 acting on ΩDM in the ambiguity averse state p12, with possible outcomes “yes” and “no”. Similarly, the actual decision between acts f3 and f4 is operationally described as a decision measurement context eD2 acting on ΩDM in the ambiguity averse state p34, with possible outcomes “yes” and “no”. These contexts give rise of the statistics of outcomes in Ellsberg-type DM tests. The representation of the DM situation above is straightforward. The DM entity ΩDM is associated with an abstract Hilbert space H . The presence of n mutually exclusive and exhaustive elementary events suggests choosing H in such a way that it is (isomorphic to) the the complex Hilbert space Cn. 6.1. Application of the SCoP Formalism to Human Probability Judgements , n, was judged as a typical example of the concept A in the typicality test above. Then, we assume that the ﬁnal state pXi of ΩA immediately after the interaction with the typicality measurement context eT is represented by the unit vector \|Ai⟩= Mi\|A⟩ ∥Mi\|A⟩∥. Of course, we agree that cognitive tests exist which do not correspond to ideal ﬁrst kind measurements of quantum theory. Nevertheless, it is reasonable to assume that such an ideal ﬁrst kind state transition occurs at least in some cases, like the ones discussed here [69]. Coming to a more concrete Hilbert space construction, in a judgement test with n distinct outcomes, we associate ΩA with a n-dimensional complex Hilbert space H , which can be chosen to be (isomorphic to) the space Cn, that is, the Hilbert space of all ordered triples of complex numbers. Then, the states of ΩA are represented by unit vectors of Cn and each orthogonal projection operator Mi projects onto the one-dimensional subspace generated by the i-th vector of the canonical orthonormal (ON) basis of Cn, i = 1, . . . , n. The mathematical construction we have put forward here provides a general framework allowing in principle to represent any probability judgement test in Hilbert space. We will apply the present Entropy 2020, 22, 738 18 of 34 quantum-theoretic framework to over- and under-extension effects and conjunctive and disjunctive fallacies and the disjunction effect in Sections 7, 7.1 and 7.2, respectively. 7. A Quantum Framework to Represent Concepts and Their Combinations Relying on a one-decade research on the foundations of quantum theory (cfr., Section 6.1), we have developed a novel framework to represent concepts and their combinations. In this framework, a conceptual entity can be expressed in terms of the SCoP formalism, hence it admits a Hilbert space representation [34,71,72]. As we have seen in Section 6, the main novelties can be summarised as follows. (i) A concept is an entity in a deﬁned state, rather than a container of items. (ii) A context is a factor that inﬂuences the concept and generally changes its state. (iii) Quantities as typicality, membership, and so forth can be measured on concepts and have different probabilistic values in different states of the concept. (iii) Quantities as typicality, membership, and so forth can be measured on concepts and have different probabilistic values in different states of the concept. We have applied the mathematical formalism of quantum theory to represent conceptual entities, states, contexts and the corresponding probabilities. The quantum-theoretic framework we have developed successfully models membership weights of concepts and their combinations [22,31,32,34,35], conceptual typicality [34,71,72] and borderline contradictions [73] in Section 2. We expose now the fundamentals of the quantum-theoretic framework, starting from the conjunction of two concepts. We refer to the membership probability judgement tests in Section 2. Let X be an item, let A and B be two concepts, and let ‘A and B’ be their conjunction. Then, let µ(A), µ(B) and µ(A and B) be the membership weights of X with respect to A, B and ‘A and B’, respectively. As seen in Section 6.1, conceptual entities are represented in an abstract Hilbert space H . When a membership judgement test is performed and a questionnaire is presented to the participants, the initial state of the concept A is represented by the unit vector \|A⟩∈H , ∥\|A⟩∥= 1, and the initial state of the concept B is represented by the unit vector \|B⟩∈H , ∥\|B⟩∥= 1. Suppose that \|A⟩and \|B⟩are orthogonal, that is, ⟨A\|B⟩= 0, for the sake of simplicity. We represent the initial state of the conjunction ‘A and B’ by the unit vector \|A and B⟩= 1 √ 2(\|A⟩+ \|B⟩). √ 2 The judgement measurement of a person who judges the membership of X with respect to A, B and ‘A and B’ is represented by the orthogonal projection operator M over H . 6.2. Application of the SCoP Formalism to Human Decisions Hence, a state pv of ΩDM is represented by a unit vector \|v⟩∈Cn. A context e is instead represented 19 of 34 19 of 34 Entropy 2020, 22, 738 by a hermitian operator or, equivalently, by a spectral family, over Cn. Thus, the context eC above is represented by the spectral family {P1, . . . , Pn}, where each Pi represents the elementary event Ei and projects onto the one-dimensional subspace generated by the i-th vector of the canonical ON basis of Cn. Then, the (subjective) probability µ(p, Ei) that the event Ei, represented by the orthogonal projection operator Pi, occurs when ΩDM is in the state pv, represented by the unit vector \|v⟩, is obtained through the Born rule of quantum probability, namely, µ(p, Ei) = ∥Pi\|v⟩∥2. An act f = (E1, x1; . . . , En, xn) is represented by the hermitian operator F = ∑n i=1 u(xi)Pi, where u(xi) is the utility value associated with the payoff xi. This suggests to ﬁnally introduce a state-dependent EU functional Wv( f ) = ⟨v\|F\|v⟩. p y gg y p f The mathematical construction we have put forward here provides a general framework allowing in principle to represent any DM test in Hilbert space. We will apply the present quantum-theoretic framework to the Ellsberg three-color and two-urn examples in Section 8. 7. A Quantum Framework to Represent Concepts and Their Combinations We calculate membership weights by means of the Born rule of quantum probability. More precisely, µ(A) = ⟨A\|M\|A⟩, (20) µ(B) = ⟨B\|M\|B⟩ (21) µ(A) = ⟨A\|M\|A⟩, (20) µ(B) = ⟨B\|M\|B⟩ (21) (20) (21) (20) (20) (21) (21) are the probabilities of membership, that is, membership weights, while are the probabilities of membership, that is, membership weights, while (22) (23) 1 −µ(A) = ⟨A\|1 −M\|A⟩, (22) 1 −µ(B) = ⟨B\|1 −M\|B⟩ (23) (22) (23) ( ) (23) (23) 20 of 34 Entropy 2020, 22, 738 are the probabilities of non-membership, in the initial states of concepts A and B. The probability of membership in the initial state of ‘A and B’ is thus given by: are the probabilities of non-membership, in the initial states of concepts A and B. The probability of membership in the initial state of ‘A and B’ is thus given by: µ(A and B) = ⟨A and B\|M\|A and B⟩= 1 2 µ(A) + µ(B) + Re(⟨A\|M\|B⟩). (24) (24) Equation (24) expresses the “quantum probability formula for the conjunction”. Different cases are possible for the interference term for the conjunction Re(⟨A\|M\|B⟩), namely, • Re(⟨A\|M\|B⟩) > 0 indicates to constructive interference; • Re(⟨A\|M\|B⟩) > 0 indicates to constructive interference; • Re(⟨A\|M\|B⟩) = 0 indicates to absence of interference; • Re(⟨A\|M\|B⟩) = 0 indicates to absence of interference; • Re(⟨A\|M\|B⟩) < 0 indicates to destructive interference. • Re(⟨A\|M\|B⟩) < 0 indicates to destructive interference. We follow the same procedure for the disjunction of two concepts. Let µ(A), µ(B) and µ(A or B) be the membership weights of the item X with respect to the concepts A, B and their disjunction ‘A and B’, respectively. When a membership judgement test is performed and a questionnaire is presented to the participants, the initial state of the concept A is represented by the unit vector \|A⟩∈H , and the initial state of the concept B is represented by the unit vector \|B⟩∈H , with ⟨A\|B⟩= 0. Then, the initial state of the disjunction ‘A or B’ is represented by the unit vector \|A or B⟩= 1 √ 2(\|A⟩+ \|B⟩). 7. A Quantum Framework to Represent Concepts and Their Combinations Indeed, if µ(A) + µ(B) > 1, these equations are satisﬁed by choosing the orthogonal projection operator M to project onto the subspace generated by the unit vectors (1, 0, 0) and (0, 1, 0) of the canonical ON basis of C3. If instead µ(A) + µ(B) ≤1, the equations are satisﬁed by choosing M to project onto the subspace generated by the unit vector (0, 0, 1) of the same basis [22]. Let us ﬁnally study the disjunction case. Let X be an item and let µ(A), µ(B) and µ(A or B) be the membership weights of X with respect to A, B and ‘A or B’, respectively. We introduce the quantities a, b and γ as in Equation (26). The quantum probability formula for the conceptual disjunction in Equation (25) then becomes satisfy Equations (20)–(24). Indeed, if µ(A) + µ(B) > 1, these equations are satisﬁed by choosing the orthogonal projection operator M to project onto the subspace generated by the unit vectors (1, 0, 0) and (0, 1, 0) of the canonical ON basis of C3. If instead µ(A) + µ(B) ≤1, the equations are satisﬁed by choosing M to project onto the subspace generated by the unit vector (0, 0, 1) of the same basis [22]. Let us ﬁnally study the disjunction case. Let X be an item and let µ(A), µ(B) and µ(A or B) be the membership weights of X with respect to A, B and ‘A or B’, respectively. We introduce the quantities a, b and γ as in Equation (26). The quantum probability formula for the conceptual disjunction in Equation (25) then becomes µ(A or B) = 1 2 µ(A) + µ(B) + q (1 −a)(1 −b) cos θd, (30) (30) where θd is the “interference angle for the conjunction”. The unit vectors \|A⟩and \|B⟩are obtained from Equations (28) and (29) by replacing θc with θd. The quantum models above enable faithful representation of several data sets, including those in References [7,8]. While a quantum-theoretic framework that models all existing cases of conceptual combinations requires an extension in Fock spaces (see, e.g., References [22,34,74]), the models presented here are anyway falsiﬁable, as they provide precise predictions on the empirical relationships between membership weights. We focus on conceptual conjunctions, for the sake of brevity. 7. A Quantum Framework to Represent Concepts and Their Combinations √ 2 Using the symbols in Equations (20)–(24), we get that the probability of membership, or membership weight, of X with respect to ‘A or B’ is given by the following “quantum probability formula for the disjunction” µ(A or B) = 1 2 µ(A) + µ(B) + Re(⟨A\|M\|B⟩), (25) (25) where Re(⟨A\|M\|B⟩) is the interference term for the disjunction. The orthogonal projection operator M in Equation (24) actually depends on the item X and the conjunction ‘and’, whereas the projection operator M in Equation (25) actually depends on the item X and the disjunction ‘or’. We have omitted such dependence here, which actually makes Equations (24) and (25) different, for the sake of simplicity. Let us now construct an explicit representation for the conjunction and the disjunction of two concepts in the complex Hilbert space C3. Let {(1, 0, 0), (0, 1, 0), (0, 0, 1)} be the canonical ON basis of C3 (details can be found in References [22,34,35]). Let us again start by the conjunction case. Let X be an item and let µ(A), µ(B) and µ(A and B) be the membership weights of X with respect to A, B and ‘A and B’, respectively. We introduce the following quantities: a = ( 1 −µ(A) if µ(A) + µ(B) ≤1 µ(A) if µ(A) + µ(B) > 1 b = ( 1 −µ(B) if µ(A) + µ(B) ≤1 µ(B) if µ(A) + µ(B) > 1 γ = ( 180◦ if µ(A) + µ(B) ≤1 0 if µ(A) + µ(B) > 1 . (26) (26) The quantum probability formula for the conceptual conjunction in Equation (24) becomes µ(A and B) = 1 2 µ(A) + µ(B) + q (1 −a)(1 −b) cos θc, (27) (27) ntropy 2020, 22, 738 21 21 of 34 Entropy 2020, 22, 738 Entropy 2020, 22, 738 where θc is the “interference angle for the conjunction”. One can easily verify that the unit vectors \|A⟩ = √ a, 0, √ 1 −a , (28) \|B⟩ =    ei(θc+γ)q (1−a)(1−b) a , q a+b−1 a , − √ 1 −b if µ(A) ̸= 0 ei(θc+γ)(0, 1, 0) if µ(A) = 0 (29) (28) (29) satisfy Equations (20)–(24). 7. A Quantum Framework to Represent Concepts and Their Combinations For example, Hampton measured the membership weights of the item Mint with respect to Food, Plant and their conjunction Food and Plant, finding µ(A) = 0.87, µ(B) = 0.81 and µ(A and B) = 0.90, that is, a case of double overextension [7]. These data are modelled by θc = 67.56◦, (39) \|A⟩ = (0.93, 0, 0.36), (40) \|B⟩ = ei67.56◦(0.17, 0.88, −0.44). (41) (39) (40) (41) (40) (41) (41) Furthermore, Hampton measured the membership weights of Ashtray with respect to Home Furnishing, Furniture and their disjunction Home Furnishing or Furniture, ﬁnding µ(A) = 0.70, µ(B) = 0.30 and µ(A or B) = 0.25, that is, a case of double underextension [8]. These data are modelled by θd = 123.06◦, (42) \|A⟩ = (0.84, 0, 0.55), (43) (42) (43) (44) θd = 123.06◦, (42) \|A⟩ = (0.84, 0, 0.55), (43) \|B⟩ = ei123.06◦(0.26, 0.58, −0.77). (44) (42) (43) (44) (44) We conclude this section with two remarks which allow us to understand the effectiveness of the quantum-theoretic framework presented here, together with its predictive and explanatory capacity. We conclude this section with two remarks which allow us to understand the effectiveness of the quantum-theoretic framework presented here, together with its predictive and explanatory capacity. Firstly, we have recently generalized Hampton’s tests, extending them to conjunctions and negations of two concepts [74]. More precisely, we have tested membership weights of items with respect to two concepts A and B, their negations ‘not A’ and ‘not B’, and all possible conjunctions ‘A and B’, ‘A and not B’, ‘not A and B’, and ‘not A and not B’. We have found systematic overextension in all conjunctions, cases of double overextension, deviations from classicality in conceptual negations, and a new unexpected and significant deviation from the marginal law. For example, we have found that (45) µ(A and B) + µ(A and not B) −µ(A) ≈0.5. (45) Looking at data in Reference [74], the left side of Equation (45) is systematically different from 0, whereas a value of 0 is what one would expect in a Kolmogorovian probability framework. But, the value is also roughly constant and independent of the item and the pair of concepts. This non-classical result is exactly predicted by the Fock space model in References [22,34,74], which thus allows to obtain new classically unexplainable results [75,76]. 7. A Quantum Framework to Represent Concepts and Their Combinations Let us set ∆ µ(A and B) 1 µ(A) + µ(B) and distinguish two cases as follows ∆AB = µ(A and B) −1 2 µ(A) + µ(B) and distinguish two cases, as follows. If µ(A) + µ(B) ≤1, µ(A), µ(B) ̸= 1, then, µ(B) ̸= 1, then, − q µ(A)µ(B) ≤∆AB ≤ q µ(A)µ(B). (31) If µ(A) + µ(B) > 1, µ(A), µ(B) ̸= 1, then, − q (1 −µ(A))(1 −µ(B)) ≤∆AB ≤ q (1 −µ(A))(1 −µ(B)). (32) − q µ(A)µ(B) ≤∆AB ≤ q µ(A)µ(B). (31) (31) − q (1 −µ(A))(1 −µ(B)) ≤∆AB ≤ q (1 −µ(A))(1 −µ(B)). (32) (32) Similar constraints have to be satisﬁed in the disjunction case for the quantum model to be able to represent empirical data. Similar constraints have to be satisﬁed in the disjunction case for the quantum model to be able to represent empirical data. Let us now consider some relevant examples. In the case of Razor with respect to Weapons, Tools and their conjunction Weapons and Tools, in Section (2), the quantum model gives θc = 64.02◦, (33) \|A⟩ = (0.79, 0, 0.61), (34) \|B⟩ = ei64.02◦(0.36, 0.81, −0.47). (35) θc = 64.02◦, (33) \|A⟩ = (0.79, 0, 0.61), (34) \|B⟩ = ei64.02◦(0.36, 0.81, −0.47). (35) (33) (34) (35) (33) (35) is, the concepts A and B “constructively interfere” in the conjunction ‘A and B’. the case of Curry with respect to Spices, Herbs and their disjunction Spices or Herbs, in Section (2), antum model gives In the case of Curry with respect to Spices, Herbs and their disjunction Spices or Herbs, in Section (2) quantum model gives θd = 65.91◦, (36) \|A⟩ = (0.95, 0, 0.32), (37) \|B⟩ = ei65.91◦(0.26, 0.58, −0.77). (38) (36) (37) (38) θd = 65.91◦, (36) \|A⟩ (0 95 0 0 32) (37) θd = 65.91◦, (36) \|A⟩ = (0.95, 0, 0.32), (37) d , ( ) \|A⟩ = (0.95, 0, 0.32), (37) \|A⟩ = (0.95, 0, 0.32), (37) \|B⟩ = ei65.91◦(0.26, 0.58, −0.77). (38) \|B⟩ = ei65.91◦(0.26, 0.58, −0.77). (38) (38) 22 of 34 Entropy 2020, 22, 738 Also in this case, the concepts A and B “constructively interfere” in the disjunction ‘A or B’. The quantum model also works in other cases that are classically highly problematical. 7. A Quantum Framework to Represent Concepts and Their Combinations Secondly, the quantum-theoretic framework presented here reveals the presence of genuine quantum effects in the formation and combination of natural concepts, namely, contextuality, interference, superposition, and emergence. In particular, to grasp the importance of the latter, let two compare the following experimental results in References [8,74] for the item Olive with respect to the natural concepts Fruits, Vegetables, Fruits and Vegetables, and Fruits or Vegetables. Hampton found µ(A) = 0.50, µ(B) = 0.10 and µ(A or B) = 0.80 in his test [8], whereas we found µ(A) = 0.56, µ(B) = 0.63 and µ(A and B) = 0.65 in ours [74]. Despite individual numerical differences, which can however be explained [74], both data exhibit non-classicality, namely, violation of Equation (16) in the disjunction and double overextension in the conjunction. More important, Olive scores similar membership weights in the disjunction and the conjunction case, that is, its membership weight is substantially independent of the combination that is considered. This clearly indicates that situations exist where people do not really take into account whether two concepts are combined through the connective ‘or’ or the connective ‘and’, but they actually judge whether the given item is a member of the new emergent concept, meant as a standalone entity, regardless 23 of 34 Entropy 2020, 22, 738 of the way it is obtained. This is a form of “conceptual emergence” and reveals the presence of a conceptual, or emergent, reasoning, which acts simultaneously with logical reasoning, in human thought [34]. We believe that it is this conceptual, or emergent, reasoning that is responsible of the other probability judgement errors studied in Section 3. In fact, the quantum conceptual framework presented here enables modelling of more general probability judgements, as we will explicitly see in the next two sections. 7.1. An Application to Conjunctive and Disjunctive Fallacies 7.1. An Application to Conjunctive and Disjunctive Fallacies We present in this section the quantum conceptual explanation we have recently elaborated for conjunctive and disjunctive fallacies—the observed empirical deviations from classicality can be interpreted as overextension effects and underextension effects, respectively. Hence, they can modelled using the quantum-theoretic framework we have developed in Section 7 for conceptual combinations. As such, these fallacies are not real errors but, rather, genuine expressions of quantum structures, in human reasoning, namely, conceptual emergence and an underlying conceptual reasoning. We will now elaborate quantum models for the conjunction and the disjunction fallacy borrowing terminology, methods and the formalism we have developed to model conceptual conjunctions and disjunctions. We refer to the probability judgement tests in Section 3. Let us denote by X the item Linda, by A the concept Feminist, by B the concept Bank Teller, by ‘A and B’ the conjunction Feminist and Bank Teller, and by ‘A or B’ the disjunction Feminist or Bank Teller. Moreover, let us denote by µ(A), µ(B), µ(A and B), and µ(A or B), the probability that Linda is judged as feminist, bank teller, feminist and bank teller, and feminist or bank teller, respectively. Finally, we use the terminology of concept theory: thus, µ(A) becomes the membership weight of Linda with respect to Feminist, µ(B) the membership weight of Linda with respect to Bank Teller, µ(A and B) the membership weight of Linda with respect to Feminist and Bank Teller, and µ(A or B) the membership weight of Linda with respect to Feminist or Bank Teller. We start by the conjunction fallacy and represent conceptual entities in an abstract Hilbert space H . Concepts A and B are represented by the mutually orthogonal unit vectors \|A⟩∈H and \|B⟩∈H , respectively. The conjunction ‘A and B’ is instead represented by the unit vector \|A and B⟩= 1 √ 2(\|A⟩+ \|B⟩). Then, we use the quantum probability formula for the conjunction of two concepts in Equation (24) and rewrite it in the following: µ(A and B) = 1 2 µ(A) + µ(B) + Re(⟨A\|M\|B⟩). (46) (46) In Reference [10], µ(A) + µ(B) = 0.83 + 0.26 = 1.09 > 1. Hence, in the C3 representation, M projects onto the subspace generated by (1, 0, 0) and (0, 1, 0), the unit vectors become In Reference [10], µ(A) + µ(B) = 0.83 + 0.26 = 1.09 > 1. 7.1. An Application to Conjunctive and Disjunctive Fallacies the concepts A and B “constructively interfere” in the disjunction ‘A or B’. The quantum-theoretic framework enables faithful representation of different data sets (see also Tables 1 and 2 in Section 3). Then, the explanation we propose for conjunctive and disjunctive fallacies is that, when the item Linda is considered, together with her story, the concepts Feminist and Bank Teller destructively interfere in the conjunction Feminist and Bank Teller, meant as a newly emerging standalone conceptual entity; the same concepts, instead constructively interfere in the disjunction Feminist or Bank Teller, again meant as a newly emerging standalone conceptual entity [34]. Conjunctive and disjunctive fallacies in human probability judgements can be accounted for genuine quantum effects, that is, contextuality, interference, superposition and emergence. The conjunction and the disjunction fallacy in decision theory are respectively the decision-theoretic counterparts of over- and under-extension effects in concept theory. In analogy with the case of double overextension in conceptual conjunctions and double underextension in conceptual disjunctions, the quantum-theoretic framework also predicts the presence of “double conjunction fallacies” and “double disjunction fallacies” [35]. 7.1. An Application to Conjunctive and Disjunctive Fallacies Hence, in the C3 representation, M projects onto the subspace generated by (1, 0, 0) and (0, 1, 0), the unit vectors become \|A⟩ = q µ(A), 0, q 1 −µ(A) , (47) \|B⟩ = eiθc s (1 −µ(A))(1 −µ(B)) µ(A) , s µ(A) + µ(B) −1 µ(A) , − q 1 −µ(B) , (48) (47) (48) and the quantum probability formula can be written as and the quantum probability formula can be written as µ(A and B) = 1 2 µ(A) + µ(B) + q (1 −µ(A))(1 −µ(B)) cos θc, (49) µ(A and B) = 1 2 µ(A) + µ(B) + q (1 −µ(A))(1 −µ(B)) cos θc, (49) (49) where θc is the interference angle for the conjunction. From the data in Reference [10]: µ(A) = 0.83, (50) µ(B) = 0.26, (51) µ(A and B) = 0.36, (52) µ(A) = 0.83, (50) µ(B) = 0.26, (51) µ(A and B) = 0.36, (52) 24 of 34 Entropy 2020, 22, 738 Entropy 2020, 22, 738 we get θc = 121.44◦, (53) \|A⟩ = (0.91, 0, 0.41), (54) \|B⟩ = ei121.44◦(0.39, 0.33, −0.86), (55) θc = 121.44◦, (53) \|A⟩ = (0.91, 0, 0.41), (54) \|B⟩ = ei121.44◦(0.39, 0.33, −0.86), (55) (53) (54) (55) (53) (54) (53) (55) that is, the concepts A and B “destructively interfere” in the conjunction ‘A and B’. Let us then come to the disjunction fallacy and follow the same steps we implemented for conceptual disjunction. In the Hilbert space H representation, formulas are the same as in the conjunction fallacy modelling, with the symbol ‘or’ replacing the symbol ‘and’. In the speciﬁc C3 representation, formulas for the disjunction fallacy are instead obtained by replacing in Equations (48) and (49) the angle θc with the interference angle for the disjunction θd. From the data in Reference [10]: From the data in Reference [10]: µ(A) = 0.83, (56) µ(B) = 0.26, (57) µ(A or B) = 0.60, (58) µ(A) = 0.83, (56) µ(B) = 0.26, (57) µ(A or B) = 0.60, (58) µ(A) = 0.83, (56) µ(B) = 0.26, (57) µ(A or B) = 0.60, (58) (56) (57) (58) (56) we get θc = 81.08◦, (59) \|A⟩ = (0.91, 0, 0.41), (60) \|B⟩ = ei81.08◦(0.39, 0.33, −0.86), (61) (59) (60) (61) (61) that is, the concepts A and B “constructively interfere” in the disjunction ‘A or B’. 7.2. An Application to the Disjunction Effect We review in this section the quantum conceptual explanation that we have recently elaborated for the disjunction effect—the empirically observed deviations from classicality can be interpreted as underextension effects, hence the disjunction effect can be modelled by using the quantum-theoretic framework we have developed in Section 7 for conceptual disjunctions. In the two-stage gamble, uncertainty aversion is just one, although an important one, of the conceptual landscapes surrounding the decision situation, playing the role of context and determining the final choice. Again, the disjunction effect reveals the presence of genuine quantum structures in human reasoning, namely, contextuality, interference, superposition and emergence. The quantum-theoretic framework for conceptual disjunctions enables modelling the disjunction effect in both the two-stage gamble and the Hawaii problem, as we have proved in Reference [35]. In this section, we apply it to the two-stage gamble. Entropy 2020, 22, 738 Entropy 2020, 22, 738 25 of 34 We refer to the DM error studied in Section 4. Let us preliminarily introduce the terminology of Section 7 and denote by A the conceptual situation of having won the ﬁrst gamble, by B the conceptual situation of having lost lost the ﬁrst gamble, and by ‘A or B’ the conceptual situation of having won “or” lost the ﬁrst gamble. Moreover, let us denote by µ(A) the probability of playing again in the conceptual situation A, by µ(B) the probability of playing again in the conceptual situation B, and by µ(A or B) the probability of playing again in the conceptual situation ‘A or B’. The terminology we use is consistent with the fact that we interpret these decision probabilities as membership weights with respect to conceptual situations and their disjunction. We represent conceptual entities in an abstract Hilbert space H . Conceptual situations A and B are represented by the mutually orthogonal unit vectors \|A⟩∈H and \|B⟩∈H , respectively, ⟨A\|B⟩= 0. The disjunction ‘A or B’ is instead represented by the unit vector \|A or B⟩= 1 √ 2(\|A⟩+ \|B⟩). Finally, the decision measurement about playing again is represented by the orthogonal projection operator over H . Then, we apply to the two-stage gamble test the quantum probability formula in for conceptual disjunctions Equation (25) and rewrite it in the following: µ(A or B) = 1 2 µ(A) + µ(B) + Re(⟨A\|M\|B⟩). (62) (62) In Reference [11], µ(A) + µ(B) = 0.69 + 0.59 = 1.28 > 1. 8. A Quantum Framework for Ellsberg-Type Paradoxes A state pv of ΩDM incorporates aspects of ambiguity and is represented by the unit vector \|v⟩∈C3, ∥\|v⟩∥= 1, that is, \|v⟩= ρReiθR\|R⟩+ ρYeiθY\|Y⟩+ ρBeiθB\|B⟩= (ρReiθR, ρYeiθY, ρBeiθB), (72) ≥0, ρ2 R + ρ2 Y + ρ2 B = 1, θR, θY, θB ∈R \|v⟩= ρReiθR\|R⟩+ ρYeiθY\|Y⟩+ ρBeiθB\|B⟩= (ρReiθR, ρYeiθY, ρBeiθB), (72) (72) e ρR, ρY, ρB ≥0, ρ2 R + ρ2 Y + ρ2 B = 1, θR, θY, θB ∈R where ρR, ρY, ρB ≥0, ρ2 R + ρ2 Y + ρ2 B = 1, θR, θY, θB ∈R where ρR, ρY, ρB ≥0, ρ2 R + ρ2 Y + ρ2 B = 1, θR, θY, θB ∈R For every i = R, Y, B, the (subjective) probability µv(Ei) that Ei occurs in the state pv of ΩDM is obtained through the Born rule of quantum probability, that is, For every i = R, Y, B, the (subjective) probability µv(Ei) that Ei occurs in the state pv of ΩDM is obtained through the Born rule of quantum probability, that is, µv(Ei) = ⟨v\|Pi\|v⟩= \|⟨i\|v⟩\|2 = ρ2 i . (73) (73) Compatibility with the three-color example requires that ρ2 R = 1 3, hence \|v⟩= ( 1 √ 3 eiθR, ρYeiθY, ρBeiθB) = ( 1 √ 3 eiθR, ρYeiθY, r 2 3 −ρ2yeiθB). (74) (74) Acts f1, f2, f3 and f4 in Table 4 are represented by the hermitian operators Acts f1, f2, f3 and f4 in Table 4 are represented by the hermitian operators F1 = u(100)PR + u(0)PY + u(0)PB, (75) F2 = u(0)PR + u(0)PY + u(100)PB, (76) F3 = u(100)PR + u(100)PY + u(0)PB, (77) F4 = u(0)PR + u(100)PY + u(100)PB. (78) F1 = u(100)PR + u(0)PY + u(0)PB, (75) F2 = u(0)PR + u(0)PY + u(100)PB, (76) F3 = u(100)PR + u(100)PY + u(0)PB, (77) F4 = u(0)PR + u(100)PY + u(100)PB. 7.2. An Application to the Disjunction Effect Hence, in the C3 representation, M projects onto the subspace generated by (1, 0, 0) and (0, 1, 0), and we have In Reference [11], µ(A) + µ(B) = 0.69 + 0.59 = 1.28 > 1. Hence, in the C3 representation, M projects onto the subspace generated by (1, 0, 0) and (0, 1, 0), and we have \|A⟩ = q µ(A), 0, q 1 −µ(A) , (63) \|B⟩ = eiθd s (1 −µ(A))(1 −µ(B)) µ(A) , s µ(A) + µ(B) −1 µ(A) , − q 1 −µ(B) , (64) (63) (64) and the quantum probability formula can be written as and the quantum probability formula can be written as µ(A or B) = 1 2 µ(A) + µ(B) + q (1 −µ(A))(1 −µ(B)) cos θd, (65) (65) where θd is the interference angle for the disjunction. where θd is the interference angle for the disjunction. From the data in Reference [11]: where θd is the interference angle for the disjunction. From the data in Reference [11]: From the data in Reference [11]: µ(A) = 0.69, (66) µ(B) = 0.59, (67) µ(A or B) = 0.36, (68) (66) (67) (68) we get we get we get we get θd = 141.76◦, (69) \|A⟩ = (0.83, 0, 0.56), (70) \|B⟩ = ei141.76◦(0.43, 0.64, −0.64), (71) (69) (70) (71) (71) that is, the conceptual situations A and B “destructively interfere” in the disjunction ‘A or B’. The quantum-theoretic framework enables faithful representation of different data sets (see also Table 3 in Section 4), explaining uncertainty aversion as one of the possible context effect produced by the overall conceptual landscape surrounding the decision situation. In the disjunction effect, a case of destructive interference occurs when the conceptual situations “winning the first gamble” and “losing the first gamble” combine in the conceptual disjunction, which is responsible of the double underextension effects observed empirically. This means that the presence of a conceptual, or emergent, reasoning can be identified in the disjunction effect too. 26 of 34 26 of 34 Entropy 2020, 22, 738 8. A Quantum Framework for Ellsberg-Type Paradoxes We have recently developed a theoretical framework, along the lines sketched in Section 6.2, that uses the mathematical formalism of quantum theory to model human DM under uncertainty. The quantum-theoretic framework: (i) provides a unitary solution for a variety of paradoxes of EUT, including Ellsberg and Machina paradoxes [33,77,78]; (ii) enables faithful representation of various sets of empirical data on these paradoxes [33,59,7 (ii) enables faithful representation of various sets of empirical data on these paradoxes [33,59,77]; (iii) successfully models shifts of attitudes towards uncertainty, from ambiguity averse to ambiguity seeking, and viceversa, as due to hope and fear effects [79]; (ii) enables faithful representation of various sets of empirical data on these paradoxes [33,59,77]; (iii) successfully models shifts of attitudes towards uncertainty, from ambiguity averse to ambiguity seeking, and viceversa, as due to hope and fear effects [79]; (iii) successfully models shifts of attitudes towards uncertainty, from ambiguity averse to ambig seeking, and viceversa, as due to hope and fear effects [79]; (iv) opens the way towards a quantum-based extension of SEUT [29,33,77]. We rely on the quantum conceptual framework in Section 6.2 and assume that, in any DM process, an interaction occurs between the conceptual entity that is the object of the decision, or DM entity, and the cognitive context, which may include the decision-maker. This interaction changes the (conceptual) state of the DM entity. Let us apply the quantum-theoretic framework to the Ellsberg three-color example presented in Section 5. The DM entity ΩDM is the urn with 30 red balls and 60 yellow and black balls in unknown proportion, and is associated with the Hilbert space C3. Let {(1,0,0), (0,1,0), (0,0,1)} be the canonical ON basis of C3. For every i = R,Y, B, the elementary event Ei is represented by the one-dimensional orthogonal projection operator Pi = \|i⟩⟨i\|, where \|R⟩= (1,0,0), \|Y⟩= (0,1,0) and \|B⟩= (0,0,1). 8. A Quantum Framework for Ellsberg-Type Paradoxes One can show that, for every α > 1 √ 3, the unit vectors the unit vectors \|w1⟩= ( 1 √ 3 , α, − r 2 3 −α2) (83) and \|w2⟩= ( 1 √ 3 , − r 2 3 −α2, α) (84) \|w1⟩= ( 1 √ 3 , α, − r 2 3 −α2) (83) \|w1⟩= ( 1 √ 3 , α, − r 2 3 −α2) (83) and \|w2⟩= ( 1 √ 3 , − r 2 3 −α2, α) (84) (84) reproduce Ellsberg preferences [80]. The orthogonality condition ⟨w1\|w2⟩= 0 implies α = ±0.7887. We choose α = +0.7887, thus getting \|w1⟩ = (0.5774, 0.7887, −0.2113), (85) \|w2⟩ = (0.5774, −0.2113, 0.7887), (86) (85) (86) (86) which completes the quantum mathematical modelling of the Ellsberg three-color example. Let us now represent in Hilbert space the data on the three-color example in Reference [59]. The decision measurement between acts f1 and f2 is represented by the spectral family {M, 1 −M}, M = \|m⟩⟨m\|, with ∥\|m⟩∥= 1. From the data in Reference [59], we have ⟨w1\|M\|w1⟩= 0.82, and one can show that M =    0.333 0.396 0.256e−i105.07◦ 0.396 0.470 0.304e−i105.07◦ 0.256ei105.07◦ 0.304ei105.07◦ 0.197    (87) M =    0.333 0.396 0.256e−i105.07◦ 0.396 0.470 0.304e−i105.07◦ 0.256ei105.07◦ 0.304ei105.07◦ 0.197    (87) (87) (see Reference [80]). The decision measurement between acts f3 and f4 is instead represented by the spectral family {N, 1 −N}, N = \|n⟩⟨n\|, with ∥\|n⟩∥= 1. From the data in Reference [59], we have ⟨w2\|N\|w2⟩= 0.78, and one can show that N =    0.333 0.276e−i102.87◦ 0.382 0.276ei102.87◦ 0.229 0.317ei102.87◦ 0.382 0.317e−i102.87◦ 0.438    (88) (88) (see Reference [80]). We have thus completed the construction of a quantum model for the Ellsberg three-color example, which also provides data representation of the DM test in Reference [59]. Ambiguity aversion is one of the conceptual landscapes interacting with the DM entity and changing its state, and genuine quantum effects, namely, contextuality, interference and superposition, occur in the DM process. The quantum-theoretic framework for human DM under uncertainty can be applied straight to the two-urn example in Section 5. In this case, we have two DM entities, entity ΩI DM, the urn with 100 red and black balls in unknown proportion, and entity ΩII DM, the urn with exactly 50 red balls and 50 black balls. 8. A Quantum Framework for Ellsberg-Type Paradoxes (78) Thus, for every i = 1, 2, 3, 4, the EU Wv( fi) of fi in the state pv is given by Thus, for every i = 1, 2, 3, 4, the EU Wv( fi) of fi in the state pv is given by Wv( f1) = ⟨v\|F1\|v⟩= 1 3u(100) + 2 3u(0), (79) Wv( f2) = ⟨v\|F2\|v⟩= (1 3 + ρ2 Y)u(0) + (2 3 −ρ2 Y)u(100), (80) Wv( f3) = ⟨v\|F3\|v⟩= (1 3 + ρ2 Y)u(100) + (2 3 −ρ2 Y)u(0), (81) Wv( f4) = ⟨v\|F4\|v⟩= 1 3u(0) + 2 3u(100) (82) (79) (82) Entropy 2020, 22, 738 27 of 34 As we can see, the EUs Wv( f1) and Wv( f4) do not depend on pv, in agreement with the fact that f1 and f4 are unambiguous acts. On the contrary, the EUs Wv( f2) and Wv( f3) do depend on pv, in agreement with the fact that f2 and f3 are ambiguous acts. As we can see, the EUs Wv( f1) and Wv( f4) do not depend on pv, in agreement with the fact that f1 and f4 are unambiguous acts. On the contrary, the EUs Wv( f2) and Wv( f3) do depend on pv, in agreement with the fact that f2 and f3 are ambiguous acts. Let us now come to the DM process. The entity ΩDM is prepared by the questionnaire in the initial state pv0 represented by the unit vector \|v0⟩= 1 √ 3(1,1,1), which leads to uniform drawing probabilities. However, in the pondering between f1 and f2, a first context operates on ΩDM and transforms pv0 into a new state pv12, depending on individual attitudes towards ambiguity; analogously, in the pondering between f3 and f4, a second context operates on ΩDM and transforms pv0 into a new state pv34, again depending on individual attitudes towards ambiguity. To reproduce Ellsberg preferences, we need to determine two ambiguity averse final states pw1 and pw2, represented by the unit vectors \|w1⟩and \|w2⟩, respectively, such that Ww1(f1) > Ww1(f2) and Ww2(f4) > Ww2(f3). 8. A Quantum Framework for Ellsberg-Type Paradoxes Both ΩI DM and ΩII DM are associated with the Hilbert space C2. Let {(1, 0), (0, 1)} be the canonical ON basis of C2. Entropy 2020, 22, 738 28 of 34 For every i = R, B, the elementary event Ei is represented by the one-dimensional orthogonal projection operator Pi = \|i⟩⟨i\|, where \|R⟩= (1, 0) and \|B⟩= (0, 1). A state pv of both ΩI DM and ΩII DM is represented by the unit vector \|v⟩∈C2, ∥\|v⟩∥= 1, that is, \|v⟩= ρReiθR\|R⟩+ ρBeiθB\|B⟩= (ρReiθR, ρBeiθB) (89) (89) re ρR, ρB ≥0, ρ2 R + ρ2 B = 1, θR, θB ∈R. where ρR, ρB ≥0, ρ2 R + ρ2 B = 1, θR, θB ∈R. where ρR, ρB ≥0, ρ2 R + ρ2 B = 1, θR, θB ∈R. For every i = R, B, the (subjective) probability µv(Ei) that the event Ei occurs in the state pv is obtained through the Born rule of quantum probability, that is, µv(Ei) = ⟨v\|Pi\|v⟩= \|⟨i\|v⟩\|2 = ρ2 i . (90) (90) Acts f1, f2, f3 and f4 in Table 5 are represented by the hermitian operators F1 = u(100)PR + u(0)PB = F2, (91) F3 = u(0)PR + u(100)PB = F4. (92) (91) (92) (91) (92) The EU of f1, f2, f3 and f4 in a state pv of both ΩI DM and ΩII DM is given by Wv( f1) = ⟨v\|F1\|v⟩= ρ2 Ru(100) + ρ2 Bu(0) = ρ2 Ru(100) + (1 −ρ2 R)u(0) = Wv( f2), (93) Wv( f3) = ⟨v\|F3\|v⟩= ρ2 Ru(0) + ρ2 Bu(100) = ρ2 Ru(0) + (1 −ρ2 R)u(100) = Wv( f4). (94) (93) (94) (94) Let us now come to the DM process. Both ΩI DM and ΩII DM are prepared by the questionnaire in the initial state pv0 represented by unit vector \|v0⟩= 1 √ 2\|e1⟩+ 1 √ 2\|e2⟩= 1 √ 2(1, 1), which leads to uniform drawing probabilities. Then, since f1 is ambiguous whereas f2 is unambiguous, a pondering between f1 and f2 will determine a change of ΩI DM from pv0 to a generally different state pw1, whereas the same pondering will leave ΩII DM in the initial state pv0. and the one-dimensional orthogonal projection operators and the one-dimensional orthogonal projection operators and the one-dimensional orthogonal projection operators M = 0.05 0.21i −0.21i 0.95 ! , (103) N = 0.93 −0.13i 0.13i 0.02 ! (104) (103) (104) reproduce empirical data (see References [70,79] for the details). The construction of a quantum model for the Ellsberg two-urn example, which reproduces data in Reference [59], is thus completed. However, the quantum model also works for shifts of ambiguity attitudes, as due to hope and fear effects, in managerial and medical decisions, as proved in Reference [79]. We conclude this section with a remark. According to SEUT, people should take decisions in such a way to maximize EU with respect to a Kolmogorovian probability measure (cfr., Section 5). The analysis provided here shows instead that the paradoxes of SEUT disappear in a theoretical framework in which people actually take decisions in such a way to maximize EU with respect to a non-Kolmogorovian, speciﬁcally quantum, probability measure. This suggests to extend EUT in a Hilbert space formalism along the lines suggested in this paper. 8. A Quantum Framework for Ellsberg-Type Paradoxes Similarly, since f3 is ambiguous whereas f4 is unambiguous, a pondering between f3 and f4 will determine a change of ΩI DM from pv0 to a generally different state pw2, whereas the same pondering will leave ΩII DM in the initial state pv0. p 2 p g DM p 0 One can then show that, for every 0 ≤α < 1 2, the unit vectors DM One can then show that, for every 0 ≤α < 1 2, the unit vectors \|w1⟩ = (√ α, √ 1 −α), (95) \|w2⟩ = ( √ 1 −α, −√ α) (96) (95) (96) (95) (96) reproduce Ellsberg preferences f2 ≻f1 and f4 ≻f3 [70]. Indeed, using Equations (93)–(96), we get Wv0( f2) = 1 2[u(100) + u(0)] > αu(100) + (1 −α)u(0) = Ww1( f1), (97) Wv0( f4) = 1 2[u(100) + u(0)] > (1 −α)u(0) + αu(100) = Ww2( f3). (98) (97) (98) Let us ﬁnally represent in Hilbert space the data on the two-urn example in Reference [59]. The decision measurement between acts f1 and f2 is represented by the spectral family {M, 1 −M}, where M = \|m⟩⟨m\| and ∥\|m⟩∥= 1. The decision measurement between acts f3 and f4 is instead represented by the spectral family {N, 1 −N}, where N = \|n⟩⟨n\| and ∥\|n⟩∥= 1. From the data in (Aerts, Geriente, Moreira, Sozzo 2018) , we have: ⟨w1\|M\|w1⟩ = 0.82, (99) ⟨w2\|N\|w2⟩ = 0.84. (100) (99) (99) (100) (100) Entropy 2020, 22, 738 29 of 34 py 2020, 22, 738 29 of 3 Entropy 2020, 22, 738 Entropy 2020, 22, 738 29 of 34 One can show that the unit vectors One can show that the unit vectors One can show that the unit vectors One can show that the unit vectors One can show that the unit vectors \|w1⟩ = (0.38, 0.92), (101) \|w2⟩ = (0.92, −0.38), (102) \|w1⟩ = (0.38, 0.92), (101) \|w2⟩ = (0.92, −0.38), (102) and the one-dimensional orthogonal projection operators 9. Conclusions In this paper, we have addressed the question of the foundations of quantum theory as a general theory for human cognition, that is, the problem of how and why we are justiﬁed in applying quantum models in cognitive domains independently of their empirical effectiveness. To this end, we have worked out a unitary theoretical framework, inspired by realistic-operational foundations of quantum physics, to represent in Hilbert space the phenomenology of human judgements and DM under uncertainty. In it, a cognitive test is described as a two-step process which involves both a preparation and a measurement on a conceptual entity, while the participant operates in the test as a measurement context for the entity and changes its state. We note the problem of the measurement with respect to the calculation in quantum theory. Consequently, both the problem of quanta observational error of the energy measurement and the inevitable perturbation of the measurement itself arise. On the subject, including Einsteinian objectivity–and–realism of a quantum theory are nowadays well identiﬁed from a historical–epistemological standpoint. A differ point of view was provided by Dirac. Both scientists essentially concluded that, at that time, the theory was not mature yet to go into a radical solution. For example, why mathematicians and physicists did not perceive that the change of energy could be per quanta? (see, e.g., References [81–84] and references therein). Whenever the conceptual entity, its states, contexts, properties, and mutual statistical relations are identiﬁed in the modelling of a given phenomenon, the quantum-theoretic framework consists in making explicit the usual Hilbert space representation of entities, states, contexts, properties, probabilities and dynamics. We have applied this general theory to long-standing cognitive fallacies, but the theory is in principle applicable to any judgement and decision. The latter remark is important, because the quantum models following from the general framework presented here work, not only in empirical situations where deviations from classicality occur, but also in those situations which admit a classical logical and probabilistic modelling, hence no fallacy occur (see, e.g., classical conjunction and disjunction data in concept combinations [22,35]). We conclude this section with some remarks that are important, in our opinion, to better grasp the content of this paper. 9. Conclusions Entropy 2020, 22, 738 Entropy 2020, 22, 738 Entropy 2020, 22, 738 30 of 34 30 of 34 Firstly, we are aware that a SCoP formalism description contains a degree of idealization and that the large variability in individual differences may play a fundamental role in cognitive processes, which may even mine the Hilbert space representation of some of these processes (see, e.g., Reference [85] for an example of a quantum model that incorporates individual differences). Nevertheless, we also think that a reﬁnement in the description of cognitive processes which takes into account individual differences is not incompatible with a SCoP formalism description where (i) each individual is confronted with a conceptual entity preliminarily prepared by a cognitive test in a deﬁned state, and (ii) the individual acts as a context for the entity, changing its state in a way that is uncontrollable and unpredictable if not at a statistical level. In this regard, it is worth mentioning that we have recently studied processes where effects of memory, evidence accumulation and response dynamics occur in sequential judgements. In these cases, we have proved that, while a general model in Hilbert space cannot be constructed which agrees with empirical data, however, a SCoP formalism description can still be worked out which suggests using a non-Kolmogorovian non-quantum model of probability, called “general tension-reduction (GTR) representation” (see, e.g., Reference [69] and references therein). Secondly, we do not claim that a “quantum, or quantum-like, theory in Hilbert space” is the only possible theory for human cognition. And, indeed, some authors have proved that suitable classical models where noise is postulated at individual level enable representing a variety of empirical situations where cognitive fallacies occur [86]. On the other side, we also believe that in these cases of empirical equivalence between different scientiﬁc paradigms, also epistemological arguments should be taken into account. In this regard, the fact that the present quantum-theoretic framework enables explaining several seemingly different cognitive phenomena in a unitary, cross-disciplinary and coherent structure, using a minimum number of theoretical hypotheses, are serious epistemological arguments in favour of a non-Kolmogorovian quantum, or quantum-like, paradigm. Thirdly, there is a tendency, mainly in empirically-based disciplines, to be critical with respect to a theory that is able to reproduce all possible situations it applies to. 9. Conclusions This is because the theory contains additional parameters, which may lead one to think that “any type of data can be modelled by allowing these parameters to get different values”. We agree that, in case we have to do with an “ad hoc model”, that is, a model especially designed for the circumstance of the situation it models, this suspicion is grounded. Adding parameters to such an “ad hoc model”, or stretching the already contained parameters to other values, does not give rise to what we call a theory. On the other hand, a theory needs to be well deﬁned, its rules, the allowed procedures, its theoretical, mathematical, and internal logical structure, “independent” of the structure of the models representing speciﬁc situations that can be coped with by the theory. Hence, the theory needs to contain a well deﬁned set of instructions of “how to produce models for speciﬁc situations”. In this regard, we distinguish between a model that is derived by a general theory, as the ones presented in this paper, and a model speciﬁcally designed to reproduce a number of empirical situations. This is connected with the important issue of the “predictive power” of quantum models. We refer here to the distinction between the scientiﬁc explanation—in certain epistemological studies also identiﬁed with a prediction—and the description of a scientiﬁc account. This distinction is linked with the presence of probabilistic models which can assume an epistemic role both for the explanatory and the descriptive. The former deals with experiments, observables, and so forth, the latter deals with data. Of course, within a scientiﬁc process, the activities are connected in a double sense with data. Thus, the explanation–and–description remains an ambiguous object to solve, especially in cognitive and social sciences where, as mentioned above, the model plays a surrogate important role in the ﬁnal decision (observable–data–information) of the theory. For example, in quantum theory a complex wave function (space and time variables) describes mathematically the state of a (physical complex vector space) system. For, probabilistic models calculate (so they do not measure) the probabilities of a state in a given space and time. This was, of course, not possible in the predictive Newtonian science until 19th century; the same remark applies to Lagrangian formulation [87,88]. 9. Conclusions Hence, a result is that it is not possible to produce simultaneous 31 of 34 Entropy 2020, 22, 738 measure (and so predictions) of position and momentum, as conjugate variables, to arbitrary precision such as established by Heisenberg’s uncertainty principle. In other words, an electron probabilistically can be located in space but without an exact (prediction of the) position. Therefore, quantum theory, which cannot assign deﬁnite values to its variables – by a probabilistic distribution – can scientiﬁcally calculate, so describes–and–explains, the values of certain observables. Models derived from a theory will generally need more data from a larger set of tests to become predictive for the outcomes of other not yet performed experiments than this is the case for “ad hoc models”. The reason is that in principle such models have to be able to faithfully represent data of all possible tests that can be performed. In quantum cognition, the relative exiguity of data, in particular, if compared to physics, prevents models from having systematic and substantial predictive power. However, we also believe that the independent applicability of the quantum-theoretic framework presented here constitutes in itself a strong argument to support this line of the quantum cognition research programme. Author Contributions: All authors contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by QUARTZ (Quantum Information Access and Retrieval Theory), the Marie Skłodowska-Curie Innovative Training Network 721321 of the European Union’s Horizon 2020 research and innovation programme. Conﬂicts of Interest: The authors declare that they have no conﬂict of interest. Conﬂicts of Interest: The authors declare that they have no conﬂict of interest. References 1. Kolmogorov, A.N. Grundbegriffe der Wahrscheinlichkeitrechnung; Ergebnisse Der Mathematik; Translated As Foundations of Probability; Chelsea Publishing Company: New York, NJ, USA, 1950. 1. Kolmogorov, A.N. Grundbegriffe der Wahrscheinlichkeitrechnung; Ergebnisse Der Mathematik; Translated As Foundations of Probability; Chelsea Publishing Company: New York, NJ, USA, 1950. 2. Savage, L. The Foundations of Statistics; John Wiley & Sons: New York, NY, USA, 1954. 3. Pitowsky, I. Quantum Probability, Quantum Logic; Lecture Notes in Physics Vol. 321; Springer: Berlin, Germany, 1989. 3. Pitowsky, I. Quantum Probability, Quantum Logic; Lecture Notes in Physics Vol. 321; Springer: Berlin, Germany, 1989. 4. Busemeyer, J.R.; Bruza, P.D. Quantum Models of Cognition and Decision; Cambridge University Press: Cambridge, UK, 2012. 4. Busemeyer, J.R.; Bruza, P.D. Quantum Models of Cognition and Decision; Cambridge University Press: Cambridge, UK, 2012. 5. Tversky, A.; Kahneman, D. Judgment under uncertainty: Heuristics and biases. Science 1974, 185, 1124–1131. [CrossRef] [PubMed] 5. Tversky, A.; Kahneman, D. Judgment under uncertainty: Heuristics and biases. Science 1974, 185, 1124–1131. [CrossRef] [PubMed] 6. Busemeyer, J.R.; Pothos, E.M.; Franco, R.; Trueblood, J.S. A quantum theoretical explanation for probability judgment errors. Psychol. Rev. 2011, 118, 193–218. [CrossRef] [PubMed] 6. Busemeyer, J.R.; Pothos, E.M.; Franco, R.; Trueblood, J.S. A quantum theoretical explanation for probability judgment errors. Psychol. Rev. 2011, 118, 193–218. [CrossRef] [PubMed] 7. Hampton, J.A. Overextension of conjunctive concepts: Evidence for a unitary model for concept typicality and class inclusion. J. Exp. Psychol. Learn. Mem. Cogn. 1988, 14, 12–32. [CrossRef] 7. Hampton, J.A. Overextension of conjunctive concepts: Evidence for a unitary model for concept typicality and class inclusion. J. Exp. Psychol. Learn. Mem. Cogn. 1988, 14, 12–32. [CrossRef] 8. Hampton, J.A. Disjunction of natural concepts. Mem. Cogn. 1988, 16, 579–591. [CrossRef] 9. Tversky, A.; Kahneman, D. Extension versus intuitive reasoning: The conjunction fallacy in pro judgment. Psychol. Rev. 1983, 90, 293–315. [CrossRef] 10. Morier, D.; Borgida, E. The conjunction fallacy: A task speciﬁc phenomenon? Personal. Soc. Psychol. Bull. 1984, 10, 243–252. [CrossRef] 11. Tversky, A.; Shaﬁr, E. The disjunction effect in choice under uncertainty. Psychol. Sci. 1992, 3, 305–309. [CrossRef] avage axioms. Q. J. Econ. 1961, 75, 643–669. [CrossRef 12. Ellsberg, D. Risk, ambiguity, and the Savage axioms. Q. J. Econ. 1961, 75, 643–669. [CrossRef] 13. Shah, A.K.; Oppenheimer, D.M. Heuristics made easy: An effort-reduction framework. Psychol. Bull. 2008, 134, 207–222. [CrossRef] 4. Simon, H.A. A behavioral model of rational choice. Q. J. Econ. 1955, 69, 99–118. [CrossRef] 15. Pisano, R. References On Principles in Sadi Carnot’s theory (1824). Epistemological Reflections. Almagest 2010, 2, 128–179. [CrossRef] 16. Pisano, R. Physics—Mathematics relationship. Historical and epistemological notes. In ESU 6 European Summer University History and Epistemology In Mathematics; Barbin, E., Kronfellner, M., Tzanakis, C., Eds.; Verlag Holzhausen GmbH–Holzhausen Publishing Ltd.: Vienna, Austria, 2011; pp. 457–472. 17. Pisano, R. On Lagrangian in Maxwell’s electromagnetic theory. In Scientiatum VI; The Federate University of Rio de Janeiro Press: Rio de Janeiro, Brazil, 2013; pp. 44–59. Entropy 2020, 22, 738 32 of 34 18. Gillispie, C.C.; Pisano, R. Lazare and Sadi Carnot. A Scientiﬁc and Filial Relationship, 2nd ed.; Springer: Dordrecht, The Netherlands, 2014. 19. Pisano, R.; Bussotti, P. The emergencies of mechanics and thermodynamics in the western society during 18th–19th Century. In A Bridge between Conceptual Frameworks, Science, Society and Technology Studies; Pisano, R., Ed.; Springer: Dordrecht, The Netherlands, 2015; pp. 399–436. 20. Pisano, R.; Anakkar, A.; Pellegrino, E.M.; Nagels, M. Thermodynamic foundations of physical chemistry. Reversible processes and thermal equilibrium into history. Found. Chem. 2019, 21, 297–323. [CrossRef] 21. Dirac, P.A.M. Quantum Mechanics, 4th ed.; Oxford University Press: Oxford, UK, 1958. 22. Aerts, D. Quantum structure in cognition. J. Math. Psychol. 2009, 53, 314–348. [CrossRef] 23. Khrennikov, A.Y. Ubiquitous Quantum Structure. From Psychology to Finance; Springer: Berlin, Germany, 2010 24. Bagarello, F. Quantum Dynamics for Classical Systems: With Applications of the Number Operator; Wiley: New York, NY, USA, 2012. 5. Haven, E.; Khrennikov, A.Y. Quantum Social Science; Cambridge University Press: Cambridge, UK, 2013 26. Melucci, M. Introduction to Information Retrieval and Quantum Mechanics; Springer: Berlin, Germany, 2015. 27. Haven, E.; Khrennikov, A.Y.; Robinson, T. Quantum Methods in Social Science: A First Course; World S Press: Singapore, 2017. 28. Wendt, A. Quantum Mind and Social Science; Cambridge University Press: Cambridge, UK, 2015. 29. Haven, E.; Khrennikov, A.Y.; Ma, C.; Sozzo, S. Introduction to quantum probability theory and its economic applications. J. Math. Econ. 2018, 78, 127–130. [CrossRef] 30. Aerts, D.; Sozzo, S. Quantum structure in cognition. Why and how concepts are entangled. In International Symposium on Quantum Interaction; Springer: Berlin, Germany, 2011; Volume 7052, pp. 116–127. 31. Aerts, D.; Broekaert, J.; Gabora, L.; Sozzo, S. Quantum structure and human thought. Behav. Brain Sci. 2013, 36, 274–276. [CrossRef] [PubMed] 32. Aerts, D.; Gabora, L.; Sozzo, S. Concepts and their dynamics: A quantum–theoretic modeling of human thought. Top. Cogn. Sci. 2013, 5, 737–772. [CrossRef] [PubMed] 33. References Cognition 1981, 9, 35 58. [CrossRef] 47. Alxatib, S.; Pelletier, J. On the psychology of truth gaps. In Vagueness in Communication; Nouwen, R., van Rooij, R., [CrossRef] 47. Alxatib, S.; Pelletier, J. On the psychology of truth gaps. In Vagueness in Communication; Nouwen, R., van Rooij, R., Sauerland, U., Schmitz, H.-C., Eds.; Springer-Verlag: Berlin/Heidelberg, Germany, 2011; pp. 13–36. 47. Alxatib, S.; Pelletier, J. On the psychology of truth gaps. In Vagueness in Communication; Nouwen, R., van R Sauerland, U., Schmitz, H.-C., Eds.; Springer-Verlag: Berlin/Heidelberg, Germany, 2011; pp. 13–36. 48. Moro, R. On the nature of the conjunction fallacy. Synthese 2009, 171, 1–24. [CrossRef] 49. Boyer-Kassem, T.; Duchêne, S.; Guerci, E. Quantum-like models cannot account for the conjunction fallacy. Theory and Decision 2016, 81, 479–510. [CrossRef] 50. Fisk, J.E.; Pidgeon, N. Component probabilities and the conjunction fallacy: Resolving signed summation and the low component model in a contingent approach. Acta Psychol. 1996, 94, 1–20. [CrossRef] 50. Fisk, J.E.; Pidgeon, N. Component probabilities and the conjunction fallacy: Resolving signed summation and the low component model in a contingent approach. Acta Psychol. 1996, 94, 1–20. [CrossRef] 51. Fisk, J.E. Judgments under uncertainty: Representativeness or potential surprise? Br. J. Psychol. 2002, 93, 431–449. [CrossRef] 52. Kühberger, A.; Kamunska, D.; Perner, J. The disjunction effect: Does it exist for two-step gambles? Organ. Behav. Hum. Decis. Process. 2001, 85, 250–264. [CrossRef] 53. Lambdin, C.; Burdsal, C. The disjunction effect reexamined: Relevant methodological issues and the fallacy of unspeciﬁed percentage comparisons. Organ. Behav. Hum. Decis. Process. 2007, 103, 268–276. [CrossRef] 54. Von Neumann, J.; Morgenstern, O. Theory of Games and Economic Behavior. Princeton University Press: 53. Lambdin, C.; Burdsal, C. The disjunction effect reexamined: Relevant methodological issues and the fallacy of unspeciﬁed percentage comparisons. Organ. Behav. Hum. Decis. Process. 2007, 103, 268–276. [CrossRef] 54. Von Neumann, J.; Morgenstern, O. Theory of Games and Economic Behavior. Princeton University Press: Princeton, NJ, USA, 1944. 54. Von Neumann, J.; Morgenstern, O. Theory of Games and Economic Behavior. Princeton Universit Princeton, NJ, USA, 1944. 55. Knight, F.H. Risk, Uncertainty and Proﬁt; Houghton Mifﬂin: Boston, MA, USA, 1921. 56. Gilboa, I.; Postlewaite, A.; Schmeidler, D. Probabilities in economic modeling. J. Econ. Perspect. 2008, 22, 173–188. [CrossRef] 57. Etner, J.; Jaleva, M.; Tallon, J.-M. Decision theory under ambiguity. J. Econ. Surv. 2012, 26, 234–270. [CrossRef] 58. Machina, M.J.; Siniscalchi, M. Ambiguity and ambiguity aversion. References Aerts, D.; Sozzo, S. From ambiguity aversion to a generalized expected utility. Modeling preferences in a quantum probabilistic framework. J. Math. Psychol. 2016, 74, 117–127. [CrossRef] 34. Aerts, D.; Sozzo, S. Quantum structure in cognition: Origins, developments, successes and expectations. In The Palgrave Handbook of Quantum Models in Social Science: Applications and Grand Challenges; Haven, E., Khrennikov, A.Y., Eds.; Palgrave & Macmillan: London, UK, 2016; pp. 157–193. 35. Aerts, D.; de Bianchi, M.S.; Sozzo, S.; Veloz, T. Modeling human decision-making: An overview of the Brussels quantum approach. Found. Sci. 2018. [CrossRef] 36. Aerts, D. Foundations of quantum physics: A general realistic and operational approach. Int. J. Theor. Phys. 1999, 38, 289–358. [CrossRef] 37. Aerts, D. Being and change: Foundations of a realistic operational formalism. In Probing the Structure of Quantum Mechanics: Nonlinearity, Nonlocality, Probability and Axiomatics; Aerts, D., Czachor, M., Durt, T., Eds.; World Scientiﬁc: Singapore, 2002; pp. 71–110. 38. Rosch, E. Natural categories. Cogn. Psychol. 1973, 4, 328–350. [CrossRef] 38. Rosch, E. Natural categories. Cogn. Psychol. 1973, 4, 328–350. [CrossRef] 39. Rosch, E. Principles of categorization. In Cognition and categorization; Rosch, E., Lloyd, B., Eds.; Lawrence Erlbaum: Hillsdale, NJ, USA, 1978; pp. 133–179. 40. Rosch, E. Prototype classification and logical classification: The two systems. In New Trends in Conceptual Representation: Challenges to Piaget Theory? Scholnick, E.K., Ed.; Lawrence Erlbaum: Hillsdale, NJ, USA, 1983; pp. 133–159. 41. Nosofsky, R. Exemplar-based accounts of relations between classiﬁcation, recognition, and typicality. J. Exp. Psychol. Learn. Mem. Cogn. 1988, 14, 700–708. [CrossRef] 42. Nosofsky, R. Exemplars, prototypes, and similarity rules. In From Learning Theory to Connectionist Theory: Essays in Honor of William K. Estes; Healy, A., Kosslyn, S., Shiffrin, R., Eds.; Erlbaum: Hillsdale, NJ, USA, 1992; pp. 149–167 43. Murphy, G.L.; Medin, D.L. The role of theories in conceptual coherence. Psychol. Rev. 1985, 92, 289–316. [CrossRef] [PubMed] 44. Rumelhart, D.E.; Norman, D.A. Representation in memory. In Stevens’ Handbook of Experimental Psychology; Atkinson, R.C., Hernsein, R.J., Lindzey, G., Duncan, R.L., Eds.; John Wiley & Sons: Hoboken, NJ, USA, 1988; pp. 511–587. 33 of 34 Entropy 2020, 22, 738 45. Zadeh, L. A note on prototype theory and fuzzy sets. Cognition 1982, 12, 291–297. [CrossRef] 46. Osherson, D.; Smith, E. On the adequacy of prototype theory as a theory of concepts. Cognition 1981, 9, 35–58. [CrossRef] 46. Osherson, D.; Smith, E. On the adequacy of prototype theory as a theory of concepts. References In Handbook of the Economics of Risk and Uncertainty; Machina, M.J., Viscusi, K., Eds.; Elsevier: New York, NJ, USA, 2014; pp. 729–807. 58. Machina, M.J.; Siniscalchi, M. Ambiguity and ambiguity aversion. In Handbook of the Economics of Risk and Uncertainty; Machina, M.J., Viscusi, K., Eds.; Elsevier: New York, NJ, USA, 2014; pp. 729–807. 59. Aerts, D.; Geriente, S.; Moreira, C.; Sozzo, S. Testing ambiguity and Machina preferences within a quantum-theoretic framework for decision-making. J. Math. Econ. 2018, 78, 176–185. [CrossRef] 60. Gilboa, I.; Marinacci, M. Ambiguity and the Bayesian paradigm. In Advances in Economics and Econometrics: Theory and Applications; Acemoglu, D., Arellano, M., Dekel, E., Eds.; Cambridge University Press: New York, NJ, USA, 2013; pp. 179–242. pp 61. Machina, M.J. Risk, ambiguity, and the dark–dependence axioms. Am. Econ. Rev. 2009, 99, 385–392. [CrossRef] 62. Baillon, A.; l’Haridon, O.; Placido, L. Ambiguity models and the Machina paradoxes. Am. Econ. Rev. 2011, 101, 1547–1560. [CrossRef] 63. L’Haridon, O.; Placido, L. Betting on Machina’s reﬂection example: An experiment on ambiguity. Theory Decis. 2010, 69, 375–393. [CrossRef] 64. Viscusi, W.K.; Chesson, H. Hopes and fears: The conﬂicting effects of risk ambiguity. J. Risk Uncertain. 1999, 47, 153–178. 65. Ho, J.; Keller, L.R.; Keltyka, P. Effects of outcome and probabilistic ambiguity on managerial choices. J. Risk Uncertain. 2002, 24, 47–74. [CrossRef] 66. Trautmann, S.T.; van de Kuilen, G. Ambiguity attitudes. In The Wiley Blackwell Handbook of Judgment and Decision Making; Keren, G., Wu, G., Eds.; John Wiley & Sons: New York, NJ, USA, 2015; pp. 89–116. 67. Jauch, J.M. Foundations of Quantum Mechanics; Addison Wesley: Boston, MA, USA, 1968. 68. Piron, C. Foundations of Quantum Physics; W.A. Benjamin: Reading, MA, USA, 1976. 69. Aerts, D.; Sassoli de Bianchi, M.; Sozzo, S. On the foundations of the Brussels operational-realistic approach to cognition. Front. Phys. 2016. [CrossRef] 70. Sozzo, S. Quantum structures in human decision-making: Towards quantum expected utility. Int. J. Theor. Phys. 2019. [CrossRef] 71. Aerts, D.; Gabora, L. A theory of concepts and their combinations I: The structure of the sets of contexts and properties. Kybernetes 2005, 34, 167–191. [CrossRef] 72. Aerts, D.; Gabora, L. A theory of concepts and their combinations II: A Hilbert space representation. Kybernetes 2005, 34, 192–221. [CrossRef] 73. Sozzo, S. A quantum probability explanation in Fock space for borderline contradictions. J. Math. Psychol. 2014, 58, 1–12. [CrossRef] 34 of 34 34 of 34 Entropy 2020, 22, 738 74. Sozzo, S. References Conjunction and negation of natural concepts: A quantum-theoretic modeling. J. Math. Psychol. 2015, 66, 83–102. [CrossRef] 75. Aerts, D.; Sozzo, S.; Veloz, T. Quantum structure of negation and conjunction in human thought. Front. Psychol. 2015. [CrossRef] 76. Aerts, D.; Sozzo, S.; Veloz, T. New fundamental evidence of non-classical structure in the combination of natural concepts. Philos. Trans. R. Soc. A 2015, 374, 20150095. [CrossRef] 77. Aerts, D.; Haven, E.; Sozzo, S. A proposal to extend expected utility in a quantum probabilistic framework. Econ. Theory 2018, 65, 1079–1109. [CrossRef] 78. Machina, M.J. Ambiguity aversion with three or more outcomes. Am. Econ. Rev. 2014, 104, 3814–3840. [CrossRef] 79. Sozzo, S. Representing attitudes towards ambiguity in Hilbert space: Foundations and applications. Found. Sci. arXiv 2020, arXiv:1907.06314. 80. Sozzo, S. Explaining versus describing human decisions. Hilbert space structures in decision theory. Soft Comput. 2019. [CrossRef] 81. Klein, M.J. Max Planck and the beginnings of the quantum theory. Arch. Hist. Exact Sci. 1962, 1, 4 [CrossRef] [ ] 82. Kuhn, T.S. Black-Body Theory and the Quantum Discontinuity, 1894–1912; Oxford University Press: Oxford, UK, 1978. 83. Kragh, H. Max Planck: The reluctant revolutionary. Phys. World 2000, 13, 31. [CrossRef] 84. Pisano, R.; Casolaro, F. Historical inquiring on geometry in relativity. Part One. Hist. Res. 2010, 1, 4 85. Mistry, P.K.; Pothos, E.M.; Vandekerckhove, J.; Trueblood, J.S. A quantum probability account of individual differences in causal reasoning. J. Math. Psychol. 2018, 87, 76–97. [CrossRef] 86. Costello, F.; Watts, P. Invariants in probabilistic reasoning. Cogn. Psychol. 2018, 100, 1–16. [CrossRef] [PubMed] 87. Pisano, R.; Capecchi, D. Conceptual and mathematical structures of mechanical science in the western civilization around 18th Century. Almagest 2013, 4, 86–121. [CrossRef] 88. Pisano, R.; Bussotti, P. Historical and foundational details on the method of inﬁnite descent: Every prime number of the form 4n+1 is the sum of two squares. Found. Sci. 2020, 1, 1–32. c⃝2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3018772150	https://jyx.jyu.fi/bitstream/123456789/70960/2/UPTP-2019-0217.R3.pdf	English	null	An adventurous learning journey. Physiotherapists’ conceptions of learning and integrating cognitive functional therapy into clinical practice	Physiotherapy theory and practice	2,020	public-domain	24,441	Year: 2022 Rights: In Copyright This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Holopainen, Riikka; Piirainen, Arja; Karppinen, Jaro; Linton, Steven James; O’Sullivan, Peter Title: An adventurous learning journey : Physiotherapists’ conceptions of learning and integrating cognitive functional therapy into clinical practice Please cite the original version: Holopainen, R., Piirainen, A., Karppinen, J., Linton, S. J., & O’Sullivan, P. (2022). An adventurous learning journey : Physiotherapists’ conceptions of learning and integrating cognitive functional therapy into clinical practice. Physiotherapy Theory and Practice, 38(2), 309-326. https://doi.org/10.1080/09593985.2020.1753271 Physiotherapy Theory and Practice For Peer Review On An adventurous learning journey. Physiotherapists’ conceptions of learning and integrating Cognitive Functional Therapy into clinical practice. Journal: Physiotherapy Theory and Practice Manuscript ID UPTP-2019-0217.R3 Manuscript Type: Qualitative Research Report Keywords: qualitative, physiotherapist, phenomenography, Cognitive Functional Therapy, Biopsychosocial, implementation or Peer Review An adventurous learning journey. Physiotherapists’ conceptions of learning and integrating Cognitive Functional Therapy into clinical practice. Journal: Physiotherapy Theory and Practice Manuscript ID UPTP-2019-0217.R3 Manuscript Type: Qualitative Research Report Keywords: qualitative, physiotherapist, phenomenography, Cognitive Functional Therapy, Biopsychosocial, implementation w O w O URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 1 1 2 An adventurous learning journey. Physiotherapists’ conceptions of learning and 3 integrating Cognitive Functional Therapy into clinical practice. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Page 1 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 1 1 2 An adventurous learning journey. Physiotherapists’ conceptions of learning and 3 integrating Cognitive Functional Therapy into clinical practice. Page 1 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 1 1 2 An adventurous learning journey. Physiotherapists’ conceptions of learning and 3 integrating Cognitive Functional Therapy into clinical practice. 4 Page 1 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 1 of 47 Page 1 of 47 1 2 An 3 integ 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Page 1 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 2 of 47 Page 2 of 47 2 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu INTRODUCTION For example, an eight-day university course on the cognitive behavioral approach for 10 physiotherapists was successful in changing physiotherapists’ beliefs and attitudes, but it did 11 not improve patient outcomes (Overmeer, Boersma, Denison, and Linton, 2011). The authors 12 concluded that the learning process requires time and experiences of practice and clinical 13 supervision, which short courses cannot provide. 14 Qualitative research provides a deeper understanding of these issues and to date, only a few 15 qualitative studies have explored physiotherapists’ views on changing their practice into a BPS 16 approach in the management of LBP (Karstens et al., 2018; Sanders, Ong, Sowden, and Foster, 17 2014). Three specific studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and 18 Dankaerts 2013; Synnott et al., 2016) have explored physiotherapists’ views on adopting a CFT 19 approach. CFT is an example of a BPS method that challenges more traditional biomechanical 20 / pathoanatomical physiotherapy approaches. It is an integrated physiotherapist-led cognitive 21 and behavioral intervention for individualizing the self-management of persistent LBP, once 22 serious and specific pathology has been excluded and has shown promising results (O’Sullivan, 23 Dankaerts, O’Sullivan, and O’Sullivan, 2015; Vibe Fersum et al., 2013). It uses a BPS clinical 24 reasoning framework to explore, identify and manage cognitive, emotional, social, physical and 25 lifestyle barriers to recovery (O’Sullivan et al., 2018; Synnott et al., 2016). CFT training aims 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 1 way to train physiotherapists to deliver psychologically informed interventions remains 2 unknown, as the results of previous attempts to direct physiotherapists’ practice behavior 3 towards the BPS approach have been conflicting. Although attending BPS-orientated 4 workshops has shown to positively change physiotherapists’ beliefs in the short term 5 (Domenech et al., 2011; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Overmeer, 6 Boersma, Denison, and Linton 2011), actual translation into a change in clinical behaviors and 7 patient outcomes has been challenging (Fritz, Söderbäck, Söderlund, and Sandborgh, 2018; 8 Overmeer, Boersma, Denison, and Linton, 2011; Sandborgh, Åsenlöf, Lindberg, and Denison, 9 2010). INTRODUCTION 1 1 2 Low back pain (LBP) is a leading cause of disability throughout the world (Hartvigsen et al., 3 2018) and current clinical practice has failed to effectively manage it (Buchbinder et al., 2018). 4 Major guidelines on the management of LBP recommend a biopsychosocial (BPS) management 5 approach (Koes et al., 2010; NICE, 2016), and despite recent studies indicating better 6 understanding of the BPS framework in physiotherapy, transferring research findings into 7 practice is a well-known problem. Unfortunately, professionals’ adherence to evidence-based 8 guidelines is poor (O'Sullivan, O’Keeffe, and O’Sullivan, 2017) and both undergraduate and 9 postgraduate curricula pay very little attention to integrating psychological (cognitive and 10 emotional), social and environmental factors into the management of LBP (Ehrström, Kettunen, 11 and Salo, 2018; Foster and Delitto 2011). As a result, many clinicians report feeling 12 inadequately skilled to treat patients with persistent pain and struggle to deal with the 13 psychosocial factors. They often also stigmatize their patients with persistent pain (Synnott et 14 al., 2015; Toye, Seers, and Barker 2017; Zangoni and Thomson, 2017). Furthermore, health 15 care professionals, including physiotherapists, often use the BPS model dualistically and 16 prioritize biomedical findings. Only when no diagnosis can be found do they turn to 17 psychosocial explanations. This indicates a clear need for training health care professionals to 18 adopt a non-dualistic biopsychosocial approach to help them better support patients with 19 persistent pain (Toye, Seers, and Barker 2017). 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 3 of 47 3 1 way to train physiotherapists to deliver psychologically informed interventions remains 2 unknown, as the results of previous attempts to direct physiotherapists’ practice behavior 3 towards the BPS approach have been conflicting. Although attending BPS-orientated 4 workshops has shown to positively change physiotherapists’ beliefs in the short term 5 (Domenech et al., 2011; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Overmeer, 6 Boersma, Denison, and Linton 2011), actual translation into a change in clinical behaviors and 7 patient outcomes has been challenging (Fritz, Söderbäck, Söderlund, and Sandborgh, 2018; 8 Overmeer, Boersma, Denison, and Linton, 2011; Sandborgh, Åsenlöf, Lindberg, and Denison, 9 2010). INTRODUCTION For example, an eight-day university course on the cognitive behavioral approach for 10 physiotherapists was successful in changing physiotherapists’ beliefs and attitudes, but it did 11 not improve patient outcomes (Overmeer, Boersma, Denison, and Linton, 2011). The authors 12 concluded that the learning process requires time and experiences of practice and clinical 13 supervision, which short courses cannot provide. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 14 Qualitative research provides a deeper understanding of these issues and to date, only a few 15 qualitative studies have explored physiotherapists’ views on changing their practice into a BPS 16 approach in the management of LBP (Karstens et al., 2018; Sanders, Ong, Sowden, and Foster, 17 2014). Three specific studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and 18 Dankaerts 2013; Synnott et al., 2016) have explored physiotherapists’ views on adopting a CFT 19 approach. CFT is an example of a BPS method that challenges more traditional biomechanical 20 / pathoanatomical physiotherapy approaches. It is an integrated physiotherapist-led cognitive 21 and behavioral intervention for individualizing the self-management of persistent LBP, once 22 serious and specific pathology has been excluded and has shown promising results (O’Sullivan, 23 Dankaerts, O’Sullivan, and O’Sullivan, 2015; Vibe Fersum et al., 2013). It uses a BPS clinical 24 reasoning framework to explore, identify and manage cognitive, emotional, social, physical and 25 lifestyle barriers to recovery (O’Sullivan et al., 2018; Synnott et al., 2016). CFT training aims 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 14 Qualitative research provides a deeper understanding of these issues and to date, only a few 15 qualitative studies have explored physiotherapists’ views on changing their practice into a BPS 16 approach in the management of LBP (Karstens et al., 2018; Sanders, Ong, Sowden, and Foster, 17 2014). Three specific studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and 18 Dankaerts 2013; Synnott et al., 2016) have explored physiotherapists’ views on adopting a CFT 19 approach. CFT is an example of a BPS method that challenges more traditional biomechanical 20 / pathoanatomical physiotherapy approaches. INTRODUCTION It is an integrated physiotherapist-led cognitive 21 and behavioral intervention for individualizing the self-management of persistent LBP, once 22 serious and specific pathology has been excluded and has shown promising results (O’Sullivan, 23 Dankaerts, O’Sullivan, and O’Sullivan, 2015; Vibe Fersum et al., 2013). It uses a BPS clinical 24 reasoning framework to explore, identify and manage cognitive, emotional, social, physical and 25 lifestyle barriers to recovery (O’Sullivan et al., 2018; Synnott et al., 2016). CFT training aims 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 4 of 47 4 1 to equip physiotherapists with these skills through a combination of written resources, training 2 workshops that include practical experimentation, and demonstrations with live patients, as well 3 as direct clinical supervision and feedback (O’Sullivan et al., 2018; Vibe Fersum et al., 2013). 4 All three qualitative studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and 5 Dankaerts, 2013; Synnott et al., 2016) found that after CFT training, physiotherapists reported 6 feeling more confident in their capacity and skills to manage the BPS dimensions of non- 7 specific persistent LBP. This included increased confidence in identifying patients’ 8 psychosocial factors and modifying their unhelpful beliefs, understanding the importance of 9 therapeutic alliance and listening skills, and increased focus on everyday functional 10 movements. However, the effects of these changes on patient outcomes are not known. The 11 physiotherapists in the Synnott et al. (2016) and Cowell et al. (2018) studies had undergone 12 intensive CFT training, which included clinical supervision, and the trainers deemed the 13 physiotherapists in the Synnott et al. (2016) study competent in delivering CFT. The twelve 14 physiotherapists in the O’Sullivan, O’Sullivan, O’Sullivan and Dankaerts (2013) study were 15 interviewed after participating in nine days of CFT workshops, but without clinical supervision. 16 As the study included physiotherapists who had changed their beliefs to a greater extent than 17 average, some of whom had previous knowledge of CFT, these findings may not represent the 18 perspective of other physiotherapists who attended the CFT workshops and reported less 19 change in their back-pain beliefs. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu INTRODUCTION 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Physiotherapy Theory and Practice Page 5 of 47 5 5 1 has been intensive and has included direct supervision and feedback (Synnott et al., 2016; Vibe 2 Fersum et al., 2015), which may be a barrier to broad implementation across the profession. It 3 is not known whether less intensive approaches are effective in training physiotherapists in CFT 4 in a non-native English-speaking country. To keep the training in line with the usual delivery 5 of continuing education courses, we conducted a training intervention that consisted of four to 6 six days of workshops combined with a web-based platform offering optional individual 7 learning tasks to support learning. Importantly, the training included no clinical supervision of 8 the physiotherapists. We investigated the views of all the physiotherapists who participated in 9 the workshops. 10 A learning process is always an individual experience and is different for each participant. 11 However, previous studies have paid little attention to the variation between the 12 physiotherapists’ different kinds of experiences of learning and integrating CFT. The aim of 13 the present study was to identify and explore physiotherapists’ conceptions of learning the 14 principles of CFT and integrating it into clinical practice. 15 METHODS 16 Study design 17 To explore the physiotherapists’ conceptions of learning about and integrating CFT into clinical 18 practice, we employed a qualitative research design (Åkerlind, 2017) involving semi-structured 19 interviews of twenty-two physiotherapists who attended a four-day CFT workshop in April 20 2016. We chose a phenomenographic approach as this allowed us to explore the variation in 21 the conceptions of the participating physiotherapists (Åkerlind, 2005; Åkerlind, 2017). INTRODUCTION 22 Physiotherapists’ training 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 has been intensive and has included direct supervision and feedback (Synnott et al., 2016; Vibe 2 Fersum et al., 2015), which may be a barrier to broad implementation across the profession. It 3 is not known whether less intensive approaches are effective in training physiotherapists in CFT 4 in a non-native English-speaking country. To keep the training in line with the usual delivery 5 of continuing education courses, we conducted a training intervention that consisted of four to 6 six days of workshops combined with a web-based platform offering optional individual 7 learning tasks to support learning. Importantly, the training included no clinical supervision of 8 the physiotherapists. We investigated the views of all the physiotherapists who participated in 9 the workshops. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 10 A learning process is always an individual experience and is different for each participant. 11 However, previous studies have paid little attention to the variation between the 12 physiotherapists’ different kinds of experiences of learning and integrating CFT. The aim of 13 the present study was to identify and explore physiotherapists’ conceptions of learning the 14 principles of CFT and integrating it into clinical practice. Physiotherapy Theory and Practice Page 6 of 47 6 6 1 The aim of the training intervention was to: 1. present a multi-dimensional framework for 2 understanding the biopsychosocial nature of LBP, 2. provide training in communication skills 3 to explore the cognitive, emotional and behavioral aspects of LBP, and 3. develop an 4 understanding of how to deliver CFT to patients with persistent LBP (O’Sullivan et al., 2018). 5 It consisted of lectures, group discussions and patient demonstrations in line with previous 6 studies, to enhance learning (O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Main 7 et al., 2012). The more detailed content of the initial training intervention is presented in Table 8 1 and the CFT approach is described in further detail in O’Sullivan et al. (2018). The 9 continuation of learning after the training sessions was supported by providing optional 10 individual learning tasks (reporting of patient cases) and additional reading materials on a web- 11 based platform. A two-day booster session was held in January 2017, which 12 of the 12 physiotherapists attended. The booster session included four patient demonstrations that we 13 used to deepen the physiotherapists’ knowledge of the application of CFT (Table 2). The 14 workshops were delivered by Peter O’Sullivan (initial workshop); Kasper Ussing (booster 15 workshop), who also delivered the CFT part of the training; and Steven J. Linton, who delivered 16 the communication and psychosocial part of the training in the initial workshop. 4 6 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 2 understanding the biopsychosocial nature of LBP, 2. provide training in communication skills 3 to explore the cognitive, emotional and behavioral aspects of LBP, and 3. develop an 4 understanding of how to deliver CFT to patients with persistent LBP (O’Sullivan et al., 2018). 5 It consisted of lectures, group discussions and patient demonstrations in line with previous 6 studies, to enhance learning (O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Main 7 et al., 2012). The more detailed content of the initial training intervention is presented in Table 8 1 and the CFT approach is described in further detail in O’Sullivan et al. (2018). Study design URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice The 9 continuation of learning after the training sessions was supported by providing optional 10 individual learning tasks (reporting of patient cases) and additional reading materials on a web- 11 based platform. A two-day booster session was held in January 2017, which 12 of the 12 physiotherapists attended. The booster session included four patient demonstrations that we 13 used to deepen the physiotherapists’ knowledge of the application of CFT (Table 2). The 14 workshops were delivered by Peter O’Sullivan (initial workshop); Kasper Ussing (booster 15 workshop), who also delivered the CFT part of the training; and Steven J. Linton, who delivered 16 the communication and psychosocial part of the training in the initial workshop. 17 18 Table 1. Content of initial four-day workshop here 19 20 Table 2. Content of two-day booster session here 21 Participants 22 The participants represented a purposive sample of 22 of the 23 physiotherapists who 23 participated in the initial CFT training workshop. One physiotherapist dropped out of the project 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Participants The interviews began by asking: “Tell me about your process of learning CFT and 22 implementing it in clinical practice” and “How do you see CFT now – what does it mean to 23 you?”, and continued dialogically according to the interviewees’ answers (see interview guide 24 Appendix 2.) The interview guide was pilot tested by a member of the research group who was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 Participants URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Page 7 of 47 Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 7 of 47 7 1 after the initial workshop because she changed jobs. We invited primary health care (public 2 health care and occupational health care) study sites from across Finland by contacting the 3 physical and rehabilitation medicine specialists of the hospital districts or the persons in charge 4 of treating musculoskeletal problems in occupational health care. The sites were only included 5 if they were interested in participating in the feasibility study and committed to reimbursing the 6 travel and salary costs of their personnel. The participating physiotherapists were selected from 7 the involved units by the persons in charge, and the researchers did not influence this selection. 8 The first author contacted all the physiotherapists by email and all 22 agreed to participate in 9 the interviews. They were from all over Finland and worked in both public outpatient clinics 10 (14) and occupational (8) health care units. Three of them were men and 19 women, with a 11 mean age of 47 years (33–61). They had an average of 20 (9–31) years of clinical experience 12 after graduation. Appendix 1 shows detailed information on the participants’ gender, age, work 13 experience, health care setting and amount of CFT training. 14 Data collection 15 The first author, who was not involved in planning and delivering the workshops, collected the 16 data in spring 2017 after the end of the training intervention. She was present during the 17 workshops to understand the process of the training but was otherwise unknown to the 18 participants. The semi-structured interviews took place at the physiotherapists’ workplaces 19 according to their wishes. They were conducted in Finnish and only the interviewer and the 20 interviewee were present. The quotations were later translated into English by a professional 21 translator. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Data collection 15 The first author, who was not involved in planning and delivering the workshops, collected the 16 data in spring 2017 after the end of the training intervention. She was present during the 17 workshops to understand the process of the training but was otherwise unknown to the 18 participants. The semi-structured interviews took place at the physiotherapists’ workplaces 19 according to their wishes. They were conducted in Finnish and only the interviewer and the 20 interviewee were present. The quotations were later translated into English by a professional 21 translator. The interviews began by asking: “Tell me about your process of learning CFT and 22 implementing it in clinical practice” and “How do you see CFT now – what does it mean to 23 you?”, and continued dialogically according to the interviewees’ answers (see interview guide 24 Appendix 2.) The interview guide was pilot tested by a member of the research group who was 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Physiotherapy Theory and Practice Page 8 of 47 Page 8 of 47 8 8 8 1 not involved in this study but had undergone CFT training. The interviews lasted 62 minutes 2 on average (47–81) and were audio-recorded and transcribed verbatim (clean, word-to-word) 3 by the first author (Åkerlind, 2008; Brinkmann, 2013). The resulting data consisted of 368 4 pages (font =Times New Roman 12, spacing = 1.5). Participant validation of the 5 transcripts/findings was not carried out (Åkerlind, 2005). 6 Data analysis 7 In the present study, we aimed to outline the variation among physiotherapists’ conceptions of 8 learning and integrating CFT into clinical work. We chose a phenomenographic approach 9 because it enables systematic identification and description of qualitatively different ways of 10 experiencing a phenomenon (Marton and Pong, 2005; Åkerlind, 2005) and the identification of 11 the variation in the physiotherapists’ conceptions and of the hierarchical structure of the 12 conceptions (Åkerlind, 2005; Åkerlind, 2008). Phenomenographic studies aim to elucidate the 13 second order perspective; to present the participants’ conceptions in categories of description 14 that illustrate the variation in how the participants understand the phenomenon in question 15 (Marton and Pong, 2005; Åkerlind, 2017). 16 The phenomenographic analysis followed the principles presented in the literature (Marton and 17 Pong, 2005; Åkerlind, 2005; Åkerlind, 2008). Data collection Phenomenography is a data-driven approach, 18 which means that all findings arise from the data (Åkerlind, 2005). Although the categories of 19 description were derived from the physiotherapists’ interviews, they do not directly represent 20 different types of individuals: They describe the variation in the physiotherapists’ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1 not involved in this study but had undergone CFT training. The interviews lasted 62 minutes 2 on average (47–81) and were audio-recorded and transcribed verbatim (clean, word-to-word) 3 by the first author (Åkerlind, 2008; Brinkmann, 2013). The resulting data consisted of 368 4 pages (font =Times New Roman 12, spacing = 1.5). Participant validation of the 5 transcripts/findings was not carried out (Åkerlind, 2005). URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Data analysis URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 9 1 one conception or may have had conceptions that belonged to different categories, related to 2 different themes. 3 The analysis process began by the first author listening to the interviews and reading the 4 transcripts several times to become familiar with the data and to identify the meaningful units. 5 The selected quotes made up the data pool from which the similarities and differences, as well 6 as the structural relationships, were identified. The initial coding of the data was conducted 7 using a Microsoft Word (Microsoft Corp, Redmond, Washington, USA) document and the 8 themes were found by sorting the printed quotes into piles and examining borderline cases (RH 9 & AP). As the themes emerged, we compared and contrasted the selected quotes in an iterative 10 manner. During this process, we observed that each theme varied hierarchically and by 11 comparing the variation of the themes, we defined the categories of the phenomenon. During 12 the analysis, the results were discussed in the larger study groupwith all of the authors and in a 13 group of qualitative researchers professionals familiar with qualitative research methods, and 14 the consistency between the original data and our findings was constantly evaluated to minimize 15 the influence of our own interpretations. The categories of description were organized 16 hierarchically – some conceptions were more complex or more complete than others. These 17 categories of description represented the expanding awareness of the phenomenon of learning 18 and integrating CFT into clinical practice. During this process, the categories’ critical aspects 19 were identified. These critical aspects of awareness highlight the transitions between the 20 categories of description and describe what is needed to move from the understanding of one Page 9 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 9 of 47 Page 9 of 47 9 1 one conception or may have had conceptions that belonged to different categories, related to 2 different themes. Data analysis The reporting 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 10 of 47 Page 10 of 47 10 1 of the study adhered to the consolidated criteria for qualitative research (COREQ) guidelines 2 (Tong, Sainsbury, and Craig, 2007). 3 4 RESULTS 5 The phenomenon of learning and integrating CFT into clinical practice, as reported by the 6 physiotherapists, was captured by five hierarchical categories of description: I) Recognizing 7 the difference of the new approach; II) The new approach challenging current practice; III) 8 Waking up to explore; IV) Commitment to the new approach; and V) Expanding the application 9 of the new approach. These categories were hierarchically structured, and the latter categories 10 represent more developed conceptions of learning and integration of the CFT approach than the 11 former categories. They varied on the basis of four themes: 1) Membership of work community; 12 2) Learning journey; 3) Transition to new working methods; and 4) Professional role as a 13 physiotherapist (Table 3, Figure 1). P Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 10 1 of the study adhered to the consolidated criteria for qualitative research (COREQ) guidelines 2 (Tong, Sainsbury, and Craig, 2007). 3 4 RESULTS 5 The phenomenon of learning and integrating CFT into clinical practice, as reported by the 6 physiotherapists, was captured by five hierarchical categories of description: I) Recognizing 7 the difference of the new approach; II) The new approach challenging current practice; III) 8 Waking up to explore; IV) Commitment to the new approach; and V) Expanding the application 9 of the new approach. These categories were hierarchically structured, and the latter categories 10 represent more developed conceptions of learning and integration of the CFT approach than the 11 former categories. Data analysis 3 The analysis process began by the first author listening to the interviews and reading the 4 transcripts several times to become familiar with the data and to identify the meaningful units. 5 The selected quotes made up the data pool from which the similarities and differences, as well 6 as the structural relationships, were identified. The initial coding of the data was conducted 7 using a Microsoft Word (Microsoft Corp, Redmond, Washington, USA) document and the 8 themes were found by sorting the printed quotes into piles and examining borderline cases (RH 9 & AP). As the themes emerged, we compared and contrasted the selected quotes in an iterative 10 manner. During this process, we observed that each theme varied hierarchically and by 11 comparing the variation of the themes, we defined the categories of the phenomenon. During 12 the analysis, the results were discussed in the larger study groupwith all of the authors and in a 13 group of qualitative researchers professionals familiar with qualitative research methods, and 14 the consistency between the original data and our findings was constantly evaluated to minimize 15 the influence of our own interpretations. The categories of description were organized 16 hierarchically – some conceptions were more complex or more complete than others. These 17 categories of description represented the expanding awareness of the phenomenon of learning 18 and integrating CFT into clinical practice. During this process, the categories’ critical aspects 19 were identified. These critical aspects of awareness highlight the transitions between the 20 categories of description and describe what is needed to move from the understanding of one 21 category of description to a more complex one (Åkerlind, 2005; Åkerlind, 2008). 22 We obtained ethical approval from the Northern Ostrobothnia Hospital District Ethics 23 Committee. Before conducting the interviews, we explained the nature of the study to the 24 participating physiotherapists. The interviewees provided their informed consent. Data analysis They varied on the basis of four themes: 1) Membership of work community; 12 2) Learning journey; 3) Transition to new working methods; and 4) Professional role as a 13 physiotherapist (Table 3, Figure 1). 14 The abbreviations at the end of the quotes identify the participating physiotherapists and page 15 number of the transcript. The variation of the themes within each category of description are 16 highlighted by the name of each theme in bold and the name of each theme of variation in 17 italics throughout the results section (see Table 3). 18 Insert Table 3 here. 19 Category I Recognizing the difference of the new approach 20 This category describes physiotherapists’ perceptions of learning about CFT and the 21 realizations that arose, especially at the beginning of the learning journey during the workshop 22 and after returning to the clinic. This highlighted the great difference of the CFT approach; the 23 physiotherapists’ previous ways of working, and the ways in which their colleagues still 24 worked. As a result, they reported feeling isolated in their work communities and resistance P Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 19 Category I Recognizing the difference of the new approach 11 1 towards the approach and the training, and insecurity about their skills. They also reported 2 doubting whether this was the right way in which to work. Some were unable to accept the 3 proposed change in their work, and their learning journey did not continue beyond the 4 conceptions described in this category. 5 The first identified theme of variation was membership of the work community, which in this 6 category consists of loneliness in one’s work community. The physiotherapists felt isolated in 7 their work communities, as others did not understand their new way of working, and they had 8 insufficient opportunities to share their thoughts due to a combination of busy workdays and a 9 lack of physiotherapists in the same unit. 10 “This is quite a lonely job; I can’t share these thoughts with anybody.” V24 11 ”In practice [during the workdays] there is not time to discuss but if there would be a forum 12 where we could share our experiences…” Q13 13 The second theme of variation, the learning journey, in this category focused on resistance 14 towards the new approach and training style. The resistance came as a surprise to some of the 15 participants and it was seen as a barrier to learning during the workshop. They understood that 16 the trainers wanted to wake them up, but a few of them found the way of training somewhat 17 abrasive and the patient demonstrations a manifestation of a “guru culture”. Most 18 physiotherapists were able to overcome the resistance, but for some it prevented their learning 19 journey from properly starting. 19 Category I Recognizing the difference of the new approach 20 “During the first two days, I shared a room with X and we had quite tough discussions in the 21 evenings and went for walks to ventilate and it felt quite surreal, I just couldn’t accept it even 22 though the patient cases were clear…my reaction was strong and it took me by surprise that I 23 reacted so strongly, when thinking about it in hindsight… I don’t know, maybe changing one’s 24 own thoughts and beliefs was just so hard…” C1 Page 11 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Physiotherapy Theory and Practice Page 11 of 47 11 towards the approach and the training, and insecurity about their skills. They also reported doubting whether this was the right way in which to work. Some were unable to accept the proposed change in their work, and their learning journey did not continue beyond the conceptions described in this category. Physiotherapy Theory and Practice Page 12 of 47 Page 12 of 47 12 1 “My memory is that we didn’t go through some of the key things in communication, but it was 2 just him [POS] showing off in front of us, just like this video presenting this man who got better, 3 which… in my opinion, diminished his credibility, with him boosting his messages with stuff 4 just like healers and preacher healers do…they have these videos as well, so it doesn’t 5 contribute anything” B17 6 In this category, the third theme, transition to new working methods was related to the 7 physiotherapists feeling insecure about applying CFT in their practice and changing their own 8 way of working. Through experience they accepted parts of it, but they felt they had insufficient 9 training and practice to become confident, because they considered most of their patient 10 population unsuitable for the CFT approach (e.g. patients with acute pain). They also reported 11 insecurity about their clinical reasoning skills in the BPS framework and about their knowledge 12 of pain science and psychosocial factors. 19 Category I Recognizing the difference of the new approach Applying certain aspects of the CFT approach was 13 perceived as difficult and the uncertainties reported by the physiotherapists varied. 14 “I just wish I had more of these [low back pain] patients, there are so many patients with 15 shoulder pain and I would need to be able to practice this broader approach” G14 16 ”Sometimes…I feel like that is this too psychological this approach that I am not ready to think 17 about these things with the patient so deeply and to reflect her thoughts like I don’t have that 18 kind of training I can’t do that” I3 19 “Well, maybe it was drawing the summary…I haven’t done it many times so far… maybe it felt 20 kind of the hardest thing…somehow I don’t trust myself to make it visible so the patient can see 21 what the cause and effect relationships are… that kind of uncertainty…” O9 22 Despite the various challenges to adopting the approach, the physiotherapists reported that their 23 professional role as a physiotherapist had changed. 19 Category I Recognizing the difference of the new approach In this category, they reported having 24 started to look at their patients in a different way, understanding that there was no one correct 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 “My memory is that we didn’t go through some of the key things in communication, but it was 2 just him [POS] showing off in front of us, just like this video presenting this man who got better, 3 which… in my opinion, diminished his credibility, with him boosting his messages with stuff 4 just like healers and preacher healers do…they have these videos as well, so it doesn’t 5 contribute anything” B17 1 “My memory is that we didn’t go through some of the key things in communication, but it was 2 just him [POS] showing off in front of us, just like this video presenting this man who got better, 3 which… in my opinion, diminished his credibility, with him boosting his messages with stuff 4 just like healers and preacher healers do…they have these videos as well, so it doesn’t 5 contribute anything” B17 “Well, maybe it was drawing the summary…I haven’t done it many times so far… maybe it felt kind of the hardest thing…somehow I don’t trust myself to make it visible so the patient can see what the cause and effect relationships are… that kind of uncertainty…” O9 Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 13 of 47 13 13 1 way to move. They noticed that many LBP patients needed relaxation rather than exercises that 2 created more tension and started to observe different aspects in their patients’ movement 3 behaviors. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 19 Category I Recognizing the difference of the new approach Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 14 of 47 14 14 1 “It is usually maximum one hour and I should also do the documentation before the next patient 2 arrives so… this lack of time…these patients would need much more time.” G8 3 ”If we do this kind of thing [CFT] we need to have some kind of [psychological] support for 4 these patients if they haven’t gotten into contact yet, like I don’t feel like a professional in those 5 things even though I feel that I communicate with the patients quite well…we have lots of small 6 groups and some pain groups and breathing groups but it’s not the same, these people need 7 individual [management]… well, I think seeing the need to get these things working was 8 certainly the biggest transformation in this process.” E3 9 “… mostly… in the beginning we had certain structures for doing this kind of preventive 10 assessment and all the materials and templates were there… with this kind of risk-based model 11 of patients’ own opportunities to influence the situation and motor control tests that I now feel 12 are less important, but I still need to give the material to the patient and I feel that I don’t want 13 to so I need to fight it because I see that the old materials are not what I would like to give the 14 patient.” C12 15 The physiotherapists faced personal challenges during the journey. They reported not being 16 able to fully engage in learning, despite recognizing the need for change, because of a lack of 17 English language skills, difficult life situations and other commitments. Their learning 18 journey was not progressing in the way they wanted. 19 Category I Recognizing the difference of the new approach 4 ”It’s kind of a relief that you don’t always need to think that you must do something but you 5 are allowed be laid-back and to relax, there is no right and wrong… no wrong kind of a body 6 and there is no right or wrong kind of sitting posture or…for example during our first day of 7 training that we had a year ago, there were many little things, just basics on how to turn relaxed 8 and lift and be relaxed…” E3 9 “Well, firstly observing the patient …with new eyes, with me immediately looking if he’s tensing 10 some part of his body or if he’s relaxed. Is there some avoidance behavior to do with his 11 tension… it starts with … observing his basic movement: how he sits, how he undresses, how 12 he is, and it already gives some direction to the whole interview part.” F2 13 14 Category II Towards integrating the new approach 15 The focus of this category was on the practical challenges that the physiotherapists faced when 16 trying to integrate their newly learned skills into their practice. There were multiple barriers to 17 the integration of these skills even though they wanted to change their practices, and in this 18 category, old and new ways of working were often combined. 19 The physiotherapists’ understanding and experiences of membership of their work 20 community expanded from the first category to the second. They started reflecting on the 21 organizational processes as barriers to integrating CFT into practice. They reported a lack of 22 capacity to deliver CFT due to short appointment times, being constantly in a hurry and unclear 23 referral pathways. Some also reported feeling the need to adhere to old protocols that they no 24 longer saw as valid. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 way to move. They noticed that many LBP patients needed relaxation rather than exercises that created more tension and started to observe different aspects in their patients’ movement behaviors. 19 Category I Recognizing the difference of the new approach 19 ”Firstly, the English language is not one of my strengths… I looked more at the examples 20 [patient demonstrations] and tried to learn from them.” S1 21 “But immersing myself and getting into the subject has now been of secondary importance 22 because of, well, being busy at work and thinking about personal stuff, so I would’ve gotten 23 much more out of this… if I had… studied it.” E17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 15 of 47 15 15 1 In this category, the transition to new working methods meant that insecurity expanded into 2 combining old and new approaches. This resulted in an understanding that their previous 3 knowledge could be still utilized and the physiotherapists expressed familiarity with and 4 relatedness to the new approach. The new approach helped them rediscover previously learned 5 but unused tools, such as relaxation exercises. 6 ”That way this approach in it was not unfamiliar…I am not trained in psychophysical 7 physiotherapy but I have used those methods because my colleagues have used them and I have 8 been in that kind of community where I have worked certain amount with psychiatric patients 9 and those methods were partially familiar like those related to relaxation and breathing and 10 generally those related to wellbeing … ”K1 11 “All the things we’ve gained…experienced so far, they’re worth utilizing in the background, 12 nothing is, nothing is kind of excluded; it’s just something extra, this approach.” T5 13 In this category, the physiotherapists reported that the new approach challenged them to change 14 their attitudes and language, positively affecting the way they practiced. They reported that 15 looking at their patients in a different way expanded to acquiring a more courageous attitude 16 towards pain, giving patients more positive messages, progressing more confidently with 17 exercises and unraveling patients’ negative beliefs, meaning a change in their professional role 18 as physiotherapists. 19 Category I Recognizing the difference of the new approach 19 “That kind of confidence, that I’ve looked at those particular red flags and I can be sure that 20 I’ve gotten the courage to encourage this patient to move, and I dare to make them bend. That’s 21 the kind of thing that really gets reinforced because before there’s always been … a bit too 22 much respect for the pain, or being afraid … with this training I’ve gained lots of courage and 23 I’ve tried to, like, communicate to the patient that there’s nothing to be afraid of, that all those 24 sensations aren’t necessarily [dangerous]…” P4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 16 of 47 Page 16 of 47 16 1 “What it means is that I aim to strengthen the person’s trust in her own body and tell that the 2 back is strong, a positive outlook on the body and it’s use. Confidence and courage… and to 3 bring up positive things about the patient, things that can help the patient move on.” V1 4 5 Category III Waking up to explore 6 In this category, the training made the physiotherapist wake up to explore their practice more 7 broadly and after starting to explore ways to make the new approach feasible in their own work 8 environments, they started to seek solutions to how they could be better help their patients. This 9 reflective process, which was painful for many, led physiotherapists outside their comfort zones 10 towards a more multidimensional approach in their practice that considered psychosocial issues 11 and scientific evidence. This was mostly new to the physiotherapists. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 16 1 “What it means is that I aim to strengthen the person’s trust in her own body and tell that the 2 back is strong, a positive outlook on the body and it’s use. 19 Category I Recognizing the difference of the new approach Confidence and courage… and to 3 bring up positive things about the patient, things that can help the patient move on.” V1 4 5 Category III Waking up to explore 6 In this category, the training made the physiotherapist wake up to explore their practice more 7 broadly and after starting to explore ways to make the new approach feasible in their own work 8 environments, they started to seek solutions to how they could be better help their patients. This 9 reflective process, which was painful for many, led physiotherapists outside their comfort zones 10 towards a more multidimensional approach in their practice that considered psychosocial issues 11 and scientific evidence. This was mostly new to the physiotherapists. 12 The membership of the work community was seen in this category as desire for a common 13 language. The physiotherapists noticed that the mixed messages that patients received from 14 different professionals, for example interpretation of MRI results, made their work more 15 difficult. They also sought more multidisciplinary work methods than those currently in use in 16 their work communities. The desire for a common language extended beyond their own work 17 community; they also wished to spread evidence-based understanding of LBP more widely 18 among health care professionals and in the media. 19 “Well, the problem was that he [a patient] had an assessment and opinion from ten different 20 experts, as well as his own… it was really hard to start helping him.” D2 21 ”If other professionals could adopt this… and maybe cooperate… in the future we should do 22 multidisciplinary work…that could maybe be fruitful if we spoke the same language and we ld h h 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 16 Physiotherapy Theory and Practice Page 17 of 47 17 17 1 “This should be in the news. This is such a wonderful message that everybody should be aware 2 of this.” V23 3 The physiotherapists stated that to get the learning journey started, a feeling of being shaken 4 was necessary. 19 Category I Recognizing the difference of the new approach It is not easy to turn one’s thinking upside down, and in the beginning, the 5 physiotherapists felt dumbfounded; the training was an eye-opening experience. For some, the 6 shock was bigger than expected. Others stated that this was the greatest change in their 7 professional thinking, their biggest upheaval since graduation. 8 ”Could I say that sometimes making a change gets easier, you know, when you get properly 9 shaken up, it might be… it can depend on a person, it may be that for me it suited well because 10 I adopted those things quite easily” V16 11 “Well, those four days, it was like being hit over the head, somehow just like a kind of stunning 12 experience, so really, really like interesting… somehow I kind of woke up and became eager to 13 sort of look at things from a totally different viewpoint.” O1 14 The transition to new working methods was linked to a critical reflection on one’s own way 15 of working. Some even felt ashamed that they had earlier unhesitatingly believed what was 16 taught at workshops. Now they discovered that some of those statements lacked evidence, 17 which led to observing their previous ways of thinking and working critically, and they hoped 18 to adopt work practices based on evidence in the future. 19 Category I Recognizing the difference of the new approach 19 ”Of course I haven’t been scientifically oriented, I have just learned by doing and I have 20 thought that what I have learned was right and I have never been able to question whether there 21 would be something wrong… so it was confusing… but it makes sense because he could explain 22 using research and the materials I have been reading…” P3 23 “I look at the way I worked earlier in a different light…I don’t mean that what I’ve done has 24 been all wrong, but with a certain patient population, with the high-risk ones there, I don’t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 “Well, those four days, it was like being hit over the head, somehow just like a kind of stunning experience, so really, really like interesting… somehow I kind of woke up and became eager to sort of look at things from a totally different viewpoint.” O1 23 “I look at the way I worked earlier in a different light…I don’t mean that what I’ve done has 24 been all wrong, but with a certain patient population, with the high-risk ones there, I don’t 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 18 of 47 18 18 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 3 The physiotherapists’ conceptions of their professional role as a physiotherapist broadened 4 further in this category and expanded beyond their previous biomedical focus, to consider 5 addressing psychosocial factors. This led them to step outside their comfort zones. Many started 6 using the Örebro Musculoskeletal Pain Screening Questionnaire (ÖMPSQ) and the STarT Back 7 Screening Tool (SBST) as shields when starting these conversations, and felt they were now 8 able to listen to distressing patient stories. They understood they did not have to be 9 psychologists to talk about all aspects of life; they could be humans to other humans and obtain 10 permission to use more time for interviews. 19 Category I Recognizing the difference of the new approach 11 ”If it works… it is because as a therapist in a situation with the skills I have, I feel that I can’t 12 handle this and progress with this, the questionnaire works as…I don’t know whether shield is 13 the right word for that” B6 14 “This phrasing of questions and then the listening itself and getting to grips with those slightly 15 trickier topics so that I’m like ready to listen to them; earlier, if the patient started to talk about 16 them, I pretty quickly tried to shift the focus away, so I was listening and hearing but then again 17 not really…” C3 18 ”After the trip to Lahti [booster session] I told the girls that now I understood that I don’t need 19 to be a psychologist, that I can ask questions, but I don’t need to be the psychologist in the way 20 it came across on that trip, you know.. it sort of woke up in me” N3 21 22 Category IV: Commitment to the new approach URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 3 The physiotherapists’ conceptions of their professional role as a physiotherapist broadened 4 further in this category and expanded beyond their previous biomedical focus, to consider 5 addressing psychosocial factors. This led them to step outside their comfort zones. Many started 6 using the Örebro Musculoskeletal Pain Screening Questionnaire (ÖMPSQ) and the STarT Back 7 Screening Tool (SBST) as shields when starting these conversations, and felt they were now 8 able to listen to distressing patient stories. They understood they did not have to be 9 psychologists to talk about all aspects of life; they could be humans to other humans and obtain 10 permission to use more time for interviews. 11 ”If it works… it is because as a therapist in a situation with the skills I have, I feel that I can’t 12 handle this and progress with this, the questionnaire works as…I don’t know whether shield is 13 the right word for that” B6 14 “This phrasing of questions and then the listening itself and getting to grips with those slightly 15 trickier topics so that I’m like ready to listen to them; earlier, if the patient started to talk about 16 them, I pretty quickly tried to shift the focus away, so I was listening and hearing but then again 17 not really…” C3 18 ”After the trip to Lahti [booster session] I told the girls that now I understood that I don’t need 19 to be a psychologist, that I can ask questions, but I don’t need to be the psychologist in the way 20 it came across on that trip, you know.. it sort of woke up in me” N3 21 22 Category IV: Commitment to the new approach URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Physiotherapy Theory and Practice Page 19 of 47 19 1 In the fourth category, the physiotherapists started to commit to the new approach with support 2 from their work communities. They reported a growing conviction in the approach, an 3 increasing feeling of competence and an understanding that a strong therapeutic alliance 4 facilitates better patient results. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 The doubts transformed into enthusiasm and many themes 5 within this category describe enablers of learning and integrating CFT rather than the barriers 6 described in the lower categories. 7 This category expanded the understanding of learning and integrating CFT into clinical practice 8 to also understanding the importance of commitment to the new approach. In this category, 9 membership of the work community meant being enhanced by a supportive work community, 10 in which supportive leadership, colleagues with a similar understanding, the opportunity to 11 share clinical experiences, and flexibility in appointment duration all helped in committing to 12 the new approach and sustaining learning as well as changing their practice behaviors. 13 “It’s already a big help that our senior physician knows about this and accepts it … it certainly 14 helps that there are others who’ve been there [on CFT course] so we can share… I’m allowed 15 to work this way and use this kind of approach, and we’re lucky we can plan our own 16 appointment lists, so in principle, when I see there’s this kind of patient then I can give them a 17 longer appointment so I won’t run out of time straightaway...” R12 18 ”It is good, it is very good to have colleagues who I can share and think together these things, 19 it is very fruitful and it helps continuing one’s own learning when I can think about this…” Q17 20 Committing to the new approach and setting out on the learning journey required becoming 21 convinced of the approach. Physiotherapists reported that this was facilitated through seeing 22 patient demonstrations. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 Moreover, watching presentations of scientific evidence, success with 23 their own patients or resolving their own back problems by experimenting and utilizing the CFT 24 approach on themselves were also seen as important, as this made the physiotherapists see that Page 19 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 1 In the fourth category, the physiotherapists started to commit to the new approach with support 2 from their work communities. They reported a growing conviction in the approach, an 3 increasing feeling of competence and an understanding that a strong therapeutic alliance 4 facilitates better patient results. The doubts transformed into enthusiasm and many themes 5 within this category describe enablers of learning and integrating CFT rather than the barriers 6 described in the lower categories. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 20 of 47 Page 20 of 47 20 1 it worked not only for the trainers, who were experienced in using the approach, but also 2 possibly for the participants. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 3 “Well, there were the patient demonstrations, they’ve been really good, and no way do I believe 4 that any kind of lectures could open up this approach this well, and there I was, amazed how 5 … this approach kind of … clicked for them, when I listened to these patients at the beginning 6 I was thinking that this is never going to work and then it was just, click, and there it was, you 7 saw that they were responsive and, well, the demonstrations were somehow quite 8 unbelievable.” O8 9 ”I have tried to reach relaxation and through relaxation my back is basically painfree…so I 10 have experienced personally this small going astray in this thing and I have personally 11 benefitted and experienced that this ideology is helpful and now I try to tell this joyous message 12 for my patients” F8 13 The physiotherapists perceived that CFT helped them gain better treatment outcomes and the 14 transition to new working methods meant that they felt more equipped to help patients with 15 persistent LBP. They saw more complex patient cases as positive challenges and no longer as 16 a source of frustration, and this helped them commit to the new approach. This also increased 17 their enthusiasm towards their work. 18 “It’s had an effect, it’s somehow given me the feeling that I can do this…it’s challenging but I 19 sort of feel much better equipped and I can, like, say that there’s a certain kind of professional 20 pride in knowing and doing something quite valuable…” U13 21 ”One is more… enthusiastic about those back pain patients, earlier I was like oh no, that one 22 comes again.. that… she has pain in the knee and all other aches and pains. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 I am more 23 courageous to meet the patient because I don’t need to think who of my colleagues could I send Pag Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20 1 it worked not only for the trainers, who were experienced in using the approach, but also 2 possibly for the participants. 1 it worked not only for the trainers, who were experienced in using the approach, but also 2 possibly for the participants. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 21 of 47 21 1 her to if I can’t do anything with her so I have more courage to handle the situation …my own 2 enthusiasm and efficacy has grown here…” J13 3 The theme of the professional role as a physiotherapist broadened as the physiotherapists 4 reported getting closer to the patients. This was possible through new-found, person-centered 5 communication skills, being present and listening to a patient’s story and using time for the 6 interview, which were seen as ways to improve treatment outcomes. 7 “Yes, well, firstly the interviews have become much longer so it takes much more time when 8 you let the patient really talk, I’ve had to hold myself back so I don’t put words in the patient’s 9 mouth; I do that sometimes because I’m kind of fast-paced, so sometimes I need to slow down 10 and tell myself to shut up and let the patient tell… their own story.” P7 11 ”My way of thinking has been enhanced, that when you listen to the patient and you have time… 12 for that patient, that you listen and that trust. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 That it is one thing I get praised for - often I have 13 been the first person who has had time to stop among that patient’s problems that ...well… 14 those seeds sometime sprout from small things I believe in that kind of on the other hand simple 15 things, in the fruitfulness of the beginning of the therapeutic alliance” K3 16 ”Now she’s been heard because it’s here that we listen so you … you just ask the question and 17 you listen and the same patient can have the experience before of coming to listen when talked 18 to, when talking about his issues and being given instructions, but he’s not necessarily asked 19 about very much … that the listening skill in this, in this approach, it’s grown, and I’ve taken 20 the skill of listening over into my everyday life and my own working community as well” T13 21 22 Category V Expanding application of the new approach 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 21 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 22 of 47 Page 22 of 47 22 22 1 In the fifth and widest category, the physiotherapists started applying their skills more broadly, 2 adapting the new approach to their own work environments. This resulted in more 3 collaboration, using the skills in the treatment of other musculoskeletal problems, 4 understanding the importance of continuous learning and using creativity in their work. This 5 was also manifested as renewed professional identity and a wider professional role. These 6 conceptions represent a good understanding of the important principles of CFT. 7 In this category, the physiotherapists understood the importance of multidisciplinarity and saw 8 the membership of their work community as playing an integral role as a member of a team. 9 Following the training, the physiotherapists reported collaborating more closely with 10 psychologists, psychiatric nurses, physical education instructors, and doctors. This enabled 11 them to help patients navigate the health care system and to know where to refer them if needed. 1 think we’ve been able to act in the right way…the approaches we’ve used earlier, they haven’t 2 been the right ones.” C17 12 “Maybe I’ve learned…to offer more conversational support, for example, through a 13 psychologist or psychiatric nurse, because here the good thing is that you can see a psychologist 14 without a referral, she’s said that I can send my patients… and in fact I’ve cooperated a fair 15 bit with the psychologist.” K4 16 ”The experience for the patient that he gets the multidisciplinarity and he gets the help for sure 17 much better in that situation” M17 18 The physiotherapists described their learning journey as a continuous adventurous journey 19 that continued beyond the official end of the training intervention, and that becoming convinced 20 about the approach was not the end of the journey. The initial four-day workshop was seen as 21 a good start to becoming familiar with the approach, but the physiotherapists stated it took time 22 and practice for things to fall into place. Those who participated in the booster session saw it as 23 important to consolidate and advance their learning. They described the journey as a wave 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 In the fifth and widest category, the physiotherapists started applying their skills more broadly, 2 adapting the new approach to their own work environments. This resulted in more 3 collaboration, using the skills in the treatment of other musculoskeletal problems, 4 understanding the importance of continuous learning and using creativity in their work. This 5 was also manifested as renewed professional identity and a wider professional role. These 6 conceptions represent a good understanding of the important principles of CFT. perience for the patient that he gets the multidisciplinarity and he gets the help for sure URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 23 1 motion: feeling tired from time to time and regressing back to old routines, but then receiving 2 support to continue the journey again. 3 “Then about the booster session, I think it was absolutely essential because it cleared up a lot 4 of thoughts and I’d managed to try it out a bit in practice… I could sort of absorb the knowledge, 5 I was at that point that, like, now I want to learn all of this, I wanted to even the first time, but 6 I just couldn’t manage it so well, only now I see it was good that the training was longer … 7 after that it’s felt like it clarified my thinking a lot more” A3 8 “Well, this year’s been a kind of a journey of adventure, the whole time I feel like I’m 9 continuously studying and learning this…” V15 10 ”If I reflect back on this period, I see it’s started with small steps and they’ve become bigger 11 as I’ve adopted the tools and in a way it’s became partly a routine, but I don’t mean I’ve 12 reached my target in any way or am somehow skilled in using this method, but I sort of feel I’ve 13 learned and made progress” K4 14 15 The transition to new working methods in this category was represented by newly learnt skills 16 taking on a personal shape. The physiotherapists felt they were given permission for creativity, 17 enabling wide use of their personality and skills. They reported feeling liberated after not having 18 to strictly work according to certain rules and formulae anymore and were instead able to be 19 more patient-centered. perience for the patient that he gets the multidisciplinarity and he gets the help for sure 20 “It’s somehow a creative space nowadays…what I find amazing is that there are no specific 21 [rules]… I don’t know beforehand what’s going to happen next… compared to earlier when we 22 had certain kinds of practices … we did certain kinds of tests with all the patients…” V13 23 ”In my opinion this [CFT] has enabled that I can even use myself creatively…” D14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 23 1 motion: feeling tired from time to time and regressing back to old routines, but then receiving 2 support to continue the journey again. Physiotherapy Theory and Practice Page 24 of 47 24 1 The physiotherapists reported that their professional role as a physiotherapist and their 2 outlook had changed from that of an “expert” to that of an “enabler”. This included helping 3 patients develop greater awareness of their cognitive processes and behaviors and helping them 4 regain body awareness while acting as a coach. The professional identity of the physiotherapists 5 was renewed. They felt motivated when the patients figured things out by themselves and the 6 physiotherapists could support their self-efficacy and saw the value of patients being able to 7 contact them if needed. 8 “Well, it’s always when you get somebody to figure things out and if I manage to do that then 9 it’s a very powerful motivating factor because so much can happen in that person’s thinking 10 about the use of the body as well… it’s these moments that are awfully motivating and I don’t 11 think those sorts of strong experiences of successfully helping someone are even necessarily 12 possible with a more traditional or other way…”A9 13 ”And also those instructions for the future and follow-up and that kind that they feel, those 14 people that they can come and always contact me if they need.. perience for the patient that he gets the multidisciplinarity and he gets the help for sure that they have somebody they 15 can trust and who they can return to” G4 16 SUMMARY OF THE FINDINGS AND CRITICAL ASPECTS BETWEEN THE 17 CATEGORIES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 1 The physiotherapists reported that their professional role as a physiotherapist and their 2 outlook had changed from that of an “expert” to that of an “enabler”. This included helping 3 patients develop greater awareness of their cognitive processes and behaviors and helping them 4 regain body awareness while acting as a coach. The professional identity of the physiotherapists 5 was renewed. They felt motivated when the patients figured things out by themselves and the 6 physiotherapists could support their self-efficacy and saw the value of patients being able to 7 contact them if needed. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu perience for the patient that he gets the multidisciplinarity and he gets the help for sure that they have somebody they 15 can trust and who they can return to” G4 16 SUMMARY OF THE FINDINGS AND CRITICAL ASPECTS BETWEEN THE 17 CATEGORIES 18 Insert Figure 1 here 19 We identified a number of key aspects that changed between the categories, which can be 20 considered essential for the learning journey towards adopting CFT and which should be 21 considered when planning support for physiotherapists during this process. The first critical 22 aspect which changed between Categories I and II, was the ability to overcome resistance and 23 to change one’s views. This included accepting new ideas and implementing parts of the new 24 approach alongside old ways of working. The process of learning and integrating CFT into 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 24 1 The physiotherapists reported that their professional role as a physiotherapist and their 2 outlook had changed from that of an “expert” to that of an “enabler”. This included helping 3 patients develop greater awareness of their cognitive processes and behaviors and helping them 4 regain body awareness while acting as a coach. The professional identity of the physiotherapists 5 was renewed. They felt motivated when the patients figured things out by themselves and the 6 physiotherapists could support their self-efficacy and saw the value of patients being able to 7 contact them if needed. 8 “Well, it’s always when you get somebody to figure things out and if I manage to do that then 9 it’s a very powerful motivating factor because so much can happen in that person’s thinking 10 about the use of the body as well… it’s these moments that are awfully motivating and I don’t 11 think those sorts of strong experiences of successfully helping someone are even necessarily 12 possible with a more traditional or other way…”A9 13 ”And also those instructions for the future and follow-up and that kind that they feel, those 14 people that they can come and always contact me if they need.. 16 SUMMARY OF THE FINDINGS AND CRITICAL ASPECTS BETWEEN THE 17 CATEGORIES NPT proposes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 clinical practice evolved further between Categories II and III, and the critical aspects that 2 changed between these categories arose from the experience of being shaken, the ability to 3 critically reflect on one’s own ways of thinking and working. This meant also stepping outside 4 one’s comfort zone to further explore the possibilities of the new approach. The critical aspects 5 that helped the learning journey continue further from Category III to IV were support from the 6 work community and gaining confidence in one’s own skills through experiential learning, 7 which led to becoming convinced of the new approach. Finally, the critical steps from Category 8 IV to V that led towards expanding the application of the new approach were multidisciplinary 9 collaboration, the use of one’s creativity at work, and understanding the importance of 10 continuous learning. Learning did not stop after the training intervention ended; it became an 11 ongoing journey. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 16 SUMMARY OF THE FINDINGS AND CRITICAL ASPECTS BETWEEN THE 17 CATEGORIES CATEGORIES URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 25 of 47 25 25 1 clinical practice evolved further between Categories II and III, and the critical aspects that 2 changed between these categories arose from the experience of being shaken, the ability to 3 critically reflect on one’s own ways of thinking and working. This meant also stepping outside 4 one’s comfort zone to further explore the possibilities of the new approach. The critical aspects 5 that helped the learning journey continue further from Category III to IV were support from the 6 work community and gaining confidence in one’s own skills through experiential learning, 7 which led to becoming convinced of the new approach. Finally, the critical steps from Category 8 IV to V that led towards expanding the application of the new approach were multidisciplinary 9 collaboration, the use of one’s creativity at work, and understanding the importance of 10 continuous learning. Learning did not stop after the training intervention ended; it became an 11 ongoing journey. 12 DISCUSSION 13 The main finding of this phenomenographic study is that physiotherapists’ conceptions of 14 learning and integrating CFT into clinical practice vary greatly. The results show that a number 15 of factors influenced the physiotherapists’ learning journey. The critical aspects between the 16 categories of description can be understood as stepping stones towards more complete 17 perceived learning and integration of CFT into clinical practice. The physiotherapists 18 participating in our study worked in different kinds of work communities, had different 19 backgrounds, levels of work experience, and opportunities to engage in learning. These factors 20 possibly contributed to the variation of the physiotherapists’ conceptions. 21 The process of learning and integrating CFT into clinical practice described by the participating 22 physiotherapists has many commonalities with the Normalization Process Theory (NPT), 23 which helps explain barriers and enablers of the adaptation of new approaches as well as the 24 activities people engage in when attempting to change their ways of working. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 26 of 47 26 1 that implementation is operationalized through four mechanisms: coherence, cognitive 2 participation, collective action and reflexive monitoring (May and Finch, 2009). These 3 mechanisms can be seen in our study in the process in which the physiotherapists attempted to 4 establish coherence between their previous practices and the new approach, described below. 5 First of all, the physiotherapists reported that they had previously received biomedically- 6 focused training, and that the CFT approach was very different to that which they had been 7 taught during their undergraduate training and further education workshops. In the beginning, 8 during the initial workshop, almost everybody felt considerably challenged, which created a 9 great deal of cognitive dissonance and resistance. Earlier studies exploring physiotherapists’ 10 experiences of adopting a BPS approach for the management of LBP have not reported this. 11 Sanders, Ong, Sowden and Foster (2014) reported thoughtful obedience from physiotherapists, 12 but no resistance. This may partially be due to the biomedical background and no previous 13 knowledge of CFT among the physiotherapists participating in our study, whereas in many 14 previous studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; 15 Synnott et al., 2016), at least some of the participants had earlier knowledge of the BPS 16 approach and were competent in CFT or had much more extensive training. Furthermore, many 17 other psychologically informed physiotherapy approaches concentrate on teaching cognitive 18 behavioral therapy methods in addition to previous skills, whereas CFT directly challenges the 19 biomedical beliefs related to physiotherapy practice. This may partly explain our findings. 20 Based on the conceptions of the physiotherapists in our study, if the resistance towards the new 21 approach and other barriers could not be overcome, no coherence was found, which led to 22 discontinuing the learning journey and not adopting the CFT approach. The first critical aspect, 23 cognitive flexibility was important for overcoming resistance and changing one’s attitudes and 24 beliefs, and required cognitive participation (May and Finch 2009). URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Physiotherapy Theory and Practice Page 27 of 47 27 27 1 were important drivers of changing their ways of working. Reflective monitoring of their own 2 practice and thinking was crucial at this level. It seems important that, in addition to the content 3 of the CFT approach, the course provided new tools to reflect on one’s own practices and to 4 critically assess information that was delivered by other professionals, the media and journals. 5 Self-reflection is seen as necessary for health care professionals treating people with 6 musculoskeletal problems (Nijs et al., 2013). This might be critical for keeping the lifelong 7 learning journey moving forward and is an important factor for all training interventions to take 8 into account. 9 In addition to earlier biomedically-oriented training, we identified a number of barriers to 10 learning and adopting CFT, some of which were similar to those in earlier studies that have 11 explored physiotherapists’ experiences of applying psychologically informed care in clinical 12 practice. For example, Karstens et al. (2018) explored physiotherapists’ views on adopting a 13 stratified treatment approach in Germany and reported a lack of clear referral pathways as a 14 barrier to adopting the approach, very like our results. Another study (Sanders, Ong, Sowden, 15 and Foster, 2014) explored the same approach in the UK and also recognized the lack of a 16 common language among health care professionals, manifested as confusing messages to 17 patients and a barrier to implementation. We, like most other qualitative studies in this field 18 (Cowell et al., 2018; Fritz, Söderbäck, Söderlund, and Sandborgh, 2018; Karstens et al., 2018; 19 Sanders, Ong, Sowden, and Foster, 2013) observed that a lack of time and a limited number of 20 patient appointments were also common barriers. The economic benefits of spending more time 21 with complex LBP patients must be demonstrated to justify spending this extra time with them. 22 Previous studies (Foster and Delitto, 2011; Nielsen, Keefe, Bennell, and Jull, 2014) also support 23 the importance of appropriate referrals to psychological health professionals. Physiotherapy Theory and Practice The physiotherapists who 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 that implementation is operationalized through four mechanisms: coherence, cognitive 2 participation, collective action and reflexive monitoring (May and Finch, 2009). These 3 mechanisms can be seen in our study in the process in which the physiotherapists attempted to 4 establish coherence between their previous practices and the new approach, described below. 3 mechanisms can be seen in our study in the process in which the physiotherapists attempted to 4 establish coherence between their previous practices and the new approach, described below. 5 First of all, the physiotherapists reported that they had previously received biomedically- 6 focused training, and that the CFT approach was very different to that which they had been 7 taught during their undergraduate training and further education workshops. In the beginning, 8 during the initial workshop, almost everybody felt considerably challenged, which created a 9 great deal of cognitive dissonance and resistance. Earlier studies exploring physiotherapists’ 10 experiences of adopting a BPS approach for the management of LBP have not reported this. 11 Sanders, Ong, Sowden and Foster (2014) reported thoughtful obedience from physiotherapists, 12 but no resistance. This may partially be due to the biomedical background and no previous 13 knowledge of CFT among the physiotherapists participating in our study, whereas in many 14 previous studies (Cowell et al., 2018; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; 15 Synnott et al., 2016), at least some of the participants had earlier knowledge of the BPS 16 approach and were competent in CFT or had much more extensive training. Furthermore, many 17 other psychologically informed physiotherapy approaches concentrate on teaching cognitive 18 behavioral therapy methods in addition to previous skills, whereas CFT directly challenges the 19 biomedical beliefs related to physiotherapy practice. This may partly explain our findings. Physiotherapy Theory and Practice In contrast to 24 these, the physiotherapists in our study also reported a lack of support and feelings of isolation 25 in their work communities, as well as the expectation to follow clinical protocols that they no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 were important drivers of changing their ways of working. Reflective monitoring of their own 2 practice and thinking was crucial at this level. It seems important that, in addition to the content 3 of the CFT approach, the course provided new tools to reflect on one’s own practices and to 4 critically assess information that was delivered by other professionals, the media and journals. 5 Self-reflection is seen as necessary for health care professionals treating people with 6 musculoskeletal problems (Nijs et al., 2013). This might be critical for keeping the lifelong 7 learning journey moving forward and is an important factor for all training interventions to take 8 into account. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 9 In addition to earlier biomedically-oriented training, we identified a number of barriers to 10 learning and adopting CFT, some of which were similar to those in earlier studies that have 11 explored physiotherapists’ experiences of applying psychologically informed care in clinical 12 practice. For example, Karstens et al. (2018) explored physiotherapists’ views on adopting a 13 stratified treatment approach in Germany and reported a lack of clear referral pathways as a 14 barrier to adopting the approach, very like our results. Another study (Sanders, Ong, Sowden, 15 and Foster, 2014) explored the same approach in the UK and also recognized the lack of a 16 common language among health care professionals, manifested as confusing messages to 17 patients and a barrier to implementation. Physiotherapy Theory and Practice We, like most other qualitative studies in this field 18 (Cowell et al., 2018; Fritz, Söderbäck, Söderlund, and Sandborgh, 2018; Karstens et al., 2018; 19 Sanders, Ong, Sowden, and Foster, 2013) observed that a lack of time and a limited number of 20 patient appointments were also common barriers. The economic benefits of spending more time 21 with complex LBP patients must be demonstrated to justify spending this extra time with them. 22 Previous studies (Foster and Delitto, 2011; Nielsen, Keefe, Bennell, and Jull, 2014) also support 23 the importance of appropriate referrals to psychological health professionals. In contrast to 24 these, the physiotherapists in our study also reported a lack of support and feelings of isolation 25 in their work communities, as well as the expectation to follow clinical protocols that they no 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 28 of 47 28 1 longer considered evidence based. Previous research has reported that clinical mentoring, which 2 our physiotherapists did not receive, is an enabler of and is crucial to changing one’s practice. 3 (Cowell et al., 2018; Synnott et al., 2016) 4 Implementation studies have identified that when change in practice happens, it also occurs at 5 the level of the whole work community and collective action is needed (May and Finch 2009), 6 whereas changing an individual’s beliefs and competences is not sufficient to bring about 7 changes in clinical behaviors. If physiotherapists feel lonely in the process of change, they 8 easily regress back to their old ways of working (Piirainen and Viitanen, 2010). Therapist drift 9 is a known phenomenon and is described in psychotherapy literature (Waller, 2009), 10 highlighting that clinicians do not always deliver therapy according to its principles, despite 11 having undergone training. The physiotherapists in our study also noticed this phenomenon. 12 When they encountered problems, they reported feeling insecure about the CFT approach and 13 went back to their old, familiar ways of working, which felt safer. Physiotherapy Theory and Practice Overmeer, Boersma, Denison 14 and Linton (2011) state that it might even be unrealistic to expect a single physiotherapist to 15 change outcomes among patients with complex problems, and that this requires collective 16 action and a broader change at an organizational level. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 longer considered evidence based. Previous research has reported that clinical mentoring, which our physiotherapists did not receive, is an enabler of and is crucial to changing one’s practice. (Cowell et al., 2018; Synnott et al., 2016) 1 longer considered evidence based. Previous research has reported that clinical mentoring, which 2 our physiotherapists did not receive, is an enabler of and is crucial to changing one’s practice. 3 (Cowell et al., 2018; Synnott et al., 2016) 4 Implementation studies have identified that when change in practice happens, it also occurs at 5 the level of the whole work community and collective action is needed (May and Finch 2009), 6 whereas changing an individual’s beliefs and competences is not sufficient to bring about 7 changes in clinical behaviors. If physiotherapists feel lonely in the process of change, they 8 easily regress back to their old ways of working (Piirainen and Viitanen, 2010). Therapist drift 9 is a known phenomenon and is described in psychotherapy literature (Waller, 2009), 10 highlighting that clinicians do not always deliver therapy according to its principles, despite 11 having undergone training. The physiotherapists in our study also noticed this phenomenon. 12 When they encountered problems, they reported feeling insecure about the CFT approach and 13 went back to their old, familiar ways of working, which felt safer. Overmeer, Boersma, Denison 14 and Linton (2011) state that it might even be unrealistic to expect a single physiotherapist to 15 change outcomes among patients with complex problems, and that this requires collective 16 action and a broader change at an organizational level. Physiotherapy Theory and Practice 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 7 Personal challenges in learning CFT, as reported by the physiotherapists in our study, and which 8 have not arisen in earlier studies, should be considered when planning support for the 9 participants of future interventions. Lack of English language skills, difficult personal situations 0 and commitment to other studies were all reported as barriers to learning. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 29 of 47 29 29 1 their new and old ways of working, and not using the new approach regularly. In contrast, others 2 reported having fully adopted the CFT approach and that it had become normalized in their 3 practice (May and Finch, 2009). Like us, Sanders, Ong, Sowden and Foster (2014) observed 4 that the physiotherapists were convinced to change their practices by trying out the new 5 approach and reflecting critically on their work. Also in line with previous studies (Nielsen, 6 Keefe, Bennell, and Jull, 2014; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; 7 Synnott et al., 2016), although most of our participants reported convincing scientific evidence 8 and live patient demonstrations as the most important enablers of learning, some 9 physiotherapists experienced these demonstrations and the style of presenting the evidence as 10 negative. 11 A more complete perceived change in one’s practice towards the CFT approach seems to have 12 been reinforced by support and collaboration in the work community, by becoming convinced 13 and gaining confidence and by successes with patients with complex problems through an 14 experiential learning process. Furthermore, the idea of being creative in one’s work and 15 continuously learning also seem to have been important. One aspect that was unique to our 16 study was that the physiotherapists reported becoming convinced through applying principles 17 of CFT to themselves (if they suffered from LBP). 18 We do not know whether the physiotherapists changed their practices. This we will explore in 19 future studies. It has been proposed that new knowledge is implemented at individual, group 20 and organizational levels (Piirainen and Viitainen, 2010; Zidarov, Thomas, and Poissant, 2013) 21 through participating in an iterative process that instead of proceeding linearly, includes phases 22 of more and less active progression. Physiotherapy Theory and Practice For example, the use of screening for psychosocial risk factors in 10 LBP is widely recommended (Lin et al., 2019), but many physiotherapists found this 11 challenging. In line with the conceptions of some of the participants in our study, many other 12 studies have reported the lack of knowledge regarding psychosocial issues and interventions as 13 a barrier to more widely implementing this perspective in physiotherapy practice (Beissner et 14 al., 2009; Foster and Delitto, 2011; Main and George, 2011; Nielsen, Keefe, Bennell, and Jull, 15 2014; Singla, Jones, Edwards, and Kumar, 2015). Even though the physiotherapists in our study 16 were taught how to use the questionnaires, many of them had not started using them or only 17 used them occasionally. This is similar to the results of a study by Sanders, Foster, and Ong 18 (2011), in which general practitioners who were taught to use a subgrouping tool reported 19 barriers to its use: time constraints and other organizational pressures, and a lack of coherence 20 related to the new way of working. However, in our study, the questionnaires were reported as 21 being enablers of discussion of psychosocial issues, as they worked as shields. 22 As shown in previous research (Matthias et al., 2010; Toye, Seers and Barker, 2017), primary 23 care providers often view caring for patients with persistent pain as burdensome, and our 24 participants stated that this had been their experience before the CFT training. However, many 25 reported that using the BPS approach to treat LBP felt professionally stimulating and rewarding 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Only 1 During the training, the physiotherapists recognized the need to change their practices into a 2 more person-centered approach to care, which closely reflects the expectations of patients with 3 LBP who seek care from health care professionals (Holopainen et al., 2018). Physiotherapy Theory and Practice The physiotherapists in our study described this process as 23 a wave motion – the adoption of CFT elements did not occur in a linear manner. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 1 their new and old ways of working, and not using the new approach regularly. In contrast, others 2 reported having fully adopted the CFT approach and that it had become normalized in their 3 practice (May and Finch, 2009). Like us, Sanders, Ong, Sowden and Foster (2014) observed 4 that the physiotherapists were convinced to change their practices by trying out the new 5 approach and reflecting critically on their work. Also in line with previous studies (Nielsen, 6 Keefe, Bennell, and Jull, 2014; O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; 7 Synnott et al., 2016), although most of our participants reported convincing scientific evidence 8 and live patient demonstrations as the most important enablers of learning, some 9 physiotherapists experienced these demonstrations and the style of presenting the evidence as 10 negative. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 30 of 47 30 For Peer Review Only 1 During the training, the physiotherapists recognized the need to change their practices into a 2 more person-centered approach to care, which closely reflects the expectations of patients with 3 LBP who seek care from health care professionals (Holopainen et al., 2018). In line with 4 previous studies (O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Synnott et al., 5 2016), most of the physiotherapists in our study reported an increased awareness of the 6 influence of cognitive, psychological and social factors on persistent LBP. Other studies that 7 have trained physiotherapists to adopt the assessment and management of psychosocial factors 8 in their work have noticed similar problems to those encountered by some of the 9 physiotherapists in our study. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice In line with 4 previous studies (O’Sullivan, O’Sullivan, O’Sullivan, and Dankaerts, 2013; Synnott et al., 5 2016), most of the physiotherapists in our study reported an increased awareness of the 6 influence of cognitive, psychological and social factors on persistent LBP. Other studies that 7 have trained physiotherapists to adopt the assessment and management of psychosocial factors 8 in their work have noticed similar problems to those encountered by some of the 9 physiotherapists in our study. For example, the use of screening for psychosocial risk factors in 10 LBP is widely recommended (Lin et al., 2019), but many physiotherapists found this 11 challenging. In line with the conceptions of some of the participants in our study, many other 12 studies have reported the lack of knowledge regarding psychosocial issues and interventions as 13 a barrier to more widely implementing this perspective in physiotherapy practice (Beissner et 14 al., 2009; Foster and Delitto, 2011; Main and George, 2011; Nielsen, Keefe, Bennell, and Jull, 15 2014; Singla, Jones, Edwards, and Kumar, 2015). Even though the physiotherapists in our study 16 were taught how to use the questionnaires, many of them had not started using them or only 17 used them occasionally. This is similar to the results of a study by Sanders, Foster, and Ong 18 (2011), in which general practitioners who were taught to use a subgrouping tool reported 19 barriers to its use: time constraints and other organizational pressures, and a lack of coherence 20 related to the new way of working. However, in our study, the questionnaires were reported as 21 being enablers of discussion of psychosocial issues, as they worked as shields. 22 As shown in previous research (Matthias et al., 2010; Toye, Seers and Barker, 2017), primary 23 care providers often view caring for patients with persistent pain as burdensome, and our 24 participants stated that this had been their experience before the CFT training However many 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Physiotherapy Theory and Practice Page 31 of 47 31 1 after receiving training and saw more complex patients as welcome challenges. Physiotherapy Theory and Practice Continuing support for using the BPS approach and creativity in one’s work is Page 31 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 31 1 after receiving training and saw more complex patients as welcome challenges. Others have 2 also reported this (Nielsen, Keefe, Bennell, and Jull, 2014; Sanders, Ong, Sowden, and Foster, 3 2014; Synnott et al., 2016). Many reported greater motivation in their work and renewed 4 professional identity. However, even after the training, some of the physiotherapists in our study 5 did not feel adequately prepared to deal with complex patients and wished for more training, as 6 in Sanders et al.’s study (Sanders, Ong, Sowden, and Foster, 2014). 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Physiotherapy Theory and Practice Others have 2 also reported this (Nielsen, Keefe, Bennell, and Jull, 2014; Sanders, Ong, Sowden, and Foster, 3 2014; Synnott et al., 2016). Many reported greater motivation in their work and renewed 4 professional identity. However, even after the training, some of the physiotherapists in our study 5 did not feel adequately prepared to deal with complex patients and wished for more training, as 6 in Sanders et al.’s study (Sanders, Ong, Sowden, and Foster, 2014). 7 Most of our participants reported using a more functional examination and management 8 approach, changing their communication to contain more positive messages and a person- 9 centered communication style, similar to previous CFT studies, (O’Sullivan, O’Sullivan, 10 O’Sullivan, and Dankaerts, 2013; Synnott et al., 2016). In our study, some physiotherapists 11 found a new role as an enabler of their patients’ own realizations. Enabling patients to find their 12 own solutions to their health issues as opposed the physiotherapist telling them what to do is 13 considered a more powerful way to change patient behaviors (Nijs et al., 2013). 14 RECOMMENDATIONS FOR THE FUTURE 15 The results of this study can be used as a tool for developing pedagogical practices in continuing 16 education in physiotherapy. The critical aspects in the learning process identified in our study 17 could be considered when planning future training interventions for physiotherapists. First, we 18 recommend providing physiotherapists with adequate support when they experience cognitive 19 dissonance and encouraging critical reflection. Secondly, including the whole workplace in the 20 training, increasing flexibility in workplace practices and supportive leadership are important. 21 Steps should also be taken to support the continuation of learning and applying the BPS 22 approach at the workplace to reinforce physiotherapists’ confidence in their skills and 23 motivation to learn more. More active collaboration between health care professionals should 24 be encouraged. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu RECOMMENDATIONS FOR THE FUTURE 15 The results of this study can be used as a tool for developing pedagogical practices in continuing 16 education in physiotherapy. The critical aspects in the learning process identified in our study 17 could be considered when planning future training interventions for physiotherapists. First, we 18 recommend providing physiotherapists with adequate support when they experience cognitive 19 dissonance and encouraging critical reflection. Secondly, including the whole workplace in the 20 training, increasing flexibility in workplace practices and supportive leadership are important. 21 Steps should also be taken to support the continuation of learning and applying the BPS 22 approach at the workplace to reinforce physiotherapists’ confidence in their skills and 23 motivation to learn more. More active collaboration between health care professionals should 24 be encouraged. Continuing support for using the BPS approach and creativity in one’s work is 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice P Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 32 of 47 Page 32 of 47 32 32 1 recommended. Steps should be taken to mandate the use of screening tools for all patients, in 2 line with best practice recommendations (Lin et al, 2019) 3 Learning about CFT and integrating it into clinical practice is a process, and many of the 4 physiotherapists in our study stated that without the booster sessions, individual learning tasks 5 and support from colleagues, the learning journey would have ended, as changing their practices 6 was not easy. The results of our study support previous research that has found that 7 physiotherapists’ professional development courses of two or three days are unlikely to be 8 sufficient for changing clinical practice. A longer process is recommended (Keefe, Main and 9 George, 2018; Mesner et al, 2016). Therefore, future studies in the field of physiotherapy should 10 also concentrate on exploring the effect of training work communities, instead of only 11 individual physiotherapists, and include auditing of clinical notes and supervision of and 12 feedback for participants. 13 Optimizing clinical training in order to help implement new knowledge and skills into clinical 14 practice is a key priority in the management of persistent LBP. RECOMMENDATIONS FOR THE FUTURE For example, funding for 15 training interventions, more effective treatment pathways and culture change in work 16 communities are needed. The current literature seems to support clinical mentoring, but more 17 research on implementation interventions is needed, as this requires considerable time and 18 effort. Whether this investment is cost-effective remains to be seen. Future studies should 19 compare training interventions with and without clinical supervision and mentoring to see 20 whether this is important for changes in practice and improved patient outcomes. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 RECOMMENDATIONS FOR THE FUTURE For example, funding for 15 training interventions, more effective treatment pathways and culture change in work 16 communities are needed. The current literature seems to support clinical mentoring, but more 17 research on implementation interventions is needed, as this requires considerable time and 18 effort. Whether this investment is cost-effective remains to be seen. Future studies should 19 compare training interventions with and without clinical supervision and mentoring to see 20 whether this is important for changes in practice and improved patient outcomes. 21 22 STRENGTHS AND LIMITATIONS 23 The information power of this study is sufficient (Malterud, Siersma, and Guassora, 2015). The 24 first author, who conducted the interviews, had training and experience in qualitative research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 recommended. Steps should be taken to mandate the use of screening tools for all patients, in 2 line with best practice recommendations (Lin et al, 2019) 3 Learning about CFT and integrating it into clinical practice is a process, and many of the 4 physiotherapists in our study stated that without the booster sessions, individual learning tasks 5 and support from colleagues, the learning journey would have ended, as changing their practices 6 was not easy. The results of our study support previous research that has found that 7 physiotherapists’ professional development courses of two or three days are unlikely to be 8 sufficient for changing clinical practice. A longer process is recommended (Keefe, Main and 9 George, 2018; Mesner et al, 2016). Therefore, future studies in the field of physiotherapy should 10 also concentrate on exploring the effect of training work communities, instead of only 11 individual physiotherapists, and include auditing of clinical notes and supervision of and 12 feedback for participants. 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 13 Optimizing clinical training in order to help implement new knowledge and skills into clinical 14 practice is a key priority in the management of persistent LBP. STRENGTHS AND LIMITATIONS The reliability of the research was also supported by one 24 author (AP) not being familiar with the CFT approach and not being included in the training g y py y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 33 1 and interviewing, and the quality of the dialogue between the interviewees and interviewer was 2 good. She is a physiotherapist and has good knowledge of the CFT approach. The aim of the 3 study was fairly specific and involved a specific group of informants – the physiotherapists who 4 participated in the CFT training intervention. The fact that the whole group of physiotherapists 5 who completed the training was included is a strength of this study. The themes raised by the 6 interviewees were rather broad, which is explained by the large number of participants, as well 7 as the analysis method, which explored the variation of understanding the phenomenon in 8 question. The study was theoretically well informed, although the feasibility of the CFT 9 approach in the context of the Finnish health care system has not previously been explored. 10 Health care systems and the basic training of physiotherapists vary in different countries and 11 this affects the organizational factors that can be perceived as barriers to or enablers of adopting 12 a BPS approach. The results cannot be directly transferred to other cultures, although many 13 findings are consistent with previous research. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 14 The illustration of the results using authentic quotations increases the validity of the study. The 15 authors are clinical and research physiotherapists, a professor of clinical psychology and a 16 professor of Physical and Rehabilitation Medicine with an interest in an individual BPS- 17 oriented approach to managing LBP. POS and SL were the trainers in this intervention and RH 18 and JK were present during the workshops. STRENGTHS AND LIMITATIONS Physiotherapy Theory and Practice Page 33 of 47 33 1 and interviewing, and the quality of the dialogue between the interviewees and interviewer was 2 good. She is a physiotherapist and has good knowledge of the CFT approach. The aim of the 3 study was fairly specific and involved a specific group of informants – the physiotherapists who 4 participated in the CFT training intervention. The fact that the whole group of physiotherapists 5 who completed the training was included is a strength of this study. The themes raised by the 6 interviewees were rather broad, which is explained by the large number of participants, as well 7 as the analysis method, which explored the variation of understanding the phenomenon in 8 question. The study was theoretically well informed, although the feasibility of the CFT 9 approach in the context of the Finnish health care system has not previously been explored. 10 Health care systems and the basic training of physiotherapists vary in different countries and 11 this affects the organizational factors that can be perceived as barriers to or enablers of adopting 12 a BPS approach. The results cannot be directly transferred to other cultures, although many 13 findings are consistent with previous research. 14 The illustration of the results using authentic quotations increases the validity of the study. The 15 authors are clinical and research physiotherapists, a professor of clinical psychology and a 16 professor of Physical and Rehabilitation Medicine with an interest in an individual BPS- 17 oriented approach to managing LBP. POS and SL were the trainers in this intervention and RH 18 and JK were present during the workshops. The diverse backgrounds of the research group 19 improved quality and rigor and subjected the analytical process to group reflexivity. The 20 credibility of the study was also strengthened by the first author writing preconceptions before 21 starting the study, as well as by discussion in the group on how professional backgrounds, 22 beliefs and attitudes towards the topic may have influenced the analysis process, and regular 23 discussions on the analysis process. STRENGTHS AND LIMITATIONS The participants reported a range of responses to the training, suggesting that for some, 21 the training was insufficient to support adequate changes in their practice behavior and that for 22 others it was a lifechanging experience. The journey was not without challenges, but where 23 they were overcome, a new way of working was possible, and physiotherapists reported 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 intervention, and by the results being discussed in the group of researchers familiar with the 2 phenomenographic research method but having no previous knowledge of the CFT approach. 1 intervention, and by the results being discussed in the group of researchers familiar with the 2 phenomenographic research method but having no previous knowledge of the CFT approach. 3 One limitation of this study was that we only arranged workshops for the participants and there 4 was no opportunity for clinical supervision and mentoring. It is also unclear whether these 5 physiotherapists actually changed their practices or whether the intervention had a positive 6 effect on patient outcomes. A strength of our study was that our training intervention was closer 7 to a normal professional development workshop setting than previous training for 8 physiotherapists that have delivered CFT interventions in randomized controlled studies (Vibe 9 Fersum et al., 2013), and that we interviewed all the participating physiotherapists, who 10 reported different kinds of responses to training. This also further enhanced the transferability 11 of the results. STRENGTHS AND LIMITATIONS The diverse backgrounds of the research group 19 improved quality and rigor and subjected the analytical process to group reflexivity. The 20 credibility of the study was also strengthened by the first author writing preconceptions before 21 starting the study, as well as by discussion in the group on how professional backgrounds, 22 beliefs and attitudes towards the topic may have influenced the analysis process, and regular 23 discussions on the analysis process. The reliability of the research was also supported by one 24 author (AP) not being familiar with the CFT approach and not being included in the training 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 34 of 47 34 1 intervention, and by the results being discussed in the group of researchers familiar with the 2 phenomenographic research method but having no previous knowledge of the CFT approach. 3 One limitation of this study was that we only arranged workshops for the participants and there 4 was no opportunity for clinical supervision and mentoring. It is also unclear whether these 5 physiotherapists actually changed their practices or whether the intervention had a positive 6 effect on patient outcomes. A strength of our study was that our training intervention was closer 7 to a normal professional development workshop setting than previous training for 8 physiotherapists that have delivered CFT interventions in randomized controlled studies (Vibe 9 Fersum et al., 2013), and that we interviewed all the participating physiotherapists, who 10 reported different kinds of responses to training. This also further enhanced the transferability 11 of the results. 12 There was a gender bias towards women in this study; however, this is reflective of the 13 workforce in Finland: in 2017 women comprised 77% of new physiotherapy students in 2007 14 (Kuusi, Jakku-Sihvonen, and Koramo, 2009) and 82% of graduate physiotherapists in Finland 15 (Valvira, 2017). 16 CONCLUSIONS 17 The participating physiotherapists’ conceptions of learning and integrating CFT into clinical 18 practice varied greatly. They reported that the CFT training intervention led them towards a 19 more biopsychosocial, multidimensional understanding and care of patients with persistent 20 LBP. CONCLUSIONS URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu 35 1 increased work motivation. However, it is not yet known whether the changes the 2 physiotherapists reported influenced their clinical practices and patient outcomes. 3 ACKNOWLEDGEMENTS 4 5 The researchers wish to thank all the physiotherapists who participated in this study, as well 6 (BLINDED), who planned and co-instructed the workshops with (BLINDED), who worked 7 hard to organize the workshops and took part in planning the whole research project. 8 9 REFERENCES 10 11 Beissner K, Henderson C, Papaleontiou M, Olkhovskaya Y, Wigglesworth J, Reid M 2009 12 Physical Therapists’ Use of Cognitive-Behavioral Therapy for Older Adults with Chronic Pain: 13 A Nationwide Survey. Physical Therapy 89:456-469. 14 Brinkmann, S 2013 Qualitative interviewing. Oxford university press. 15 Buchbinder R, van Tulder M, Öberg B, Menezes Costa L, Woolf A, Schoene M, Croft P 2018 16 Low back pain: a call for action. The Lancet 391:2384–2388. 17 Cowell I, O'Sullivan P, O'Sullivan K, Poyton R, McGregor A, Murtagh, G 2018 The 18 perspectives of physiotherapists on managing nonspecific low back pain following a training Page 35 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Physiotherapy Theory and Practice 1 increased 2 physiother 3 4 5 The resear 6 (BLINDE 7 hard to org 8 9 10 11 Beissner K 12 Physical T 13 A Nationw 14 Brinkman 15 Buchbinde 16 Low back 17 Cowell I, 18 perspectiv 19 programm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 35 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu REFERENCES 14 Brinkmann, S 2013 Qualitative interviewing. Oxford university press. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 36 of 47 36 For Peer Review Only 1 Domenech J, Sanchez-Zuriaga D, Segura-Orti E, Espejo-Tort B, Lisón J 2011 Impact of 2 biomedical and biopsychosocial training sessions on the attitudes, beliefs and recommendations 3 of health care providers about low back pain: A randomised clinical trial. Pain 152: 2557-2563. 4 Ehrström J, Kettunen J, Salo P 2018 Physiotherapy pain curricula in Finland: a faculty survey. 5 Scandinavian Journal of Pain, 18:593-601. 6 Foster N, Delitto A 2011 Embedding psychosocial perspectives within clinical management of 7 low back pain: integration of psychosocially informed management principles into physical 8 therapist practice - challenges and opportunities. Physical Therapy 91:790–803. 9 Fritz J, Söderbäck M, Söderlund A, Sandborgh M 2018 The complexity of integrating a 10 behavioral medicine approach into physiotherapy clinical practice. Physiotherapy Theory and 11 Practice. [in press] 12 Gilbert D 1991 How mental systems believe. American Psychologist 46:107–119. 13 Guerrero A, Maujean A, Campbell L, Sterling M 2018 A Systematic Review and Meta-Analysis 14 of the Effectiveness of Psychological Interventions Delivered by Physiotherapists on Pain, 15 Disability and Psychological Outcomes in Musculoskeletal Pain Conditions. Clinical Journal 16 of Pain 34: 838-857. 17 Hartvigsen J, Hancock M, Kongsted A, Louw Q, Ferreira M, Genevay S, Hoy D, Karppinen J, 18 Pransky G, Sieper J, et al. for the Lancet Low Back Pain Series Working Group 2018 What is 19 low back pain and why we need to pay attention. The Lancet 391: 2356–2367. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 Domenech J, Sanchez-Zuriaga D, Segura-Orti E, Espejo-Tort B, Lisón J 2011 Impact of 2 biomedical and biopsychosocial training sessions on the attitudes, beliefs and recommendations 3 of health care providers about low back pain: A randomised clinical trial. Pain 152: 2557-2563. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu REFERENCES URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 37 of 47 For Peer Review Only 37 1 Holopainen R, Piirainen A, Heinonen A, Karppinen J, O'Sullivan, P 2018 From “Non- 2 encounters” to autonomic agency. Conceptions of patients with low back pain about their 3 encounters in the health care system. Musculoskeletal Care 16: 269-277. 4 Keefe, Main C, George S 2018 Advancing psychologically informed practice for patients with 5 persistent musculoskeletal pain: promise, pitfalls, and solutions. Physical Therapy 98:398–407. 6 Karstens S, Kuithan P, Joos S, Hill J, Wensing M, SteinHäuser J, Krug K, Szecsenyi J 2018 7 Physiotherapists’ views of implementing a stratified treatment approach for patients with low 8 back pain in Germany: a qualitative study. BMC Health Services Research 18:214. 9 Koes B, Lin C, Macedo L, McAuley J, Maher C 2010 An updated overview of clinical 10 guidelines for the management of nonspecific low back pain in primary care. European Spine 11 Journal 19:2075–2094. 12 Kuusi H, Jakku-Sihvonen R, Koramo M 2009 Koulutus ja sukupuolten tasa-arvo. Sosiaali- ja 13 terveysministeriön selvityksiä 2009:57. 14 Lin I, Wiles L, Waller R, Goucke R, Nagree Y, Gibberd M, Straker L, Maher C, O’Sullivan P 15 2019 What does best practice care for musculoskeletal pain look like? Eleven consistent 16 recommendations from high-quality clinical practice guidelines: systematic review. British 17 Journal of Sports Medicine. Published Online First: 02 March 2019. doi: 10.1136/bjsports- 18 2018-099878 19 Main C, George S 2011 Psychologically Informed Practice for Management of Low Back Pain: 20 Future Directions in Practice and Research. Physical therapy 91:1-5. Page 37 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 37 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 38 of 47 Page 38 of 47 38 1 Main C, Sowden G, Hill J, Watson P, Hay E 2012 Integrating physical and psychological 2 approaches to treatment in low back pain: The development and content of the STarT Back 3 trial's 'high‐risk' intervention (StarT Back; ISRCTN 37113406). Physiotherapy 98:110–116. REFERENCES 4 Malterud K, Siersma V, Guassora A 2016 Sample size in qualitative interview studies: guided 5 by information power. Qualitative Health Research 26: 1753–1760. 6 Marton F, Booth S 2009 Learning and Awareness. New York: Routledge. Taylor & Francis 7 Group. 8 Marton F, Pong W 2005 On the unit of description in phenomenography. Higher Education 9 Research and Development 24:335-348. 10 Matthias M, Parpart A, Nyland K, Huffman M, Stubbs D, Sargent C, Bair M 2010 The patient- 11 provider relationship in chronic pain care: Providers’ perspectives. Pain Medicine 12 11:1688-1697.’ 13 May C, Finch T 2009 Implementing, Embedding, and Integrating Practices: An Outline of 14 Normalization Process Theory. Sociology 43:535-554. 15 Mesner S, Foster N, French S 2016 Implementation interventions to improve the management 16 of non-specific low back pain: a systematic review. BMC Musculoskeletal Disorders 17 17:258. 18 NICE National Institute for Health and Care Excellence 2016 Low back pain and sciatica in 19 over 16s: Assessment and Management. National Institute for Health and Care Excellence: 20 Clinical Guidelines. London: National Institute for Health and Care Excellence (UK). 21 Nielsen M, Keefe F, Bennell K, Jull G 2014 Physical therapist – delivered cognitive -behavioral 22 therapy: a qualitative study of physical therapists’ perceptions and experiences. Physical 23 therapy 94:197-207. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Main C, Sowden G, Hill J, Watson P, Hay E 2012 Integrating physical and psychological 2 approaches to treatment in low back pain: The development and content of the STarT Back 3 trial's 'high‐risk' intervention (StarT Back; ISRCTN 37113406). Physiotherapy 98:110–116. 1 Main C, Sowden G, Hill J, Watson P, Hay E 2012 Integrating physical and psychological 2 approaches to treatment in low back pain: The development and content of the STarT Back 3 trial's 'high‐risk' intervention (StarT Back; ISRCTN 37113406). Physiotherapy 98:110–116. 4 Malterud K, Siersma V, Guassora A 2016 Sample size in qualitative interview studies: guided 5 by information power. Qualitative Health Research 26: 1753–1760. REFERENCES URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 39 of 47 39 39 1 Nijs J, Roussel N, Paul van Wilgen C, Koke A, Smeets R 2013 Thinking beyond muscles and 2 joints: Therapists’ and patients’ attitudes and beliefs regarding chronic musculoskeletal pain 3 are key to applying effective treatment. Manual Therapy 18: 96–102. 4 O’Sullivan K, O’Sullivan P, O’Sullivan L, Dankaerts W 2013 Back pain beliefs among 5 physiotherapists are more positive after biopsychosocially orientated workshops. 6 Physiotherapy Practice and Research 34:37–45. 7 O’Sullivan K, Dankaerts W, O’Sullivan L, O’Sullivan P 2015 Cognitive Functional Therapy 8 for disabling, nonspecific chronic low back pain: multiple case-cohort study. Physical Therapy 9 95:1478-1488. 10 O'Sullivan K, O'Keeffe M, O'Sullivan P 2017 NICE low back pain guidelines: Opportunities 11 and obstacles to change practice. British Journal of Sports Medicine, 51:1632–1633. 12 O’Sullivan P, Caneiro JP, O’Keeffe M, Smith A, Dankaerts W, Fersum K, O’Sullivan K 2018 13 Cognitive Functional Therapy: An Integrated Behavioral Approach for the Targeted 14 Management of Disabling Low Back Pain. Physical therapy 98:408-423. 15 Overmeer T, Boersma K, Denison E, Linton S 2011 Does teaching physical therapists to deliver 16 a biopsychosocial treatment program result in better patient outcomes? A randomized 17 controlled trial. Physical Therapy 91:804–819. 18 Piirainen A, Viitanen E 2010 Transforming expertise from individual to regional community 19 expertise: a four-year study of an education intervention. International Journal of Lifelong 20 Education 29:581-506. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 39 10 O'Sullivan K, O'Keeffe M, O'Sullivan P 2017 NICE low back pain guidelines: Opportunities 11 and obstacles to change practice. British Journal of Sports Medicine, 51:1632–1633. 6 7 8 9 0 1 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 40 of 47 Page 40 of 47 40 1 Sandborgh M, Åsenlöf P, Lindberg P, Denison E 2010 Implementing behavioural medicine in 2 physiotherapy treatment. Part II: adherence to treatment protocol. Advances in Physiotherapy 3 12: 13–23. REFERENCES 4 Sanders T, Ong B, Sowden G, Foster N 2014 Implementing change in physiotherapy: 5 professions, contexts and interventions. Journal of Health Organization and Management. 6 28:96-114. 7 Singla M, Jones M, Edwards I, Kumar S 2015 Physiotherapists’ assessment of patients’ 8 psychosocial status: Are we standing on thin ice? A qualitative descriptive study. Manual 9 Therapy 20:328-334. 10 Synnott A, O’Keeffe M, Bunzli S, Dankaerts W, O’Sullivan P, Robinson K, O’Sullivan K 2016 11 Physiotherapists report improved understanding of and attitude toward the cognitive, 12 psychological and social dimensions of chronic low back pain after Cognitive Functional 13 Therapy training: a qualitative study. Journal of Physiotherapy 62:215-221. 14 15 Tong A, Sainsbury P, Craig J 2007 Consolidated criteria for reporting qualitative research. 16 (COREQ): a 32-item checklist for interviews and focus groups. International Journal for 17 Quality in Health Care 19:249-253. 18 Toye F, Seers K, Barker KL 2017 Meta-ethnography to understand healthcare professionals’ 19 experience of treating adults with chronic non-malignant pain BMJ Open 7:e018411. 20 Waller G 2009 Evidence-based treatment and therapist drift. Behaviour Research and Therapy 21 47:119-127. 22 Valvira 2017 Registers of social welfare and healthcare professionals (Terhikki) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 1 Sandborgh M, Åsenlöf P, Lindberg P, Denison E 2010 Implementing behavioural medicine in 2 physiotherapy treatment. Part II: adherence to treatment protocol. Advances in Physiotherapy 3 12: 13–23. 1 Sandborgh M, Åsenlöf P, Lindberg P, Denison E 2010 Implementing behavioural medicine in 2 physiotherapy treatment. Part II: adherence to treatment protocol. Advances in Physiotherapy 3 12: 13–23. 4 Sanders T, Ong B, Sowden G, Foster N 2014 Implementing change in physiotherapy: 5 professions, contexts and interventions. Journal of Health Organization and Management. 6 28:96-114. 18 Toye F, Seers K, Barker KL 2017 Meta-ethnography to understand healthcare professionals’ 19 experience of treating adults with chronic non-malignant pain BMJ Open 7:e018411. 20 Waller G 2009 Evidence-based treatment and therapist drift. Behaviour Research and Therapy 21 47:119-127. REFERENCES 22 Valvira 2017 Registers of social welfare and healthcare professionals (Terhikki) URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Physiotherapy Theory and Practice Page 41 of 47 41 1 Vibe Fersum K, O’Sullivan P, Skouen J, Smith A, Kvåle A 2013 Efficacy of classification- 2 based cognitive functional therapy in patients with non-specific chronic low back pain: a 3 randomized controlled trial. European Journal of Pain 17:916–928. 4 Zangoni G, Thomson O 2017 'I need to do another course' - Italian physiotherapists' knowledge 5 and beliefs when assessing psychosocial factors in patients presenting with chronic low back 6 pain. Musculoskeletal Science and Practice 27):71-77. 7 Zidarov D, Thomas A, Poissant L 2013 Knowledge translation in physical therapy: from theory 8 to practice. Disability and Rehabilitation 35:1571-1577. 9 Åkerlind G 2005 Variation and commonality in phenomenographic research methods. Higher 10 Education Research & Development 24:321-334. 11 12 Åkerlind G 2008 An academic perspective on research and being a researcher: an integration 13 of the literature. Studies in Higher Education 33:17-31. 14 15 Åkerlind G 2017 What future for phenomenographic research? On continuity and development 16 in the phenomenography and variation theory research tradition. Scandinavian Journal of 17 Educational Research 62:949-958. 18 Page 41 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 41 URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 42 of 47 1h 45min Overview of evidence of management of low back pain (LBP), multidimensional framework for understanding and exploring the biopsychosocial nature of LBP, beliefs and attitudes. (Lecture) 2h 45min Physical, psychosocial and lifestyle risk factors (Lecture), Utilization of screening tools to identify psychosocial risk factors 1h 30min Interview and examination 1h 30 min Communication training 7h Management planning, interventions (including management of fear avoidance behaviour, mal-adaptive movement patterns, pain behaviours, graded activity, graded exposure), problem solving, complex cases. (Lecture + group discussions, practicing the use of clinical reasoning form) 1h 30 min Case studies 7h Patient demonstrations (4 patients with 2 follow-up visits) Table 1. Content of the initial four-day workshop Table 1. Content of the initial four-day workshop Table 1. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu REFERENCES Content of the initial four-day workshop URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Table 2. Content of the two-day booster session 1h 30min Questions and answers session, discussion on participants’ difficulties / obstacles and successes in integrating cognitive functional therapy into clinical practice 2h Management of low risk patients, movement patterns, challenging beliefs (lecture, group discussion) 7h 30min Patient demonstrations (4 patients) with discussion and practice of the use of the clinical reasoning form Page 43 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Physiotherapy Theory and Practice Page 43 of 47 Page 43 of 47 Table 2. Content of the two-day booster session 1h 30min Questions and answers session, discussion on participants’ difficulties / obstacles and successes in integrating cognitive functional therapy into clinical practice 2h Management of low risk patients, movement patterns, challenging beliefs (lecture, group discussion) 7h 30min Patient demonstrations (4 patients) with discussion and practice of the use of the clinical reasoning form 0 1 2 3 4 5 6 Table 2. Content of the two-day booster session URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 44 of 47 Page 44 of 47 Table 3. Themes of variation and categories of description of phenomenon of learning and integrating Cognitive Functional therapy into clinical practice Categories Themes of variation I Recognizing difference of new approach II Towards integrating the new approach III Waking up to explore IV Commitment to new approach V Expanding application of new approach Membership of work community Loneliness in work community Organizational traditions as barriers Desire for common language Supportive work community Importance of multidisciplinari ty Learning journey Resistance Personal challenges during journey Being shaken Becoming convinced Continuous adventurous journey Transition to new working methods Insecurity Combining old and new approach Critical reflection on one’s own work Better equipped to help Permission for creativity Professional role as a physiotherapist Looking at patients in different way Changing attitudes and language Stepping outside one’s comfort zone Closer to patient Renewed professional identity able 3. REFERENCES Themes of variation and categories of description of phenomenon of learning and tegrating Cognitive Functional therapy into clinical practice URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Figure 1: The critical aspects between the categories of description of the phenomenon of learning and integrating CFT into clinical practice. IV Commitment to the new approach III Awakening to explore II The new approach challenges current understanding I Recognizing the difference of the new approach Creativity, multidisciplinary collaboration and continuous learning Support from the work community Becoming convinced Being shaken Ability to critically reflect on one’s work Ability to overcome resistance and to change views I I Recognizing difference of new approach II Towards integrating the new approach III Waking up to explore IV Commitment to new approach V Expanding application of new approach Page 45 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Page 45 of 47 IV Commitment to the new approach III Awakening to explore II The new approach challenges current understanding I Recognizing the difference of the new approach Creativity, multidisciplinary collaboration and continuous learning Support from the work community Becoming convinced Being shaken Ability to critically reflect on one’s work Ability to overcome resistance and to change views II Towards integrating the new approach III Waking up to explore IV Commitment to new approach V Expanding application of new approach Page 45 of 47 Physiotherapy Theory and Practice 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 roach Creativity, multidisciplinary collaboration and continuous learning mmitment to the new Support from the work community II The Being shaken Ability to critically reflect on one’s work t t V Expanding application of new approach IV Commitment to new approach Ability to overcome resistance and to change views II Towards integrating the new approach I Recognizing difference of new approach Figure 1: The critical aspects between the categories of description of the phenomenon of learning and integrating CFT into clinical practice. Figure 1: The critical aspects between the categories of description of the phenomenon of learning and integrating CFT into clinical practice. REFERENCES Figure 1: The critical aspects between the categories of description of the phenomenon of learning and integrating CFT into clinical practice. URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 46 of 47 Page 46 of 47 Appendix 1. Participants’ characteristics *OHC=occupational health care, PHC=primary health care, outpatient clinic Physiotherapist Female /Male Age Years of practice Type of health care unit Days of Cognitive Functional Therapy training by the time of interview A F 37 15 OHC 6 B M 34 9 OHC 4 C F 35 10 OHC 6 D F 52 25 OHC 6 E F 52 27 PHC 4 F M 55 31 PHC 6 G F 55 30 PHC 4 H F 47 28 PHC 4 I F 43 19 PHC 4 J F 34 10 PHC 4 K F 55 30 PHC 4 L M 40 15 PHC 4 M F 39 16 OHC 6 N F 38 12 OHC 6 O F 52 29 PHC 6 P F 50 24 PHC 4 Q F 33 11 PHC 6 R F 40 16 PHC 6 S F 61 30 OHC 6 T F 52 15 OHC 6 U F 45 15 PHC 4 V F 53 27 PHC 6 Appendix 1. Participants’ characteristics URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu Physiotherapy Theory and Practice Page 47 of 47 Key questions: How do you see CFT now – what does it mean to you? Tell me about your process of learning CFT and implementing it in practice. How do you see CFT now – what does it mean to you? Tell me about your process of learning CFT and implementing it in practice. Following themes were flexibly addressed if not otherwise brought up by the interviewee Expectations of training – were they realized? How? Did the training change beliefs and attitudes, understanding of low back pain? Were your beliefs challenged? How? Did the training influence your working methods? Communication, interview, assessment, treatment? Did it affect the skills for building a therapeutic alliance? How? Did your patients notice a change? Did this training affect your patients? How? What promoted the learning/implementation of the CFT approach? What hindered the learning/implementation of the CFT approach? Did it affect your personal well-being / professional identity? How? Did it increase your confidence in assessing psychosocial factors with your patients? And treating according to the assessment? Did it affect your confidence in your skills for treating patients with persistent pain (low back pain)? What was easy/helpful? What was difficult/unhelpful? What promoted learning? Was the amount of training sufficient? What was the impact of the booster session/ web-based tasks? Are you willing to develop further? What is needed to develop further? Following themes were flexibly addressed if not otherwise brought up by the interviewee Expectations of training – were they realized? How? URL: http://mc.manuscriptcental.com/uptp Email: shasson@gru.edu
https://openalex.org/W4246343425	https://copim.pubpub.org/pub/academic-libraries-and-oa-books-france/download/pdf	English	null	France	Community-led Open Publication Infrastructures for Monographs (COPIM) eBooks	2,021	cc-by	1,423	General library system for e-content and OA publications The higher education system in France has three main contributors: public universities, research centres such as the CNRS, and Grandes Écoles. The country has historically relied on a strongly centralised academic libraries system, with the Ministry of Education at its core. Although academic and research libraries still fall under its aegis, they are also gaining more autonomy. Academic libraries are organised around institutions. In the case of public universities, each of them has its own central system with smaller faculty libraries. In order to facilitate the exchange between universities, some of them come together to form mergers of three or four institutions. Negotiations on access to electronic e-content in France are managed by the Couperin consortium (Unified Consortium of Higher Education and Research Organisations for Access to Digital Publications), founded in 1999. It unites academic and research institutions in France and negotiates deals with publishers on their behalf. Currently Couperin has 264 members: among them are universities, research institutes and national libraries (Liste Des Membres - Membres, n.d.). ABES (Bibliographic Agency for Higher Education) is a body responsible for the development of cataloguing standards used across libraries in France, eBooks included. Key findings The academic library system is centralised around the Ministry of Education at its The academic library system is centralised around the Ministry of Education at its core The Couperin consortium represents French libraries on the national level The Couperin consortium represents French libraries on the national level The National Plan for Open Science, introduced in 2018, calls for open access to publicly funded publications for journal articles and books alike The National Plan for Open Science, introduced in 2018, calls for open access to publicly funded publications for journal articles and books alike There are no OA book-specific funds in France; OA book publishing is mainly supported b grants No library-led OA book publishing initiatives were identified y p g OpenEdition acts as an open access platform for journals and books in HSS OpenEdition acts as an open access platform for journals and books in HSS Libraries rely on ABES for OA books coverage Libraries rely on ABES for OA books coverage COPIM • Academic Libraries and Open Access Books in Europe: a Landscape Study France France COPIM • Academic Libraries and Open Access Books in Europe: a Landscape Study Library community and open access ADBU (the French Association of Academic Libraries and Documentation) is a well-established organisation uniting academic libraries across the country since the 1970s, when ADBU was officially formed. The organisation takes pride in working on strategical development of the academic library, which is seen as an institution with a strongly pronounced societal role: that of innovating and providing access to knowledge (Swiatek, 2020). Open Access is one of the questions that sits high on 2 COPIM • Academic Libraries and Open Access Books in Europe: a Landscape Study France France the discussion list in the ADBU, which supports Plan S and organises numerous roundtables and conferences supporting developments in open science. the discussion list in the ADBU, which supports Plan S and organises numerous roundtables and conferences supporting developments in open science. Open Science Committee, an organisation that came to life in 2018 under the auspice of the Ministry of Education, is a collection of working groups dealing with a spectrum of topics that have to do with open science. Librarians constitute a large part of the working groups, alongside researchers and publishing experts. Open Science Committee defines its mission as that of leading the way and indicating directions in which open science should evolve, as well as educating people about its benefits and animating the open science community. It serves as one of the main platforms where librarians engage in discussions on open access (Ouvrir La Science - The Committee for Open Science, n.d.). Library/scholar-led OA book publishing Academic libraries in France do not act as publishers per se. There are several examples of libraries being involved in journal publishing (for example, at the universities of Nice, Bordeaux, Clermont and Lyon), but none involved in OA books publishing. Some libraries might be engaged in depositing theses in institutional repositories, yet they do not strictly take publishing initiatives themselves, leaving this job to a well-developed network of university presses. OpenEdition provides university presses with an infrastructure for hosting open access books. The platform unites open access journals, books, blogs and academic announcements. Currently there are over 10,000 OA books accessible through OpenEdition. OA book funding There are no OA book-specific funds for researchers available in France. OA publications are mainly covered by research grants. Several French libraries participate in the freemium programs available from the OpenEdition platform, therefore allocating their budgets to OA initiatives. OA book policies In 2018, France published the National Plan for Open Science, in which it committed to generalising open access for publications using public funding, structuring research data and making it available OA, and being an active part in the OA international community. The Plan pertains to journals and books alike. While it outlines roadmaps for all three commitments, it does not give any specific dates by which the policy should be implemented (National Plan for Open Science, 2018). The same year saw the creation of the Jussieu Call for open science and bibliodiversity, a declaration crafted by French researchers and academic publishers. In the document they express the need for a more diverse publishing landscape, supporting the development of innovative scientific publishing models (Jussieu Call, n.d.). Currently, there are 24 institutions with recorded OA policies in the ROAR map. Among them, nine specifically mention books: Currently, there are 24 institutions with recorded OA policies in the ROAR map. Among them, nine specifically mention books: Agence National de la Recherche (HSS branch), Arts et Metiers ParisTech, Arts et Metiers ParisTech, CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), CNRS, CentraleSupélec, INRIA (National Institute for Research in Computer Sciences & Control), Ifsttar: Institut français des sciences et technologies des transports, de l’aménagement et des réseaux, Institut français de recherche pour l'exploitation de la mer (Ifremer). The most important public funder in France, the French National Research Agency (ANR), supports the National Plan recommendations mandating deposits of publications coming out of funded projects into The most important public funder in France, the French National Research Agency (ANR), supports the National Plan recommendations mandating deposits of publications coming out of funded projects into 3 COPIM • Academic Libraries and Open Access Books in Europe: a Landscape Study France France institutional or national open archives (HAL). ANR encourages publication in open access journals (Open Science, n.d.). Integration of OA books in library systems ABES (Bibliographic Agency of Higher Education) plays a crucial role in the process of cataloguing OA books and integrating them with library discovery systems on a national level. Some universities, in order to make sure that OA books are included in their discovery systems additionally rely on the DOAB metadata. COUPERIN https://www.couperin.org COUPERIN https://www.couperin.org References Liste des membres - Membres. (n.d.). Retrieved December 3, 2020, from ADBU (Association française des directeurs et personnels de direction des bibliothèques universitaires et de la documentation) https://adbu.fr Bibliographic Agency of Higher Education (ABES) https://abes.fr/en/ Open Science Committee https://www.ouvrirlascience.fr/open-science/ Open Archives for Academic Publications HAL https://hal.archives-ouvertes.fr/?lang=en OpenEdition https://www.openedition.org ADBU (Association française des directeurs et personnels de direction des bibliothèques universitaires et de la documentation) https://adbu.fr Bibliographic Agency of Higher Education (ABES) https://abes.fr/en/ Open Science Committee https://www.ouvrirlascience.fr/open-science/ Open Archives for Academic Publications HAL https://hal.archives-ouvertes.fr/?lang=en OpenEdition https://www.openedition.org References Liste des membres - Membres. (n.d.). Retrieved December 3, 2020, from https://www.couperin.org/presentation/membres France France COPIM • Academic Libraries and Open Access Books in Europe: a Landscape Study Photo by Marie-Lan Nguyen, CC BY 2.0. National Plan for Open Science. (2018). https://cache.media.enseignementsup- recherche.gouv.fr/file/Recherche/50/1/SO_A4_2018_EN_01_leger_982501.pdf National Plan for Open Science. (2018). https://cache.media.enseignementsup- recherche.gouv.fr/file/Recherche/50/1/SO_A4_2018_EN_01_leger_982501.pdf Open Science. (n.d.). Agence Nationale de La Recherche. Retrieved January 20, 2021, from https://anr.fr/en/anrs-role-in-research/values-and-commitments/open-science/ Open Science. (n.d.). Agence Nationale de La Recherche. Retrieved January 20, 2021, from https://anr.fr/en/anrs-role-in-research/values-and-commitments/open-science/ ABES. Projet d’établissement ABES 2018-2022. https://abes.fr/wp-content/uploads/2019/07/projet- etablissement-abes-2018-2022.pdf ABES. Projet d’établissement ABES 2018-2022. https://abes.fr/wp-content/uploads/2019/07/projet- etablissement-abes-2018-2022.pdf Jussieu Call. (n.d.). Retrieved January 5, 2021, from https://jussieucall.org/jussieu-call/ Ouvrir la Science - The Committee for Open Science. (n.d.). Retrieved January 8, 2021, from https://www.ouvrirlascience.fr/the-committee-for-open-science/ Swiatek, C. (2020, March 20). ADBU: Qui sommes-nous ? / About us. ADBU – Association des directeurs et des personnels de direction des bibliothèques universitaires. https://adbu.fr/about/ Swiatek, C. (2020, March 20). ADBU: Qui sommes-nous ? / About us. ADBU – Association des directeurs et des personnels de direction des bibliothèques universitaires. https://adbu.fr/about/ 5
https://openalex.org/W2560052278	https://www.rasi.vr.uff.br/index.php/rasi/article/download/22/pdf	Portuguese	null	Capacidades de Inovação e Indicadores Não Convencionais: um estudo exploratório	Revista de Administração, Sociedade e Inovação	2,016	cc-by	9,636	Capacidades de Inovação e Indicadores Não Convencionais: um estudo exploratório Josiane Couto Silva Roana de Toledo Leite Murilo Alvarenga Oliveira (malvarenga@id.uff.br) RESUMO O presente artigo tem como propósito elaborar um quadro teórico-analítico que integre o processo de inovação das firmas através de indicadores não convencionais e das capacidades de inovação, especialmente para análise deste processo em empresas localizadas em economias emergentes como o Brasil. O estudo com características exploratórias foi realizado por meio de pesquisa bibliográfica sobre os temas gestão e capacidade de inovação, indicadores tradicionais e não tradicionais, além de um grupo de foco com especialistas no assunto, onde se aplicou uma matriz G.U.T. adaptada e posteriormente um fator de importância. O quadro teórico-analítico associou os indicadores priorizados às capacidades organizacionais. PALAVRAS CHAVE: Capacidade de inovação; Gestão da Inovação; Indicadores não convencionais. 1. Introdução O mercado mundial é altamente competitivo e para se manter à frente as empresas precisam se antecipar às necessidades dos consumidores. A inovação se tornou, portanto, um diferencial dentro desse cenário, pois ela está voltada para criação de ideias e sua aplicação trazendo melhoria para os produtos e processos. No que diz respeito à criação de tecnologias, há uma distinção entre países desenvolvidos e países em desenvolvimento. Estudos mostram que existe uma tendência a acreditar que esse processo de criação de tecnologia de inovação é maior em países desenvolvidos e que os países emergentes seriam receptores desta tecnologia, apenas adaptando-a ao seu contexto. Segundo Kobal, Lázaro e Santos (2012) e Marins e Zawislak (2010) essas empresas são de caráter passivo em relação à criação e por isso não desenvolvem a inovação em seu sentido exato, importando a tecnologia dos países desenvolvidos. ç p g p Uma pesquisa realizada pela Thomson Reuters divulgada em março de 2013 revela que o cenário de produções cientificas em países em desenvolvimento vem se alterando nos últimos anos. Na década de 70 dois terços das publicações científicas eram realizados por países pertencentes ao G7, grupo que reúne os sete países mais desenvolvidos economicamente, sendo eles, Estados Unidos, Alemanha, Canadá, França, Itália, Japão e Reino Unido. Atualmente estes países correspondem por menos da metade do total de publicações. Essa mudança decorreu do aumento do investimento em pesquisa dos principais países em desenvolvimento, Brasil, Rússia, Índia, China e Coréia do Sul. O Brasil, por exemplo, “possui hoje grande estrutura de pesquisa, forma milhares de pesquisadores, que produzem e publicam os resultados das descobertas em revistas científicas” (Senado Federal do Brasil, 2012, p.18). ( p Este dado corrobora com a pesquisa publicada pelo IBGE (Instituto Brasileiro de Geografia e Estatística) em 2012 que demonstra um crescimento significativo de 273,9%, entre 2000 e 2011, superior ao crescimento de artigos do mundo (160,5%) o que levou o Brasil a ser notado por sua taxa de crescimento expressiva, ou seja, os artigos brasileiros representavam 1,15% em 2000 e passou para 2,39% em 2011 do total mundial. O IBGE em 2013 mostrou que o número de pesquisadores, grupos de pesquisa e de doutores, cresceram respectivamente 24%, 21% e 22% no ano de 2011. Capacidades de Inovação e Indicadores Não Convencionais: um estudo exploratório Capacidades de Inovação e Indicadores Não Convencionais: um estudo exploratório ABSTRACT This article aims to develop a theoretical and analytical framework that integrates the process of innovation of companies through non-conventional indicators and innovation capabilities, especially for analysis of this process in companies located in emerging economies such as Brazil. The study of exploratory characteristics was performed by bibliographic research on the subjects of management and innovation capabilities, traditional and non-traditional indicators, as well an expert group on this subject, a G.U.T. matrix was applied, adapted and subsequently a factor of importance. The theoretical and analytical framework associated the prioritized indicators to organizational capabilities. KEYWORDS: Innovation capability; Innovative Management; Non-conventional indicators. R. Desembargador Ellis Hermydio Figueira, 783, Bloco A, sl. 218, Aterrado. 27213-415 - Volta Redonda, RJ – Brasil www.uff.br Copyright © 2016 RASI. Todos os direitos, até mesmo de tradução, são reservados. É permitido citar parte de artigos sem autorização prévia, desde que seja identificada a fonte. Recebido em 10/10/2016 Aceito em 18/11/2016 Recebido em 10/10/2016 Aceito em 18/11/2016 Recebido em 10/10/2016 Aceito em 18/11/2016 Silva, Leite & Oliveira\| 168 https://doi.org/10.20401/rasi.2.2.110 http://www.rasi.uff.br 1. Introdução Isto demonstra o empenho em investir em fatores que possibilitem o desenvolvimento da inovação no Brasil, pois o investimento na qualificação profissional é a principal ferramenta que viabiliza o desenvolvimento de novas tecnologias, ou seja, o país que desenvolve estes fatores deixa de ser exclusivamente passivo e passa a criá-las. Assim, o aumento da produção interna de conhecimento, muda o caráter do país de apenas receptor para autor de suas próprias tecnologias. O investimento em inovação tem ganhado importância no cenário nacional. Mesmo com uma distância significativa no quesito inovação entre o Brasil, país em desenvolvimento, e economias desenvolvidas como Estados Unidos, Europa e Japão, há um movimento positivo do Brasil em aumentar seus investimentos em inovação. Oliveira e Sorgi (2008) em artigo publicado pelo Conselho Federal de Economia (COFECON) expõem que essa realidade surgiu a partir da década de 1990 com a abertura do mercado para empresas estrangeiras, gerando uma competitividade e uma RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 169 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 169 https://doi.org/10.20401/rasi.2.2.110 necessidade de transformação contínua. Desde então, as empresas nacionais vem buscando aprimorar a qualidade dos seus produtos e processos através da inovação, com investimentos em P&D, mão-de-obra qualificada dentre outros. necessidade de transformação contínua. Desde então, as empresas nacionais vem buscando aprimorar a qualidade dos seus produtos e processos através da inovação, com investimentos em P&D, mão-de-obra qualificada dentre outros. O potencial de crescimento e desenvolvimento nas economias emergentes é grande e deve ser incentivado. Os países que compõem o BRIC (Brasil, Rússia, Índia e China) são economias que despontam dentre os países em crescimento. A WIPO (World Intellectual Property Organization), através do Índice Global de Inovação de 2014, mostra o Brasil em uma pontuação de 0 a 100 com 36,29 pontos ocupando a posição de 61° entre 143 países, a Rússia com uma pontuação de 39,14 pontos e posição 49º, a Índia alcançou 33,70 pontos atingindo a posição 76º e a China 46, 57pontos e posição 29º. O Brasil é um exemplo do esforço das economias emergentes de alavancar a inovação dentro de suas limitações. Um parâmetro relevante de evidenciar este crescimento é acompanhar o investimento realizado em P&D com base em um percentual relativo ao PIB (Produto Interno Bruto), pois o PIB representa toda a riqueza produzida no país durante um determinado período. Silva, Leite & Oliveira\| 170 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 170 https://doi.org/10.20401/rasi.2.2.110 teórico-analítico para integrar o processo de inovação das firmas através das capacidades de inovação e indicadores não convencionais. teórico-analítico para integrar o processo de inovação das firmas através das capacidades de inovação e indicadores não convencionais. O estudo apresenta além da introdução, mais quatro seções. Num primeiro momento a fundamentação teórica aborda os temas gestão da inovação e fatores importantes relacionados a este assunto, além da discussão entre autores, capacidades de inovação onde se procura descrever as quatro capacidades de inovação propostas por Zawislak (2014) e indicadores de inovação onde são descritos os indicadores tradicionais de inovação e a importância do desenvolvimento de indicadores não tradicionais. Em sequência apresenta-se o método de pesquisa juntamente com o tratamento das informações, o desenho do fluxo de estudo e a apresentação do quadro teórico-analítico e por fim a discussão dos resultados e as considerações finais. 2.1. Gestão da Inovação Esta seção tem por finalidade apresentar a inovação como um processo gerencial articulando as ideias de autores que se complementam produzindo um sistema de geração de soluções para a organização se adaptar as incertezas do mercado. Isto é possível, pois Tidd, Bessant e Pavitt (2008) definem inovação como um processo e não um evento isolado, podendo, portanto, ser gerenciada como tal. Existe sobre o processo de inovação influências que podem ser manipuladas a fim de afetar os resultados. Como o processo de inovação não ocorre isoladamente, ele está submetido a uma série de influências internas e externas, e que permite identificar o que é possível e o que realmente acontece (Tidd; Bessant & Pavitt, 2008). De acordo com Tidd (2001) o ambiente externo, repleto de incertezas e complexidade, afeta o grau, tipo, o modo de organizar e a gestão da inovação, sendo assim, o mais coerente é o ajuste destes fatores para se chegar a um melhor desempenho. Van de Ven (1986) afirma que o contexto extra organizacional inclui os grandes dotes culturais e de recursos que a sociedade oferece, incluindo as leis, regras governamentais, distribuições de conhecimentos e recursos, bem como a estrutura da indústria em que a inovação é localizada. Todos estes fatores influenciam no modo em que a empresa irá gerenciar a inovação também em seu interior. A cultura, as estratégias, os valores, estruturas, processos e principalmente as pessoas são peculiares a cada organização. Quadros (2008) aborda esses elementos defendendo que a diferenciação quanto a qualidade do processo de inovação e a capacidade de inovar estão diretamente relacionados a cultura voltada para inovação, através de ações que busquem o aprendizado, a criatividade, a administração da tolerância ao risco e o comprometimento em construir um processo de inovação baseado na estratégia de diferenciação competitiva sustentada. Outro aspecto interno de importância para a gestão da inovação são as rotinas praticadas pela organização. Levitt e March (1988) expõem que as empresas são construídas e operam através de rotinas. Os autores as definem como um conjunto de regras, procedimentos, convenções, estratégias e tecnologias, estruturas, crenças, paradigmas, códigos, culturas e conhecimentos. As rotinas são consideradas importantes, pois elas demonstram segundo Tidd, Bessant e Pavitt (2008) o comportamento e o jeito de realizar as tarefas, sendo este peculiar a cada organização, ou seja, as rotinas são o que tornam uma empresa distinta das outras, quando praticada a RASI, Volta Redonda/RJ, v. 2, n. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. 1. Introdução A pesquisa publicada pelo IBGE em 2013 mostrou que 2009 os investimentos corresponderam a 1,17% do PIB e 1,16% em 2010. Apesar desta pequena redução, em 2011 alcançou um crescimento expressivo de 1,21% sendo o maior valor investido desde 2000. No caso do Brasil, a indústria ainda participa pouco no que compete à inovação se comparada aos países mais desenvolvidos. Segundo, Marins e Zawislak (2010), as empresas em países em desenvolvimento precisam ser analisadas, para se entender o processo de inovação, sob uma ótica diferenciada. Os indicadores tradicionais levam um modelo linear de inovação baseado em entradas (inputs) e saídas (outputs), ou seja, tem o foco nos resultados obtidos com o processo de inovação sendo eles número de patentes, número de doutores e investimentos em P&D, estes “encontram-se consagrados e são referência em estudos e pesquisas que buscam mensurar a inovação” (Marins & Zawislak, 2010, p.2). A realidade evidenciada pelos indicadores tradicionais mostra que, em comparação com os países desenvolvidos, os índices de investimento em P&D, registro de patentes e número de doutores é superior aos países em desenvolvimento. Porém, essa realidade não leva em consideração o processo de inovação ocorrido dentro das empresas no cenário emergente. “Assim, apesar dos méritos e da relevância dos indicadores tradicionais de inovação, seu escopo de análise se mostra limitado, especialmente em firmas de economias emergentes.” (Marins & Zawislak, 2010, p.4). A realidade de como o processo de inovação ocorre dentro das empresas emergentes requer, por tanto, indicadores que exponham o processo e dissertem sobre as ações e as estratégias para alcançar um resultado, não apenas quantitativo, mas qualitativo. O estudo de Marins e Zawislak (2010) propõem indicadores não convencionais para compreender o processo de inovação dentro de empresas de países emergentes. Como apoio ao mapeamento proposto por esses indicadores alternativos, vinculou-se o estudo de Zawislak (2014) que analisa a empresa através de quatro capacidades, sendo elas, capacidade de desenvolvimento tecnológico, capacidade operacional, capacidade gerencial e capacidade transacional, divididas em dois condutores: tecnológico e de negócios. Para auxiliar a pesquisa foi feita uma análise sobre a gestão da inovação e sua importância para as organizações, bem como seu efeito sobre a competitividade e diferenciação de cada empresa. O objetivo desse artigo foi a elaboração de um quadro RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 171 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 171 https://doi.org/10.20401/rasi.2.2.110 mesma tarefa básica. Ainda segundo os autores, questões de estruturação e gestão da empresa são respondidas pelas rotinas que compõe o gerenciamento eficaz da inovação resultando em uma habilidade competitiva diferenciada. mesma tarefa básica. Ainda segundo os autores, questões de estruturação e gestão da empresa são respondidas pelas rotinas que compõe o gerenciamento eficaz da inovação resultando em uma habilidade competitiva diferenciada. De acordo com Fuck e Vilha (2011), a gestão da inovação consiste em elaborar uma estrutura sistemática de rotinas e ferramentas, com base em uma perspectiva estratégica, e durante um determinado período, de forma que a inovação não seja espontânea e desarticulada na organização. Porém, Quadros (2008) afirma que a efetividade da inovação não se limita apenas as ferramentas e processos de gestão e sim aos profissionais que utilizam tais ferramentas e realizam estes processos. Já para Terra (2012) a empresa precisa ter mais do que ideias, laboratórios, recursos financeiros para investir em P&D e um bom projeto de inovação para se tornar uma organização inovadora. Ela precisa se diferenciar das demais, quanto a processos, produtos, gestão e modelos de negócios direcionando estes para a inovação. Para Gavira et al. (2007) a gestão da inovação auxilia o gestor a tomar decisões sobre como criar valor por meio da inovação, além de organizar a geração desta em um processo de renovação para a empresa. Longanezi (2008) amplia o que Gavira et al. (2007) esboçaram, afirmando que no processo de gestão da inovação é necessário que o gestor tenha um entendimento amplo e aprofundado do assunto e um alto grau de disciplina. Dentre o que já foi discorrido Tidd, Bessant e Pavitt (2008) ressaltam outro ponto importante sobre a gestão da inovação. É assumido por eles que toda organização possui dificuldades, como erros, barreiras técnicas e problemas em geral, a maioria das falhas, no entanto, advém de inconsistências na gestão do processo de inovação. Isso corrobora com Gavira et al. (2007) e Longanezi (2008), pois há uma associação entre a responsabilidade do gestor com a execução da gestão da inovação. Este contexto está relacionado com as condições de incerteza do ambiente em que a empresa inserida. 2.1. Gestão da Inovação 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br 2.2. Capacidades de Inovação A inovação num contexto de competição pode caracterizar-se como um diferencial para a organização. Segundo Tidd, Bessant e Pavitt (2008) a inovação deve ser entendida como um processo central da organização já que esta se associa a renovação da mesma, ou seja, a partir desta compreensão, a inovação é vista como uma atividade genérica, associada a sobrevivência e crescimento. Assim, o conceito de inovação na prática é muito mais amplo e complexo. De acordo com Sen e Egelhoff (2000), a inovação de sucesso é resultado de uma combinação entre as variedades de bens pertencentes à empresa, seus recursos e capacidades. Guan (2003) define capacidade de inovação como um recurso especial da organização, sendo esta tácita, além, de está intimamente ligada às experiências recebidas pela empresa. Para o autor a capacidade de inovação deve acompanhar a estratégia da empresa e se acomodar em condições especiais de competição. Dito isto, torna-se claro que a capacidade de inovação se constitui em um conjunto de atividades de inovação, sendo estas no nível multidimensional com uma abordagem complexa e interativa (Zawislak, 2008). Sob esse ponto de vista, pode-se dizer que o modelo elaborado por Zawislak, Fracasso e Tello-Gamarra (2013) expõem o esquema organizacional dividido em dois condutores e as capacidades que as compreende. p A empresa para poder colocar em andamento suas atividades mercadológicas ela necessita de dois condutores: o tecnológico e o de negócios. O primeiro condutor é responsável pelo desenvolvimento tecnológico que abrange a criação de novos produtos e todas as operações subsequentes vinculadas a essa elaboração. O condutor de negócios tem como função integrar as diferentes áreas da empresa e transacionar os bens e serviços para o mercado. Ambos, então, possuem um perfil de contribuição e apoio para o desempenho da inovação, que irá ser construído ao longo das atividades imersas nas capacidades que compõe esses condutores. No condutor tecnológico estão inseridas as capacidades de desenvolvimento tecnológico e operacional e no condutor de negócios estão inseridas as capacidades gerencial e transacional. (Zawislak; Fracasso & Tello-Gamarra, 2013). p g Segundo Zawislak (2014), um dos motivos que justifica o sucesso de empresas em países emergentes no mercado, é o fato delas desenvolverem bem uma dessas capacidades, mesmo que o seu investimento em inovação não seja elevado. O resultado diferenciado da empresa, por tanto, não necessariamente depende de todas as áreas, recursos e capacidades internas que a empresa possui. Por isso, Tidd, Bessant e Pavitt (2005), defendem que para a organização ter um desempenho diferenciado, ela precisa saber explorar todas as fontes de informações e dados a fim de gerar conhecimento significativo e estratégico para organização. Com base nesse esforço, a empresa possui o desafio de buscar a solução mais apropriada para os mais diversos problemas, fazendo a escolha que traz ao processo a eficiência. p Ainda de acordo com esses autores, o resultado final da inovação é cheio de incertezas, porém o gestor poderá diminuir estas por meio do gerenciamento adequado do processo de inovação. Portanto, a gestão da inovação, através da mobilização dos recursos adequados, consegue transformar estas incertezas em conhecimento e reduzi-la a uma situação de equilíbrio. Kline e Rosenberg (1986) também apontam sobre este nível de incerteza e afirmam que a gestão da inovação tem como propósito justamente esta tentativa de redução e acrescentam que a gestão da inovação demanda a criação de sistemas que evitem a perda da capacidade inovadora. Todas essas variáveis influenciam na maneira como cada empresa gerencia seu processo de criação de valor. Segundo isso, o processo de gestão da inovação irá se delinear de acordo com o resultado que empresa deseja, pois segundo Pradella (2013) a inovação envolve a organização como um todo proporcionando a mesma vantagem competitiva e agregação de valor. Neste cenário, Stefanovitz e Nagano (2014) enfatizam também que é fundamental que os modelos de gestão da inovação utilizados sejam avaliados de acordo com a realidade empresarial. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 172 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 172 https://doi.org/10.20401/rasi.2.2.110 Sendo a realidade empresarial complexa não se pode uniformizar um único tipo de processo de inovação. (Rothwell, 1994). Não existe, portanto, um modelo único de gestão da inovação, pois este depende do ambiente interno e externo da mesma, se adequando segundo seus objetivos quanto à geração de valor. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. 2.3. Indicadores de Inovação Os primeiros indicadores desenvolvidos para mensurar a inovação têm como propósito trazer à empresa uma visão com foco nos resultados. Para alcançar esse objetivo, os indicadores foram estruturados sob uma lógica pautada no modelo linear, que tem como finalidade a análise das entradas (inputs) e saídas (outputs) das organizações (Godin, 2002). Segundo Marins (2010), os indicadores tradicionais são consagrados e vistos como referência quando se trata de mensurar a inovação e eles englobam gastos com P&D, as patentes e também o grau de qualificação do corpo de funcionários. Trabalhos como da Associação Nacional de Pesquisa, Desenvolvimento e Engenharia das Empresas Inovadoras [ANPEI], Pesquisa Industrial de Inovação Tecnológica[PINTEC], realizada pelo Instituto Brasileiro de Geografia e Estatística [IBGE], e o estudo do Instituto de Pesquisa Econômica Aplicada [IPEA] desdobramento da própria PINTEC, utilizam os indicadores tradicionais como ferramenta para mensurar a inovação dentro das empresas brasileiras. Porém apesar de sua grande relevância, estes estudos, segundo Figueiredo (2006), estão muito arraigados aos indicadores tradicionais de inovação e mesmo quando se propõem a analisar o processo de inovação dentro destas firmas deixam passar características importantes que poderiam contribuir para seu melhor entendimento, limitando-se a uma perspectiva mais agregada e não a uma visão mais aprofundada dos aspectos intra-organizacionais. De acordo com Katz (2004), as empresas em economias emergentes nem sempre possuem práticas intra-organizacionais formalizadas o que dificulta o entendimento de como o processo de inovação ocorre no interior das firmas, já que os indicadores tradicionais se limitam a compreender os resultados obtidos pelas entradas e saídas. Além disso, o autor afirma que para se entender o processo de inovação em uma firma deve-se levar em consideração o contexto que essas estão inseridas, pois como enfatizado por ele, analisar as empresas em economias emergentes sob a mesma ótica das economias desenvolvidas é subestimar o desempenho da inovação, já que há uma disponibilidade de recursos, em especial financeiros, menor. Albuquerque (2006), também afirma que os países desenvolvidos apresentam um sistema de inovação consolidado e articulados se comparado aos países em desenvolvimento. Arocena e Sutz (2006) também analisam a inovação, porém ressaltam que a primeira coisa que deve ser levada em consideração é a natureza informal que as empresas inseridas no contexto das economias emergentes apresentam, não negligenciando seu impacto, pois segundo eles existe uma necessidade de averiguar a existência real da inovação nesses países. 2.2. Capacidades de Inovação http://www.rasi.uff.br Silva, Leite & Oliveira\| 174 https://doi.org/10.20401/rasi.2.2.110 2.2. Capacidades de Inovação As capacidades levam a inovação, logo o desenvolvimento eficaz de uma ou mais delas já conseguem diferenciar a empresa e sua atuação no mercado, cenário esse, comum em empresas de países em desenvolvimento. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 173 https://doi.org/10.20401/rasi.2.2.110 Figura 1. Capacidades da empresa para o desempenho inovador Fonte: Adaptado de Zawislak, Tello-Gamarra, Alves, Barbieux & Reichert (2014). Figura 1. Capacidades da empresa para o desempenho inovador Fonte: Adaptado de Zawislak, Tello-Gamarra, Alves, Barbieux & Reichert (2014). A figura 1 mostra a relação entre as capacidades e seus respectivos condutores. A capacidade de desenvolvimento tecnológico, da ênfase na construção de todo o processo de imaginar e construir novas soluções de valor (novas tecnologias e novos produtos) para os consumidores. Além da criação e desenvolvimento ela é responsável pela adaptação das tecnologias já existentes como meio para manter sua oferta inovadora. A capacidade operacional dá ênfase para a produção em escala comercial de bens e serviços projetados pela capacidade de desenvolvimento tecnológico. Para isto, a tecnologia absorvida pela organização e os sistemas devem ser levados em consideração no planejamento das decisões, tanto para seu controle quanto para sua capacidade de produção. A empresa deve considerar a qualidade, a confiabilidade, o custo, a entrega, capacidade de resposta, rotinas, estabilidade, eficiência e padronização no processo de produção de bens e serviços. Na capacidade gerencial a empresa tem para coordenar de forma eficaz e eficiente todas as demais áreas da empresa, também integrando os recursos internos de forma coerente melhorando a sua utilização e evitando a escassez. Coordenar e integrar todas as áreas da empresa significa, portanto, administrar todo e qualquer conflito gerado entre essas áreas, tendo flexibilidade quanto à resolução dos possíveis problemas decorrentes de atritos. A capacidade transacional aborda o processo de compra e venda de produtos, ou seja, esta capacidade cuida das relações externas que a organização possui com fornecedores e clientes. São necessárias informações que auxiliem tanto na minimização dos custos para a produção em escala comercial, através da busca pelas melhores condições de seus fornecedores em relação à qualidade e preço, quanto à descoberta do mercado que a empresa irá atender. Ela busca que todas as transações necessárias envolvendo logística, marketing, comércio e distribuição cheguem ao mercado com o menor custo. http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. Silva, Leite & Oliveira\| 175 https://doi.org/10.20401/rasi.2.2.110 o objeto de estudo são empresas localizadas em países em desenvolvimento. A solução para este problema segundo Arundel (2006) é desenvolver indicadores que possam auxiliar na descrição do processo de inovação no interior destas empresas. o objeto de estudo são empresas localizadas em países em desenvolvimento. A solução para este problema segundo Arundel (2006) é desenvolver indicadores que possam auxiliar na descrição do processo de inovação no interior destas empresas. Para este estudo utilizou-se os indicadores alternativos propostos por Marins (2010). A princípio seu estudo foi estrutura a partir de quatro dimensões de inovação embasadas em teorias econômicas. A dimensão Empreendedorismo foi pautada nas teorias Schumpeteriana (Schumpeter, 1942) e da Firma de Coase (1937) e é importante, pois segundo a autora o empreendedorismo é relevante para provocar mudanças que sejam agregadoras de valor e assim se tornem inovação. Mesmo sendo fundamentais, os indicadores tradicionais não conseguem mensurar o empreendedorismo dentro das firmas. Para isso Marins (2010) propõe quatro novos indicadores. O primeiro indicador é o de Criatividade e consiste em contabilizar o número de ideias em determinado espaço de tempo, geradas pelos funcionários das empresas, mas que sejam convertidas em projetos de inovação. O Segundo indicador chamado Project champions mensura a partir destas ideias geradas o número de funcionários responsáveis por impulsionar o inicio do projeto de inovação. A Capacidade de realização é o indicador que mede a quantidade de projetos de inovação que obtiveram sucesso quando concluídos e partir deles foi produzido e lançado um novo produto em um determinado tempo. E por último, o indicador Controle de erros analisa o grau de formalização das práticas que a empresa utiliza para controlar os erros ocorridos através de uma escala que avalia se existe relatório de não conformidade e se este é formal ou informal. A Estrutura, segunda dimensão, baseada na Teoria da Firma (Coase, 1937) busca mensurar os aspectos que envolvem a base de conhecimento existente quanto a sua administração e as informações disponíveis, ambas em prol das modificações necessárias no processo para que este amplie a sua resolução de problemas, propiciando a firma mais inovações. Para avaliar esta dimensão Marins (2010) propõe outros quatros indicadores. O primeiro é chamado de Externalização, busca realizar a análise se há ou não distribuição de atividades externas de inovação da empresa quanto ao desenvolvimento de novos produtos em um período determinado. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br 2.3. Indicadores de Inovação Como justificativa a esse cenário, Marins (2010) reafirma o que Arocena e Sutz (2006) dizem a respeito da informalidade no interior das empresas, porém acrescenta que os indicadores tradicionais não são adequados para entender as atividades de inovação. Por isso, os indicadores tradicionais precisam ser relativizados ou, ainda, complementados por indicadores alternativos que demonstrem como o processo de inovação ocorre dentro destas firmas. Os indicadores tradicionais, para Katz (2004) são limitados quanto a sua finalidade não captando a atividade de inovação como um todo o que prejudica o amplo entendimento da mesma. Segundo, Marins e Zawislak (2010) há uma carência de indicadores que possam avaliar de forma mais ampla a inovação, principalmente quando RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 176 https://doi.org/10.20401/rasi.2.2.110 estratégia da firma, atribuindo o mesmo grau quanto a existência ou não de estratégia de inovação e se esta está formalmente deliberada e se está alinhada com a estratégia da empresa. O próximo indicador é o Portfólio de projetos, possui um foco no levantamento dos projetos desenvolvidos quanto o seu propósito e identificar o perfil predominante. Podendo estes ser, projetos de rotina, incremental de curto ou longo prazo e radical de curto ou longo prazo. é considerado um projeto predominante quando ele é mais de 50% do total de projetos realizados. O indicador Cadência procura mensurar a quantidade de projetos de inovação realizados simultaneamente por uma empresa com recursos financeiros, físicos e humanos próprios. O quarto indicador é Parcerias estratégicas, o qual faz uma proporção entre o número de parceiros estratégicos voltados às atividades de inovação em relação ao total de parceiros vinculados a empresa, apresentando ao final um percentual. estratégia da firma, atribuindo o mesmo grau quanto a existência ou não de estratégia de inovação e se esta está formalmente deliberada e se está alinhada com a estratégia da empresa. O próximo indicador é o Portfólio de projetos, possui um foco no levantamento dos projetos desenvolvidos quanto o seu propósito e identificar o perfil predominante. Podendo estes ser, projetos de rotina, incremental de curto ou longo prazo e radical de curto ou longo prazo. é considerado um projeto predominante quando ele é mais de 50% do total de projetos realizados. O indicador Cadência procura mensurar a quantidade de projetos de inovação realizados simultaneamente por uma empresa com recursos financeiros, físicos e humanos próprios. O quarto indicador é Parcerias estratégicas, o qual faz uma proporção entre o número de parceiros estratégicos voltados às atividades de inovação em relação ao total de parceiros vinculados a empresa, apresentando ao final um percentual. A quarta e última dimensão, possui o nome Valor que deve ser mensurado, pois os indicadores tradicionais não conseguem mensurar aspectos que vão além do número total de novos produtos e processos, novas patentes. Sendo assim, a empresa precisa gerar não somente o novo, mas também o novo que lhe agregue valor. Diante disto, Marins (2010) apresenta quatro novos indicadores para analisar esta dimensão. O primeiro indicador é o Lucro operacional inovativo que seria a porcentagem do lucro operacional gerada pela criação de novos produtos em um período específico. Time to market é o indicador que visa à mensuração do tempo médio entre a geração de uma nova ideia e a disponibilização da mesma em forma de produto no mercado. Em complemento, o indicador Time to profit, irá mensurar o tempo médio entre a elaboração da ideia para a criação de um novo produto até o ganho com o mesmo. Por último, o indicador Valor agregado que visa mensurar em um determinado período a porcentagem de valor agregado à firma advindo das atividades de inovação. O segundo indicador é Interatividade que mensura o percentual correspondente ao número de novos produtos gerados a partir da interação de pelo menos dois departamentos ou unidades de negócios em um determinado período. O indicador Recursos físicos tangíveis, realiza o percentual total da quantidade investida em atividade de inovação pela empresa, levando em consideração período determinado de tempo. Por último, o indicador Aplicações tecnológicas visa mensurar a quantidade de produtos novos produzidos em um determinado período de tempo a partir da utilização de uma nova tecnologia. A próxima dimensão é a Coordenação que busca coordenar e organizar os recursos da melhor maneira a firma de resolver os problemas de seu mercado consumidor. Esta dimensão foi fundamentada na Teoria da Firma de Coase que parte do princípio de que a atividade de inovação precisa de um conjunto de ações contínuas voltadas para a mesma. A coordenação empreendedora irá gerenciar estas ações, porém os indicadores de atividade inovativa desta dimensão terá como finalidade identificar o esforço, baseado nas ações efetivas ao longo do tempo. Sendo assim, Marins (2010) propõe outros quatro indicadores relacionados a esta dimensão. O primeiro indicador é chamado Estratégia de inovação, e busca identificar dentro da organização estratégias de inovação que estejam alinhadas com a RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 177 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 177 https://doi.org/10.20401/rasi.2.2.110 que complementassem o estudo já iniciado. Na Tabela 1 há a descrição dos especialistas sobre o tema quanto a sua formação, experiência acadêmica e profissional, além de linhas de pesquisa em que atuam. que complementassem o estudo já iniciado. Na Tabela 1 há a descrição dos especialistas sobre o tema quanto a sua formação, experiência acadêmica e profissional, além de linhas de pesquisa em que atuam. Tabela 1 Tabela 1 Perfil dos Especialistas Participantes da Pesquisa Esp. Formação e Titulação Experiência Acadêmica Experiência Profissional Linhas de Pesquisa 1 Graduação em Administração; Mestrado em Eng de Produção; Doutorado em Economia Docente na área de Gestão de Projeto em Instituições Públicas e Privada – 11 anos Gerente da Incubadora de empresas de base tecnológica (Incubadora de um IFES) – 3 anos; Diretor de Inovação e Projetos (Consultoria) – 2 anos; Gerente de Qualidade (Indústria) – 2 anos. As políticas públicas de Ciência, Tecnologia e Inovação 2 Graduação, Mestrado e Doutorado em Administração. Docente na área de Estratégia Empresarial e Gestão de Projeto em Instituições Públicas e Privada – 15 anos Analista de Desenvolvimento de Novos Produtos (Indústria) – 2 anos; Coordenador de P&D (Indústria) – 2 anos; Gerente de Soluções em TI (Consultoria) – 3 anos. Estratégia, Tecnologia e Inovação 3 Graduação, Mestrado e Doutorado em Administração. Docente na área de Gestão da Inovação e Projeto em Instituições Públicas e Privada – 13 anos Gerente de Tecnologia (Consultoria) – 5 anos; Coordenador de Fundo de apoio Inovações Tecnológica (CRIATEC) – 3 anos. Gestão de Tecnologia, Qualidade e Competitividade Nota: Elaborada pelos autores. Nota: Elaborada pelos autores. 3.1. Tratamento das Informações 3. Método de Pesquisa Foi realizada uma pesquisa exploratória a fim de identificar os estudos que tratassem de indicadores de inovação, capacidade de inovação para ampliar o conhecimento sobre o tema. A pesquisa exploratória tem como função “proporcionar maior familiaridade com o problema, com vistas a torná-lo mais explícito ou a constituir hipóteses (...) têm como objetivo principal o aprimoramento de ideias.” (Gil, 2002, p.41). O objetivo de utilizar a pesquisa exploratória foi para enriquecer a delimitação inicial do estudo a fim de averiguar possíveis bibliografias que abordassem indicadores não tradicionais que apoiassem a pesquisa. Além disso, entender como a inovação ocorre em empresas nos países emergentes. Para auxiliar a pesquisa também foi realizado um levantamento a partir da técnica grupo focal com especialistas da área. A técnica de caráter qualitativo busca obter dados por meio de reuniões em grupo tendo como finalidade a interação entre os profissionais convidados e o pesquisador, ambos direcionados a promover uma discussão focada na coleta de dados a partir de tópicos específicos (Lervolino & Pelicioni, 2001). Como critério, foram escolhidos profissionais da área acadêmica que pesquisassem sobre o assunto. O grupo foi direcionado a pontuar um quadro que auxiliaria na escolha dos melhores indicadores alternativos a serem utilizados de acordo com critérios pré-estabelecidos. Além disso, este teve como objetivo indicar literaturas RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br 3.1. Tratamento das Informações A partir da pesquisa exploratória foi possível identificar autores que tratam dos indicadores não tradicionais como um tema de relevância quando se trata de processo de inovação com o foco na compreensão do mesmo. Apesar da afirmativa, a análise apresentou carência quanto a propostas de conjunto de novos indicadores que servissem como complementares aos indicadores já existentes, os tradicionais. Paralelamente, buscou-se literaturas que tratassem sobre capacidades e processo de inovação no interior das empresas. Por ser um tema amplamente discutido, foram encontrados diversos estudos, sendo o mais apropriado para esta pesquisa, o estudo de Zawislak (2014) sobre as capacidades de inovação da firma, pois foi visualizada a possibilidade de relacionar estas capacidades com os indicadores alternativos de inovação. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 178 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 178 https://doi.org/10.20401/rasi.2.2.110 Sendo assim, este artigo baseia-se em dois estudos específicos do tema no país, o primeiro foi realizado por Zawislak (2014) acerca das capacidades de inovação das firmas e o segundo por Marins (2010) sobre indicadores alternativos de inovação. Zawislak (2014) expõe em seu estudo que a firma é composta por quatro capacidades, sendo elas, capacidade de desenvolvimento tecnológico e operacional, capacidades gerencial e transacional anteriormente abordadas. Ele afirma que a caracterização de uma empresa inovadora não é necessariamente resultado do bom desempenho das quatro capacidades juntas, podendo a empresas desenvolver bem apenas uma delas. A pesquisa de Marins (2010) consiste em apresentar um conjunto de novos indicadores de inovação que seriam complementares aos indicadores tradicionais, já que Marins (2010) alega que o último possui um foco no resultado, modelo linear, não sendo este capaz de compreender o processo de inovação no interior de empresas em economias emergentes, já que estas empresas não possuem uma estrutura de investimento em inovação formalizada. A partir de ambos os estudos foi elaborado um arcabouço teórico onde se relaciona as capacidades de inovação com os indicadores alternativos. Acredita-se que é possível entender o processo de inovação dentro das empresas através das capacidades associadas a informações que partiriam dos indicadores alternativos. Zawislak (2014) aponta que uma empresa caracterizada como inovadora, possui pelo menos uma dessas quatro capacidades bem desenvolvida. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br 3.1. Tratamento das Informações Neste sentido a empresa, primeiramente, deve compreender a existência dessas capacidades e assim construir uma visão clara e alinhada em relação a elas, a fim de analisá-las e por consequência alcançar uma melhor eficiência na utilização de seus recursos para a inovação. Simultaneamente a este processo, os indicadores têm como função servir de apoio para o levantamento de informações para conhecimento e acompanhamento das capacidades. Vale ressaltar, que este processo não é estático, competindo à empresa o exercício contínuo dessa análise. A partir desta análise foi possível elaborar um quadro em que pudesse ser feita a associação entre as capacidades descritas por Zawislak (2014) e os indicadores alternativos propostos por Marins (2010). Inicialmente, buscou-se compreender as definições expostas pela autora sobre os indicadores, analisando quais possíveis informações poderiam ser obtidas quando este fosse aplicado na empresa. Posteriormente o mesmo procedimento foi realizado para as definições das capacidades propostas por Zawislak (2014), visando apenas o entendimento das mesmas. Com isso, foi possível alocar os indicadores apropriados a cada capacidade, não sendo feita nenhuma alteração nos conceitos apresentados pelos autores. Com o propósito de embasar o quadro anteriormente descrito, usou-se outro trabalho, Zawislak, Fracasso e Tello-Gamarra (2013), no qual os autores apresentam um framework com o intuito de fundamentar as capacidades por eles definidas, utilizando autores que serviram como um referencial teórico. Para este estudo foi utilizado a mesma base teórica apresentada, acrescentando ao mesmo os indicadores alternativos propostos por Marins (2010) como mostra a Tabela 2: RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 179 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 179 https://doi.org/10.20401/rasi.2.2.110 Tabela 2 Relação entre Indicadores e Capacidades das firmas Capacidades Indicadores Autores Capacidade de desenvolvimento tecnológico Criatividade Davila (2000), Wong et al. (1998), Rush et al. (2007), Huergo(2006), Zawislak et al. (2012b), Marins (2010) Project champions Cadência Capacidade operacional Capacidade de realização Capon (1990), Roth e Miler (1992), Zawislak et al. (2012b), Marins (2010) Controle de erros Interatividade Aplicações tecnológicas Capacidade de gerenciamento Estratégia de inovação Barnard (1966), Penrose (1959), Ansoff (1965), Marins (2010) Portfólio de projetos de inovação Recursos físicos tangíveis Lucro operacional inovativo Time to Market Time to profit Valor agregado Capacidade transacional Parcerias estratégicas Bosse e Alvarez (2010), Williamson (1985 - 1999), Marins (2010) Externalização Fonte: Adaptado de Zawislak, Fracasso; Tello-Gamarra, 2013, p. 8. 3.1. Tratamento das Informações Com base nesta relação realizou-se um grupo de foco com o objetivo de trazer uma visão de especialistas no assunto ao trabalho. O objetivo foi priorizar os indicadores que fossem importantes para a análise do processo de inovação, além de averiguar aqueles que seriam exequíveis de medição e a sua associação teórica dentro do campo de pesquisa, atribuindo a estes notas de 1 a 5. Para tanto, utilizou-se a matriz G.U.T. que segundo Meireles (2001) consiste em uma ferramenta com a finalidade de priorizar dentre alternativas de ação. Esta organiza as atividades por ordem de importância analisando a Gravidade, Urgência e Tendência, de modo a escolher racionalmente a ação menos prejudicial. Nesse estudo, adaptou-se a matriz G.U.T. utilizando Importância, Execução e Associação Teórica, sendo esses critérios foram submetidos à análise de especialistas na área de pesquisa. Ao final, as notas atribuídas a cada indicador foram contabilizadas pela multiplicação dos critérios. A aplicação e realização do grupo de foco contribuíram para priorizar os indicadores que fossem exequíveis dentro da organização, através de uma análise dos indicadores, da atribuição de notas pelos especialistas e a utilização de um fator de prioridade. Este fator foi calculado a partir da soma das notas dos especialistas atribuída a cada indicador multiplicada por (1-CV), sendo CV o coeficiente de variação das notas de cada indicador. Assim, (1-CV) é a ponderação que serve como um critério de convergência das opiniões. Quanto menor a variação das opiniões dos especialistas melhor. Por fim, definiu-se que a média do fator de prioridade por capacidade como sendo o critério que selecionou os indicadores que foram mantidos. O fator de prioridade (FP) representa o produto entre o resultado da soma dos itens da matriz G.U.T. adaptada e o inverso do coeficiente de variação conforme a equação 1. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 180 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 180 https://doi.org/10.20401/rasi.2.2.110 𝐹𝑃= 𝐶𝑖 𝑥 (1 −𝐶𝑉) 𝑛=3 𝑖=3 1 1 onde: onde: onde: C = critérios da matriz G.U.T. adaptada i = número de critérios n = respondentes (1-CV) = índice de convergência onde: C = critérios da matriz G.U.T. 3.1. Tratamento das Informações adaptada i = número de critérios n = respondentes (1-CV) = índice de convergência A Figura 2 representa em forma de esquema os passos que organizam o estudo em suas etapas dando origem a um protocolo que pode servir de base para estudos futuros. Figura 2. Protocolo do Estudo Nota. Fonte: Elaborado pelos autores. Figura 2. Protocolo do Estudo Figura 2. Protocolo do Estudo Nota. Fonte: Elaborado pelos autores. Silva, Leite & Oliveira\| 181 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 181 https://doi.org/10.20401/rasi.2.2.110 necessários para o entendimento do processo de inovação. Por isso, ao alocar cada indicador as capacidades buscou-se relacionar àqueles que poderiam auxiliar no entendimento do processo de inovação associado a cada capacidade da organização. necessários para o entendimento do processo de inovação. Por isso, ao alocar cada indicador as capacidades buscou-se relacionar àqueles que poderiam auxiliar no entendimento do processo de inovação associado a cada capacidade da organização. Deste modo, foi apenas alocado, sem alteração de sentido, os indicadores Criatividade, Project champions e Cadência à capacidade de desenvolvimento tecnológico; os indicadores capacidade de realização, Controle de erros, Interatividade e Aplicações tecnológicas à capacidade operacional; os indicadores Estratégia de inovação, Portifólio de projetos de inovação, Recursos físicos tangíveis, Lucro operacional inovativo, Time do market, Time do profit e Valor agregado à capacidade gerencial e por último os indicadores Parcerias estratégicas e Externalização à capacidade transacional, como já visto no Quadro 1. p j Através da análise do Quadro 1 foi possível por meio do grupo de foco, priorizar alguns indicadores e também retirar aqueles que não seriam exequíveis, segundo a opinião dos especialistas. Deste modo, através da avaliação dos especialistas ficou decidido retirar o indicador Controle de erros, antes mesmo da aplicação da matriz de priorização, pois na opinião dos mesmos, este indicador não teria adequação para a avaliação do desempenho inovativo. A justificativa foi que, segundo a definição do indicador, este não atenderia de maneira satisfatória ao objetivo de tentar entender como ocorre a inovação dentro das empresas. O resultado deste estudo foi a junção dos indicadores com as capacidades, mas a fim de chegar a uma proposta final de indicadores que atendesse ao objetivo foi utilizada a matriz G.U.T. adaptada com o intuito de priorizar os indicadores alocados a cada capacidade. Essa priorização foi realizada por especialistas através do grupo de foco no qual foi apresentado a cada um a definição dos indicadores de Marins (2010) e das capacidades de Zawislak (2014) fazendo uma discussão do entendimento destes sobre o significado e a alocação dos mesmos. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br 4. Análise de Discussão dos Resultados Sendo assim, após análise da pesquisa de Marins (2010), buscou-se neste estudo associar os indicadores não convencionais propostos pela autora às capacidades de inovação explicitadas por Zawislak (2014). Isto foi possível, pois ao estudar as capacidades de inovação das firmas, principalmente aquelas em economias emergentes, observa-se a necessidade de indicadores que consigam mensurar os elementos RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br Como resultado da associação entre capacidade de inovação e indicadores não convencionais discutido no grupo de foco foram feitos ajustes aos modelos originais como por exemplo a alteração do indicador Externalização, originalmente colocado na capacidade de desenvolvimento tecnológico e assim movido para a capacidade transacional e a exclusão do indicador Controle de erros. Com o resultado da multiplicação dos critérios estabelecidos foi possível a partir do fator de importância elencar os indicadores pertencentes à proposta final. Sendo assim, os números de 1 a 6 na Tabela 3 indicam os indicadores priorizados a partir da Equação 1. http://www.rasi.uff.br Tabela 3. Resultado Final da Priorização dos Especialistas Fonte: Elaborado pelos autores. Desempenho Técnico Capacidades Indicadores Esp. 1 Esp. 2 Esp. 3 SOMA CV Fator de Prioridade Capacidade de desenvolvimento tecnológico Criatividade (1) 20 15 40 75 0,529 35,3 Project champions 10 15 40 65 0,741 16,8 Cadência 6 60 24 90 0,916 7,5 Total 19,9 Capacidade operacional Capacidade de realização (2) 15 15 50 80 0,757 19,4 Controle de erros - - - 0 0 0,0 Interatividade 3 16 5 24 0,875 3,0 Aplicações tecnológicas (3) 30 100 20 150 0,871 19,2 Total 10,4 Desempenho Econômico Capacidade de gerenciamento Estratégia de inovação (4) 32 50 25 107 0,361 68,3 Portfólio de projetos de inovação 4 45 25 74 0,831 12,5 Recursos físicos tangíveis (5) 64 24 40 128 0,471 67,6 Lucro operacional inovativo 12 5 25 42 0,724 11,6 Time to Market 8 5 20 33 0,721 9,2 Time to profit 8 4 6 18 0,333 12,0 Valor agregado 12 5 6 23 0,493 11,6 Total 27,5 Capacidade transacional Parcerias estratégicas 48 125 30 203 0,745 51,6 Externalização (6) 60 125 36 221 0,625 82,9 Total 67,2 Tabela 3. Tabela 3. Resultado Final da Priorização dos Especialistas Resultado Final da Priorização dos Especialistas Na Tabela 4 está representada, assim, a versão final do estudo. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br Tabela 4. Proposta final dos indicadores não convencionais de inovação Capacidades Indicadores Desempenho Técnico Capacidade de desenvolvimento tecnológico Criatividade Capacidade operacional Capacidade de realização Aplicações tecnológicas Desempenho Econômico Capacidade de gerenciamento Estratégia de inovação Recursos físicos tangíveis Capacidade transacional Externalização Fonte: Elaboração própria. Silva, Leite & Oliveira\| 183 https://doi.org/10.20401/rasi.2.2.110 Silva, Leite & Oliveira\| 183 https://doi.org/10.20401/rasi.2.2.110 5. Considerações Finais Após a análise e associação dos trabalhos de Marins (2010) e Zawislak (2014) e o complemento sobre a priorização dos indicadores mais exequíveis dentro da organização por meio do auxílio de especialistas na área de inovação e desempenho ocorrido no grupo de foco e da aplicação de um fator de importância a cada indicador foi possível elaborar um quadro teórico-analítico para integrar o processo de inovação das organizações através da associação das capacidades de inovação e indicadores não convencionais. Com isso, o estudo sugere como proposta alguns indicadores não convencionais relacionados às capacidades de inovação da firma para análise do processo de inovação. Este trabalho propõe a manutenção dos indicadores: Criatividade, Capacidade de realização, Aplicações tecnológicas, Estratégia de inovação, Recursos físicos tangíveis e Externalização. Deste modo, pode-se dizer que o objetivo do trabalho foi alcançado, já que através de algumas técnicas de análise e priorização foi possível elaborar um quadro teórico-analítico com os indicadores não convencionais associados às capacidades para avaliação do processo de inovação dentro das empresas. Além disso, esta pesquisa vem a contribuir para estudos futuros relacionados ao entendimento do processo de inovação de empresas em economias emergentes, como o Brasil, pois o desenvolvimento de indicadores de inovação focados nos processos internos das empresas e não somente nos resultados, pode representar uma alternativa aos tradicionais, assim contribuir para a mensuração da inovação em países hoje considerados pouco inovadores. A pesquisa demonstra algumas limitações, no que diz respeito ao pequeno número de especialistas entrevistados para o grupo de foco, não evidenciada por estudos empíricos nas organizações, por isso, para avanços são propostos novos estudos a respeito. Neste sentido sugere-se que sejam realizados novos levantamentos com especialistas com a utilização do método Delphi para a melhor análise dos indicadores propostos por Marins (2010), além disso, poderiam ser realizadas entrevistas com especialistas de diversos seguimentos para haver maior diversidade de opiniões melhor e embasamento teórico. Outra sugestão seria a aplicação da proposta final de indicadores não convencionais em empresas adotando-se como procedimento técnico o estudo de caso, possibilitando a análise empírica da proposta dentro das organizações. Referências Albuquerque, E. M. (2006). Sistemas nacionais de inovação e desenvolvimento. Revista da Universidade Federal de Minas Gerais, ano 5, n. 10, out. Ansoff, H. I. (1965). Corporate strategy: an analytic approach to business policy forgrowth and expansion. New York: McGraw - Hill, p.241. Arocena, R., & Sutz, J. (2006). El estudio de la innovación desde el Sur y las perspectivas de un nuevo desarrollo. Revista Iberoamericana de Ciencia, Tecnología, Sociedad e Innovación, n. 7, 2006. Recuperado em 10 de novembro de 2014 de http://www.oei.es/revistactsi/numero7/articulo01.htm#1. Arocena, R., & Sutz, J. (2006). El estudio de la innovación desde el Sur y las perspectivas de un nuevo desarrollo. Revista Iberoamericana de Ciencia, Tecnología, Sociedad e Innovación, n. 7, 2006. Recuperado em 10 de novembro de 2014 de http://www.oei.es/revistactsi/numero7/articulo01.htm#1. Arundel, A. (2006). Innovation survey indicators: any progress since 1996? G20 at the Leader's level (L20 Workshop). Maastricht, UNU-MERIT, (Background Paper). Arundel, A. (2006). Innovation survey indicators: any progress since 1996? G20 at the Leader's level (L20 Workshop). Maastricht, UNU-MERIT, (Background Paper). Arundel, A. (2006). Innovation survey indicators: any progress since 1996? G20 at the Leader's level (L20 Workshop). Maastricht, UNU-MERIT, (Background Paper). RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br Silva, Leite & Oliveira\| 184 https://doi.org/10.20401/rasi.2.2.110 Bosse, D., & Alvarez, S. (2010). Bargaining power in alliance governance negotiations: evidence from the biotechnology industry. Technovation, v.30, n.5 - 6, p. 367 - 375. Capon, N., Farley, J. U. & Hoenig, S. (1990). Determinants of financial performance: a meta-analysis. Management Science, v 36, n. 6, p.1143 -1159. Coase, R. (1937). The nature of the firm. Economica, 4(16), 386-405. Davila, T. (2000). An empirical study on the drivers of management control systems' design in new product development. Accounting Organizations and Society, v. 25, n. 4 - 5. p. 383 - 409. Figueiredo. P. (2006). Introduction. In: International Journal of Technology Management, v. 36, nºs 1-3, p. 1-13. Fuck, M. P., Vilha, A. M. (2012). Inovação Tecnológica: da definição à ação. Revista Contemporâneos. n. 9, nov. 2011 a abr. Gavira, M. O., Ferro, A. F. P., Rohrich, S. S. & Quadroset, R. (2007). Gestão da inovação tecnológica: uma análise da aplicação do funil de inovação em uma organização de bens de consumo. RAM – Revista de Administração Mackenzie. v. 8, n.1, p. 77-107. Gil, A. C. (2002). Como elaborar projetos de pesquisa. 4. ed. - São Paulo: Atlas. Godin, B. (2002). Referências The rise of innovation surveys: measuring a fuzzy concept. Canadian Science and Innovation Indicators Consortium, Project on the History and Sociology of S&T Statistics: Montreal, Canadá, (Working Papernº16). Guan, J.; M. A, N. (2003). Innovative capability and export performance of Chinese firms. Technovation, v. 23, n. 9, p. 737-747. Huergo, E. (2006). The role of technological management as a source of innovation: Evidence from Spanish manufacturing firms, Research Policy, v. 35, n. 9, p. 1377-1388. Instituto Brasileiro de Geografia e Estatística. (2012). Brasil em números. Rio de Janeiro: IBGE, v. 20, 2012. ___ (2013). Rio de Janeiro: IBGE, v. 21, 2013. _ (2013). Rio de Janeiro: IBGE, v. 21, 2013. Katz, J. (2004). The limits of the prevailing orthodoxy: technology and education as restrictions to productivity growth and international competitiveness in Latin America. DRUID Summer Conference on Industrial Dynamics, Innovation and Development: Elsinore, Dinamarca. (Anais). Kline, S. J. & Rosenberg, N. (1986). An Overview of Innovation. Recuperado 23 de setembro de 2014 de ftp://ftp.ige.unicamp.br/pub/CT010/aula%202/KlineRosenberg(1986).pdf. Kobal, A. B.; Lázaro, J. C. & Santos, S. M. (2012). O perfil do crescimento da inovação brasileira, baseado em indicadores segundo as pesquisas acadêmicas. Revista Estudos do CEPE, Santa Cruz do Sul, n36, p.195-227, jul./dez. 2012. Lervolino, S. A. & Pelicioni, M. C. F. (2001). A utilização do grupo focal como metodologia qualitativa na promoção da saúde. Rev Esc Enf USP, v. 35, n.2, p.115-21, jun. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br Silva, Leite & Oliveira\| 185 https://doi.org/10.20401/rasi.2.2.110 Levitt, B. & March, J (1988). Organizational Learning. Annual Review of Sociology, Stanford, v. 14, p. 319-338, ago. Longanezi, T (2008). Os sistemas de gestão da inovação e a capacidade inovadora das empresas. Tese de Doutorado em Engenharia Química. Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil. Marins, L. M. (2010). Atividade de inovação em firmas de economias emergentes: proposta de um conjunto de novos indicadores. Tese de Doutorado em Administração. Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil. Marins, L. M.; & Zawislak, P. A. (2010). O Desempenho Inovativo de Sete Firmas Brasileiras à Luz de um Conjunto de Novos Indicadores de Inovação. Anais do Encontro Associação Nacional de Pós-Graduação e Pesquisa em Administração, Rio de Janeiro, 34. Meireles, M. (2001). Ferramentas Administrativas para identificar, observar e analisar problemas: organizações com foco no cliente. 1 ed. v. 2. São Paulo: Arte & Ciência. Oliveira, L. C., & Sorgi, F. A. (2014). O poder da inovação no Brasil. COFECON. Conselho Federal e Economia. Recuperado em 06 de setembro de 2014 de http://www.cofecon.org.br/noticias/artigos/16-artigo/1357-artigo-o-poder-da-inovacao- no-brasil. Penrose, E. (1959). The theory of the growth of the firm. New York: Oxford University Press. First published. Pradella. S. (2014) Gestão da Inovação Tecnológica. Anais do Congresso Nacional de Excelência em Gestão, 10. Recuperado em 30 setembro de 2014 de http://www.excelenciaemgestao.org/portals/2/documents/cneg9/anais/t13_0655_3149.p df. Quadros, R. (2008). Aprendendo a inovar: Padrões de Gestão da Inovação Tecnológica em Empresas Industriais Brasileiras. (Relatório de Pesquisa). GEMPI – Grupo de Estudos de Empresas e Inovação. _ (2013). Rio de Janeiro: IBGE, v. 21, 2013. Recuperado em 30 de setembro de 2014 de http://www.extecamp.unicamp.br/gestaodainovacao/biblioteca/Quadros_(2008).pdf. Roth, A., & Miller, J. (1992). Success factors in manufacturing. Business Horizons, v. 35, n. 4, p. 73 - 81, July/Aug. Rothwell. R. (1994). Towards the Fifth generation Innovation Process. International Marketing Review, v. 11, Iss 1, pp. 7- 31. Rush, H.; Bessant, J. & Hobday, M. (2007). Assessing the technological capabilities of firms:developing a policy tool. British Journal Management, v. 37, n.3, p.221-236. Sen, F. K. & Egelhoff, W. G. (2000). Innovative capabilities of a firm and the use of technical alliances. IEE Transactions on Engineering Management. v.47, n. 2, p. 174- 183. Schumpeter, J. A. ([1942] 2008). Capitalism, socialism and democracy. New York: Harper Perennial Modern Thought. Stefanovitz, J. P., & Nagano, M. S. (2014). Gestão da inovação: análise e síntese dos conceitos. Produto & Produção, Porto Alegre, v. 15, n. 2, p.11-23, jun. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br Silva, Leite & Oliveira\| 186 https://doi.org/10.20401/rasi.2.2.110 Senado Federal do Brasil. (2012). País ainda constrói bases para inovação. Em Discussão: Revista de audiências públicas de Senado Federal. ano 3, n. 12, set. 2012. Recuperado em 06 de setembro de 2014 de http://www senado gov br/NOTICIAS/JORNAL/EMDISCUSSAO/inovacao aspx Senado Federal do Brasil. (2012). País ainda constrói bases para inovação. Em Discussão: Revista de audiências públicas de Senado Federal. ano 3, n. 12, set. 2012. Senado Federal do Brasil. (2012). País ainda constrói bases para inovação. Em Discussão: Revista de audiências públicas de Senado Federal. ano 3, n. 12, set. 2012. Recuperado em 06 de setembro de 2014 de http://www.senado.gov.br/NOTICIAS/JORNAL/EMDISCUSSAO/inovacao.aspx. p , , Recuperado em 06 de setembro de 2014 de http://www.senado.gov.br/NOTICIAS/JORNAL/EMDISCUSSAO/inovacao.aspx. Terra, J. C. (Org.), (2012). Dez Dimensões da gestão da inovação: uma abordagem para a transformação organizacional. Rio de Janeiro: Elsevier, 2012. Tidd, J. (2001). Innovation management in context: environment, organization and performance. International Journal of Management Reviews, Brighton, v. 3, Issue 3, p. 169–183, set. Tidd, J., Bessant, J. & Pavitt, K. (2008). Gestão da Inovação. 3. ed. São Paulo: Bookman. Thomson Reuters. (2012). Building BRICKS: Exploring the global research and innovation impact of Brazil, Russia, India, China and South Korea. Recuperado em 22 de agosto de 2014 de http://sciencewatch.com/sites/sw/files/sw-article/media/grr- brick.pdf. Van de Ven, A. H. (1986). Central Problems in the Management of Innovation. Management Science, Minneapolis, v. 32, n. 5, p. 590-607, mar. Williamson, O. (1985). The economic institutions of capitalism. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. RASI, Volta Redonda/RJ, v. 2, n. 2, pp. 167-186, jul./dez. 2016. http://www.rasi.uff.br http://www.rasi.uff.br _ (2013). Rio de Janeiro: IBGE, v. 21, 2013. New York: Free Press. ______. (1999). Strategic research: governance and competence. Strategic Managerial Journal, v. 20, n. 12, p. 1087 - 1108. World Intellectual Property Organization. WIPO. (2014). The Global Innovation Index 2014. Recuperado em 06 de setembro de 2014 de http://www.wipo.int/export/sites/www/econ_stat/en/economics/gii/pdf/2014/gii_2014_r ankings.pdf#page=2. Wong, V.; Shaw, V. & Sher, J. (1998). Effective organization and management of technology assimilation the case of taiwanese information technology firms. Industrial Marketing Management, v.27 , n. 3, p. 213 - 227. Zawislak, P. A., Fracasso, E. & Tello-Gamarra, J. (2013). Intensidade tecnológica e capacidade de inovação de firmas industriais. Anais do Congresso Latino- Iberoamericana de Gestão de Tecnologia, 2013, Porto, Portugal. Zawislak, P. A., Tello-Gamarra, J., Alves, A. C., Barbieux, D., & Reichert, F. M. (2014). The different innovation capabilities of the firm: further remarks upon the Brazilian experience. Journal of Innovation Economics, 13, 129-150. Zawislak, P. A, Borges, M., Wegner, D., Santos, A., & Castro-Lucas, C. (2008). Towards the Innovation Function. Journal of technology management & innovation, 3(4), 17-30. https://dx.doi.org/10.4067/S0718-27242008000200002. http://www.rasi.uff.br
https://openalex.org/W4291923930	https://zenodo.org/records/2666371/files/10459.pdf	English	null	Effect of Thickness on Structural and Electrical Properties of CuAlS2 Thin Films Grown by Two Stage Vacuum Thermal Evaporation Technique	Zenodo (CERN European Organization for Nuclear Research)	2,014	cc-by	401	World Academy of Science, Engineering and Technology International Journal of Energy and Power Engineering Vol:8, No:6, 2014 World Academy of Science, Engineering and Technology International Journal of Energy and Power Engineering Vol:8, No:6, 2014 International Scholarly and Scientific Research & Innovation 8(6) 2014 Effect of Thickness on Structural and Electrical Properties of CuAlS2 Thin Films Grown by Two Stage Vacuum Thermal Evaporation Technique Authors : A. U. Moreh, M. Momoh, H. N. Yahya, B. Hamza, I. G. Saidu, S. Abdullahi Authors : A. U. Moreh, M. Momoh, H. N. Yahya, B. Hamza, I. G. Saidu, S. Abdullahi Abstract : This work studies the effect of thickness on structural and electrical properties of CuAlS2 thin films grown by two stage vacuum thermal evaporation technique. CuAlS2 thin films of thicknesses 50nm, 100nm and 200nm were deposited on suitably cleaned corning 7059 glass substrate at room temperature (RT). In the first stage Cu-Al precursors were grown at room temperature by thermal evaporation and in the second stage Cu-Al precursors were converted to CuAlS2 thin films by sulfurisation under sulfur atmosphere at the temperature of 673K. The structural properties of the films were examined by X- ray diffraction (XRD) technique while electrical properties of the specimens were studied using four point probe method. The XRD studies revealed that the films are of crystalline in nature having tetragonal structure. The variations of the micro- structural parameters, such as crystallite size (D), dislocation density ( ), and micro-strain ( ), with film thickness were investigated. The results showed that the crystallite sizes increase as the thickness of the film increases. The dislocation density and micro-strain decreases as the thickness increases. The resistivity (  ) of CuAlS2 film is found to decrease with increase in film thickness, which is related to the increase of carrier concentration with film thickness. Thus thicker films exhibit the lowest resistivity and high carrier concentration, implying these are the most conductive films. Low electrical resistivity and high carrier concentration are widely used as the essential components in various optoelectronic devices such as light-emitting diode and photovoltaic cells. pen Science Index, Energy and Power Engineering Vol:8, No:6, 2014 waset.org/abstracts/10459 Keywords : CuAlS2, evaporation, sulfurisation, thickness, resistivity, crystalline Conference Title : ICRESA 2014 : International Conference on Renewable Energy Systems and Applications Conference Location : Paris, France Conference Dates : June 26-27, 2014 International Scholarly and Scientific Research & Innovation 8(6) 2014 ISNI:0000000091950263 1
https://openalex.org/W2002400144	https://link.springer.com/content/pdf/10.1007/s12274-014-0683-9.pdf	English	null	Tracking morphologies at the nanoscale: Self-assembly of an amphiphilic designer peptide into a double helix superstructure	Nano research	2,015	cc-by	7,793	Tracking morphologies at the nanoscale: Self-assembly of an amphiphilic designer peptide into a double helix superstructure p Karin Kornmueller1, Ilse Letofsky-Papst2, Kerstin Gradauer1, Christian Mikl1, Fernando Cacho-Nerin3,†, Mario Leypold4, Walter Keller5, Gerd Leitinger6,7, Heinz Amenitsch3, and Ruth Prassl1 () 1 Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, 8010 Graz, Austria 2 Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology and Graz Centre for Electron Microscopy, 8010 Graz, Austria 3 Institute of Inorganic Chemistry, Graz University of Technology, 8010 Graz, Austria 4 Institute of Organic Chemistry, Graz University of Technology, 8010 Graz, Austria 5 Institute for Molecular Biosciences, Karl-Franzens-University Graz, 8010 Graz, Austria 6 Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 8010 Graz, Austria 7 Center for Medical Research, Medical University of Graz, 8010 Graz, Austria † Present address: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom Karin Kornmueller1, Ilse Letofsky-Papst2, Kerstin Gradauer1, Christian Mikl1, Fernando Cacho-Nerin3,†, Mario Leypold4, Walter Keller5, Gerd Leitinger6,7, Heinz Amenitsch3, and Ruth Prassl1 () 1 Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, 8010 Graz, Austria 2 Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology and Graz Centre for Electron Microscopy, 8010 Graz, Austria 7 Center for Medical Research, Medical University of Graz, 8010 Graz, Austria † Present address: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom Received: 18 August 2014 Revised: 05 December 2014 Accepted: 07 December 2014 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014 KEYWORDS double helix, amphiphilic designer peptide, self-assembly, SAXS, TEM, spectroscopy Nano Research 2015, 8(6): 1822–1833 DOI 10.1007/s12274-014-0683-9 Nano Research 2015, 8(6): 1822–1833 DOI 10.1007/s12274-014-0683-9 Nano Research 2015, 8(6): 1822–1833 DOI 10.1007/s12274-014-0683-9 Nano Research 2015, 8(6): 1822–1833 DOI 10.1007/s12274-014-0683-9 Tracking morphologies at the nanoscale: Self-assembly of an amphiphilic designer peptide into a double helix superstructure Karin Kornmueller1, Ilse Letofsky-Papst2, Kerstin Gradauer1, Christian Mikl1, Fernando Cacho-Nerin3,†, Mario Leypold4, Walter Keller5, Gerd Leitinger6,7, Heinz Amenitsch3, and Ruth Prassl1 () 1 Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, 8010 Graz, Austria 2 Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology and Graz Centre for Electron Microscopy, 8010 Graz, Austria 3 Institute of Inorganic Chemistry, Graz University of Technology, 8010 Graz, Austria 4 Institute of Organic Chemistry, Graz University of Technology, 8010 Graz, Austria 5 Institute for Molecular Biosciences, Karl-Franzens-University Graz, 8010 Graz, Austria 6 Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 8010 Graz, Austria 7 Center for Medical Research, Medical University of Graz, 8010 Graz, Austria † Present address: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom Address correspondence to ruth.prassl@medunigraz.at ABSTRACT Hierarchical self-assembly is a fundamental principle in nature, which gives rise to astonishing supramolecular architectures that are an inspiration for the development of innovative materials in nanotechnology. Here, we present the unique structure of a cone-shaped amphiphilic designer peptide. While tracking its concentration-dependent morphologies, we observed elongated bilayered single tapes at the beginning of the assembly process, which further developed into novel double-helix-like superstructures at high concentrations. This architecture is characterized by a tight intertwisting of two individual helices, resulting in a periodic pitch size over their total lengths of several hundred nanometers. Solution X-ray scattering data revealed a marked 2-layered internal organization. All these characteristics remained unaltered for the investigated period of almost three months. In their collective morphology, the assemblies are integrated into a network with hydrogel characteristics. Such a peptide-based structure holds promise as a building block for next-generation nanostructured biomaterials. © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014 Address correspondence to ruth.prassl@medunigraz.at Address correspondence to ruth.prassl@medunigraz.at Nano Res. 2015, 8(6): 1822–1833 1823 1 Introduction in different length scales, and therefore give insights into different parts of the assembled nanostructures. SAXS allows monitoring of the self-assembly of biomolecules in solution, without the need of immobilization, labeling, or any other modification. It provides a detailed insight into the internal organization of structures in the low-nanometer range (~3–60 nm), and therefore can be used to describe the cross section of extended structures in high details. In contrast, longitudinal extensions, which often exceed the nanometer range, are well resolved with TEM. When combined, both techniques yield a detailed picture of the individual structure, as well as the collective morphologies in solution. Nature offers elegant and efficient ways to create highly organized systems over several orders of magnitude by hierarchical self-assembly. This basic principle has been mimicked for the design of synthetic amphiphilic molecules in order to create novel materials [1–3]. Our study focused on short amphiphilic designer peptides. These molecules are exclusively composed of amino acids, and exhibit lengths of 4–10 amino acid residues. As their basic characteristic, they contain anta- gonistic hydrophilic and hydrophobic residues within the same molecule [4]. Self-assembling amphiphilic designer peptides have been explored as building blocks for the development of controllable, tailor- made, and biocompatible nanomaterials only recently. Because their structure is simple, yet variable, they have a huge scientific as well as economic potential. Their applications span the range from material sciences to chemistry and nanotechnology, and are of particular interest in nanomedicine due to the peptides’ biocompatibility and biodegradability [5–7]. A constantly increasing global demand in innovative materials makes the elucidation of their assembly mechanism highly important. Our data indicates that the peptide concentration directly relates to the morphology, order, and com- plexity of the assembled structure. We monitored the transitions from monomeric systems to elongated single and double tapes, and finally to double-helix-like structures by increasing the peptide concentration. 2.1 SAXS analysis of the low-concentration regime SAXS measurements were complemented with TEM, which allows direct visualization of the assemblies. TEM pictures reveal a quite heterogeneous distri- bution of structures in the intermediate-concentration regime: Double-helix-like structures are the dominant species, along with extended tapes and double tapes (Fig. 2 (d1–d3)). Uniformity in tape width is observed by TEM throughout the intermediate morphologies. First, we examined the peptide morphology at concentrations below or close to the CAC. This data could be modeled with the theoretical form factor of the peptide monomer, adding a hard-sphere interaction term [24]. The form factor was calculated with the program FoXS [25, 26], from a theoretical atomic model of the monomer (Fig. S2 in the ESM). Figure 2(a) shows the scattering curves of the peptide at con- centrations ranging from 10 to 45 mM, with their respective fits (green curves) shown as a function of the scattering vector (q). All curves show the same characteristics with an almost linear region in the low q-range, followed by a broad shoulder at q = 1.3 nm–1. The parameters to describe molecular interactions are obtained from the fitting function. As seen in Fig. 2(b), the hard-sphere radius (Rs) and the volume fraction () remain constant for the measured con- centrations. The averaged value of Rs, 1.6 nm, is in good agreement with the monomer’s theoretical total length of 3 nm (≈ 2Rs). The linear increase in the Figure 1 Molecular model of the amphiphilic designer-peptide, Ac-GAAVILRR-NH2. The hydrophobic tail is composed of amino acids with consecutively increasing hydrophobicity and size. Two arginine residues provide the positively charged hydrophilic head. Peptide modifications include acetylation of the N-terminus and amidation of the C-terminus. The monomer length is approximately 3 nm, and its widest lateral extension is around 1.5 nm. The overall structure of the molecule looks like a cone. Color code: green: carbon, blue: nitrogen, gray: oxygen, and white: hydrogen. 2.2 Structural heterogeneity dominates the intermediate-concentration regime A pronounced change in the structure is observed when the peptide concentration is increased well above the CAC. Here, we distinguish between a transition structure (55–65 mM, Figs. 2(c) and 2(d)) and the final structure (75 mM, Figs. 2(e) and 2(f)). The SAXS pattern of the 55 mM sample shows a large increase in the intensity in the lower q-range, indicating the first steps of peptide assembly. At a slightly elevated concentration (60 mM), we observe a SAXS pattern typical of elongated structures. Increasing the concentration to 65 mM leads to a SAXS pattern that already shows distinct features of the endpoint structure, but does not show the high viscosity of the 75 mM sample. A pronounced change in the structure is observed when the peptide concentration is increased well above the CAC. Here, we distinguish between a transition structure (55–65 mM, Figs. 2(c) and 2(d)) and the final structure (75 mM, Figs. 2(e) and 2(f)). Synchrotron SAXS was applied to investigate the self-assembly of GAAVILRR in solution as a function of peptide concentration. The individual scattering curves show concentration-dependent characteristics, and can broadly be divided into three groups (Fig. 2): low-concentration regime, intermediate-concentration regime, and high-concentration regime. 2 Results and discussion The newly designed amphiphilic peptide, GAAVILRR, is composed of eight amino acid residues. The hydro- philic head consists of two bulky, positively charged arginine residues, whereas the hydrophobic tail is composed of aliphatic amino acids with consecuti- vely increasing hydrophobicity. The N-terminus is acetylated, keeping it uncharged. The C-terminus is amidated, resulting in an overall charge distribution of two positive net charges. The counterion is trifluoroa- cetate. Figure 1 illustrates the structure of a monomer. Lyophilized samples of varying concentrations were dissolved in water, resulting in optically clear, homo- geneous, colorless solutions. These samples in highest concentration (75 mM) exhibited a gel-like appearance. In order to obtain fully developed structures, the samples were aged for 1 week. The molecule’s amphi- philic nature endows a strong self-assembling ability in aqueous solutions above its critical aggregation concentration (CAC). For GAAVILRR, the CAC was estimated by fluorescence depolarization [23] to be about 30 mM in water (see Fig. S1 in Electronic Supplementary Material (ESM)). A variety of supramolecular peptide-based architec- tures have been reported so far, including vesicles [8–10], spherical micelles [11–13], bilayered tapes [14, 15], donuts [16], helical and twisted ribbons [11, 12, 17, 18], fibers [12–14, 18, 19], rods [14, 19, 20], and tubes [8–11, 17, 21, 22]. Here, we report a new structural motif that can be added to the list of assem- blies; for the first time a double-helix-like morphology was observed. Only a few studies have investigated the eluci- dation of supramolecular peptide structures with scattering techniques [11, 17–19, 21], and none of the reported peptides have shown the propensity to form double helices. We used small angle X-ray scattering (SAXS), along with transmission electron microscopy (TEM), to investigate the different morphologies of nanostructures derived from varying concentrations of the amphiphilic designer peptide GAAVILRR. It is important to emphasize that these techniques operate www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research Nano Res. 2015, 8(6): 1822–1833 1824 extrapolated intensity at zero angle (I0) is directly related to the increasing peptide concentration, and is also visible in the constant value when I0 is normalized to the concentration. Based on these characteristics we conclude that the transition from a dilute, noninteracting colloidal system (10 mM) to a more concentrated system (45 mM) occurs without supramolecular structure formation. \| www.editorialmanager.com/nare/default.asp 2.3 Model development to fit the scattering curve of the highest peptide concentration Increasing the peptide concentration to 75 mM results in a highly viscous solution, typical of hydrogels that are frequently reported in the context of amphiphilic peptides [12, 27]. Its scattering pattern, as shown in Fig. 2(e), exhibits four distinct features: a broad shoulder at q ≈ 0.1–0.35 nm–1, and three side maxima with consecutively decreasing intensities at q = 0.5, 1.18 and 2.4 nm–1. In a first approach and for com- pleteness, we tested various torus-like models to fit \| www.editorialmanager.com/nare/default.asp 1825 Nano Res. 2015, 8(6): 1822–1833 gure 2 Concentration-dependent self-assembly of GAAVILRR. Scattering curves of GAAVILRR at different conce week of aging are shown with their respective fits. Schematic illustrations of the assembled structures and the yo-TEM images. (a) Low-concentration regime (10–45 mM). The fitting function that was used combines a hard-sphe theoretical form factor of the peptide monomer; the fits are represented by the green curves. (b) Concentration-depe ed hard-sphere parameters, showing that the structures do not change over the investigated concentration regime. Th dius (Rs) and volume fraction ( remain constant, whereas the extrapolated intensity at zero angle (I0) increases ncentration. Normalizing I0 to the concentration results in a constant value. (c) Intermediate-concentration regime veral models obtained equally good fits (see Table S5 and Fig. S6 in ESM). This is in agreement with the observatio ermediate structures using Cryo-TEM. (d) Selection of possible intermediates found at 60 mM. Both the single t rallel-aligned double tapes (d2) are found. Double-helix-like structures are the dominant species (d3), and are chara riodicity between the intersection points. The double helices are several hundreds of nm in length, and 15–20 nm in ermediates have a related (single) tape width. (e) Hydrogel-forming sample (75 mM). The scattering curve was fitted ix model combined with the theoretical form factor of the peptide monomer. (f) Cryo-TEM image of the double-helix- Figure 2 Concentration-dependent self-assembly of GAAVILRR. Scattering curves of GAAVILRR at different concentrations after 1 week of aging are shown with their respective fits. Schematic illustrations of the assembled structures and the corresponding Cryo-TEM images. (a) Low-concentration regime (10–45 mM). The fitting function that was used combines a hard-sphere model with the theoretical form factor of the peptide monomer; the fits are represented by the green curves. (b) Concentration-dependence of the fitted hard-sphere parameters, showing that the structures do not change over the investigated concentration regime. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 2.3 Model development to fit the scattering curve of the highest peptide concentration The amplitudes of the single layer lines given by the values of tape width and  discriminate between single and double helices. Equally good fits are achieved by using single and double helix models for the intermediate structures from 55 to 65 mM (for details, Table S5 and Fig. S6 in ESM). As stated above, this concentration regime represents an intermediate state of a dynamic structure evolution process, where the coexistence of different morphologies is seen. Both the single helical tapes and double-helix-like structures are present. Even the existence of twisted ribbons, often described as precursors in helix formation, cannot be excluded. This heterogeneity is represented by the imperfect fit of any single model to the SAXS data at intermediate concentrations. Ensemble analysis shows a distribution of approximately 65% single helices and 35% double helices (Fig. S7 and Table S6 in ESM). The situation is different for the highest peptide concentration; in this region, the population of structures is more homogenous. The assemblies can be best described as two individual ribbons that are tightly intertwined (i e not loosely connected) and homogeneity in their total widths, an interaction between the two strands seems likely. Therefore, we describe them as double-helix-like structures, and indeed, fitting with the developed 2-layered double helix model results in an excellent fit to the experi- mentally obtained scattering data. 2.3 Model development to fit the scattering curve of the highest peptide concentration The hard-sphere radius (Rs) and volume fraction ( remain constant, whereas the extrapolated intensity at zero angle (I0) increases linearly with concentration. Normalizing I0 to the concentration results in a constant value. (c) Intermediate-concentration regime (55–65 mM). Several models obtained equally good fits (see Table S5 and Fig. S6 in ESM). This is in agreement with the observation of different intermediate structures using Cryo-TEM. (d) Selection of possible intermediates found at 60 mM. Both the single tapes (d1) and parallel-aligned double tapes (d2) are found. Double-helix-like structures are the dominant species (d3), and are characterized by a periodicity between the intersection points. The double helices are several hundreds of nm in length, and 15–20 nm in width. All the intermediates have a related (single) tape width. (e) Hydrogel-forming sample (75 mM). The scattering curve was fitted with a double helix model combined with the theoretical form factor of the peptide monomer. (f) Cryo-TEM image of the double-helix-like structures at 75 mM. They are defined by using parameters identical to those of the assemblies in d3, except for a slightly shorter distance of ~80 nm between the intersection points. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1826 Nano Res. 2015, 8(6): 1822–1833 this curve, although TEM images suggested elongated structures. The rationale behind these tests was that Khoe et al. [16] investigated a peptide with a similar sequence, lacking just one alanine residue, which formed donut-like structures in solution. However, our scattering curve cannot be properly described by a torus, a torus with an elliptical cross section, shell- models thereof [28], or elongated stacked tori (for a description of the fitting functions and the respective fits, see Fig. S3 and Table S1 in ESM). Also, a twisted ribbon model [29, 30] can be excluded due to its imperfect fit (see Fig. S4 and Table S2 in ESM). Application of a helical fitting function greatly im- proves the fit (Fig. S5 and Table S3 in ESM). However, it is only after extending the helical model [30] to a layered double helix, based on the equations of Pringle et al. [31], that the fitting results can be obtained with a minimal chi-square value [32]. In our study, a double helix is defined as two single helices that fit into each other, and are displaced by angle . This formalism introduces a coupling of the different layer lines, which is typical of the helical diffraction pattern [33]. 2.4 SAXS-derived double helical model The X-ray derived parameters describing the novel 2-layered double helical model are illustrated in Fig. 3 (see also Table S4 in ESM). The double helix exhibits a pitch (P) of ~60 nm and a mean radius (R) of 6 nm, resulting in a helical twist angle of  ~ 60°. The tape height along the longitudinal axis (wh) is ~23 nm (Fig. 3(a)). The diameter of the double helix is ~16 nm (Fig. 3(b)), indicating that no change in the overall width of the tape has occurred upon hydrogel formation. Most importantly, the electron density profile () derived from the SAXS data allows for a sectioning of the tape into several layers (Fig. 3(c)). Accordingly, the peptide-containing region accounts for ~3.5 nm of one tape. The inner hydrophobic core has the highest electron density and spans approximately 1 nm (D1). In this innermost region, we expect antiparallel stacking of the peptide monomers due to intermolecular hydrogen bonding. Given a monomer length of ~3 nm, we assume a very tight packing and interdigitation of the two monomers, and we suggest that this organization determines the high stability of the supramolecular structures. Figure 3(d) shows one sterically permitted arrangement for the stacking of a peptide pair, and the respective hydrogen bond distribution. The hydrophobic core region is flanked by a region providing the space for the hydrophilic head groups. A decrease in the electron density, from 1 = 1.4 to 2 = 0.8 electrons/Å3, points to a less densely packed zone, which indicates a higher flexibility of the head groups. In addition, the electron density distribution map shows an area that is characterized by a diffuse scattering contribution (3). We attribute this region to a low fraction of twisted ribbons (below 5%) that coexist in the sample. They are characterized by the same tape width (12 nm) as those forming the double-helix-like structures. The structural morphology of the highest peptide con- centration was also approached by three different \| www.editorialmanager.com/nare/default.asp \| www.editorialmanager.com/nare/default.asp 1827 Nano Res. 2015, 8(6): 1822–1833 TEM techniques: freeze-fracture, negative-staining, and Cryo-TEM. All three techniques support our model of two individual tapes that intertwine to form the double-helix-like structures. The images, as well as a discussion on the strengths and limitations of either technique, can be found (Fig. S10 in the ESM). organization of the structures and to confirm our model of antiparallel organized monomers (as pro- posed in Fig. 2.4 SAXS-derived double helical model 3(d)), we applied circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. In principle, the obtained CD spectra demonstrate a transition from mainly random-coil (1–45 mM, Fig. 4(a)) to pre- dominantly -type structures (60 mM, Fig. 4(b)). The lowest peptide concentrations show a large negative p→p* transition at 198–202 nm, followed by a negative 2.5 Spectroscopic techniques to monitor the structure’s internal organization To obtain additional information about the internal Figure 3 Structural parameters of the double helical model. Values are based on the 75 mM sample fit presented in Fig. 2(e). (a) Side-view of the double helix composed of GAAVILRR molecules. Two individual strands are intertwisted to form a double-helix-like structure. The illustrated parameters are the pitch (P), helical twist angle (), tape thickness (w), tape height (wh), free space between one tape (h), and corresponding free space in the bilayer plane (d). (b) The cross section shows the mean radius (R) and angle (by which the two tapes are displaced with respect to each other. (c) Electron density distribution of the cross section, which can be divided into individual shells, with D1 and D2 being the shell distances with electron densities, 1 and 2. Electron density3 (gray mesh) reflects the contribution of a low fraction of twisted ribbons in the sample. The dashed line represents the symmetry line. In the innermost region (highlighted in green), we expect hydrogen bonding between antiparallel aligned peptide molecules. (d) The hypothesized arrangement of two peptide molecules, together with a proposed hydrogen bond distribution pattern depicted in the chemical formulas. Color code of the molecular model of GAAVILRR: green: carbon, blue: nitrogen, gray: oxygen, and white: hydrogen. Figure 3 Structural parameters of the double helical model. Values are based on the 75 mM sample fit presented in Fig. 2(e). (a) Side-view of the double helix composed of GAAVILRR molecules. Two individual strands are intertwisted to form a double-helix-like structure. The illustrated parameters are the pitch (P), helical twist angle (), tape thickness (w), tape height (wh), free space between one tape (h), and corresponding free space in the bilayer plane (d). (b) The cross section shows the mean radius (R) and angle (by which the two tapes are displaced with respect to each other. (c) Electron density distribution of the cross section, which can be divided into individual shells, with D1 and D2 being the shell distances with electron densities, 1 and 2. Electron density3 (gray mesh) reflects the contribution of a low fraction of twisted ribbons in the sample. The dashed line represents the symmetry line. In the innermost region (highlighted in green), we expect hydrogen bonding between antiparallel aligned peptide molecules. (d) The hypothesized arrangement of two peptide molecules, together with a proposed hydrogen bond distribution pattern depicted in the chemical formulas. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 2.5 Spectroscopic techniques to monitor the structure’s internal organization Color code of the molecular model of GAAVILRR: green: carbon, blue: nitrogen, gray: oxygen, and white: hydrogen. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research Nano Res. 2015, 8(6): 1822–1833 1828 Figure 4 Transition from random-coil (1–45 mM) to -ty structures (60–75 mM), measured by circular dichroism a infrared spectroscopy. (a) CD spectra of the low-concentrati samples recorded after 10 days of incubation. (b) CD spectra the 60 mM sample recorded at different time points over incubation time of 4 days. Dotted lines represent parts of t spectra where a threshold of 600 V in the high-tension (HT) sign was exceeded. (c) ATR-FTIR spectroscopy confirms the presen of antiparallel -sheets (peaks at 1,620 and 1,690 cm–1), the pe at 1,673 nm–1 is indicative of residual TFA. a rapid increase in the high-tension (HT) voltage, and therefore, these parts of the spectra (dotted lines) should be considered with caution. Nevertheless, all spectra of the low-concentration regime point to a random-coil structure. In the 60 mM sample, the -strand content significantly increases, and the spectroscopic characteristics develop towards a negative maximum at 230 nm (Fig. 4(b)). Although very few reports exist on how to interpret this large negative peak at 230 nm, there is a consensus that it is related to -type structures [18, 34, 35]. A red shift in the signal has been associated with a higher degree of twisting in -sheets than that of planar -sheets [27]. CD spectra with striking similarity to ours were reported in the literature [36, 37]; interestingly, all these spectra were related to double helices. We now hypothesize that a CD spectrum like ours— characterized by the absence of the typical positive peak at 190–200 nm, and a single large negative band around 230 nm—could be directly associated with the double helix morphology formed when -strands are present. ATR-FTIR measurements confirmed the presence of -sheet structures (Fig. 4(c)). The major amide I peak at 1,620 nm–1 is characteristic of -sheet interactions. The weak band at 1,690 nm–1 can be assigned to an antiparallel component, whereas the contribution at 1,673 nm–1 arises from residual trifluoroacetic acid (TFA) in the sample. In comparison, both the 60 mM and 75 mM samples show the same spectroscopic characteristics, as well as the typical scaling of the signal with concentration. Figure 4 Transition from random-coil (1–45 mM) to -type structures (60–75 mM), measured by circular dichroism and infrared spectroscopy. 2.5 Spectroscopic techniques to monitor the structure’s internal organization (a) CD spectra of the low-concentration samples recorded after 10 days of incubation. (b) CD spectra of the 60 mM sample recorded at different time points over an incubation time of 4 days. Dotted lines represent parts of the spectra where a threshold of 600 V in the high-tension (HT) signal was exceeded. (c) ATR-FTIR spectroscopy confirms the presence of antiparallel -sheets (peaks at 1,620 and 1,690 cm–1), the peak at 1,673 nm–1 is indicative of residual TFA. The results of CD and ATR-FTIR spectroscopy are consistent with our proposed model of tightly packed, interdigitated peptide molecules that are held together by hydrogen bonding due to antiparallel stacking. 2.6 Evolution of structure over time To determine whether the double-helix-like structures are intermediates that develop for the often- described tubular structures, or whether they are a final state of the assembly, we performed a series of SAXS and Cryo-TEM measurements at different time points over 11 weeks. The SAXS pattern of the 75 mM sample remains unchanged over the observed band with a maximum at 220 nm, which can be assigned to the n→p* transition. These characteristics are associated with a mainly random-coil structure. The large negative peak becomes less pronounced with increasing peptide concentration, and is shifted to higher wavelengths. This change is consistent with \| www.editorialmanager.com/nare/default.asp 1829 Nano Res. 2015, 8(6): 1822–1833 period (1, 4, and 11 weeks), revealing that the overall structure and the internal organization do not change (see Fig. S8 in the ESM). These results are further supported by Cryo-TEM micrographs, in which stable double-helix-like structures are observed at every time point (Fig. 5). Only 30 min after dissolution, a double- helix-like morphology develops and shows the final widths of ~15–20 nm, and a mean distance between periodic intersection points of ~80 nm (see white arrows in Fig. 5). These characteristics remain unaltered for at least 11 weeks. single amino acids in a given sequence are a matter of intense research [12, 13, 18, 27, 38, 39]. A deeper understanding of the role of every single amino acid opens up the opportunity for tunable structures. They are highly desirable for the multifaceted application fields of supramolecular self-assembled nano- structures. Whether the double helical arrangement of amphiphilic designer peptides has previously been overlooked due to experimental limitations, or is a unique novel feature related to peptide sequence remains unresolved. An interesting point that needs to be addressed is the strikingly different morphology of GAAVILRR compared to the structure of the peptide GAVILRR reported by Khoe et al. [16], which differs from our sequence in lacking just one alanine residue. While GAVILRR forms donut-like structures in solution, we have found double-helix-like assemblies for GAAVILRR. These results highlight the fact that minor differences in the amino acid composition can have a huge influence on the resulting structures. In fact, this feature is not unique, and systematic variations of the tail length or the substitution of www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 3 Conclusions We have shown that the structure of supramolecular peptide assemblies in solution strongly depends on the peptide concentration. Below the CAC, the peptides are dispersed as monomers and do not display superstructural features. According to Cenker et al. [40], as soon as lyophilized peptides are dispersed in water above their CAC, one part of the molecules is present in the form of aggregates, whereas the other part (equal to the concentration of the CAC) is in its molecularly dissolved state. In a time-dependent crystal growth process, the aggregates grow by uniaxial extension through the assembly of monomers. In a dissolution-reassembly mechanism, structures with a high level of perfection are grown within minutes to a few hours. At this stage, the structures already exhibit a definite cross section with their equilibrium diameters [40]. We observed bilayered tapes, in which we expect that hydrophilic arginine residues exposed to the solvent and the hydrophobic tail residues interdigitated and tightly packed in the interior were stabilized by intermolecular hydrogen bonding. Because of the peptides’ intrinsic chirality, the monomers do not assemble in a strictly parallel manner, but slightly tilt with each other, leading to twisted structures. In addition, a nonplanar alignment of the monomers leads to bending. This accounts for a twisted-to-helical transition on the one hand, and on the other hand limits the lateral growth, thus favoring uniaxial elongation [29, 41, 42]. Hence, double-helix-like structures, with several hundred nanometers to micrometers in length but only around 16 nm in width, can develop. Because of the high Figure 5 Cryo-TEM of 75 mM GAAVILRR at different time points, (a) freshly dissolved, (b) 1 week, (c) 4 weeks, and (d) 11 weeks, show no alteration in the global morphology. The peptide forms double helices at every time point, with similar structural parameters: width ~15–20 nm and intersection points every ~80 nm (see white arrows). Figure 5 Cryo-TEM of 75 mM GAAVILRR at different time points, (a) freshly dissolved, (b) 1 week, (c) 4 weeks, and (d) 11 weeks, show no alteration in the global morphology. The peptide forms double helices at every time point, with similar structural parameters: width ~15–20 nm and intersection points every ~80 nm (see white arrows). 1830 Nano Res. 2015, 8(6): 1822–1833 in Table S7 and Fig. S8 in the ESM. Data analysis was performed using Fit2D [49] and Igor Pro (Version 6.22A, WaveMetrics Inc., USA). 3 Conclusions Experimental intensities were normalized to sample transmission and corrected for background. Absolute calibration was performed using water as the secondary standard, using the method described by Orthaber [50]. regularity of pitch distances, the orientation of the two individual strands seems to be the consequence of a sensitive interplay between geometrical constraints, intertape hydrogen bonding interactions, hydrophobic affinity of the tail residues, and electrostatic repulsion of the positively charged arginine residues. Finally, the double-helix-like superstructures assemble into a network displaying gel-like features. As these intertwined assemblies are stable for several months, this architecture seems energetically and sterically highly favorable. Keeping in mind the manifold app- lications for supramolecular self-assembled structures [4, 5, 43–47], a double-helix-like tape might be the building block for the next-generation bioinspired nanomaterials. 4.2 Small angle X-Ray scattering (SAXS) Synchrotron X-ray scattering data was collected at the Austrian SAXS beamline at ELETTRA (Trieste, Italy) [48]. The measurements were carried out at a wavelength of 0.154 nm and a sample-detector distance of 1.1 m. The photon energy was 8 keV. The data were recorded with a Pilatus detector (PILATUS 100 K, DECTRIS Ltd., Villigen PSI, Switzerland), cali- brated with silver behenate. The scattering intensity was measured as a function of the scattering vector (q), 4.3 Formal description of a helix and a double helix: fitting parameters The mean radius (R) is the distance between the helix center to the center of the innermost shell. The cross section of our model comprises two to three layers with their respective distances (D1–D3) and electron densities (1–3). Parameter solvent is the electron density of the solvent, which was maintained constant at 0.335 e/Å3. The mean radius and pitch (P) provide the helical twist angle , where  = arctan[P/(2R)]. It describes the angle of the helix with respect to the equatorial plane. The rotation angle ( reflects the degree of opening of the helix. The thickness of the tape (w) is defined as w = D3sin, the tape height (wh) is defined as wh = w/cos, and the free space between the tapes (h) is defined as h = P – wh. The free distance (d) between the tapes in the bilayer plane is defined as d = (wh)/wh [30, 51]. 4.1 Peptide preparation Ac-GAAVILRR-NH2 was custom synthesized and purified by piCHEM GmbH (Graz, Austria) following the general procedure for Fmoc solid-phase peptide synthesis. The samples were prepared by dissolving the lyophilized peptide in double distilled water to obtain the desired concentrations. The samples were incubated at 4 °C under an argon atmosphere for 1 week prior to SAXS and TEM measurements. A double helix results from two single helical tapes (with the same properties as described above) that are displaced with respect to each other by angle . The pitch of a single helix should be greater than the tape height (wh) in order to leave room for the second tape. 4.4 Cryogenic transmission electron microscopy (Cryo-TEM) The samples were adsorbed onto a glow-discharged carbon-coated copper grid, blotted to create a thin film, plunged into liquid ethane, and transferred to liquid nitrogen. Vitrified specimens were transferred onto a Gatan 626-DH cryo transfer specimen holder and imaged at cryogenic temperatures in a Tecnai T12 (FEI, The Netherlands) microscope, operated at 120 kV. q = 4 (sin)/, where 2 is the scattering angle and  is the wavelength. The samples were measured either in a 1.5 mm glass capillary (10–60 mM) or in a gel sample holder (75 mM). A discussion on the influence of shearing forces when filling the capillary can be found \| www.editorialmanager.com/nare/default.asp 4.7 Artwork Illustrations of the assembled structures were created with Blender (Version 2.66, Blender Foundation) [53] and Adobe Photoshop CS5 Extended (Version 12.0.3 × 32, Adobe Systems Inc., USA). Chemical formulas were drawn with ChemDraw (Version 6.0.1, Perkin Elmer Informatics, USA). [5] Ellis-Behnke, R. G.; Liang, Y.-X.; You, S.-W.; Tay, D. K.; Zhang, S. G.; So, K.-F.; Schneider, G. E. Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5054–5059. [6] Ellis-Behnke, R. G.; Schneider, G. E. Peptide amphiphiles and porous biodegradable scaffolds for tissue regeneration in the brain and spinal cord. Methods Mol. Biol. 2011, 726, 259–281. 4.5 Circular dichroism (CD) spectroscopy Measurements were conducted on a JASCO J-715 circular dichroism spectrometer (Jasco Inc., Easton, \| www.editorialmanager.com/nare/default.asp 1831 Nano Res. 2015, 8(6): 1822–1833 MD) using a 0.1 mm path length cell. The spectra were recorded between 185 and 260 nm in the interval scan mode with 0.2 nm resolution and an average time of 1 s. The scan speed was 50 nm/min. Spectra of the low-concentration samples (10–45 mM) were recorded after 10 days of incubation. The 60 mM sample was filled into the capillary directly after dissolution, and its structure evolution was monitored over 4 days. Because of the high viscosity of the 75 mM sample, it was not possible to fill the sample into a measurement cell. The final spectra were corrected for background by subtracting the corresponding buffer spectra obtained under identical conditions. Data processing and analysis were performed with CDtool [52] and Igor Pro (Version 6.22A, WaveMetrics Inc., USA). The threshold of the high-tension (HT) voltage signal was set to 600 V. Parts of the CD spectra exceeding this threshold are marked by dotted lines. Electronic Supplementary Material: Supplementary material (experimental details; determination of the CAC; SAXS; fitting functions: hard-sphere model, theoretical form factor of a peptide monomer, torus, torus with elliptical cross section, core-shell model of a torus and a torus with elliptical cross section, stacked models thereof, helix, double helix; fits and derived parameters of the torus-, twisted ribbon- and helix- models; ensemble analysis for coexisting structures, time evolution of the 75 mM sample. The influence of shearing forces on the resulting SAXS pattern when filling the capillary. Comparison of freeze-fracture, negative-staining and Cryo-TEM techniques for the direct visualization of superstructures) is available in the online version of this article at http://dx.doi.org/ 10.1007/s12274-014-0683-9. Electronic Supplementary Material: Supplementary material (experimental details; determination of the CAC; SAXS; fitting functions: hard-sphere model, theoretical form factor of a peptide monomer, torus, torus with elliptical cross section, core-shell model of a torus and a torus with elliptical cross section, stacked models thereof, helix, double helix; fits and derived parameters of the torus-, twisted ribbon- and helix- models; ensemble analysis for coexisting structures, time evolution of the 75 mM sample. The influence of shearing forces on the resulting SAXS pattern when filling the capillary. Comparison of freeze-fracture, negative-staining and Cryo-TEM techniques for the direct visualization of superstructures) is available in the online version of this article at http://dx.doi.org/ 10.1007/s12274-014-0683-9. 4.6 Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy [1] Luo, Z. L.; Zhang, S. G. Designer nanomaterials using chiral self-assembling peptide systems and their emerging benefit for society. Chem. Soc. Rev. 2012, 41, 4736–4754. Infrared spectra were recorded between 4,000 and 600 cm–1 on a Bruker TENSOR 37 FTIR spectrometer, equipped with an ATR accessory (Bruker Optics Inc., Billerica, USA). The spectra were the averages of 128 scans with a resolution of 4 cm–1. Data analysis was performed with the OPUS software (Version 6.5, Bruker Optics Inc., Billerica, USA). [2] Zelzer, M.; Ulijn, R. V. Next-generation peptide nanomaterials: Molecular networks, interfaces and supramolecular func- tionality. Chem. Soc. Rev. 2010, 39, 3351–3357. [3] Aida, T.; Meijer, E. W.; Stupp, S. I. Functional supramolecular polymers. Science 2012, 335, 813–817. [4] Zhao, X. B.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H. H.; Hauser, C. A.; Zhang, S. G.; Lu, J. R. Molecular self-assembly and applications of designer peptide amphiphiles. Chem. Soc. Rev. 2010, 39, 3480–3498. Acknowledgements [7] Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001, 294, 1684–1688. This work has been supported by the Austrian Science Fund (FWF Project No. I 1109-N28 to R. P.). We thank Elisabeth Bock and Gertrud Havlicek for technical assistance with electron microscopy. We gratefully acknowledge Rolf Breinbauer for providing access to infrared spectroscopy instrumentation. [8] Vauthey, S.; Santoso, S.; Gong, H. Y.; Watson, N.; Zhang, S. G. Molecular self-assembly of surfactant-like peptides to form nanotubes and nanovesicles. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5355–5360. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research 1832 Nano Res. 2015, 8(6): 1822–1833 [9] Santoso, S.; Hwang, W.; Hartman, H.; Zhang, S. G. Self- assembly of surfactant-like peptides with variable glycine tails to form nanotubes and nanovesicles. Nano Lett. 2002, 2, 687–691. 7645–7647. [21] Bucak, S.; Cenker, C.; Nasir, I.; Olsson, U.; Zackrisson, M. Peptide nanotube nematic phase. Langmuir 2009, 25, 4262– 4265. [22] Wang, S. J.; Ge, X.; Xue, J. Y.; Fan, H. M.; Mu, L. J.; Li, Y. P.; Xu, H.; Lu, J. R. Mechanistic processes underlying biomimetic synthesis of silica nanotubes from self-assembled ultrashort peptide templates. Chem. Mater. 2011, 23, 2466– 2474. [10] von Maltzahn, G.; Vauthey, S.; Santoso, S.; Zhang, S. G. Positively charged surfactant-like peptides self-assemble into nanostructures. Langmuir 2003, 19, 4332–4337. [11] Xu, H.; Wang, Y. M.; Ge, X.; Han, S. Y.; Wang, S. J.; Zhou, P.; Shan, H. H.; Zhao, X. B.; Lu, J. R. Twisted nanotubes formed from ultrashort amphiphilic peptide I3K and their templating for the fabrication of silica nanotubes. Chem. Mater. 2010, 22, 5165–5173. [23] Yang, S. J.; Zhang, S. G. Self-assembling behavior of designer lipid-like peptides. Supramol. Chem. 2006, 18, 389–396. [24] Pontoni, D.; Finet, S.; Narayanan, T.; Rennie, A. R. Interactions and kinetic arrest in an adhesive hard-sphere colloidal system. J. Chem. Phys. 2003, 119, 6157–6165. [12] Lakshmanan, A.; Hauser, C. A. E. Ultrasmall peptides self-assemble into diverse nanostructures: Morphological evaluation and potential implications. Int. J. Mol. Sci. 2011, 12, 5736–5746. [25] Schneidman-Duhovny, D.; Hammel, M.; Tainer, J. A.; Sali, A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys. J. 2013, 105, 962–974. [13] Han, S. Y.; Cao, S. S.; Wang, Y. M.; Wang, J. Q.; Xia, D. H.; Xu, H.; Zhao, X. B.; Lu, J. R. Self-assembly of short peptide amphiphiles: The cooperative effect of hydrophobic interaction and hydrogen bonding. Chem.-Eur. Acknowledgements J. 2011, 17, 13095–13102. [26] Schneidman-Duhovny, D.; Hammel, M.; Sali, A. FoXS: A web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 2010, 38, W540–W544. [14] Xu, H.; Wang, J.; Han, S. Y.; Wang, J. Q.; Yu, D. Y.; Zhang, H. Y.; Xia, D. H.; Zhao, X. B.; Waigh, T. A.; Lu, J. R. Hydrophobic-region-induced transitions in self-assembled peptide nanostructures. Langmuir 2009, 25, 4115–4123. [27] Pashuck, E. T.; Cui, H. G.; Stupp, S. I. Tuning supramole- cular rigidity of peptide fibers through molecular structure. J. Am. Chem. Soc. 2010, 132, 6041–6046. [28] Kawaguchi, T. Radii of gyration and scattering functions of a torus and its derivatives. J. Appl. Crystallogr. 2001, 34, 580–584. [15] Pan, F.; Zhao, X. B.; Perumal, S.; Waigh, T. A.; Lu, J. R.; Webster, J. R. P. Interfacial dynamic adsorption and structure of molecular layers of peptide surfactants. Langmuir 2009, 26, 5690–5696. [29] Selinger, J. V.; Spector, M. S.; Schnur, J. M. Theory of self-assembled tubules and helical ribbons. J. Phys. Chem. B 2001, 105, 7157–7169. [16] Khoe, U.; Yang, Y. L.; Zhang, S. G. Self-assembly of nanodonut structure from a cone-shaped designer lipid-like peptide surfactant. Langmuir 2009, 25, 4111–4114. [30] Teixeira, C. V.; Amenitsch, H.; Fukushima, T.; Hill, J. P.; Jin, W.; Aida, T.; Hotokka, M.; Lindén, M. Form factor of an N-layered helical tape and its application to nanotube formation of hexa-peri-hexabenzocoronene-based molecules. J. Appl. Crystallogr. 2010, 43, 850–857. [17] Castelletto, V.; Nutt, D. R.; Hamley, I. W.; Bucak, S.; Cenker, C.; Olsson, U. Structure of single-wall peptide nanotubes: In situ flow aligning X-ray diffraction. Chem. Commun. 2010, 46, 6270–6272. [31] Pringle, O. A.; Schmidt, P. W. Small-angle X-ray scattering from helical macromolecules. J. Appl. Crystallogr. 1971, 4, 290–293. [18] Hauser, C. A.; Deng, R. S.; Mishra, A.; Loo, Y.; Khoe, U.; Zhuang, F. R.; Cheong, D. W.; Accardo, A.; Sullivan, M. B.; Riekel, C.; et al. Natural tri- to hexapeptides self-assemble in water to amyloid β-type fiber aggregates by unexpected α-helical intermediate structures. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1361–1366. [32] Pedersen, J. S. Analysis of small-angle scattering data from colloid and polymer solutions: Modeling and least-square fitting. Adv. Colloid Interface Sci. 1997, 70, 171–210. [33] Hamley, I. W. Form factor of helical ribbons. Macromolecules 2008, 41, 8948–8950. [19] Wang, J.; Han, S. Y.; Meng, G.; Xu, H.; Xia, D. H.; Zhao, X. Nano Res. 2015, 8(6): 1822–1833 [36] Hicks, M. R.; Damianoglou, A.; Rodger, A.; Dafforn, T. R. Folding and membrane insertion of the pore-forming peptide gramicidin occur as a concerted process. J. Mol. Biol. 2008, 383, 358–366. An amphiphilic pillar[5]arene: Synthesis, controllable self- assembly in water, and application in calcein release and TNT adsorption. J. Am. Chem. Soc. 2012, 134, 15712–15715. [46] Yao, Y.; Xue, M.; Zhang, Z. B.; Zhang, M. M.; Wang, Y.; Huang, F. H. Gold nanoparticles stabilized by an amphiphilic pillar[5]arene: Preparation, self-assembly into composite microtubes in water and application in green catalysis. Chem. Sci. 2013, 4, 3667–3672. [37] Chen, C.-L.; Zhang, P. J.; Rosi, N. L. A new peptide-based method for the design and synthesis of nanoparticle super- structures: Construction of highly ordered gold nanoparticle double helices. J. Am. Chem. Soc. 2008, 130, 13555–13557. [38] Paramonov, S. E.; Jun, H.-W.; Hartgerink, J. D. Self- assembly of peptide-amphiphile nanofibers: The roles of hydrogen bonding and amphiphilic packing. J. Am. Chem. Soc. 2006, 128, 7291–7298. [47] Yu, G. C.; Ma, Y. J.; Han, C. Y.; Yao, Y.; Tang, G. P.; Mao, Z. W.; Gao, C. Y.; Huang, F. H A sugar-functionalized amphiphilic pillar[5]arene: Synthesis, self-assembly in water, and application in bacterial cell agglutination. J. Am. Chem. Soc. 2013, 135, 10310–10313. [39] Dong, H.; Paramonov, S. E.; Aulisa, L.; Bakota, E. L.; Hartgerink, J. D. Self-assembly of multidomain peptides: Balancing molecular frustration controls conformation and nanostructure. J. Am. Chem. Soc. 2007, 129, 12468–12472. [48] Amenitsch, H.; Rappolt, M.; Kriechbaum, M.; Mio, H.; Laggner, P.; Bernstorff, S. First performance assessment of the small-angle X-ray scattering beamline at ELETTRA. J. Synchrotron Rad. 1998, 5, 506–508. [40] Cenker, Ç. Ç.; Bomans, P. H. H.; Friedrich, H.; Dedeoglu, B.; Aviyente, V.; Olsson, U.; Sommerdijk, N. A. J. M.; Bucak, S. Peptide nanotube formation: A crystal growth process. Soft Matter 2012, 8, 7463–7470. [49] European Synchrotron Radiation Facility, Fit2D. http://www.esrf.eu/computing/scientific/FIT2D/ (accessed August, 2014). [50] Orthaber, D.; Bergmann, A.; Glatter, O. SAXS experiments on absolute scale with Kratky systems using water as a secondary standard. J. Appl. Crystallogr. 2000, 33, 218–225. [41] Fishwick, C. W. G.; Beevers, A. J.; Carrick, L. M.; Whitehouse, C. D.; Aggeli, A.; Boden, N. Structures of helical -tapes and twisted ribbons: The role of side-chain interactions on twist and bend behavior. Nano Lett. 2003, 3, 1475–1479. [51] Chung, D. S.; Benedek, G. B.; Konikoff, F. M.; Donovan, J. M. Acknowledgements B.; Schweins, R.; Lu, J. R. Dynamic self-assembly of surfactant-like peptides A6K and A9K. Soft Matter 2009, 5, 3870–3878. [34] Rudra, J. S.; Tian, Y. F.; Jung, J. P.; Collier, J. H. A self- assembling peptide acting as an immune adjuvant. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 622–627. [20] Baumann, M. K.; Textor, M.; Reimhult, E. Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity. Langmuir 2008, 24, [35] Collier, J. H.; Messersmith, P. B. Enzymatic modification of self-assembled peptide structures with tissue transglutaminase. Bioconjugate Chem. 2003, 14, 748–755. \| www.editorialmanager.com/nare/default.asp Nano Res. 2015, 8(6): 1822–1833 1833 Nano Res. 2015, 8(6): 1822–1833 Elastic free energy of anisotropic helical ribbons as metastable intermediates in the crystallization of cholesterol. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 11341–11345. [42] Armon, S.; Aharoni, H.; Moshe, M.; Sharon, E. Shape selection in chiral ribbons: From seed pods to supramolecular assemblies. Soft Matter 2014, 10, 2733–2740. [43] Cui, H. G.; Webber, M. J.; Stupp, S. I. Self-assembly of peptide amphiphiles: From molecules to nanostructures to biomaterials. Peptide Sci. 2010, 94, 1–18. [52] Lees, J. G.; Smith, B. R.; Wien, F.; Miles, A. J.; Wallace, B. A. CDtool—An integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving. Anal. Biochem. 2004, 332, 285–289. [44] Hamley, I. W. Self-assembly of amphiphilic peptides. Soft Matter 2011, 7, 4122–4138. [53] Blender Foundation. http://www.blender.org/ (accessed August, 2014). [45] Yao, Y.; Xue, M.; Chen, J. Z.; Zhang, M. M.; Huang, F. H. www.theNanoResearch.com∣www.Springer.com/journal/12274 \| Nano Research
https://openalex.org/W4241282462	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0192795&type=printable	English	null	Correction: Exploring the Relationship between Experiential Avoidance, Coping Functions and the Recency and Frequency of Self-Harm	PloS one	2,018	cc-by	275	CORRECTION 1. Nielsen E, Sayal K, Townsend E (2016) Exploring the Relationship between Experiential Avoidance, Coping Functions and the Recency and Frequency of Self-Harm. PLoS ONE 11(7): e0159854. https:// doi.org/10.1371/journal.pone.0159854 PMID: 27442036 OPEN ACCESS Citation: Nielsen E, Sayal K, Townsend E (2018) Correction: Exploring the Relationship between Experiential Avoidance, Coping Functions and the Recency and Frequency of Self-Harm. PLoS ONE 13(2): e0192795. https://doi.org/10.1371/journal. pone.0192795 Correction: Exploring the Relationship between Experiential Avoidance, Coping Functions and the Recency and Frequency of Self-Harm Emma Nielsen, Kapil Sayal, Ellen Townsend There is an error in the first sentence of the fourth paragraph of the results section. The correct sentence is as follows: The relationships between experiential avoidance, coping functions and demographic/ contextual factors were explored (see Table 3). The last paragraph of the Limitations section is incorrect. The correct paragraph is as fol- lows: Notwithstanding these limitations, the study offers novel insight into the relationship between self-harm, experiential avoidance and coping function, expanding on the existing lit- erature to consider the recency and frequency of self-harm engagement. There are errors in the Author Contributions. The correct contributions are: Conceived and designed the experiments: EN KS ET. Performed the experiments: EN. Analyzed the data: EN KS ET. Wrote the paper: EN KS ET. Reference a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Published: February 14, 2018 Copyright: © 2018 Nielsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0192795 February 14, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0192795 February 14, 2018
https://openalex.org/W4378981283	https://commonplace.knowledgefutures.org/pub/y3psgahk/download/pdf	English	null	5 Tools for a Post-Truth World	Commonplace	2,023	cc-by	1,428	Ruby Thelot 5 Tools for a Post-Truth World Commonplace • Series 3.1: Tech, Tools, & Media Transcript Then she got into the lift, for the good reason that the door stood open; and was shot smoothly upwards. The very fabric of life now, she thought as she rose, is magic. In the eighteenth century, we knew how everything was done; but here I rise through the air; I listen to voices in America; I see men flying – but how it's done I can't even begin to wonder. So my belief in magic returns. ABSTRACT This is a video-essay broaching the topic of information resistance in our post-truth world. First, it addresses the vested interest technology conglomerates have in maintaining the mystification of technology. Arthur C. Clarke once said “Any sufficiently advanced technology is indistinguishable from magic.” At this current juncture, complaints about technology are often akin to cloud-shouting. We’ve become increasingly suspicious of magic but have no points of leverage against it. It may seem like that is a consequence of technological progress, but in the video-essay, I make the case that the mystification is embedded and desired by the conglomerates since it keeps the power of criticism in the hands of technologists. Second, it proposes a series of 5 tips, a mini-manifesto for dealing with technological “magic.” Visit the web version of this article to view interactive content. Virginia Woolf, Orlando: A Biography (1928) The disenchanted world has become, by its patented opaqueness, enchanted. However, it is not the enchantment of the primitive man, where one can hypothesize the probable causes of phenomena, where the natural world remains to be discovered, it is technological enchantment, less like magic and more like illusion. As Czech philosopher Villem Flusser, puts it, “The difference between ancient and modern magic can be stated as follows: Prehistoric magic is a ritualization of models known as ‘myths’; current magic is a ritualization of models known as ‘programs’.” “How it’s done I can’t even begin to wonder” “How it’s done I can’t even begin to wonder” How am I typing on this wireless keyboard? How am I typing on this wireless keyboard? How are my words being recorded? How can you hear me? How can you hear me? 2 Commonplace • Series 3.1: Tech, Tools, & Media 5 Tools for a Post-Truth World Commonplace • Series 3.1: Tech, Tools, & Media “How it’s done I can’t even begin to wonder” Arthur C. Clarke wrote, in his “Hazards of Prophecy: The Failure of Imagination” essay, that “Any sufficiently advanced technology is indistinguishable from magic.” The quote is somewhat misleading as it is not that technology itself inching closer magic, rather it is that technology is mystified by its makers in order to render it indistinguishable from magic. Its mystification is part of a techno-optimist agenda to sever it from the range of human influence. An illusion is composed of three parts: the pledge, the turn, and the prestige. In the first, the magician takes an ordinary object. In the first, the magician takes an ordinary object. In the first, the magician takes an ordinary object. In the second, the object does something extraordinary. In the second, the object does something extraordinary. In the third, the object returns to normal, but with a smell of magic still lingering on. Every society has its magician, its illusionist. The shaman who can read the stars. The sibyl connected to the gods. The oracle who can traverse to the netherworlds. This function in modern society is now occupied by the Techno-Evangelist. The individual capable, for their own gain, of transfiguring an ordinary object into a magical one through the power of “narrative”. They have the capacity to imbue desirable, quasi-super-natural characteristics to ordinary things. The technological object is intentionally shrouded in technical jargon to alienate its user from its inner workings. Early appliances were accessible to the modern individual. Homeowners could repair their own ovens or fix their fridges. But oligopolic conglomerates purposefully prevent access to our tool’s innards through proprietary screws and glued components. The Techno-Evangelist says “This will change everything”. The act of claiming engenders progress. The act of claiming engenders progress. Progress can be summoned through hyperstition, the summoning of futures through the techno-cultural discourse. Hyperstition is a positive feedback circuit including culture as a component. It can be defined as the experimental (techno-)science of self-fulfilling prophecies. Superstitions are merely false beliefs, but hyperstitions — by their very existence as ideas — function causally to bring about their own reality. 3 Commonplace • Series 3.1: Tech, Tools, & Media 5 Tools for a Post-Truth World Capitalist economics is extremely sensitive to hyperstition, where confidence acts as an effective tonic, and inversely. The (fictional) idea of Cyberspace contributed to the influx of investment that rapidly converted it into a technosocial reality. Nick Land, interview with Delphi Carstens (2009) This mystification enables technological progress to advance unchecked until it reaches a state of naturalization. This predicament we enter is akin to the one Mark Fisher describes when he speaks of Capitalist Realism. Similarly, through the invocation of magic, technology entrenches itself within society, governments, and infrastructure to the point where its removal becomes unthinkable. To borrow from philosopher Frederic Jameson, “It is easier to imagine the end of the world than the end of technological progress.” The technical language that shrouds technology acts as its armor. When enacting criticism towards it, one is accused of not possessing the proper technical background to understand it. Isn’t it fortuitous that with every technological advance the general public is told that this new thing is highly technical and complex? Move fast and break things What should we do now? What should we do now? Move fast and break things Speed is a feature not a bug, it is speed that allows technology to evade regulation and scrutiny. Speed allows it to entrench itself. Reality is slow to respond to the assaults of speed. Allegedly, only the Techno-Evangelist holds the required knowledge to critique the fast moving limbs of technology as it attaches itself onto the body-reality and changes its composition. We are never asked. We suffer the second-order consequences. We are the collateral damage of technological progress. As technological magic permeates our daily lives, they become re-enchanted. Rapidly, estranged from the real, Truth itself becomes under attack. 4 Commonplace • Series 3.1: Tech, Tools, & Media 5 Tools for a Post-Truth World We used to ask “Is this an ad?” but, in the age of Post-Truth, now we ask “Is this real?” We used to ask “Is this an ad?” but, in the age of Post-Truth, now we ask “Is this real? New technologies further the assault. The proliferation of computer-generated images is accelerating. The generative technologies are self-regenerating. Even as we run out of human-made images, models can now train on the creations of other models. Manifesto for a Post-Truth World 1. Demand clarity from techno-evangelists --- Object to marketing speak and statistical predictions. Ask what is this? What does it do now, today? Who can use it? Clarity is grounded in the present. Clarity is the antidote against hyperstition. 2. Become a skeptic --- The general philosophical stance to adopt towards techno-futuristic claims should be skepticism. No post unless of a mutual borne should be trusted. 2. Become a skeptic --- The general philosophical stance to adopt towards techno-futuristic claims should be skepticism. No post unless of a mutual borne should be trusted. 3. Exercise memory --- Memory is a radical act. Evanescent shapes cannot hold a people together. Memory is the safeguard against the perversions of History. We cannot claim to know if we do not remember. 4. Embrace the unmediated --- Now is the time to embrace experiences that resist mediation. A rolling meadow is more beautiful than an AI-generated image of the Victory of Samothrace. 5. Learn to love the unknown --- There still remains a myriad of mysteries, jeremiads of things ineffable. Refuse meters, metrics, even words. Some things are better left uncounted and unsaid. 5. Learn to love the unknown --- There still remains a myriad of mysteries, jeremiads of things ineffable. Refuse meters, metrics, even words. Some things are better left uncounted and unsaid. Ruby Thelot is a designer and researcher based in New York. He is the founder of the award-winning creative research and design studio 13101401 inc. His work focuses on the interactions between humans and artificial intelligence, the metaverse and the implications of being-on-line. He has given talks and shown works in Tallin, Berlin and Abuja, amongst other places.
W2802954628.txt	https://zenodo.org/records/1651985/files/article.pdf	de		Neozenkerina Mildbr. nov. gen. Eine neue Scrophulariaceen-Gattung aus Kamerun	Notizblatt des Königl. botanischen Gartens und Museums zu Berlin	1,921	public-domain	937	Neozenkerina Mildbr. nov. gen. Eine neue Scrophulariaceen-Gattung aus Kamerun Author(s): J. Mildbraed Source: Notizblatt des Königl. botanischen Gartens und Museums zu Berlin, Bd. 7, Nr. 70 (Mar. 1, 1921), pp. 491-493 Published by: Botanischer Garten und Botanisches Museum, Berlin-Dahlem Stable URL: http://www.jstor.org/stable/3994511 . Accessed: 14/06/2014 09:38 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. . Botanischer Garten und Botanisches Museum, Berlin-Dahlem is collaborating with JSTOR to digitize, preserve and extend access to Notizblatt des Königl. botanischen Gartens und Museums zu Berlin. http://www.jstor.org This content downloaded from 188.72.126.35 on Sat, 14 Jun 2014 09:38:46 AM All use subject to JSTOR Terms and Conditions IV. NeozenkerinaMildbr.nov. gen. aus Kamerun. Eineneue Scrophulariaceen-Gattung Von J. Mildbraed. Neozenkerina Mildbr. nov. gen. - Sepala omnia basi usque ad 1/4 totius longitud. calycis connata, 2 antica inter sese usque ad medium vel paulo altius connata ideoque labium inferum bilobum lobis acutis efformantia, sepalum posticum medianum quam antica paulo longius et latius, cum lateralibus posticis multo minoribus labium superum efformans. Corollae calycem subduplo superantis tubus inferior ca. 1/4 totius longitud. aequans cylindricus, intus parte superiore pilis longissimis albidis ornatus, tubus superior sensim campanulato-tubulosus, laciniae ca. 1/, longitud. corollae aequantes posticae suborbiculares late rotundatae laterales anticas late ovatas subaequilongas aestivatione tegentes, lacinia antica lateralibus similis. Stamina supra zonam pilosam inserta didynamia 2 antica paulo longiora, thecae inter sese paralleles et omnino separatae ramificationibus brevibus horizontaliter patentibus connectivi dibrachiatodilatati vel malleiformis insidentes apice tantum rima brevi aperientes; staminodium posticum minutum lineare apice dilatato-incrassatum. Ovarium ovato-oblongum in stylum longum filiformem stamina paulo superantem stigmate bilobo lobo postico abbreviato transverse bicornuto vel semilunato antico linguiformi coronatumbreviter attenuatum; ovula numerosa complanata placentis medio dissepimenti affixis ? manifeste 3-seriatim insidentia. Capsula tenuissime membranacea maturitate iam destructa seminibus numerosis subglobosis calycis fundo inclusis. - Herba repens, folia opposita, inflorescentiae terminales abbreviato-spicatae,flores bracteis et prophyllis magnis suffulti. Neozenkerina bicolor Mildbr. x. spec. - Herba repens caule 2-3 mm crasso pilis exsiccatione collapsis albidis villoso internodiis This content downloaded from 188.72.126.35 on Sat, 14 Jun 2014 09:38:46 AM All use subject to JSTOR Terms and Conditions 492 inforioribus 8-10 cm longis. Folia opposita longe petiolata petiolo 1,5-3 cm longo eodem indumento ut caulis vestito, lamina ovalis usque oblonga basi subacuta apice obtusa supra atro-olivacea glabra, subtus pallida et ad costam latam, nervos laterales utrinque 6-8 et ad venas laxe reticulatas pilis villosis instructa, 5-11, plerumque 8-9 cm longa, 3,5-5, plerumque 4 cm lata. Flores in spicam terminalem abbreviatam subcapituliformem dispositi bractea magna e basi cuneata ovata subacuta 15 mm longa et 8 mm lata et prophyllis 2 similibus lanceolatis 15 mm longis et 5-6 mm latis pilis longis parce ciliatis et hinc inde adspersis pallide viridibus herbaceis suffulti; sepala 2 antica prophyllis aequilonga usque ad medium vel paulo altius inter sese connata ideoque labium inferum bilobatum lobis acutis efformantia, sepalum posticum impar ovatum subacuminatum basin cuneatam versus cum sepalis posticis lateralibus multo minoribus fere linearibus 1 cm tota longitudine aequantibus usque ad 5 mm connatum, 18 mm longum et 7-8 mm latum. Corollae extus pilis capitatis brevibus paulo conspicuis instructae 25 mm tota longitud. aequantis pars inferior tubulosus ca. 6 mm longa apicem versus pilis longis albidis instructa, laciniae 2 posticae suborbiculares late rotundatae pallidae 4 mm longae, laciniae 3 anticae late ovatae obtusae brunneo-rubraeposticis subaequilongae. Stamina didynamia 2 anteriora paulo longiora filamentis ca. 8 mm longis, staminodium posticum minutum apice dilatato paulo emarginatum vix 1 mm longum. Ovarium e basi lata ovato-oblongum glabrum in stylum filiformem antheras paulo superantem breviter angustatum. Capsula tenuissime membranacea sub maturitate jam destructa, semina subglobosa ca. 0,75 mm diam. atrocastanea pilis brevibus albidis singulis adspersa. Siidkameruner Waldgebiet: Unweit Ebolowa am Wege nach Akom im Niederwuchs eines primiiren Waldstuickes. (Mit Blilten und reifen Samen Ende Dezember 1913 - MILDBRAED n. 7619.) Die neue Gattungerinnertim Wuchs, in den Bliitenstinden,Brakteen undVorblatternsehr an Zenkerina,unterscheidetsich aberdurchdenKelch, besondersdurch die Verwachsungder beiden vorderenZipfel, durch die AusbildungderAntherenundnamentlichdurchdie Beschaffenheit der Frucht. Die Kapselwandist so dunn und vergiinglich,daB ich die reifen Samenin allen untersuchtenBluten stets nur lose im Grundedes Kelchesfand, an demFetzen der zerstortenKapselwandklebten,gewohnlichwarenauch noch die Plazentenals 2 diinneLeisten vorhanden. Dabei ist das Materialwohl erhalten,so dal nicht etwa eine nachtraglicheVerrottungserscheinung in Frage kommt. Sehr eigentiimlichsind ferner die Antheren. Das Filament ist amEnde kurz zweiarmig-hammerformig verbreitertund auf diesenkurzen Asten (aber nicht an ihren auuerstenEnden)sitzen parallel nebeneinander, This content downloaded from 188.72.126.35 on Sat, 14 Jun 2014 09:38:46 AM All use subject to JSTOR Terms and Conditions - 493 aber mehr oder minder senkrecht zur Richtung des Staubfadens, also gegen die Mittelachse der Bliitenrohre gekehrt, v6llig voneinander getrennt die beiden sackartigen Theken, die sich an der Spitze auf den gegeneinander gekehrten Seiten mit einem kurzen RiB 5ffnen. Am meisten entspricht dieses Verhalten noch dem von Gratiola. Wenn bei Zenkerina Zweifel an der Zugehorigkeit zu den Scrophulariaceen oder Acanthaceen moglich sind, so kann Neozenkerina wegen der Bildung der Frucht und der Antheren kaum noch zu der letzten Familie gerechnet werden, wegen der Antherenbildung scheint mir eine Stellung bei den Gratioleacnoch am ungezwungensten, wenngleich ich eine niihere Verwandtschaftnicht anzugeben weiB; eine solche scheint mir aber zu Zenkerina zu bestehen. vielleicht sind beides sehr alte Gattungen, die Reste einer friuher deutlichen Verbindungsreihe zwischen Scrophulariaceen und Acanthaceen darstellen. Cystolithen fehlen bei beiden This content downloaded from 188.72.126.35 on Sat, 14 Jun 2014 09:38:46 AM All use subject to JSTOR Terms and Conditions
https://openalex.org/W2605665221	https://ijpds.org/article/download/158/141	English	null	The effect of cross-jurisdictional linked hospital and death data on estimating risk-adjusted grouped hospital standardised mortality ratios in Australia	International journal of population data science	2,017	cc-by	663	International Journal of Population Data Science (2017) 1:139 International Journal of Population Data Science (2017) 1:139 ∗Corresponding Author: Email Address: katrina.spilsbury@curtin.edu.au (K. Spilsbury) The eﬀect of cross-jurisdictional linked hospital and death data on estimating risk-adjusted grouped hospital standardised mortality ratios in Australia Spilsbury, Katrina1*, Ferrante, Anna2, Rosman, Diana3, Alan, Janine1, Boyd, James2, and Sem 1Centre for Population Health Research, Faculty of Health Sciences, Curtin University 2PHRN Centre for Data Linkage, Centre for Population Health Research, Curtin University 3Data Linkage, Department of Health WA, Perth, Western Australia Approach In Australia, administrative hospital and death data are collected by individual state governments. The newly established Centre for Data Linkage created a cross-jurisdictional linkage key that brought together hospital and death records belonging to indi- viduals across four Australian states over a ﬁve year period (1st July 2004 - 30th June 2009). Hospital inpatient records from public, psychiatric and private hospitals and private day surgery centres were provided by New South Wales, Western Australia and Queensland. South Australia provided public hospital inpa- tient records only. The linked data underwent extensive cleaning and standardisation to improve the validity of interstate compar- isons. Objectives this study. Death registrations from all four states were obtained from state-based registries of births, deaths and marriages. this study. Death registrations from all four states were obtained from state-based registries of births, deaths and marriages. The Population Health Research Network (PHRN) was estab- lished to increase data linkage capacity in Australia. A proof of concept study investigating cross border hospital use and hospi- tal mortality was undertaken to demonstrate the eﬀectiveness of increased data linkage capacity in supporting nationally signiﬁ- cant health research. The objective of this study was to evaluate whether cross-jurisdictional linkage of hospital and death records across Australian states could reﬁne estimation of Hospital Stan- dardised Mortality Ratios (HSMRs). Results Cross-jurisdictional linkage identiﬁed 11,116 cross-border hospi- tal transfers of which 170 resulted in a cross-border in-hospital death. An additional 496 cross-border deaths occurred within 30 day of hospital discharge. The inclusion of cross-jurisdictional person-level links to unlinked hospital records reduced the co- eﬃcient of variation amongst the grouped HSMRs from 0.19 to 0.15; the inclusion of 30 day deaths reduced the coeﬃ- cient of variation further to 0.11. There were minor changes in grouped HSMRs between cross-jurisdictional and within- jurisdictional linkages, although the impact of cross-jurisdictional linkage increased when restricted to geographic regions with high cross-border hospital use such as the New South Wales and Queensland border area. http://dx.doi.org/10.23889/ijpds.v1i1.158 August 2016 c⃝The Authors. Open Access under CC BY-NC-ND 4.0 (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en) Journal Website: www.ijpds.org Journal Website: www.ijpds.org http://dx.doi.org/10.23889/ijpds.v1i1.158 August 2016 c⃝The Authors. Open Access under CC BY-NC-ND 4.0 (https://creativecommons.org/licen Conclusion Cross-jurisdictional data linkage modiﬁed estimates of grouped HSMRs, particularly for hospitals groups that were likely to re- ceive a high proportion of cross-border users. The ﬁnal cohort comprised 7.7 million hospital patients. In- hospital deaths and deaths within 30 days of hospital discharge from the four state jurisdictions were used to estimate the SMR of hospital groups deﬁned by geography and type of hospital (grouped HSMR) under three record linkage scenarios; 1) cross- jurisdictional person-level linkage, 2) within-jurisdictional (state- based) person-level linkage and 3) unlinked records. All public and private hospitals in New South Wales, Queensland, Western Australia and public hospitals in South Australia were included in hospital deaths and deaths within 30 days of hospital discharge from the four state jurisdictions were used to estimate the SMR of hospital groups deﬁned by geography and type of hospital (grouped HSMR) under three record linkage scenarios; 1) cross- jurisdictional person-level linkage, 2) within-jurisdictional (state- based) person-level linkage and 3) unlinked records. All public and private hospitals in New South Wales, Queensland, Western Australia and public hospitals in South Australia were included in ∗Corresponding Author: Email Address: katrina.spilsbury@curtin.edu.au (K. Spilsbury) http://dx.doi.org/10.23889/ijpds.v1i1.158 August 2016 c⃝The Authors. Open Access under CC BY-NC-ND 4.0 (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en) MR ital oss- ate- blic ern d in (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en) http://dx.doi.org/10.23889/ijpds.v1i1.158 August 2016 c⃝The Authors. Open Access under CC BY-NC-ND 4.0 (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en)
https://openalex.org/W4313196702	https://www.epj-conferences.org/articles/epjconf/pdf/2022/10/epjconf_eosam2022_10021.pdf	English	null	Fabrication influences on a miniaturised stokes polarimeter consisting of stacked nano-optical wire grid polarizer and retarders	EPJ web of conferences	2,022	cc-by	1,716	1 Introduction purpose, the grating period must be sufficiently small, such that only the zeroth diffraction order is transmitted. Depending on the utilized material either artificial birefringence or di-attenuation can be observed. For an absorbing grating material an exponential damping occurs for TE polarized light (TE-transversal electric, electric field vector parallel to ridges) while TM-polarized light is mostly transmitted [7]. Hence, the transmittance of such structures strongly depend on the polarisation direction, so that these elements can be used as linear polarizers (LP). If the grating material is dielectric the effective refractive index depends on the polarisation direction, hence such structures can be used as quarter wave plates (QWP) [8]. Nowadays optics technology is an important driving force in many aspects of our everyday life. Highly developed optical systems are enabling self-driving cars [1,2], high- resolution images with smartphone cameras and advanced medical services [3,4]. To achieve that, modern fabrication technology strives to ever smaller and more demanding structures. This is only achievable by closed loop process control. While many solutions are existing for that purpose, often elaborate equipment is required. As an example, ellipsometry can be utilized to retrieve information about thickness and refractive index of a deposited thin film [5]. While the achievable results are excellent with very small uncertainties, the required devices are large and expensive. By more sophisticated devices, it is even possible to deduce detailed information about the geometry of nanostructures [6]. Unfortunately, the size of such tools reduces their application range drastically. We propose a miniaturized polarimeter consisting of pixelated and stacked polarizer and retarder structures on an image sensor. In order to measure the Stokes parameter of incident light a QWP and a LP are typically utilized. Classically, four (or six) subsequent measurements at different rotation angles of the linear polarizer, with and without the quarter wave plate, are performed to deduce the full set of stokes parameters[9]. In the here shown concept the LP and QWP are stacked in different layers of the optical element on a microscopic scale. Since the structures are fabricated by lithography, their direction can be locally changed (see Fig. 2). Thereby, multiple channels can be achieved on an image sensor. Combining the information of all channels, the stokes parameters can be evaluated. Fig. 1. Schematic grating structure for polarisation control. Direction for TE and TM polarized light is marked. Fig. 2. Fabrication influences on a miniaturised stokes polarimeter consisting of stacked nano-optical wire grid polarizer and retarders Thomas Siefke, Markus Walther, Carsten Stock and Uwe D. Zeitner Friedrich Schiller University Jena, Institute of Applied Physics, Albert-Einstein-Str. 15, 07745 Jena, Germany Abstract. The polarization properties of light can be fully controlled with nano-optical wire grid polarizers and artificial birefringent grating structures. We demonstrate an integrated polarimeter based on stacked layers of such elements. However, the optical performance of such elements is fundamentally limited and may be further altered by deviations arising from the fabrication processes. In this contribution we investigate the influences on the polarimetry performance for such a device. Corresponding author: thomas.siefke@uni-jena.de © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 266, 10021 (2022) EOSAM 2022 EPJ Web of Conferences 266, 10021 (2022) EOSAM 2022 https://doi.org/10.1051/epjconf/202226610021 Conclusion 2 Nano-optical wire grid polarizers and retarders allow the full control of the polarization properties of light. Furthermore, since these elements are fabricated by lithography, they can be arranged on an image sensor in a pixelated manner. Thereby, multiple polarization channels are achieved enabling the single-shot evaluation of the Stokes parameter of incident light. However, the optical performance of such elements is limited. In this contribution, we investigate the influence on the performance to miniaturised polarimetry. L. Ghazaryan, E.-B. Kley, A. Tünnermann, and A. Szeghalmi, "Nanoporous SiO 2 made by atomic layer deposition and atomic layer etching," Nanotechnology 27(25), 96270P (2015). y g, gy ( ), (2015). "DE102016100907A1 - Verfahren zur Herstellung eines reflexionsmindernden Schichtsystems und reflexionsminderndes Schichtsystem - Google Patents," https://patents.google.com/patent/DE102016100907A1/fr. 13. "DE102016100907A1 - Verfahren zur Herstellung eines reflexionsmindernden Schichtsystems und reflexionsminderndes Schichtsystem - Google Patents," reflexionsmindernden Schichtsystems und reflexionsminderndes Schichtsystem - Google Patents," https://patents.google.com/patent/DE102016100907A1/fr. T. Siefke, M. Heusinger, C. B. R. Hurtado, J. Dickmann, U. Zeitner, A. Tünnermann, and S. Kroker, "Line-edge roughness as a challenge for high-performance wire grid polarizers in the far ultraviolet and beyond," Opt. Express 26(15), 19534 (2018). 15. T. Siefke, D. Lehr, T. Weber, D. Voigt, E.-B. Kley, and A. Tünnermann, "Fabrication influences on deep-ultraviolet tungsten wire grid polarizers manufactured by double patterning," Opt. Lett. 39(22), (2014). 1 Introduction Schematic of an integrated Stokes polarimeter consisting of a (top to bottom) birefringent grating as QWP, wire grid polarizer as LP and an image sensor. Fig. 1. Schematic grating structure for polarisation control. Direction for TE and TM polarized light is marked. Fig. 2. Schematic of an integrated Stokes polarimeter consisting of a (top to bottom) birefringent grating as QWP, wire grid polarizer as LP and an image sensor. Zero-order grating structures (see Fig. 1) can be utilized to control the polarisation state of light. For this EPJ Web of Conferences 266, 10021 (2022) EOSAM 2022 https://doi.org/10.1051/epjconf/202226610021 In Fig. 3, an SEM image of a cross section of one pixel of such a stacked nano-optical device is shown. The lower grating layer is the QWP structure consisting of titanium dioxide [8,10]. The upper layer is the LP structure consisting of iridium [11]. The intermediate layer is fabricated by filling the underlying grating with a sacrificial polymer and over coating of porous silicon oxide [12,13]. The sacrificial layer is removed by oxygen plasma ashing, creating a void in between the grating ridges. Thereby a high refractive index contrast is achieved. The wire grid polarizer is capped by over coating with ion beam deposition under an oblique angle. Opt. Soc. Am. B 36(5), D97 (2019). ( ) ( ) A. Forbes, A. Dudley, K. Singh, and N. Tabebordbar, "Digital Stokes polarimetry and its application to structured light: tutorial," JOSA A, Vol. 37, Issue 11, pp. C33-C44 37(11), C33–C44 (2020). T. Siefke, S. Kroker, K. Pfeiffer, O. Puffky, K. Dietrich, D. Franta, I. Ohlídal, A. Szeghalmi, E. Kley, and A. Tünnermann, "Materials Pushing the Application Limits of Wire Grid Polarizers further into the Deep Ultraviolet Spectral Range," Adv. Opt. Mater. 4, 1780–1786 (2016). 11. T. Weber, T. Käsebier, A. Szeghalmi, M. Knez, E.-B. Kley, and A. Tünnermann, "Iridium wire grid polarizer fabricated using atomic layer deposition," Nanoscale Res. Lett. 6(1), 558 (2011). References 1. C. Häne, L. Heng, G. H. Lee, F. Fraundorfer, P. Furgale, T. Sattler, and M. Pollefeys, "3D visual perception for self- driving cars using a multi-camera system: Calibration, mapping, localization, and obstacle detection," Image Vis. Comput. 68, 14–27 (2017). p , ( ) 2. C. Badue, R. Guidolini, R. V. Carneiro, P. Azevedo, V. B. Cardoso, A. Forechi, L. Jesus, R. Berriel, T. M. Paixão, F. Mutz, L. de Paula Veronese, T. Oliveira-Santos, and A. F. De Souza, "Self-driving cars: A survey," Expert Syst. Appl. 165, 113816 (2021). ( ) 3. D. Qiao, F. Zulkernine, R. Masroor, R. Rasool, and N. Jaffar, "Measuring Heart Rate and Heart Rate Variability with Smartphone Camera," Proc. - IEEE Int. Conf. Mob. Data Manag. 2021-June, 248–249 (2021). K. Kaur, W. Chelangat, S. I. Druzhinin, N. W. Karuri, M. Müller, and H. Schönherr, "Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera," Biosens. 2021, Vol. 11, Page 25 11(1), 25 (2021). A. Gottwald, K. Wiese, T. Siefke, and M. Richter, "Validation of thin film TiO <inf>2</inf> optical constants by reflectometry and ellipsometry in the VUV spectral range," Meas. Sci. Technol. 30(4), (2019). Fig. 3. Cross sectional SEM image of a layered grating structure. The upper layer consists of Ir and serves as linear polarizer, the lower layer consists of titanium dioxide and serves as retarder. Fig. 3. Cross sectional SEM image of a layered grating structure. The upper layer consists of Ir and serves as linear polarizer, the lower layer consists of titanium dioxide and serves as retarder. ( ) ( ) T. Käseberg, J. DIckmann, T. Siefke, M. Wurm, S. Kroker, and B. Bodermann, "Mueller matrix ellipsometry for enhanced optical form metrology of sub-lambda structures," in Proceedings of SPIE The International Society for Optical T. Käseberg, J. DIckmann, T. Siefke, M. Wurm, S. Kroker, and B. Bodermann, "Mueller matrix ellipsometry for enhanced optical form metrology of sub-lambda structures," in Proceedings of SPIE - The International Society for Optical Engineering (2019), 11057. in Proceedings of SPIE - The International Society for Optical Engineering (2019), 11057. Typically the optical performance achievable with such nano-optical elements is limited compared to their classical counterparts [14,15]. That means the phase of the QWP may deviate from the ideal value and the extinction ratio (ratio between transmittance for TE and TM polarized light) of the LP is limited to values <1000 [16]. In this contribution we investigate the influence of these deviations on the optical performance of such a Stokes polarimetry by rigorous coupled wave analysis, Monte Carlo simulations and ray tracing. Furthermore, we propose advanced designs exploiting the unique possibilities available due to the lithographic fabrication techniques. T. Siefke, E.-B. Kley, A. Tünnermann, and S. Kroker, "Design and fabrication of titanium dioxide wire grid polarizer for the far ultraviolet spectral range," in Proceedings of SPIE - The International Society for Optical Engineering (2016), 9927. T. Siefke, E.-B. Kley, A. Tünnermann, and S. Kroker, "Design and fabrication of titanium dioxide wire grid polarizer for the far ultraviolet spectral range," in Proceedings of SPIE - The International Society for Optical Engineering (2016), 9927. C. Stock, T. Siefke, and U. Zeitner, "Metasurface-based patterned wave plates for VIS applications," J. Opt. Soc. Am. B 36(5), D97 (2019). y f p g g ( ), C. Stock, T. Siefke, and U. Zeitner, "Metasurface-based patterned wave plates for VIS applications," J. Acknowledgement 3 This work is funded through the project 20FUN02 POlight within the Programme EMPIR. The EMPIR initiative is co-founded by the European Unions Horizon 2020 research and innovation program and the EMPIR Participating Countries. T. Siefke and S. Kroker, "Polarization Control by Deep Ultra Violet Wire Grid Polarizers," in Optical Chaacterization of Thin Solid Films, O. Stenzel and M. Ohlidal, eds. (Springer, 2018), pp. 359–374. 2 2
https://openalex.org/W3190015723	https://journal.ibrahimy.ac.id/index.php/assidanah/article/download/1216/959	Indonesian	null	PENDAMPINGAN PEMUDA DESA SUMBEREJO DALAM PENGUATAN MENTAL SPIRITUAL MELALUI MAJELIS DZIKIR BASMALAH	As-Sidanah	2,021	cc-by-sa	5,940	As-sidanah As-sidanah V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 13 Jurnal Pengabdian Masyarakat da’wah of Islam rahmatan lil ‘alamin. Keyword: Mentoring, strengthening mental-spirituality, Majelis Dzikir Basmalah PENDAMPINGAN PEMUDA DESA SUMBEREJO DALAM PENGUATAN MENTAL SPIRITUAL MELALUI MAJELIS DZIKIR BASMALAH Ainun Najib Universitas Ibrahimy Situbondo, Indonesia adjie245@gmail.com Abstract: The role of pesantren colleges has a big responsibility in fostering and developing the potential of the surrounding community, especially for the younger generation who become assets to continue struggles in the future. The threat of drying up spiritual values among the younger generation is getting away from behavior that is in accordance with the expected morality. So the youth mentoring program in strengthening mental-spirituality is one of the efforts to care for geranerasi through participatory transfromative social change actions. Preventive efforts to equip adolescents with religious knowledge and mental and spiritual strengthening, not only have a positive impact personally, but will also benefit the community around them. Together with stakeholders who have a significant role, this program emphasizes the assistance and empowerment of the community in understanding and resolving their own social problems, especially in the religious field. The continuity of the program which is carried out simultaneously will continue to be directed at developing the potential of future generations, while still integrating the roles and functions of the youth of the village of Sumberejo, which play an active role in spreading the Ainun Najib \| 13 Jurnal Pengabdian Masyarakat Ainun Najib \| 13 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah As-sidanah da’wah of Islam rahmatan lil ‘alamin. da’wah of Islam rahmatan lil ‘alamin. Isu dan Fokus Pengabdian Dunia remaja merupakan masa-masa yang sangat menentukan dalam memberi banyak pengalaman hidup. Masa remaja adalah masa- masa labil bagi seseorang yang tumbuh dewasa untuk mencari jati dirinya. Mereka ingin mengetahui yang terbaik untuk masa depannya, namun tidak menutup kemungkinan pula justeru terjerumus dalam pergaulan yang dapat menghncurkan masa depan mereka. Gaya hidup yang berkaitan dengan perkembangan zaman dan teknologi, berpengaruh terhadap perkembangan kelangsungan hidup kaum remaja. Mereka menjadi sasaran ancaman peredaran narkoba, minum- minuman keras, pergaulan bebas yang semakin hari semakin menghawatirkan. Keringnya nilai-nilai spiritualitas bagi para remaja menjadi salah satu faktor yang memudahkan mereka terjerumus dalam pergaulan yang tidak baik. Sehingga membawa dampak pada perilaku atau tindakan amoral dan asusila yang semakin meresahkan masyarakat. Maraknya penyimpangan perilaku generasi muda tersebut, dapat ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah membahayakan keberlangsungan hidup bangsa ini di kemudian hari. Karena pemuda sebagai generasi yang diharapkan menjadi penerus bangsa, semakin hari semakin rapuh. Oleh karena itu, menyelmatkan generasi muda sama halnya dengan menyelamatkan bangsa di masa yang akan datang dari ancaman dekadensi moral penerus perjuangannnya. Tidak terkecuali dengan masyarakat di Desa Sumberejo Kecamatan Bnayuputih Situbondo. Salah satu upaya yang mungkin dilakukan adalah upaya prefentif dengan membekali para remaja dengan pengetahuan keagamaan serta penguatan mental spiritual, tidak hanya memberikan dampak positif secara pribadi, melainkan juga akan memberikan maslahat kepada masyarakat di sekitarnya. Fakta tersebut menjadi tantangan serius bagi Universitas Ibrahimy untuk mengkaji dan mengembangkan pola pengabdian masyarakat agar dapat menghasilkan sesuatu yang benar-benar bermanfaat dan dirasa nyata oleh masyarakat. Upaya yang dikembangkan adalah bentuk pengabdian yang diarahkan kepada pendampingan dan pemberdayaan masyarakat dalam memahami dan menyeselesaikan problem sosialnya, khususnya dibidang keagamaan. Untuk mewujudkan ide tersebut, dosen melakukan pengabdian pada masyarakat dengan memperkuat pendampingan di tengah-tengah masyarakat khususnya wilayah yang V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah menjadi Desa binaan Universitas Ibrahimy. ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah menjadi Desa binaan Universitas Ibrahimy. menjadi Desa binaan Universitas Ibrahimy. Program ini adalah pendampingan masyarakat dalam penguatan pembangunan mental siritual. Hal yang menjadi fokus pendampingan adalah: Bagaimanakah pendampingan terhadap masyarakat pemuda Desa Sumberejo Melalui Majelis Dzikir Basmalah? Alasan Memilih Dampingan Penyusunan perencanaan program pengbadian kepada masyarakat adalah hal pertama yang dilakukan, yaitu dengan menyusun perencanaan yang akan dilaksanakan tim pengabdian selama selama proses pelaksanaan pendampingan. Penyusunan program didasarkan atas hasil observasi lapangan yang dilaksanakan bersama- sama untuk mengetahui kondisi dan situasi serta masyarakat dampingan. Sumberejo merupakan salah satu desa yang berada di kecamatan Banyuputih Situbondo Jawa Timur. Sebelum bernama Sumberejo, desa ini bernama Sumberjoyo. Desa Sumberejo terbagi ke dalam delapan dusun, yatiu Dusun Sukorejo Utara, Dusun Sukorejo Seatan, Dusun Krajan, Dusun Karangrejo, Dusun Sodung, Dusun Leduk, Dusun Lesong, dan Dusun Bendera. Rata-rata penduduknya bekerja sebagai petani, nelayan dan peternak. Selain itu, masyarakat desa Sumberejo juga banya yang menjadi pegawai swasta. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 16 Jurnal Pengabdian Masyarakat Ainun Najib \| 16 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Desa Sumberejo terdapat salah suatu Pondok Pesantren tertua dan terbesar di Indonesia, yaitu Pondok Pesantren Syalafiyah Syafi’iyah Sukorejo, yang merupakan salah satu pondok pesantren terbesar di dikabupaten Situbondo. Secara sosio historis Pondok Salafiyah Syafi`iyah Sukorejo ini awalnya berupa hutan belantara yang membentang dari gunung baluran sampai wilayah Asembagus. Hutan belantara itu dikenal sangat angker karena dihuni oleh binatang buas dan para dedemit karenanya tidak ada satupun masyarakat yang berani memasuki hutan tersebut. Pada tahun 1328 H/ 1908 M, Kiai Syamsul Arifin atas saran Habib Musawa dan Kiai Asadullah dari semarang, membabat hutan lebat tersebut untuk kemudian dijadikan sebuah pesantren. Dipilihnya hutan tersebut bukan serta merta karena kehendak K.H.R. As’ad akan tetapi hasil dari Istikhoroh. Pondok ini menempati area seluas 11,9 ha dengan ciri kekhasannya yaitu memadukan antara sistem salaf dan sistem modern. Upaya keras Kiai Syamsul Arifin akhirnya terwujud. Berdirilah sebuah pesantren kecil terdiri dari beberapa gubuk untuk difungsikan sebagai rumah, mushollah dan asrama santri yang waktu itu hanya ada beberapa orang. Pada masa perjuangan kemerdekaan, pesantren Sukorejo tidak hanya menjadi pusat belajar tapi juga sebagai pusat perjuangan kemerdekaan. Para pejuang banyak ditampung dipesantren, V o l . 3 N o . Alasan Memilih Dampingan 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah sekaligus sebagai markas penyusunan strategi melawan penjajah Sejak tahun 1914, pesantren kecil itu berkembang bersamaan dengan datangnya para santri dari wilayah sekitar keresidenan Besuki. Pada tahun itu pula, masyarakat pun mulai berdatangan untuk kemudian menetap didesa itu. Hutan yang telah dirambah itu berubah menjadi areal pertanian ladang dan kebun yang hasilnya mulai bisa dirasakan penduduk. Pergaulan penduduk dengan pesantren pun berlangsung harmonis Dalam upaya mewujudkan pendidikan modern sesuai dengan kebutuhan zaman, berbagai lembaga pendidikan kejuruan dan keahlian pun didirikan seperti sekolah menengah kejuruan (SMK), lembaga kader ahli fiqh, Ma’had aly dan Madrasatul Qur’an yang dijadikan sebagai lembaga kajian keislaman dan pendalaman ilmu- ilmu al- Qur’an. Sebagai lembaga pendidikan keagamaan yang memiliki karakteristik khas Nusantara, secara substansial institusi pondok pesantren tidak mungkin bisa dilepaskan dari masyarakat, khususnya masyarakat pedesaan. Pondok pesantren tumbuh dan berkembang dari, oleh dan untuk masyarakat, dengan posisinya sebagai institusi yang berperan melakukan transformasi sosial bagi masyarakat yang berada di lingkungannya. Demikian halnya dengan eksistensi Pondok Pesantren Salafiyah Syafi’iyah Sukorejo, dengan figur Kiai yang tidak hanya V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah sebagai figur tokoh agama, melainkan juga sekaligus sebagai tokoh masyarakat. ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah sebagai figur tokoh agama melainkan juga sekaligus sebagai tokoh sebagai figur tokoh agama, melainkan juga sekaligus sebagai tokoh masyarakat. Kondisi Dampingan Saat Ini Desa Sumberejo adalah salah satu desa yang menjadi bagian dari kecamatan Banyuputih, dengan luas wilayah mencapai 23.24 Km2 dengan tingkat kepadatan penduduk mencapai 980 jiwa per Km2.1 Sumberejo merupakan desa yang memiliki penduduk paling besar diantara desa-desa lainnya di kecamatan Banyuputih, yaitu berjumlah 22.766 Jiwa, dengan rincian 11.189 Laki-laki, dan 11.577 perempuan.2 Secara kuantitas penduduk desa Sumberejo 99,89% beragama Islam, namun secara kualitas keberagamaannya masih sangat beragam. Hal ini dibuktikan dengan perilaku sosial yang dapat mencerminkan nilai-nilai religiusitas masih relatif rendah. Khususnya di kalangan kaum muda yang masih cenderung ekspresif dalam setiap gaya hidup pergaulannya. Para pemuda ini butuh perhatian dan binaan untuk dapat diarahkan kepada hal-hal yang beroirntasi lebih positif. Maka sudah sepatutnya menyelamatkan pergaulan mereka melalui pendampingan yang dapat mengarahkan kepada hal yang lebih baik dan bermanfaat. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 19 Jurnal Pengabdian Masyarakat 1 Kecamatan Banyuputih dalam Angka 2019, (Badan Pusat Statistik Kabupaten Situbondo). hlm. 31 2 Ibid. hlm. 33 V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 19 Jurnal Pengabdian Masyarakat 1 Kecamatan Banyuputih dalam Angka 2019, (Badan Pusat Statistik Kabupaten Situbondo). hlm. 31 2 Ibid. hlm. 33 Ainun Najib \| 19 Jurnal Pengabdian Masyarakat ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Melalui program pengabdian kepada masyarakat yang diorientasikan kepada pemuda di desa Sumberejo, diharapkan akan menumbuhkan peningkatan tingkat keberagamaan mental piritual. Strategi yang Digunakan Guna mengantisipasi dan menanggulangi probem-problem praktis di kalangan pemuda terhadap perubahan tatanan sosial masyarakat, maka proses pelaksanaan program pengabdian masyarakat ini dilaksanakan melalui beberapa strategi dengan siklus yang diarahkan pada peningkatan pemahaman tentang proses perubahan tatanan sosial yang dijalankan oleh kerangka kerja etik melalui cara-cara partisipatoris. Maka model program pengabdian kepada masyarakat ini menggunakan Participatory Action Research (PAR) dalam bentuk penguatan masyarakat melalui kesadaran diri guna melakukan tindakan perubahan yang efektif menuju kondisi kehidupan sosial yang lebih baik.3 Metode Strategi yang Digunakan Kondisi Dampingan yang Diharapkan Secara umum kondisi dampingan yang diharapkan pada program pengabdian masyarakat ini adalah keberdayaan masyarakat dalam meningkatkan kualitas spiritualitas pemuda melalui majelis dzikir. Oleh karenanya, kondisi dampingan yang diharapkan dalam pengabdian ini adalah: a) Penguatan pemahaman akan pentingnya mematuhi hukum-hukum agama Islam, nilai-nilai badah, berakhlaq yang baik, serta kepekaan sosial terhadap masyarakat sekitar bagi para pemuda. Pemuda merupakan generasi emas yang perlu diselamatkan, sebagai generasi penerus perjuangan bangsa di masa yang akan datang. a) Penguatan pemahaman akan pentingnya mematuhi hukum-hukum agama Islam, nilai-nilai badah, berakhlaq yang baik, serta kepekaan sosial terhadap masyarakat sekitar bagi para pemuda. Pemuda merupakan generasi emas yang perlu diselamatkan, sebagai generasi penerus perjuangan bangsa di masa yang akan datang. b) Terbentuknya kelompok/simpul-simpul pemuda yang gemar untuk terus menigkatkan pengetahuan keagamaan, serta kegiatan-kegiatan sosial yang berorientasi pada kemaslahatan ummat, serta memiliki nilai-nilai ibadah. c) Terbentuknya masyarakat yang bermartabat dengan bekal pengetahuan agama yang kuat. Sehingga tercipta suatu bangsa yang baldatun thoyyibatun wa robbun ghafur. c) Terbentuknya masyarakat yang bermartabat dengan bekal pengetahuan agama yang kuat. Sehingga tercipta suatu bangsa yang baldatun thoyyibatun wa robbun ghafur. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 20 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 20 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Metode ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah 3 Abdullah Faisal, dkk., Metode & Teknik Kuliah Kerja Nyata Transformatif; Implementasi Participatory Action Research (PAR) & Participatory Rural Appraisal (PRA) untuk Aksi Perubahan Sosial, (P3M STAIN Surakarta, 2006), hlm. 69 V o l . 3 N o . 1 , A p r i l 2 0 2 1 Stakeholders yang Berperan stekeholders yang memilii peran signifikan dalam proses pelaksanaan program, yaitu Pemerintah Desa, Organisasi Sosial Keagamaan, sejumlah tokoh masyarakat, pondok pesantren Salafiyah Syafi’iyah Sukorejo, dan Universitas Ibrahimy. 3 Abdullah Faisal, dkk., Metode & Teknik Kuliah Kerja Nyata Transformatif; Implementasi Participatory Action Research (PAR) & Participatory Rural Appraisal (PRA) untuk Aksi Perubahan Sosial, (P3M STAIN Surakarta, 2006), hlm. 69 Ainun Najib \| 21 Jurnal Pengabdian Masyarakat ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah a. Unsur pemerintah desa, diharapkan membantu memfasilitasi para pemuda desa agar memiliki orientasi pada peningkatan nilai-nilai religiusitas, agar lebih menjamin terselenggaranya program pegabdian kepada masyarakat yang optimal sesuai dengan apa yang telah diproramkan dengan baik. b. Organisasi Sosial Keagamaan, seperti IPNU, GP. Ansor, MWC NU Kecamatan Banyuputih, juga diharapkan dapat mensinergikan beberapa program yang memiliki kesesuaian tema dengan program pengabdian masyarakat ini, khususnya dalam bidang keagamaan dan mental spiritual. b. Organisasi Sosial Keagamaan, seperti IPNU, GP. Ansor, MWC NU Kecamatan Banyuputih, juga diharapkan dapat mensinergikan beberapa program yang memiliki kesesuaian tema dengan program pengabdian masyarakat ini, khususnya dalam bidang keagamaan dan mental spiritual. c. Tokoh masyarakat yang memiliki pengaruh, diharapkan dapat membantu terlaksananya program dengan baik, serta terus menjaga kesinambungan dari apa yang telah dilakukan melalui program pengabdian masyarakat ini. d. Pondok Pesantren Salafiyah Syafi’iyah Sukorejo Situbondo. Bentuk- bentuk keterlibatannya adalah, mendukung secara moril terhadap kegiatan pengabdian, memberikan masukan, saran, serta arahan kepada tim yang berkaitan dengan sosial keagamaan. d. Pondok Pesantren Salafiyah Syafi’iyah Sukorejo Situbondo. Bentuk- bentuk keterlibatannya adalah, mendukung secara moril terhadap kegiatan pengabdian, memberikan masukan, saran, serta arahan kepada tim yang berkaitan dengan sosial keagamaan. e. Universitas Ibrahimy, bentuk-bentuk keterlibatannya adalah: merencanakan, melaksanakan, dan memfasilitasi pelaksanaan program; melakukan pendampingan selama pelaksanaan program; e. Universitas Ibrahimy, bentuk-bentuk keterlibatannya adalah: merencanakan, melaksanakan, dan memfasilitasi pelaksanaan program; melakukan pendampingan selama pelaksanaan program; V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 22 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 As-sidanah melakukan evaluasi program; penyusunan laporan dan mendeseminasikan hasil kegiatan agar tetap terus berkesinambungan. Penyusunan Rencana Aksi Partisipatoris Setalah melakukan proses pemetaan awal yang juga melibatkan pihak- pihak terkait dalam pendampingan ini, maka kegiatan pengabdian kepada masayarakat pemuda desa Sumberejo antara lain: a. Melakukan Focus Group Discussion (FGD) dengan pengurus pondok Pesantren Salafiyah Syafi’iyah Sukorejo, aparat pemerintahan desa Sumberejo, beserta sejumlah tokoh masarakat yang menjadi rujukan dalam proses pelaksanaan pengabdian ini. b. Membangun komunikasi dengan komunitas-komunitas pemuda dan masayarakat desa Sumberejo. Tahapan ini tim merancang format kegiatan yang menyesuaikan degan permasalahan yang ditemukan selama proses pemetaan awal, sehingga mudah mendeskripsikan arah program yang akan dilaksanakan. Langkah-langkah dalam Pendampingan Rancangan kegiatan yang akan dijalankan disinergikan dengan hasil observasi lapangan dengan mengkaji lokasi dampingan, serta potensi-potensi yang dimiliki, dengan melibatkan stakeholders yang dipandang perlu. melalui tahapan ini dapat digambarkan perencanaan kegiatan yang akan dilakukan untuk tahap selanjutnya. Kedua, tim melaksakan pengkajian secara mendalam terhadap problem rendahnya pemahaman dan pengamalan nilai-nilai agama di masyarakat, yang disesuaikan dengan hasil observasi terdahulu dan memetakannya dengan potensi atau kekuatan yang ada. Langkah berikutnya adalah melakukan pemetaan berdasarkan kemampuan Sumber Daya Manusia yang dimiliki, untuk melakukan persuasi di kalangan pemuda agar program pengabdian yang direncakan dapat terlaksana dengan baik dan memperoleh hasisl sesuai dengan yang direncanakan. V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Melaksanakan Aksi Dalam melaksanakan program pengabdian masyarakat ini, aksi yang dilaksanakan adalah memberikan orientasi kapada narasumber pendamping tentang topik problem yang dihadapi masayarakat pemuda desa Sumberejo. a. Memberikan argumentasi-argumentasi tentang pentingnya peran pemuda dalam mengawal lahirnya generasi terbaik di masa yang akan datang. a. Memberikan argumentasi-argumentasi tentang pentingnya peran pemuda dalam mengawal lahirnya generasi terbaik di masa yang akan datang. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 24 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 24 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah b. Melakukan pendampingan dalam proses-proses pengambilan keputusan bersama, dan memfasilitasi pengambilan keputusan secara patisipatif.4 b. Melakukan pendampingan dalam proses-proses pengambilan keputusan bersama, dan memfasilitasi pengambilan keputusan secara patisipatif.4 c. Melakukan pendampingan kepada narasumber pendamping, dalam penyampaian materi dan kegiatan aksi lainnya selama kegiatan pengabdian berlangsung. c. Melakukan pendampingan kepada narasumber pendamping, dalam penyampaian materi dan kegiatan aksi lainnya selama kegiatan pengabdian berlangsung. 4 Sudiyono, dkk., Strategi Pembelajaran Partisipatori, (UIN-Malang Press, Malang, 2006), hlm. 53 V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 25 Jurnal Pengabdian Masyarakat 2006), hlm. 53 5 Anju Dwivedi, Metodologi Pelatihan Partisipatif, (Pondok Edukasi, Yogyakarta, 2003), hlm. 2. Bentuk Kegiatan Pendampingan dan Pembinaan 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Hizb Basmalah, yaitu setiap 41 hari sekali, yang dibacakan langsung oleh KHR. Ahmad Azaim Ibrahimy. ISSN : 2656-5161 e-ISSN : 2686-0643 Hizb Basmalah, yaitu setiap 41 hari sekali, yang dibacakan langsung oleh KHR. Ahmad Azaim Ibrahimy. Pembacaan kitab al-Mukhtar min Kalami al-Akhyar disajikan dengan bahasa yang lugas, mengingat jamaah Majelis Dzikir Basmalah brsifat umum, serta memiliki tingkat pendidikan yang berbeda- beda. Penyampaian materi diselingi dengan rangkaian hikmah lainnya yang dikontekstualisasikan dengan situasi terkini yang berkembang di masyarakat, baik yang berhubungan dengan isu regional, nasional, maupun global. Bentuk Kegiatan Pendampingan dan Pembinaan Terdapat beberapa program kegiatan yang dilaksanakan dalam program pengabdian kepada masyarakat bersama pemuda desa Sumberejo yang fokus dan pengembangannuya dapat diklasifikasikan melalui tiga penguatan. Yaitu ranah kognitif (intelektual), afektif (emosional), dan psikomotorik (keterampilan),5 antara lain:: j Hizb Basmalah adalah salah satu aurad pembacaan kalimat-kalimat Basmalah, yang didahului dengan wasilah serta ditutup dengan do’a. pembacaan Hizb Basmalah berjamaah ini dilaksanakan dalam dua majlis, yaitu Majelis Dzikir Basmalah yang dilaksanakan setiap 41 hari, dan Majelis Dzikir Basmalah yang dilaksanakan secara rutin setiap dua pekan sekali. Majelis Dzikir Basmalah yang dilaksanakan 4 Sudiyono, dkk., Strategi Pembelajaran Partisipatori, (UIN-Malang Press, Malang, 2006), hlm. 53 Ainun Najib \| 25 Jurnal Pengabdian Masyarakat Ainun Najib \| 25 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah setiap 41 hari dipimpin dan dipandu langsung oleh KHR. Ahmad Azaim Ibrahimy, sedangkan Majelis Dzikir Basmalah yang dilaksanakan dua pekan sekali dipimpin secara bergiliran oleh jamaah majlis. ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah setiap 41 hari dipimpin dan dipandu langsung oleh KHR. Ahmad Azaim Ibrahimy, sedangkan Majelis Dzikir Basmalah yang dilaksanakan dua pekan sekali dipimpin secara bergiliran oleh jamaah majlis. Lokasi pelaksanaan pembacaan Hizb Basmalah berjamaah ini, baik pembacaan Hizb Basmalah yang 41 hari sekali maupun yang dua pekan sekali, dilakukan secara anjangsana bergilir di kediaman masing-masing jamaah. Sesekali pembacaan Hizb Basmalah berjamaah dilaksanakan di lokasi alam bebas, di pantai, pegunungan, dan lain sebagainya. Tujuannya adalah untuk lebih meningkatkan kecintaan serta mengagungkan Allah melalui semesta ciptaan-Nya. b. Pengajian Kitab al-Mukhtar min Kalami al-Akhyar Kitab al-Mukhtar min Kalam al-Akhyar merupakan salah satu karya terbaik Syaikh Muhammad bin Alawi Al-Maliki Al-Hasani Al- Makki, ulama besar dari kalangan Ahlussunnah di kota Makkah. Kitab berbahasa Arab tersebut merupakan kumpulan kisah teladan para nabi serta orangorang shaleh yang penuh dengan hikmah. Pengajian Kitab al-Mukhtar min Kalami al-Akhyar bagi jamaah Majelis Dzikir basmalah dilaksanakan pada waktu yang sama dengan Dzikir V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 26 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 26 Jurnal Pengabdian Masyarakat V o l . 3 N o . c. Pengajian Online Program Donasi Anak Yatim menyalurkan bantuan berupa dana pendidikan, khususnya bagi anakanak yatim yang masih duduk di bangku SD dan SLTP. Secara bertahap program ini tidak hanya mengelola santunan berupa uang dan untuk anak yatim saja, namun juga barang-barang berharga lainnya yang dapat dihibahkan kepada para fakir miskin, khususnya warga desa Sumberejo Kecamatan Banyuputih Kabupaten Situbondo. Program Donasi Anak Yatim menyalurkan bantuan berupa dana pendidikan, khususnya bagi anakanak yatim yang masih duduk di bangku SD dan SLTP. Secara bertahap program ini tidak hanya mengelola santunan berupa uang dan untuk anak yatim saja, namun juga barang-barang berharga lainnya yang dapat dihibahkan kepada para fakir miskin, khususnya warga desa Sumberejo Kecamatan Banyuputih Kabupaten Situbondo. c. Pengajian Online Maraknya dakwah-dakwah yang berorentasi pada radikalisme umat Islam, menjadi persoalan serius yang perlu terus dicarikan solusi. Gerakan dakwah kelompok-kelompok tersebut banyak menyasar kalangan kaum muslim muda, sehingga media dakwah yang digunakan adalah melalui media-media elektronik yang banyak menyebar melalui jejaring media sosial. Ketika kelompok ini telah berhasil menguasai media, maka tidak menutup kemungkinan di masa-masa yang akan datang berhasil mengikis integrasi nasionalisme kebangsaan di kalangan kaum muda akibat pemahaman keagamaan yang instant. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 27 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Untuk membentengi gerakan-gerakan tersebut, perlu ada penyeimbang terhadap gerakan dakwah yang berorientasi pada radikalisme fundamental. Atas dasar itulah Majelis Dzikir Basmalah berinisiatif membentuk program dakwah melalui multimedia yang bertajuk Pengajian Online Majelis Dzikir Basmalah. Seluruh awak kru media Program Pengajian Online ini terdiri dari pemuda desa Sumberejo. Pengajian Online dibina oleh KHR. Ahmad Azaim Ibrahimy, dan secara berkesinambungan juga akan menghadirkan tokoh-tokoh agama lainnya. Pengajian Online Majelis Dzikir Basmalah ini dapat diikuti melalui: 1) Situs Internet : mdbasmalah.com : mdbasmalah.com 2) Yotube Channel : Majelis Dzikir Basmalah : Majelis Dzikir Basmalah 3) Akun Facebook : Majlis Dzikir Basmalah 4) Akun Twitter : md_basmalah 5) Akun Instagram : mdbasmalah d. Donasi Anak Yatim Sebagai bentuk kepedulian terhadap sesama, Majelis Dzikir Basmalah juga memiliki program santunan bagi anak-anak yatim yang bertajuk Donasi Anak Yatim (DAY). Melalui program tersebut Majelis Dzikir Basmalah melakukan penggalangan dana secara berkala, yang tidak hanya terbatas kepada jamaah majlis saja, V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 28 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah melainkan bagi masyarakat secara umum yang hendak mendermakan sebagian hartanya untuk anak yatim. e. Usaha Ekonomi Kreatif Kreatifitas pemuda pada biasanya selalu berkembang, mereka butuh sarana penyaluran agar tetap dapat berkreasi dan terus meningkat kreatifitasnya. Demikian halnya dengan para pemuda yang tergabung dalam Majelis Dzikir Basmalah, bakat dan ide-ide mereka sangat baik untuk diwujudkan. Maka kreatifitas mereka difasilitasi dalam berbagai usaha yang juga bisa mendatangkan keuntungan bersama. kegiatan ini juga laboratorium untuk melatih kemandirian dan kecakapan para jamaah dalam mengembangkan usaha nantinya. Bentuk-bentuk usaha ekonomi kreatif yang dilakukan seperti: usaha sablon, sticker cutting, gantungan kunci, kopyah bordir, lampu hias, V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah hiasan dinding, dan lain-lain. Hasil dari kreatifitas merekalah yang terus dikembangkan dalam bidang pengembangan ekonomi majelis, sehingga kegiatan-kegiatan Majelis Dzikir Basmalah dapat berjalan secara mandiri tanpa menggantungkan bantuan dari pihak lain. Hasil Dan Diskusi ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah hiasan dinding, dan lain-lain. Hasil dari kreatifitas merekalah yang terus dikembangkan dalam bidang pengembangan ekonomi majelis, sehingga kegiatan-kegiatan Majelis Dzikir Basmalah dapat berjalan secara mandiri tanpa menggantungkan bantuan dari pihak lain. Dampak Perubahan Program pengadian masyarakat dirasa telah membawa dampak perubahan kepada arah yang lebih baik, khususnya terhadap pemuda desa Sumberejo. Baik perilaku sosial maupun perilaku mental spiritual. Proses pelaksanaan pengabdian masyarakat berjalan sesuai dengan yang telah direncanakan, dan akan terus dikembangkan di masa-masa yang akan datang. Kecintaan pemuda kepada majelis dzikir secara simultan akan membawa dampak kepada arah perubahan yang lebih positif. Pemuda yang identik dengan perkumpulan yang lebih berorientasi kepada hal- hal yang negatif, dapat terhindarkan dengan bergabungnya ke dalam majelis dzikir dengan berbagai program yang memberikan peningkatan pemahan keagamaan, serta diharapkan membawa perubahan pada perilaku mental spiritual sehari-hari, hari ini dan di masa yang akan datang. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 30 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 30 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Keberlanjutan program ini akan terus diperhatikan, mengingat dampak positifnya telah dapat dirasakan secara nyata, baik bagi masyarakat desa Sumberejo khususnya di kalangan para pemuda, maupun bagi masyarakat luas yang dapat mengikuti program pengajian online yang diselenggarakan oleh Majelis Dzikir Basmalah. Bersama stakeholders yang memiliki peran signifikan, beberapa program telah dicanangkan untuk pengembangan program di masa yang akan datang, dengan tetap mengintegrasikan peran serta fungsi pemuda desa Sumberejo dalam menyebarkan dakwah Islam rahmatan lil ‘alamin. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 32 Jurnal Pengabdian Masyarakat 6 Hasyim Asy’ari, Adab al-‘Alim wa al-Muta’allim, (Maktabah At-Turats Al-Islamy, Tebuireng Jombang, 1415 H), hlm. 10. Diskusi Keilmuan Dalam ajaran agama sangat diperhatikan aspek pendidikan moral atau pembentukan kepribadian. Karena terbentuknya watak dan kepribadian inilah yang sesungguhnya merupakan inti tujuan dari semua proses pergaulan antar sesama manusia di muka bumi. Kecerdasan intelektual akan menjadi tidak ada artinya jika tidak didukung dengan moral, watak dan kepribadian yang luhur. Hal ini sebagaimana dijelaskan oleh KH. Hasyim Asy’ari di dalam pengantar kitabnya Adab al-’Alim wa al-Muta’allim yang ia kutip dari Ibnu al- Mubarak: nahnu ila qalîl min al-adab ahwaj minnâ ila kathîr min al-‘ilm (kami lebih membutuhkan adab [etika, moral, budi pekerti] meskipun V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 31 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah sedikit dari pada [memiliki] banyak ilmu pengetahuan [tetapi tidak beradab]).6 sedikit dari pada [memiliki] banyak ilmu pengetahuan [tetapi tidak beradab]).6 Bagi masyarakat desa Sumberejo, Kiai merupakan “tokoh sentral” yang dikonsepsikan sebagai guru ideal yang patut untuk dijadikan teladan bagi segenap masyarakat, baik dalam masalah keilmuan maupun perilaku dan kepribadiannya. Dengan demikian, peran kyai tidak hanya terbatas pada memberikan pengajaran yang berorientasi pada pengembangan intelektualitas (kognisi) saja, tetapi juga memberikan bimbingan ruhani serta prilaku yang baik, membangun karakter yang kuat, berperilaku mulia sesuai dengan norma-norma ajaran agama (Islam) dan nilai-nilai etika, serta menghargai tradisi agar mereka dapat menjadi manusia seutuhnya di tengah-tengah masyarakat sesuai fitrah kemanusiaannya baik sebagai hamba Allah maupun selaku khalifah-Nya di muka bumi. Masyarakat juga menjadikan Kiai sebagai sumber rujukan dalam memberikan legitimasi dalam segenap tindakan warganya, sudah barang tentu mempunyai dasar pijakan yang bersifat keagamaan dalam memberikan penilaian. Hal ini karena watak pemimpin agama dan warga masyarakatnya yang fiqh oreinted, selalu meletakkan kegiatan ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah yang dilakukan dalam pola hitam-putih atau salah-benar menurut hukum Islam.7 yang dilakukan dalam pola hitam-putih atau salah-benar menurut hukum Islam.7 Sebagai salah satu desa yang di dalamnya terdapat pondok pesantren, maka sudah sepatutnya masyarakat Sumberejo dapat mencerminkan nilai-nilai sebagaimana yang diajarkan dalam pendidikan pesantren, yang secara garis besar mengandung muatan- muatan yang dibedakan dalam 2 (dua) kategori. V o l . 3 N o . 1 , A p r i l 2 0 2 1 Ainun Najib \| 33 Jurnal Pengabdian Masyarakat 7 KH. MA. Sahal Mahfudh, Nuansa Fiqih Sosial, (LKiS, Yogyakarta, 2003), hlm. 349. Diskusi Keilmuan Kategori pertama adalah ajaran dasar yang merupakan referensi bagi landasan hidup berikut penyelesaiannya dalam mengatasi seluruh problematika kehidupan di tengah dinamika sosial-budaya, yang mempunyai nilai kebenaran bersifat mutlak dan tidak runtuh dalam segala perubahan zaman. Ajaran dasar ini mempunyai muatan-muatan nilai universal, yang mempunyai daya relevansi dalam segala tataran ruang dan waktu. Bahkan, ajaran ini memiliki legalitas sakral dan telah secara tuntas dikodifikasikan oleh para ulama salafus shalih, yakni berupa Al-Qur’an dan Sunnah Nabi. Kategori kedua adalah ajaran-ajaran yang merupakan hasil interpretasi dan derivasi dari ajaran dasar di atas. Ajaran ini mengelaborasi muatan ajaran dasar dengan kecenderungan pada aspek- aspek praktis-aplikatif. Maka masyarakat di sekitar pesantren menjadi ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah garda terdepan yang dipercaya mampu mengakomodir kebutuhan penyelesaian persoalan-persoalan sosial-keagamaan di lingkungannya. Tak hanya identik dengan makna keislaman, tetapi karena karakteristik eksistensial pesantren yang mengandung arti keaslian Indonesia (indigenous). Sebagai indigenous, pesantren dan masyarakat di sekitarnya muncul dan berkembang dari pengalaman sosiologis di lingkungannya. Terdapat suatu hipotesa bahwa jika saja Indonesia tidak mengalami penjajahan, mungkin pertumbuhan sistem pendidikan di negeri ini akan mewarisi corak pendidikan ala pesantren. Proses modernisasi sering tampak sebagai suatu perubahan yang memiliki dampak sangat mendasar dan mendalam pada struktur nilai yang telah berlaku dalam kehidupan di masyarakat. Tidak mustahil sering terjadi pergeseran nilai atas perkembangan modernisasi. Dalam kaitannya dengan permasalahan ini, kalangan kademis bersama pihak pesantren harus mampu memberikan motivasi dan arahan terhadap perkembangan yang ada, dan berupaya merangkum realitas kehidupan dalam jalinan nilai-nilai spiritual dan moralitas yang Islami. Sebagai implikasi dari kepeduliannya terhadap permasalahan masyarakat ini, kalangan akademisi bersama pesantren akan dapat memberi arah perkembangan masyarakat dari aspek sosial, budaya, ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah politik bahkan hingga ekonomi, yang ditindaklanjuti dengan kerja nyata dalam rangka “pencerahan” dan pemecahan masalah yang ditentukan dalam masyarakat. Terbukti, berbagai fatwa ataupun keputusan masa’il fiqhiyah yang lahir dari hasil pemikiran orang-orang pesantren tidak saja memberikan keputusan halal-haram, tetapi juga melihat realitas perkembangan kehidupan dan mencarikan jalan keluar, sehingga masyarakat tidak terus terperangkap dalam “kegelapan” dan keharaman. 8 Civil Society disamping dimaknai masyarakat sipil, padanan kata lainnya yang sering digunakan adalah masyarakat beradab atau masyarakat berbudaya, masyarakat kewargaan dan masyarakat madani. Maksud masyarakat berbudaya sebagai padanan kata Civil Society adalah sebagai lawan masyarakat liar. Masyarakat berbudaya merujuk pada masyarakat yang saling menghargai nilai-nilai sosial kemanusiaan (termasuk dalam kehidupan sosial, budaya, politik dan ekonomi). Sementara, istilah masyarakat madani merujuk pada Madinah, sebuah kota yang sebelumnya bernama Yastrib di wilayah Arab, di mana masyarakat Islam di bawah kepemimpinan Nabi Muhammad di masa lalu pernah membangun peradaban yang tinggi. Lihat, Muhammad A. S. Hikam, Demokrasi dan Civil Society, (LP3ES, Jakarta, 1999), hlm. 15. Diskusi Keilmuan Maka, simbiosis antara pesantren dengan masyarakat ini kemudian dikenal sebagai konsep penguatan civil society,8 dimana tugas pokok Kiai dan pesantren adalah mendorong masyarakat agar lebih memahami dirinya, karena pemahaman tersebut akan menghantarkan mereka untuk membangun dan menemukan jati diri dan dunia ini, sesuai dengan konsep yang dikehendaki oleh Sang Maha Pencipta, dan yang pasti juga selaras dengan kemaslahatan masyarakat/sosial. 8 Civil Society disamping dimaknai masyarakat sipil, padanan kata lainnya yang sering digunakan adalah masyarakat beradab atau masyarakat berbudaya, masyarakat kewargaan dan masyarakat madani. Maksud masyarakat berbudaya sebagai padanan kata Civil Society adalah sebagai lawan masyarakat liar. Masyarakat berbudaya merujuk pada masyarakat yang saling menghargai nilai-nilai sosial kemanusiaan (termasuk dalam kehidupan sosial, budaya, politik dan ekonomi). Sementara, istilah masyarakat madani merujuk pada Madinah, sebuah kota yang sebelumnya bernama Yastrib di wilayah Arab, di mana masyarakat Islam di bawah kepemimpinan Nabi Muhammad di masa lalu pernah membangun peradaban yang tinggi. Lihat, Muhammad A. S. Hikam, Demokrasi dan Civil Society, (LP3ES, Jakarta, 1999), hlm. 15. Ainun Najib \| 35 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Kesimpulan Melalui kegiatan pendampingan ini, diharapkan agar Universitas Ibrahimy Sukorejo tetap mengembangkan pengabdian pada masyarakat, khususnya yang berkaitan dengan penguatan pembangunan mental spiritual masyarakat, khususnya di kalangan para pemuda, sebagai generasi perjuangan bangsa. Dengan tetap menjalin keterlibatan stakeholders dari berbagai pihak yang dapat mempermudah terlaksananya program pengabdian kepada masyarakat secara baik dan terus berkesinambungan. Demikian sebagai bahan laporan dan bukti tertulis pelaksanaan kegiatan pengabdian kepada masyarakat di desa Sumberejo Kecamatan Banyuputih Kabupaten Situbondo yang dilaksanakan dalam bentuk pendampingan bagi para pemuda desa Sumberejo untuk meningkatkan mental spiritual melalui Majelis Dzikir Basmalah. Sudiyono, dkk., Strategi Pembelajaran Partisipatori, UIN-Malang Press, Malang, 2006 Daftar Pustaka A. S. Hikam, Demokrasi dan Civil Society, LP3ES Jakarta:, 1999. Abdullah Faisal, dkk., Metode & Teknik Kuliah Kerja Nyata Transformatif; Implementasi Participatory Action Research (PAR) & Participatory Rural Appraisal (PRA) untuk Aksi Perubahan Sosial, P3M STAIN Surakarta, 2006. Ainun Najib, dkk. Peradaban Sukorejo, Ikhtisar Perjalanan Satu Abad Pondok Pesantren Salafiyah Syafi’iyah Sukorejo Situbondo, Tim Penelitian Panitia Satu Abad P2S2, Sukorejo Situbondo, 2014. Ainun Najib, dkk. Peradaban Sukorejo, Ikhtisar Perjalanan Satu Abad Pondok Pesantren Salafiyah Syafi’iyah Sukorejo Situbondo, Tim Penelitian Panitia Satu Abad P2S2, Sukorejo Situbondo, 2014. Ainun Najib \| 36 Jurnal Pengabdian Masyarakat V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah Ainun Najib, Pengembaraan Terakhir, Sejarah dan Perjuangan KHR. Syamsul Arifin, Pena Salsabila, Jember, 2016. Anju Dwivedi, Metodologi Pelatihan Partisipatif, Pondok Edukasi, Yogyakarta, 2003. Hasyim Asy’ari, Adab al-‘Alim wa al-Muta’allim, Maktabah At-Turats Al- Islamy, Tebuireng Jombang: 1415 H. Kecamatan Banyuputih dalam Angka 2019, Badan Pusat Statistik Kabupaten Situbondo. KH. MA. Sahal Mahfudh, Nuansa Fiqih Sosial, LKiS, Yogyakarta: 2003. M. Hasan Basri, KHR. As’ad Syamsul Arifin, Riwayat Hidup dan Perjuangannya, CV. Sahabat Ilmu, Surabaya, 1994. Profil Desa Sumberejo Banyuputih Situbondo, https://opensid.sumberejo.id/. Sudiyono, dkk., Strategi Pembelajaran Partisipatori, UIN-Malang Press, Malang, 2006 V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1 ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah ISSN : 2656-5161 e-ISSN : 2686-0643 As-sidanah As-sidanah JURNAL V o l . 3 N o . 1 , A p r i l 2 0 2 1 V o l . 3 N o . 1 , A p r i l 2 0 2 1
https://openalex.org/W4213092191	https://www.researchsquare.com/article/rs-1334726/latest.pdf	English	null	Identification of molecular subtypes and a novel prognostic model of diffuse large B-cell lymphoma based on a metabolism-associated gene signature	Research Square (Research Square)	2,022	cc-by	10,945	Identification of molecular subtypes and a novel prognostic model of diffuse large B-cell lymphoma based on a metabolism-associated gene signature Jing He Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Ziwei Chen Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Qingfeng Xue Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Pingping Sun Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Yuan Wang Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Cindy（Xinyi） Zhu UCLA: University of California Los Angeles Wenyu Shi (  shiwenyu@hotmail.com ) Affiliated Hospital of Nantong University https://orcid.org/0000-0002-2371-3332 Research Article Keywords: diffuse large B-cell lymphoma, metabolism, molecular subtype, prognosis, immune microenvironment Posted Date: February 18th, 2022 DOI: https://doi org/10 21203/rs 3 rs-1334726/v1 Identification of molecular subtypes and a novel prognostic model of diffuse large B-cell lymphoma based on a metabolism-associated gene signature Jing He Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Ziwei Chen Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Qingfeng Xue Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Pingping Sun Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Yuan Wang Nantong University Affiliated Hospital: Affiliated Hospital of Nantong University Cindy（Xinyi） Zhu UCLA: University of California Los Angeles Wenyu Shi (  shiwenyu@hotmail.com ) Affiliated Hospital of Nantong University https://orcid.org/0000-0002-2371-3332 Research Article Keywords: diffuse large B-cell lymphoma, metabolism, molecular subtype, prognosis, immune microenvironment Posted Date: February 18th, 2022 DOI: https://doi org/10 21203/rs 3 rs-1334726/v1 Background: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in adults. Metabolic reprogramming in tumors is closely related to the immune microenvironment. This study aimed to explore the interactions between metabolism-associated genes (MAGs) and DLBCL prognosis and their potential associations with the immune microenvironment. Conclusions: The molecular subtypes and a risk model based on MAGs proposed in our study are both promising prognostic stratification tools in DLBCL, which may provide novel insights for developing accurate targeted cancer therapies. Results： Consensus clustering divided DLBCL patients into two metabolic subtypes with significant differences in prognosis and the immune microenvironment. Poor prognosis was associated with an immunosuppressive microenvironment. A prognostic risk model was constructed based on 14 MAGs and it was used to classify the patients into two risk groups; the high-risk group had poorer prognosis and an immunosuppressive microenvironment characterized by low immune score, low immune status, high abundance of immunosuppressive cells, and high expression of immune checkpoints. Cox regression, ROC curve analysis, and a nomogram indicated that the risk model was an independent prognostic factor and had a better prognostic value than the International Prognostic Index (IPI) score. The risk model underwent multiple validations and the verification of the two hub genes in TMA indicated consistent results with the bioinformatics analyses. Methods: Gene expression and clinical data on DLBCL patients were obtained from the GEO database. Metabolism- associated molecular subtypes were identified by consensus clustering. A prognostic risk model containing 14 MAGs was established using Lasso-Cox regression in the GEO training cohort. It was then validated in the GEO internal testing cohort and TCGA external validation cohort. GO, KEGG and GSVA were used to explore the differences in enriched pathways between high- and low-risk groups. ESTIMATE, CIBERSORT, and ssGSEA analyses were used to assess the immune microenvironment. Finally, WGCNA analysis was used to identify two hub genes among the 14 model MAGs, and they were preliminarily verified in our tissue microarray (TMA) using multiple fluorescence immunohistochemistry (mIHC). Research Article Keywords: diffuse large B-cell lymphoma, metabolism, molecular subtype, prognosis, immune microenvironment Posted Date: February 18th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1334726/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/28 Page 1/28 Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. About 60% of DLBCL patients experience effective remission after standard R-CHOP regimens. However, Page 2/28 approximately 30–40% of patients eventually relapse and 10% are primary refractory cases [1]. The International Prognostic Index (IPI), which is widely used to evaluate the prognosis of DLBCL, mainly depends on traditional clinicopathological features but does not consider the molecular characteristics and microenvironmental differences in lymphoma. Given that DLBCL is a highly heterogeneous tumor, having the same clinicopathological features does not always lead to the same prognosis [2]. Therefore, IPI score is not sufficient to accurately predict the prognosis [3]. It is necessary for us to develop new strategies to identify risk among DLBCL patients more reliably, so as to personalize treatment strategies. Recent studies have shown that risk models based on multi-gene expression are a reliable choice [4–6]. Metabolic reprogramming is regarded as a key hallmark of tumor malignancy. Tumors reprogram metabolic pathways to meet the high metabolic needs of the malignant cells and maintain tumor growth and proliferation [7]. Growing data suggest that metabolism-associated genes (MAGs) are highly expressed in a variety of tumor tissues and are directly involved in tumorigenesis, progression, and treatment responses [8, 9]. Therefore, MAGs are considered promising diagnostic markers and potential therapeutic targets. In addition, recent studies have focused on the relationship between metabolism and survival: pan-cancer studies have indicated that tumor subtypes with different MAG expression patterns lead to significantly different survival [10, 11]. Moreover, several risk models based on MAGs have been proposed for breast cancer [6], colorectal cancer [12], gastric cancer [13] and osteosarcoma [14]. However, the value of MAGs in DLBCL subtype identification and prognostic prediction remains unclear. The tumor microenvironment, as the hotbed of the tumor, has significant immune cell infiltration. In accordance with the complexity of the tumor microenvironment, immune cells recruited to the tumor tissues have dual tumor-promoting and tumor-antagonizing characteristics [15]. The immune microenvironment plays a key role in tumor development and treatment. Studies have shown that metabolic reprogramming is closely related to the tumor immune microenvironment [16]. Metabolites derived from tumor cells can influence the composition and distribution of cells in the immune microenvironment in many ways, ultimately leading to immune dysfunction and tumor progression [17]. Introduction For example, metabolic reprogramming can affect the differentiation subtypes and functions of T cells and the polarization and function of macrophages [18]. However, studies on the relationship between MAGs and the immune microenvironment in DLBCL remain limited. In the present study, we used multiple bioinformatics methods to comprehensively analyze MAGs, identified metabolism-associated molecular subtypes in DLBCL patients, constructed a novel MAG-based risk model evaluating the prognostic value of MAGs in DLBCL, and explored the relationships between MAGs and the immune microenvironment. Finally, two hub genes in the risk model were selected and verified using our own tissue microarray (TMA), and their potential utility as therapeutic targets and diagnostic markers was discussed. Our study may provide new clues on mechanisms and metabolic targets in DLBCL, and it may lay the foundation for accurate immunotherapy that targets metabolic pathways in DLBCL. Construction and validation of a MAG-based risk model The 92 candidate MAGs related to prognosis were selected, and a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression. The risk score formula (based on the expression of each included gene weighted by its LASSO regression coefficient) was constructed using the following format: risk score=∑n i=1coef ∗geneexpression. Thereafter, the risk score of each The 92 candidate MAGs related to prognosis were selected, and a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression. The risk score formula (based on the expression of each included gene weighted by its LASSO regression coefficient) was constructed using the following format: risk score=∑n i=1coef ∗geneexpression. Thereafter, the risk score of each patient was calculated. Using the median risk score as the cutoff, the training cohort was divided into low- and high-risk groups. Survival curves were generated by the Kaplan–Meier method and the two groups were compared using the log-rank test. A time-dependent receiver operating characteristic (ROC) curve analysis was used to study the model prediction accuracy. Cox regression was used to assess the independent prognostic value of the risk score and other clinicopathological features. To provide a reference for predicting the prognosis of DLBCL patients, we used the "rms" R package to construct a nomogram based on the risk score and clinicopathological features, and a calibration plot was used to assess the prognostic ability of the nomogram. using the following format: risk score=∑i=1coef ∗geneexpression. Thereafter, the risk score of each patient was calculated. Using the median risk score as the cutoff, the training cohort was divided into low- and high-risk groups. Survival curves were generated by the Kaplan–Meier method and the two groups were compared using the log-rank test. A time-dependent receiver operating characteristic (ROC) curve analysis was used to study the model prediction accuracy. Cox regression was used to assess the independent prognostic value of the risk score and other clinicopathological features. To provide a reference for predicting the prognosis of DLBCL patients, we used the "rms" R package to construct a nomogram based on the risk score and clinicopathological features, and a calibration plot was used to assess the prognostic ability of the nomogram. Methods Page 3/28 Page 3/28 Data sources and preprocessing The GSE10846 Series Matrix File data were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (the annotation platform was GPL570). The data of 412 DLBCL patients with a complete mRNA expression profile and survival time >0 was extracted from the GSE10846 dataset. Additionally, data on DLBC were downloaded from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/), and 47 DLBCL cases with a complete mRNA expression profile and survival time >0 were obtained from the TCGA database. We randomly divided (4:1 ratio) the 412 cases extracted from the GSE10846 dataset into a training cohort (n=330) and a testing cohort (n=82). We used the GEO testing cohort as an internal validation cohort and the TCGA dataset (n=47) as an external validation cohort to evaluate the predictive efficacy and robustness of the prognosis-associated risk model. Relevant grouping information and clinicopathological features are shown in Table 1. Identification of metabolism-associated subtypes MAGs were obtained from the GeneCards database (https://www.genecards.org/). We identified 92 candidate prognosis-related MAGs in GSE10846 by univariate Cox regression. Based on the 92 MAGs, the 412 patients were divided into subgroups with different metabolic expression patterns by consensus clustering using the "ConsensusClusterPlus" R package, and unbiased and unsupervised outcomes were obtained. Immune analyses The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) method was performed to calculate the stromal score, immune score, ESTIMATE score, and tumor purity. Next, the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm was used to analyze the RNA-Seq data of DLBCL patients in order to determine Page 4/28 Page 4/28 the relative proportions of 22 infiltrating immune cells. Furthermore, to quantify the immune cell infiltration in each sample, single-sample Gene Set Enrichment Analysis (ssGSEA) was used to assess the enrichment of 28 immune cells in the tumor samples. We then calculated the correlations between the risk score and immune regulatory genes, especially immune checkpoints. the relative proportions of 22 infiltrating immune cells. Furthermore, to quantify the immune cell infiltration in each sample, single-sample Gene Set Enrichment Analysis (ssGSEA) was used to assess the enrichment of 28 immune cells in the tumor samples. We then calculated the correlations between the risk score and immune regulatory genes, especially immune checkpoints. Drug sensitivity analysis and construction of competing endogenouse RNA (ceRNA) network Based on the Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org/), which is the largest pharmacogenomics database, we used the "pRRophetic" R package to predict the chemotherapy sensitivity of each tumor sample. The estimated half-maximal inhibitory concentration (IC50) value of each chemotherapy drug was obtained by regression, and the accuracy of regression and prediction was tested by cross-validation with GDSC training set for 10 times. All parameters were selected as default values, including "combat" for removing batch effect and the average value of repeated gene expression. Furthermore, we used FunRich (v3.1.3) and NPInter (v4.0) to construct a ceRNA network based on the model genes. Gene Set Variation Analysis (GSVA) and functional enrichment analyses GSVA is a non-parametric and unsupervised method for evaluating the enrichment of gene sets in relation to mRNA expression data. In this study, gene sets were downloaded from the Molecular Signatures Database (v7.0). Each gene set was comprehensively scored by the GSVA algorithm, and the potential differences in biological functions between the high- and low-risk groups were evaluated. Additionally, to explore the functions of the prognosis-associated MAGs, the "ClusterProfiler" R package was used to annotate the genes with their predicted functions based on Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. GO terms and KEGG pathways with p and q values <0.05 were deemed statistically significant. Fluorescence-based multiplex immunohistochemistry (mIHC) staining The DLBCL TMA slides were stained with multiplex fluorescence by using the Opal 7-color Manual IHC Kit (PerkinElmer, MA). After dewaxing by xylene and rehydration by ethanol, slides were heated in a microwave with AR6 Buffer (AR600, AKOYA) and AR9 Buffer (AR900, AKOYA) for antigen retrieval. The slides were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibody for 10min at room temperature. At last, we used 4',6-diamidino-2-phenylindole (DAPI; F6057, Sigma) to stain the nuclei and seal the slides. Imaging was achieved using the Vectra 3.0 Automated Quantitative Pathology Imaging System. Tumor and stroma images were captured at ×20 magnification. Finally, the staining was scored by inForm® Cell Analysis software based on the intensity and degree of staining. The primary antibodies used in this study were as follows: rabbit anti-PHKA1 (24279-1-AP, Proteintech), rabbit anti-PLTP (ab282456, Abcam), rabbit anti-CD163 (93498, Cell Signaling Technology), rabbit anti- CD68 (76437, Cell Signaling Technology), rabbit anti-CD11B (49420, Cell Signaling Technology), mouse anti-CD66b (ARG66287, Arigobio), rabbit anti-PD-1 (86163, Cell Signaling Technology) and rabbit anti-PD- L1 (13684, Cell Signaling Technology). The secondary antibody was Opal™ polymer HRP Ms+Rb (ARH1001EA, Perkin Elmer). Weighted Gene Co-expression Network Analysis (WGCNA) Weighted Gene Co-expression Network Analysis (WGCNA) To identify the hub genes among the 14 model genes, we used the WGCNA algorithm. After constructing a weighted gene co-expression network, the gene co-expression modules were identified, and the correlations between gene network and clinical phenotype were explored. The WGCNA-R package was used to construct the co-expression network of all genes in the GSE10846 dataset, and the genes with variance within the first 5000 were identified by the algorithm for subsequent analysis. The soft-threshold β was determined by the function " sft$powerEstimate". The weighted adjacency matrix was transformed into a topological overlap matrix (TOM) to estimate the network connectivity, with hierarchical clustering being used to construct the clustering tree structure of the TOM. Different branches of the clustering tree represented different gene modules, and different colors represented different modules. Tens of thousands of genes were classified into modules based on having similar expression patterns (using their weighted correlation coefficients). thousands of genes were classified into modules based on having similar expression patterns (using their weighted correlation coefficients). Page 5/28 Page 5/28 Page 5/28 TMA tissue samples The DLBCL TMA contained 104 DLBCL tissues and 28 reactive hyperplasia tissues (from cases with the same gender ratio and age range) collected from 2008 to 2015. It was prepared by the Department of Clinical Biobank of the Affiliated Hospital of Nantong University. Clinicopathological data, including gender, age, B symptoms, Ann Arbor stage, hemoglobin (Hb) level, serum lactate dehydrogenase (LDH) level, IPI score were collected. In addition, X-tile 3.6.1 software was performed to determine the optimal cutoff values for two hub genes expression. This study was a retrospective study, and the informed consent of all patients was obtained before the study. The Ethics Committee of the Affiliated Hospital of Nantong University approved this research Statistical analysis Survival curves were generated by the Kaplan–Meier method and compared using the log-rank test. Multivariate Cox proportional hazards regression was used to identify independent prognostic factors. All statistical analyses were performed in R software (v4.0). All statistical tests were two tailed, and P<0.05 was considered statistically significant. Identification of prognosis-associated MAGs in DLBCL Page 6/28 The whole study process is depicted in the flow chart in Fig. 1. First, we obtained 958 MAGs from the GeneCards database by using “metabolism” as the search term and setting the relevance score >5. Second, DLBC mRNA expression data (Fragments Per Kilobase of transcript per Million mapped reads [FPKM]) in the GSE10846 dataset was downloaded, and 877 MAGs were extracted (based on the 958 MAGs identified using GeneCards). To identify the prognosis-associated genes among the 877 MAGs, we used prognostic data on DLBCL patients and obtained 92 prognosis-associated MAGs by univariate Cox regression (p<0.001) (Fig. S1). Identifying metabolism-associated molecular subgroups and differences in prognosis and the immune microenvironment between subgroups Identifying metabolism-associated molecular subgroups and differences in prognosis and the immune microenvironment between subgroups Based on the expression patterns of 92 prognosis-associated MAGs, we used consensus clustering to cluster the 412 patients in the GSE10846 dataset into different metabolism-associated molecular subgroups. By increasing the clustering variable (k) from 2 to 5, we found that consensus clustering was most stable when k=2 (Fig. 2A). This indicated that DLBCL patients could be readily divided into two clusters, with 183 patients in cluster 1 and 229 patients in cluster 2. Heatmap visualization showed significant differences in the expression of the 92 MAGs between the two clusters (Fig. 2B). Survival analysis showed that the overall survival in the two clusters was different, with cluster 1 patients having a significantly worse prognosis than cluster 2 patients (Fig. 2C). In addition, the CIBERSORT algorithm was used to evaluate the differences in immune cell infiltration between the two clusters. The abundances of infiltrating immune cells between clusters are shown in Fig. S2, and the quantitative analysis demonstrated that there were significant differences. Patients in cluster 1 had a high infiltration of immunosuppressive cells (Tregs, NK cells resting, mast cells activated) and a low infiltration of immunopositive cells (T cells gamma delta, T cells CD4 memory activated, monocytes, dendritic cells resting), while cluster 2 showed the opposite trends (Fig. 3A). Analysis of the differences in the expression of immune checkpoints between the two clusters showed that ADORA2A, CD244, CD274, CSF1R, CTLA4, HAVCR2, KIR2DL1, KIR2DL3, LAG3, LGALS9, PDCD1, TGFB1, TGFBR1, and VTCN1 expression levels were significantly higher in cluster 1 than in cluster 2 (Fig. 3B). Cluster 1 patients had an immunosuppressive tumor microenvironment, which was in accordance with their poor prognosis. These findings indicate that the expression of MAGs is related to the prognosis and the immunosuppressive microenvironment in DLBCL patients. Construction and validation of a prognosis-associated risk model composed of 14 MAGs Based on the MAGs identified in univariate Cox regression analyses, we selected 14 MAGs by LASSO regression in order to construct a metabolism-associated prognosis risk model (Fig. 5A–C and Table 2). The 412 GEO patients were randomly divided (4:1 ratio) into a training cohort (n=330) and a testing cohort (n=82). After constructing the model, the risk score of each DLBCL patient in the training cohort was computed based on the following formula: risk score = NR3C1×(-0.285969433071478) + IGFBP3×(-0.16695536054869) + RARRES2×(-0.14291303044122) + F5×(-0.0961037965837689) + APOC1×(-0.0768487272489624) + CSF2RA×(-0.056110125649913) + ENPP1×(-0.0221430402174049)+ GYG1×0.0449620982512186 + PHKA1×0.0693510636252306 + CPT1A×0.0752116074259248 + PDK4×0.0767229743647787 + CLOCK×0.0858851468938173 + CTH×0.108708800807851 + PLTP×0.16013617077787. Patients were divided into high- and low-risk groups according to the median risk score. The distribution of risk score, survival status, and the expression of the 14 MAGs in the training cohort are depicted in Fig. 5D. The results indicated that patients in the high-risk group were more likely to exhibit high expression of risk genes (hazard ratio >1; GYG1, PHKA1, CPT1A, PDK4, CLOCK, CTH, and PLTP), while patients in the low-risk group tended to exhibit high expression of protective genes (hazard ratio <1; NR3C1, IGFBP3, RARRES2, F5, APOC1, CSF2RA, and ENPP1). Additionally, Kaplan–Meier curves indicated that the DLBCL patients in the high-risk group had significantly worse overall survival (OS) (Fig. 5F). The area under the curve (AUC) values of the ROC curves for 1-, 2-, and 3-year OS were 0.79, 0.81, and 0.81, respectively (Fig. 5H), demonstrating the great predictive performance of the prognosis- associated risk model. Next, we used the GEO testing cohort as an internal validation cohort and the TCGA dataset as an external validation cohort to evaluate the prediction performance and robustness of the prognosis- associated risk model. Using the same formula, we obtained consistent results in the testing cohort, which confirmed the robustness of the risk model. The risk score distribution and gene expression profiles are shown in Fig. 5E. OS was significantly worse in the high-risk group than the low-risk group (Fig. 5G). The AUCs for 1-, 2-, and 3-year OS were 0.79, 0.83 and 0.81, respectively (Fig. 5I). In addition, the TCGA results concurred with the GEO training cohort results. The risk score distribution and gene expression profile are shown in Fig. 6A. Patients with higher risk scores had worse OS (Fig. 6B). Functional enrichment analyses of prognosis-associated MAGs and construction of transcriptional regulatory network By performing GO and KEGG analyses of the 92 prognosis-associated MAGs, we found that they were significantly enriched in many key metabolic and biosynthetic pathways. GO analysis indicated that the MAGs were enriched in small molecule catabolic process, cofactor biosynthetic process, cofactor metabolic process, vitamin metabolic process, and monosaccharide metabolic process (Fig. 4A). KEGG Page 7/28 Page 7/28 analysis revealed similar pathways, including biosynthesis of cofactors, starch and sucrose metabolism, central carbon metabolism in cancer, fatty acid degradation, and biosynthesis of amino acids (Fig. 4B). Furthermore, we used Cytoscape software to construct a protein–protein interaction network of these prognostic MAGs (Fig. 4C). analysis revealed similar pathways, including biosynthesis of cofactors, starch and sucrose metabolism, central carbon metabolism in cancer, fatty acid degradation, and biosynthesis of amino acids (Fig. 4B). Furthermore, we used Cytoscape software to construct a protein–protein interaction network of these prognostic MAGs (Fig. 4C). Clinical correlations and independent prognosis analysis of risk score To identify whether the risk score was related to clinicopathological features, we compared the risk scores between groups divided based on clinical features, as shown in box plots in Fig. 7A–G. Using Kruskal– Wallis rank sum tests, we found that the risk score significantly differed by age, Eastern Cooperative Oncology Group (ECOG) status, stage, lactate dehydrogenase (LDH) level, and IPI score (Fig. 7A–E), but it was not related to gender or extranodal sites (Fig. 7F–G). These results indicated that the risk score had good clinical value for classifying DLBCL samples. In addition, univariate and multivariate Cox regression indicated that risk score was an independent prognostic factor in DLBCL (Fig. 8A). Next, we constructed a prognostic nomogram that integrated the risk score and all significant clinical features. Nomograms can be used to quantitatively predict the prognosis of patients, providing a reference for clinical decision-making. The nomogram demonstrated that the risk score contributed the most to the prognosis, more than the IPI score and other clinical features (such as age, stage, ECOG status, and LDH level) (Fig. 8B). Additionally, the calibration curves for 3- and 5-year OS indicated high consistency between the nomogram predictions and actual observations (Fig. 8C). These findings confirmed that the risk score was a satisfactory prognostic tool for use in DLBCL. Instinctively, we would compare it with the current widely used scoring system, IPI score. Time- dependent ROC curve analysis was further used to determine which scoring system best predicted the OS of DLBCL patients. The AUC of the risk score for predicting 1-year OS (AUC=0.799) was significantly higher than that of IPI score (AUC=0.631) (Fig. 8D). This demonstrated that the risk score was superior to the IPI score regarding survival prediction accuracy in DLBCL. Construction and validation of a prognosis-associated risk model composed of 14 MAGs The AUCs for 1-, 2-, and 3-year OS were 0.78, 0.61, and 0.61, respectively, indicating that the risk model had a strong prognostic value for DLBCL patients in the TCGA validation cohort (Fig. 6C). In conclusion, these results confirmed that the risk model had a robust and accurate ability to predict OS. Page 8/28 Page 8/28 Relationship between risk score and immune microenvironment The tumor immune microenvironment significantly affects the therapeutic effect and prognosis of tumor. We assessed the relationship between the risk score and the tumor immune microenvironment in DLBCL by multiple immune analyses. The ESTIMATE results indicated that the patients in the high-risk group had significantly lower stromal and ESTIMATE scores, and higher tumor purity, than those in the low-risk group (Fig. 9A). The ssGSEA was used to assess the immune status of the two groups, which suggesting that the DLBCL patients in the high-risk group had a relatively low immune status (Fig. 9B), which was consistent with the ESTIMATE results. In addition, the results of the CIBERSORT analysis revealed that there were significant differences in most infiltrating immune cells (Fig. S3). The high-risk group was associated with significantly increased abundances of B cells naive, monocytes, macrophages M2, NK cells resting, NK cells activated, and significantly decreased abundances of T cells CD4 naive, T cells follicular helper, T cells gamma delta, macrophages M0 and dendritic cells resting, while the low-risk group showed the opposite trends (Fig. 9C). Higher abundance of immunosuppressive cells and lower abundance of immunopositive cells in the high-risk group indicated an immunosuppressive tumor microenvironment, which was consistent with the poor prognosis. Page 9/28 Page 9/28 Additionally, we further explored the correlations between the risk score and immune cell content. The results showed that the risk score was positively correlated with macrophages M2, T cells CD4 naive, monocytes, B cells naïve, NK cells resting, and negatively correlated with T cells gamma delta, macrophages M0 and T cells follicular helper (Fig. 9D). Additionally, we further explored the correlations between the risk score and immune cell content. The results showed that the risk score was positively correlated with macrophages M2, T cells CD4 naive, monocytes, B cells naïve, NK cells resting, and negatively correlated with T cells gamma delta, macrophages M0 and T cells follicular helper (Fig. 9D). Thereafter, the immune-regulatory genes were further analyzed, and the differences in the expression levels of immune-related chemokines, immunosuppressants, immunostimulants, major histocompatibility complex (MHC) factors, and immune receptors between the high- and low-risk groups are shown in a heatmap (Fig. 10A). As immune cell dysfunction and immunosuppressive microenvironments are characterized by high expression of immune checkpoint-related transcripts, we subsequently focused on the differences in immune checkpoint expression levels and their correlations with the risk score. Relationship between risk score and immune microenvironment The results showed that the expression levels of ADORA2A, CD274, CSF1R, IL10, KIR2DL1, KIR2DL3, LGALS9, and TGFB1 were significantly higher in the high-risk group than the low-risk group (Fig. 10B). The correlations between the risk score and the expression of immune checkpoints showed that the risk score was positively correlated with the expression of CD274, CSF1R, KIR2DL1, KIR2DL3, LGALS9, and PVRL2, and negatively correlated with the expression of CD96, PDCD1LG2, TGFBR1, and TIGIT (Fig. 10C). The significantly increased expression of most immune checkpoints in the high-risk group further confirmed that the poor prognosis of high-risk patients was partly related to the immunosuppressive microenvironment. Based on these results, we can reasonably assume that the immunosuppressive microenvironments (characterized by low immune scores, low immune status, high abundance of immunosuppressive cells, and high expression of immune checkpoints) led to poor prognosis in the high-risk group. This is also consistent with our immune analysis of metabolism- associated molecular subtypes. Therefore, the risk model based on 14 MAGs is related to the immunosuppressive microenvironment of DLBCL, and abnormal immune cell infiltration and differential expression of immune checkpoints can be used as prognostic indicators and targets of immunotherapy, which is clinically significant. Screening for hub genes in the risk model by WGCNA To identify the most critical genes among the 14 model genes for further experimental verification, the WGCNA algorithm was applied. We constructed a weighted gene co-expression network based on the GSE10846 dataset to explore the important regulatory genes (Fig. 13A) in DLBCL. The soft-threshold was set to 1 (Fig. 13B). Next, based on the TOM, three gene modules were identified, namely, the blue (208), brown (151) and turquoise (4641) modules. Correlation analyses between the modules and clinical phenotypes (using the high- and low-risk groups based on patients' risk scores) showed that the turquoise module had the highest correlation (cor=0.4, p=2e-17) (Fig. 13C). To identify the hub genes among the 14 model genes, we identified the overlapping genes between the turquoise module and the 14 model genes. This led to two hub genes being identified: PLTP and PHKA1 (Fig. 13D). We then explored the relevance of these two hub genes regarding infiltrating immune cells and immune checkpoints, and found that both hub genes were positively correlated with the infiltration of M2 macrophages and the expression of most immune checkpoints (such as PD-1, PD-L1 and LAG3) (Fig. 16A and Fig. 17A). The potential mechanism of these two hub genes in DLBCL deserves further verification and discussion. Heterogeneity of drug sensitivity and signaling pathways in high- and low- risk groups Based on the drug sensitivity data from the GDSC database, we used the "pRRophetic" R package to predict the drug sensitivity of each patient. Gemcitabine, vinblastine, and metformin had lower IC50 values in the high-risk group, while cisplatin and etoposide had higher IC50 values in the high-risk group (Fig. 11A). Furthermore, to explore the discrepancies in signaling pathways between the high- and low-risk groups, GSVA was performed. As shown in Fig. 11B, the differentially enriched signaling pathways between the two groups mainly involved the unfolded protein response, xenobiotic metabolism, KRAS signaling, glycolysis, TGF beta signaling, epithelial–mesenchymal transition, and heme metabolism. The GSVA results suggest that disturbances in these signaling pathways may worsen the prognosis of DLBCL patients in the high-risk group compared to the low-risk group. Page 10/28 To further understand how the 14 MAGs in the risk model regulate mRNA expression by acting as miRNA sponges in DLBCL, we constructed a ceRNA network based on the 14 MAGs. Using FunRich to reverse predict miRNAs based on the 14 MAGs, 57 miRNAs and 130 mRNA-miRNA interactions were identified. Subsequently, using NPInter to reverse predict lncRNAs, 7395 mRNA-miRNA-lncRNA interactions were obtained. Finally, a ceRNA network related to the 14 genes was successfully constructed (Fig. 12A). These data may provide clues for identifying the regulatory mechanism of the 14 MAGs in DLBCL. Screening for hub genes in the risk model by WGCNA Experimental verification of hub gene expression and their relationships with prognosis and the immune microenvironment in the DLBCL TMA cohort Next, we preliminarily verified the two hub genes using mIHC in our own TMA cohort. First, we compared DLBCL tissues with benign reactive hyperplasia tissues, and we found that the expression levels of PLTP and PHKA1 were both significantly increased in DLBCL patients (Fig. 14A and Fig. 15A). Next, X-tile analysis of 5-year OS was performed using the TMA cohort to determine the optimal cutoff values for PLTP and PHKA1 expression. According to the optimal cutoff value for each gene, we divided the 104 DLBCL samples in the TMA cohort into high- and low-expression groups. Relevant grouping information and clinicopathological features are shown in Tables 3-4. We then studied the differences in the tumor immune microenvironment between the pairs of groups. Regarding PLTP, the high-expression group had worse prognosis and higher infiltration of M2 macrophages and tumor-associated macrophages (TAMs) compared to the low-expression group (Fig. 14B-D and Fig. 16B, D). Moreover, PLTP expression was positively correlated with clinical stage (Table 3) and immune checkpoints PD-1 and PD-L1, but not significantly correlated with LAG3 (Fig. 14E-G and Fig. 17B). Likewise, regarding PHKA1, the high- expression group had worse prognosis and higher infiltration of M2 macrophages and TAMs compared Page 11/28 Page 11/28 to the low-expression group (Fig. 15B-D and Fig. 16C-D). Moreover, PHKA1 expression was positively correlated with immune checkpoints PD-1 and PD-L1, but not significantly correlated with clinical features and LAG3 (Fig. 15E-G and Fig. 17C). Finally, univariate and multivariate Cox regression analyses indicated that PLTP and PHKA1 were both independent prognostic factors for patients with DLBCL (Fig. 14H and Fig. 15H). Our verification results confirmed that these two genes were both overexpressed in DLBCL tissues, and their expression levels were closely related to the prognosis and immunosuppressive microenvironment of DLBCL. The conclusions were basically consistent with the results of our bioinformatics analyses. Discussion As previously mentioned, in view of the close relationship between metabolic reprogramming and the tumor immune microenvironment, we performed multiple immune analyses Page 12/28 Page 12/28 (ESTIMATE, ssGSEA, and CIBERSORT) to explore the differences in the immune landscape between the high- and low- risk groups. As expected, the high-risk group had a poor prognosis and an immunosuppressive microenvironment characterized by low immune score, low immune status, high abundance of immunosuppressive cells, and high expression of immune checkpoints. The low-risk group showed the opposite trend, with a better prognosis and a relatively immunopositive microenvironment. This is also consistent with our immune analysis of metabolism-associated molecular subtypes. An increased risk score indicates a “cold tumor” [22], with attenuated immunotherapy effectiveness and an immunosuppressive tumor microenvironment caused by metabolic reprogramming, which is consistent with poor prognosis. These conclusions further indicated that MAGs might play important roles in the altered immune response in DLBCL. (ESTIMATE, ssGSEA, and CIBERSORT) to explore the differences in the immune landscape between the high- and low- risk groups. As expected, the high-risk group had a poor prognosis and an immunosuppressive microenvironment characterized by low immune score, low immune status, high abundance of immunosuppressive cells, and high expression of immune checkpoints. The low-risk group showed the opposite trend, with a better prognosis and a relatively immunopositive microenvironment. This is also consistent with our immune analysis of metabolism-associated molecular subtypes. An increased risk score indicates a “cold tumor” [22], with attenuated immunotherapy effectiveness and an immunosuppressive tumor microenvironment caused by metabolic reprogramming, which is consistent with poor prognosis. These conclusions further indicated that MAGs might play important roles in the altered immune response in DLBCL. Notably, in the two groups with poor prognosis (cluster 1 and the high-risk group), in addition to the increase in the abundance of immunosuppressive cells and the expression of immune checkpoints, there was a significant increase in the infiltration of resting and activated NK cells. This is consistent with the results of previous studies, that is, an increased abundance of activated NK cells is associated with poor prognosis [23]. NK cell dysfunction is common in hematological cancer, and it is related to tumor immune escape [24]. We also found that KIR2DL1 and KIR2DL3 [25], the common immune checkpoints on NK cells, were also significantly overexpressed in cluster 1 and the high-risk group. Discussion The molecular heterogeneity of DLBCL brings great challenges to precision therapy. It is generally accepted that the traditional IPI score cannot adequately predict the prognosis of DLBCL, and developing more reliable strategies for subtype identification and risk stratification is urgent [3, 19]. In this study, we identified two metabolism-associated molecular subtypes, and there were significant differences in prognosis and the immune microenvironment between these two subtypes. In addition, we developed a prognostic risk model based on 14 MAGs. We found that it was a powerful independent prognostic tool with better predictive performance than the IPI score and was closely related to the immunosuppressive microenvironment. Finally, we identified two hub genes among the model genes, and preliminarily verified them in our own TMA cohort using mIHC. Our results may contribute to the development of accurate immunotherapy for DLBCL that targets metabolic pathways. Consensus clustering is an unsupervised clustering method that can identify different molecular subtypes according to a gene expression matrix [20]. Using consensus clustering, we identified two metabolism-associated molecular subtypes, which had significant differences in prognosis and the immune microenvironment. Compared to cluster 2, the prognosis of the patients in cluster 1 was poor, accompanied by a high abundance of immunosuppressive cells and a general increase in the expression of immune checkpoints, indicating an immunosuppressive microenvironment. This is consistent with findings regarding other malignancies [6, 13, 14, 21]. As the consensus clustering was based on a MAG expression matrix, we inferred that the expression of MAGs was related to the prognosis and immunosuppressive microenvironment of DLBCL patients. To further evaluate the prognostic value of the MAGs, we established a 14-gene risk model in the GEO training cohort by univariate Cox regression and LASSO regression. We then constructed a prognostic nomogram that integrated the risk score based on this model and all significant clinical features. The risk score effectively predicted prognosis in the GEO training cohort and was validated in a GEO internal validation cohort and a TCGA external validation cohort. ROC curve analysis confirmed that the risk score was superior to the traditional IPI score. Multiple validation methods indicated the robustness of the risk model, and it is reasonable to believe that this risk model will be broadly applicable for individualized risk management. Discussion In the future, immunotherapy that blocks KIR2DL1/KIR2DL3 might reduce the abundance of activated NK cells. Most of the MAGs in the risk model have been reported to be associated with cancer. To identify the most critical genes, i.e., the hub genes, among the 14 model genes for further experimental verification, we used the WGCNA algorithm to select key genes and then identified the overlapping genes among these genes and the model genes. As a result, we identified two hub genes: PLTP and PHKA1. The potential mechanisms of these two hub genes in DLBCL deserve further discussion. Phospholipid transfer protein (PLTP) is a widely expressed lipid transfer protein that belongs to the lipopolysaccharide (LPS)-binding/lipid transfer gene family. PLTP can promote the transfer of a series of lipid molecules, including diacylglycerol, phosphatidic acid, sphingomyelin, phosphatidylcholine, phosphatidylglycerol, brain glycosides, and phosphatidylethanolamine. These transport functions play an important role in lipid and lipoprotein metabolism [26, 27]. PLTP is differentially expressed in many kinds of tumors, such as prostate cancer [27], ovarian cancer [28], breast cancer [29], lung cancer [30], gastric cancer [31] and glioma [32]. Such a wide range of cancer types with differential expression of PLTP indicate that PLTP may be an important regulator of some common processes related to tumors. The phosphorylase kinase regulatory subunit alpha 1 (PHKA1) gene encodes the muscle-type isoform of the PHK alpha subunit [33]. PHKA1 plays a key role in glycogen metabolism [34] and PHKA1 mutations cause glycogen storage disease type 9D, also known as X-linked muscle glycogenosis [35]. However, research on PHKA1 in tumors is still limited. Research has shown that PHKA1, as an important gene related to glycogen metabolism, is related to the metastasis of prostate cancer [36]. In addition, the Page 13/28 increased expression of PHKA1 was associated with younger ages of gastrointestinal stromal tumor patients [37]. We further preliminarily validated the two hub genes in our TMA cohort using mIHC, which can quantify immune cells in the tumor microenvironment more objectively than traditional semi-quantitative methods [38]. Our verification results confirmed that the two hub genes were both overexpressed in DLBCL tissues. Thereafter, using X-tile (a valuable tool for outcome-based cutoff optimization) [39] and the 5-year OS of patients, we determined the optimal cutoff value for PLTP and PHKA1 expression. Discussion Based on each cutoff value, we subdivided the DLBCL patients into high- and low-expression groups, and further studied the differences in the tumor immune microenvironment between the pairs of groups. We found that the prognosis of the high-expression groups was poorer, accompanied by an immunosuppressive microenvironment characterized by higher abundances of immunosuppressive cells (M2 macrophages and TAMs) and higher expression of immune checkpoints (PD-L1 and PD-1). Finally, univariate and multivariate Cox regression analyses indicated that PLTP and PHKA1 were both independent prognostic factors in DLBCL. These experimental results showed that high expression of the hub genes was closely related to the prognosis and immunosuppressive microenvironment of DLBCL, which was consistent with our bioinformatics analyses, and further verified the stability and accuracy of the risk model. Studies have shown that metabolic reprogramming is an important feature of immune cell activation. Immune cells have different metabolic characteristics, which affect their immune function [16, 18]. Macrophages, as the main immune-infiltrating cells in solid tumors, can polarize into inflammatory (M1) or immunosuppressive (M2) phenotypes based on external stimuli. M1 macrophages have pro- inflammatory and anti-tumor effects, while M2 macrophages have anti-inflammatory and pro-tumor effects [40]. The metabolic reprogramming of tumors can affect the polarization process of macrophages [41, 42]. For example, hypoxia and lactic acid accumulation can promote the production of immunosuppressive M2 macrophages. The increase in tumor glycolysis produces a large amount of lactic acid, and the accumulation of lactic acid drives macrophages toward the M2 phenotype. M2 macrophages overexpress arginase 1 (ARG1). ARG1 consumes L-arginine, which is necessary for cytotoxic T lymphocytes to exert anti-tumor activity, and produces polyamines with strong immunosuppressive effects [18, 43]. Additionally, hypoxia promotes tumor development by inducing the production of angiogenic factors, mitogenic factors, and cytokines related to tumor metastasis in macrophages [9]. Additionally, macrophages can undergo lipid-based metabolic reprogramming to promote tumor progression via increased membrane cholesterol efflux [44, 45]. Moreover, M2 macrophages up-regulate fatty acid oxidation, mitochondrial respiration, and angiogenesis, thereby promoting tumor progression [9, 46]. Our mIHC results also confirmed that M2 macrophages in DLBCL patients with high metabolic gene expression were significantly increased. Therefore, M2 macrophages may have potential as immunotherapy targets. Our study also showed that the expression of most immune checkpoints significantly increased with increasing risk score, indicating an immunosuppressive microenvironment that was consistent with poor prognosis. Recent research has shown that checkpoint signals regulate metabolism [18]. Conclusions In this study, we identified two metabolism-associated DLBCL subtypes and constructed a risk model based on MAGs. Our risk model is related to the tumor immune microenvironment and prognosis of DLBCL. This study provides a new 14-gene signature for predicting the prognosis of DLBCL, which may facilitate personalized treatment strategies, and provides an important basis for further study on MAGs and the immune microenvironment. Discussion For example, Page 14/28 Page 14/28 PD-L1 in tumor cells can activate the PI3K-Akt-mTOR pathway, stimulate glycolysis, and enhance glucose uptake by the tumor cells [47]. CD155-TIGIT signaling in T cells of human gastric cancer inhibits glucose uptake, lactic acid production, and glycolytic enzyme expression [48]. Therefore, combining metabolic inhibitors with checkpoint inhibitors is expected to improve the efficacy of checkpoint blockade [49, 50]. Powell's team showed that a glutamine metabolism inhibitor not only improved the immunosuppressive microenvironment, but also effectively reversed PD-1 inhibitor resistance when combined with a PD-1 inhibitor [50]. Our research has some unique advantages. In this study, two metabolism-associated DLBCL subtypes were identified, and a risk model based on MAGs was constructed. We used multiple validation methods to evaluate the model: first, we tested the model in a GEO internal testing cohort, then in a TCGA external validation cohort, and finally we identified two hub genes and carried out preliminary verification in our own TMA cohort. Satisfactory results were obtained from the multiple validation methods, confirming the robustness and accuracy of the risk model. In addition, we not only studied the predictive performance of the risk model, but also explored the effect of MAG expression on the tumor immune microenvironment in DLBCL. Limitations Although our findings have potential clinical significance, there are still some limitations. First, the clinical features extracted from the GEO and TCGA databases were limited, as they did not include potential prognostic factors such as smoking and background diseases. Second, this is a retrospective study, and an independent prospective cohort is needed to verify the risk model established in this study. Finally, the value of the two hub genes as potential pharmacological targets needs to be further investigated. Funding This study was supported by the National Natural Science Foundation of China Grants (No. 81570184 to WS). This study was supported by the National Natural Science Foundation of China Grants (No. 81570184 to WS). Availability of data and materials All the data corresponding to the DLBCL series used in this study are available in GEO (https://www.ncbi.nlm.nih.gov/geo) and TCGA (https://portal.gdc.cancer.gov/), which are public functional genomics data repositories. Abbreviations DLBCL, diffuse large B cell lymphoma; MAGs, metabolism-associated genes; GEO, Gene Expression Omnibus; LASSO, least absolute shrinkage and selection operator; TCGA, The Cancer Genome Atlas; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSVA, gene set variation analysis; ESTIMATE, Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data; CIBERSORT, Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts; ssGSEA, single- sample Gene Set Enrichment Analysis; ceRNA, competing endogenouse RNA; WGCNA, weighted gene co- Page 15/28 Page 15/28 expression network analysis; TMA, tissue microarray; mIHC, multiple fluorescence immunohistochemistry; ROC, receiver operating characteristic; IPI, International Prognostic Index; NCBI, National Center for Biotechnology Information; IC50, half maximal inhibitory concentration; AUC, area under the curve; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; MHC, major histocompatibility complex; PD-1, programmed death-1; PD-L1, programmed cell death-1 ligand 1. expression network analysis; TMA, tissue microarray; mIHC, multiple fluorescence immunohistochemistry; ROC, receiver operating characteristic; IPI, International Prognostic Index; NCBI, National Center for Biotechnology Information; IC50, half maximal inhibitory concentration; AUC, area under the curve; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; MHC, major histocompatibility complex; PD-1, programmed death-1; PD-L1, programmed cell death-1 ligand 1. Acknowledgments We are grateful to the patients for their contributions to this study. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The informed consent of all patients was obtained before the study. The Ethics Committee of the Affiliated Hospital of Nantong University approved this research. Authors’ contributions WS, CZ and JH designed the study. WS, JH, ZC, QX, PS, YW and CZ conducted the experiments and data analysis. WS and JH wrote the manuscript. All authors read and approve the final manuscript. References Page 16/28 1. Patriarca A, Gaidano G. Investigational drugs for the treatment of diffuse large B-cell lymphoma. Expert Opin Investig Drugs. 2021;30(1):25–38. Page 16/28 2. Ennishi D, Hsi ED, Steidl C, Scott DW. Toward a new molecular taxonomy of diffuse large B-cell lymphoma. Cancer Discov. 2020;10(9):1267–281. 3. Wight JC, Chong G, Grigg AP, Hawkes EA. Prognostication of diffuse large B-cell lymphoma in the molecular era: moving beyond the IPI. Blood Rev. 2018;32(5):400–15. 4. Kamel HFM, Al-Amodi H. Exploitation of gene expression and cancer biomarkers in paving the path to era of personalized medicine. Genom Proteom Bioinform. 2017;15(4):220–35. 5. Ahluwalia P, Kolhe R, Gahlay GK. The clinical relevance of gene expression based prognostic signatures in colorectal cancer. Biochim Biophys Acta Rev Cancer. 2021;1875(2):188513. 6. Ye Z, Zou S, Niu Z, Xu Z, Hu Y. A novel risk model based on lipid metabolism-associated genes predicts prognosis and indicates immune microenvironment in breast cancer. Front Cell Dev Biol. 2021;9:691676. 7. Ohshima K, Morii E. Metabolic reprogramming of cancer cells during tumor progression and metastasis. Metabolites. 2021;11(1):28. 8. Faubert B, Solmonson A, DeBerardinis RJ. Metabolic reprogramming and cancer progression. Science (New York). 2020;368(6487):eaaw5473. 9. Tong Y, Gao WQ, Liu Y. Metabolic heterogeneity in cancer: an overview and therapeutic implications. Biochim Biophys Acta Rev Cancer. 2020;1874(2):188421. 10. Peng X, Chen Z, Farshidfar F, Xu X, Lorenzi PL, Wang Y, et al. Molecular characterization and clinical relevance of metabolic expression subtypes in human cancers. Cell Rep. 2018;23(1):255–69.e4. 11. Sinkala M, Mulder N, Patrick Martin D. Metabolic gene alterations impact the clinical aggressiveness and drug responses of 32 human cancers. Commun Biol. 2019;2:414. 12. Lin D, Fan W, Zhang R, Zhao E, Li P, Zhou W, et al. Molecular subtype identification and prognosis stratification by a metabolism-related gene expression signature in colorectal cancer. J Transl Med. 2021;19(1):279. 13. Yu S, Hu C, Cai L, Du X, Lin F, Yu Q, et al. Seven-gene signature based on glycolysis is closely related to the prognosis and tumor immune infiltration of patients with gastric cancer. Front Oncol. 2020;10:1778. 14. Qian H, Lei T, Hu Y, Lei P. Expression of lipid-metabolism genes is correlated with immune microenvironment and predicts prognosis in osteosarcoma. Front Cell Dev Biol. 2021;9:673827. 14. Qian H, Lei T, Hu Y, Lei P. Expression of lipid-metabolism genes is correlated with immune microenvironment and predicts prognosis in osteosarcoma. Front Cell Dev Biol. 2021;9:673827. 15. References Lei X, Lei Y, Li JK, Du WX, Li RG, Yang J, et al. Immune cells within the tumor microenvironment: biological functions and roles in cancer immunotherapy. Cancer Lett. 2020;470:126–33. 15. Lei X, Lei Y, Li JK, Du WX, Li RG, Yang J, et al. Immune cells within the tumor microenvironment: biological functions and roles in cancer immunotherapy. Cancer Lett. 2020;470:126–33. 16. Li X, Wenes M, Romero P, Huang SC, Fendt SM, Ho PC. Navigating metabolic pathways to enhance antitumour immunity and immunotherapy. Nat Rev Clin Oncol. 2019;16(7):425–41. 16. Li X, Wenes M, Romero P, Huang SC, Fendt SM, Ho PC. Navigating metabolic pathways to enhance antitumour immunity and immunotherapy. Nat Rev Clin Oncol. 2019;16(7):425–41. 17. Wang JX, Choi SYC, Niu X, Kang N, Xue H, Killam J, et al. Lactic acid and an acidic tumor microenvironment suppress anticancer immunity. Int J Mol Sci. 2020;21(21):8363. 17. Wang JX, Choi SYC, Niu X, Kang N, Xue H, Killam J, et al. Lactic acid and an acidic tumor microenvironment suppress anticancer immunity. Int J Mol Sci. 2020;21(21):8363. 18. Leone RD, Powell JD. Metabolism of immune cells in cancer. Nat Rev Cancer. 2020;20(9):516–31. 18. Leone RD, Powell JD. Metabolism of immune cells in cancer. Nat Rev Cancer. 2020;20(9):516–31. Page 17/28 Page 17/28 19. Wright GW, Huang DW, Phelan JD, Coulibaly ZA, Roulland S, Young RM, et al. A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications. Cancer Cell. 2020;37(4):551–68.e14. 20. Wilkerson MD, Hayes DN. ConsensusClusterPlus: a class discovery tool with confidence assessments and item tracking. Bioinf (Oxford England). 2010;26(12):1572–73. 21. Qi J, Liu Y, Hu J, Lu L, Dou Z, Dai H, et al. Identification of FPR3 as a unique biomarker for targeted therapy in the immune microenvironment of breast cancer. Front Pharmacol. 2020;11:593247. 22. Galon J, Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov. 2019;18(3):197–218. 23. Zhou H, Zheng C, Huang DS. A prognostic gene model of immune cell infiltration in diffuse large B- cell lymphoma. PeerJ. 2020;8:e9658. 24. Vo DN, Alexia C, Allende-Vega N, Morschhauser F, Houot R, Menard C, et al. NK cell activation and recovery of NK cell subsets in lymphoma patients after obinutuzumab and lenalidomide treatment. Oncoimmunology. 2018;7(4):e1409322. 25. Sim MJ, Stowell J, Sergeant R, Altmann DM, Long EO, Boyton RJ. References Myopathy and phosphorylase kinase deficiency caused by a mutation in the PHKA1 gene. Am J Med Genet A. 2005;133a(1):82–4. 35. Herbert M, Goldstein JL, Rehder C, Austin S, Kishnani PS, Bali DS. Phosphorylase kinase deficiency. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. GeneReviews®. Seattle WA: University of Washington;1993-2021. 36. Chen J, Cao S, Situ B, et al. Metabolic reprogramming-based characterization of circulating tumor cells in prostate cancer. J Exp Clin Cancer Res. 2018;37(1):127. 37. Prakash S, Sarran L, Socci N, DeMatteo RP, Eisenstat J, Greco AM, et al. Gastrointestinal stromal tumors in children and young adults: a clinicopathologic, molecular, and genomic study of 15 cases and review of the literature. J Pediatr Hematol Oncol. 2005;27(4):179–87. 38. Schalper KA, Brown J, Carvajal-Hausdorf D, McLaughlin J, Velcheti V, Syrigos KN, et al. Objective measurement and clinical significance of TILs in non-small cell lung cancer. J Natl Cancer Inst. 2015;107(3):dju435. 39. Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 2004;10(21):7252–59. 40. Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol. 2020;15:123–47. 41. Mehla K, Singh PK. Metabolic regulation of macrophage polarization in cancer. Trends Cancer. 2019;5(12):822–34. 42. Biswas SK. Metabolic reprogramming of immune cells in cancer progression. Immunity. 2015;43(3):435–49. 43. Paolini L, Adam C, Beauvillain C, Preisser L, Blanchard S, Pignon P, et al. Lactic acidosis together with GM-CSF and M-CSF induces human macrophages toward an inflammatory protumor phenotype. Cancer Immunol Res. 2020;8(3):383–95. 44. Xiang Y, Miao H. Lipid metabolism in tumor-associated macrophages. Adv Exp Med Biol. 2021;1316:87–101. 45. Goossens P, Rodriguez-Vita J, Etzerodt A, Masse M, Rastoin O, Gouirand V, et al. Membrane cholesterol efflux drives tumor-associated macrophage reprogramming and tumor progression. Cell Metab. 2019;29(6):1376–89.e4. 46. Liu Y, Xu R, Gu H, Zhang E, Qu J, Cao W, et al. Metabolic reprogramming in macrophage responses. Biomark Res. 2021;9(1):1. 47. Chang CH, Qiu J, O'Sullivan D, Buck MD, Noguchi T, Curtis JD, et al. Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell. 2015;162(6):1229–41. 47. Chang CH, Qiu J, O'Sullivan D, Buck MD, Noguchi T, Curtis JD, et al. Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell. 2015;162(6):1229–41. 48. He W, Zhang H, Han F, Chen X, Lin R, Wang W, et al. References KIR2DL3 and KIR2DL1 show similar impact on licensing of human NK cells. Eur J Immunol. 2016;46(1):185–91. 26. Albers JJ, Vuletic S, Cheung MC. Role of plasma phospholipid transfer protein in lipid and lipoprotein metabolism. Biochim Biophys Acta. 2012;1821(3):345–57. 27. Yazdanyar A, Yeang C, Jiang XC. Role of phospholipid transfer protein in high-density lipoprotein- mediated reverse cholesterol transport. Curr Atheroscler Rep. 2011;13(3):242–48. 28. Kuk C, Kulasingam V, Gunawardana CG, Smith CR, Batruch I, Diamandis EP. Mining the ovarian cancer ascites proteome for potential ovarian cancer biomarkers. Mol Cell Proteomics. 2009;8(4):661–69. 29. Bianchini G, Qi Y, Alvarez RH, et al. Molecular anatomy of breast cancer stroma and its prognostic value in estrogen receptor-positive and -negative cancers. J Clin Oncol. 2010;28(28):4316–23. 30. Rohrbeck A, Borlak J. Cancer genomics identifies regulatory gene networks associated with the transition from dysplasia to advanced lung adenocarcinomas induced by c-Raf-1. PLoS ONE. 2009;4(10):e7315. 31. Huang K, Chen S, Xie R, Jiang P, Yu C, Fang J, et al. Identification of three predictors of gastric cancer progression and prognosis. FEBS Open Bio. 2020;10(9):1891–99. 31. Huang K, Chen S, Xie R, Jiang P, Yu C, Fang J, et al. Identification of three predictors of gastric cancer progression and prognosis. FEBS Open Bio. 2020;10(9):1891–99. 32. Dong W, Gong H, Zhang G, Vuletic S, Albers J, Zhang J, et al. Lipoprotein lipase and phospholipid transfer protein overexpression in human glioma cells and their effect on cell growth, apoptosis, and migration. Acta Biochim Biophys Sin (Shanghai). 2017;49(1):62–73. 32. Dong W, Gong H, Zhang G, Vuletic S, Albers J, Zhang J, et al. Lipoprotein lipase and phospholipid transfer protein overexpression in human glioma cells and their effect on cell growth, apoptosis, and migration. Acta Biochim Biophys Sin (Shanghai). 2017;49(1):62–73. 33. Davidson JJ, Ozçelik T, Hamacher C, Willems PJ, Francke U, Kilimann MW. cDNA cloning of a liver isoform of the phosphorylase kinase alpha subunit and mapping of the gene to Xp22.2-p22.1, the region of human X-linked liver glycogenosis. Proc Natl Acad Sci U S A. 1992;89(6):2096–100. 33. Davidson JJ, Ozçelik T, Hamacher C, Willems PJ, Francke U, Kilimann MW. cDNA cloning of a liver isoform of the phosphorylase kinase alpha subunit and mapping of the gene to Xp22.2-p22.1, the region of human X-linked liver glycogenosis. Proc Natl Acad Sci U S A. 1992;89(6):2096–100. Page 18/28 34. Wuyts W, Reyniers E, Ceuterick C, Storm K, de Barsy T, Martin JJ. References CD155T/TIGIT signaling regulates CD8(+) T-cell metabolism and promotes tumor progression in human gastric cancer. Cancer Res. 2017;77(22):6375–88. Page 19/28 Page 19/28 49. Schaer DA, Geeganage S, Amaladas N, Lu ZH, Rasmussen ER, Sonyi A, et al. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy. Clin Cancer Res. 2019;25(23):7175–88. 50. Leone RD, Zhao L, Englert JM, Sun IM, Oh MH, Sun IH, et al. Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science. 2019;366(6468):1013–21. 50. Leone RD, Zhao L, Englert JM, Sun IM, Oh MH, Sun IH, et al. Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science. 2019;366(6468):1013–21. 50. Leone RD, Zhao L, Englert JM, Sun IM, Oh MH, Sun IH, et al. Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science. 2019;366(6468):1013–21. Tables Tables 1-4 are available in the Supplementary Files section. Tables 1-4 are available in the Supplementary Files section. Figures Figure 1 Figure 1 Flow chart of the data analyzing process Page 20/28 Figure 2 Consensus clustering. (A) Consensus matrix heatmap indicating that the optimal value for consensus clustering is K=2. (B) Heatmap visualizing the different expression pattern of the 92 MAGs in the two subgroups. (C) Survival curve of the patients in the two subgroups. Figure 2 Consensus clustering. (A) Consensus matrix heatmap indicating that the optimal value for consensus clustering is K=2. (B) Heatmap visualizing the different expression pattern of the 92 MAGs in the two subgroups. (C) Survival curve of the patients in the two subgroups. Consensus clustering. (A) Consensus matrix heatmap indicating that the optimal value for consensus clustering is K=2. (B) Heatmap visualizing the different expression pattern of the 92 MAGs in the two subgroups. (C) Survival curve of the patients in the two subgroups. Page 21/28 Page 21/28 Figure 3 Comparison of the immune infiltration and the expression of immune checkpoints among two clusters (A) CIBERSORT analysis in the two clusters. (B) the expression of immune checkpoints among two clusters. P values were showed as: ns not significant; P < 0.05; P < 0.01; *P < 0.001. Figure 3 Comparison of the immune infiltration and the expression of immune checkpoints among two clusters. (A) CIBERSORT analysis in the two clusters. (B) the expression of immune checkpoints among two clusters. P values were showed as: ns not significant; P < 0.05; P < 0.01; P < 0.001. Figure 3 Figure 3 Comparison of the immune infiltration and the expression of immune checkpoints among two clusters. (A) CIBERSORT analysis in the two clusters. (B) the expression of immune checkpoints among two clusters. P values were showed as: ns not significant; P < 0.05; P < 0.01; P < 0.001. Comparison of the immune infiltration and the expression of immune checkpoints among two clusters. (A) CIBERSORT analysis in the two clusters. (B) the expression of immune checkpoints among two clusters. P values were showed as: ns not significant; P < 0.05; P < 0.01; P < 0.001. Page 22/28 Figure 4 Functional analyses based on 92 prognostic MAGs. (A) Lollipop Chart for GO enrichment (BP, biological process; CC, cellularcomponent; MF, molecular function). (B) Barplot graph for KEGG pathways. (C) PPI analysis showing the protein interaction networkof MAGs. Page 23/28 Page 23/28 Figure 5 Construction and validation of the metabolic risk model in the GSE10846 cohort. (A-C) LASS analysis revealingthe minimum criteria (A, B) and coefficients (C). (D, E) The distribution of ri survival status, and 14 MAGs expression profilesfor the training cohort (D) and testing cohor Kaplan–Meier curves indicated that the high-risk group had worse overall survival than the lo Figure 8 Construction and validation of a predictive nomogram. (A) Cox univariate and multivariate regression analysis revealed that the risk score was an independent prognostic factor for DLBCL patients. (B) Nomogram for predicting the 3- and 5-year OSof DLBCL patients. (C) Calibration curves of the nomogram for OS prediction at 3- and 5- year. (D) ROC curves indicating the comparisons of the risk score and the IPI score in predicting 1-year OS. Figure 7 (A-G) Relationships between the risk score and clinicopathological features (including age, ECOG status, stage, LDH level, IPI score, gender and extranodal sites). Figure 5 Construction and validation of the metabolic risk model in the GSE10846 cohort. (A-C) LASSO regression analysis revealingthe minimum criteria (A, B) and coefficients (C). (D, E) The distribution of risk score, survival status, and 14 MAGs expression profilesfor the training cohort (D) and testing cohort (E). (F, G) Kaplan–Meier curves indicated that the high-risk group had worse overall survival than the low-risk group Page 24/28 Page 24/28 in the training cohort (F) and testing cohort (G). (H, I) Receiver operating characteristic (ROC) curvesfor predicting the 1/2/3-year overall survival in the training cohort (H) and testing cohort (I). Figure 6 External validation of the robustness of the metabolic risk model in the TCGA cohort. (A) The distribution of risk score, survival status, and 14 MAGs expression profiles for the TCGA cohort. (B) Kaplan–Meier curves indicated that the high-risk group had worse overall survival than the low-risk group in the TCGA cohort. (C) ROC curves for predicting the 1/2/3-year overall survival in the TCGA cohort. Figure 9 The tumor immune microenvironment in the training cohort. (A) ESTIMATE algorithm. (B) ssGSEA analysis. (C) CIBERSORT analysis. (D) Correlation between risk score and immune cell content. p values were showed as: ns not significant; p < 0.05; p < 0.01; p < 0.001. The tumor immune microenvironment in the training cohort. (A) ESTIMATE algorithm. (B) ssGSEA analysis. (C) CIBERSORT analysis. (D) Correlation between risk score and immune cell content. p values were showed as: ns not significant; p < 0.05; p < 0.01; p < 0.001. The tumor immune microenvironment in the training cohort. (A) ESTIMATE algorithm. (B) ssGSEA analysis. (C) CIBERSORT analysis. (D) Correlation between risk score and immune cell content. p values were showed as: ns not significant; p < 0.05; p < 0.01; p < 0.001. Figure 10 Figure 10 Page 25/28 The expression of immune-regulatory genes and the correlation betweeTAPBP n the risk score and immune checkpoints in the training cohort. (A) Immune-regulatory genes expression in the high- and low- rBTLA isk group. (B) Immune checkpoint expression and (C) its correlation with the risk score. p values were showed as: ns not significaCD160CD244 nt; p < 0.05; p < 0.01; p < 0.001. The expression of immune-regulatory genes and the correlation betweeTAPBP n the risk score and immune checkpoints in the training cohort. (A) Immune-regulatory genes expression in the high- and low- rBTLA isk group. (B) Immune checkpoint expression and (C) its correlation with the risk score. p values were showed as: ns not significaCD160CD244 nt; p < 0.05; p < 0.01; *p < 0.001. Figure 12 (A) The ceRNA network associated with the model genes. The red dots represent mRNAs, the green dots represent miRNAs and the blue dots represent lncRNAs. Figure 11 Prediction of drug sensitivity and signaling pathways in high- and low- risk groups. (A) Sensitivity analysis of common therapeutic drugs in patients of two groups. (B) GSVA analysis for differentially expressed signaling pathways between two groups. Page 26/28 Page 26/28 Figure 13 Construction of weighted co-expression network and identification of the hub genes. (A) Dendrogram of patients in GSE10846 dataset clustered according to risk group. (B) Soft-thresholding power selection in the WGCNA analysis. (C) Correlation between modules and traits. Blue represents the negative correlation and red represents the positive correlation. (D) Venn diagram analysis: overlapping MAGs from WGCNA analysis and LASSO model. Figure 14 Experimental verification of PLTP expression and its relationship with prognosis and the immune microenvironment in the DLBCL TMA cohort. (A) Differences in PLTP expression between reactive hyperplasia tissues and DLBCL tissues with mIHC. (B) Kaplan–Meier curve for the PLTP high- and low- expression groups in our TMA cohort. The optimal cutoff point was obtained from X-tile 3.6.1 software. (C, D) Immune cell content in the PLTP high- and low- expression groups. CD68+CD163+ indicated the content of M2 macrophages (C) and CD11B+ indicated the content of TAMs (D). (E-G) Correlations between PLTP expression and immune checkpoints (including PD-1, PD-L1, and LAG3). (H) Cox univariate and multivariate regression analysis revealed that PLTP was an independent prognostic factor for DLBCL patients. Figure 17 Prediction and TMA verification of the relationships between the two hub genes and immune checkpoints. (A) Prediction ofcorrelations between hub genes and immune checkpoints. (B,C) Characterization of cell immunophenotypes with mIHC. A staining panel was developed to visualize DAPI, PD-1, PD-L1, LAG3 and PLTP simultaneously on the same tissue slide(B). A staining panelwas developed to visualize DAPI, PD-1, PD-L1, LAG3 and PHKA1 simultaneously on the same tissue slide(C).Scale bar=100 µm. CD274 indicated the content of PD-L1 and PDCD1 indicated the content of PD-1. Figure 16 Prediction and TMA verification of the relationships between the two hub genes and immune cell content. (A) Prediction of correlations between hub genes and immune cell content. (B-D) Characterization of cell immunophenotypes with mIHC. A staining panel was developed to visualize DAPI, CD68, CD163 and PLTP simultaneously on the same tissue slide (B). A staining panel wasdeveloped to visualize DAPI, CD68, CD163 and PHKA1 simultaneously on the same tissue slide (C). A staining panel was developedto visualize DAPI, CD11B, PLTP and PHKA1 simultaneously on the same tissue slide (D). Scale bar=100 µm. CD68+CD163+ indicated the content of M2 macrophages and CD11B+ indicated the content of TAMs. Figure 15 Experimental verification of PHKA1 expression and its relationship with prognosis and the immune microenvironment in the DLBCL TMA cohort. (A) Differences in PHKA1 expression between reactive hyperplasia tissues and DLBCL tissues with mIHC. (B) Kaplan–Meier curve for the PHKA1 high- and low- expression groups in our TMA cohort. The optimal cutoff point was obtained from X-tile 3.6.1 software. (C, D) Immune cell content in the PHKA1 high- and low- expression groups. CD68+CD163+ indicated the content of M2 macrophages (C) and CD11B+ indicated the content of TAMs (D). (E-G) Correlations between PHKA1 expression and immune checkpoints (including PD-1, PD-L1, and LAG3). (H) Cox Page 27/28 univariate and multivariate regression analysis revealed that PHKA1 was an independent prognostic factor for DLBCL patients. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table1.pdf Table2.pdf Table3.pdf Table4.pdf FigS1.pdf FigS2.pdf FigS3.pdf Table1.pdf Table2.pdf Page 28/28
https://openalex.org/W4381051928	https://www.qeios.com/read/OBGF08/pdf	English	null	Review of: "Richir’s Phenomenological and Symbolical as a Model for the Relationship between Phenomenology and Theology"	null	2,023	cc-by	144	Qeios, CC-BY 4.0 · Review, June 17, 2023 Review of: "Richir’s Phenomenological and Symbolical as a Model for the Relationship between Phenomenology and Theology" Jorge Roggero1 1 University of Buenos Aires Potential competing interests: No potential competing interests to declare. Qeios ID: OBGF08 · https://doi.org/10.32388/OBGF08 Potential competing interests: No potential competing interests to declare. The article presents an original and sound hypothesis based on Gondek and Tengelyi's suggestion. It is well structured and very clear about its aims and objectives. The arguments are well supported. Nevertheless the text can be improved with references to the secondary literature (there are no references to articles on Richir's phenomenology). On the other hand, it can also be improved with a more detailed analyses of the relevance of Janicaud's thesis on Marion, who actually assumes a "methodological atheism" strategy in Being given. Qeios ID: OBGF08 · https://doi.org/10.32388/OBGF08 1/1
https://openalex.org/W2751388403	https://serval.unil.ch/resource/serval:BIB_6469004280EE.P001/REF.pdf	English	null	Phylogenetic approaches to identifying fragments of the same gene, with application to the wheat genome	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	8,681	Abstract Motivation: As the time and cost of sequencing decrease, the number of available genomes and transcriptomes rapidly increases. Yet the quality of the assemblies and the gene annotations varies considerably and often remains poor, affecting downstream analyses. This is particularly true when fragments of the same gene are annotated as distinct genes, which may cause them to be mistaken as paralogs. Results: In this study, we introduce two novel phylogenetic tests to infer non-overlapping or partial- ly overlapping genes that are in fact parts of the same gene. One approach collapses branches with low bootstrap support and the other computes a likelihood ratio test. We extensively validated these methods by (i) introducing and recovering fragmentation on the bread wheat, Triticum aesti- vum cv. Chinese Spring, chromosome 3B; (ii) by applying the methods to the low-quality 3B as- sembly and validating predictions against the high-quality 3B assembly; and (iii) by comparing the performance of the proposed methods to the performance of existing methods, namely Ensembl Compara and ESPRIT. Application of this combination to a draft shotgun assembly of the entire bread wheat genome revealed 1221 pairs of genes that are highly likely to be fragments of the same gene. Our approach demonstrates the power of ﬁne-grained evolutionary inferences across multiple species to improving genome assemblies and annotations. g g Availability and implementation: An open source software tool is available at https://github.com/ DessimozLab/esprit2. Contact: c.dessimoz@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. Ivana Pilizota1,2, Cle´ment-Marie Train3,4,5, Adrian Altenhoff5, Henning Redestig6,† and Christophe Dessimoz 1,2,3,4,5,* 1Department of Genetics Evolution & Environment and 2Department of Computer Science, University College London, UK, 3Department of Computational Biology and 4Center for Integrative Genomics University of Lausanne, Lausanne, Switzerland, 5Swiss Institute of Bioinformatics, Biophore Building, Lausanne, Switzerland and 6Bayer CropScience NV, Ghent, Belgium To whom correspondence should be addressed †Present address: Genencor International BV, Leiden, The Netherlands Associate Editor: Janet Kelso To whom correspondence should be addressed Received on September 20, 2017; revised on July 30, 2018; editorial decision on August 29, 2018; accepted on August 31, 2018 well-established short-read sequencing protocols are cost effective and widely applied. However, without reads that are long enough to span repetitive regions, the assembly step remains a challenge with negative consequences on downstream analyses (Jiao and Schneeberger, 2017; Lee and Tang 2012). well-established short-read sequencing protocols are cost effective and widely applied. However, without reads that are long enough to span repetitive regions, the assembly step remains a challenge with negative consequences on downstream analyses (Jiao and Schneeberger, 2017; Lee and Tang 2012). y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Bioinformatics, 35(7), 2019, 1159–1166 doi: 10.1093/bioinformatics/bty772 Advance Access Publication Date: 1 September 2018 Original Paper Bioinformatics, 35(7), 2019, 1159–1166 doi: 10.1093/bioinformatics/bty772 Advance Access Publication Date: 1 September 2018 Original Paper Phylogenetics Downloaded from https://academic.oup.com/bioinformatics/article-abstract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 2.1 Phylogenetic tests of split genes Given a genome assembly with a large number of annotated contigs, the task we face is to figure out which annotated genes actually be- long to the same gene, due to annotation mistakes or where the as- sembler failed to concatenate collinear contigs. Consider therefore two non-overlapping fragments of the same gene sequence. If we perform a multiple sequence alignment of the two fragments to- gether with full-length homologs from other species, and infer a tree based on the alignment, we can expect that the two fragments: (i) align to different regions of the multiple sequence alignment (since they are non-overlapping) and (ii) have a similar evolutionary dis- tance to homologous sequences in other genomes. One problem in low-quality genome assemblies is that fragments of the same gene can be annotated as distinct entries in genome data- bases; thus such fragments may be wrongly taken to be paralogs. However, it is possible to use homologous proteins conserved in other genomes to detect fragments that are likely to be part of the same gene. To our knowledge, four such approaches have been pro- posed. First, the Ensembl Compara pipeline (Herrero et al., 2016; Vilella et al., 2009) infers pairs of apparent paralogs as a ‘gene_split’, if they lie within one megabase on the same strand of the same region of the assembly and do not overlap in the multiple sequence alignment of the family. Restricting these predictions to genes belonging to the same contig greatly reduces the risk of false positive split gene calling, but particularly for fragmented assemblies with many short contigs, this approach detects only a fraction of all splits. Second, ESPRIT (Dessimoz et al., 2011) uses pairwise com- parisons to identify non-overlapping pairs of paralogs that have a similar evolutionary distance to homologous sequences in other genomes. The third approach, SWiPS (Li and Copley, 2013) is con- ceptually similar in that it also works based on pairwise align- ments—by identifying sets of non-overlapping candidate sequences that have a maximal sum of score with homologous sequences in other genomes. The fourth approach is the computationally efficient PEP_scaffolder (Zhu et al., 2016), which relies on high-identity matches of reference proteins to multiple contigs. Thus, like ESPRIT and SWiPS, the approach relies on pairwise alignments. It also has the strength of considering a maximum intron length to avoid com- bining gene fragments that are unrealistically far apart. 1 Introduction Thanks to rapid developments in sequencing technology (reviewed in Goodwin et al., 2016), individual laboratories now routinely se- quence and assemble entire genomes and transcriptomes. The most 1159 V C The Author(s) 2018. Published by Oxford University Press. V C The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/license unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1160 I.Pilizota et al. 2.2 Test #1: collapsing ‘insignificant’ branches Tree branch support measures are commonly used to gauge the reli- ability of a branch. Since fragments of the same genes can be expected to be separated by insignificant internal branches on the tree, collapsing branches with low support should result in frag- ments becoming sister leaves. Thus, for a given threshold, the test collapses all branches below that threshold and infers all candidates that are sister leaves as fragments of the same gene. Here, to address this problem, we present two complementary phylogenetic methods to identify non-overlapping or partially over- lapping fragments of the same gene that exploit evolutionary rela- tionships across gene families. The first one exploits bootstrap support, and the second relies on likelihood ratio tests. We evaluate their performance on an artificially fragmented version of the refer- ence sequence assembly of the hexaploid bread wheat chromosome 3B (Choulet et al., 2014). We also compare the two methods, and a meta-approach combining the two methods with ESPRIT (Dessimoz et al., 2011), with the Ensembl Compara pipeline and ESPRIT. Finally, we apply new phylogeny-based methods to the early, highly fragmented, draft release of the entire hexaploid wheat genome (International Wheat Genome Sequencing Consortium (IWGSC), 2014) and identify 1221 high-confidence pairs of split genes. 2 Algorithm The challenge of genome assembly is particularly acute in plants, which tend to have large and heavily redundant genomes (Claros et al., 2012; Jiao and Schneeberger, 2017). Data from such genomes frequently result in fragmentary assemblies with overestimated gene counts (Denton et al., 2014) and limited utility for downstream pur- poses such as creation of physical maps used in marker assisted breeding. Fragmentary genes not only lack sequence information— they have been shown to cause problems in downstream analyses, such as in tree inference (Sayyari et al., 2017) and orthology infer- ence (Dalquen et al., 2013; Train et al., 2017). We first introduce our phylogenetic tests of split genes, then proceed to describe the datasets analysed and the evaluation methods. Note that we provide the fine implementations details in the Supplementary materials. 2.3 Test #2: likelihood ratio test The second test to infer fragments of the same gene is a likelihood ratio test (LRT). Our null hypothesis (labelled ‘s’ for split) is that fragments come from the same gene, and thus can be concatenated into one sequence. The alternative hypothesis (called ‘p’ for paral- ogs) is that the two fragments belong to paralogous genes. Hs: n 1 genes (split gene) 2.1 Phylogenetic tests of split genes However, perhaps surprisingly at first sight, although these frag- ments will generally be close to one another on the tree, they will al- most never be inferred as sister leaves. The reason for this is that since they have no character in common, they cannot be directly compared with each other, only with the other genes in the tree. Thus, there is no phylogenetic information available to infer the re- lationship between them. The location of the split between the two sequences is therefore undetermined. Furthermore, recall that evolu- tion is modelled as a stochastic process on a tree, with each column in the alignment being a realization of the process. Due to this sto- chastic nature, the two fragments will almost never attach to the exact same place on the tree. act/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 Under the correct model of evolution, however, if the two frag- ments originate from the same sequence, the difference in the place these are attached to the tree should not be significant. Here, we introduce two tests to infer whether two non- overlapping or partially overlapping sequences from the same gen- ome are fragments of the same gene: collapsing branches with low bootstrap support (Efron et al., 1996) and a likelihood ratio test (Wilks, 1938). Yet for all of these methods, the correct identification of split genes heavily depends on their ability to distinguish fragments of the same gene from fragments of paralogous ones. Ensembl Compara and PEP_scaffolder make no attempt to distinguish between the two. As for ESPRIT and SWiPS, although they attempt to identify fragments that match reference proteins consistently—either by requiring consistent evolutionary distances to the reference for all fragments or by requiring consistent best matches for all frag- ments—these comparisons are inherently limited to pairwise com- parison, which loses out on phylogenetic information available in a multiple-sequence and tree setting. 3 Implementation As input candidate pairs, we identify, among all the protein sequen- ces of a target genome, those that belong to the same gene family— either established by Ensembl Compara or defined as deepest hier- archical orthologous groups (HOGs) as inferred by OMA (Altenhoff et al., 2013). We further require that fragments be non-overlapping, or overlapping with less than 10% residues of both fragment being aligned in the same alignment column, using Mafft v7.164b (Katoh and Standley, 2013). In other words, we require that a12 < 0.1l1 AND a12 < 0.1l2, where l1 and l2 are the number of residues in the two fragments, and a12 is the number of these residues that are aligned. Thus, for each gene family, we align the sequences, enumer- ate all possible pairs of sequences belonging to the target genome and retain as candidate pairs those that satisfy the aforementioned overlap requirement. Fig. 1. Conceptual overview of the likelihood ratio test. The null hypothesis is that the two fragments come from the same gene (Hs) while the alternative hypothesis is that the two fragments come from different paralogous copies (Hp). a refers to the signiﬁcance level, which is the area under the curve above the rejection threshold. This setup is motivated by the fact that the split gene hypothesis has fewer parameters. However, it is unusual in that failure to re- ject the test leads to a prediction, and not the other way round. Furthermore, because the two models are not nested, we estimate the null distribution empirically Fig. 1. Conceptual overview of the likelihood ratio test. The null hypothesis is that the two fragments come from the same gene (Hs) while the alternative hypothesis is that the two fragments come from different paralogous copies (Hp). a refers to the signiﬁcance level, which is the area under the curve above the rejection threshold. This setup is motivated by the fact that the split gene hypothesis has fewer parameters. However, it is unusual in that failure to re- ject the test leads to a prediction, and not the other way round. Furthermore, because the two models are not nested, we estimate the null distribution empirically The LRT requires computing maximum likelihood estimates, i.e. finding an optimal tree under both Hs and Hp. Under the Hs hypothesis, fragments are part of the same gene. 3.1 Datasets and evaluation methodology A f l i d li i f 2. Since we have no prior knowledge on the distribution of the test statistic under the null hypothesis, we estimate the distribution using non-parametric bootstrapping. First, from the multiple se- quence alignment used under the Hs, we generate n artiﬁcial alignments of the same length, i.e. n bootstrap samples by sam- pling columns with replacement. Second, we create alignments to be used under the Hp by splitting a target full-length gene (i.e. the one made up of two candidate fragments) at the same pos- ition as in the original alignment. Finally, we compute the test statistic for each of the n samples; let’s denote them by T1, T2, . . ., Tn. If the sampling is correct, the distribution of Ti, i ¼ 1, 2,. . ., n will converge to the true distribution of the test statistic when n ﬁ‘. Hence, if repeated many times, the distribution of the bootstrap sample test statistic values will approximate the distribution of the unknown test statistic. Throughout this pro- ject, we set n to 100 unless otherwise stated. 2. Since we have no prior knowledge on the distribution of the test statistic under the null hypothesis, we estimate the distribution using non-parametric bootstrapping. First, from the multiple se- quence alignment used under the Hs, we generate n artiﬁcial alignments of the same length, i.e. n bootstrap samples by sam- pling columns with replacement. Second, we create alignments to be used under the Hp by splitting a target full-length gene (i.e. the one made up of two candidate fragments) at the same pos- ition as in the original alignment. Finally, we compute the test statistic for each of the n samples; let’s denote them by T1, T2, 2. Since we have no prior knowledge on the distribution of the test statistic under the null hypothesis, we estimate the distribution using non-parametric bootstrapping. First, from the multiple se- quence alignment used under the Hs, we generate n artiﬁcial alignments of the same length, i.e. n bootstrap samples by sam- pling columns with replacement. Second, we create alignments to be used under the Hp by splitting a target full-length gene (i.e. the one made up of two candidate fragments) at the same pos- ition as in the original alignment. Hp: n genes (paralogous genes) The test statistic is defined as T ¼ 2ln LðHPÞ LðHSÞ ; where L() denotes the maximum estimator under each hypothesis (Fig. 1). Phylogenetic approaches to identifying fragments of the same gene 1161 Fig. 1. Conceptual overview of the likelihood ratio test. The null hypothesis is that the two fragments come from the same gene (Hs) while the alternative hypothesis is that the two fragments come from different paralogous copies (Hp). a refers to the signiﬁcance level, which is the area under the curve above the rejection threshold. This setup is motivated by the fact that the split gene hypothesis has fewer parameters. However, it is unusual in that failure to re- ject the test leads to a prediction, and not the other way round. Furthermore, because the two models are not nested, we estimate the null distribution empirically 3. Compute bootstrap p value as the proportion of samples with likelihood equal or above that of the input data: pB ¼ fNo: of Ti T0g n 3. Compute bootstrap p value as the proportion of samples with likelihood equal or above that of the input data: pB ¼ fNo: of Ti T0g n 3 Implementation Compute the value of the test statistic; let’s denote it by T0 3 Implementation Hence, in order to find a maximum-likelihood tree under the Hs, we concat- enate the candidate fragments into a single sequence. To correct for some cases when a tree-building method gives a suboptimal tree, which may result in the estimated T0 < 0, we performed two tree searches under the Hp model; a tree search without providing an input topology, and a tree search with an input topology start- ing with the best tree under Hs with the two hypothetical frag- ments as sister leaves), and proceeded with the tree with higher likelihood. This setup is unusual in two respects. First, considering that we aim to predict split genes, it may come as a little suspect that we use Hs as null model. Our motivation for this is however that the model with split genes is more constrained (the two fragments sit at the same place in the tree) and thus has fewer free parameters. It is the ‘simpler’ model. For this reason, in absence of evidence to the con- trary, we deem it reasonable to favour Hs. Second, in a typical set- ting of the likelihood ratio test, the null model is a special case of the alternative model. The models are said to be ‘nested’, and theory tells us that the test statistic—twice the difference in log likeli- hood—is v2 distributed. Since our models are not nested, the distri- bution of the test statistic given under the assumption that Hs is true is unknown. We can bypass this problem by estimating the empirical distribution under the null using bootstrapping (Efron and Tibshirani 1993; Goldman 1993). Hence, for a particular sam- ple, we: ract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 Some genes might be involved in multiple predictions, i.e. in more than one pair of fragments coming from a split gene. If all these multiple predictions span different parts of the sequences, we conclude that the gene is split in more than two pieces and consider these predictions as non-ambiguous. If in contrast more than one prediction spans over a common part of the sequence (which might be the case if the fragments come from very closely related paralogs, or if alternative splicing variants of the same gene are erroneously annotated as separate genes), we report the overlapping predictions as ambiguous. 1. Compute the value of the test statistic; let’s denote it by T0 1. 3.1 Datasets and evaluation methodology A f l i d li i f Finally, we compute the test statistic for each of the n samples; let’s denote them by T1, T2, As a test case for evaluation and application of the methods, we used the proteome of bread wheat, i.e. Triticum aestivum cv. Chinese Spring. The bread wheat genome is notoriously large (17 Gbp) and redundant: it is a hexaploid genome which arose from two recent allopolyploidization events—with the three subgenomes referred to as A, B and D. Because of this large size and redundancy, the wheat genome is proving very difficult to assemble and annotate. In 2014, the International Wheat Genome Sequencing Consortium (IWGSC) published a highly fragmented chromosome-by- chromosome survey sequence of the bread wheat genome (International Wheat Genome Sequencing Consortium (IWGSC), 2014). The same year, Choulet et al. (2014) published a high-quality reference sequence of bread wheat chromosome 3B (third chromo- some of subgenome B). The two provide a good basis to evaluate our methodology on a challenging dataset. . . ., Tn. If the sampling is correct, the distribution of Ti, i ¼ 1, 2,. . ., n will converge to the true distribution of the test statistic when n ﬁ‘. Hence, if repeated many times, the distribution of the bootstrap sample test statistic values will approximate the distribution of the unknown test statistic. Throughout this pro- ject, we set n to 100 unless otherwise stated. 1162 I.Pilizota et al. We also tested our method on the cassava genome, i.e. Manihot esculenta, draft version 4.1 (Prochnik et al., 2012) retrieved from Phytozome v7 (Goodstein et al., 2012). rather distant species, we proceeded with families containing no more than 100 genes where at least two of them were cassava genes and other genes were assigned to at least nine distinct reference spe- cies. To calculate recall and precision, we randomly chose 200 cas- sava genes and 200 pairs of cassava paralogous genes, each of them being at least 200 amino-acids long, which we fragmented the same way as in the case of wheat. All alignments and trees were recon- structed with the same software and settings as for the wheat. As customary in the field, we determine the quality of the meth- ods by measuring the precision and recall. Here the recall measures the proportion of fragmented genes that the methods can identify. The precision penalizes for erroneous predictions by measuring the proportion of predictions that are indeed fragmented genes. 3.3 Introducing non-overlapping paralogs in wheat 3B reference assembly (precision) To inspect cases where the methods incorrectly predict split genes, we simulated fragments from pairs of paralogs assigned to the bread wheat 3B reference sequence using the same datasets as above. We chose 100 pairs of same-species paralogs, cut them at a random pos- ition and took two complementary fragments (one from each initial gene) each being at least 50 amino-acids long. Again, MSAs were obtained by Mafft v7.164b (default parameters) and gene trees by FastTree v2.1.8 (default parameters). 3.2 Random fragmentation of the wheat 3B reference assembly (recall) For the predictions, we used the same reference species as in the simulations on HOGs (see sections 3.2 and 3.3) which we again exported from OMA Browser (Supplementary Table S3). We com- puted gene families by the GETHOGs algorithm with a default set of parameters. We generated 500 bootstrap samples for each family and performed both tests on fragments overlapping less than 10%. Sequences were aligned with Mafft v7.164b (default parameters) and trees built with FastTree v2.1.8 (default parameters) as above. In addition, we also computed HOGs with a different set of parame- ters and repeated the rest of the experiment. To determine the recall of the methods, we simulated fragmentation on genes assigned to a high-quality assembly of bread wheat chromosome 3B (3B reference sequence). All genes and their gene families were obtained from Ensembl Plants, release 31. We ran- domly chose 100 genes, each at least 100 amino-acids long, and split them at a random position such that both fragments are at least 50 amino-acids long. All alignments were performed using Mafft v7.164b with default parameters. Gene trees were built by FastTree v2.1.8 (Price et al. 2010), also with a default set of parameters. e-abstract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 For the assessment, the mapping of sequences between the survey and high-quality genomes was not straightforward because the two differ not only in the degree of fragmentation, but also in some of the sequences themselves due to sequencing error, contamination etc. To allow for a bit of tolerance while still maintaining unambigu- ous mapping between the two, we required hits to cover at least 95% of the corresponding query, the percentage identity in these matching regions to be at least 95%, and the hit to be unambiguous. As a stringent control, we also performed a validation where, in addition to these two requirements, we only allowed mismatches to occur at the ends of a query sequence. In addition, we simulated fragmentation in a more challenging setting, i.e. on small gene families typically containing only evolu- tionarily very close paralogs. As a source of homologous groups, we used HOGs. They were computed by the GETHOGs algorithm with a default set of parameters on the input dataset comprised of 13 plants: bread wheat and 12 flowering plants exported from OMA Browser (Altenhoff et al. 2014) (Supplementary Table S1). 3.5 Validation on 3B survey assembly To assess predictions on the real data containing fragmented genes, we applied our approaches to a low-quality assembly of bread wheat chromosome 3B, the 3B survey sequence (IWGSP1; 2013-11-MIPS), and compared the predictions with the high-quality assembly of chromosome 3B (‘3B reference sequence’) downloaded from URGI (https://urgi.versailles.inra.fr). As a gold standard, we mapped sequences between the two assemblies using BLASTþ v2.2.30 (Camacho et al. 2009). Finally, we applied the methods to the rest of the wheat survey sequence to infer split genes in the bread wheat proteome. 3.6 Comparison to established methods and meta-approach As a point of comparison, we employed the Ensembl Compara pipe- line and ESPRIT on the same 3B survey sequence as above. Again, the obtained predictions from each method were mapped to the 3B reference sequence by BLASTþ v2.2.30 to inspect if predicted pairs belong to the same gene or not, requiring both coverage and percent- age identity to be at least 95%. Validated predictions were com- pared to the results from Validation experiment on 3B survey sequence with the same BLASTþ criteria. Similarly as above, we also simulated more challenging cases of fragmentation. We used the same set of HOGs as in the previous section. To obtain a comparable set of predictions on the 3B survey se- quence using public results available from the Ensembl Compara pipeline, we filtered ‘gene_split’ pairs from their homologies file (re- lease 21). We took only pairs where both genes were at least 50 amino-acids long and such that, when its corresponding gene family was aligned with Mafft v7.164b, candidate genes overlapped for less than 10%. We also included cases where more than two genes were inferred as a part of the same gene given that no two genes involved overlapped for 10% or more. Since some of the sequences could not be found in the OMA Browser dataset used for validating Collapsing and LRT approach, we classified Ensembl predictions 3.1 Datasets and evaluation methodology A f l i d li i f For both measures, we simulated fragmentation on the wheat 3B refer- ence sequence and cassava genome. In a subsequent experiment, we applied the tests to the wheat 3B survey sequence and validated pre- dictions using the wheat 3B reference sequence. In this case the total number of fragmented genes is unknown, so we could only count the number of correct and wrong predictions, and calculate the precision. 3.4 Introducing fragmentation in cassava assembly (recall, precision) To inspect behavior of the tests on species other than wheat, we introduced random fragmentation in cassava draft proteome (v4.1) downloaded from Phytozome v7. As reference species, we used the 16 other dicot species available in OMA Browser, Dec 2017 release (Supplementary Table S2), of which the closest species is the western balsam poplar, which has diverged from cassava approximately 80 MYA (Fawcett et al., 2009). Gene families were again obtained by running GETHOGs algorithm with default settings. Having a set of 1163 Phylogenetic approaches to identifying fragments of the same gene into two groups: those that could be found in the OMA Browser dataset, and hence, included in the comparison, and those that could not. two phylogenetic methods: one based on collapsing branches with low bootstrap support and the other relying on a likelihood ratio test (LRT). To evaluate the methods and determine parameters for predictions on the bread wheat assembly, we took two approaches. First, we simulated fragmentation on the real data to calculate recall and precision. Then, we applied both methods to the bread wheat chromosome 3B survey sequence and validated predictions with re- spect to the 3B reference sequence. Finally, based on the best param- eters obtained from these analyses, we applied the method to infer split genes in the 20 other chromosomes of the survey wheat genome assembly. Another set of predictions was obtained by running ESPRIT on the same 3B survey sequence data using 12 reference plants (the same dataset as in the Validation section, Supplementary Table S3) keeping all parameters default but increasing the required length of the candidate genes to be at least 50 amino-acids (option ‘MinSeqLenContig ¼ 50’). We only considered a confident unam- biguous set of predictions (reported in the hits.txt output file). In addition, we considered a meta-approach ESPRIT 2.0, which encompasses ESPRIT and the new combined approach. It takes the union of predictions made by ESPRIT and our joint method (col- lapsing branches with support lower than 0.95 and likelihood ratio test with significance of 0.01). 4.1 Artificial fragmentation of the wheat 3B reference assembly To assess our methods, we first simulated fragmentation in 100 pro- tein sequences from the high-quality wheat 3B reference assembly and tried to recover these pairs. Our simulations also included 100 pairs of non-overlapping fragments generated from pairs of random- ly selected paralogous genes—which can be very difficult negative cases if the paralogs are near-identical. 4 Results Recall that we aim to identify fragments of the same gene wrongly annotated as separate genes in a genome of interest, leveraging genomes of related species. In the previous section, we introduced (a) (b) (c) (a) (b) (c) Fig. 2. Evaluation of the methods. (a) Wheat genes from the high-quality wheat 3B chromosome were artiﬁcially fragmented and recovered by the collapsing, like- lihood ratio test (LRT) and a combination of the two. Numbers indicate the threshold used for each datapoint. (b) Split genes inferred on the low-quality (‘survey’) wheat genome were validated using the high-quality wheat 3B, and comparison with three other approaches (Ensembl Compara, ESPRIT and the meta-method). Numbers indicate the threshold used for each datapoint. The meta-method takes union of ESPRIT’s and the predictions inferred when combining collapsing ap- proach (threshold 0.95) and LRT (signiﬁcance 0.01). (c) The number of predictions on 3B survey sequence classiﬁed as correct in the BLASTþ validation. ’New ap- proach’ denotes a combination (intersection) of collapsing approach (threshold 0.95) and LRT (signiﬁcance 0.01) (a) (c) (b) (c) Fig. 2. Evaluation of the methods. (a) Wheat genes from the high-quality wheat 3B chromosome were artiﬁcially fragmented and recovered by the collapsing, like- lihood ratio test (LRT) and a combination of the two. Numbers indicate the threshold used for each datapoint. (b) Split genes inferred on the low-quality (‘survey’) wheat genome were validated using the high-quality wheat 3B, and comparison with three other approaches (Ensembl Compara, ESPRIT and the meta-method). Numbers indicate the threshold used for each datapoint. The meta-method takes union of ESPRIT’s and the predictions inferred when combining collapsing ap- proach (threshold 0.95) and LRT (signiﬁcance 0.01). (c) The number of predictions on 3B survey sequence classiﬁed as correct in the BLASTþ validation. ’New ap- proach’ denotes a combination (intersection) of collapsing approach (threshold 0.95) and LRT (signiﬁcance 0.01) Fig. 2. Evaluation of the methods. (a) Wheat genes from the high-quality wheat 3B chromosome were artiﬁcially fragmented and recovered by the collapsing, like- lihood ratio test (LRT) and a combination of the two. Numbers indicate the threshold used for each datapoint. (b) Split genes inferred on the low-quality (‘survey’) wheat genome were validated using the high-quality wheat 3B, and comparison with three other approaches (Ensembl Compara, ESPRIT and the meta-method). Numbers indicate the threshold used for each datapoint. 3.7 Inferring split genes on the rest of the wheat survey assembly Finally, we employed the tests to infer fragmented genes in the first draft release of the predicted genes in whole bread wheat genome, i.e. T. aestivum cv. Chinese Spring proteome (IWGSP1; 2013-11- MIPS). We considered only candidate fragments assigned to the same chromosome and the same chromosome arm. We used the same reference genomes as in the previous analyses with HOGs (see above). Based on simulations and validation on the 3B survey se- quence, we determined a set of parameters used for predictions. In particular, we ran GETHOGs with default parameters and allowed candidate fragments to mutually overlap less than 10% in the corre- sponding MSA. We used Mafft v7.164b to get alignments and FastTree v2.1.8 to construct trees, both with their default set of parameters. Finally, we chose 0.95 as a threshold for collapsing and set the significance threshold of the LRT to 0.01. On these challenging simulations, the collapsing test yielded high precision (0.85–0.88) and moderate recall (0.20–0.58), while the LRT performed the other way round, yielding moderate precision (0.56–0.64) and high recall (0.81–0.99) (Fig. 2a; Supplementary File S1, Supplementary File esprit2_simulations.tar.gz). We also evaluated an approach that combines our two methods. A split gene was inferred if both methods were in agreement. This approach resembled the recall and precision of the collapsing ap- proach (with the same threshold) but with slightly higher precision (Fig. 2a, Supplementary File S1). As a control, we performed another set of simulations using a different set of input homologous sequences—OMA HOGs contain- ing protein sequences from thirteen plants including wheat (Supplementary File S1, Supplementary File esprit2_simulation- s.tar.gz). Precision of the collapsing test was again high (0.73–0.81) while recall varied between 0.30 and 0.78. Precision of the LRT was moderate to high (0.51–0.89) and the recall was high (0.70– 0.75) (Supplementary Fig. S3a). As additional controls, we also 4 Results The meta-method takes union of ESPRIT’s and the predictions inferred when combining collapsing ap- proach (threshold 0.95) and LRT (signiﬁcance 0.01). (c) The number of predictions on 3B survey sequence classiﬁed as correct in the BLASTþ validation. ’New ap- proach’ denotes a combination (intersection) of collapsing approach (threshold 0.95) and LRT (signiﬁcance 0.01) I.Pilizota et al. 1164 repeated the analysis by changing one parameter of the pipeline at a time: repeated the analysis by changing one parameter of the pipeline at a time: sequencing errors or 10% contamination from other chromosomes (International Wheat Genome Sequencing Consortium (IWGSC), 2014). If we only consider fragments that can be perfectly mapped between the draft whole-genome assembly and the reference assem- bly (no mismatch in their central part, see Supplementary materials), the number of predictions that could be validated diminishes, but on the remaining set, our approaches showed even higher precision (Supplementary Fig. S3c and d), indicating that the performance reported in Figure 2b is conservative. 1. alignment mode in Mafft that relies on local alignments, which could conceivably deal better with fragments (L-INS-I instead of FFT-NS-2) 2. special mode in FastTree that reportedly deals better with frag- mentary genes (option ‘-pseudo’) 3. increasing the number of bootstrap replicates from 100 to 500 in the likelihood ratio test Control experiments also gave consistent results (Supplementary File S2, Supplementary File esprit2_validation.tar.gz). As expected, relaxing parameters yielded more predicted split genes, but at a cost of lower precision (Fig. 2b versus Supplementary Fig. S3b). 4. increasing the number of artiﬁcially introduced split genes from 100 to 500. All these variants yielded qualitatively similar results (Supplementary Fig. S4). 4.2 Artificial fragmentation of the cassava genome To assess whether our approach also works on a different set of spe- cies, we performed the artificial fragmentation analysis on 200 sin- gle and 200 paralogous pairs of randomly selected the cassava genes. As reference species, we used the 16 other dicot species avail- able in OMA version December 2017, of which the closest species is the Western balsam poplar, which has diverged from cassava ap- proximately 80 MYA (Fawcett et al., 2009). To gain further insights into the performance of the proposed approaches, we compared them to two existing methods, namely Ensembl Compara pipeline (which however cannot easily be run on custom genome data) and ESPRIT, as described in Section 3. Both methods were applied to the 3B survey sequence and then validated against the 3B reference sequence using BLASTþ (Supplementary File esprit2_comparison.tar.gz). We also considered a meta- approach, which we call ESPRIT 2.0, comprising ESPRIT and a combination of the collapsing approach (threshold 0.95) and LRT (significance 0.01). Using the combined approach of collapsing (threshold 0.95)þLRT(significance 0.01), we observed a recall of 63% and a pre- cision of 40% (Supplementary File esprit2_simulations_cassava.tar.gz). Inspection of the false positives revealed that nearly all mistakes were due to fragmentation of close (i.e. species-specific) paralogs, which to our method are indistinguishable from split genes. Thus, the lower pre- cision is explained by the much higher frequency of species-specific paralogs in cassava (75% of sampled paralogs in cassava versus 13% in wheat). Indeed, when we repeated the analysis excluding artificial fragmentation of such species-specific paralogs, the precision increased to 65%, in line with the results on wheat (Supplementary Fig. S4c). In terms of the number of correct predictions, Ensembl Compara and ESPRIT performed equally well or better than our approaches e-abstract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 (a) (b) Fig. 3. High-conﬁdence inferred gene splits on the wheat genome. A, B and D refer to the three subgenomes of the hexaploid wheat genome. (a) Number of unambiguous predictions for each chromosome arm. (b) Number of am- biguous predictions (i.e. for which there are more than two candidate frag- ments for a single juncture). Pairs of fragments are inferred separately for each chromosome arm of ﬂow-sorted Triticum aestivum cv. Chinese Spring, except chromosome 3B, for which the analysis was performed on the entire chromosome (a) (a) (b) (b) 5 Discussion and outlook Despite technological and algorithmic advances, genome assembly and annotation remain a challenge, especially for large polyploid genomes with complex evolutionary histories. Genes often remain fragmented and fragments get annotated as separate genes. Our work demonstrates that using available assemblies of related species can provide enough information to recognize some of those cases and obtain full-length genes. tract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 We developed two approaches and showcase their good per- formance on a challenging proteome of hexaploid bread wheat (T. aestivum cv. Chinese Spring). In simulations and validation, both of which were performed on the real data taking into account all its complexities, the approach relying on collapsing gene tree branches showed lower recall and higher precision than a likelihood ratio test (Fig. 2). As a trade-off between precision and recall, we propose tak- ing an intersection of their predictions, as we did in the quest for fragmented genes in the wheat survey sequence dataset. As our strin- gent simulation and real data assessment shows, the inferred split genes are highly specific. The performance is even better when we combine the new phylogeny-based tests to our earlier pairwise ap- proach ‘ESPRIT’. Throughout this project, we fixed some of the parameters. First, we considered only genes at least 50 amino-acids long. Shorter sequences contain less information thus make phylogeny reconstruc- tion more challenging; at the same time, the benefit of putting to- gether short fragments is also more limited. Second, we required candidate fragments to overlap less than 10%. Increasing the over- lap increases the number of candidate pairs and, consequently, the number of predictions including false positive and ambiguous pre- dictions. Finally, we used Mafft v7.164b to align gene families and FastTree v2.1.8 to reconstruct gene trees, both with their default parameters due to their convenience and speed. Exploring their par- ameter space or using more suitable tools for the dataset of interest could contribute to higher precision and recall. The two main inherent challenges of in silico split gene inference are the confounding effect of close paralogs and the variation in the rate of evolution along the sequences. Indeed, sometimes fragments come from identical or nearly identical paralogs, and there is not enough information to distinguish fragments belonging to one gene from another. Hence, we are more likely to make a false positive prediction (Supplementary Fig. S6). 4.3 Validation on 3B survey assembly To further assess the tests and identify suitable parameters, we applied our methods on the chromosome 3B of the draft-quality bread wheat survey genome (International Wheat Genome Sequencing Consortium (IWGSC), 2014). We focused on this chromosome arm because it was one for which a much higher- quality reference assembly was available, obtained through pains- taking piecewise sequencing and assembly using 8452 bacterial artificial chromosomes (Choulet et al., 2014). To give an idea of the improvement between the two, the N50 statistic, which measures the assembly quality by reporting the minimum contig/scaffold length needed to cover 50% of the assembly, is 2.7 kb for the draft genome 3B chromosome versus 892 kb for the high-quality 3B chromosome. Thus, the latter could be treated as ground truth— thus enabling us to gauge, in a realistic setup, how well our approaches can infer split genes in a highly fragmented genome with abundant potential paralogs. Overall, the methods achieved higher precision than when applied to simulated fragmentation (Fig. 2b). The analysis showed particularly high precision with the collapsing approach. The abso- lute recall rate could not be easily assessed on these real data; in- stead, we considered the number of correctly predicted HOG annotations as a surrogate for recall, yielding results highly consist- ent with the simulations (Fig. 2b). Fig. 3. High-conﬁdence inferred gene splits on the wheat genome. A, B and D refer to the three subgenomes of the hexaploid wheat genome. (a) Number of unambiguous predictions for each chromosome arm. (b) Number of am- biguous predictions (i.e. for which there are more than two candidate frag- ments for a single juncture). Pairs of fragments are inferred separately for each chromosome arm of ﬂow-sorted Triticum aestivum cv. Chinese Spring, except chromosome 3B, for which the analysis was performed on the entire chromosome One challenge with this setup was the fact that the draft survey sequence assembly contains other types of problems, such as Phylogenetic approaches to identifying fragments of the same gene 1165 displaying high precision (Fig. 2b and Supplementary Table S4). Further analysis showed that predictions from different methods are rather complementary and worthwhile to take into account (Supplementary Fig. S5). Hence, the meta-approach, ESPRIT 2.0, inferred by far the biggest number of correct predictions with high precision (Fig. 2b and Supplementary Table S4). sequences. Consider for instance a protein composed of two domains—one slowly evolving and one fast evolving. 4.5 Predictions on the rest of the survey assembly Finally, we applied our tests to infer split genes on the rest of the bread wheat genome, i.e. all chromosomes other than 3B. Based on the analyses on simulated fragmentation and between two assem- blies (see above), we determined parameters for the tests. For each chromosome arm, we obtained gene families by running OMA GETHOGs with default parameters. In the collapsing approach, we collapsed all branches with bootstrap support less than 0.95, and we performed the likelihood ratio test with the significance level of 0.01. The intersection of predictions identified 1442 pairs in total: 1221 unambiguous and 221 ambiguous cases. The distribution of the number of predictions per chromosome is shown in Figure 3 (see also Supplementary File S3) while fragment IDs are provided in Supplementary file esprit2_predictions_wheat.tar.gz. At a practical level, predictions heavily depend on the choice of two parameters: a threshold for collapsing branches and a signifi- cance level for the likelihood ratio test. Lower, more stringent thresh- olds for collapsing yield more confident predictions, while higher, less conserved thresholds will produce more predictions but less con- fident. Similarly, a higher significance of the likelihood ratio test will result with less but more confident predictions. Obtaining more pre- dictions can be achieved by lowering the significance of the test at the cost of their lower confidence. Overall, it is important to choose thresholds depending on the application. For example, a higher num- ber of predictions can be favourable for comparison with other data. Predictions also depend on the input families. Bigger gene families facilitate more predictions (Fig. 2, Supplementary Fig. S3) but also re- sult in more ambiguous calls, i.e. cases where a fragment is involved in multiple predictions (Supplementary File S2). We observed fewer false positive predictions when we simulated fragmentation on bigger gene families where we were more likely to randomly split a pair of more distant paralogs in comparison to small gene families which are more likely to contain only very close paralogs (Fig. 2a, Supplementary Fig. S3a). However, the results of validation indicate that the methods are still able to identify a reasonable number of split genes with high precision even when small gene families are used. 4.3 Validation on 3B survey assembly If we consider each domain as a distinct sequence and look at their position in a gene tree including full-length homologous counterparts, the branch lengths connecting these fragments to the rest of the tree may have markedly different lengths. As a consequence, the increase in likeli- hood obtained by having distinct branches for each fragment may be sufficiently large for our test to erroneously infer that the fragments come from distinct sequences (see Supplementary Table S5 for an ac- tual example). It may be possible to address this problem by more ex- plicitly modelling variation of rate among sites. Downloaded from https://academic.oup.com/bioinformatics/article-abstract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 5 Discussion and outlook , ( ) p g pp g p g regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii l h l h d h h f f regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes). Brief. Bioinform., 12, 474–484. (Holocephali, Chondrichthyes). Brief. Bioinform., 12, 474–484. (Holocephali, Chondrichthyes). Brief. Bioinform., 12, 474–484. Efron,B. et al. (1996) Bootstrap conﬁdence levels for phylogenetic trees. Proc. Natl. Acad. Sci. USA, 93, 7085–7090. Downloaded from https://academic.oup.com/bioinformatics/article-abstract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2 Efron,B., and Tibshirani,R.J. (eds) (1993) Hypothesis testing with the boot- I A I d i h B S i B MA But already in its present form, as the large number of detected split genes in the wheat genome illustrates, our approach is already proving highly useful. All computer code is available for reuse as a user-friendly package named ‘ESPRIT 2.0’ (https://github.com/ DessimozLab/esprit2) that we hope will help make phylogeny-based detection of split genes a routine step in genome assembly and annotation. strap. In: An Introduction to the Bootstrap. Springer, Boston, MA, pp. 220–236. Fawcett,J.A. et al. (2009) Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proc. Natl. Acad. Sci. USA, 106, 5737–5742. Goodstein,D.M. et al. (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res., 40, D1178–D1186. Goldman,N. (1993) Statistical tests of models of DNA substitution. J. Mol. Evol., 36, 182–198. Goodwin,S. et al. (2016) Coming of age: ten years of next-generation sequenc- ing technologies. Nat. Rev. Genet., 17, 333–351. Funding This work was supported by Bayer CropScience NV, University College London, the Swiss National Science Foundation [PP00P3_150654 to C.D.]; and the UK Biotechnology and Biological Sciences Research Council [BB/ L018241/1 to C.D.]. Katoh,K. and Standley,D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol., 30, 772–780. Lee,H. and Tang,H. (2012) Next-generation sequencing technologies and fragment assembly algorithms. Methods Mol. Biol., 855, 155–174. Conﬂict of Interest: none declared. stract/35/7/1159/5089230 by Universite and EPFL Lausanne user on 09 May 2019 Li,Y.I. and Copley,R.R. (2013) Scaffolding low quality genomes using orthol- ogous protein sequences. Bioinformatics, 29, 160–165. Acknowledgements Herrero,J. et al. (2016) Ensembl comparative genomics resources. Database, 2016, baw053. We thank Dr Natasha Glover for helpful feedback on the manuscript. Computations were performed on the University College London Computer Science cluster and at the high-performance computing center Vital-IT. International Wheat Genome Sequencing Consortium (IWGSC) (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science, 345, 1251788. Jiao,W.-B. and Schneeberger,K. (2017) The impact of third generation genom- ic technologies on plant genome assembly. Curr. Opin. Plant Biol., 36, 64–70. 5 Discussion and outlook This was particularly salient in the artificial fragmentation analyses on the cassava genome. Indeed, cassava is known to have undergone a whole-genome duplication around 35–47 MYA ago (Bredeson et al., 2016), which is not shared by another reference species in our analysis. As a result, many pairs of paralogs are specific to this species, and thus virtually indistin- guishable from fragments of the same gene by our method. As often with new approaches, the likelihood ratio test still has room for improvement. Currently, we compute the distribution of the test statistic empirically, via resampling. We computed up to 500 samples per test which, given the simulations and validation, seems to be enough here; yet the convergence of the distribution could be explored. Increasing the number of samples might lead to signifi- cantly better approximation of the distribution and more accurate results. In addition, parameterizing the distribution of the test statis- tic would reduce computational time and memory usage. Since both tests rely on evolutionary relationships, some of the mistakes could be avoided by implementing a more realistic evolution- ary model. This is of particular importance for cases which are missed due to differences in evolutionary rates across the length of the gene. As for the second main challenge, evolutionary rate heterogeneity across the protein length, this can pose problem because fragments of the same genes can wrongly appear to be coming from distinct To further improve the performance, one could try to find opti- mal parameters for the dataset of interest and application in 1166 I.Pilizota et al. Denton,J.F. et al. (2014) Extensive error in the number of genes inferred from draft genome assemblies. PLoS Comput. Biol., 10, e1003998. question. Different strategies could be used to obtain input families as well as alternative tools for alignments and methods with more exhaustive optimal tree search. For datasets with relatively close lev- els of divergence, tree inference based on nucleotide instead of amino-acid sequences might confer more statistical power to our tests. It may also be possible to exploit transcriptome data as add- itional source of information (Zhang et al., 2016). draft genome assemblies. PLoS Comput. Biol., 10, e1003998. Dessimoz,C. et al. (2011) Comparative genomics approach to detecting split-coding Dessimoz,C. et al. (2011) Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes). Brief. Bioinform., 12, 474–484. References Price,M.N. et al. (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One, 5, e9490. Altenhoff,A.M. et al. (2013) Inferring hierarchical orthologous groups from orthologous gene pairs. PLoS One, 8, e53786. Prochnik,S. et al. (2012) The cassava genome: current progress, future direc- tions. Trop. Plant Biol., 5, 88–94. Altenhoff,A.M. et al. (2014) The OMA orthology database in 2015: function predictions, better plant support, synteny view and other improvements. Nucleic Acids Res., 43, D1, D240–D249. Sayyari,E. et al. (2017) Fragmentary gene sequences negatively impact gene tree and species tree reconstruction. Mol. Biol. Evol., 34, 3279–3291. Train,C.-M. et al. (2017) Orthologous Matrix (OMA) algorithm 2.0: more ro- bust to asymmetric evolutionary rates and more scalable hierarchical orthol- ogous group inference. Bioinformatics, 33, i75–i82. Bredeson,J.V. et al. (2016) Sequencing wild and cultivated cassava and related species reveals extensive interspeciﬁc hybridization and genetic diversity. Nat. Biotechnol., 34, 562–570. Vilella,A.J. et al. (2009) EnsemblCompara GeneTrees: complete, duplication-aware phylogenetic trees in vertebrates. Genome Res., 19, 327–335. Camacho,C. et al. (2009) BLASTþ: architecture and applications. BMC Bioinformatics, 10, 421. Choulet,F. et al. (2014) Structural and functional partitioning of bread wheat chromosome 3B. Science, 345, 1249721. Wilks,S.S. (1938) The large-sample distribution of the likelihood ratio for test- ing composite hypotheses. Ann. Math. Stat., 9, 60–62. Claros,M.G. et al. (2012) Why assembling plant genome sequences is so chal- lenging. Biology, 1, 439–459. Zhang,S.V. et al. (2016) AGOUTI: improving genome assembly and annota- tion using transcriptome data. Gigascience, 5, 31. tion using transcriptome data. Gigascience, 5, 31. Dalquen,D.A. et al. (2013) The impact of gene duplication, insertion, deletion, lateral gene transfer and sequencing error on orthology inference: a simula- tion study. PLoS One, 8, e56925. Zhu,B.-H. et al. (2016) PEP_scaffolder: using (homologous) proteins to scaf- fold genomes. Bioinformatics, 32, 3193–3195.
https://openalex.org/W1574420429	http://old.scielo.br/pdf/reeusp/v24n3/0080-6234-reeusp-24-3-359.pdf	Portuguese	null	"ASSISTÊNCIA DE ENFERMAGEM PERIOPERATÓRIA — UM MODELO CONCEPTUAL"	Revista da Escola de Enfermagem da USP	1,990	cc-by	5,137	* Prof. Dr. do Departamento de Enfermagem em Saúde Coletiva. ** Prof. Dr. do Departamento de Enfermagem Médico-Cirúrgica. Escola de Enfermagem da U S P - S.P. "ASSISTÊNCIA DE ENFERMAGEM PERIOPERATÓRIA — UM MODELO CONCEPTUAL" Briguita Elza P. Castellanos * Vanda Maria Galvão Jouclas ** Briguita Elza P. Castellanos * Vanda Maria Galvão Jouclas ** CASTELLANOS, B.E.P. & JOUCLAS, V.M.G. Assistência de enfermagem periope- ratória: um modelo conceituai. Rev. Esc. Enf. USP, São Paulo, 24(3):359-370, dez. 1990. Este trabalho apresenta uma proposta de modelo conceituai de assistência de enfermagem perioperatória, modelo este utilizado no desenvolvimento do processo ensino-aprendizagem da disciplina Enfermagem em Centro Cirúrgico da Escola de Enfermagem da USP, em fase de operacionalização dentro do Programa de Integração Docente-Assistencial no Hospital Universitário da USP. O modelo é descrito a partir de suas bases filosóficas, as finalidades, os objetivos e sua operacionalização. UN1TERMOS: Enfermagem em centro cirúrgico. Cuidados perioperatórios. Kev. Esc. Enf U8P, São Paulo, 2£(3) :359-370, dez. 1990 INTRODUÇÃO Dentro da literatura de enfermagem nacional, já podemos contar com vários trabalhos na área da assistência transoperatória, que relacio- nam os problemas de enfermagem mais freqüentemente encontrados nesta fase KAMIYAMA (1972), SOUZA (1976), PANZA (1977), ME- NEZES (1978), RODRIGUES (1979) e FERRAZ (1981). Estes trabalhos apresentam com primeira prioridade, em relação aos "problemas sentidos" e às "expectativas de cuidado", o desenvolvi- mento do papel expressivo do enfermeiro do centro-cirúrgico, sendo a "segurança emocional" a necessidade básica mais afetada- concretizada por expressões do tipo: "falta e preocupação com a famüia; medo da anestesia, da cirurgia e da morte; medo do desconhecido; desatenção por parte dos elementos da equipe cirúrgica; necessidade de maiores orien* tações sobre as normas de vida pós-alta" e pelas expectativas de "ser tratado com delicadeza' receber mais amor e carinho, haver diálogo en- tre enfermeiro e cliente e obter mais companhia". Como segunda prioridade, aparecem nestes mesmos trabalhos, o de- senvolvimento do papel instrumental do enfermeiro do centro-cirúrgico, sendo a "segurança física" a necessidade básica mais afetada. Assim, a Kev. Esc. Enf U8P, São Paulo, 2£(3) :359-370, dez. 1990 359 competência profissional do enfermeiro estava expressa em termos de "receber os cuidados necessários", "ser observado adequadamente" e "receber bons cuidados". competência profissional do enfermeiro estava expressa em termos de "receber os cuidados necessários", "ser observado adequadamente" e "receber bons cuidados". A necessidade da intervenção profissional de enfermagem no centro cirúrgico frente a estas necessidades básicas afetadas, portanto, é fato comprovado não só pelos inúmeros trabalhos na área psicossomática do paciente cirúrgico, como também por estes estudos mencionados an- teriormente. Neste sentido, a assistência de enfermagem sistematizada é de re- levante importância no período perioperatório, como referem CASTEL- LANOS (1978) e CASTELLANOS & MANDELBAUM (1985) quando propõem a aplicação do processo de enfermagem ao cuidado do paciente durante a fase transoperatória tanto no que se refere ao preparo físico do paciente para o ato anestésico-cirúrgico, como também com o intuito de prepará-lo emocionalmente para enfrentar esta agressão. Para que isto ocorra o enfermeiro de centro cirúrgico necessita levar em conside- ração dois componentes básicos da enfermagem profissional: o modelo conceituai de enfermagem e a determinação da competência profissional do enfermeiro. INTRODUÇÃO O primeiro pode ser descrito como o guia que levará o enfermeiro determinar que dados coletar para fins de identificar o diagnóstico de enfermagem e a conseqüente intervenção e o segundo é caracterizado pelos conhecimentos cognitivos, afetivos e psicomotores ne- cessários para o atendimento das necessidades afetadas dos pacientes decorrentes do ato anestésico-cirúrgico propriamente dito. O enfermeiro, ser social que é, desenvolve seu exercício profissional em determinado espaço social- fazendo uso dos instrumentos da cultura da sua sociedade e produzindo conhecimentos e representações com fina- lidades determinadas. O enfoque do corpo de conhecimentos da enfermagem ao longo de sua história sempre esteve relacionado com a natureza do seu serviço, com os objetivos almejados, com a concreticidade de seu ambiente de trabalho e de seus clientes, bem como com a sua congruência com o sistema ideológico vigente. Os modelos de intervenção de enfermagem, ou seja, as maneiras como os fatores no componente "conhecimento" são estudados, dão significado à visão do cuidado de enfermagem, expli- citando o enfoque de sua prática na medida que deixam transparecer a concepção de homem, de enfermagem, do processo saúde-doença e das relações sociais e institucionais. Considerados como instrumentos de trabalho, os modelos de inter- venção têm um caráter mediador entre o enfermeiro e seu cliente e pressupõem um projeto que, através do trabalho (a própria intervenção de enfermagem) transforma o pensamento em ação. A maioria destes modelos surgiram- e continuam surgindo, em resposta a como os clientes são vistos pelos enfermeiros e pelas suas relações supra e infra estrutu- rais. Assim temos, entre outros- os modelos de: "resposta ao estresse" de LITTLE& CARNEVALI (1976) e NEUMAN (1979); "interação" de PEPLAU (1952); TRAVELBEE (1971); ORLANDO (1978); OREM (1980) e KING (1971); "resolução de problemas" de LEVTNE (1973), WATSON & MAYERS (1981) e STEVENS (1984); "necessidades hu- manas básicas" de YURA & WALSH (1967) e HORTA (1970) e de "sis- temas" de ROY (1976) e PAIM (1978) e aqueles mais corriqueiramente encontrados na maioria de nossos serviços de enfermagem, os modelos "médicos" e "hospitalares". INTRODUÇÃO Apesar de que a maioria dos enfermeiros de centro cirúrgico con- corde que o "modelo médico", orientado para sistemas biológicos e esta- dos patológicos "que hegemonia a autoridade do médico, induz o enfer- meiro a promover a confiança na relação médico-paciente e objetiva a manutenção de uma relação harmoniosa e confiante entre o enfermeiro e o médico, mesmo às expensas da relação enfermeiro^aciente'' MUR- FHY (1979) e o "modelo Jwspitalar", burocrático, funcionalista, reite- rativo, cujo objetivo é "manter a ordem social da instituição, mesma às expensas do bem estar individual do paciente e cuja ênfase é no tra-balho em equipe e na responsabilidade da equipe com desconsideração à interação enferm/eim-paciente e à responsabilidade individual". MUR- PHY (1979), e que não estimula e apoia a interação dos diferentes pro- fissionais e ocupacionais, não são adequados para a prática profissional da Enfermagem- estes são- ainda, os modelos mais utilizados na realida- de dos centros cirúrgicos de nossos hospitais. MODELO CONCEITUAL PROPOSTO A fim de contrapor este enfoque da prática do enfermeiro de centro cirúrgico caracterizada quase exclusivamente por atividades ditas admi- nistrativas, mas que se limitam às atividades burocráticas, médico-de- legadas e de outras áreas, tais como de Zeladoria, do Serviço de Manu- tenção, do Serviço de Limpeza, de Secretaria e assim por diante, e de intervenções de enfermagem baseadas nas disfunções sistêmicas ou or- gânicas, no diagnóstico e no procedimento terapêutico-médico, faz-se necessário que nós, enfermeiros de centro cirúrgico paremos para refle- tir e repensar nossa prática diária afim de que a mesma vá de encontro aos seus componentes profissionais. Como ponto de partida para esta reflexão, propomos um modelo conceitual-filosófico de assistência de enfermagem perioperatória, modelo este utilizado no desenvolvimento do processo ensino-aprendizagem da disciplina Enfermagem em Centro-Cirúrgico da Escola de Enfermagem da Universidade de São Paulo e em fase de operacionalização, dentro do Programa de Integração Docente-Assistencial no Hospital Universitário da USP. O alvo de atenção da enfermagem perioperatória- ou seja- o domí- nio de sua prática, origina-se das situações, condições ou fenômenos re- lacionados ao processo saúde-doença e conseqüentes à terapêutica mé- dica-cirúrgica (procedimento anestésico-cirúrgico) como um evento da vida do paciente. Esses problemas de saúde geralmente refletem a ne- cessidade do indivíduo alterar e/ou reforçar seu comportamento ou es- tilo de vida a fim de conseguir lidar com esta condição e melhorar seu estado de saúde. O enfermeiro de centro-cirúrgico para ser realmente útil ao paciente cirúrgico, deve ter algumas idéias e pensamentos acerca do serviço que presta (assistência de enfermagem perioperatória). tilo de vida a fim de conseguir lidar com esta condição e melhorar seu estado de saúde. O enfermeiro de centro-cirúrgico para ser realmente útil ao paciente cirúrgico, deve ter algumas idéias e pensamentos acerca do serviço que presta (assistência de enfermagem perioperatória). De acordo com CHIN & JACOBS (1983), no exercício da profissão temos um estilo de pensar acerca de situações de enfermagem, estilo este que geralmente utiliza conceitos para classificar eventos. Estes conceitos e suas pressuposições básicas podem proporcionar um modo de descrever o que os enfermeiros observam e vivenciam. Pela utilização de um sistema de conceitos, portanto, o enfermeiro observará, analisará e modificará os conceitos abrangidos e, assim, au- mentará a sua competência profissional. MODELO CONCEITUAL PROPOSTO Apesar da maioria dos enfermeiros nunca pensar na sua prática em termos de modelos de atuação, é inquestionável que todos têm um mo- delo a partir do qual desenvolvem seu exercício profissional. As bases destes modelos derivam, como já vimos, principalmente das crenças acerca do Homem e da Vida (p. ex. a saúde é de responsabilidade pessoal ou profissional?; o nosso enfoque é a quantidade ou a qualidade da vida?). Para definirmos um modelo para a nossa prática como docentes de Enfermagem em Centro Cirúrgico exploramos' primeiro- nossa filosofia pessoal acerca dos componentes essenciais da Enfermagem e suas rela- ções, os quais incluem, como indica FAWCETT (1978), o Homem, o ambiente, a saúde e a Enfermagem. Em seguida, elaboramos um modelo de atuação baseado nos conceitos de assistência integral, continuada, participativa, individualizada, documentada e avaliada, que é utilizado por nossos alunos do 3" ano do Curso de Graduação em Enfermagem (120h teóricas e 150h práticas). A aplicação deste modelo à atuação dos enfermeiros do Centro Ci- rúrgico do Hospital Universitário da Universidade de São Paulo se deu como conseqüência de nossa participação, como membros da disciplina Enfermagem em Centro Cirúrgico da EEUSP, na organização e implan- tação da referida unidade e da coerência dos nossos valores e pressupos- tos com os do Departamento de Assistência de Enfermagem do Hospital em questão. Assim sendo* iniciamos a sua operacionalização num tra- balho conjunto enfermeiros de campo e enfermeiros docentes. Faz-se necessário salientar que as bases conceituais e filosóficas propostas neste trabalho só poderão ser convertidas em atividades se estas forem consideradas como um determinado ato ou conjunto deles em virtude do qual um sujeito ativo- "agente", modifica uma determi- nada situação e que por "agente", (no caso, nós enfermeiros de centro cirúrgico), se defina aquele que age, o que atua e não apenas tem possi- bilidade e ou está disponível para atuar ou agir em atos que se estrutu- ram dentro de um processo total, como recomenda VASQUES (1977) que caracteriza, ainda, esta prática como uma "praxis reflexiva" (sen- sibilidade de agir sobre o concreto diante de um grau elevado de cons- ciencia que se tem da atividade prática). DEFINIÇÃO DE TERMOS Periodo perioperatório, compreende os períodos pré-operatório ime- diato, transoperatório- intra-operatório, recuperação pós anestésica e pós-operatório imediato. Período pré-operatório imediato, compreendido desde a véspera da cirurgia até o momento em que o paciente é recebido no centro cirúrgico. Período transoperatório, compreendido desde o momento em que o paciente é recebido no centro cirúrgico até o momento em que é enca- minhado para a sala de recuperação pós-anestésica. Período intra-operatório, compreendido desde o momento do início até o final da anestesia. Período de recuperação pós-anestésica, compreendido desde o mo- mento da alta do paciente da sala de operações até sua alta da sala de recuperação pós-anestésica. Período de pós-operatório imediato, compreendido desde a alta do paciente da sala de recuperação pós-anestésica até as primeiras 48 horas pós operatorias. * O termo «sistemática de assistência de enfermagem perioperatória», se refere a denomina- nfto utilizada pelo Departamento de Enfermagem do Hospital Universitário - U S P ( S A E ) acrescida da letra « P » para designar o período «perioperatório» ( S A E P ) . MODELO CONCEITUAL PROPOSTO Assim, de acordo com um modelo gerado a partir de componentes profissionais próprios, poderemos garantir a melhoria da qualidade da nossa prática, esta decorrente do compromisso social de oferecer ao cliente que vai passar pela experiência de um ato anestésico-cirúrgico uma assistência de enfermagem eficien- te que atenda às suas reais necessidades, combinando ações tanto ex- pressivas como instrumentais. Colocaremos em seguida as bases teóricas que desenvolvidas, po- dem oferecer aos enfermeiros de centro cirúrgico subsídios para uma reflexão profunda sobre a operacionalização da assistência de enferma- gem perioperatória. A SAEP tem como finalidades: — proporcionar assistência integral (holística), individualizada, participativa e continuada por meio da avaliação e preparos pré-opera- tórios imediatos, intervenção de enfermagem transoperatória e avalia- ção pós-operatória; — propiciar- por meio da integração das ações inter-institucionais, inter-disciplinares e multiprofissionais, condições favoráveis para o en- sino da enfermagem perioperatória, nos níveis de graduação, pós-gra- duação e educação continuada; — estimular, planejar e desenvolver investigações e pesquisas na área da assistência e educação da enfermagem perioperatória e, — estimular, planejar e desenvolver investigações e pesquisas na área da assistência e educação da enfermagem perioperatória e, — participar do desenvolvimento de investigações e pesquisa na área da assistência e educação de enfermagem planejadas com enfer- meiros de outras áreas. — participar do desenvolvimento de investigações e pesquisa na área da assistência e educação de enfermagem planejadas com enfer- meiros de outras áreas. FILOSOFIA DA ASSISTÊNCIA DE ENFERMAGEM PERIOPERATÓRIA Nosso sistema referencial da assistência de enfermagem periopera- tória, abrange os conceitos da assistência holística, continuada, partici- pativa, individualizada, documentada e avaliada, operacionalizada por meio da "sistemática de assistência de enfermagem perioperatória" (SAEP) . Assim, acreditamos que: — o paciente é singular e tem direito à assistência de enfermagem integral, individualizada e participativa; — a qualidade da assistência de enfermagem no período periopera- tório, está vinculada a uma intervenção conjunta dos enfermeiros das unidades de internação e do centro cirúrgico; — o sistema da assistência de enfermagem promove a continuida- de dessa assistência, a participação do paciente, família e condições de documentação e de avaliação da assistência prestada; — o sistema da assistência de enfermagem promove a continuida- de dessa assistência, a participação do paciente, família e condições de documentação e de avaliação da assistência prestada; — os padrões de assistência de enfermagem promovem a educação continuada do pessoal de enfermagem; — os padrões de assistência de enfermagem promovem a educação continuada do pessoal de enfermagem; — a avaliação da assistência prestada proporciona subsídios para a melhoria da qualidade da assistência de enfermagem e para o desenvol- vimento da profissão; — a avaliação da assistência prestada proporciona subsídios para a melhoria da qualidade da assistência de enfermagem e para o desenvol- vimento da profissão; — a assistência de enfermagem perioperatória ao paciente cons- ciente, semi-consciente e inconsciente deve proporcionar respeito pelo indivíduo com proteção a seus direitos humanos e à sua dignidade pes- soal, satisfação das suas necessidades sentidas, prevenção de acidentes e lesões passíveis de acontecer por negligência, impericia ou omissão, e estado de alerta e proteção contra perigos peculiares ao ambiente do centro cirúrgico, sala de recuperação pós-anestésica e/ou procedimen- tos específicos. Adaptada para ser operacloraalizada no H U / U S P . FINALIDADES DA SISTEMÁTICA DA ASSISTÊNCIA DE ENFERMAGEM PERIOPERATÓRIA* A SAEP tem como finalidades: OBJETIVO DA ASSISTÊNCIA DE ENFERMAGEM PERIOPERATÓRIA Na assistência de enfermagem .,o paciente no período perioperató- rio, o enfermeiro do centro cirúrgico, como um dos agentes da assistên- cia à saúde do paciente cirúrgico visa: — ajudar o paciente, e sua família, a compreender seu problema de saúde, a preparar-se para o tratamento anestésico-cirúrgico proposto e suas conseqüências e a utilizar seus mecanismos de defesa, fisiológicos p psicológicos, durante este período e, — diminuir ao máximo os riscos inerentes ao ambiente específico do centro cirúrgico e da sala de recuperação pós-anestésica e à utiliza- ção dos materiais e equipamentos necessários ao desenvolvimento dos procedimentos anestésicos-cirúrgicos, bem como colaborar na consecu- ção destes procedimentos, pela previsão, provisão e controle de recursos humanos, em qualidade e quantidade necessários. OPERACIONAL1ZAÇÃO DA SAEP Para que a filosofia, as finalidades e objetivos expostos sejam colo- cados em prática cinco (5) momentos devem ser considerados, como podemos observar no Fluxo Operacional da SAEP, apresentado no Anexo 1: 1 — Avaliação pré-operatória — através do desenvolvimento de dois procedimentos básicos: coleta de dados para a implementação da visita pré-operatória e a entrevista com o paciente a ser operado e sua família (visita pré-operatória) que têm como objetivos principais a pro- moção da continuidade da assistência de enfermagem entre a unidade de internação e o centro cirúrgico, a nível de cuidados pré-operatórios imediatos e mediatos e cuidados transoperatórios, o respeito ao holísmo e à individualidade do paciente e a redução da ansiedade do paciente/ família. 2 — Identificação de problemas — a partir da conceituação de "problema de paciente" como sendo "condição, situação, dificuldade, questão ou fenômeno apresentado pelo paciente e/ou sua família, neces- sidade não atendida adequadamente" e de "problema de enfermagem" como sendo "problema do paciente cujo atendimento está dentro do âm- bito de atuação da enfermagem" FRIEDLANDER (1977), procuramos identificar os problemas sentidos pelo paciente a ser operado e os evi- denciados pelo enfermeiro durante a visita pré operatoria e no momento do recebimento do paciente no Centro Cirúrgico. 2 — Identificação de problemas — a partir da conceituação de "problema de paciente" como sendo "condição, situação, dificuldade, questão ou fenômeno apresentado pelo paciente e/ou sua família, neces- sidade não atendida adequadamente" e de "problema de enfermagem" como sendo "problema do paciente cujo atendimento está dentro do âm- bito de atuação da enfermagem" FRIEDLANDER (1977), procuramos identificar os problemas sentidos pelo paciente a ser operado e os evi- denciados pelo enfermeiro durante a visita pré operatoria e no momento do recebimento do paciente no Centro Cirúrgico. 3 — Planejamento de cuidados — análise dos problemas de enfer- magem detectados e elaboração da evolução e da prescrição de enfer- magem para o período transoperatório, considerando-se a participação do paciente e família como um elemento de fortalecimento do indivíduo. 3 — Planejamento de cuidados — análise dos problemas de enfer- magem detectados e elaboração da evolução e da prescrição de enfer- magem para o período transoperatório, considerando-se a participação do paciente e família como um elemento de fortalecimento do indivíduo. 4 — Implementação da assistência de enfermagem para o período transoperatório — a nivel de fazer, ajudar, orientar, supervisionar e encaminhar, são implementados os cuidados prescritos. OPERACIONAL1ZAÇÃO DA SAEP Neste momento é imprescindível, além de toda a documentação dos cuidados oferecidos, a evolução de enfermagem, ao final da cirurgia para a efetuação de nova prescrição para a fase de recuperação pós-anestésica, bem como a evo- lução e a prescrição ao ser dada a alta do paciente na SRPA. 4 — Implementação da assistência de enfermagem para o período transoperatório — a nivel de fazer, ajudar, orientar, supervisionar e encaminhar, são implementados os cuidados prescritos. Neste momento é imprescindível, além de toda a documentação dos cuidados oferecidos, a evolução de enfermagem, ao final da cirurgia para a efetuação de nova prescrição para a fase de recuperação pós-anestésica, bem como a evo- lução e a prescrição ao ser dada a alta do paciente na SRPA. 5 — Avaliação pós-operatória — através da visita pós-operatória, o enfermeiro de centro cirúrgico tem condições de avaliar a assistência planejada e implementada, e assim corrigir as falhas detectadas. Esta visita é efetuada entre 24 e 48 h após o ato anestésico cirúrgico. Para finalizar o fluxo operacional proposto, e assegurar sua vali- dade- é imprescindível que o enfermeiro do centro cirúrgico comunique, a todas as pessoas envolvidas na assistência ao paciente, os dados resul- tantes da avaliação pós-operatória (equipes de enfermagem da SRPA, do Centro Cirúrgico, da Unidade de origem ou encaminhamento do paciente). Todos os momentos descritos devem ser anotados no Prontuário do Paciente para, assim, assegurar o registro, tido como documento le- gal, de todos os cuidados de enfermagem oferecidos bem como para constituirem-se em fontes de informações para o planejamento da as- sistência na sala de recuperação pós-anestésica e na unidade de origem ou de encaminhamento do paciente. CONSIDERAÇÕES FINAIS Acreditando que nossa intervenção de enfermagem no período pe- rioperatório deva trilhar o caminho de uma "ação do Homem sobre a matéria e criação — através dela — de uma realidade humanizada" (VASQUES, 1977), o modelo conceitual-filosófico exposto é uma alter- nativa teórica para assegurar a prestação da assistência de enfermagem reflexiva, criadora e, portanto, profissional. Como tal, está sendo ope- racionalizada no processo ensino-aprendizagem da disciplina Enferma- gem em Centro Cirúrgico da EEUSP e na SAEP do Centro Cirúrgico do Hospital Universitário da USP. Nesta relação teoria-prática é necessário enfatizar que temos total consciência do perigo de burocratizar esta práxis, isto é, "converter o formal (o fluxo operacional) no seu próprio conteúdo} sacrificando as- sim o conteúdo à forma, o real ao ideal e o particular concreto ao uni- versal abstrato" (VASQUES, 1977) bem como de torná-la uma práxis me- cânica, reiterativa, em que "a sua repetição infinita se alcança mediante sua extrema formalização, ou seja, mediante a negação do papel do conteúdo para sujeitá-la inteiramente a uma forma que lhe é exterior" (VASQUES, 1977). Neste sentido temos nos preocupado com o grau de penetração da consciência do enfermeiro no desenvolvimento de sua prática e com o grau de criação e humanização do cuidado de enfermagem. Procuramos, assim, continuamente discutir esta relação entre a atividade da cons- ciência dos alunos e dos enfermeiros e a suas respectivas atuações pois "no processo verdadeiramente criador, a unidade de arribos osi lados do processo — o subjetivo e objetivo, o interior e o exterior se apresenta de modo indissolúvel... na criação temos a atividade consciente do sujeito sobre uma determinada matéria, que é trabalhada ou estruturada de acordo com a finalidade ou o projeto que a consciência traça" (VAS- QUES' 1977). Q ) Como modelo conceitual-filosófico sabemos, também, que ele tem* "no tempo, certa esfera de validade, dai a possibilidade e necessidade de generalizá-lo e estendê-lo, isto é, de repeti-lo, enquanto esta validade se mantiver... a repetição se justifica, enquanto a própria vida não re- clama uma nova criação" (VASQUES, 1977). CASTELLANOS, B.E.P. & JOUCLAS, V.M.G. Perioperative nursing care: a concep- tual model. Rev. Esc. Enf. USP, São Paulo, 24(3):359-370, Dec. 1990. This study shows a proposed conceptual model of perioperative nursing care. This model has been used during the teaching-learning process of students in the operating room course of the School of Nursing State of University of São Paulo. REFERÊNCIAS BIBLIOGRÁFICAS C A S T E L L A N O S , B.E.P. Aplicação do processo de enfermagem na unidade de centro cirúrgico. Rev. Esc. Enf. USP., São Paulo, 12(3):170-86, dez. 1978. C A S T E L L A N O S , B.E.P. Aplicação do processo de enfermagem na unidade de centro cirúrgico. Rev. Esc. Enf. USP., São Paulo, 12(3):170-86, dez. 1978. & M A N D E L B A U M , M.H.S. Uma proposta para discussão: o papel do enfermeiro na unidade de centro cirúrgico. Rev. Paul. Enf., São Paulo, 5(1):16-20, jan./mar. 1985. CHIN, P. & JACOBS, M. Theory and nursing: a sistema tic approach. St. Louis, Mosby, 1983. 222p. F A W C E T T , J. The relationship between theory and research: double helix. Gaithsburg, 1(1):49-62, Oct. 1978. F E R R A Z , E.R. O paciente cirúrgico e suas expectativas e opiniões quanto ao cuidado de enfermagem no periodo transoperatório. Trabalho apresentado no CONGRESSO B R A S I - L E I R O D E E N F E R M A G E M , 33., M A N A U S , 1981. 21p. F R I E D L A N D E R , M.R. Problemas de enfermagem e sua conceituação. Rev. Esc. Enf. USP., São Paulo, 11(3):325-30, dez. 1977. H O R T A , W . de A. Contribuição para uma teoria de enfermagem Rev. Bras. Enf., Rio de Janeiro, 23(3-6):119-25, jul./dez. 1970. K A M I Y A M A , Y. O doente hospitalizado e sua percepção quanto à prioridade de seus proble» mas. São Paulo, 1972. l l l p . (Tese de doutorado — Escola de Enfermagem da U S P ) . K I N G , I.M. Toward a theory for nursing: general concepts of human behavior. New York, John Wiley & Sons, 1971. 204p. E V I N S , M.E. Introduction to clinical nursing. 2. ed. Philadelphia, Davis, 1973. L I T T L E , D. & C A R N E V A U , D. Diagnostic statement: problem defined. Tn: W A L - TER, J.B. et alii. Dynamics of problem oriented approaches: patient care and documen- tation. Philadelphia, Lippincott, 1976. p. 213. MENEZES, A . R . CONSIDERAÇÕES FINAIS The development of perioperative nursing care in the "University Hospital" is based in this model and it is in the implementation phase as an activity of "Assistencial- Teaching Program". The model demonstrates the philosophical concepts, finalities and its performance. UNITERMS: Operating room nursing. Perioperative care. UNITERMS: Operating room nursing. Perioperative care. UNITERMS: Operating room nursing. Perioperative care. MENEZES, A . R . A problemática da enfermagem dos pacientes no periodo transoperatório: um estudo dos problemas sentidos e observados. São Paulo, 1978. 81p. (Dissertação de mestrado — Escola de Enfermagem da U S P ) . Y U R A , H . & W A L S H , M.B. Human needs and the nursing process. New York, Appleton- Century-Crofts, 1967. 330p. REFERÊNCIAS BIBLIOGRÁFICAS A problemática da enfermagem dos pacientes no periodo transoperatório: um estudo dos problemas sentidos e observados. São Paulo, 1978. 81p. (Dissertação de mestrado — Escola de Enfermagem da U S P ) . M U R P H Y , C.C. Ethical aspects of decision making. In: N A T I O N A L L E A G U E F O R N U R - SING. Political, social and education forces: impact of social forces. New York, 1979. p. 17-25. (Publ. n» 15-1774). N E U M A N , M. Theory development in nursing. Philadelphia, Davis, 1979. 94p. OREM, D.E. Nursing: concepts of practice. 2. cd. New York, McGraw-Hill. 1980. 232p. O R L A N D O , I.J. O relacionamento dinâmico enfernieiro-paciente. São Paulo, E P U , 1 P A I M , R. Problemas de enfermagem e terapia centrada nas necessidades do paciente. Rio de Janeiro, União dos Cursos Cariocas, 1978. 283p. P A N Z A , A . M . M . Efeito da visita pré-operatória da enfermeira dc centro cirúrgico sobre o estresse do paciente no pré-operatório, no dia da cirurgia e nos pó-operatório. São Paulo, 1977. 75p. (Dissertação do mostrado — Escola do Enfermagem da UfciP). P E P L A U , H. Interpersonal relations in nursing. 3. ed. New York, Putman's, 1952. 330p. RODRIGUES, A . I . O paciente no sistema centro-cirúrgico: um estudo sobre as percepções e opiniões dos pacientes em relação ao período trans-operatório. São Paulo, 1979. 150p. (Dis- sertação de mestrado —• Escola de Enfermagem da U S P ) . ROY, S.C Introduction to nursing: an adaptation model. Englcwood Cliffs, Prentice-Hall, 1976. 402p. SOUZA, M.F. Efeito da interação enfermeira-paciente como método de atendimento à neces- sidade de segurança do paciente cirúrgico. Porto Alegre, 1976. 43p. (Tese de livre-do- cência — Escola de Enfermagem da U F R S ) . STEVENS, B.J. Nursing theory: analyses, application, evaluation. 2. ed. Boston, Little- Brow, 1984. 298p. T I Í A V E L B E E , .) Interpersonal aspects of nursing- 2. ed. Philadelphia, Davis, 1971. 128p. T I Í A V E L B E E , .) Interpersonal aspects of nursing- 2. ed. Philadelphia, Davis, 1971. 128p. VASQUES, A.S. Filosofia da praxis. 2. ed. Rio de Janeiro, Paz e Terra, 1977. 454p. VASQUES, A.S. Filosofia da praxis. 2. ed. REFERÊNCIAS BIBLIOGRÁFICAS Rio de Janeiro, Paz e Terra, 1977. 454p. W A T S O N , A . B . & MAYERS, M.G. Assesment and documentation: nursing theories in action. New Jersey, Charles B. Slack, 1981. 373p. Y U R A , H . & W A L S H , M.B. Human needs and the nursing process. New York, Appleton- Century-Crofts, 1967. 330p. A N E X O I A N E X O I F L U X O O P E R A C I O N A L D A S A E P SISTEMA REFERENCI AL~ jsjstenc. ía i n t e g r a l ^Assistência continuada - A s s i s t ê n c i a p a r t i c i p a t i v a - A s s i s t ê n c i a i n d i v i d u a l i z a d a A s s i s t ê n c i a documentada A s s i s t ê n c i a a v a l i a d a ENFERMEIRA DA UNIDADE PRONTUARIO DO PACIENTE ENTREVISTA PACIENTE/FAMILIA ENF. SRPA ENF. U I / U T I EQUIPE ENF. C . C . V i s i t a p r e - o p e r a t o r i a v ~ * Intervenção enf. Anotações e/ou evoluções e p r e s c r i ç õ e s I d e n t i f i c a ç ã o de problemas sentidos e evidenciados Evolução e p r e s c r i ç ã o de enf. para o t r a n s , op. F . A . 0 . S . E . Anotações e/ou evolução e p r e s c r i ç ã o t r a n s , e de RPA V i s i t a pos op . Intervenção enf. Anotações e/ou evolução e prese ri çao F L U X O O P E R A C I O N A L D A S A E P F L U X O O P E R A C I O N A L D A S A E P jsjstenc. REFERÊNCIAS BIBLIOGRÁFICAS ía i n t e g r a l ^Assistência continuada - A s s i s t ê n c i a p a r t i c i p a t i v a - A s s i s t ê n c i a i n d i v i d u a l i z a d a A s s i s t ê n c i a documentada A s s i s t ê n c i a a v a l i a d a SISTEMA REFERENCI AL~ V i s i t a p r e - o p e r a t o r i a Intervenção enf. Anotações e/ou evoluções e p r e s c r i ç õ e s Recebido em 28/11/89
https://openalex.org/W4308653805	https://trialsjournal.biomedcentral.com/counter/pdf/10.1186/s13063-022-06875-9	English	null	The effectiveness of a tablet-based video game that stimulates cognitive, emotional, and social skills in developing academic skills among preschoolers: study protocol for a randomized controlled trial	Trials	2,022	cc-by	9,823	Abstract Background: Evidence suggests that children from low-income families begin the preschool stage with less aca‑ demic and non-academic skills development compared to higher-income families. There are several successful expe‑ riences of early stimulation of cognitive and social-emotional skills; however, there is scarce evidence of the effec‑ tiveness of a video game that incorporates the stimulation of these skills simultaneously. This study aims to evaluate the effectiveness of a video game in stimulating cognitive, emotional, and social competence skills in developing academic skills in socioeconomically disadvantaged preschool children. Methods: A cluster-randomized controlled trial design will be used. A tablet-based video game that stimulates cognitive and socio-emotional skills to improve the development of academic skills is compared with a tablet-based game where students draw and paint with no explicit stimulation of cognitive and socio-emotional skills. Eighteen schools and 750 Chilean preschool students will be recruited. The effectiveness of the intervention will be assessed using a direct evaluation of children on literacy learning and pre-calculation skills at baseline, immediately after stimu‑ lation, and at 6, 12, 18, and 24 months post-intervention. The mediating effect of working memory, inhibitory control, emotion recognition, and prosocial behaviours will be assessed on the effectiveness of the intervention. Discussion: The proposed study will be the first to test the effectiveness of a tablet-based video game stimulating cognitive and social-emotional skills to improve academic skills in socioeconomically disadvantaged preschool chil‑ dren in Chile, controlling for gender, age (in months), mental health, and baseline conditions of stimulated skills. Trial registration: ClinicalTrials.gov NCT05224700. Registered on February 2022 Keywords: Cognitive skills, Emotional skills, Academic skills, Tablet play, Preschools, Randomized con © The Author(s) 2022. Open Access This article is l permits use, sharing, adaptation, distribution and r original author(s) and the source, provide a link to t other third party material in this article are included to the material. If material is not included in the art regulation or exceeds the permitted use, you will n licence, visit http://creativecommons.org/licenses/ mmons.org/publicdomain/zero/1.0/) applies to th p jg @ 2 Research Center for Students Mental Health (ISME), Faculty of Education, Universidad de los Andes, Santiago, Chile Full list of author information is available at the end of the article © The Author(s) 2022. The effectiveness of a tablet‑based video game that stimulates cognitive, emotional, and social skills in developing academic skills among preschoolers: study protocol for a randomized controlled trial Cristian A. Rojas‑Barahona1, Jorge Gaete2,3* , Mauricio Véliz4, Ramón D. Castillo1, Saray Ramírez2,3 and Ricardo Araya5 Rojas‑Barahona et al. Trials (2022) 23:936 https://doi.org/10.1186/s13063-022-06875-9 Open Access Background and rationale {6a} Contact information: E-mail: c.rojas@ utalca.cl; Phone +71 2201747; Address: Avenida Lircay S/N, Campus Lircay, Faculty of Psychology, Universidad de Talca, Maule Region. Role of sponsor {5c} Funders played no role in the design of the study, collection, management, analysis, and interpretation of the report. They will not have ultimate authority over any of these activities. The effectiveness of a tablet-based video game that stimulates cognitive, emotional, and social skills on develop‑ ing academic skills among preschool‑ ers: study protocol for a randomized controlled trial. The effectiveness of a tablet-based video game that stimulates cognitive, emotional, and social skills on develop‑ ing academic skills among preschool‑ ers: study protocol for a randomized controlled trial. Trial identifier NCT05224700 in Clinical Trials [ClinicalTrials.gov] under the registry name: “Early Development of Academic Skills in the Classroom: Short- and Mid-term Effects of a Tablet- based Game That Stimulates Cogni‑ tive, Emotional, and Social Skills for Pre-kindergarten Children”; [registered in February 2022] g y The EFs are integrated by different components par- ticipating in goal-directed behaviour, where inhibitory response, attention, and working memory seem criti- cal [6]. In recent years, the development of EFs has been a significant focus of research, among other reasons, because [6] (a) there is growing evidence that EFs are crucial to school success; (b) EFs are deficient in several learning/developmental disorders; (c) children in socio- economically disadvantaged context seem to have lower EF development before entering formal education when compared when their counterparts from more affluent families; and (d) there is increasing evidence that EFs can be stimulated in children with typical and atypical development. This research is funded by the National Research and Development Agency [ANID]. Unique ID: Fondecyt Regular 1210989; and by ANID – Millennium Sci‑ ence Initiative Program – NCS2021_081. J. Gaete: Research Center for Students Mental Health (ISME), Faculty of Educa‑ tion, Universidad de los Andes. Chile. Millennium Nucleus to Improve the Mental Health of Adolescents and Youths, Millennium Science Initiative, Chile. Within EFs, working memory (WM) and inhibitory control (IC) have been identified as critical components in the development of academic skills such as early liter- acy and precalculus [7], in addition to the evidence that they can be stimulated early in development [8]. Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Rojas‑Barahona et al. Trials (2022) 23:936 This research is funded by the National Research and Development Agency [ANID]. Unique ID: Fondecyt Regular 1210989; and by ANID – Millennium Sci‑ ence Initiative Program – NCS2021_081. Background and rationale {6a} Several authors [1] have expressed that improving learn- ing and socialization skills has many advantages in the prevention of behaviour problems, the quality of life of children, and long-term health and economic outcomes. Evidence suggests that children from low-income families may begin the preschool stage with lower development of academic skills (such as precalculus and initial literacy) and non-academic skills (such as emotional and social competence), when compared to children from higher- income families [2]. Such differences could increase long- term social-emotional, educational, and health disparities [3]. Between the ages of 3 and 7, critical aspects of human development happen, such as emotion recognition, con- trol of behaviour [4], and the development of Executive Functions (EF) [5]. Therefore, it becomes essential to stimulate these skills among socially vulnerable children. 2013-statement-defining-standard-protocol-items-for- clinical-trials/). Title {1} The effectiveness of a tablet-based video game that stimulates cognitive, emotional, and social skills on develop‑ ing academic skills among preschool‑ ers: study protocol for a randomized controlled trial. Trial registration {2a and 2b}. Trial identifier NCT05224700 in Clinical Trials [ClinicalTrials.gov] under the registry name: “Early Development of Academic Skills in the Classroom: Short- and Mid-term Effects of a Tablet- based Game That Stimulates Cogni‑ tive, Emotional, and Social Skills for Pre-kindergarten Children”; [registered in February 2022] Protocol version {3} Version 1 of 01-25-2022 Funding {4} This research is funded by the National Research and Development Agency [ANID]. Unique ID: Fondecyt Regular 1210989; and by ANID – Millennium Sci‑ ence Initiative Program – NCS2021_081. Author details {5a} C. Rojas: Faculty of Psychology, Research Center on Cognitive Sciences, Universi‑ dad de Talca-Chile. J. Gaete: Research Center for Students Mental Health (ISME), Faculty of Educa‑ tion, Universidad de los Andes. Chile. Millennium Nucleus to Improve the Mental Health of Adolescents and Youths, Millennium Science Initiative, Chile. M. Veliz: Facultad de Ciencias de la Edu‑ cación, Universidad de Talca-Chile. R. Castillo: Faculty of Psychology, Research Center on Cognitive Sciences, Universidad de Talca-Chile. S. Ramírez: Research Center for Students Mental Health (ISME), Faculty of Educa‑ tion, Universidad de los Andes. Chile. Millennium Nucleus to Improve the Mental Health of Adolescents and Youths, Millennium Science Initiative, Chile. R. Araya: Department of Health Service & Population Research, King’s College London, London, United Name and contact information for the trial sponsor {5b} Investigator initiated clinical trial; Cristian Rojas (Principal Investigator). Introduction Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http:// www.equator-network.org/reporting-guidelines/spirit- 2013-statement-defining-standard-protocol-items-for- clinical-trials/). Name and contact information for the trial sponsor {5b} C. Rojas: Faculty of Psychology, Research Center on Cognitive Sciences, Universi‑ dad de Talca-Chile. Objectives {7}h The main objective of this study is to assess the effec- tiveness of a programme of stimulating cognitive skills (working memory and inhibitory control), emotional skills (recognition of emotions), and social competence (choice of prosocial behaviours), in the development of academic skills of initial literacy and precalculus in soci- oeconomically disadvantaged preschool children. The outcomes will be assessed immediately after the inter- vention, at 6, 12, 18, and 24 months post-intervention, controlling for gender, age (in months), mental health, and baseline conditions of the stimulated skills. In general, interventions that stimulate emotional and social skills include varied strategies and practices that are usually not limited to promoting a particular skill. Therefore, it is difficult to isolate the effect of the stimu- lation of a specific skill. A meta-analysis by Blewitt et al. [19], which included the revision of programmes for children between 2 and 6 years old, found that children exposed to social and emotional stimulation interven- tions showed a significant improvement in social and emotional competencies, learning, behavioural regula- tion, and decreased their emotional and behavioural problems. Most of the revised interventions have been studied in developed countries, are complex, sustained in time, and require a lot of resources in personnel and material that are not readily available in vulnerable socio- economic contexts. Objectives {7}h The specific objectives are (a) to determine the accept- ability and ease of use of the programme of stimulation of cognitive, emotional, and social competence skills in socioeconomically disadvantaged preschool chil- dren; (b) to evaluate the changes in the variables work- ing memory, inhibitory control, emotion recognition, and the choice of prosocial behaviours, in both groups (intervention and comparison group), immediately after the intervention and at 6, 12, 18, and 24 months post- intervention; (c) to determine the effectiveness of the programme of stimulation of cognitive, emotional, and social competence skills in the development of the aca- demic ability of early literacy and in the development of the academic skill of precalculus in socioeconomically disadvantaged preschool children, immediately after the intervention and at 6, 12, 18, and 24 months post- intervention, controlling for gender, age (in months), mental health, and baseline conditions of the stimulated skills; and (d) to explore the influence of potential medi- ators (working memory, inhibitory control, emotion recognition, and choice of prosocial behaviours) on the observed effects of the cognitive, emotional, and social competence skill stimulation programme, controlling for gender, age (in months), mental health, and basal condi- tions of the stimulated skills. In recent years, there has been a growing use of devices with touch screens by children. It has been observed that their use in an educational context for academic purposes has favourable evidence [20], especially when these appli- cations have been created with careful designs and con- tent selection [21]. A recent systematic review by Griffith et al. [22] concludes that there is evidence that interactive applications can be helpful and accessible tools to sup- port early academic development. Herodotou [23] found that most of the research involving technological devices evaluated cognitive aspects and, in general, neglected the socio-emotional aspects. In addition, of the studies ana- lysed, which report results in various areas of children’s development, eight present findings in the development of academic skills, evidencing advances that guide the contribution of devices with touch screens in these skills, that is, in learning. Finally, there are several successful experiences of early stimulation of cognitive skills using technologi- cal devices, such as COGMED for children aged 7–17 years with ADHD by Klingberg et al. [24], and Memory Booster for children aged 5 to 8 years of St Clair-Thomp- son and collaborators [8]. Background and rationale {6a} WM and IC can be developed if they are stimulated directly; how- ever, it is less clear, especially for WM, the impact of their stimulation on the development of other skills such as lit- eracy and precalculus [9–11]. Studies have also found the relationship between EFs and other behavioural and non- cognitive skills [12], such as social [13] and emotional development [14]. M. Veliz: Facultad de Ciencias de la Edu‑ cación, Universidad de Talca-Chile. R. Castillo: Faculty of Psychology, Research Center on Cognitive Sciences, Universidad de Talca-Chile. Investigator initiated clinical trial; Cristian Rojas (Principal Investigator). Contact information: E-mail: c.rojas@ utalca.cl; Phone +71 2201747; Address: Avenida Lircay S/N, Campus Lircay, Faculty of Psychology, Universidad de Talca, Maule Region. Emotional understanding refers to the fashion chil- dren identify, predict, and explain emotions regarding themselves and others. Around 3 years of age, children can start labelling emotional expressions. Then, they rapidly continue to advance in the development of emo- tion recognition. Rosnay et al. [15] have reported that children who are more successful in understanding emo- tional cues are more likely to develop social skills and prosocial responses to their peers and to form positive Funders played no role in the design of the study, collection, management, analysis, and interpretation of the report. They will not have ultimate authority over any of these activities. Rojas‑Barahona et al. Trials (2022) 23:936 Rojas‑Barahona et al. Trials (2022) 23:936 Page 3 of 12 interpersonal relationships that encourage adaptation to social situations. With regard specifically to social ability and behaviour involving the other, it becomes necessary to divide components into those inherent to the person, with those that are dyadic. We will dwell on the precursor elements of a personal level, such as the ability to identify and monitor related behaviours, closely linked to emo- tional and executive functions [16]. In this sense, several studies have observed that the development early in the life of early emotional and social competencies facilitates the successful interactions of children with others and favours better academic achievements and mental health [17, 18]. strategies have also accumulated good evidence, such as PATHS created by Greenberg and collaborators [25] and Incredible Years by Carolyn Webster-Stratton [26]. Background and rationale {6a} How- ever, we did not find an evaluation of the effectiveness of a programme that incorporates the stimulation of cogni- tive and social-emotional skills using an interactive video game on a tablet for preschool children, followed up in the medium term. Eligibility criteria {10} Inclusion criteria Eligibility criteria {10} Inclusion criteria greater development in the intervention group, which will be maintained throughout the different measure- ments. The same will happen in developing early lit- eracy and precalculus academic skills. The effect of the programme is mediated by working memory, inhibi- tory control, emotion recognition, and choice of proso- cial behaviours, controlling for gender, age (in months), mental health, and baseline conditions of stimulated skills. greater development in the intervention group, which will be maintained throughout the different measure- ments. The same will happen in developing early lit- eracy and precalculus academic skills. The effect of the programme is mediated by working memory, inhibi- tory control, emotion recognition, and choice of proso- cial behaviours, controlling for gender, age (in months), mental health, and baseline conditions of stimulated skills. – Establishments with an index of school vulnerability (ISV) ≥70% (based on the information provided by participating families) – Establishment that offers preschool and elementary education – Establishment located in the region of Maule, Chile – Establishment located in the region of Maule, Chile – Mixed-sex schools. – Mixed-sex schools. – Mixed-sex schools. – Classes with a minimum of 15 students each Trial design {8}h This is a cluster randomized controlled trial, parallel- group type, where the tablet-based video game stimulat- ing cognitive, emotional, and social skills is compared to a tablet-based video game involving only painting. The effectiveness of this programme will be evaluated by one pre-stimulation measurement, and five post-stimulation measures, immediately after stimulation, at 6, 12, 18, and 24 months. The patient allocation is randomized with a ratio of 1:1. See Fig. 1. Exclusion criteria – School is implementing a similar programme. – Special education school. – Children with a reported cognitive disability. Objectives {7}h Other interventions promot- ing socio-emotional skills using interactive teaching We hypothesize that the programme will have high acceptability among socioeconomically disadvantaged preschool children. Immediately after the end of the intervention, there will be an improvement in the vari- ables working memory, inhibitory control, recognition of emotions and choice of prosocial behaviours, with Rojas‑Barahona et al. Trials (2022) 23:936 Rojas‑Barahona et al. Trials (2022) 23:936 Page 4 of 12 Methods: participants, interventions, and outcomes Study setting {9}h All schools in Region del Maule will be assessed for eli- gibility. The eligibility of schools to participate in this study will be evaluated based on the abovementioned criteria. After the schools have been considered eligible, all eligible schools will be invited. Then, the principal of the selected schools will receive the study information. Study setting {9}h This study will be carried out in schools with preschool education located in two cities in the Maule region (Talca and Linares), Chile. Fig. 1 Flowchart Fig. 1 Flowchart Page 5 of 12 Rojas‑Barahona et al. Trials (2022) 23:936 Rojas‑Barahona et al. Trials (2022) 23:936 After school officials agree to participate in the study and sign an authorization form, preschool educators and pre- kindergarten assistants will be invited to participate (in conjunction with kindergarten and first-grade educators as part of the longitudinal study). Subsequently, the par- ents or guardians of the students will be informed about the study in a meeting and invited to participate. Along with this information, parents will receive the informed consent form and must sign it if they agree to have their children participate in the study. Finally, all authorized children will be asked for their willingness to participate in the study. Even if you have the consent of children, if the child does not confirm that they want to participate, they will not be forced. through a tablet game programme that promotes cogni- tive skills (working memory and inhibitory control), emo- tional (recognition of emotions), and social competence (choice of prosocial behaviours) individually in the class- room context. This programme will be held during the academic year during school hours and has 20 sessions of 30 min, two times a week (in total two and a half months), where students will be in groups in the room, but each child will work with a tablet with headphones individu- ally. At the end of the activity, each child will receive a sticker for participation. All activities are games with the following structure: an introduction with instructions and selection of the protagonist (avatar: which can be modified according to hair colour, skin, sex, and clothes, who speaks directly to the child), followed by a practice exercise to make sure that the children understand what they should do (it is repeated until the child performs the routine correctly), and finally, the stimulation activity (4 different activities with five exercises each). In the first ten sessions, working memory and emotion recognition are stimulated together (4 activities for each skill in each session). In the remaining ten sessions, inhibitory control and the choice of prosocial behaviours are stimulated. Additional consent provisions for collection and use of participant data and biological specimens {26b} No samples were collected. Explanation for the choice of comparators (6b) The control group (active) will not work with the pro- gramme of stimulation of cognitive skills and socio-emo- tional; instead, this group will use a tablet-based game where they must paint during the 20 sessions of 30 min, two times a week. At the end of the activity, you will be given a sticker for your participation. In this group, the educators will have the same training in cognitive, emo- tional and social competence skills as those in the inter- vention group. They will also be trained in using the game to paint, and meetings will be held every 15 days to evaluate possible doubts. This way, an attempt to avoid the effects of the programme dependent on the educators is made since, in both groups, a similar treatment would be received. The knowledge will remain within the edu- cational institution for future work in the area. Like the intervention group, the group will be guided by the kin- dergarten educator responsible for the course and will work with the entire course group simultaneously (each child will have an individual tablet with headphones). All children who wish to play will be able to participate (to avoid discrimination); however, only children who have parental/guardian consent and the consent of the child themselves will be evaluated. A research team member will be present at each session to ensure that the session flows correctly. Strategies to improve adherence to interventions {11c} A record of the children’s attendance and percentage of participation in each session (for both groups) will be made. In addition, a research team member will regis- ter each session’s incidents and observations. For exam- ple, there will be a registry of the number of children Study setting {9}h There are no plans for future studies using data collected in this trial. Criteria for discontinuing or modifying allocated interventions {11b}h The students of the intervention and comparison group will participate in all the programme sessions; however, the data of the children with cognitive disabilities (with previous diagnosis) will not be considered for the analy- ses. Children with cognitive disabilities, in the context of this study, are those children who have been identified by a trained school professional indicating that they cannot clearly understand the instructions of the instruments. The main reasons for excluding these children from the analyses are that the measurement tools are not validated for children with cognitive disabilities. These children may have difficulty understanding the instructions mak- ing the results less reliable. In addition, while students cannot leave the class- room if they do not want to participate in the sessions, they can leave the study at any time and for any reason, if they wish, without any consequences. If children leave the study, their information and collected data will not be analysed. Intervention description {11a}h The intervention group will use the tablet-based video game to stimulate cognitive and emotional skills and social competence. The intervention will be implemented Rojas‑Barahona et al. Trials (2022) 23:936 Page 6 of 12 Rojas‑Barahona et al. Trials (2022) 23:936 will be used, and it was also validated for this age group by Medina et al. [28] (a =.79). Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by base- line mean scores. playing every 5 min, and the reasons why the children are not performing the task will also be noted (e.g. going to the bathroom, a problem with the tablet, etc.). Finally, once every 15 days, a meeting will be held with all the educators to evaluate the possible doubts and difficulties in developing the programme. Secondary outcomes 2. Mental health problems: Goodman’s “Strengths and Difficulties Questionnaire” (SDQ) [31] will be uti- lized. It consists of a series of emotional and behav- ioural problems experienced by children between 4 and 16 years old. It has four subscales of psycho- logical difficulties (of 5 items each): emotional symp- toms, behavioural problems, hyperactivity/attention problems, peer problems, and prosocial behaviour. These subscales can be combined into a Difficul- ties subscale with a total score. The version for chil- dren and young people in Chile was validated by the team of this project Gaete et al. [32], with an inter- nal consistency through the McDonald’s Omega index, which fluctuates between .76 and .85 (report from main caregivers). We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. 2. Mental health problems: Goodman’s “Strengths and Difficulties Questionnaire” (SDQ) [31] will be uti- lized. It consists of a series of emotional and behav- ioural problems experienced by children between 4 and 16 years old. It has four subscales of psycho- logical difficulties (of 5 items each): emotional symp- toms, behavioural problems, hyperactivity/attention problems, peer problems, and prosocial behaviour. These subscales can be combined into a Difficul- ties subscale with a total score. The version for chil- dren and young people in Chile was validated by the team of this project Gaete et al. [32], with an inter- nal consistency through the McDonald’s Omega index, which fluctuates between .76 and .85 (report from main caregivers). We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. Provisions for post‑trial care {30}h There is no harm nor potential harm in this trial. The intervention group will receive an intervention built under the best evidence available to stimulate pre- schoolers’ cognitive, social, and emotional skills. The active control group will receive a tablet game pro- gramme where they will only paint. We have included several benefits for children and schools participating in the study: (1) A report with the data collected will be provided to all schools (without the children’s iden- tity) to help the institutions plan future measures to be included in the regular teaching activities to stimulate development. (2) Additionally, those parents or main caregivers who request information about the results of the measures carried out on their children will be granted. (3) We have offered to all schools some train- ing opportunities for early educators to gain new skills related to promoting cognitive and non-cognitive skills among children. This training will be implemented after the final follow-up assessment. Relevant concomitant care permitted or prohibited during the trial {11d}h 1.2. Pre-calculation skills: Woodcock-Muñoz (Battery III [30]), includes the following subtests of the achievement test: Quanti- fication, Verbal Counting, Identification of numbers, and Applied Problems. Split-half reliability for Spanish-speaking children ages 4 and 5 ranges from .93 to .95. Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the differ- ence between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. 1.2. Pre-calculation skills: Woodcock-Muñoz (Battery III [30]), includes the following subtests of the achievement test: Quanti- fication, Verbal Counting, Identification of numbers, and Applied Problems. Split-half reliability for Spanish-speaking children ages 4 and 5 ranges from .93 to .95. Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the differ- ence between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. The schools participating in the study cannot imple- ment other programmes stimulating cognitive and socio-emotional skills during the trial. Outcomes {12} Primary outcomes These subscales can be combined into a Difficul- ties subscale with a total score. The version for chil- dren and young people in Chile was validated by the team of this project Gaete et al. [32], with an inter- nal consistency through the McDonald’s Omega index, which fluctuates between .76 and .85 (report from main caregivers). We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. 1. Tests of academic skills: 1. Tests of academic skills: 1.1. Initial literacy learning: Two tests will be used: Texas Lee, designed by the Texas Education Agency [27] in collaboration with the Univer- sity of Houston to evaluate reading in Spanish- speaking children. This instrument has been validated for Chile by Medina et al. [28]. It con- siders the following dimensions: Knowledge of the printed, Knowledge of the alphabet, Phonological/phonemic awareness, and Oral comprehension (a =.82). To evaluate vocabu- lary, the Spanish adaptation of the Kaufman Assessment Battery for Children (K-ABC) [29] 3. Cognitive Skills Tests: 3.1. Working memory: 2 memory tests will be applied: visual-spatial and phonological. For the visuospatial area, the “Corsi Cube test” [33] will be administered. It involves repeating a Rojas‑Barahona et al. Trials (2022) 23:936 Rojas‑Barahona et al. Trials (2022) 23:936 Page 7 of 12 4.2. Selection of prosocial behaviours: the “Chal- lenging Situations Task” (CST) by Den- ham et al. [36] will be employed, reporting a Cronbach alpha of .83. The instrument assesses children’s ability to solve problems. Children are presented with peer problems, and their responses are classified into four possibilities: prosocial, aggressive, crying, and avoidant. Each option selected by chil- dren will be summed in a subsequent sub- scale. The prosocial subscale will be used as the main comparator between groups. We will assess the difference between groups at each point in time (immediately after stim- ulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. sequence of up to nine spatially separated iden- tical blocks on a screen. The series starts simple but becomes more complex until the subject’s performance decreases (internal consistency between .75 and .78. For the phonological area, the auditive memory subtest of Woodcock- Muñoz (Battery III [30]) (internal consistency .80) will be used. A sequence of audio messages is presented from lowest to greatest difficulty, and the child is asked to remember these mes- sages. Each correct answer will be registered as one point. The total score means the num- ber of correct answers. We will assess the dif- ference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. j g y 3.2. 1. Tests of academic skills: Inhibitory control: the “Hearts & Flowers” task [34] will be used, which is a hybrid that combines elements of Simon and spa- tial tasks of Stroop and contains congru- ent and incongruous essays. For congruent trials, subjects must obey the rule: “Press on the same side as the stimulus (Hearts)”. For incongruous trials, subjects should fol- low the rule: “Press on the side opposite the stimulus (Flowers)”. The test showed a Cronbach alpha of .83. Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the difference between groups at each point in time (immedi- ately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by baseline mean scores. 4. Socio-emotional Skills Tests: 4. Socio-emotional Skills Tests: 4.1. Emotion recognition: the “Assessment of Chil- dren’s Emotional Skills” (ACES) by Schultz et al. [35] will be administered. The test com- prises three subscales: facial expressions, social situations, and social behaviour. The current project will administer the subscale of facial expressions (a =.68). Each child will need to identify correctly 16 facial expressions. Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by base- line mean scores. 4.1. Emotion recognition: the “Assessment of Chil- dren’s Emotional Skills” (ACES) by Schultz et al. [35] will be administered. The test com- prises three subscales: facial expressions, social situations, and social behaviour. The current project will administer the subscale of facial expressions (a =.68). Each child will need to identify correctly 16 facial expressions. Each correct answer will be registered as one point. The total score means the number of correct answers. We will assess the difference between groups at each point in time (immediately after stimulation, at 6, 12, 18, and 24 months), using the mean score of the group, adjusting by base- line mean scores. Sample size {14} To determine a significant difference (alpha =0.05) in the measure of the primary outcome (academic skills: early liter- acy/precalculus), between the comparison and intervention groups, with a power of 80%, we need to recruit six schools with a total of 264 students per group, with an average of 44 students per school (distributed in two courses). For the sam- ple calculation, the Stata statistical package was used, and the “clustersampsi” command, which indicates the differences between groups concerning an average score and consid- ers the correlation effect between the subjects within the same school through the intracluster correlation coefficient (ICC = 0.02) [11]. The information to feed this equation was extracted from a previous study [37], where the control group in the early literacy variable post-intervention meas- ure had a score of 20.06 (DS = 8.32). Assuming a difference in favour of the intervention group of 0.4 DS, the sample size was calculated for this study. In addition, it is estimated that 10% of eligible students might not want to participate, and estimating a 20% loss in follow-up, each group should initially recruit 377 students. This implies that four extra schools will be recruited per group, totalling 16. Recruitment {15}h The research team already has good collaboration net- works with municipalities and schools in Chile. Strate- gies to achieve adequate school enrolment to achieve the target sample size will consist of contacting and present- ing to the municipal study authorities who are expected to help establish contact with school authorities. In addi- tion, research assistants will communicate directly and Rojas‑Barahona et al. Trials (2022) 23:936 Page 8 of 12 Table 1 Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) diagram Table 1 Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) diagram inform school authorities about the study’s purpose, requirements and duration. give this information to a research assistant. Later, the research assistant will inform schools by email and con- firm by telephone. Plans for assessment and collection of outcomes {18a} Each participant will be evaluated individually on six occasions, one before the intervention and five after the intervention, using the tasks focused on working memory, inhibitory control, emotion recognition, choice of proso- cial behaviours and precalculus skills and early literacy, Assignment of interventions: blinding Who will be blinded {17a}h Schools will be randomly distributed to either group with an allocation ratio of 1:1 through an automated proce- dure using Excel (Microsoft) and the command “RAND”. A statistician independent of the research team will per- form the randomization. This is a double-blinded trial, blinded to the outcome evaluators and the data analyst. Outcome evaluators will not be informed about the group of belonging and will be instructed not to ask the students or the school authorities about the school’s condition. A data analyst will work with the final dataset, where the group condition will be masked. Concealment mechanism {16b} After the randomization and allocation, schools will be informed by a research assistant of the group belonging by email and confirmed by telephone. Each school will only receive their information of allocation. Additionally, this information will not be disclosed to the assessment research team (outcome evaluators) to keep them blind to the schools’ allocation. Procedure for unblinding if needed {17b} For the nature of the intervention, the participants will know the allocation of the school. Data collection and management Plans for assessment and collection of outcomes {18a} Statistical methods for primary and secondary outcomes {20a} First, descriptive analyses will be performed to evaluate the balance of variables between groups at the begin- ning of the study. The primary analysis will be conducted based on intention-to-treat. This analysis includes all ran- domized subjects in the groups, regardless of whether or not they complete the treatment or follow-up period. Multivariate regression models of mixed effects will be carried out considering as a dependent variable the per- formance in the tests of academic skills (initial literacy and precalculus) and as an independent variable belong- ing to the intervention or comparison group, control- ling for gender, age (in months), mental health, and performance in the same tests in the baseline evaluation. Regression models will consider the nested nature of the data (clustering), since students’ performance within the same educational establishment tends to be correlated. While primary outcomes will be considered as those obtained after the intervention, analyses will be repeated for follow-up at 6, 12, 18, and 24 months. This will allow for evaluation of the maintenance of the effect of the intervention until first grade. We will use mixed-effect regression models of repeated measures to assess conver- gence and divergence between groups in follow-up times. Subgroup analyses will be performed for the primary out- come variable using interaction terms in the regression models between the randomized groups and the gender variable. This will allow us to evaluate the moderating effect. Sensitivity analyses will also be performed to assess the impact of lost data using multiple imputations. The results with and without imputed data will be presented and compared. The tests will be done with Stata 15.0. Data management {19} After the participants have completed the questionnaires, the data will be entered into a secure platform without identifying information (each participant will be assigned an encrypted ID number). The original copies of the instruments will be filed and stored, under lock and key, in a self-storage, along with the list linking the partici- pants’ names and ID numbers. Only the principal investi- gator, research assistants in charge of data entry, and the statistician will have access to the database. h Finally, multilevel models of mediation will be adjusted using Mplus 6, entering all the hypothesized mediators simultaneously: working memory, inhibitory control, recognition of emotions, and choice of prosocial behav- iours. We will calculate the effect of the intervention on each mediator, the effect of each mediator on the primary outcome, and the effect of the intervention that each Plans to promote participant retention and complete follow‑up {18b} To ensure greater rigour in the research design, a record of the children’s attendance and percentage of participa- tion of the children in each session will be made (for both groups: intervention and comparison). An observation record will be kept, made by a member of the research team, of each session carried out, where the number of children who are playing will be recorded every 5 min, and the reasons why the children are not performing the task will be noted (e.g. went to the bathroom, a problem with the tablet). In addition, the observation protocol will include multiple-choice items that will try to capture the main difficulties encountered during the programme implementation and how the educator and the assistant are responsible for supporting the children during the session to carry out the activity. Students will not have access to the software between sessions. Implementation {16c} An independent statistician will perform the randomiza- tion to assign to the study arms, and this statistician will Rojas‑Barahona et al. Trials (2022) 23:936 Page 9 of 12 in 2 sessions lasting approximately 40 minutes each (with breaks in each). Trained psychologists will conduct the children’s assessment in a 1:1 ratio. Each trained psycholo- gist will arrange the evaluation time during school hours, which will occur in a pre-determined location on the school premises. The conditions for the evaluation will be ensured: privacy, no distractions, and safety. The trained psychologist will be responsible for the well-being of the children in the assessment, and a supervisor may be in place or on call for any support if needed. To ensure data quality recollection, evaluators will fulfil a daily report of incidents with the children, and a supervisor will solve the problems. In addition, the supervisor will make field visits to check if these assessments follow the evaluation proto- col. On the other hand, the parents will answer a mental health questionnaire of the child during the periods of the children’s assessments. The instruments to be used in the measurements are described below. code list will only be available to the research team men- tioned above. It will be documented and safeguarded according to research guidelines after the completion of the study. No participant identification details will be reported in publications or any study report. Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} Not applicable. There are no biological samples in this assay. Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} Not applicable. There are no biological samples in this assay. Composition of the coordinating centre and trial steering committee {5d}h Composition of the coordinating centre and trial steering committee {5d}h Confidentiality {27} Research data will be stored using a study identification code for each participant. The key to the identification Rojas‑Barahona et al. Trials (2022) 23:936 Page 10 of 12 Page 10 of 12 mediator mediated. For each model, we will calculate the intervention’s indirect effects and the unique indirect effect for each mediator. Then, we will calculate the direct effect of the programme. Because randomization will be at the school level, we will understand the school as sec- ond-level and individuals as first-level. To control vari- ability across schools, we will use the Mplus stratification option. Then, we will separate the models for initial lit- eracy and prequel skills. All models will be adjusted for gender, mental health, and values of the mediating vari- ables in the baseline evaluation. Covariations between all variables within time will be allowed. study coordinator helped in trial registration and will coordinate study visits and reports. Study research assis- tants will help to identify potential participating schools, collect informed consent, ensure follow-up accord- ing to protocol, and resolve doubts from school staff or participants. The study team will meet weekly during the whole duration of the study. There is no trial steering committee or stakeholder and public involvement group. The Ethical Scientific Committee of the University of Talca will check the presence and completeness of the investigation. Methods in analysis to handle protocol non‑adherence and any statistical methods to handle missing data {20c}h y The primary outcome will be assessed using an intention- to-treat analysis. Every participant randomized to the trial will enter the primary analysis. Accordingly, partici- pants who drop out prematurely, or are non-compliant with the study intervention, will be included in the pri- mary analysis within the respective group they have been assigned to at randomization. Multiple imputations will be used to handle missing data in the primary and sec- ondary analyses. Interim analyses {21b}h Not applicable. There will not be interim analyses because the data will be analysed at the end of the trial. A monitor from the Ethical Scientific Committee of the University of Talca will check at least twice during the investigation of the presence and completeness of the research. This committee is independent of the sponsor and has no competing interests; further details about its charter can be asked via email: cec@utalca.cl. Adverse event reporting and harms {22}h The intervention or the data collection procedure does not infer harm among the participants. However, any situation that compromises participants’ physi- cal and psychological integrity during all different actions related to the project will be registered. Addi- tionally, any incident of discomfort or harm reported by the child or educator will be recorded through a form that a research assistant will fill out, both in the evaluations and in the intervention. This information will be managed by the Principal Investigator and Pro- ject Coordinator and reported to the Ethical Scientific Committee, and, if necessary, they will contact school authorities and main caregivers. Any adverse events will be reported in the trial publications. Frequency and plans for auditing trial conduct {23}i q y p g A monitor from the Ethical Scientific Committee of the University of Talca will check at least twice during the project the presence and completeness of the investiga- tion files, such as informed consents, inclusion and exclu- sion criteria, and data collection and storage. The datasets produced during the current study will be available in an international database repository called UK Data Service. Methods for additional analyses (e.g. subgroup analyses) {20b} Subgroup analyses will be performed to explore dif- ferences between males and females. The statistical approach will follow the procedures described above. Ethics approval and consent to participate {24} Approval was obtained from the ethics committee of the University of Talca (Folio: 12-2021, April 28, 2021). The authorization of the directors of the schools will be obtained, and the written informed consent of the parents or legal guardians of the participants, in addition to the consent of the students to participate in the study, will be obtained. Author details 1 1 Faculty of Psychology, Research Center on Cognitive Sciences, Universidad de Talca, Talca, Chile. 2 Research Center for Students Mental Health (ISME), Faculty of Education, Universidad de los Andes, Santiago, Chile. 3 Millennium Nucleus to Improve the Mental Health of Adolescents and Youths, Millennium Science Initiative, Santiago, Chile. 4 Facultad de Ciencias de la Educación, Universidad de Talca, Talca, Chile. 5 Department of Health Service & Population Research, King’s College London, London, UK. Received: 4 July 2022 Accepted: 29 October 2022 Received: 4 July 2022 Accepted: 29 October 2022 Trial status R i Recruitment will begin in April–June 2022. The cur- rent protocol is version 1 of April 2022. It is estimated that patient recruitment will be completed around July– August 2022. All items included in this protocol can be found on Clinical Trial Id NCT05224700 [ClinicalTrials. gov]. Abbreviations EF E i f 3. Durlak JA, Weissberg RP, Dymnicki AB, Taylor RD, Schellinger KB. The impact of enhancing students’ social and emotional learning: a meta- analysis of school-based universal interventions. Spec Issue Raising Healthy Child. 2011;82(1):405–32. EF: Executive functions; WM: Working memory; IC: Inhibitory control; ACES: Assessment of Children’s Emotions Skills; CST: Challenging Situations Task; SDQ: Strengths and Difficulties Questionnaire; K-ABC: Kaufman Assessment Battery for Children. 4. Baird JA, Moses LJ. Do preschoolers appreciate that identical actions may be motivated by different intentions? J Cogn Dev. 2001;2:413–48.f be motivated by different intentions? J Cogn Dev. 2001;2:413–48. 5. Caffarena Barcenilla C, Rojas-Barahona CA. Self-regulation in early child‑ hood: advances from research. Ecuadorian J Neurol. 2019;28(2):37–49. 6. Rojas-Barahona CA, editor. Executive functions and education. Under‑ standing key skills for learning. Santiago: Ediciones UC; 2017. Availability of data and materials {29}i It is known that there are several successful experiences of early stimulation of cognitive skills and social-emo- tional skills. However, we did not find an evaluation of the effectiveness of a programme that incorporates both skills together, even more so using tablets and for pre- school children, followed up in the medium term. With the proposed study, he hopes there will be evidence for the first time of a tablet-based cognitive and social- emotional skills stimulation programme that improves academic skills in socioeconomically disadvantaged pre- school children, controlling for gender, age (in months), mental health, and baseline conditions of the stimulated skills. The final trial dataset will be available in an international data repository called UK Data Service. Authors’ contributions {31b} about the changes. If needed, additional consent will be requested and registered. Also, online trial registries will be updated accordingly. CR is the principal investigator; CR and JG have conceived the study and led the proposal, protocol development, study design, and methodology. MV, RC, SR, and RA contributed to the development of the protocol and method. All authors read and approved the final manuscript. In addition, JG, CRB, and RA will be included in all future publications. Each of the authors will lead different roles for each publication. Other authors can be included in future publications regarding the specific support provided for those authors (e.g. statistician) Funding {4} This research is funded by the National Research and Development Agency [ANID]. Unique ID: Fondecyt Regular 1210989, and by ANID – Millennium Sci‑ ence Initiative Program – NCS2021_081. There is no role of the funding body in the study design; collection, analysis, interpretation of data; and writing of the manuscript. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}i This is a monocentre study designed, performed, and coordinated at the University of Talca, Chile. Daily sup- port for the trial is provided by the principal investiga- tor, who supervises the trial. Additionally, a data manager organizes data collection and assures data quality. The All substantial amendments will be notified to the eth- ics committee of the University of Talca. If amendments concern or affect participants, they will be informed Rojas‑Barahona et al. Trials (2022) 23:936 Page 11 of 12 Page 11 of 12 References 1. Reynolds AJ, Temple JA. Economic returns of investments in preschool education. In: A vision for universal preschool education: Cambridge University Press; 2006. p. 37–68. https://doi.org/10.1017/CBO9781139 167284.004. 1. Reynolds AJ, Temple JA. Economic returns of investments in preschool education. In: A vision for universal preschool education: Cambridge University Press; 2006. p. 37–68. https://doi.org/10.1017/CBO9781139 167284.004. 2. Qi CH, Kaiser AP. Behaviour problems of preschool children from low- income families: review of the literature. Topics Early Child Spec Educ. 2003;23:188–216. 2. Qi CH, Kaiser AP. Behaviour problems of preschool children from low- income families: review of the literature. Topics Early Child Spec Educ. 2003;23:188–216. Dissemination plans {31a}h The results of this research will be entirely disclosed in international peer-reviewed journals. Both positive and negative results will be reported. A summary of the results will be given to school authorities. 1. Reynolds AJ, Temple JA. Economic returns of investments in preschool education. In: A vision for universal preschool education: Cambridge University Press; 2006. p. 37–68. https://doi.org/10.1017/CBO9781139 167284.004. 2. Qi CH, Kaiser AP. Behaviour problems of preschool children from low- income families: review of the literature. Topics Early Child Spec Educ. 2003;23:188–216. 3. Durlak JA, Weissberg RP, Dymnicki AB, Taylor RD, Schellinger KB. The impact of enhancing students’ social and emotional learning: a meta- analysis of school-based universal interventions. Spec Issue Raising Healthy Child. 2011;82(1):405–32. 4. Baird JA, Moses LJ. Do preschoolers appreciate that identical actions may be motivated by different intentions? J Cogn Dev. 2001;2:413–48. 5. Caffarena Barcenilla C, Rojas-Barahona CA. Self-regulation in early child‑ hood: advances from research. Ecuadorian J Neurol. 2019;28(2):37–49. 6. Rojas-Barahona CA, editor. Executive functions and education. Under‑ standing key skills for learning. Santiago: Ediciones UC; 2017. Consent for publication {32} Not applicable, this manuscript does not contain individual personal data from participants. Declarations Ethics approval and consent to participate {24} 2. Qi CH, Kaiser AP. Behaviour problems of preschool children from low- income families: review of the literature. Topics Early Child Spec Educ. 2003;23:188–216. 3. Durlak JA, Weissberg RP, Dymnicki AB, Taylor RD, Schellinger KB. The impact of enhancing students’ social and emotional learning: a meta- analysis of school-based universal interventions. Spec Issue Raising Healthy Child. 2011;82(1):405–32. 4. Baird JA, Moses LJ. Do preschoolers appreciate that identical actions may be motivated by different intentions? J Cogn Dev. 2001;2:413–48. 5. Caffarena Barcenilla C, Rojas-Barahona CA. Self-regulation in early child‑ hood: advances from research. Ecuadorian J Neurol. 2019;28(2):37–49. 6. Rojas-Barahona CA, editor. Executive functions and education. Under‑ standing key skills for learning. Santiago: Ediciones UC; 2017. Competing interests {28} There are some potential limitations to keep in mind. There would be an unknown impact of the COVID-19 pandemic on preschool students because the pandemic and its implemented public health measures have dras- tically changed the way children could interact socially with their peers. There is also a risk that the implementa- tion will be lost due to potential school closures due to COVID-19 infections. Acknowledgements The effect of inhibitory control on general mathematics achievement and fraction comparison in middle school children. ZDM Math Educ. 2015;47:801–11 34. Diamond A, Barnett S, Thomas J, Munro S. Preschool program improves cognitive control. Science. 2007;318:1387–8. 34. Diamond A, Barnett S, Thomas J, Munro S. Preschool program improves cognitive control. Science. 2007;318:1387–8. 11. McClelland MM, Cameron CE, Connor CM, Farris CL, Jewkes AM, Mor‑ rison FJ. Links between behavioral regulation and preschoolers’ literacy, vocabulary and math skills. Dev Psychol. 2007;43(4):947–59. https://doi. org/10.1037/0012-1649.43.4.947. 35. Schultz D, Izard CE, Bear G. Children’s emotion processing: relations to emotionality and aggression. Dev Psychopathol. 2004;16(2):371–87. https://doi.org/10.1017/S0954579404044566. 11. McClelland MM, Cameron CE, Connor CM, Farris CL, Jewkes AM, Mor‑ rison FJ. Links between behavioral regulation and preschoolers’ literacy, vocabulary and math skills. Dev Psychol. 2007;43(4):947–59. https://doi. org/10.1037/0012-1649.43.4.947. 35. Schultz D, Izard CE, Bear G. Children’s emotion processing: relations to emotionality and aggression. Dev Psychopathol. 2004;16(2):371–87. https://doi.org/10.1017/S0954579404044566. 36. Denham SA, Bouril B, Belouad F. Preschoolers’ affect and cognition about challenging peer situations. Child Study J. 1994;24:1–21. 12. Blair C. School readiness: integrating cognition and emotion in a neuro‑ biological conceptualization of children’s functioning at school entry. Am Psychol. 2002;57:111–27. 37. Rojas-Barahona CA, Förster Marín CE, Moreno-Ríos S, McClelland MM. Improvement of working memory in preschoolers and its impact on early literacy skills: a study in deprived communities of rural and urban areas. Early Educ Dev. 2015;26(5-6):871–92. https://doi.org/10.1080/10409 289.2015.1036346. 37. Rojas-Barahona CA, Förster Marín CE, Moreno-Ríos S, McClelland MM. Improvement of working memory in preschoolers and its impact on early literacy skills: a study in deprived communities of rural and urban areas. Early Educ Dev. 2015;26(5-6):871–92. https://doi.org/10.1080/10409 289.2015.1036346. y 13. Jacobson L, Williford A, Pianta RC. The role of executive function in children’s competent adjustment to middle school. Child Neuropsychol. 2011;17(3):255–80. 14. Costa EM, Osório A, Veríssimo M, Martins C. Emotion understanding in preschool children: the role of executive functions. Int J Behav Dev. 2016;40(1):1–10. Acknowledgements We appreciate the participation of the students, their parents/primary caregivers, kindergarten educators, kindergarten assistants, and the schools contributing to this research project. Page 12 of 12 Rojas‑Barahona et al. Trials (2022) 23:936 Rojas‑Barahona et al. Trials (2022) 23:936 7. Montoya MF, Susperreguy MI, Dinarte L, Morrison F, San Martin E, Rojas- Barahona CA, et al. Executive function in Chilean preschool children: do short-term memory, working memory, and response inhibition contrib‑ ute differentially to early academic skills? Early Child Res Q. 2019;46:187– 200. https://doi.org/10.1016/j.ecresq.2018.02.009. 7. Montoya MF, Susperreguy MI, Dinarte L, Morrison F, San Martin E, Rojas- Barahona CA, et al. Executive function in Chilean preschool children: do short-term memory, working memory, and response inhibition contrib‑ ute differentially to early academic skills? Early Child Res Q. 2019;46:187– 200. https://doi.org/10.1016/j.ecresq.2018.02.009. 7. Montoya MF, Susperreguy MI, Dinarte L, Morrison F, San Martin E, Rojas- Barahona CA, et al. Executive function in Chilean preschool children: do short-term memory, working memory, and response inhibition contrib‑ ute differentially to early academic skills? Early Child Res Q. 2019;46:187– 200. https://doi.org/10.1016/j.ecresq.2018.02.009. 29. Kaufman A, Kaufman N. Kaufman Evaluation Battery for k–abc children. Madrid: TEA Ediciones; 1997. 30. Muñoz-Sandoval AF, Woodcock RW, McGrew KS, Mather N. Drums III Woodcock-Muñoz. Itasca: Riverside Publishing; 2005. Woodcock-Muñoz. Itasca: Riverside Publishing; 2005. 31. Goodman R. The Strengths and Difficulties Questionnaire: a research note. J Child Psychol Psychiatry. 1997;38(5):581–6. 8. St Clair-Thompson HL, Holmes J. Improving short-term and working memory: methods of memory training. In: Johansen NB, editor. New research on short-term memory. New York: Nova Science; 2008. p. 125–54. 8. St Clair-Thompson HL, Holmes J. Improving short-term and working memory: methods of memory training. In: Johansen NB, editor. New research on short-term memory. New York: Nova Science; 2008. p. 125–54. 32. Gaete J, Montero-Marin J, Valenzuela D, Rojas-Barahona CA, Olivares E, Araya R. Mental health among children and adolescents: construct valid‑ ity, reliability, and parent-adolescent agreement on the ‘Strengths and Difficulties Questionnaire’ in Chile. PLoS One. 2018;13(2):e0191809. 9. Sala G, Gobet F. Working memory training in typically developing chil‑ dren: a multilevel meta-analysis. Psychon Bull Rev. 2020;27:423–34. fi 33. Corsi PM. Human memory and the medial temporal region of the brain. Diss Abstr Int. 1972;34(2):819B. fi 33. Corsi PM. Human memory and the medial temporal region of the brain. Diss Abstr Int. 1972;34(2):819B. 10. Gómez D, Jiménez A, Bobadilla R, Reyes C, Dartnell P. Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 15. Rosnay M, Harris LP, Pons F. Emotion understanding and developmental psychopathology in young children. In: Fonagy P, Goodyer I, editors. Social cognition and developmental psychopathology. UK: Oxford Uni‑ versity Press; 2008. p. 343–86. 16. Calkins S, Fox N. Self-regulatory processes in early personality develop‑ ment: a multilevel approach to the study of childhood social withdrawal and aggression. Dev Psychopathol. 2002;14:477–98. 17. Halle TG, Darling-Churchill KE. Review of measures of social and emo‑ tional development. J Appl Dev Psychol. 2016;45:8–18. 18. Kalvin CB, Bierman KL, Gatzke-Kopp LM. Emotional reactivity, behavior problems, and social adjustment at school entry in a high-risk sample. J Abnorm Child Psychol. 2016;44(8):1527–41. y 19. Blewitt C, Fuller-Tyszkiewicz M, Nolan A, Bergmeier H, Vicary D, Huang T, et al. Social and emotional learning associated with universal curriculum- based interventions in early childhood education and care centers. A systematic review and meta-analysis. JAMA. 2018;1(8):e185727. y y 20. Rojas-Barahona CA (Coord.). Early childhood in the age of digital transfor‑ mation. An Ibero-American look. Madrid: Organization of Ibero-American States for Education, Science and Culture (OEI); 2022. 21. Papadakis S, Kalogiannakis M, Zaranis N. Educational apps from the Android Google Play for Greek preschoolers: a systematic review. Comput Educ. 2018;116:139–60. 22. Griffith SF, Hagan MB, Heymann P, Heflin BH, Bagner DM. Apps as learning tools: a systematic review. Pediatrics. 2020;145(1):E20191579. https://doi. org/10.1542/peds.2019-1579. 23. Herodotou C. Young children and tablets: a systematic review of effects on learning and development. J Comput Assist Learn. 2017;34:1–9. 24. Klingberg T, Fernell E, Olesen PJ, Johnson M, Gustafsson P, Dahlström K, et al. Computerized training of working memory in children with ADHD: a randomized, controlled trial. J Am Acad Child Adolesc Psychiatry. 2005;44(2):177–86. 25. Greenberg MT, Kusche CA, Cook ET, Quamma JP. Promoting emotional competence in school-aged children: the effects of the PATHS Curricu‑ lum. Dev Psychopathol. 1995;7:117–36. 26. Webster-Stratton C, Reid MJ. Treating conduct problems and strength‑ ening social and emotional competence in young children: the Dina Dinosaur treatment program. J Emot Behav Disord. 2003;11(3):130–43. 27. Texas Education Agency. Texas reading first initiative, 2008-2009 commis‑ sioner’s list of reading instruments. 2009. http://ritter.tea.state.tx.us/curri culum/readingfirst. 28. Medina L, Villalón M, Meneses A, Valdivia A, Ruiz M, San Martín E, et al. Literacy in subsidized Chilean establishments. Final report. Santiago de Chile: Ministry of Education; 2011.
https://openalex.org/W4287864737	https://kar.kent.ac.uk/81176/17/Lemmens2022_Article_AMetricVersionOfPoincar%C3%A9STheor.pdf	English	null	A metric version of Poincaré's theorem concerning biholomorphic inequivalence of domains	arXiv (Cornell University)	2,020	cc-by	11,805	Licence for this version CC BY (Attribution) Additional information Kent Academic Repository Lemmens, Bas (2022) A Metric Version of Poincaré’s Theorem Concerning Biholomorphic Inequivalence of Domains. Journal of Geometric Analysis, 32 . ISSN 1050-6926. Downloaded from https://kar.kent.ac.uk/81176/ The University of Kent's Academic Repository KAR https://kar.kent.ac.uk/81176/ The University of Kent's Academic Repository KAR Author Accepted Manuscripts If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding. Cite as Surname, Initial. (Year) 'Title of article'. 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The Journal of Geometric Analysis (2022) 32:160 https://doi.org/10.1007/s12220-022-00893-4 Mathematics Subject Classiﬁcation Primary 32F45 · Secondary 51F99 Mathematics Subject Classiﬁcation Primary 32F45 · Secondary 51F99 Mathematics Subject Classiﬁcation Primary 32F45 · Secondary 51F99 A Metric Version of Poincaré’s Theorem Concerning Biholomorphic Inequivalence of Domains Bas Lemmens1 Received: 7 September 2020 / Accepted: 9 February 2022 © The Author(s) 2022 Abstract Abstract We show that if Y j ⊂Cn j is a bounded strongly convex domain with C3-boundary for j = 1, . . . , q, and X j ⊂Cm j is a bounded convex domain for j = 1, . . . , p, then the product domain p j=1 X j ⊂Cm cannot be isometrically embedded into q j=1 Y j ⊂Cn under the Kobayashi distance, if p > q. This result generalises Poincaré’s theorem which says that there is no biholomorphic map from the polydisc onto the Euclidean ball in Cn for n ≥2. The method of proof only relies on the metric geometry of the spaces and will be derived from a more general result for products of proper geodesic metric spaces with the sup-metric. In fact, the main goal of the paper is to establish a general criterion, in terms of certain asymptotic geometric properties of the individual metric spaces, that yields an obstruction for the existence of an isometric embedding between product metric spaces. Keywords Product metric spaces · Product domains · Kobayashi distance · Isometric embeddings · Metric compactiﬁcation · Busemann points · Detour distance B Bas Lemmens B.Lemmens@kent.ac.uk 1 Introduction Numerous theorems in several complex variables are instances of results in metric geometry. In this paper, we shall see that a classic theorem due to Poincaré [22], which says that there is no biholomorphic map from the polydisc n onto the (open) Euclidean ball Bn in Cn if n ≥2, is a case in point. In fact, it is known [19, 29, 30] that there exists no surjective Kobayashi distance isometry of n onto Bn if n ≥2. More The author gratefully acknowledges the support of the EPSRC (Grant EP/R044228/1). B Bas Lemmens B.Lemmens@kent.ac.uk 1 School of Mathematics, Statistics & Actuarial Science, University of Kent, Canterbury CT2 7NX, U 123 Page 2 of 20 160 B. Lemmens generally, one may wonder when it is possible to isometrically embed a product domain p j=1 X j ⊂Cm into another product domain q j=1 Y j ⊂Cn under the Kobayashi distance. In this paper, we show, among other results, the following theorem. Theorem 1.1 Suppose that X j ⊂Cm j is a bounded convex domain for j = 1, . . . , p, and Y j ⊂Cn j is a bounded strongly convex domain with C3-boundary for j = 1, . . . , q.. If p > q, then there is no isometric embedding of p j=1 X j into q j=1 Y j under the Kobayashi distance. Note that Poincaré’s theorem is a special case where p = n ≥2 and q = 1, as the boundary of the Euclidean ball is smooth. The case where j m j = j n j and the isometry is surjective was analysed by Zwonek [29, Theorem 2.2.5] who used different methods. A key property of the Kobayashi distance is the product property, see [13, Theorem 3.1.9]. Indeed, if X j ⊂Cm j is a bounded convex domain for j = 1, . . . , p, then the Kobayashi distance, kX, on the product domain X := p j=1 X j satisﬁes kX(w, z) = max j=1,...,p kX j (w j, z j) for all w = (w1, . . . , wp), z = (z1, . . . , z p) ∈X. kX(w, z) = max j=1,...,p kX j (w j, z j) for all w = (w1, . . . , wp), z = (z1, . . . , z p) ∈X. 1 Introduction metric spaces, such as ﬁnite dimensional normed spaces with smooth norms [24], Hilbert geometries on bounded strictly convex domains with C1-boundary [25], and, as we shall see in Lemma 3.3, Kobayashi metric spaces (D, kD), where D ⊂Cn is a bounded strongly convex domain with C3-boundary. It turns out that the parts of the horofunction boundary and the detour distance in product metric spaces have a special structure that is closely linked to a quotient space of (Rn, 2∥· ∥∞), where ∥x∥∞= max j \|x j\|. More precisely, if we let Sp(1) := {λ(1, . . . , 1) ∈Rn : λ ∈R}, then the quotient space Rn/Sp(1) with respect to 2∥· ∥∞ has the variation norm as the quotient norm, which is given by ∥x∥var := max j x j + max j (−x j) for x ∈Rn/Sp(1), (1.1) (1.1) see [17, Sect. 4]. It is known, e.g. [14, Proposition 2.2.4], that (Rn/Sp(1), ∥· ∥var) is isometric to the Hilbert metric space on the open (n −1)-dimensional simplex. We show in Theorem 2.8 that if, for j = 1, . . . , q, we have that (N j, ρ j) is a proper geodesic metric space such that all its horofunctions are Busemann points, and δ(h j, h′ j) = ∞for all h j ̸= h′ j Busemann points of (N j, ρ j), then each part of (q j=1 N j, d∞) is isometric to (Rn/Sp(1), ∥· ∥var) for some 1 ≤n ≤q. j The horofunctions for the product of two metric spaces have been considered by Walsh [26, Sect. 8]. Some of our results are extensions of his work to arbitrary ﬁnite products, and the ideas of some of the proofs are quite similar. For the reader’s con- venience, we give full proofs and provide comments on the relation with the work in [26] where relevant. The work in this paper has links to work by Bracci and Gaussier [7] who studied the interaction between topological properties and the metric geometry of hyperbolic complex spaces. It is also worth mentioning that various other aspects of the metric geometry of product metric spaces have been studied in context of Teichmüller space in [9, 20]. 1 Introduction In view of the product property, it is natural to consider product metric spaces with the sup-metric. Given metric spaces (M j, d j), j = 1, . . . , p, the product metric space (p j=1 M j, d∞) is given by d∞(x, y) := max j d j(x j, y j) for x = (x1, . . . , x p), y = (y1, . . . , yp) ∈ p j=1 M j. In this general context, it is interesting to understand when one can isometrically embed a product metric space into another one. The main goal of this paper is to establish a general criterion, in terms of certain asymptotic geometric properties of the individual metric spaces, that yields an obstruction for the existence of an isometric embedding between product metric spaces, and to show how this criterion can be used to derive Theorem 1.1. The key concepts from metric geometry involved are as follows: the horofunction boundary of proper geodesic metric spaces, almost geodesics, Busemann points, the detour distance, and the parts of the horofunction boundary, which will all be recalled in the next section. Our main result is the following. Theorem 1.2 Suppose that (M j, d j) is a proper geodesic metric space containing an almost geodesic sequence for j = 1, . . . , p, and (N j, ρ j) is a proper geodesic metric space such that all its horofunctions are Busemann points, and δ(h j, h′ j) = ∞for all h j ̸= h′ j Busemann points of (N j, ρ j), for j = 1, . . . , q. If p > q, then there exists no isometric embedding of (p j=1 M j, d∞) into (q j=1 N j, d∞). The assumption that each horofunction is a Busemann point and that any two distinct Busemann points lie at inﬁnite detour distance from each other is a type of regularity condition on the asymptotic geometry of the space, which is satisﬁed by numerous 123 A Metric Version of Poincaré’s Theorem Concerning... Page 3 of 20 160 metric spaces, such as ﬁnite dimensional normed spaces with smooth norms [24], Hilbert geometries on bounded strictly convex domains with C1-boundary [25], and, as we shall see in Lemma 3.3, Kobayashi metric spaces (D, kD), where D ⊂Cn is a bounded strongly convex domain with C3-boundary. 2 The Metric Compactiﬁcation of Product Spaces In our set-up, we will follow the terminology in [12], which contains further references and background on the metric compactiﬁcation. Let (M, d) be a metric space, and let RM be the space of all real functions on M equipped with the topology of pointwise convergence. Fix b ∈M, which is called the basepoint. Let Lip1 b(M) denote the set of all functions h ∈RM such that h(b) = 0 and h is 1-Lipschitz, i.e. \|h(x) −h(y)\| ≤d(x, y) for all x, y ∈M. Then Lip1 b(M) is a closed subset of RM. Moreover, as \|h(x)\| = \|h(x) −h(b)\| ≤d(x, b) for all h ∈Lip1 b(M) and x ∈M, we get that Lip1 b(M) ⊆[−d(x, b), d(x, b)]M, which is compact by Tychonoff’s theorem. Thus, Lip1 b(M) is a compact subset of RM. for all h ∈Lip1 b(M) and x ∈M, we get that Lip1 b(M) ⊆[−d(x, b), d(x, b)]M, which is compact by Tychonoff’s theorem. Thus, Lip1 b(M) is a compact subset of RM. 123 160 Page 4 of 20 Page 4 of 20 160 B. Lemmens Now for y ∈M consider the real-valued function Now for y ∈M consider the real-valued function hy(z) := d(z, y) −d(b, y) with z ∈M. Then hy(b) = 0 and \|hy(z) −hy(w)\| = \|d(z, y) −d(w, y)\| ≤d(z, w). Thus, hy ∈Lip1 b(M) for all y ∈M. The closure of {hy : y ∈M} is called the metric compactiﬁcation of M and is denoted Mh. The boundary ∂Mh := Mh \ {hy : y ∈M} is called the horofunction boundary of M, and its elements are called horofunctions. Given a horofunction h and r ∈R, the set H(h,r) := {x ∈M : h(x) < r} is a called a horoball. We will assume that the metric space (M, d) is proper, meaning that all closed balls are compact. Such metric spaces are separable, since every compact metric space is separable. It is known that if (M, d) is separable, then the topology of pointwise convergence on Lip1 b(M) is metrizable, and hence each horofunction is the limit of a sequence of functions (hyn) with yn ∈M for all n ≥1. In general, however, horofunctions are limits of nets. A curve γ : I →(M, d), where I is a possibly unbounded interval in R, is called a geodesic path if d(γ (s), γ (t)) = \|s −t\| for all s, t ∈I. The metric space (M, d) is said to be a geodesic space if for each x, y ∈M there exists a geodesic path γ : [a, b] →M with γ (a) = x and γ (b) = y. A proof of the following well-known fact can be found in [16, Lemma 2.1]. Lemma 2.1 If (M, d) is a proper geodesic metric space, then h ∈∂Mh if and only if there exists a sequence (yn) in M such that hyn →h and d(yn, b) →∞as n →∞. It should be noted that in the previous lemma, it is necessary to assume that the metric space is proper. Indeed, consider the star graph with centre vertex b and edges En = {b, vn} of length n for n ∈N. Then the sequence (vn) in the resulting path metric space, with basepoint b, satisﬁes lim n→∞hvn(x) = lim n→∞d(x, vn) −d(b, vn) = d(x, b) = hb(x) for all x, and hence does not yield a horofunction. Now for y ∈M consider the real-valued function A sequence (yn) in (M, d) is called an almost geodesic sequence if d(yn, y0) →∞ as n →∞, and for each ε > 0, there exists N ≥0 such that A sequence (yn) in (M, d) is called an almost geodesic sequence if d(yn, y0) →∞ as n →∞, and for each ε > 0, there exists N ≥0 such that d(ym, yk) + d(yk, y0) −d(ym, y0) < ε for all m ≥k ≥N. The notion of an almost geodesic sequence goes back to Rieffel [23] and was further developed in [5, 15, 24, 27]. In particular, any almost geodesic sequence yields a horofunction, see [23, Lemma 4.5]. The notion of an almost geodesic sequence goes back to Rieffel [23] and was further developed in [5, 15, 24, 27]. In particular, any almost geodesic sequence yields a horofunction, see [23, Lemma 4.5]. 123 Page 5 of 20 A Metric Version of Poincaré’s Theorem Concerning... 160 Lemma 2.2 Let (M, d) be a proper geodesic metric space. If (yn) is an almost geodesic sequence in M, then Lemma 2.2 Let (M, d) be a proper geodesic metric space. If (yn) is an almost geodesic sequence in M, then h(x) = lim n→∞d(x, yn) −d(b, yn) exists for all x ∈M and, moreover, h ∈∂Mh. Given a proper geodesic metric space (M, d), a horofunction h ∈Mh is called a Busemann point if there exists an almost geodesic sequence (yn) in M such that h(x) = limn→∞d(x, yn) −d(b, yn) for all x ∈M. We denote the collection of all Busemann points by BM. It is known that a product metric space (p j=1 M j, d∞), where d∞(x, y) = max j d j(x j, y j) for x = (x1, . . . , x p), y = (y1, . . . , yp) ∈ j=1 M j, is a proper geodesic metric space, if each (M j, d j) is a proper geodesic metric space, see for instance [21, Proposition 2.6.6]. The horofunctions of a product proper geodesic metric spaces have a special form, as the following theorem shows. This theorem is an extension of [26, Proposition 8.1], and the basic idea of the proof is the same. Theorem 2.3 For j = 1, . . . , p let (M j, d j) be proper geodesic metric spaces. Sup- pose that h is a horofunction of (p j=1 M j, d∞) with basepoint b = (b1, . . . , bp). If (yn) is a sequence in p j=1 M j converging to h, then there exist J ⊆{1, . . . , p} non-empty, horofunctions h j in M j h with basepoint b j for j ∈J, α ∈RJ with min j∈J α j = 0, and a subsequence (ynk) such that (1) d∞(b, ynk) −dk(b j, ynk j ) →α j for all j ∈J, (2) d∞(b, ynk) −dk(bi, ynk i ) →∞for all i /∈J, (3) hynk j →h j for all j ∈J. (1) d∞(b, ynk) −dk(b j, ynk j ) →α j for all j ∈J, (2) d∞(b, ynk) −dk(bi, ynk i ) →∞for all i /∈J, (3) hynk j →h j for all j ∈J. i (3) hynk j →h j for all j ∈J. Moreover, h is of the form, Moreover, h is of the form, h(x) = max j∈J h j(x j) −α j for x = (x1, . . . , x p) ∈ p j=1 M j. (2.1) (2.1) Proof Let (yn) be a sequence in p j=1 M j such that (hyn) converges to a horo- function h. exists for all x ∈M and, moreover, h ∈∂Mh. So h(x) = limn→∞d∞(x, yn) −d∞(b, yn) for all x ∈p j=1 M j. As the product metric space is a proper geodesic metric space, it follows from Lemma 2.1 that d∞(b, yn) →∞as n →∞. Write yn := (yn 1, . . . , yn p) and let αn j := d∞(b, yn) −d j(b j, yn j ) ≥0 for all j = 1, . . . , p and n ≥0. Proof Let (yn) be a sequence in p j=1 M j such that (hyn) converges to a horo- function h. So h(x) = limn→∞d∞(x, yn) −d∞(b, yn) for all x ∈p j=1 M j. As the product metric space is a proper geodesic metric space, it follows from Lemma 2.1 that d∞(b, yn) →∞as n →∞. Write yn := (yn 1, . . . , yn p) and let αn j := d∞(b, yn) −d j(b j, yn j ) ≥0 for all j = 1, . . . , p and n ≥0. j j We may assume, after taking a subsequence, that hyn j (·) := d j(·, yn j ) −d j(b j, yn j ) converges to h j ∈M j h and αn j →α j ∈[0, ∞] for all j ∈{1, . . . , p}, and αn j0 = 0 123 Page 6 of 20 160 B. Lemmens for all n ≥0 for some ﬁxed j0 ∈{1, . . . , p}. Let J := { j : α j < ∞} and note that j0 ∈J. So, h(x) = lim n→∞d∞(x, yn) −d∞(b, yn) = lim n→∞max j (d j(x j, yn j ) −d j(b j, yn j ) −αn j ) = max j∈J h j(x j) −α j. To complete the proof note that α j < ∞implies that d j(b j, yn j ) →∞, and hence by Lemma 2.1, we ﬁnd that h j is a horofunction of (M j, d j) for j ∈J. ⊓⊔ To complete the proof note that α j < ∞implies that d j(b j, yn j ) →∞, and hence by Lemma 2.1, we ﬁnd that h j is a horofunction of (M j, d j) for j ∈J. ⊓⊔ ⊓⊔ The following notion will be useful in the sequel. exists for all x ∈M and, moreover, h ∈∂Mh. A path γ : [0, ∞) →(M, d) is called an almost geodesic ray if d(γ (t), γ (0)) →∞, and for each ε > 0, there exists T ≥0 such that d(γ (t), γ (s)) + d(γ (s), γ (0)) −d(γ (t), γ (0)) < ε for all t ≥s ≥T . Let (yn) be an almost geodesic sequence in a geodesic metric space (M, d) and assume that Let (yn) be an almost geodesic sequence in a geodesic metric space (M, d) and assume that d(yn, y0) < d(yn+1, y0) for all n ≥0. (2.2) (2.2) For simplicity, we write n := d(yn, y0) and we let γn : [0, d(yn+1, yn)] →(M, d) be a geodesic path connecting yn and yn+1, i.e. γn(0) = yn and γn(d(yn+1, yn)) = yn+1 for all n ≥0 For simplicity, we write n := d(yn, y0) and we let γn : [0, d(yn+1, yn)] →(M, d) be a geodesic path connecting yn and yn+1, i.e. γn(0) = yn and γn(d(yn+1, yn)) = yn+1. for all n ≥0. Wewrite In := [n, n+1]andlet ¯γn : In →(M, d)betheafﬁnereparametrisation of γn given by ¯γn(t) := γn d(yn+1, yn) n+1 −n (t −n) for all t ∈In. We call the path ¯γ : [0, ∞) →(M, d) given by ¯γ (t) := ¯γn(t) for t ∈In a ray induced by (yn). Note that ¯γ is well deﬁned for all t ≥0 by (2.2). a ray induced by (yn). Note that ¯γ is well deﬁned for all t ≥0 by (2.2). Then by using (2.3), we know that for all n and k large, Suppose that t > s are such that t ∈In and s ∈Ik with n > k. Then by using (2.3), we know that for all n and k large, d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) ≤d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), yk) + d(yk, y0) −d( ¯γ (t), y0) ≤d( ¯γ (t), yn) + d(yn, ¯γ (s)) + d( ¯γ (s), yk) +d(yk, y0) −d( ¯γ (t), y0) < −d(yn, y0) + d(yn, ¯γ (s)) + d( ¯γ (s), yk) +d(yk, y0) + ε ≤−d(yn, y0) + d(yn, yk+1) + d(yk+1, ¯γ (s)) +d( ¯γ (s), yk) + d(yk, y0) + ε = −d(yn, y0) + d(yn, yk+1) + d(yk+1, yk) +d(yk, y0) + ε < −d(yn, y0) + d(yn, yk+1) +d(yk+1, y0) + 2ε < 3ε. d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) ≤d( ¯γ (t), ¯γ (s)) −d( ¯γ (t), y0) ≤d( ¯γ (t), yn) + d(yn, ¯γ (s)) + d( ¯γ (s), yk) +d(yk, y0) −d( ¯γ (t), y0) < −d(yn, y0) + d(yn, ¯γ (s)) + d( ¯γ (s), yk) +d(yk, y0) + ε ≤−d(yn, y0) + d(yn, yk+1) + d(yk+1, ¯γ (s)) +d( ¯γ (s), yk) + d(yk, y0) + ε = −d(yn, y0) + d(yn, yk+1) + d(yk+1, yk) +d(yk, y0) + ε < −d(yn, y0) + d(yn, yk+1) +d(yk+1, y0) + 2ε < 3ε. Finally suppose that t ≥s are such that t, s ∈In. Then for all n ≥0 large we have that d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) = d( ¯γ (t), yn) −d(yn, ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) ≤d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) < ε. d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) = d( ¯γ (t), yn) −d(yn, ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) ≤d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) < ε. As ¯γ is an almost geodesic ray, we know by [23, Lemma 4.5] that h ¯γ (t) →h′, where h′ is a horofunction. a ray induced by (yn). Note that ¯γ is well deﬁned for all t ≥0 by (2.2). Lemma 2.4 If (yn) is an almost geodesic sequence in a geodesic metric space (M, d) converging to a horofunction h and satisfying (2.2), then each ray, ¯γ , induced by (yn) satisﬁes: (i) ¯γ is an almost geodesic ray and h ¯γ (t) →h as t →∞, (ii) the map t →d( ¯γ (t), ¯γ (0)) is continuous on [0, ∞). (i) ¯γ is an almost geodesic ray and h ¯γ (t) →h as t →∞, (ii) the map t →d( ¯γ (t), ¯γ (0)) is continuous on [0, ∞). (i) ¯γ is an almost geodesic ray and h ¯γ (t) →h as t →∞, (ii) the map t →d( ¯γ (t), ¯γ (0)) is continuous on [0, ∞). Proof We ﬁrst show that for each ε > 0, there exists T ≥0 such that Proof We ﬁrst show that for each ε > 0, there exists T ≥0 such that d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) < ε for all t ≥T and n ≥0 with t ∈In. (2.3) d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) < ε for all t ≥T and n ≥0 with t ∈In. (2.3) 123 123 12 123 Page 7 of 20 16 A Metric Version of Poincaré’s Theorem Concerning... 160 To get this inequality, just note that there exists N ≥0 such that To get this inequality, just note that there exists N ≥0 such that d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) = d(yn+1, ¯γ (t)) + d( ¯γ (t), yn) + d(yn, y0) −d( ¯γ (t), y0) −d(yn+1, ¯γ (t)) ≤d(yn+1, yn)+d(yn, y0)−d(yn+1, y0)<ε, ≤d(yn+1, yn)+d(yn, y0)−d(yn+1, y0)<ε, for all t ∈In and n ≥N, as (yn) is an almost geodesic sequence. So to get (2.3), we can take T = N. for all t ∈In and n ≥N, as (yn) is an almost geodesic sequence. So to get (2.3), we can take T = N. To prove that ¯γ is an almost geodesic ray, we need to show that for each ε > 0, there exists S ≥0 such that d( ¯γ (t), ¯γ (s)) + d( ¯γ (s), ¯γ (0)) −d( ¯γ (t), ¯γ (0)) < ε for all t ≥s ≥S. Suppose that t > s are such that t ∈In and s ∈Ik with n > k. which completes the proof. which completes the proof. ⊓⊔ Lemma 2.5 If (yn) is an almost geodesic sequence in a geodesic metric space (M, d) satisfying (2.2) and ¯γ is a ray induced by (yn), then for each sequence (βn) in [0, ∞) with βn+1 > βn for all n ≥0, there exists sequence (tn) in [0, ∞) with tn+1 > tn for all n ≥0 such that d( ¯γ (tn), ¯γ (0)) = βn for all n ≥0. Proof Note that as γ : [0, ∞) →M is an almost geodesic by Lemma 2.4(i), we know that d(γ (t), γ (0)) →∞as t →∞. From Lemma 2.4(ii) we know that γ is continuous on [0, ∞), so there exists tn 0 ≥0 such that d(γ (tn 0 ), γ (0)) = βn. Now if we let tn := inf{t ≥0: d(γ (t), γ (0)) = βn}, then by continuity of γ we have that d(γ (tn), γ (0)) = βn and tn+1 > tn for all n ≥0. ⊓⊔ a ray induced by (yn). Note that ¯γ is well deﬁned for all t ≥0 by (2.2). As ¯γ (n) = yn for all n, we get that h′ = h. To prove the second assertion, we note that the afﬁne map t →d(yn+1, yn) n+1 −n (t −n) 123 123 Page 8 of 20 B. Lemmens 160 is a continuous map from In onto [0, d(yn1, yn)], and the map γn : [0, d(yn+1, yn)] → (M, d) is continuous, as γn is a geodesic. Thus, the map t →d( ¯γ (t), ¯γ (0)) is contin- uous on the interior of the interval In for all n ≥0. To get continuity at the endpoints, we simply note that for all n ≥0, lim t→−n d( ¯γ (t), ¯γ (0)) = d(yn, ¯γ (0)) = lim t→+n d( ¯γ (t), ¯γ (0)), which completes the proof. 2.1 Detour Distance Suppose that (M, d) is a proper geodesic metric space. Given two Busemann points h1, h2 ∈∂Mh, the detour cost is given by H(h1, h2) := inf (zn) lim inf n d(b, zn) + h2(zn), (2.4) (2.4) where the inﬁmum is taken over all sequences (zn) such that hzn converges to h1. It is known, see [15, Lemma 3.1], that if (zn) is an almost geodesic sequence converging to h1 and (wm) converges to h2, then where the inﬁmum is taken over all sequences (zn) such that hzn converges to h1. It is known, see [15, Lemma 3.1], that if (zn) is an almost geodesic sequence converging to h1 and (wm) converges to h2, then H(h1, h2) = lim n→∞ d(b, zn) + lim m→∞d(zn, wm) −d(b, wm) = lim n→∞d(b, zn) + h2(zn). The detour distance is given by The detour distance is given by δ(h1, h2) := H(h1, h2) + H(h2, h1). Note that for all m, n ≥0, we have that Note that for all m, n ≥0, we have that d(b, zn) + d(zn, wm) −d(b, wm) ≥0, so that H(h1, h2) ≥0 for all h1, h2 ∈∂Mh. It is, however, possible for H(h1, h2) to be inﬁnite. It can be shown, see [15, Sect. 3] or [27, Sect. 2], that the detour distance is independent of the basepoint. so that H(h1, h2) ≥0 for all h1, h2 ∈∂Mh. It is, however, possible for H(h1, h2) to be inﬁnite. It can be shown, see [15, Sect. 3] or [27, Sect. 2], that the detour distance is independent of the basepoint. 123 12 A Metric Version of Poincaré’s Theorem Concerning... Page 9 of 20 160 The detour distance was introduced in [5] and has been exploited and further devel- oped in [15, 27]. It is known, see for instance [15, Sect. 3] or [27, Sect. 2], that on BM ⊆∂Mh, the detour distance is symmetric, satisﬁes the triangle inequality, and δ(h1, h2) = 0 if and only if h1 = h2. This yields a partition of BM into equivalence The detour distance was introduced in [5] and has been exploited and further devel- oped in [15, 27]. It is known, see for instance [15, Sect. 3] or [27, Sect. 2], that on BM ⊆∂Mh, the detour distance is symmetric, satisﬁes the triangle inequality, and δ(h1, h2) = 0 if and only if h1 = h2. 2.1 Detour Distance This yields a partition of BM into equivalence classes, where h1 and h2 are said to be equivalent if δ(h1, h2) < ∞. The equivalence class of h will be denoted by P(h). Thus, the set of Busemann points, BM, is the disjoint union of metric spaces under the detour distance, which are called parts of BM. Isometric embeddings between proper geodesic metric spaces can be extended to the parts of the metric spaces as detour distance isometries. Indeed, suppose that ϕ : (M, d) →(N, ρ) is an isometric embedding, i.e. ρ(ϕ(x), ϕ(y)) = d(x, y) for all x, y ∈M. (Note that ϕ need not be onto.) If h is a Busemann point of (M, d) with basepoint b and (zn) is an almost geodesic sequence such that (hzn) converges to h, then (un), with un := ϕ(zn) for n ≥0, is an almost geodesic sequence in (N, ρ), and hence (hun) converges to a Busemann point, say ϕ(h), of (N, ρ) with basepoint ϕ(b). We note that ϕ(h) is independent of the almost geodesic sequence (zn). To see this, let (wn) be another almost geodesic such that (hwn) converges to h. Write vn := ϕ(wn) for n ≥0 and let ϕ(h)′ be the limit of (hvn). Then H(h, h) = lim n→∞d(wn, b) + lim m→∞d(wn, zm) −d(b, zm) = lim n→∞ρ(vn, ϕ(b)) + lim m→∞ρ(vn, um) −ρ(ϕ(b), um) = H(ϕ(h)′, ϕ(h)). Likewise, H(ϕ(h), ϕ(h)′) = H(h, h), and we deduce that δ(ϕ(h)′, ϕ(h)) = H(ϕ(h)′, ϕ(h)) + H(ϕ(h), ϕ(h)′) = δ(h, h) = 0, which shows that ϕ(h)′ = ϕ(h), as ϕ(h)′ and ϕ(h) are Busemann points. Thus, there exists a well-deﬁned map : BM →BN given by (h) := ϕ(h). Lemma 2.6 If ϕ : (M, d) →(N, ρ) is an isometric embedding, then (P(h)) ⊆ P(ϕ(h)) for all Busemann points h of (M, d) and Lemma 2.6 If ϕ : (M, d) →(N, ρ) is an isometric embedding, then (P(h)) ⊆ P(ϕ(h)) for all Busemann points h of (M, d) and δ(h′, h) = δ((h′), (h)) for all h, h′ ∈BM. Proof Let (zn) and (wn) be almost geodesic sequences such that (hzn) converges to h and (hwn) converges to h′ in (M, d) with basepoint b. 2.1 Detour Distance Moreover, there exists an almost geodesic sequence (zn) converging to h, where (zn j) is an almost geodesic converging to h j for j ∈J such that for all n ≥1, we have that d∞(zn, y0) − d j(zn j, y0 j ) = α j for j ∈J, and di(zn i , y0 i ) = 0 for all i /∈J. is a Busemann point with basepoint y0 = (y0 1, . . . , y0 p). Moreover, there exists an almost geodesic sequence (zn) converging to h, where (zn j) is an almost geodesic converging to h j for j ∈J such that for all n ≥1, we have that d∞(zn, y0) − d j(zn j, y0 j ) = α j for j ∈J, and di(zn i , y0 i ) = 0 for all i /∈J. j j (ii) If β ∈RJ with min j∈J β j = 0 and h′ is a Busemann point with basepoint y0 of the form, j j (ii) If β ∈RJ with min j∈J β j = 0 and h′ is a Busemann point with basepoint y0 of the form, h′(x) = max j∈J h j(x j) −β j, for x ∈ p j=1 M j, then δ(h, h′) = ∥α −β∥var. J then δ(h, h′) = ∥α −β∥var. then δ(h, h′) = ∥α −β∥var. then δ(h, h ) ∥α β∥var. (iii) For h as in (2.5) the part (P(h), δ) contains an isometric copy of (RJ/Sp(1), ∥· ∥var). (iii) For h as in (2.5) the part (P(h), δ) contains an isometric copy of (RJ/Sp(1), ∥· ∥var). Proof We know there exists an almost geodesic sequence (yn j ) in (M j, d j) such that hyn j →h j for each j ∈J. As d j(yn j , b j) →∞, we can take a subsequence and assume that d j(yn+1 j , y0 j ) > d j(yn j , y0 j ) > α j for all n ≥1. Let ¯γ j be a ray induced by (yn j ). j For j ∈J we get from Lemma 2.5 a sequence (tn j ) in [0, ∞) with t0 j = 0 and d j( ¯γ j(tn j ), y0 j ) = (max i∈J di(yn i , y0 i )) −α j ≥0 for all n ≥1. Let z0 := (y0 1, . . . 2.1 Detour Distance Then Proof Let (zn) and (wn) be almost geodesic sequences such that (hzn) converges to h and (hwn) converges to h′ in (M, d) with basepoint b. Then H(h′, h) = lim n→∞d(wn, b) + lim m→∞d(wn, zm) −d(b, zm) = lim n→∞ρ(vn, ϕ(b)) + lim m→∞ρ(vn, um) −ρ(ϕ(b), um) H(h′, h) = lim n→∞d(wn, b) + lim m→∞d(wn, zm) −d(b, zm) = lim n→∞ρ(vn, ϕ(b)) + lim m→∞ρ(vn, um) −ρ(ϕ(b), um) = H(ϕ(h)′, ϕ(h)). = H(ϕ(h)′, ϕ(h)). Likewise, H(h, h′) = H(ϕ(h), ϕ(h)′), so that δ(h′, h) = δ((h′), (h)), which completes the proof. ⊓⊔ Likewise, H(h, h′) = H(ϕ(h), ϕ(h)′), so that δ(h′, h) = δ((h′), (h)), which completes the proof. ⊓⊔ It could happen that all parts consist of a single Busemann point, but there are also natural instances where there are nontrivial parts. In case of products of metric spaces 123 Page 10 of 20 160 B. Lemmens coming from proper geodesic metric spaces, it turns out that the parts and the detour distance have a special structure that is linked to the quotient space, (Rn/Sp(1), ∥·∥var) given in (1.1), as shown by the following proposition. coming from proper geodesic metric spaces, it turns out that the parts and the detour distance have a special structure that is linked to the quotient space, (Rn/Sp(1), ∥·∥var) given in (1.1), as shown by the following proposition. Proposition 2.7 If, for j = 1, . . . , p, (M j, d j) is a proper geodesic metric space with almost geodesic sequence (yn j ) and corresponding Busemann point h j with basepoint y0 j , and J ⊆{1, . . . , p} is non-empty, then the following assertions hold: (i) For α ∈RJ with min j∈J α j = 0 the function h : (p j=1 M j, d∞) →R given by, h(x) = max j∈J h j(x j) −α j, for x ∈ p j=1 M j, (2.5) (2.5) is a Busemann point with basepoint y0 = (y0 1, . . . , y0 p). Moreover, there exists an almost geodesic sequence (zn) converging to h, where (zn j) is an almost geodesic is a Busemann point with basepoint y0 = (y0 1, . . . , y0 p). 2.1 Detour Distance As J is non-empty, we ﬁnd for all n ≥k large that d∞(zn, zk) + d∞(zk, z0) −d∞(zn, z0) = d j(zn j, zk j) + d j(zk j, z0 j) + α j −d j(zn j, z0 j) −α j < ε. d∞(zn, zk) + d∞(zk, z0) −d∞(zn, z0) = d j(zn j, zk j) + d j(zk j, z0 j) + α j −d j(zn j, z0 j) −α j < ε Also for n ≥0 large and x ∈p j=1 M j, we have that hzn(x) = max j∈J (d j(x j, zn j) −d∞(zn, z0)) = max j∈J (d j(x j, zn j) −d j(zn j, z0 j) −α j). Letting n →∞gives h(x) = max j∈J h j(x j) −α j for all x ∈ p j=1 M j and shows that h is a Busemann point with basepoint y0 = (y0 1, . . . , y0 p). This com- pletes the proof of assertion (i). and shows that h is a Busemann point with basepoint y0 = (y0 1, . . . , y0 p). This com- pletes the proof of assertion (i). To prove the second assertion, we know from the ﬁrst assertion that there exists an almost geodesic sequence (wn) converging to h′, where (wn j ) is an almost geodesic converging to h j and d∞(wn, y0) −d j(wn j , y0 j ) = β j for j ∈J. So, we get that max j∈J (β j −α j) = max j∈J (H(h j, h j) + β j −α j) = max j∈J ( lim n→∞d j(wn j , y0 j ) + β j + h j(wn j ) −α j) = max j∈J ( lim n→∞d∞(wn, y0) + h j(wn j ) −α j) = lim n→∞max j∈J (d∞(wn, y0) + h j(wn j ) −α j) = lim n→∞d∞(wn, y0) + h(wn). = max j∈J ( lim n→∞d j(wn j , y0 j ) + β j + h j(wn j ) −α j) = max j∈J ( lim n→∞d∞(wn, y0) + h j(wn j ) −α j) = lim n→∞max j∈J (d∞(wn, y0) + h j(wn j ) −α j) = lim n→∞d∞(wn, y0) + h(wn). 2.1 Detour Distance , y0 p) and for n ≥1 deﬁne zn = (zn 1, . . . , zn p) ∈p j=1 M j by zn j := ¯γ j(tn j ) if j ∈J, and zn j := y0 j otherwise. Let z0 := (y0 1, . . . , y0 p) and for n ≥1 deﬁne zn = (zn 1, . . . , zn p) ∈p j=1 M j by zn j := ¯γ j(tn j ) if j ∈J, and zn j := y0 j otherwise. As min α 0 we get by construction that j j J α j = 0, we get by construction that j j j j j As min j∈J α j = 0, we get by construction that j j j j j As min j∈J α j = 0, we get by construction that d∞(zn, z0) = max i∈J di(yn i , y0 i ) = d j(zn j, z0 j) + α j for all n ≥1 and j ∈J. Moreover, it follows from Lemma 2.4 that (zn j) is an almost geodesic converging to h j for j ∈J. Moreover, it follows from Lemma 2.4 that (zn j) is an almost geodesic converging to h j for j ∈J. 123 A Metric Version of Poincaré’s Theorem Concerning... A Metric Version of Poincaré’s Theorem Concerning... Page 11 of 20 160 We claim that (zn) is an almost geodesic sequence in (p j=1 M j, d∞). Indeed, note that for n ≥k ≥0, we have that d∞(zn, zk) + d∞(zk, z0) −d∞(zn, z0) = d j(zn j, zk j) + d∞(zk, z0) −d∞(zn, z0) for some j = j(n, k) ∈J, as d j(zn j, zk j) = 0 for all j /∈J. As J is non-empty, we ﬁnd for all n ≥k large that d∞(zn, zk) + d∞(zk, z0) −d∞(zn, z0) = d j(zn j, zk j) + d∞(zk, z0) −d∞(zn, z0) f j j( k) J d ( n k) 0 f ll j / J A J i t d∞(zn, zk) + d∞(zk, z0) −d∞(zn, z0) = d j(zn j, zk j) + d∞(zk, z0) −d∞(zn, z0) for some j = j(n, k) ∈J, as d j(zn j, zk j) = 0 for all j /∈J. 2.1 Detour Distance Interchanging the roles of h and h′, we ﬁnd that Interchanging the roles of h and h′, we ﬁnd that δ(h′, h) = H(h′, h) + H(h, h′) = max j∈J (β j −α j) + max j∈J (α j −β j) = ∥α −β∥var. The ﬁnal assertion is a direct consequence of the previous two, as (S, ∥· ∥var) with S := {α ∈RJ : min j∈J α j = 0} is isometric to (RJ/Sp(1), ∥· ∥var). ⊓⊔ The ﬁnal assertion is a direct consequence of the previous two, as (S, ∥· ∥var) with S := {α ∈RJ : min j∈J α j = 0} is isometric to (RJ/Sp(1), ∥· ∥var). ⊓⊔ 123 Page 12 of 20 160 B. Lemmens Proposition 2.7 is related to [26, Propositions 8.3 and 8.4], where the Busemann points for the product of two metric spaces are characterised and the detour cost is determined. Proposition 2.7 is related to [26, Propositions 8.3 and 8.4], where the Busemann points for the product of two metric spaces are characterised and the detour cost is determined. It is interesting to understand when a part (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥ ) It is interesting to understand when a part (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥var). Theorem 2.8 If, for j = 1, . . . , q, (N j, ρ j) is a proper geodesic metric space such that all horofunctions are Busemann points, and δ(h j, h′ j) = ∞for all h j ̸= h′ j Busemann points of (N j, ρ j), then every horofunction h of (q j=1 N j, d∞) is a Busemann point, and (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥var) for some J ⊆{1, . . . , q}. Proof Let h be a horofunction of (q j=1 N j, d∞) with respect to basepoint b = (b1, . . . , bq). By Theorem 2.3 we know that h is of the form h(x) = max j∈J h j(x j) −α j for x ∈ q j=1 N j, and h j is a horofunction of (N j, ρ j) with respect to basepoint b j for each j ∈J. As each horofunction of (N j, ρ j), is a Busemann point, there exists an almost geodesic sequence (yn j ) such that (hyn j ) converges to h j with basepoint b j. 2.1 Detour Distance Page 13 of 20 160 160 by Theorem 2.3, we know h′ is of the form by Theorem 2.3, we know h′ is of the form 2.3, we know h′ is of the form h′(x) = max j∈J ′ h′ j(x j) −β j, for x ∈ q j=1 N j, (2.6) (2.6) and, after taking a subsequence, we may assume that d∞(wn j , y0)−dk(wn j , y0 j ) →β j for all j ∈J ′, d∞(wn, y0) −di(wn i , y0 i ) →∞for all i /∈J ′, and hwn j →h′ j ∈∂N j h for all j ∈J ′. and, after taking a subsequence, we may assume that d∞(wn j , y0)−dk(wn j , y0 j ) →β j for all j ∈J ′, d∞(wn, y0) −di(wn i , y0 i ) →∞for all i /∈J ′, and hwn j →h′ j ∈∂N j h for all j ∈J ′. j We claim that if J ̸= J ′, or, J = J ′ and hk ̸= h′ k for some k ∈J, then δ(h∗, h′) = ∞. Suppose that J ̸= J ′ and k ∈J, but k /∈J ′. Then lim m→∞d∞(wn, zm) −d∞(y0, zm) = lim m→∞d∞(wn, zm) −dk(y0 k , zm k ) −γk ≥lim m→∞dk(wn k , zm k ) −dk(y0 k , zm k ) −γk ≥−dk(wn k , y0 k ) −γk, so that lim n→∞ d∞(wn, y0) + lim m→∞d∞(wn, zm) −d∞(y0, zm) ≥lim n→∞d∞(wn, y0) −dk(wn k , y0 k ) −γk = ∞ lim n→∞ d∞(wn, y0) + lim m→∞d∞(wn, zm) −d∞(y0, zm) ≥lim n→∞d∞(wn, y0) −dk(wn k , y0 k ) −γk = ∞. Thus, H(h′, h∗) = ∞and hence δ(h∗, h′) = ∞. The case where k ∈J ′ and k /∈J can be shown in the same way. y Now suppose that h∗ k ̸= h′ k for some k ∈J ∩J ′. By assumption we know that δ(h∗ k, h′ k) = ∞. Note that lim n→∞d∞(wn, y0) + h∗(wn) = lim n→∞d∞(wn, y0) + max j∈J h∗ j(wn j ) −γ j ≥lim inf n→∞dk(wn k , y0 k ) + h∗ k(wn k ) −γk. It now follows from (2.4) that H(h′, h∗) ≥H(h′ k, h∗ k) −γk. 2.1 Detour Distance j For j /∈J let y0 j = b j and deﬁne y0 := (y0 1, . . . , y0 q). Let h∗ j be the Busemann point obtained by changing the basepoint of h j to y0 j , so h∗ j(x j) := h j(x j) −h j(y0 j ). Now note that if we change the basepoint for h to y0, we get the Busemann point h∗(x) := h(x) −h(y0) = max j∈J (h j(x j) −α j) −max i∈J (hi(y0 i ) −αi) = max j∈J (h∗ j(x j) + h j(y0 j ) −α j −max i∈J (hi(y0 i ) −αi)) = max j∈J h∗ j(x j) −γ j, where γ j := maxi∈J(hi(y0 i )−αi)−(h j(y0 j )−α j) ≥0 for j ∈J and min j∈J γ j = 0. It now follows from Proposition 2.7(i) that h∗is a Busemann point of (q j=1 N j, d∞) with respect to basepoint y0, and hence h is a Busemann point (q j=1 N j, d∞) with respect to basepoint b. Moreover, there exists an almost geodesic sequence (zm) con- verging to h∗, where (zm j ) is an almost geodesic converging to h∗ j for j ∈J, and for all m ≥1 we have that d∞(zm, y0) −d j(zm j , y0 j ) = γ j for j ∈J, and di(zm i , y0 i ) = 0 for all i /∈J. To prove the second assertion we note that (P(h), δ) is isometric to (P(h∗), δ), since δ is independent of the basepoint. Let h′ is a Busemann point of (q j=1 N j, d∞) with respect to basepoint y0 and (wn) be an almost geodesic converging to h′. Then 123 A Metric Version of Poincaré’s Theorem Concerning... Page 13 of 20 160 on of Poincaré’s Theorem Concerning... Page 13 of 20 160 A Metric Version of Poincaré’s Theorem Concerning... 2.1 Detour Distance , n} non-empty and α ∈RJ with min j∈J α j = 0, where the sign is ﬁxed for each j ∈J, see also [10, Theorem 5.2]. Moreover, (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥var). We are now in position to prove Theorem 1.2. Proof of Theorem 1.2 As each (M j, d j) contains an almost geodesic sequence (yn j ), it has a Busemann point, say h j. We know from Proposition 2.7(i) that the function h of the form, h(x) = max j=1,...,p h j(x j), is a Busemann point of (p j=1 M j, d∞). Moreover, it follows from the third part of the same proposition that (P(h), δ) contains an isometric copy of (Rp/Sp(1), ∥· ∥var). Now suppose, for the sake of contradiction, that there exists an isometric embedding ϕ : (p j=1 M j, d∞) →(q j=1 N j, d∞). Then it follows from Lemma 2.6 that the restrictionoftoP(h)yieldsanisometricembeddingof(P(h), δ)into(P((h)), δ′), where δ′ is the detour distance on P((h)). It now follows from Theorem 2.8 that (P((h)), δ′) is isometric to (Rn/Sp(1), ∥· ∥var) for some n ∈{1, . . . , q}. So, yields an isometric embedding of (Rp/Sp(1), ∥· ∥var) into (Rn/Sp(1), ∥· ∥var) with n < p, which contradicts Brouwer’s invariance of domains theorem [8]. ⊓⊔ 2.1 Detour Distance Interchanging the roles of h∗and h′, we also get that H(h∗, h′) ≥H(h∗ k, h′ k) −βk, and hence δ(h∗, h′) ≥ δ(h∗ k, h′ k) −(γk + βk) = ∞. It now follows from (2.4) that H(h′, h∗) ≥H(h′ k, h∗ k) −γk. Interchanging the roles of h∗and h′, we also get that H(h∗, h′) ≥H(h∗ k, h′ k) −βk, and hence δ(h∗, h′) ≥ δ(h∗ k, h′ k) −(γk + βk) = ∞. k k On the other hand, if J = J ′ and h∗ j = h′ j for all j ∈J, then it follows from Proposition 2.7(ii) that δ(h∗, h′) = ∥α −β∥var. Moreover, it follows from that Propo- sition 2.7(i) that for each β ∈RJ with min j∈J β j = 0, there exists a Busemann point in the part of h∗of the form (2.6), and hence P(h∗) consists of all h′ of the form (2.6), where min j∈J β j = 0. So if we let S := {β ∈RJ : min j∈J β j = 0}, then (P(h∗), δ) is isometric to (S, ∥· ∥var), which in turn is isometric to the quotient space (RJ/Sp(1), ∥· ∥var). ⊓⊔ An elementary example is the product space (Rn, d∞) where d∞(x, y) = max j \|x j −y j\|. It is easy to verify that (R, \| · \|) with basepoint 0 has only two horo- functions, namely h+ : x →x and h−: x →−x, both of which are Busemann points 123 Page 14 of 20 160 B. Lemmens and δ(h+, h−) = ∞. So, in this case, we see that the horofunctions h of (Rn, d∞) are all Busemann points and of the form, and δ(h+, h−) = ∞. So, in this case, we see that the horofunctions h of (Rn, d∞) are all Busemann points and of the form, h(x) = max j∈J ±x j −α j, h(x) = max j∈J ±x j −α j, for some J ⊆{1, . . . , n} non-empty and α ∈RJ with min j∈J α j = 0, where the sign is ﬁxed for each j ∈J, see also [10, Theorem 5.2]. Moreover, (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥var). for some J ⊆{1, . . . 3 Product Domains in Cn Before we show how we can use Theorem 1.2 to derive Theorem 1.1, we ﬁrst recall some basic facts concerning the Kobayashi distance, see [13, Chapter 4] for more details. On the disc, := {z ∈C: \|z\| < 1}, the hyperbolic distance is given by ρ(z, w) := log 1 + w−z 1−¯zw 1 − w−z 1−¯zw =2 tanh−1 1 −(1 −\|w\|2)(1 −\|z\|2) \|1 −w¯z\|2 1/2 for z, w∈. Given a convex domain D ⊆Cn, the Kobayashi distance is given by Given a convex domain D ⊆Cn, the Kobayashi distance is given by kD(z, w) := inf{ρ(ζ, η): ∃f : →D holomorphic with f (ζ) = z and f (η) = w} for all z, w ∈D. It was shown by Lempert [18] that on bounded convex domains, the Kobayashi distance coincides with the Caratheodory distance, which is given by kD(z, w) := inf{ρ(ζ, η): ∃f : →D holomorphic with f (ζ) = z and f (η) = w} for all z, w ∈D. It was shown by Lempert [18] that on bounded convex domains, the Kobayashi distance coincides with the Caratheodory distance which is given by kD(z, w) := inf{ρ(ζ, η): ∃f : →D holomorphic with f (ζ) = z and f (η) = w} D(z, w) := inf{ρ(ζ, η): ∃f : →D holomorphic with f (ζ) = z and f (η) = w} for all z, w ∈D. It was shown by Lempert [18] that on bounded convex domains, the Kobayashi distance coincides with the Caratheodory distance, which is given by cD(z, w) := sup f ρ( f (z), f (w)) for all z, w ∈D, cD(z, w) := sup f ρ( f (z), f (w)) for all z, w ∈D, cD(z, w) := sup f ρ( f (z), f (w)) for all z, w ∈D, where the sup is taken over all holomorphic maps f : D →. 12 A Metric Version of Poincaré’s Theorem Concerning... Page 15 of 20 160 It is known, see [2, Proposition 2.3.10], that if D ⊂Cn is bounded convex domain, then (D, kD) is a proper metric space, whose topology coincides with the usual topol- ogy on Cn. Moreover, (D, kD) is a geodesic metric space containing geodesics rays, see [2, Theorem 2.6.19] or [13, Theorem 4.8.6]. In the case of the Euclidean ball Bn := {(z1, . . . 3 Product Domains in Cn , zn) ∈Cn : ∥z∥2 < 1}, where ∥z∥2 = i \|zi\|2, the Kobayashi distance has an explicit formula: kBn(z, w) = 2 tanh−1 1 −(1 −∥w∥2)(1 −∥z∥2) \|1 −⟨z, w⟩\|2 1/2 for all z, w ∈Bn, see [2, Chapters 2.2 and 2.3]. for all z, w ∈Bn, see [2, Chapters 2.2 and 2.3]. On the other hand, on the polydisc n := {(z1, . . . , zn) ∈Cn : maxi \|zi\| < 1}, the Kobayashi distance satisﬁes kn(z, w) = max i ρ(zi, wi) for all w = (w1, . . . , wn), z = (z1, . . . , zn) ∈n, kn(z, w) = max i ρ(zi, wi) for all w = (w1, . . . , wn), z = (z1, . . . , zn) ∈n, by the product property, see [13, Theorem 3.1.9]. by the product property, see [13, Theorem 3.1.9]. To determine the horofunctions of (Bn, kBn), with basepoint b = 0, it sufﬁces to consider limits of sequences (hwn), where wn →ξ ∈∂Bn in norm. As kBn(z, wn) = log \|1 −⟨z, wn⟩\| + (\|1 −⟨z, wn⟩\|2 −(1 −∥z∥2)(1 −∥wn∥2))1/22 (1 −∥z∥2)(1 −∥wn∥2) , and kBn(0, wn) = log (1 + ∥wn∥)2 1 −∥wn∥2 , it follows that it follows that it follows that h(z) = lim n→∞kBn(z, wn) −kBn(0, wn) = log (\|1 −⟨z, ξ⟩\| + \|1 −⟨z, ξ⟩\|)2 (1 −∥z∥2)(1 + ∥ξ∥)2 = log \|1 −⟨z, ξ⟩\|2 1 −∥z∥2 . h(z) = lim n→∞kBn(z, wn) −kBn(0, wn) = log (\|1 −⟨z, ξ⟩\| + \|1 −⟨z, ξ⟩\|)2 (1 −∥z∥2)(1 + ∥ξ∥)2 = log \|1 −⟨z, ξ⟩\|2 1 −∥z∥2 . for all z ∈Bn. Thus, if we write hξ(z) := log \|1 −⟨z, ξ⟩\|2 1 −∥z∥2 for all z ∈Bn, (3.1) (3.1) then we obtain ∂Bnh = {hξ : ξ ∈∂Bn}, see also [3, Lemma 2.28] and [11, Remark 3.1]. Moreover, each hξ is a Busemann point, as it is the limit induced by the geodesic ray t →et−1 et+1ξ, for 0 ≤t < ∞. 123 160 Page 16 of 20 B. Lemmens 160 Corollary 3.1 If hξ and hη are distinct horofunctions of (Bn, kBn), then δ(hξ, hη) = ∞. 3 Product Domains in Cn Proof If ξ ̸= η in ∂Bn, then Proof If ξ ̸= η in ∂Bn, then lim z→η kBn(z, 0) + hξ(z) = lim z→η log 1 + ∥z∥ 1 −∥z∥+ log \|1 −⟨z, ξ⟩\|2 1 −∥z∥2 = ∞, lim z→η kBn(z, 0) + hξ(z) = lim z→η log 1 + ∥z∥ 1 −∥z∥+ log \|1 −⟨z, ξ⟩\|2 1 −∥z∥2 = ∞, which implies that δ(hξ h ) = ∞ h implies that δ(hξ, hη) = ∞. ⊓⊔ ⊓⊔ Note that if n = 1, we recover the well-known expression for the horofunctions of the hyperbolic distance on : hξ(z) = log \|1 −zξ\|2 1 −\|z\|2 = log \|ξ −z\|2 1 −\|z\|2 for all z ∈. Combining (3.1) with Theorem 2.3 and Proposition 2.7, we get the following. Combining (3.1) with Theorem 2.3 and Proposition 2.7, we get the following. Corollary 3.2 For Bn1 × · · · × Bnq, the Kobayashi distance horofunctions with base- point b = 0 are precisely the functions of the form, h(z) = max j∈J log \|1 −⟨z j, ξ j⟩\|2 1 −∥z j∥2 −α j , where J ⊆{1, . . . , q} non-empty, ξ j ∈∂Bn j for j ∈J, and min j∈J α j = 0. Moreover, each horofunction is a Busemann point, and (P(h), δ) is isometric to (RJ/Sp(1), ∥· ∥var). Corollary 3.2 should be compared with [2, Proposition 2.4.12]. Asimilarresultholdsformoregeneralproductdomains.Weknowfrom[2,Theorem 2.6.45] that for each ξ ∈∂D, there exists a unique geodesic ray γξ : [0, ∞) →D such that γξ(0) = b and limt→∞γξ(t) = ξ, if D ⊂C is bounded strongly convex domain with C3-boundary. We will denote the corresponding Busemann point by hξ : D →R, so hξ(z) = lim t→∞kD(z, γξ(t)) −kD(b, γξ(t)). Lemma 3.3 If D ⊂Cn is a bounded strongly convex domain with C3-boundary, then each horofunction of (D, kD) is a Busemann point and of the form hξ for some ξ ∈∂D. Moreover, δ(hξ, hη) = ∞if ξ ̸= η. If D = r i=1 Di, where each Di is a bounded strongly convex domain with C3-boundary, then the horofunctions h of (D, kD) are Busemann points and precisely the functions of the form, h(z) = max j∈J hξ j (z j) −α j, where J ⊆{1, . . . ,r} non-empty, ξ j ∈∂D j for j ∈J, and min j∈J α j = 0. 3 Product Domains in Cn 123 12 A Metric Version of Poincaré’s Theorem Concerning... Page 17 of 20 160 Proof To prove the ﬁrst assertion let h ̸= h′ be horofunctions of (D, kD). As (D, kD) is a proper geodesic metric space, we know there exist sequences (wn) and (zn) in D such that hwn →h and hzn →h′. After taking subsequences, we may assume that wn →ξ ∈∂D and zn →η ∈∂D, since D has a compact norm closure and h and h′ are horofunctions. We claim that ξ ̸= η. To prove this, we need the assumption that D ⊂C is a bounded strongly convex domain with C3-boundary and use results by Abate [1] concerning the so-called small and large horospheres. These are deﬁned as follows: for R > 0, the small horosphere with centre ζ ∈∂D (and basepoint b ∈D) is given by E(ζ, R) := x ∈D : lim sup z→ζ kD(x, z) −kD(b, z) < 1 2 log R and the large horosphere with centre ζ ∈∂D (and basepoint b ∈D) is given by F(ζ, R) := x ∈D : lim inf z→ζ kD(x, z) −kD(b, z) < 1 2 log R . We note that the horoballs We note that the horoballs H(h, 1 2 log R) = x ∈D : lim n→∞kD(x, wn) −kD(b, wn) < 1 2 log R and H(h′, 1 2 log R) = x ∈D : lim n→∞kD(x, zn) −kD(b, zn) < 1 2 log R satisfy satisfy satisfy satisfy E(ξ, R) ⊆H(h, 1 2 log R) ⊆F(ξ, R)and E(η, R) ⊆H(h′, 1 2 log R) ⊆F(η, R). It follows from [2, Theorem 2.6.47] (see also [1]) that E(ξ, R) = H(h, 1 2 log R) = F(ξ, R) and E(η, R) = H(h′, 1 2 log R) = F(η, R), as D is strongly convex and has C3-boundary. Thus, if ξ = η, then h = h′, since the horoballs, H(h,r) and H(h′,r) for r ∈R, completely determine the horofunctions. This shows that ξ ̸= η. It follows that each horofunction is of the form hξ, with ξ ∈∂D, and hence a Busemann point. 1 2 Now suppose that h1 and h2 are horofunctions, with (z1 n) converging to h1 and (z2 n) converging to h2. After taking a subsequence, we may assume that z1 n →ξ1 ∈∂D and z2 n →ξ2 ∈∂D. 3 Product Domains in Cn We now show that if ξ1 ̸= ξ2, then h1 ̸= h2. This implies that there is a one-to-one correspondence between the horofunctions of (D, kD) and ξ ∈∂D. To prove this, we note that as D is strongly convex, D is strictly convex, i.e. for each ν ̸= μ in ∂D, the open straight-line segment (ν, μ) ⊂D. By [1, Theorem 1.7], we know that ∂D ∩cl(F(h1, R)) = {ξ1} and ∂D ∩cl(F(h2, R)) = {ξ2} for 123 123 Page 18 of 20 B. Lemmens 160 all R > 0. This implies that ∂D ∩cl(H(h1,r)) = {ξ1} and ∂D ∩cl(H(h2,r)) = {ξ2} for all r ∈R. Moreover, from [4, Lemma 5], we know that the straight-line segment [b, ξ1] ⊂cl(H(h1, 0)). There exists a neighbourhood W ⊂Cn of ξ1 such that W ∩cl(H(h2, 0)) = ∅. If we let w ∈[b, ξ1)∩W, then h1(w) ≤0, but h2(w) > 0, and hence h1 ̸= h2. Now suppose that ξ ̸= η in ∂D. We know that ∂D ∩cl(H(hη, 0)) = {η} and γξ(t) /∈cl(H(hη, 0)) for all t > 0 large. So, H(hξ, hη) = lim t→∞kD(γξ(t), b) + hη(γξ(t)) ≥lim inf t→∞kD(γξ(t), b) = ∞, since hη(γξ(t)) ≥0 for all t large. This implies that δ(hξ, hη) = ∞. nce hη(γξ(t)) ≥0 for all t large. This implies that δ(hξ, hη) = ∞. ⊓⊔ η ξ ξ η The ﬁnal part follows directly from Theorem 2.3 and Proposition 2.7. The proof of Theorem 1.1 is now elementary. Proof of Theorem 1.1 If X j ⊂Cm j is a bounded convex domain, then (X j, kX j ) is proper geodesic metric space which contains a geodesic ray by [2, Theorem 2.6.19]. Moreover, if Y j ⊂Cn j is a bounded strongly convex domain with C3-boundary, then by Lemma 3.3, all the horofunctions of (Y j, kY j ) are Busemann points and any two distinct Busemann points have inﬁnite detour distance. So, Theorem 1.2 applies and gives the desired result. ⊓⊔ ⊓⊔ Remark 3.4 I am grateful to Andrew Zimmer for sharing the following observations with me. In the case where q = 1, Theorem 1.1 can be strengthened and shown in a variety of other ways. Acknowledgements The author wishes to thank the referee for their useful feedback and careful reading of the paper. 3 Product Domains in Cn Indeed, it was shown by Balogh and Bonk [6] that the Kobayashi distance is Gromov hyperbolic on a strongly pseudo-convex domain with C2-boundary, but the Kobayashi distance on a product domain is clearly not Gromov hyperbolic. This immediately implies Theorem 1.1 for q = 1 in the more general case where the image domain is strongly pseudo-convex and has C2-boundary. In fact, if q = 1 there exists a further strengthening of Theorem 1.1 which only requires the image domain to be strictly convex by using a local argument. The iso- metric embedding is a locally Lipschitz map with respect to the Euclidean norm, and hence differentiable almost everywhere by Rademacher’s theorem. This implies that the embedding is also an isometric embedding under the Kobayashi inﬁnitesimal met- ric. On strictly convex domains, the unit balls in the tangent spaces are strictly convex and in product domains, they are not, which yields a contradiction. Finally, for holomorphic isometric embeddings and q = 1, Theorem 1.1 can be extended to the case where the image domain is convex with C1,α-boundary, see [28, Theorem 2.22]. Looking at the conditions required in Theorem 1.2, it seems likely the regularity conditions on the domains Y j in Theorem 1.1 can be relaxed considerably. In particular, one may speculate that it sufﬁces to assume that each domain Y j is strictly convex and has a C1-boundary. Acknowledgements The author wishes to thank the referee for their useful feedback and careful reading of the paper. 123 A Metric Version of Poincaré’s Theorem Concerning... Page 19 of 20 160 OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References 1. Abate, M.: Horospheres and iterates of holomorphic maps. Math. Z. 198, 225–238 (1988) 2. Abate, M.: Iteration Theory of Holomorphic Maps on Taut Manifolds. Research and Lecture Notes Mathematics. Complex Analysis and Geometry. Mediterranean Press, Rende (1989) 3. Abate, M.: The Kobayashi distance in holomorphic dynamics and operator theory. In: Blanc-Centi, L. (ed.) Metrics and Dynamical Aspects in Complex Analysis. Springer, New York (2017) 3. Abate, M.: The Kobayashi distance in holomorphic dynamics and operator theory. In: Blan (ed.) Metrics and Dynamical Aspects in Complex Analysis. Springer, New York (2017) 4. Abate, M., Raissy, J.: Wolff-Denjoy theorems in nonsmooth convex domains. Ann. Mat. Pura Appl. (4) 193(5), 1503–1518 (2014) 5. Akian, M., Gaubert, S., Walsh, C.: The max-plus Martin boundary. Doc. Math. 14, 195–240 (2009) 5. Akian, M., Gaubert, S., Walsh, C.: The max-plus Martin boundary. Doc. Math. 14, 195–240 (2009) 6 Balogh Z M Bonk M : Gromov hyperbolicity and the Kobayashi metric on strictly pseudoconvex 5. Akian, M., Gaubert, S., Walsh, C.: The max-plus Martin boundary. Doc. Math. 14, 195–240 (2009) 6. Balogh, Z.M., Bonk, M.: Gromov hyperbolicity and the Kobayashi metric on strictly pseudoconvex domains Comment Math Helv 75(3) 504–533 (2000) 6. Balogh, Z.M., Bonk, M.: Gromov hyperbolicity and the Kobayashi metric on strictly pseudoconve domains. Comment. Math. Helv. 75(3), 504–533 (2000) 7. Bracci, F., Gaussier, H.: Horosphere topology. Ann. Sc. Norm. Super. Pisa Cl. Sci. (5) 20(1), 239–289 (2020) 8. Brouwer, L.E.J.: Beweis der Invarianz des n-dimensionalen Gebiets. Math. Ann. 71, 305–315 (1912) 8. Brouwer, L.E.J.: Beweis der Invarianz des n dimensionalen Gebiets. Math. Ann. 71, 305 315 (191 9. Duchin, M., Fisher, N.: Stars at inﬁnity in Teichmüller space. Geom. Dedicata 213, 531–545 (2021 9. Duchin, M., Fisher, N.: Stars at inﬁnity in Teichmüller space. Geom. Dedicata 213, 531–545 (2021) 10. Gutiérrez, A.W.: The horofunction boundary of ﬁnite-dimensional ℓp spaces. Colloq. Math. 155(1), 51–65 (2019) 10. Gutiérrez, A.W.: The horofunction boundary of ﬁnite-dimensional ℓp spaces. Colloq. Math. 155(1), 51–65 (2019) 11. Kapeluszny, J., Kuczumow, T., Reich, S.: The Denjoy-Wolff theorem in the open unit ball of a strictly convex Banach space. Adv. Math. 143(1), 111–123 (1999) p 12. Karlsson, A.: Elements of a metric spectral theory (2019). arXiv:1904.01398 13. Kobayashi, S.: Hyperbolic complex spaces. In: Grundlehren der Mathematischen Wissenschaften, vol. 318. Springer, Berlin (1998) p g 14. Lemmens, B., Nussbaum, R.: Nonlinear Perron-Frobenius Theory, Cambridge Tracts in Mathematics 189. Cambridge University, Press (2012) 15. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 28. Zimmer, A.M.: Characterizing domains by the limit set of their automorphism group (2017). arXiv:1506.07852 26. Walsh, C.: Gauge-reversing maps on cones, and Hilbert and Thompson isometries. Geom. Topol. 22(1), 55–104 (2018) 27 W l h C Hilb t d Th t i i t i t i ﬁit di i l B h A 29. Zwonek, W.: Izometrie Zespolone, PhD Dissertation, Jagiellonian University, Kraków, (1994) 30. Zwonek, W.: The Carathéodory isometries between the products of balls. Arch. Math. (Basel) 65, 434–443 (1995) 27. Walsh, C.: Hilbert and Thompson geometries isometric to inﬁnite-dimensional Banach spaces. An Inst. Fourier (Grenoble) 68(5), 1831–1877 (2018) References Lemmens, B., Walsh, C.: Isometries of polyhedral Hilbert geometries. J. Topol. Anal. 3(2), 213–241 (2011) 16. Lemmens, B., Lins, B., Nussbaum, R.: Detecting ﬁxed points of nonexpansive maps by illuminating the unit ball. Isr. J. Math. 224(1), 231–262 (2018) 17. Lemmens, B., Roelands, M., Wortel, M.: Isometries of inﬁnite dimensional Hilbert geometries. Topol. Anal. 10(4), 941–959 (2018) 18. Lempert, L.: La métrique de Kobayashi et la représentation des domaines sur la boule. Bull. Soc. Mat France 109, 427–474 (1981) 19. Mahajan, P.: On isometries of the Kobayashi and Caratheodory metrics. Ann. Polon. Math. 104(2), 121–151 (2012) 20. Minsky, Y.N.: Extremal length estimates and product regions in Teichmüller space. Duke Math. J. 83(2), 249–286 (1996) 21. Papadopoulos, A.: Metric spaces, convexity and nonpositive curvature. In: IRMA Lectures in Mathe- matics and Theoretical Physics, 6. European Mathematical Society (EMS), Zürich (2005) 22. Poincaré, H.: Les fonctions analytiques de deux variables et la répresentation conforme. Rend. Circ. Mat. Palermo 23, 185–220 (1907) ( ) 23. Rieffel, M.A.: Group C∗-algebras as compact quantum metric spaces. Doc. Math. 7, 605–651 (200 3. Rieffel, M.A.: Group C∗-algebras as compact qua 23. Rieffel, M.A.: Group C∗-algebras as compact quantum metric spaces. Doc. Math. 7, 605–651 (2002) 24. Walsh, C.: The horofunction boundary of ﬁnite-dimensional normed spaces. Math. Proc. Camb. Philos. Soc. 142(3), 497–507 (2007) 24. Walsh, C.: The horofunction boundary of ﬁnite-dimensional normed spaces. Math. Proc. Camb. Philos. Soc. 142(3), 497–507 (2007) 25. Walsh, C.: The horofunction boundary of the Hilbert geometry. Adv. Geom. 8(4), 503–529 (2008) 12 3 160 Page 20 of 20 160 B. Lemmens 27. Walsh, C.: Hilbert and Thompson geometries isometric to inﬁnite-dimensional Banach spaces. An Inst. Fourier (Grenoble) 68(5), 1831–1877 (2018) 28. Zimmer, A.M.: Characterizing domains by the limit set of their automorphism group (2017 arXiv:1506.07852 29. Zwonek, W.: Izometrie Zespolone, PhD Dissertation, Jagiellonian University, Kraków, (1994) 30. Zwonek, W.: The Carathéodory isometries between the products of balls. Arch. Math. (Basel) 6 434–443 (1995) 123
https://openalex.org/W2108414867	https://dspace.mit.edu/bitstream/1721.1/82924/1/Woodfield-2013-Electron%20acceleratio.pdf	English	null	Electron acceleration at Jupiter: input from cyclotron-resonant interaction with whistler-mode chorus waves	Annales geophysicae	2,013	cc-by	9,139	MIT Open Access Articles Electron acceleration at Jupiter: input from cyclotron- resonant interaction with whistler-mode chorus waves The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. tation: Woodfield, E. E., R. B. Horne, S. A. Glauert, J. D. Menietti, and Y. Y. Shprits. “Electron cceleration at Jupiter: input from cyclotron-resonant interaction with whistler-mode chorus aves.” Annales Geophysicae 31, no. 10 (October 2, 2013): 1619-1630. s Published: http://dx.doi.org/10.5194/angeo-31-1619-2013 ublisher: Copernicus GmbH ersistent URL: http://hdl.handle.net/1721.1/82924 MIT Open Access Articles Electron acceleration at Jupiter: input from cyclotron- resonant interaction with whistler-mode chorus waves The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation: Woodfield, E. E., R. B. Horne, S. A. Glauert, J. D. Menietti, and Y. Y. Shprits. “Electron acceleration at Jupiter: input from cyclotron-resonant interaction with whistler-mode chorus waves.” Annales Geophysicae 31, no. 10 (October 2, 2013): 1619-1630. As Published: http://dx.doi.org/10.5194/angeo-31-1619-2013 Publisher: Copernicus GmbH MIT Open Access Articles The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation: Woodfield, E. E., R. B. Horne, S. A. Glauert, J. D. Menietti, and Y. Y. Shprits. “Electron acceleration at Jupiter: input from cyclotron-resonant interaction with whistler-mode chorus waves.” Annales Geophysicae 31, no. 10 (October 2, 2013): 1619-1630. Version: Final published version: final published article, as it appeared in a journal, conference proceedings, or other formally published context 1 Introduction Abstract. Jupiter has the most intense radiation belts of all the outer planets. It is not yet known how electrons can be accelerated to energies of 10 MeV or more. It has been sug- gested that cyclotron-resonant wave-particle interactions by chorus waves could accelerate electrons to a few MeV near the orbit of Io. Here we use the chorus wave intensities ob- served by the Galileo spacecraft to calculate the changes in electron ﬂux as a result of pitch angle and energy diffusion. We show that, when the bandwidth of the waves and its vari- ation with L are taken into account, pitch angle and energy diffusion due to chorus waves is a factor of 8 larger at L- shells greater than 10 than previously shown. We have used the latitudinal wave intensity proﬁle from Galileo data to model the time evolution of the electron ﬂux using the British Antarctic Survey Radiation Belt (BAS) model. This pro- ﬁle conﬁnes intense chorus waves near the magnetic equa- tor with a peak intensity at ∼5◦latitude. Electron ﬂuxes in the BAS model increase by an order of magnitude for ener- gies around 3 MeV. Extending our results to L = 14 shows that cyclotron-resonant interactions with chorus waves are equally important for electron acceleration beyond L = 10. These results suggest that there is signiﬁcant electron ac- celeration by cyclotron-resonant interactions at Jupiter con- tributing to the creation of Jupiter’s radiation belts and also increasing the range of L-shells over which this mechanism should be considered. Jupiter has the most intense radiation belts of all the mag- netized planets, with electron energies in excess of 50 MeV at L = 1.4 (Bolton et al., 2002; de Pater and Dunn, 2003). These belts are believed to be formed by inward radial trans- port of electrons from a source beyond the orbit of the moon Io (L = 6.6) on the assumption that the ﬁrst adiabatic invari- ant remains conserved (Santos-Costa and Bourdarie, 2001; Sicard and Bourdarie, 2004). However, this idea requires a source of electrons in excess of 1 MeV for L > 6.6 (Woch et al., 2004); the ﬁrst such indications of a source were given in Horne et al. (2008). Correspondence to: E. E. Woodﬁeld (emmwoo@bas.ac.uk) Correspondence to: E. E. Woodﬁeld (emmwoo@bas.ac.uk) Received: 4 June 2013 – Revised: 9 August 2013 – Accepted: 12 August 2013 – Published: 2 October 2013 1 Introduction Strong whistler-mode waves have been observed outside the orbit of Io (Gurnett et al., 1996; Menietti et al., 2008) in association with magnetic ﬂux interchange instabilities (Kivelson et al., 1997; Thorne et al., 1997; Xiao et al., 2003). These waves are thought to be generated by the in- ﬂow of relatively warm electrons at energies of typically a few keV resulting from the interchange instability (Hill et al., 1981; Kivelson et al., 1997). The interchange instabil- ity moves overdense ﬂux tubes, created by ionization of ma- terial ejected from Io, outwards to be replaced with an inﬂow of hotter but less dense plasma originating further away from the planet. The potential energy released during this process both drives the instability and adiabatically heats the incom- ing plasma (Thorne et al., 1997). At Earth chorus waves are very effective in accelerating electrons to MeV (Summers et al., 1998; Horne et al., 2005; Chen et al., 2007), and thus, by analogy, it has been suggested that electron acceleration Keywords. Magnetospheric physics (energetic particles, trapped; planetary magnetospheres) – Space plasma physics (wave–particle interactions) Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ doi:10.5194/angeo-31-1619-2013 © Author(s) 2013. CC Attribution 3.0 License. Electron acceleration at Jupiter: input from cyclotron-resonant interaction with whistler-mode chorus waves E. E. Woodﬁeld1, R. B. Horne1, S. A. Glauert1, J. D. Menietti2, and Y. Y. S 1British Antarctic Survey, Cambridge, UK 2Department of Physics and Astronomy, University of Iowa, USA 3Skolkovo Institute of Science and Technology, Moscow, Russian Federation 4Massachusetts Institute of Technology, Cambridge, MA, USA 5University of California, Los Angeles, CA, USA E. E. Woodﬁeld1, R. B. Horne1, S. A. Glauert1, J. D. Menietti2, and Y. Y. Shprits3,4,5 1British Antarctic Survey, Cambridge, UK 2Department of Physics and Astronomy, University of Iowa, USA 3Skolkovo Institute of Science and Technology, Moscow, Russian Federation 4Massachusetts Institute of Technology, Cambridge, MA, USA 5University of California, Los Angeles, CA, USA 1British Antarctic Survey, Cambridge, UK 2Department of Physics and Astronomy, University of Iowa, USA 3Skolkovo Institute of Science and Technology, Moscow, Russian Federation 4Massachusetts Institute of Technology, Cambridge, MA, USA 5University of California, Los Angeles, CA, USA E. E. Woodﬁeld et al.: Electron acceleration at Jupiter E. E. Woodﬁeld et al.: Electron acceleration at Jupiter by whistler-mode chorus waves could provide the source of MeV electrons for Jupiter’s radiation belts (Horne et al., 2008). Recent work shows that Jupiter is the most favorable planet for this type of acceleration (Shprits et al., 2012). effects of chorus on the electron distribution over a period of several hours or days due to computer limitations. It is therefore necessary to use an approximation. One of the most widely used approximations is quasi-linear theory, which has been used extensively in radiation belt models at the Earth (e.g., Varotsou et al., 2005, 2008; Albert et al., 2009; Shprits et al., 2009a, b; Fok et al., 2008; Su et al., 2010). Quasi-linear theory does not include phase trapping or phase bunching of electrons but assumes a broad band of waves and uses the time-averaged wave power. Quasi-linear theory treats the in- teraction as a diffusion process where the electrons are dif- fused in pitch angle α and energy E by the waves (e.g., Glauert and Horne, 2005). Test particle simulations (Tao et al., 2012) and comparisons between particle scattering in the fully nonlinear and diffusive regimes (Albert, 2010) pro- vide a remarkable level of agreement for small-amplitude waves, and also when quasi-linear diffusion rates are aver- aged over wave frequency and direction of propagation in a non-homogeneous plasma (Albert, 2010). Wave acceleration is most effective when the ratio of plasma frequency to gyrofrequency (fpe/fce) is low, typi- cally < 4 (Horne et al., 2005). At Jupiter wave observations have only been made for latitudes ≲10◦in the region out- side Io; previous work to calculate the electron acceleration due to chorus waves assumed the waves only extended to 10◦(Horne et al., 2008). However, observations at Earth and Saturn show that waves are observed up to latitudes of 45◦ (Meredith et al., 2001, 2012; Bunch et al., 2012; Menietti et al., 2012). Since the plasma density drops signiﬁcantly with increasing latitude (Bagenal, 1994), the conditions for acceleration may be more favorable at higher latitudes. Sh- prits et al. (2012) showed that changing the theoretical lati- tudinal extent of the chorus waves plays a major role in the effectiveness of wave–particle interactions at Jupiter and Sat- urn. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter In this study we use data from the Galileo spacecraft to investigate in more detail the acceleration of electrons by wave–particle interactions at Jupiter in the region outside Io. Changes in electron distribution due to wave–particle in- teractions can be calculated from a diffusion equation given by Schulz and Lanzerotti (1974); Subbotin and Shprits (2009), ∂f ∂t = 1 g(α) ∂ ∂α E g(α)Dαα ∂f ∂α E + 1 A(E) ∂ ∂E α A(E)DEE ∂f ∂E α −f τ (1) g(α) = sin2α 1.3802 −0.3198(sinα + (sinα) 1 2 ) (2) A(E) = (E + E0)(E(E + 2E◦)) 1 2 , (3) ∂f ∂t = 1 g(α) ∂ ∂α E g(α)Dαα ∂f ∂α E + 1 A(E) ∂ ∂E α A(E)DEE ∂f ∂E α −f τ (1) g(α) = sin2α 1.3802 −0.3198(sinα + (sinα) 1 2 ) (2) A(E) = (E + E0)(E(E + 2E◦)) 1 2 , (3) 2 Calculating diffusion coefﬁcients When whistler-mode chorus waves are examined with high time resolution they are usually characterized by short- duration bursts of radiation below the local electron gyrofre- quency, fce, which rise or fall rapidly in frequency. This type of frequency time structure is observed at Jupiter (Gur- nett and Scarf, 1983) as well as the Earth (e.g., Burtis and Helliwell, 1969; Tsurutani and Smith, 1974; Santolík et al., 2003) and Saturn (Hospodarsky et al., 2008). Individual cho- rus bursts may only last a few milliseconds, but they often overlap in time and occur repeatedly for periods of hours and longer. For example, chorus waves were observed for several hours by Galileo each time the spacecraft orbited in- side 12 RJ, and this occurred on several orbits (e.g., Fig. 4 of Menietti et al., 2008). The frequency-time characteris- tics and discrete bursty nature of the signals suggests that the waves are generated by nonlinear wave–particle interac- tions (Trakhtengerts, 1999; Nunn et al., 1997; Omura et al., 2009). The general concept is that an electron temperature anisotropy causes linear wave growth and some of the res- onant electrons become trapped and phase bunched by the waves via nonlinear effects. The trapped particles then act as a resonant current and re-radiate at a higher frequency with a nonlinear growth rate (for rising tones). These non- linear wave–particle interactions and the formation of rising frequency elements have been shown in simulations (Omura et al., 2009; Katoh and Omura, 2007, 2011) which require large amounts of computing resources. α g(α) = sin2α 1.3802 −0.3198(sinα + (sinα) 1 2 ) (2) A(E) = (E + E0)(E(E + 2E◦)) 1 2 , (3) (3) where f is the phase space density, α is the equatorial pitch angle, Dαα and DEE are the pitch angle and energy diffu- sion coefﬁcients respectively, E0 is the electron rest energy and τ is the loss timescale which is only non-zero inside the loss cone. The ﬁrst term represents diffusion in pitch angle, the second diffusion in energy and the third represents losses to the atmosphere. The diffusion coefﬁcients are calculated separately before solving Eq. (1) by using a code such as the PADIE code (Pitch Angle and energy Diffusion of Ions and Electrons; Glauert and Horne, 2005) to generate a value of Dαα and DEE for each electron energy and L-shell. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1620 www.ann-geophys.net/31/1619/2013/ E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1621 Fig. 1. Mean energy diffusion coefﬁcients (averaged over all pitch angles) for our new results and those of Horne et al. (2008). Fig. 1. Mean energy diffusion coefﬁcients (averaged over all pitch to Horne et al. (2008). A Gaussian power spectrum for the chorus waves is assumed with a frequency of the peak or maximum given by fmax = 0.15fce and a half width at half maximum of 0.05fce. We have assumed that the chorus wave power is dominated by lower-band chorus since upper-band chorus has rarely been seen at Jupiter (Menietti et al., 2012). The direction of propagation (wave normal angle, ψ) is as- sumed to be described by a Gaussian distribution of X = tan(ψ), ﬁeld aligned with a width of Xw = tan(30◦) in com- mon with studies of chorus at the Earth (Horne et al., 2003). When wave propagation at an angle to the magnetic ﬁeld, B, is considered, electron diffusion by both Landau (n = 0) and higher-order cyclotron harmonic resonances are impor- tant. We use n = 0 and n = ±1,±2,±3,±4,±5 resonances in calculating the diffusion coefﬁcients. We start by assuming that the resonant interactions occur within ±10◦of the mag- netic equator following Horne et al. (2008). The diffusion coefﬁcients from the PADIE code are bounce averaged; i.e., the bouncing of the electrons along the magnetic ﬁeld line and the corresponding changes in resonances, plasma den- sity, magnetic ﬁeld, and wave power are included in the ﬁnal diffusion coefﬁcients. We use the plasma density model from Bagenal (1994) and a dipole magnetic ﬁeld (with equatorial ﬁeld strength 409.113 µT). Fig. 1. Mean energy diffusion coefﬁcients (averaged over all pitch angles) for our new results and those of Horne et al. (2008). chorus is observed up to 45◦(Meredith et al., 2012; Bunch et al., 2012; Menietti et al., 2012), suggest that chorus may be present at Jupiter far from the equator. Recalling that acceleration due to chorus waves is depen- dent on the ratio fpe/fce, and is most efﬁcient when this ratio is ≤4 (Horne et al., 2003, 2005), it is important to consider fpe/fce over the whole range of L and latitudes for which the waves are present. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter The strength of the background mag- netic ﬁeld is also important; when the background ﬁeld is large, scattering is inefﬁcient since the ratio of the wave mag- netic ﬁeld to the background ﬁeld, Bw/B, becomes smaller (Lyons, 1974). We use the plasma density model of Bage- nal (1994) and a dipole magnetic ﬁeld to calculate the vari- ation of fpe/fce shown in Fig. 2a. This shows the region close to Jupiter where X is the distance from the planet in the magnetic equatorial plane in Jovian radii, RJ, and Z is the distance above the same plane. Overlaid as dashed lines on Fig. 2a are selected lines of constant latitude. Figure 2b shows how fpe/fce varies along the ﬁeld lines. The ability of chorus to accelerate electrons should be suppressed by the Io plasma torus between X ∼6 and 10 RJ and for Z ≲2 RJ where fpe/fce > 4. Jupiter’s plasma sheet is thinned by the rapid rotation of the planet, and this inﬂuences the values of fpe/fce close to the equator until X ≈15 RJ where the decreased magnetic ﬁeld dominates the frequency ratio. Be- yond L ∼10 and for λ ≳5◦there is a region where fpe/fce is small, which suggests that chorus waves could be important for electron acceleration and loss to the atmosphere. An important input to the PADIE code is the amplitude of the chorus wave magnetic ﬁeld Bw. In previous work (Horne et al., 2008) the bounce-averaged diffusion rates were calcu- lated using PADIE for L = 10 and then scaled by the wave electric ﬁeld E2 w to obtain the diffusion rates over the range 6 ≤L ≤18 so that Bw2(L) = Bw2(L = 10) Ew2(L) Ew2(L = 10) , (4) (4) where Bw2(L) and Ew2(L) are the magnetic and electric wave intensities measured by Galileo at different L. This scaling was used since only wave electric ﬁeld measurements were available. However, the chorus wave bandwidth was not scaled by fce in the calculations of Horne et al. (2008) (they assumed a bandwidth of 0.05fce, but the factor of fce was missing in the calculations). We have therefore re-computed the bounce-averaged diffusion rates at each L taking into account the variation in bandwidth and show the results in Fig. 1. Here the diffusion rates have been averaged over all pitch angles at a given L. 2 Calculating diffusion coefﬁcients We then assume that the underlying cold plasma conditions on which the diffusion coefﬁcient calculations are based do not change while solving Eq. (1) so the calculated values of Dαα and DEE also do not change with time. Radial diffusion has been omitted as the focus of this paper is to examine the role of wave–particle interactions. The cross diffusion terms, which tend to be important at pitch angles near 10◦–45◦(Tao et al., 2009), have also been omitted, but their importance is described in relation to the results below. where f is the phase space density, α is the equatorial pitch angle, Dαα and DEE are the pitch angle and energy diffu- sion coefﬁcients respectively, E0 is the electron rest energy and τ is the loss timescale which is only non-zero inside the loss cone. The ﬁrst term represents diffusion in pitch angle, the second diffusion in energy and the third represents losses to the atmosphere. The diffusion coefﬁcients are calculated separately before solving Eq. (1) by using a code such as the PADIE code (Pitch Angle and energy Diffusion of Ions and Electrons; Glauert and Horne, 2005) to generate a value of Dαα and DEE for each electron energy and L-shell. We then assume that the underlying cold plasma conditions on which the diffusion coefﬁcient calculations are based do not change while solving Eq. (1) so the calculated values of Dαα and DEE also do not change with time. Radial diffusion has been omitted as the focus of this paper is to examine the role of wave–particle interactions. The cross diffusion terms, which tend to be important at pitch angles near 10◦–45◦(Tao et al., 2009), have also been omitted, but their importance is described in relation to the results below. The energy diffusion coefﬁcients in Horne et al. (2008) were calculated using the PADIE code at L = 10. We use the same wave parameters here to allow a direct comparison While nonlinear effects are very important for the gener- ation of chorus waves, it is not yet possible to calculate the Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ E. E. Woodﬁeld et al.: Electron acceleration at Jupiter The correct scaling increases the diffusion rates by up to a factor of 8 for L > 10 and decreases them for L < 10, and suggests that cyclotron-resonant inter- actions should be more effective over a wider range of L. The survey of chorus wave activity as seen by the Galileo spacecraft (Menietti et al., 2008) shows that intense whistler- mode chorus is observed at a wide range of L from the orbit of Io out to beyond L = 15. The orbit of Galileo was such that it covered regions close to the magnetic equator, but the quantity of wave data falls off dramatically at latitudes λ ≳10◦. Evidence from both the Earth and Saturn, where 4 Evolution of electron ﬂux Boundary Condition α = 0◦ ∂f ∂α = 0 α = 90◦ ∂f ∂α = 0 Emin = 20 keV f = constant Emax = 85 MeV f = constant In order to assess the effect of the combined pitch angle and energy diffusion on the electron ﬂux we have used the British Antarctic Survey Radiation Belt (BAS) model which has been adapted for Jupiter. We have switched off the radial diffusion term and only included pitch angle and energy dif- fusion so as to focus on the effects of chorus waves. The BAS model uses an unconditionally stable fully implicit numeri- cal scheme to solve the modiﬁed Fokker–Planck equation, which in the absence of radial diffusion is given by Eq. (1). ⟨Dαα⟩, and energy, ⟨DEE⟩, diffusion rates using the PADIE code for chorus waves extending to different latitudes λw. For example, if λw = 10◦then the waves are present only from −10◦to +10◦magnetic latitude. Figure 3a shows that as the latitude distribution of the waves is extended from λw = 10◦ to 30◦, pitch angle diffusion at 3 MeV increases at smaller pitch angles and into the loss cone near ∼2◦. In both pan- els (a) and (b) it is apparent that increasing the latitudinal range of the waves increases the diffusion rates at small pitch angles as there is a larger range of latitudes where electrons can resonate with the chorus waves and fpe/fce is smaller at higher latitudes. The increase in pitch angle diffusion with increasing λw (Fig. 3a) should result in electrons of all pitch angles being scattered towards the loss cone if intense chorus waves extend up to 30◦. The energy diffusion rate in Fig. 3b peaks near 40◦and is over an order of magnitude larger than the diffusion rate at 70◦to 90◦pitch angle. We have used a ﬁxed loss cone angle of 2◦and included a loss term for particles inside the loss cone where the loss timescale τ is a quarter of the bounce time and is calculated using the method from Schulz and Lanzerotti (1974). The boundary conditions used in the model are listed in Table 1. The initial pitch angle distribution of phase space density is set to (5) f (α) = f (α = 90◦)sinα f (α) = f (α = 90◦)sinα (5) to provide a value of f that decreases towards the loss cone (Subbotin and Shprits, 2009). Table 1. BAS model boundary conditions. Table 1. BAS model boundary conditions. 3 Latitude distribution of chorus Following the work of Horne et al. (2008), we ﬁrst assume that the power of the chorus waves remains constant with latitude and then calculate the bounce-averaged pitch angle, Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ 1622 E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 2. Plots of fpe/fce: (a) in the magnetic xz plane, with white on black lines showing 3 latitudes; (b) along selected magnetic L-shells. 1622 Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ E. E. Woodﬁeld et al.: Electron acceleration at Jupiter E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 2. Plots of fpe/fce: (a) in the magnetic xz plane, with white on black lines showing 3 latitudes; (b) along selected magnetic L-shells. Table 1. BAS model boundary conditions. Boundary Condition α = 0◦ ∂f ∂α = 0 α = 90◦ ∂f ∂α = 0 Emin = 20 keV f = constant Emax = 85 MeV f = constant Table 1. BAS model boundary conditions. Boundary Condition α = 0◦ ∂f ∂α = 0 α = 90◦ ∂f ∂α = 0 Emin = 20 keV f = constant Emax = 85 MeV f = constant E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 3. Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of constant intensity as a function of latitude and or different latitude ranges; left column: (a) shows < D > /p2 at 3 MeV (c) variation of < D > /p2 with energy for λ = ±10◦and Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of constant intensity as a function of latitude and erent latitude ranges; left column: (a) shows < Dαα > /p2 at 3 MeV, (c) variation of < Dαα > /p2 with energy for λw = ±10◦and me for λw = ±30◦. Right column (b, d and f): same but for < DEE > /E2. Fig. 3. Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of constant intensity as a function of latitude and for different latitude ranges; left column: (a) shows < Dαα > /p2 at 3 MeV, (c) variation of < Dαα > /p2 with energy for λw = ±10◦and ( ) f λ ±30◦Ri ht l (b d d f) b t f D /E2 Fig. 3. Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of constant intensity as a function of latitude and for different latitude ranges; left column: (a) shows < Dαα > /p2 at 3 MeV, (c) variation of < Dαα > /p2 with energy for λw = ±10◦and (e) same for λw = ±30◦. Right column (b, d and f): same but for < DEE > /E2. useful discussion of this effect can be found in Shprits et al. (2012). Figure 4a shows the electron spectrum at α = 84◦at 0 days (dashed line) and then at 30 days (solid line); Fig. 4b shows the corresponding pitch angle distributions. A dipole magnetic ﬁeld was used in the model, which is a reasonable assumption for distances up to approximately 14 RJ along the equator. The change from dipole magnetic ﬁeld to current-sheet-dominated magnetic ﬁeld occurs be- tween approximately 12 and 20 RJ (Tomás et al., 2004). Fig- ure 4 shows the results of the BAS model at L = 10 where λw takes values of 10◦, 20◦and 30◦. The intensity of the cho- rus waves is assumed to be constant with latitude in this case. The ﬂux evolution is similar to that of Horne et al. 4 Evolution of electron ﬂux The constant values for the boundaries and initial values are taken from the Galileo In- terim Radiation Environment model (GIRE) for Jupiter (Gar- rett et al., 2003), which is based on averaged measurements from Galileo combined with the earlier Divine model (Di- vine and Garrett, 1983). The minimum energy boundary we use (Emin = 20 keV) is much lower than that used previously (300 keV) (Horne et al., 2008) so that we can investigate a wider energy range. We use the GIRE model to set f at the Emin boundary. The values of f are kept constant on the as- sumption that there is a balance of sources and losses at this low energy. The GIRE model based on measurements from Galileo shows that at 85 MeV there is a very small ﬂux of electrons, so we have set f at the maximum energy Emax to be constant. In reality the initial conditions may already include acceleration and loss due to cyclotron-resonant in- teractions with chorus and other waves, but at present this contribution cannot be separated out of the observations. A The variation of the diffusion rates with electron energy is shown in panels (c) to (f) of Fig. 3. The effect of increasing the latitude distribution of chorus is to increase ⟨Dαα⟩/p2 for electrons of a few MeV and to increase ⟨DEE⟩/E2 at pitch angles between 20◦and 60◦. The combination of these increases implies that although much more energy diffusion will occur, the electrons will also be scattered rapidly towards the loss cone and depending on the gradient of the distribu- tion function may be lost before they can be accelerated. Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ 1623 E. E. Woodﬁeld et al.: Electron acceleration at Jupiter (2008), where for λw = 10◦the ﬂux at energies of a few MeV in- creases by an order of magnitude over a timescale of 30 days (comparable to the timescale for transport near the Io torus for thermal plasma (Delamere and Bagenal, 2003)). How- ever, as the latitude range is increased to 20◦(blue) and 30◦ (red) there is a signiﬁcant loss of ﬂux at lower energies and 5 Wave intensity with latitude Although the path of Galileo was restricted close to the equa- tor there is some suggestion in the data that the wave inten- sity decreases with latitude λ (Menietti et al., 2008). There- fore we have used the data in Fig. 6 of Menietti et al. (2008) to produce a Gaussian power proﬁle of Bw with latitude (Fig. 5). Based on the symmetry of the data in Fig. 6 of Meni- etti et al. (2008) we assume the wave intensity is symmetrical about the magnetic equator, and we use all the data from both sides of the equator to produce a Gaussian model of the vari- ation of wave intensity with latitude Bw = Bw0e−z2/2 0.48 (6) z = θ −4.80 3.94 , (7) (6) (7) where Bw0 is the magnetic wave amplitude at the equator and θ is the magnetic latitude in degrees. We note that the wave intensity is highest about 5◦off the equator; a similar effect has also been observed at Saturn which may be linked to weak or linear wave growth close to the equator (Meni- etti et al., 2013). We assume the same intensity proﬁle model for all L-shells. Figure 5 shows that the waves become very weak by λ ∼20◦. Although chorus waves at Jupiter may ex- tend beyond this latitude as they do at the Earth and Saturn, there are no measurements at high latitudes at Jupiter. Ex- trapolation of the data using our model indicates that wave power is likely to be low at high latitudes (Fig. 5). Fig. 5. Magnetic wave amplitude derived from electric ﬁeld energy density from the Galileo spacecraft from Menietti et al. (2008) and an equatorial value of Bw as shown at L = 10. In red, a Gaussian ﬁt to that data. the energy where enhanced ﬂux is seen has increased towards 10 MeV. The pitch angle distributions have a ﬂat top between 45◦and 90◦which becomes broader for waves which extend to higher latitudes. The distribution also becomes anisotropic as the electron distribution at large pitch angles is much higher than that at small pitch angles. The shape of the pitch angle distribution is highly dependent on the value of λw. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1624 E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 4. Electron ﬂuxes after 30 days for constant wave intensity extending to ±λw. Dashed line shows initial condition. Fig. 4. Electron ﬂuxes after 30 days for constant wave intensity extending to ±λw. Dashed line shows initial condition. Fig. 5. Magnetic wave amplitude derived from electric ﬁeld energy density from the Galileo spacecraft from Menietti et al. (2008) and an equatorial value of Bw as shown at L = 10. In red, a Gaussian ﬁt to that data. www.ann-geophys.net/31/1619/2013/ www.ann-geophys.net/31/1619/2013/ Ann. Geophys., 31, 1619–1630, 2013 1624 Ann. Geophys., 31, 1619–1630, 2013 5 Wave intensity with latitude Although cross diffusion terms are not included in our cal- culations, simulations show that they are important at pitch angles in the region 10◦–40◦(Tao et al., 2009) and would lead to more rapid diffusion in this region, which would tend to make the anisotropy more apparent. Using the model of the wave intensity distribution shown in Fig. 5, we have calculated new diffusion coefﬁcients (Fig. 6). The large reduction in wave intensity by λw = 20◦ results in almost identical diffusion coefﬁcients for the maxi- mum latitude of the waves λw = 20◦and λw = 30◦such that the red lines in Fig. 6a and b overlay the blue λw = 20◦lines. The diffusion coefﬁcients are increased somewhat at large pitch angles compared to Fig. 3 due to the increase in wave Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ www.ann-geophys.net/31/1619/2013/ 1625 E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 7. Electron ﬂuxes after 30 days using Galileo-based wave intensity extending to ±λw. Please note the red and blue lines are overlapping. Dashed line shows initial condition. Fig. 7. Electron ﬂuxes after 30 days using Galileo-based wave intensity extending to ±λw. Please note the red and blue lines are overlapping. Dashed line shows initial condition. Fig. 8. Electron ﬂuxes at 0 days (a) and after 30 days (b) using BAS model calculation based on the varying intensity as a function of latitude proﬁle from Galileo data for λw = 30◦. Fig. 8. Electron ﬂuxes at 0 days (a) and after 30 days (b) using BAS model calculation based on the varying intensity as a function of latitude proﬁle from Galileo data for λw = 30◦. The increases in energy diffusion at L > 10 shown in Fig. 1 suggests that we should see strong acceleration at L > 10. We test this hypothesis as far as L = 14RJ, where a dipole magnetic ﬁeld model should still provide a reason- able approximation to the magnetic ﬁeld. The results at both L = 12 (blue line) and L = 14 (red line) in Fig. 9 show that there is still an order of magnitude increase in the ﬂux of electrons at ∼3 MeV at L = 14. There is a similarly shaped response in the electron ﬂux at these larger L-shells to the results at L = 10, with losses for electrons below ∼1 MeV and gains above this value. at 30 days using λw = ±30◦and the diffusion coefﬁcients based on Galileo data. Note the electrons very close to 90◦ pitch angle at energies less than 1 MeV remain trapped be- cause the pitch angle diffusion rate in this limited range of pitch angles is very small when considering chorus waves alone (see Fig. 6). At very low pitch angles electrons are assumed to be lost into the Jovian atmosphere within one quarter of the bounce time; this results in emptying of the loss cone at energies greater than approximately 0.3 MeV (weak diffusion regime), but at lower energies strong diffu- sion maintains the ﬂux levels at these low pitch angles. The decreases in ﬂux from the initial condition at ener- gies between approximately 0.1 and 0.6 MeV are seen to be consistent across almost all pitch angles in Fig. 8b. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Fig. 6. Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of varying intensity as a function of latitude and for different latitude ranges; left column: (a) shows < Dαα > /p2 at 3 MeV (please note that the red and blue lines are overlapping), (c) variation 2 2 Fig. 6. Pitch angle and energy diffusion coefﬁcients at L = 10 produced with chorus waves of varying intensity as a function of latitude and for different latitude ranges; left column: (a) shows < Dαα > /p2 at 3 MeV (please note that the red and blue lines are overlapping), (c) variation of < Dαα > /p2 with energy for λw = ±10◦and (e) same for λw = ±30◦. Right column (b, d and f): same but for < DEE > /E2. gests that acceleration can extend to a wider range of energies with little loss. intensity from 0◦to ∼5◦, which was not taken into account before. Whereas in Fig. 3 at lower pitch angles with λw = 30◦ the pitch angle diffusion rates promoted pitch angle scatter- ing across all pitch angles, with the modeled wave intensity distribution this no longer occurs, allowing electrons to be more easily trapped and accelerated. The diffusion coefﬁ- cients calculated using the intensity proﬁle model show in- creases in pitch angle diffusion across a smaller range of en- ergies (∼0.4 to 1 MeV, Fig. 6) than with the constant inten- sity with latitude model (∼0.4 to 6 MeV, Fig. 3). This sug- Figure 7 shows the effect the new diffusion coefﬁcients have on the evolution of the electron ﬂux after 30 days. This is very similar to the λw = 10◦results in Fig. 4 although there are now greater increases of ﬂux for both λw = 10◦and λw = 30◦in Fig. 7. For the λw = 30◦case, the peak ﬂux at 30 days has moved to an energy slightly lower than 3 MeV. Figure 8 shows how the electron ﬂux evolves over both energy and pitch angle from the initial condition to the ﬂux www.ann-geophys.net/31/1619/2013/ Ann. Geophys., 31, 1619–1630, 2013 1626 6 Discussion waves have been observed at Jupiter (Menietti et al., 2012) and are thought to be an important acceleration mechanism at Saturn (Gu et al., 2013). The results in the previous two sections show the critical dif- ference the location and intensity of whistler-mode chorus makes to the evolution of electron ﬂux through wave–particle interactions. One common feature present in the output of the BAS model throughout our investigations is a plateau in the electron ﬂux as a function of energy. The plateau occurs at varying energies depending on the wave intensity latitude proﬁle. For example in Fig. 9, the ﬂux at 30 days close to 1 MeV remained the same as the initial value for all three L- shells, whereas at lower energies the ﬂux has deviated below the electron spectrum from the GIRE model and at higher energies it is higher. Although we have included losses within the loss cone there are other potential loss mechanisms that we have ne- glected in our model runs. For example broadband hiss and electromagnetic ion cyclotron (EMIC) waves are known to cause pitch angle scattering and particle losses; we have also not included any losses due to the orbits of Jupiter’s moons Europa (at 9.4 RJ) and Ganymede (at 14.97 RJ). The inclu- sion of cross terms in the BAS model could also address the excess of electrons at higher energies since these terms tend to reduce local acceleration (Shprits et al., 2012). Tao et al. (2009) have assessed the importance of cross terms on elec- tron acceleration by chorus and concluded that for pitch an- gles close to 90◦the cross terms have a very limited effect on the acceleration, but at high energies (∼few MeV) and low pitch angles the ﬂux can be signiﬁcantly overestimated (by 2 orders of magnitude) if the cross terms are not included. Our results emphasize the importance of electron accelera- tion at large pitch angles, and so we do not expect the omis- sion of cross terms to affect our results signiﬁcantly. In fact the results in Tao et al. (2009) suggest that the pitch angle anisotropies in our results may in fact be an underestimate of the level of anisotropy since cross diffusion terms should reduce the acceleration at lower pitch angles. Several factors may lead to these deviations from the GIRE model. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1627 Fig. 9. Evolution of electron ﬂuxes at selected L-shells using diffusion coefﬁcients from varying wave intensity proﬁle with latitude up to λw = 30◦; solid lines are ﬂux after 30 days, dashed lines are the starting spectra at each L-shell, and vertical dotted line in (a) shows energy used for (b). Fig. 9. Evolution of electron ﬂuxes at selected L-shells using diffusion coefﬁcients from varying wave intensity proﬁle with latitude up to λw = 30◦; solid lines are ﬂux after 30 days, dashed lines are the starting spectra at each L-shell, and vertical dotted line in (a) shows energy used for (b). E. E. Woodﬁeld et al.: Electron acceleration at Jupiter The ﬂux increases observed at energies of a few MeV in Fig. 7a are also observed over a very wide range of pitch angles, with the minimum pitch angle of the elevated ﬂux level increasing with increasing energy. The pitch angle distributions for all 3 L-shells shown in Fig. 9 are remarkably similar, all showing an increase in the electron ﬂux at pitch angles ≳30◦by an order of magnitude or more. All the distributions in Fig. 9b show a very pro- nounced pitch angle anisotropy peaked near 90◦, where the distributions at energies of a few MeV have a ﬂat top. Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ www.ann-geophys.net/31/1619/2013/ 1627 www.ann-geophys.net/31/1619/2013/ Ann. Geophys., 31, 1619–1630, 2013 7 Conclusions Albert, J. M., Meredith, N. P., and Horne, R. B.: Three-dimensional diffusion simulation of outer radiation belt electrons during the 9 October 1990 magnetic storm, J. Geophys. Res., 114, A09214, doi:10.1029/2009JA014336, 2009. The radiation belts at Jupiter are the most intense in the Solar System. Previous work has suggested that cyclotron-resonant interactions with chorus waves are a signiﬁcant factor in cre- ating these radiation belts. In this paper we have used Galileo spacecraft data to show the importance of electron accelera- tion by resonant interaction with chorus waves as a source of high-energy electrons in the region outside of Io at Jupiter. Bagenal, F.: Empirical-model of the Io plasma Torus – Voy- ager Measurements, J. Geophys. Res., 99, 11043–11062, doi:10.1029/93JA02908, 1994. Bolton, S. J., Janssen, M., Thorne, R., Levin, S., Klein, M., Gulkis, S., Bastian, T., Sault, R., Elachi, C., Hofstadter, M., Bunker, A., Dulk, G., Gudim, E., Hamilton, G., Johnson, W. T. K., Leblanc, Y., Liepack, O., McLeod, R., Roller, J., Roth, L., and West, R.: Ultra-relativistic electrons in Jupiter’s radiation belts, Nature, 415, 987–991, doi:10.1038/415987a, 2002. Three important ﬁndings have been reported here: 1. By taking into account the bandwidth of the power spectrum of chorus waves and how it varies with L- shell, we ﬁnd that energy diffusion rates due to chorus waves are signiﬁcantly larger by up to a factor of 8 at L-shells > 10 and signiﬁcantly smaller at L-shells < 10 than previously reported by Horne et al. (2008). Bunch, N. L., Spasojevic, M., and Shprits, Y. Y.: Off-equatorial chorus occurrence and wave amplitude distributions as observed by the Polar Plasma Wave Instrument, J. Geophys. Res., 117, A04205, doi:10.1029/2011JA017228, 2012. Burtis, W. J. and Helliwell, R. A.: Banded chorus – A new type of VLF radiation observed in the magnetosphere by OGO 1 and OGO 3, J. Geophys. Res., 11, 3002–3010, 1969. 2. Data show that chorus wave intensities peak near λ ∼ 5◦and fall with increasing magnetic latitude. Model- ing of this latitude-dependent intensity proﬁle shows that cyclotron-resonant wave acceleration via chorus waves can increase the electron ﬂux at ∼3 MeV by a factor of 10 or more over a period of 30 days. Further- more, this should result in an anisotropic distribution peak between ∼40 and 90◦in pitch angle. Chen, Y., Reeves, G. D., and Friedel, R. H. References Albert, J. M.: Diffusion by one wave and by many waves, J. Geo- phys. Res., 115, A00F05, doi:10.1029/2009JA014732, 2010. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1628 produced by electron acceleration by chorus waves outside the orbit of the moon Io. 6 Discussion Firstly, the GIRE model ﬁts a functional form to the accumulated Galileo electron data from many individ- ual orbits, so one might expect a relatively smooth spectrum where some of the variations have been averaged out. An- other important factor which could explain why the ﬂux at lower energies is less than the GIRE model is the omission of particle sources in the BAS model (Shprits et al., 2012). For example particle injections are known to occur at Jupiter with a timescale of approximately 3 days (Woch et al., 2004; Mauk et al., 1999; Kronberg et al., 2012). These tend to oc- cur at energies of a few tens of keV to a few hundred keV, whereas we have assumed that the ﬂux is constant at our low energy boundary at 20 keV. It is difﬁcult to say with- out very detailed modeling of this source whether the in- jections provide enough electron ﬂux to compensate for the losses at energies of a few hundred keV, and this needs to be investigated in the future. Another possibility is that other waves, such as Z-mode waves (Glauert and Horne, 2005) and magnetosonic waves (Horne et al., 2007), could contribute to electron acceleration and increase the electron ﬂux. Z-mode As a ﬁnal comment, we have omitted radial diffusion that will transport electrons towards and away from Jupiter with associated acceleration and deceleration of the particles. Ra- dial diffusion would be effective over a range of energies and would tend to smooth the radial proﬁle of the ﬂux. Never- theless, we point out that anisotropic pitch angle distribu- tions have been observed at Jupiter and have been associated with radial diffusion. Here we show that they could also be Ann. Geophys., 31, 1619–1630, 2013 Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ 7 Conclusions W.: The energization of relativistic electrons in the outer Van Allen radiation belt, Nature Physics, 3, 614–617, doi:10.1038/nphys655, 2007. Delamere, P. A. and Bagenal, F.: Modeling variability of plasma conditions in the Io torus, J. Geophys. Res., 108, 1276, doi:10.1029/2002JA009706, 2003. de Pater, I. and Dunn, D.: VLA observations of Jupiter’s syn- chrotron radiation at 15 and 22 GHz, ICARUS, 163, 449–455, doi:10.1016/S0019-1035(03)00068-X, 2003. 3. Taking into account the observed distribution of cho- rus wave power with L-shell, we ﬁnd that this should result in an increase in the electron ﬂux in the region 10 < L < 14. Acceleration may also extend to larger L-shells where non-dipole magnetic ﬁeld effects be- come important, but this has yet to be conﬁrmed. Divine, N. and Garrett, H. B.: Charged particle distributions in Jupiter’s magnetosphere, J. Geophys. Res., 88, 6889, doi:10.1029/JA088iA09p06889, 1983. Fok, M.-C., Horne, R. B., Meredith, N. P., and Glauert, S. A.: Radiation belt environment model: Application to space weather nowcasting, J. Geophys. Res., 113, A03S08, doi:10.1029/2007JA012558, 2008. We conclude that cyclotron-resonant wave acceleration is an important process for providing a population of few MeV electrons in the radiation belt at 10 to 14 RJ, and which pro- vides the source of particles for transportation to ∼1.5 RJ where the most intense radiation belts are located. Garrett, H. B., Jun, I., Ratliff, J. M., Evans, R. W., Clough, G. A., and McEntire, R. W.: Galileo Interim Radiation Elec- tron (GIRE) Model, JPL Publication, pp. 3–6, http://www. openchannelfoundation.org/projects/GIRE/, 2003. Glauert, S. A. and Horne, R. B.: Calculation of pitch angle and energy diffusion coefﬁcients with the PADIE code, J. Geophys. Res., 110, A04206, doi:10.1029/2004JA010851, 2005. Acknowledgements. E. E. Woodﬁeld and R. B. Horne are funded through STFC grant number ST/I001727/1. R. B. Horne and S. A. Glauert are funded in the UK by NERC. J. D. Menietti is funded by NASA grant NNX11AM36G. The authors wish to thank F. Bagenal for the plasma density model and H. B. Garrett and B. Mauk for electron ﬂux data. Gu, X., Thorne, R. M., Ni, B., and Ye, S.-Y.: Resonant diffusion of energetic electrons by narrowband Z mode waves in Sat- urn’s inner magnetosphere, Geophys. Res. Lett., 40, 255–261, doi:10.1029/2012GL054330, 2013. Gurnett, D. A. and Scarf, F. L.: Plasma waves in the Jovian mag- netosphere, in: Physics of the Jovian Magnetosphere, edited by: Dessler, A. J., Cambridge University Press, Cambridge, 1983. Topical Editor L. Ann. Geophys., 31, 1619–1630, 2013 Topical Editor L. Blomberg thanks two anonymous referees for their help in evaluating this paper. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Res., 113, A12206, doi:10.1029/2008JA013237, 2008. Schulz, M. and Lanzerotti, L. J.: Particle diffusion in the radiation belts, Physics and chemistry in space, Springer-Verlag, 1974. Katoh, Y. and Omura, Y.: Computer simulation of chorus wave gen- eration in the Earth’s inner magnetosphere, Geophys. Res. Lett., 34, L03102, doi:10.1029/2006GL028594, 2007. Shprits, Y. Y., Chen, L., and Thorne, R. M.: Simulations of pitch angle scattering of relativistic electrons with MLT- dependent diffusion coefﬁcients, J. Geophys. Res., 114, A03219, doi:10.1029/2008JA013695, 2009a. Katoh, Y. and Omura, Y.: Amplitude dependence of frequency sweep rates of whistler mode chorus emissions, J. Geophys. Res., 116, A07201, doi:10.1029/2011JA016496, 2011. Shprits, Y. Y., Subbotin, D., and Ni, B.: Evolution of electron ﬂuxes in the outer radiation belt computed with the VERB code, J. Geo- phys. Res., 114, A11209, doi:10.1029/2008JA013784, 2009b. Kivelson, M. G., Khurana, K. K., Russell, C. T., and Walker, R. J.: Intermittent short-duration magnetic ﬁeld anomalies in the Io torus: Evidence for plasma interchange?, Geophys. Res. Lett., 24, 2127–2130, doi:10.1029/97GL02202, 1997. Shprits, Y. Y., Menietti, J. D., Gu, X., Kim, K. C., and Horne, R. B.: Gyroresonant interactions between the radiation belt electrons and whistler mode chorus waves in the radiation environments of Earth, Jupiter, and Saturn: A comparative study, J. Geophys. Res., 117, A11216, doi:10.1029/2012JA018031, 2012. Kronberg, E. A., Kasahara, S., Krupp, N., and Woch, J.: Field- aligned beams and reconnection in the jovian magnetotail, Icarus, 217, 55–65, doi:10.1016/j.icarus.2011.10.011, 2012. Lyons, L. R.: Pitch angle and energy diffusion-coefﬁcients from resonant interactions with ion-cyclotron and whistler waves, J. Plasma Phys., 12, 417–432, 1974. Sicard, A. and Bourdarie, S.: Physical Electron Belt Model from Jupiter’s surface to the orbit of Europa, J. Geophys. Res., 109, A02216, doi:10.1029/2003JA010203, 2004. Su, Z., Xiao, F., Zheng, H., and Wang, S.: STEERB: A three-dimensional code for storm-time evolution of electron radiation belt, J. Geophys. Res., 115, A09208, doi:10.1029/2009JA015210, 2010. Mauk, B. H., Williams, D. J., McEntire, R. W., Khurana, K. K., and Roederer, J. G.: Storm-like dynamics of Jupiter’s in- ner and middle magnetosphere, J. Geophys. Res., 104, 22759, doi:10.1029/1999JA900097, 1999. Subbotin, D. A. and Shprits, Y. Y.: Three-dimensional mod- eling of the radiation belts using the Versatile Electron Radiation Belt (VERB) code, Space Weather, 7, S10001, doi:10.1029/2008SW000452, 2009. Menietti, J. D., Horne, R. B., Gurnett, D. A., Hospodarsky, G. B., Piker, C. W., and Groene, J. B.: A survey of Galileo plasma wave instrument observations of Jovian whistler-mode chorus, Ann. E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1629 Meredith, N. P., Horne, R. B., and Anderson, R. R.: Substorm de- pendence of chorus amplitudes: Implications for the accelera- tion of electrons to relativistic energies, J. Geophys. Res., 106, 13165–13178, doi:10.1029/2000JA900156, 2001. Hill, T. W., Dessler, A. J., and Maher, L. J.: Corotating mag- netospheric convection, J. Geophys. Res., 86, 9020–9028, doi:10.1029/JA086iA11p09020, 1981. Horne, R. B., Glauert, S. A., and Thorne, R. M.: Resonant diffusion of radiation belt electrons by whistler-mode chorus, Geophys. Res. Lett., 30, 1493, doi:10.1029/2003GL016963, 2003. Meredith, N. P., Horne, R. B., Sicard-Piet, A., Boscher, D., Yearby, K. H., Li, W., and Thorne, R. M.: Global model of lower band and upper band chorus from multiple satellite observations, J. Geophys. Res., 117, A10225, doi:10.1029/2012JA017978, 2012. Horne, R. B., Thorne, R. M., Shprits, Y. Y., Meredith, N. P., Glauert, S. A., Smith, A. J., Kanekal, S. G., Baker, D. N., Engebretson, M. J., Posch, J. L., Spasojevic, M., Inan, U. S., Pickett, J. S., and De- creau, P. M. E.: Wave acceleration of electrons in the Van Allen radiation belts, Nature, 437, 227–230, doi:10.1038/nature03939, 2005. Nunn, D., Omura, Y., Matsumoto, H., Nagano, I., and Yagitani, S.: The numerical simulation of VLF chorus and discrete emissions observed on the Geotail satellite using a Vlasov code, J. Geo- phys. Res., 102, 27083–27097, doi:10.1029/97JA02518, 1997. Horne, R. B., Thorne, R. M., Glauert, S. A., Meredith, N. P., Pokhotelov, D., and Santolik, O.: Electron acceleration in the Van Allen radiation belts by fast magnetosonic waves, Geophys. Res. Lett., 34, L17107, doi:10.1029/2007GL030267, 2007. Omura, Y., Hikishima, M., Katoh, Y., Summers, D., and Yagitani, S.: Nonlinear mechanisms of lower-band and upper-band VLF chorus emissions in the magnetosphere, J. Geophys. Res., 114, A07217, doi:10.1029/2009JA014206, 2009. Santolík, O., Gurnett, D. A., Pickett, J. S., Parrot, M., and Cornilleau-Wehrlin, N.: Spatio-temporal structure of storm-time chorus, J. Geophys. Res., 108, 1278, doi:10.1029/2002JA009791, 2003. Horne, R. B., Thorne, R. M., Glauert, S. A., Menietti, J. D., Shprits, Y. Y., and Gurnett, D. A.: Gyro-resonant electron acceleration at Jupiter, Nature Physics, 4, 301–304, doi:10.1038/nphys897, 2008. Santos-Costa, D. and Bourdarie, S. A.: Modeling the inner Jovian electron radiation belt including non-equatorial particles, Planet. Sp. Sci., 49, 303–312, doi:10.1016/S0032-0633(00)00151-3, 2001. Hospodarsky, G. B., Averkamp, T. F., Kurth, W. S., Gurnett, D. A., Menietti, J. D., Santolik, O., and Dougherty, M. K.: Observa- tions of chorus at Saturn using the Cassini Radio and Plasma Wave Science instrument, J. Geophys. 7 Conclusions Blomberg thanks two anonymous referees for their help in evaluating this paper. Gurnett, D. A., Kurth, W. S., Roux, A., Bolton, S. J., and Kennel, C. F.: Galileo plasma wave observations in the Io plasma torus and near Io, Science, 274, 391–392, doi:10.1126/science.274.5286.391, 1996. Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ E. E. Woodﬁeld et al.: Electron acceleration at Jupiter Geophys., 26, 1819–1828, doi:10.5194/angeo-26-1819- 2008, 2008. Summers, D., Thorne, R. M., and Xiao, F. L.: Relativistic theory of wave-particle resonant diffusion with application to electron ac- celeration in the magnetosphere, J. Geophys. Res., 103, 20487– 20500, doi:10.1029/98JA01740, 1998. Menietti, J. D., Shprits, Y. Y., Horne, R. B., Woodﬁeld, E. E., Hospodarsky, G. B., and Gurnett, D. A.: Chorus, ECH, and Z-mode emissions observed at Jupiter and Saturn and pos- sible electron acceleration, J. Geophys. Res., 117, A12214, doi:10.1029/2012JA018187, 2012. Tao, X., Albert, J. M., and Chan, A. A.: Numerical mod- eling of multidimensional diffusion in the radiation belts using layer methods, J. Geophys. Res., 114, A02215, doi:10.1029/2008JA013826, 2009. Menietti, J. D., Katoh, Y., Hospodarsky, G. B., and Gurnett, D. A.: Frequency drift of Saturn chorus emission compared to nonlinear theory, J. Geophys. Res., 118, 982–990, doi:10.1002/jgra.50165, 2013. Tao, X., Bortnik, J., Albert, J. M., and Thorne, R. M.: Com- parison of bounce-averaged quasi-linear diffusion coefﬁcients Ann. Geophys., 31, 1619–1630, 2013 www.ann-geophys.net/31/1619/2013/ www.ann-geophys.net/31/1619/2013/ Ann. Geophys., 31, 1619–1630, 2013 E. E. Woodﬁeld et al.: Electron acceleration at Jupiter 1630 for parallel propagating whistler mode waves with test particle simulations, J. Geophys. Res., 117, A10205, doi:10.1029/2012JA017931, 2012. Varotsou, A., Boscher, D., Bourdarie, S., Horne, R., Glauert, S., and Meredith, N.: Simulation of the outer radiation belt elec- trons near geosynchronous orbit including both radial diffusion and resonant interaction with Whistler-mode chorus waves, Geo- phys. Res. Lett., 32, L19106, doi:10.1029/2005GL023282, 2005. Thorne, R. M., Armstrong, T. P., Stone, S., Williams, D. J., McEn- tire, R. W., Bolton, S. J., Gurnett, D. A., and Kivelson, M. G.: Galileo evidence for rapid interchange transport in the Io torus, Geophys. Res. Lett., 24, 2131–2134, doi:10.1029/97GL01788, 1997. Varotsou, A., Boscher, D., Bourdarie, S., Horne, R. B., Meredith, N. P., Glauert, S. A., and Friedel, R. H.: Three-dimensional test simulations of the outer radiation belt electron dynamics includ- ing electron-chorus resonant interactions, J. Geophys. Res., 113, A12212, doi:10.1029/2007JA012862, 2008. Tomás, A., Woch, J., Krupp, N., Lagg, A., Glassmeier, K. H., Dougherty, M. K., and Hanlon, P. G.: Changes of the energetic particles characteristics in the inner part of the Jovian mag- netosphere: a topological study, Planet. Sp. Sci., 52, 491–498, doi:10.1016/j.pss.2003.06.011, 2004. Woch, J., Krupp, N., Lagg, A., and Tomas, A.: The structure and dy- namics of the Jovian energetic particle distribution, in: Compara- tive Magnetospheres, edited by: BlancoCano, X. and Russell, C. T., vol. 33 of Adv. Sp. Res., pp. 2030–2038, Comm. Space Res., doi:10.1016/j.asr.2003.04.050, 2004. Trakhtengerts, V. Y.: A generation mechanism for chorus emission, Ann. Geophys., 17, 95–100, doi:10.1007/s00585-999-0095-4, 1999. Xiao, F., Thorne, R. M., Gurnett, D. A., and Williams, D. J.: Whistler-mode excitation and electron scattering during an interchange event near Io, Geophys. Res. Lett., 30, 1749, doi:10.1029/2003GL017123, 2003. Tsurutani, B. T. and Smith, E. J.: Postmidnight chorus: A substorm phenomenon, J. Geophys. Res., 79, 118–127, 1974. Ann. Geophys., 31, 1619–1630, 2013
https://openalex.org/W4319289682	https://zenodo.org/records/7608470/files/223-231.pdf	Russian	null	ЖАДИД ПЕДАГОГИКАСИНИНГ ИЛМИЙ – НАЗАРИЙ АСОСЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,667	Volume 2\| Issue 1 ISSN: 2181-3639 Volume 2\| Issue 1 ISSN: 2181-3639 Аннотация Ушбу мақолада, XX асрда Туркистон ижтимоий-сиёсий ҳаётида муҳим рол ўйнаган жадид маьрифатпарварларининг маьрифий- педагогик фаолияти ҳақида сўз юритилади. Жадид дарсликлари ҳақида атрофлича маьлумотлар берилиб, уларнинг дидактик мазмуни очиб берилади. Дарсликлар таҳлилида Маҳмудхўжа Беҳбудийнинг "Жуғрофия " дарслигига алоҳида тўхталиб, унинг мазмун моҳияти таҳлил этилади. Калит сўзлар: жадидчилик, маьрифатпарварлик, педагогик фаолият, жадид дарсликлари, мактаб, маориф, миллий педагогика. Аннотация В данной статье рассказывается о просветительско- педагогической деятельности современной интеллигенции, сыгравшей важную роль в общественно-политической жизни Туркестана в ХХ веке. Будет освещена подробная информация о джадидских учебниках и раскрыто их дидактическое содержание.При анализе учебников будет проанализирован учебник Махмудходжи Бехбуди “География”. Ключевые слова: модернизм, просвещение, педагогическая деятельность, современные учебники, школа, образование, воспитание, народная педагогика. Abstract This article describes the educational and pedagogical activities of the jadid intelligentsia, which played an important role in the socio-political life of Turkestan in the ХХ century. Detailed information about the Jadid textbooks will be https://academicsresearch.com/index.php/rnsr/index Results of National Scientific Research International Journal 2023 Volume 2\| Issue 1 SJIF- 4.431 ISSN: 2181-3639 covered and their didactic content will be revealed. When analyzing the textbooks, the textbook of Mahmudhoji Behbudi “Geography” will be analyzed. Results of National Scientific Research International Journal 2023 Volume 2\| Issue 1 SJIF- 4.431 ISSN: 2181-3639 covered and their didactic content will be revealed. When analyzing the textbooks, the textbook of Mahmudhoji Behbudi “Geography” will be analyzed. Results of National Scientific Research International Journal 2023 SJIF 4 431 Key words: modernism, education, pedagogical activity, modern textbooks, school, education, folk pedagogy. КИРИШ Жадид маърифатпарвар зиёлилари орасида маориф , мактаб ишларини ташкил этиш ва унинг асосларини яратиш ишини бир бутун ҳолда қараб, бу йўлда ҳам кўп мувафаққиятларга эришган. Ушбу асослар эса уларнинг усули жадид мактаблари учун яратган китоб ва қўлланмаларида акс этади. Жадид маърифатпарвар педагоглари орасида Маҳмудхўжа Беҳбудий яратган усули жадид руҳидаги ўзбекча ва форсча дарсликлари ўз замонасидаѐқ жуда машҳур бўлиб кетган ва улардан баъзилари қайта нашр этилган. Беҳбудий яратган дарсликлар ҳақида кейинги вақтларда олимларимиз томонидан эълон қилинаѐтган мақола ва рисолаларда номлари қайд этилиб келинди. Булар 1990 йилда нашр қилинган Б. Қосимов ва У. Долимовларнинг "Маърифат дарғалари" номли китобида, Ш.Турдиевнинг 1994 йили "Мулоқот" журналининг 3-4 қўшма сонларида эълон қилинган "Маҳмудхўжа Беҳбудий" сарлавҳали мақоласида, А.Алиевнинг "Маърифат" газетаси 1993 йил 16 июнда "Маҳмудхўжа Беҳбудий таълим-тарбия хусусида" мақоласи, Н.Авазовнинг 1993 йил чоп этилган "Маҳмудхўжа Беҳбудий – маърифатпарвар" номли рисоласида тилга олиниб ўтилган. АСОСИЙ ҚИСМ Олимларимиз томонидан келтирилган Беҳбудий дарсликлари рўйхатига суянган ҳолда, адибнинг ўз муҳаррирлигидаги "Самарқанд" газети ва "Оина" журналларида доимо сўнгги саҳифадан жой олган "Усули жадид мактаби учун тайѐр китоблар" сарлавҳаси остида санаб ўтилган китоблар рўйхатини келтириш мумкин. Булар қуйидагилар: https://academicsresearch.com/index.php/rnsr/index https://academicsresearch.com/index.php/rnsr/index 223 223 https://academicsresearch.com/index.php/rnsr/index https://academicsresearch.com/index.php/rnsr/index 224 224 https://academicsresearch.com/index.php/rnsr/index Results of National Scientific Research International Journal 2023 SJIF- 4.431 Volume 2\| Issue 1 ISSN: 2181-3639 1. Алифбо мактаби исломия - форсча. 1. Алифбо мактаби исломия - форсча. 2. Китобат ул-атфол (Болалар учун китоб) - турк ва форсча тилларда. 3. Амалиѐти ислом - туркий тилда 4. Мухтасари тарихи ислом - ўзбек тилида. 4. Мухтасари тарихи ислом - ўзбек тилида. 5. Мунтаҳаби жуғрофияи умумий - туркий тилда. 6. Мадхали жуғрофияи имроний - Мухтасари жуғрофияи Русий- форс ва ўзбек тилларида. 6. Мадхали жуғрофияи имроний - Мухтасари жуғрофияи Русий- форс ва ўзбек тилларида. Ушбу рўйхатдан ташқари Б.Қосимовнинг "Ёшлик" журналида эълон қилинган "Карвонбоши" мақоласида татар ѐзувчиси Олимжон Иброҳимовнинг сўзларини келтиради. Унда шундай дейилган: "...дарсликларимиз оламинда Истанбулли Аҳмад Мидхатнинг "Хожа аввал"и, бизнинг "Муаллими аввал"имиз ўрнина аларнинг Хўжа Беҳбудийлари "Адиби аввал"лар ѐзғонлиғин..." айтиб ўтади. Бундан ташқари Н.Авазов ўзининг "Махмудхўжа Беҳбудийнинг ижодий мероси" номли диссертациясида Беҳбудий китоблари рўйха- тига 1918 йилда яратилган "Ҳисоб" номли китобни ҳам келтиради. Юқоридаги далилларга суянган ҳолда айтиш мумкинки, Беҳбудий- нинг "Адиби аввал" ва "Ҳисоб" номли дарсликлари ҳам мавжуд бўлган. Лекин ҳалигача бу китоблар илмий жамоатчилик томонидан топилган эмас. Беҳбудий дарсликлари тартибини юқорида номлари келтирилган олимларимиз томонидан тузилган рўйхатларга ва Беҳбудийнинг ўзи санаб ўтган китоблар рўйхатига суяниб, уларни қуйидагича келтириш мумкин: 1. Алифбо мактаби исломия. - 1904 йил. форс тилида. 40 саҳифа, 15 тийин. 2. Мадхали жуғрофияи имроний - Мухтасари жуғрофияи Русий (Аҳоли жуғрофиясига кириш). - 1905 йил, форс тилида, 88 саҳифа, 45 тийин. 3. Мунтахаби жуғрофияи умумий (+исқача умумий жуғрофия). - 1906 йил, 124 саҳифа, 75 тийин. 4. Китобат ул-атфол (Болалар учун китоб). - 1908 йил. биринчи нашри, 225 Volume 2\| Issue 1 ISSN: 2181-3639 SJIF- 4.431 ISSN: 2181-3639 китоби саналади. Китоб муаллиф таъкидлаганидек, усули жадид мактаблари шогирдлари учун яратилган. Дарслик муқовасида "Китоб. Мунтаҳаби жуғрофияи умумий ва намунаи жуғрофия. Риѐ- зий, тарихий, имроний, русий, сиѐсий ва татбиқот диния ва фанния" деган сўзлар билан бошланади. Ушбу ѐзувлардан шуни англаш мумкинки, китоб мактаб шогирдларига содда ва тушунарли тилда энг умумий жуғрофий билим бериш учун яратилган. "Бисмиллоҳир Раҳмонир Раҳийм", деб бошланувчи сўз бошида муаллиф ушбу дарсликни яратиш учун "...туркий, арабий, форсий, русий лафзларға тасниф бўлган ўтуз қадар қадим ва жадид китоб ва арасоил жуғрофия, ҳайъат, риѐзий, тарихий, табиийлардан мўътабар нусха" ларини кўриб чиқиб, улардан ижодий фойдаланиб, "жам ва тартиб" қилганлигини баѐн этади. Муаллиф жуғрофий китобини ўз сарф-харажати эвазига яратилганлигини қайд этиб ўтади ва бу ишдан мақсади аҳли ватанга кўрсатган бир хизматимдир, дейди. Китобхонларга мурожаат этиб, агар китобда бирор бир камчилик кўрсангиз, албатта, бизга мурожаат этинг, шояд бу сизларнинг маориф тараққиѐтига хизматингиздир, дейди. Беҳбудийнинг жуғрофий китоби 103 бетдан иборат бўлиб, унга ер шарининг хариталари илова қилинган. Мавзулар бирин-кетин оддий жуғрофий тушунчалардан аста-секин ривожланиб боради ва кичик- кичик мақолалар тарзида халқона услубда баѐн этилади. +олаверса, китоб усули жадид шогирдларига мўлжалланиб яратилганлиги учун ҳам оддий иборалардан, ўринли ўхшатишлардан фойдаланишга ҳаракат қилинган. Буни биз мавзуларга қўйилган сарлавҳалардан ҳам кўрамиз. Булар: "Жуғрофия қачон пайдо бўлган?", "Туркистон донишмандлари", "Жуғрофия ўқимоқнинг нафиқ, "Жуғрофия ўқумоқ мусулмонларға лозимдур", "Эски ва янги донишмандлар", "Янгиҳуқамоларнинг сўзларидан намуналар", "Ерни шакли", "Ҳукамолар қавлича ерни юмалоқлигини аломатлари", "Тоғ ва сойлар ерни юмалоқлигига зарар қилмайдур", "Ер айланганда устидагиларнинг йиқилмаслиги" каби. Ушбу сарлавҳалар остида эса бу илмнинг афзаллиги, уни J китоби саналади. Китоб муаллиф таъкидлаганидек, усули жадид мактаблари шогирдлари учун яратилган. Дарслик муқовасида "Китоб. Мунтаҳаби жуғрофияи умумий ва намунаи жуғрофия. Риѐ- зий, тарихий, имроний, русий, сиѐсий ва татбиқот диния ва фанния" деган сўзлар билан бошланади. Ушбу ѐзувлардан шуни англаш мумкинки, китоб мактаб шогирдларига содда ва тушунарли тилда энг умумий жуғрофий билим бериш учун яратилган. "Бисмиллоҳир Раҳмонир Раҳийм", деб бошланувчи сўз бошида муаллиф ушбу дарсликни яратиш учун "...туркий, арабий, форсий, русий лафзларға тасниф бўлган ўтуз қадар қадим ва жадид китоб ва арасоил жуғрофия, ҳайъат, риѐзий, тарихий, табиийлардан мўътабар нусха" ларини кўриб чиқиб, улардан ижодий фойдаланиб, "жам ва тартиб" қилганлигини баѐн этади. Муаллиф жуғрофий китобини ўз сарф-харажати эвазига яратилганлигини қайд этиб ўтади ва бу ишдан мақсади аҳли ватанга кўрсатган бир хизматимдир, дейди. Китобхонларга мурожаат этиб, агар китобда бирор бир камчилик кўрсангиз, албатта, бизга мурожаат этинг, шояд бу сизларнинг маориф тараққиѐтига хизматингиздир, дейди. Беҳбудийнинг жуғрофий китоби 103 бетдан иборат бўлиб, унга ер шарининг хариталари илова қилинган. Results of National Scientific Research International Journal 2023 SJIF- 4.431 1914 йил, иккинчи нашри, турк ва форс тилларида, 36 саҳифа, 15 тийин. 5. Амалиѐти исломия - 1908 йил, турк тилида, 36 саҳифа, 15 тийин. 5. Амалиѐти исломия - 1908 йил, турк тилида, 36 саҳифа, 15 тийин. 6. Мухтасари тарихи ислом (Исломнинг қисқача тарихи). - 1909 йил, ўзбек тилида, 38 саҳифа, 15 тийин. 6. Мухтасари тарихи ислом (Исломнинг қисқача тарихи). - 1909 йил, ўзбек тилида, 38 саҳифа, 15 тийин. 7. Ҳисоб. - 1918 йил. (Ушбу "Ҳисоб" номли китоб ҳақидаги маълумотни Н.Авазовнинг "М.Беҳбудийнинг ижодий мероси" диссертациясига ѐзилган авторефератидан олдик. 1995 йил, 17 февраль, 12-бет.) 7. Ҳисоб. - 1918 йил. (Ушбу "Ҳисоб" номли китоб ҳақидаги маълумотни Н.Авазовнинг "М.Беҳбудийнинг ижодий мероси" диссертациясига ѐзилган авторефератидан олдик. 1995 йил, 17 февраль, 12-бет.) Юқорида Б.Қосимовнинг "Карвонбоши" мақоласида айтилган "Адиби аввал" дарслигини етарли даражада асосланмаганлиги, Беҳбудийнинг ўзи ва бошқа олимлар томонидан қайд этилмаганлиги учун уни китоблар рўйхатига онгли суратда киритмадик. Муаллиф- нинг ўз китоблар рўйхатида "Мадхали жуғрофияи имроний ва Мухтасари жуғрофияи Русий китобларини тире билан битта тартибда беради. Демак, ушбу китоблар икки номдаги битта китоб дейиш мумкин. Биз олиб борган изланишларимиз натижасида Россиянинг Москва шаҳридаги бош кутубхона фондида сақланаѐтган Беҳбудийнинг "Мунтахаби жуғрофияи умумий" китобидан нусха кўчиртириб келтирдик ва Беҳбудийнинг набираси, Самарқанд Давлат университети профессори Надимхон Беҳбудий шахсий кутубхонасида сақланаѐтган "Китобат ул-атфол" номли дарсликларини топиб, тадқиқ қилишга муваффақ бўлдик. қолган дарсликлар ҳақида эса Беҳбудийнинг ўзи "Оина" журнали ва "Самарқанд" газетида берган таърифларига суянган ҳолда ўз мулоҳазаларимизни баѐн этамиз. М.Беҳбудий жуғрофияга оид икки китоб ва бир неча мақоллар ѐзган. Булар "Мунтахаби жуғрофияи умумий" ва "Мадхали жуғрофи- яи имроний", деб аталади. Ушбу мақолага оид "Жуғрофия илми", "Жуғрофия чист" , "Тарих ва жуғрофия" каби йирик мақолалари мавжуд. Бундан ташқари, муаллиф бу мавзуда ўша давр матбуотида тез-тез мурожаат қилиб турган. "Мунтахаби жуғрофияи умумий" Беҳбудийнинг биринчи жуғрофий 226 https://academicsresearch.com/index.php/rnsr/index Volume 2\| Issue 1 ISSN: 2181-3639 SJIF- 4.431 ISSN: 2181-3639 билмоқ дунѐни билишнинг калитларидан бири эканлигини барча далилу исботлар билан баѐн этади. Муаллиф жуғрофия илмида шуҳрат қозонган олимлар ҳақида гапирганда, албатта, Туркистон уламоларини меҳру муҳаббат билан тилга олади. "Туркистон уламоларини ҳаммасини аҳволи ва таснифотини ѐзмоқ лозим бўлса, ушбу китобимиздан ҳам каттароқ китоб тайѐрламоқ керак бўладур", дейди. Ундан сўнг эса Туркистон уламоларининг номларини келтириб ўтади. Булар: Кўрагоний Самарқандий (Мирзо Улуғбек), Муҳаммад Хўжандий, Али бин Можаров ўғли Ҳасан, Абдулла Фарғоний, Абу Райҳон Хоразмий, Ғиѐсиддин Самарқандий, Ғиѐсиддин Муҳаммад Ҳиротий, Муҳаммад Фарғоний, Ҳофиз Абру Ҳиротий, Абу Саъд Самъоний, Абдураззоқ Самарқандий, Мир Абдулкарим Нажарий.... Муаллифнинг ушбу фузалоларни тилга олишдан мақсади, Туркистон аҳлининг шу кунгача на қадар илму урфонга ошнолигини ва ҳатто бутун дунѐи олам уларнинг ижодларидан баҳраманд бўлганлигини кўз-кўз қилиш эди. Бу жуғрофия илми биз учун янгилик эмас, балки у бизнинг бобомерос илмимиздир, деган фикрни илгари суради. Бугунги илмсизлик аҳволи эса мустабидлик сиѐсати сабаб эканлигини ишоралар билан кўрсатишга ҳаракат қилади. "Оврупо ѐ Русияни 15 яшар бир шогирди бизни 50 яшар савдогар ѐ муллаларимиздан дунѐ илми тўғрисиға яхши хабардордир", дейди муаллиф куйиниб. Бугун аҳли дунѐ телеграф, телефон, ҳар хил машиналар ихтиро қилиб, тараққиѐтга юз тутмоқдаки, бизнинг Туркистон мусулмонлари ҳали ҳам "Ер ҳўкуз шоҳига, ҳўкуз балиғга, балиғ сувга, сув ҳавоға, казо-казо" , деган авомона хаѐл билан юриб- дилар, дейди. Беҳбудийнинг бу сўзлари нафақат ушбу китоб мўлжалланган мактаб шогирдларига айтган бу нарса балки, бутун Туркистон аҳлини ўйлантирадиган масалалардан бири эди. Ер ҳақидаги қарашлар доимо икки хил ихтилоф билан давом этиб келган. Муаллиф буни қуйидагича келтиради: "Баъзи уламоларни китобларига ер сокин, янги уламоларни китоблариға ер айланадур, деб таъкид қиладур", дейди. Ушбу масалани ойдинлаштириш учун муаллиф диний SJIF 4.431 ISSN: 2181 3639 билмоқ дунѐни билишнинг калитларидан бири эканлигини барча далилу исботлар билан баѐн этади. Муаллиф жуғрофия илмида шуҳрат қозонган олимлар ҳақида гапирганда, албатта, Туркистон уламоларини меҳру муҳаббат билан тилга олади. "Туркистон уламоларини ҳаммасини аҳволи ва таснифотини ѐзмоқ лозим бўлса, ушбу китобимиздан ҳам каттароқ китоб тайѐрламоқ керак бўладур", дейди. Ундан сўнг эса Туркистон уламоларининг номларини келтириб ўтади. Булар: Кўрагоний Самарқандий (Мирзо Улуғбек), Муҳаммад Хўжандий, Али бин Можаров ўғли Ҳасан, Абдулла Фарғоний, Абу Райҳон Хоразмий, Ғиѐсиддин Самарқандий, Ғиѐсиддин Муҳаммад Ҳиротий, Муҳаммад Фарғоний, Ҳофиз Абру Ҳиротий, Абу Саъд Самъоний, Абдураззоқ Самарқандий, Мир Абдулкарим Нажарий.... Муаллифнинг ушбу фузалоларни тилга олишдан мақсади, Туркистон аҳлининг шу кунгача на қадар илму урфонга ошнолигини ва ҳатто бутун дунѐи олам уларнинг ижодларидан баҳраманд бўлганлигини кўз-кўз қилиш эди. Бу жуғрофия илми биз учун янгилик эмас, балки у бизнинг бобомерос илмимиздир, деган фикрни илгари суради. Volume 2\| Issue 1 ISSN: 2181-3639 Мавзулар бирин-кетин оддий жуғрофий тушунчалардан аста-секин ривожланиб боради ва кичик- кичик мақолалар тарзида халқона услубда баѐн этилади. +олаверса, китоб усули жадид шогирдларига мўлжалланиб яратилганлиги учун ҳам оддий иборалардан, ўринли ўхшатишлардан фойдаланишга ҳаракат қилинган. Буни биз мавзуларга қўйилган сарлавҳалардан ҳам кўрамиз. Булар: "Жуғрофия қачон пайдо бўлган?", "Туркистон донишмандлари", "Жуғрофия ўқимоқнинг нафиқ, "Жуғрофия ўқумоқ мусулмонларға лозимдур", "Эски ва янги донишмандлар", "Янгиҳуқамоларнинг сўзларидан намуналар", "Ерни шакли", "Ҳукамолар қавлича ерни юмалоқлигини аломатлари", "Тоғ ва сойлар ерни юмалоқлигига зарар қилмайдур", "Ер айланганда устидагиларнинг йиқилмаслиги" каби. Ушбу сарлавҳалар остида эса бу илмнинг афзаллиги, уни 227 https://academicsresearch.com/index.php/rnsr/index Volume 2\| Issue 1 ISSN: 2181-3639 Бугунги илмсизлик аҳволи эса мустабидлик сиѐсати сабаб эканлигини ишоралар билан кўрсатишга ҳаракат қилади. "Оврупо ѐ Русияни 15 яшар бир шогирди бизни 50 яшар савдогар ѐ муллаларимиздан дунѐ илми тўғрисиға яхши хабардордир", дейди муаллиф куйиниб. Бугун аҳли дунѐ телеграф, телефон, ҳар хил машиналар ихтиро қилиб, тараққиѐтга юз тутмоқдаки, бизнинг Туркистон мусулмонлари ҳали ҳам "Ер ҳўкуз шоҳига, ҳўкуз балиғга, балиғ сувга, сув ҳавоға, казо-казо" , деган авомона хаѐл билан юриб- дилар, дейди. Беҳбудийнинг бу сўзлари нафақат ушбу китоб мўлжалланган мактаб шогирдларига айтган бу нарса балки, бутун Туркистон аҳлини ўйлантирадиган масалалардан бири эди. Ер ҳақидаги қарашлар доимо икки хил ихтилоф билан давом этиб келган. Муаллиф буни қуйидагича келтиради: "Баъзи уламоларни китобларига ер сокин, янги уламоларни китоблариға ер айланадур, деб таъкид қиладур", дейди. Ушбу масалани ойдинлаштириш учун муаллиф диний 228 https://academicsresearch.com/index.php/rnsr/index Volume 2\| Issue 1 ISSN: 2181-3639 - Ҳинд, рупия (кумуш) - Афғон, Қирон (кумуш) - Эрон, Ян (кумуш) - Япон, Лира (олтин) - Усмоний, фунт (стерлинг) - Англия, Франк (кумуш) - Франсия, Марк(кумуш) - Германия, Дуллар (кумуш) - Америка ... каби. - Ҳинд, рупия (кумуш) - Афғон, Қирон (кумуш) - Эрон, Ян (кумуш) - Япон, Лира (олтин) - Усмоний, фунт (стерлинг) - Англия, Франк (кумуш) - Франсия, Марк(кумуш) - Германия, Дуллар (кумуш) - Америка ... каби. Дарслик сўнгроғида "Муаллифдан эълон" ѐзуви остида Беҳбу- дийнинг ўзи яратган китоблари ҳақида мълумотлар берилган. "Алифбо мактаби исломия" эълонида "Болалар учун усул жадида узра форсий тилға қирқ саҳифалик бир китобчадур. Риқъий хат ила 4-5 қалам хат ўткарилиб ўзимиз табъ ва таҳрир этганмиз" , деб ѐзади ва унинг баҳосини ҳам кўрсатиб ўтади. Бундан шу нарса кўринадики, Беҳбудий ўзи яратган китоблари тарғиботида, ушбу дарсликларидан ҳамунумли фойдалана билган. Китобнингэнг сўнгги саҳифаларида Шимолий ва Жанубий Африка, Оврупо, Австралия, Русия каби мамлакатларнинг хариталари илова қилинган. Volume 2\| Issue 1 ISSN: 2181-3639 билимларнинг пирларидан бўлган ҳазрат имом Ғаззолийга мурожаат этади,унинг асарларида ҳам ер ҳўкуз устида туради, деган гапни асло учратмай, балки, ер ҳавога муаллақ турганлигини нисбат берганини айтиб ўтади. Беҳбудий жуғрофий ҳодисаларни баѐн этар экан, унинг тушу- нарли бўлиши учун ўринли ва қизиқарли ўхшатишлар топади. Оқибатда, бу ўхшатишлар болалар онгига тез етиб боришига сабаб бўлади. Масалан, "Тоғ ва сойлар ерни юмолоқлигига зарар қил- майдур" сарлавҳаси остида берилган мақолада "Ер ила тоғни нисбати шундайдирки, бир катта тарвуз устига бир дона қора қумни қўйилганидек, яъни ерни бир тарвуз фараз қилса ўн минг газлик баланд тоғ они олдиға биро дона қора қумдек..." , деган қизиқарли ўхшатиш қилади. "Ер айланганда устидагиларни йиқилмаслиги" сарлавҳаси остида баѐн қилинган фикрларда эса, қозуқға боғлаб қўйилган отдек, еримиз марказ шамсия атрофиға оний жозиба арқонини ўзунликиға қараб айланиб узоқ кетармайдур" , деган ўринли ҳам мантиқли ўхшатиш ясайди. "Харита" сарлавҳаси остида, бу жуғрофий атамани тушунтирар экан, уни, "Одамни шакли ва ҳайъати нақадар катта ѐ ниҳоят кичик қилиб кўрсатмоқ суврати кишиларга мумкинлигидек илм аҳлига ерни шаклияти истаганларича кичик қилиб кўрсатмоқ мумкиндир" , деб харита ҳақида тушунча беради. Беҳбудий ибтидоий даврдан бошлаб инсоният тарихида рўй берган машҳур воқеалар ҳақида маълумотлар беришга ҳаракат қилади ва саналар асносида бу воқеаларнинг тартибини тузиб чиқади. Масалан, 2920 санада деҳқонлик асбоби ясалгани, 2723 санада зайтун дарахти экилгани, 2725 санада либос тикилмоқлик бошлангани... Худди мана шундай воқеалар батафсил баѐн этилади. Муаллиф ушбу дарсликла дунѐнинг турли туман ҳодисалари ҳақида кўпроқ маълумотлар беришга ҳаракат қилади. "Дунѐнинг оқчалари" сарлавҳасида дунѐ пуллари ҳақида қизиқарли маълумот беради. Масалан, Танга (кумуш), тилла (олтун)- Бухоро, танга (кумуш) - Хива, Килдор (кумуш) 229 229 ХУЛОСА Беҳбудийнинг "Мунтахаби жуғрофияи умумий" дарслиги хусусида шуни таъкидлаб ўтиш лозимки, муаллиф ўз олдига қўйган усули жадид мактабларининг ўрта синф шогирдларига энг умумий жуғрофий маълумот бериш вазифасини ўз ўрнида бажара олган. Юқорида кўриб ўтганимиздек, бу китоб нафақат мактаб шогирдлари, балки Туркистоннинг барча афкор оммаси учун ҳам қулай бир қўлланма эканини кўрамиз. https://academicsresearch.com/index.php/rnsr/index Results of National Scientific Research International Journal 2023 SJIF- 4.431 Results of National Scientific Research International Journal 2023 SJIF- 4.431 Фойдаланилган адабиётлар рўйҳати 1. З. Б. Аҳрарова (2021). УЗЛУКСИЗ ТАЪЛИМДА ПЕДАГОГИК ТЕХНОЛОГИЯЛАРНИ ҚЎЛЛАШ ЖАРАЁНЛАРИ. Academic research in educational sciences, 2 (NUU Conference 1), 190-194. 2. Беҳбудий М. Мунтаҳаби жуғрофияи умумий ва намунаи жуғрофия.- Самарқанд:- Типография Г.И. Демурова.1906 йил,103-бет. 3. Беҳбудий М. Тарих ва жуғрофия. Туркистон вилоятининг газети.- 1908йил.-1декабрь.-№ 90. 3. Беҳбудий М. Тарих ва жуғрофия. Туркистон вилоятининг газети.- 1908йил.-1декабрь.-№ 90. 4. Беҳбудий М. Қасди сафар.Оина.-1914 йил-№ 31.-598-600 б. https://academicsresearch.com/index.php/rnsr/index 230 https://academicsresearch.com/index.php/rnsr/index Results of National Scientific Research International Journal 2023 SJIF- 4.431 Volume 2\| Issue 1 ISSN: 2181-3639 J 5. Шахноза Эркиновна Хамрокулова (2021). Ўзбек маърифатпарвари Абдурауф Фитрат асарларида экологик тарбия масалалари. Science and Education, 2 (11), 1116-1119. 5. Шахноза Эркиновна Хамрокулова (2021). Ўзбек маърифатпарвари Абдурауф Фитрат асарларида экологик тарбия масалалари. Science and Education, 2 (11), 1116-1119. 5. Шахноза Эркиновна Хамрокулова (2021). Ўзбек маърифатпарвари Абдурауф Фитрат асарларида экологик тарбия масалалари. Science and Education, 2 (11), 1116-1119. 231 231 https://academicsresearch.com/index.php/rnsr/index
W1579567843.txt	https://publications.goettingen-research-online.de/bitstream/2/60647/1/2005-noubactep-grundwasser.pdf	de		Laboruntersuchungen zur Freisetzung von Unat aus einem Gestein unter oxischen naturnahen Bedingungen	Grundwasser	2,005	cc-by	6,080	1 Überarbeitetes Manuskript für Grundwasser Laboruntersuchungen zur Freisetzung von Unat aus einem Gestein unter oxischen naturnahen Bedingungen. Chicgoua Noubactep, Jürgen Sonnefeld, Martin Sauter C. Noubactep, M. Sauter Geowissenschaftliches Zentrum der Universität Göttingen; Goldschmidtstrasse 3, 37077 Göttingen Telefon 0551 39 3191 (7911), Telefax: 0551 399379 Email: cnoubac@gwdg.de J. Sonnefeld Institut für Physikalische Chemie der Friedrich-Schiller-Universität Jena; Lessingstrasse 10; 07743 Jena Telefon 03641 948341 (948347), Telefax: 03641 948302 Email: cjs@pc02.chemie.uni-jena.de Abstract Investigations for the release of Unat from ore bearing rock under natural near oxic conditions The effects of carbonate concentration and the presence of iron hydroxide phases on uranium release into the environment were investigated under oxic conditions and in the pH range from 6 to 9. For this purpose not-shaken batch experiments were conducted with a constant amount (8, 10 or 40 g/L) of a uranium bearing rock and different types of water (deionised, tap and mineral water). For comparison parallel experiments were conducted with 0.1 M Na2CO3 and 0.1 M H2SO4. The use of dolomite confirmed the favourable role of carbonate bearing minerals for U transport while the presence of pyrite on Uranium mobilisation was shown to be considerably more complex. This study shows that the approach of equilibrium conditions can be strongly delayed by sorption processes. Zusammenfassung Im Labor wurden verschiedene Größen (pH-Wert, HCO 3--Gehalt, Präsenz von Fe(OH) 3) untersucht, von denen angenommen werden konnte, dass sie die umweltrelevante Freisetzung von Uran aus Gesteinen (z.B. Haufwerken, Halden, Tailings) im neutralen und schwach basischen pH-Bereich (6 bis 9) beeinflussen. Hierzu wurde der Austrag von Unat aus einem schwach vererzten Urangestein (2.31 %) in Batchversuchen untersucht. Diese Versuche wurden mit 8, 10 oder 40 g/L Gestein und Leitungs- bzw. Mineralwasser durchgeführt. Zum Vergleich wurden parallele Standversuche in deionisiertem Wasser, 0,1 M Na 2CO 3 und 0,1 M H2SO4 durchgeführt. Die Ergebnisse zeigen eine Begünstigung der Uranfreisetzung in Anwesenheit von Carbonat (gelöst oder in-situ durch Dolomitauflösung entstanden), während die Rolle des Pyrits auf die Freisetzung deutlich komplexer ist. Das Erreichen eines Gleichgewichtszustands kann unter Umständen durch sorptive Einflüsse verzögert werden. 2 Einleitung Infolge des nuklearen Wettrüstens wurden in zahlreichen Ländern Uran ohne Rücksicht auf die Umwelt und die Gesundheit von Menschen abgebaut. Uran besteht im wesentlichen aus dem Isotop 238, welches nur eine geringe Strahlenwirkung hat. Jedoch stellt das Uran vor allem wegen seiner Chemotoxizität eine Gefahr für die Gesundheit des Menschen weltweit dar (US EPA 2002, MERKEL 2002). Dies betrifft gerade Regionen in denen sich Abraumhalden und Tailings in der Nähe von Städten und Gemeinden befinden, da die Urangehalte auf diesen Abraumhalden immer noch deutlich erhöht sind. Der Wind kann radioaktive Partikel verwehen. Kontaminiertes Wasser infiltriert in den Boden bzw. ins Grundwasser oder gelangt direkt in die Flüsse. Ein Beispiel für Regionen, wo Menschen dieser Gefahr ausgesetzt sind, stellen die ehemaligen Uranabbaugebiete in Sachsen und Thüringen dar (GATZWEILER & MAGER 1995, HURST ET AL. 2002, JUNGHANS & HELLING 1998). Zur Urangewinnung für den o.g. Zweck stand bis Anfang der 80er Jahre weltweit die Erzgewinnung (d.h. Uranlaugung aus dem Gestein mit technischen Lösungen wie NaHCO3 oder H2SO 4) im Zentrum der Uranchemie (C LARK ET AL. 1995). Es ist bisher kaum untersucht worden, inwieweit natürliches Wasser zur Uranmobilisierung aus Halden und anderen Haufwerken beitragen kann. Es gibt noch deutliche Wissenslücken über die Umweltchemie des Urans (z.B. mangelnde Daten für U(V)-Spezies bzw. zur Komplexbildung von U(IV)Spezies - BERNHARD ET AL. 1996, MEINRATH ET AL. 1999, NOUBACTEP ET AL. 2003). Die Verbesserung der Datenlage ist von großer Bedeutung für z.B. weitere Sanierungsentscheidungen, da Millionen Tonnen an Haldenmaterial in ehemaligen Uranbergbaugebieten exponiert sind (GATZWEILER & MAGER 1995, JUNGHANS & HELLING 1998, HELLING 1999, OHLENDORF 1999, NEITZEL ET AL. 2000, SCHNEIDER ET AL. 2000, HURST ET AL. 2002, MEINRATH ET AL. 2003). Bis zum Ende der 80er Jahre war nur wenig über das Langzeitverhalten von Radionukliden in der Umwelt bekannt. Diese Lücken wurden 3 zum Teil durch Analogstudien im Rahmen von Untersuchungen zum Verständnis des Urantransports in die Umwelt behoben (z.B. C HAPMAN ET AL. 1992, IVANOVICH & HARMON 1992, READ 1992, R EYNOLDS & GOLDHABER 1978, ROMERO ET AL. 1992). Zurzeit wird anwendungsorientierte Grundlagenforschung zur Entwicklung neuartiger Sanierungskonzepte für uranbelastete Grund- und Oberflächenwässer bzw. Boden betrieben (BAYER ET AL. 2004, C ATCHPOLE & KIRCHNER 1995, MORRISON & SPRANGLER 1993, MORRISON ET AL. 2001, NEITZEL ET AL. 2000, NOUBACTEP 2003). Nach dem heutigen Wissensstand werden in-situ Sanierungsverfahren in vielen Fällen favorisiert (EDEL & VOIGT 2001, KÖRBER ET AL. 2001, NOUBACTEP 2003). Hierfür ist, unabhängig von den wirkenden Rückhalteprozessen in der reaktiven Zone, die Abschätzung der Uranfracht von besonderer Bedeutung. Die bisherige Vorgehensweise zur Quantifizierung der Uranausbreitung in der Umwelt ähnelt der, für die Optimierung technologischer Prozesse eingesetzten. In der Regel geht es darum zu verstehen, unter welchen Bedingungen und auf welche Weise sich Uran ausbreitet, und welche Prozesse für die Fixierung und die Freisetzung von Uran relevant sind (LANGMUIR 1978, LANGMUIR 1997). Die so gewonnenen Erkenntnisse werden auf mehr oder weniger komplexe Systeme angewandt, um Modellprognosen über Jahrzehnte und Jahrhunderte machen zu können (z.B. BAIN ET AL. 2001). Basierend auf der chemischen Zusammensetzung eines Sandes und den Arbeiten von KIRCHHEIMER (1959A; 1959B) zur Uransorption an Kieselsäurebildungen wurde z.B. angenommen, dass Quarz keine bedeutende sorptive Eigenschaften gegenüber Uran besitzt. Seit READ ET AL. (1993) weiß man, dass die Annahme nicht zulässig ist. Neben der Erarbeitung eines Prozessverständnisses ist die Untersuchung der Mobilisierung von Uran durch natürliche Wässer bzw. relevante naturähnliche Wässer (BAASBECKING ET AL. 1960) aus Halden und Gesteinen von Bedeutung. Die vergleichende Auswertung der Ergebnisse aus den hier beschriebenen Versuchsreihen können helfen ein realitätsnäheres Bild der natürlichen Prozesse zu gewinnen. 4 Mit dieser Arbeit soll ein Beitrag zum Verständnis des Mechanismus der Uranentfreisetzung aus einem Gestein in die Wasserphase durch natürliche Wässer in neutralen pH-Bereich geleistet werden. Dabei kommt der Untersuchung der Rolle der verschiedenen Gesteinsbestandteile auf den Freisetzungsprozess eine besondere Bedeutung zu. Zur Lösung der Zielstellung wurde ein uranhaltiges Gestein (U-Gestein) gemahlen und mit Wässern unterschiedlichen Carbonatgehaltes bzw. zwei technischen Laugungslösungen (Na 2CO 3 und H2SO4) in Kontakt gebracht. Weitere Versuche dienten dazu, den Einfluss von in-situ entstandenem Eisenoxid (zur Vereinfachung als Fe(OH) 3 bezeichnet) (Einsatz von Pyrit) bzw. in-situ freigesetzten Carbonationen (Einsatz von Dolomit) auf die Uranfreisetzung zu charakterisieren. Experimenteller Teil Materialien Das eingesetzte U-Gestein wurde mittels eines wellenlängen-dispersiven Röntgenfluoreszenzspektrometer - RFA [Philips / Typ PW2400] analysiert und zeigte folgende Zusammensetzung: 2,31 % U; 81,25 % SiO2; 0,14% TiO2; 7,36 % Al 2O3; 1 % Fe2O3; 0,01% MnO; 0,48 % MgO; 0,67 % CaO; 1,19 % Na 2O; 1,48 % K2O; 0,36 % P 2O5 und 0,01% SO3. Die Analyse wurde am Institut für Geowissenschaften der Friedrich-SchillerUniversität Jena durchgeführt. Eine REM-Analyse zeigte, dass es sich um ein Uranerz handelt, welches geringe Mengen an mehreren Uranmineralen enthielt (u.a. Uraninite). Eine XRD-Analyse zeigte keine kristalline Uranphase. Aus dem gemahlenen Material wurden die gewünschten Partikelgrößen durch Sieben gewonnen und ohne weitere Behandlung verwendet. Ein zweites U-Gestein mit ca. 1.2 % U wurde in einigen Versuchen eingesetzt. Die eingesetzte Partikelgrößen sind in Tabelle 1 zusammengestellt. < Tabelle 1> 5 Pyrit (d 3) mit der Zusammensetzung Fe: 40%; S: 31,4%; Si: 6,7%; Cl: 0,5%; C: 0,15% and Ca < 0.01% wurde zur Senkung des pH bzw. zur in-situ Herstellung von Eisen(hydr)oxiden (zur Vereinfachung als Fe(OH) 3 in der Folge bezeichnet) eingesetzt. Dolomit (CaMg(CO 3)2 - d4) mit der Zusammensetzung SiO2: 1,2 %; TiO2: 0,03 %; Al2O3: 0,4 %; Fe2O3 0,6 %; MgO: 20,24 %; CaO: 30,94; Na 2O: 0.04% wurde zur Erhöhung des Carbonatgehalts verwendet. Durch seinen Einsatz (Auflösung) wird eine erhöhte Kinetik der Uranfreisetzung erhofft. Zur Nachbildung natürlicher Verhältnisse wurde mit verschiedenen Wässern unterschiedlicher Carbonatgehalte gearbeitet. Tabelle 2 fasst die Carbonatgehalte und die simulierten Bedingungen zusammen. Alle diese Wässer enthielten kaum Phosphat und die jeweilige Calciumkonzentration betrug 0, 36 und 99 mg/L für deionisiertes (DW), Leitungs(LW: Cl-: 15,3; NO 3-: 10,0; SO42-: 72,1; HCO3-: 268,0; Na+: 8,7; K+: 5,3; Mg2+: 29,3; Ca 2+: 80,9) und Mineralwasser (MW: Cl-: 129; NO 3-: <0,01; SO42-: 37,0; HCO3-: 1854,0; Na +: 574; K+: 14,5; Mg2+: 60,5; Ca2+: 99,0). Außerdem wurden zwei gängige technische Laugungslösungen des Uranabbaus (Na 2CO3 und H2SO 4) zum Vergleich eingesetzt. < Tabelle 2> Uranfreisetzungsexperimente Zur Charakterisierung des Potentials der reaktiven Materialien zur Uranfreisetzung aus dem Gestein stehen für Batchversuche prinzipiell Stand- und Rührversuche zur Auswahl. Beide Techniken ergänzen sich in der Regel, wobei das Rühren verschiedene Prozesse einleiten bzw. beschleunigen kann (NOUBACTEP 2003). Das Rühren (Rührversuche) kann insbesondere zur mechanischen Zerkleinerung der Reaktanden und somit zu einer erhöhten Reaktivität (größere Oberfläche) führen. Dieses Phänomen wird bei fast stagnierenden Grundwässern nicht vorherrschen. Um dieser Tatsache Rechnung zu tragen, wurden in dieser Arbeit zwei Typen von Experimenten durchgeführt, bei denen reaktiven Materialien mechanisch nicht beansprucht werden: 6 Nicht geschüttelte Batchversuche oder Standversuche: Die gewünschte Menge an reaktiven Materialien (U-Gestein und ggf. Dolomit oder Pyrit) wurde in mit 13 mL Laugungslösung (DW, LW, MW, Na 2CO 3 oder H2SO4) gefüllte Reagenzgläser gegeben. Nach der gewünschten Reaktionszeit wurden 500 µL der Lösung zur Uranbestimmung sorgfältig abpipettiert (nicht filtriert). Eine nähere Beschreibung der Vorgehensweise befindet sich in NOUBACTEP ET AL. (2004). Luft-homogenisierte Batchversuche oder belüftete Standversuche: Bei dieser Versuchsreihe wurde Luftzutritt (Atmosphärendruck) ermöglicht. Die Experimente erfolgten in geschlossenen Glasgefäßen von ca. 100 mL Volumen, die mit Gaszu- und -ableitungen sowie zwei Schliffen versehen waren, um Lösungen entnehmen oder zugeben zu können. Die Luft wurde durch vorgeschaltete Waschflaschen geleitet und so befeuchtet bzw. von Staubpartikeln befreit (NOUBACTEP 2003). Jedes Gefäß enthielt zu Beginn des Experiments 100 mL Leitungswasser, 1,00 g U-Gestein und ggf. 0,75 g Zuschlagsstoffe (Dolomit oder Pyrit). Zu gegebenen Zeiten wurden von den Gefäßen je 1,5 mL Lösung zur Uranbestimmung abpipettiert und das gleiche Volumen an Leitungswasser zugegeben. Redoxpotential und pHWert wurden zu ausgewählten Zeitpunkten gemessen. Analytische Methoden Die Bestimmung der Urankonzentration erfolgte mittels ICP-MS [ThermoElemental Winsford, U.K./PQ3-S] am Institut für Geowissenschaften der Universität Jena. Alle verwendeten Chemikalien waren von analytischem Reinheitsgrad. Die pH-Bestimmung erfolgte mit einer SenTix 50 pH-Elektrode und einem pH-Meter WTW pH 325. Zur Messung des pH-Wertes wurde das Elektrodenpotential gegen fünf Standard-Pufferlösungen (Fa. Roth) gemäß der neuen IUPAC-Empfehlungen (BUCK ET AL. 2002) kalibriert. Die Messung ist auf ± 0.02 pH-Einheiten reproduzierbar (NOUBACTEP 2003). Krypton-Adsorptions-Isothermen bei 77 K wurden mit Autosorb-1-Instrument (QUANTACHROME) gemessen und die spezifischen 7 Oberflächen mithilfe des BET-Mehrpunktverfahrens von BRUNAUER ET AL. (1938) abgeleitet. Vor den Messungen wurden die Proben eine Stunde lang bei 300 °C evakuiert. Die Standversuche wurden in drei parallelen Versuchsreihen durchgeführt. Die Fehlerbalken in den Abbildungen zeigen die Standardabweichung der Triplikate. Alle Experimente erfolgten bei Raumtemperatur (20 ± 3 °C). Ergebnisse und Diskussion Als Maß für die Charakterisierung der Freisetzung diente die wässrige Urankonzentration (in µg/L) oder die umgerechnete prozentuale Freisetzung (in %) bzw. die umgerechnete Masse (in mg). Sie bezieht sich entweder auf die ausgerechnete Uranmasse im eingewogenen Gestein oder auf die operationell definierte „maximale auslaugbare Uranmenge“ unter gegebenen experimentellen Bedingungen. Einfluss der Partikelgröße Zur Charakterisierung des Einflusses der Partikelgröße auf die Uranfreisetzung wurde eine Siebanalyse durchgeführt (Tabelle 1). Die Partikelgröße hat eine große Bedeutung für die Auflösung von Mineralien (z.B. R EICHE P. 1950). Die spezifische Oberfläche von Partikeln stellt ein gebräuchliches Feinheitsmerkmal dar. Sie ist umgekehrt proportional zur Partikelgröße. Nach der gängigen Annahme gilt: Je feiner die Partikel sind, desto einfacher ist die Auflösung (chemische Verwitterung durch Wasser und/oder Luft). In dieser Arbeit wurden vier Korngrößenklassen (alle < 1 mm, Tabelle 1) in Standversuchen für zwei Wochen eingesetzt. Tabelle 3 fasst die Ergebnisse zusammen. < Tabelle 3> Nach der obigen Annahme sollte die Reaktivitätsreihe wie folgt abnehmen: d1 > d2 > d3 > d4. Diese Reihenfolge gilt strenggenommen nur im Experiment mit 0.1 N Na 2CO 3 (Tabelle 3). 8 In den Systemen mit Leitungswasser und 0.1 N H2SO 4 war die Reaktivitätsreihe: d1 > d2 ≅ d3 > d4 (Tabelle 3). Tabelle 1 zeigte schon eine Abnormalität in der Änderung der spezifischen Oberfläche mit der Partikelgröße für das zweite Gestein (S2). Dieses weist auf andere Effekte hin (z.B. mineralogischer Art), welche die Auflösung des Gesteins beeinflussen. Weitere Untersuchungen waren aufgrund der begrenzten Probenmenge nicht möglich. Es ist jedoch bekannt, dass bei abnehmender Partikelgröße, i.d.R. unterhalb von ca. 50 µm, die durch Schwingungen übertragbaren Massenkräfte meist durch Adhäsions- bzw. Kohäsionskräfte dominiert werden. Dadurch haften die Partikel aneinander. Derartige Effekte können z.B. bedingt durch Feuchtigkeit auch bei wesentlich höheren Partikelgrößen (hier > 150 µm) auftreten. Tabelle 3 zeigt, dass in allen Fällen weniger als 50 % der ursprünglichen Uranmenge ausgelaugt werden konnte, selbst mit einem starken Laugungsmittel wie 0.1 N H2SO 4 (43 %). Die Laugungsrate im Leitungswasser, welches natürliche Sickerwässer (Regen) simulieren sollte, variierte zwischen 8 und 13 %. Diese Beobachtung veranschaulicht die Ineffizienz der Schadstofflaugung aus natürlichen Materialien (z.B. Erzgestein, Sediment) durch natürliche Wässer und damit auch die begrenzte Wirksamkeit der sogenannten „pump-and-treat“Methode (BAYER ET AL. 2004, MACKAY & CHERRY 1989, MCMURTY & ELTON 1985, NYER ET AL. 1996). Bei diesem Verfahren ist die Effizienz noch geringer als im Laborversuch, da nicht davon ausgegangen werden kann, dass sämtliches Wasser im Kontakt mit uranführendem Material wegen der Heterogenität in der Durchlässigkeit quantitativ ausgetauscht wird. Ferner verringern in der Natur die Präsenz anderer Minerale und organischer Substanzen auf Grund der Sorption die Laugungsfähigkeit des Wassers. Die Wasserzusammensetzung, insbesondere der Carbonatgehalt ([HCO3-] oder PCO2) wird dabei die ausschlaggebende Rolle spielen. 9 Einfluss des Carbonatgehalts Gezielte Prozesse, welche die Urangewinnung aus dem Erzkörper ermöglichen, sind bekannt und werden in der Urangewinnung eingesetzt (z.B. PETERS 1978, SMIRNOV & MATVEEV 2001). Um die Uranbindungsstärke an Geomaterialien zu charakterisieren, werden verschiedene technische Laugungslösungen bestimmt und eingesetzt (e.g. BRAITHWAITE ET AL. 1997, DUFF ET AL. 1997, FREDERIKSON ET AL. 2000, KAPLAN & SERKIZ 2001, LIU ET AL. 2004, POSEY-DOWTY ET AL., 1987). Alle diese operationellen Lösungen sind aggressiver als natürliches Wasser. Um die Laugungsfähigkeit verschiedener natürlicher Wässer für das eingesetzte Gestein zu charakterisieren, wurden drei Wassertypen in dieser Arbeit getestet (Tabelle 2); die Ergebnisse wurden mit den technischen Laugungen (0.1 N Na 2CO3 und 0.1 N H2SO4 ) verglichen (Tabelle 4, Abbildung 1). < Tabelle 4> Die Ergebnisse in Tabelle 4 können wie folgt zusammengefasst werden: • Die Auflösung des Gesteins bewirkt eine geringfügige pH-Anhebung, bei pHi < 7 und eine geringfügige Senkung für pH i > 7. Diese Beobachtung deutet darauf hin, dass die Gesteinsauflösung keine größere Auswirkung auf den pH-Wert hat. Die chemische Zusammensetzung des Gesteins (81,3 % SiO2) unterstützt diese Aussage. < Abbildung 1> • Die Laugungsrate in natürlichen Wässern variiert zwischen 0,3 und 5,0 %, während diese Rate für Na 2CO3 und H2SO 4 2,2 bzw. 24 % beträgt (Abbildung 1). Es ist bemerkenswert, dass die Uranlaugungsrate im eingesetzten Mineralwasser (MW; pH i 6,87) größer als in Na 2CO 3 (pH i 11,47) ist, obwohl der Vergleich der Molarverhältnisse [CO32-] zu [U] in beiden Systemen darauf hinweist, dass im System mit Na 2CO 3 viermal mehr Carbonationen zur Urankomplexierung zur Verfügung stehen als im Mineralwasser (MW). Dieses Ergebnis kann mit dem Trend zur Uran-Mitfällung bei höheren pH-Werten begründet werden. Es ist anzunehmen, dass ein Teil des in Na 2CO 3 aufgelösten Urans z.B. als Na-Uranat 10 (Na 2UO4) wieder ausfällt. Diese Annahme wird durch die Auflösungsrate des SiO2 in verschiedenen Lösungen bekräftigt (Tabelle 4). Demnach löst sich das Gestein (> 80 % SiO2) mit zunehmendem pH-Wert besser. Angesichts der besseren Reproduzierbarkeit der Ergebnisse im Mineralwasser (MW; 11 % zu 69% Standardabweichung in Abbildung 1) können ausgewählte HCO3--reiche Wässer (Mineralwässer und sogenannte Heilwässer nach ZANGER 2002) als Alternative zu technischen Carbonatlösungen für Laugungsexperimente für Umweltzwecke vorgeschlagen werden. Die Laugungsrate in H2SO4 ist deutlich höher, hat jedoch keine Umweltrelevanz. Daher wurde diese in den jeweiligen gegebenen experimentellen Bedingungen als Bezugsgröße für die Definition der relativen prozentualen Uranfreisetzung eingesetzt (Abbildung 1). Einfluss des Sauerstoffs Zur Abschätzung des Sauerstoffeinflusses wurden zwei Experimente durchgeführt. Das erste bestand in einem Standversuch im Leitungswasser mit einer frischen Gesteinsprobe und einer Probe, die ein Jahr unter Laborbedingungen aufbewahrt worden war. Es wurde angenommen, dass unter letzteren Bedingungen der Luftsauerstoff vorhandenes Uran (IV) teilweise oxidiert und dessen Auflösung begünstigt (LANGMUIR 1978). Die Ergebnisse zeigen eine ca. achtmal größere Laugungsrate für die „gealterte Probe“ (PU = 2,2 % gegen 0,3 % für die „frische Probe“). Es wird jedoch ausdrücklich betont, dass diese Beobachtung keinen klaren Nachweis für U(IV)-Oxidation darstellt; vielmehr handelt es sich hierbei um Komplexprozesse der Verwitterung an der Luft. Der zweite Versuch bestand in parallelen belüfteten Experimenten (PO2 = 20 %; PCO2 = 0,035 %) und in einer Glove-Box (PO2 = 2 %; PCO2 = 0,0035 %). Der CO2-Druck in der Glove-Box wurde nicht gemessen. Da der PO2 jedoch durch Spülen der Box mit N2 eingestellt wurde, reduzierte sich der CO2-Druck im gleichen Verhältnis wie der O2-Druck. Der Versuch 11 wurde mit einer frisch gemahlenen Gesteinsprobe 2 (S 2 in Tabelle 1) im Jenaer Leitungswasser ([HCO3-] = 270 mg/L) durchgeführt. Abbildung 2 zeigt die Ergebnisse. < Abbildung 2> Aus Abbildung 2 ist zu entnehmen, dass die Laugungsrate bei 20 % O2 (an der Luft) deutlich höher ist als bei 2 %. Nach ca. zwei Wochen (300 Stunden) erreicht die Lösung in der GloveBox einen stationären Zustand (Plateau; Abb. 2), während die Urankonzentration im Experiment an der Luft weiter zunimmt. Allerdings muss auch in diesem Fall berücksichtigt werden, dass das Experiment in der Glove-Box praktisch unter Ausschluss von Kohlendioxid (CO2) stattfindet, so dass ebenfalls von keiner klaren U(IV)-Oxidation durch Luftsauerstoff gesprochen werden kann. Mit dieser Versuchsreihe sollten wichtige Aspekte der Uranfreisetzung unter oxischen Bedingungen charakterisiert werden. Diese beiden Experimente zeigen jedoch, wie wichtig es ist, die Einflüsse von O2 und CO 2 getrennt zu betrachten. In der gängigen Literatur sind diese Effekte nicht immer eindeutig unterschieden, zumal, wie oben erwähnt, Erkenntnisse aus der Uranchemie zum Uranabbau direkt auf die Sanierung von urankontaminierten Standorten übertragt werden. Beim Uranabbau (z.B. Untertage) wird dem Grundwasser eine chemische Lösung beigegeben, welche das Uranerz aus dem Nebengestein löst. Bei der Sanierung durch Reaktionswände geht es darum, durch Sickerwasser mobilisiertes Uran in den reaktiven Materialien der Wand zu immobilisieren. Einfluss der eingesetzten Zusatzstoffe (Dolomit und Pyrit) Eine weitere Methode zur Charakterisierung des Einflusses der relevanten Größen auf die Uranfreisetzung besteht darin, das U-Gestein und den jeweiligen reaktiven Zusatzstoff (Dolomit und Pyrit) in belüfteten Batchversuchen ins Gleichgewicht zu setzen. Unter solchen Bedingungen entstehen allmählich gelöstes Uran und reaktive Spezies, die miteinander reagieren. Abbildung 3 stellt die Ergebnisse dar, die sich wie folgt zusammenfassen lassen: < Abbildung 3> 12 • Die Änderungen des pH- bzw. EH-Wertes wurden an anderer Stelle diskutiert (NOUBACTEP ET AL. 2004) und zeigten, dass im Referenzsystem (U-Gestein allein) und im System mit Dolomit der pH-Wert während des ganzen Experiments mehr oder weniger konstant und gleich dem des Lösungsmittels (LW; ca. pH 8,3) blieb. Im System mit Pyrit dagegen betrug den Anfangs-pH 6,38 und stieg nach 70 Tagen auf 8,00 an. Das Redoxpotential zeigte denselben Trend mit einem Endwert von 430 mV (auf die Normalwasserstoffelektrode bezogen) in sämtlichen Experimenten. • Die pH-Abnahme im System mit Pyrit ist eine Folge der Pyritoxidation (z.B. WILLIAMSON & RIMSTIDT 1994). Dieser Vorgang bewirkt in der Natur wegen der Versauerung eine Zunahme der Uranlöslichkeit (NOUBACTEP ET AL. 2002). Unter den gegebenen experimentellen Bedingungen jedoch (begrenzte Pyritmenge, praktisch neutraler pH-Wert) werden aufgelöste Fe 2+-Ionen durch Luftsauerstoff zu Fe3+ aufoxidiert, welches sich in verschiedene Hydroxide (Fe(OH) 3(am) ) mit bekannten Sorptionseigenschaften für Uran (HO & MILLER 1986) umwandelt. Dies erklärt die geringe Urankonzentration im System während der ersten fünf Tage des Experiments. Nach dieser Phase lässt die Fe(OH) 3-Produktion nach und das freigesetzte Uran geht in Lösung (stationärer Zustand bei der Pyritoxidation und progressive U-Gesteinsauflösung). Die Urankonzentration steigt folglich kontinuierlich an und gleicht dem Wert des Referenzsystems am Ende des Versuchs. • Während der ersten 14 Tage des Experiments ändert sich die Urankonzentration gleichmäßig im Referenzsystem und im System mit Dolomit; danach steigt die Urankonzentration im letztgenannten System kontinuierlich bis zum Ende des Versuchs (70 Tage) an, während das Referenzsystem einen Gleichgewichtszustand nach ca. 50 Tagen erreicht. Diese Beobachtung deutet auf eine langsame Kinetik der Dolomitauflösung hin. Die Erhöhung der Urankonzentration ist eindeutig eine Folge der Dolomitauflösung. Diese bewirkt bekanntlich die Bildung von UO 22+-(CO 32-)-Spezies. Unter vergleichbaren experimentellen Bedingungen (pH 8,1) konnten B ERNHARD ET AL. (1996) das Vorhandensein 13 von ungeladenen löslichen Calcium-Uranyl-Komplexen der Formel Ca2[UO 2(CO 3)3] x 10H2O nachweisen. • Die Tatsache, dass nach 70 Tagen das System mit Pyrit eine Urankonzentration aufweist, welche sich im Referenzsystem seit zwei Wochen eingestellt hatte, zeigt, dass beide einen Gleichgewichtszustand erreicht haben. Somit ist [U] eq = 8300 µg/L (8,3 mg/L) die Gleichgewichtskonzentration des Referenzsystems unter diesen experimentellen Bedingungen, welche durch sorptive Eigenschaften des in-situ entstandenen Fe(OH) 3 erst verzögert im System mit Pyrit erreicht werden könnte. • Im System mit Dolomit beträgt Urankonzentration nach 70 Tagen 11700 µg/L (11,7 mg/L) und steigt weiter an. Einfluss der Stillegung Gängige Strategien bei der Risikoabschätzung und Grundwassersanierung bestehen u. a. darin, natürliche Prozesse mit einzubeziehen (natural attenuation) ggf. durch gezielte Unterstützung (enhanced natural attenuation) zu verstärken. Dabei spielen insbesondere mikrobiologische Prozesse beim Abbau organischen Materials und Lösungs- /Fällungsprozessen eine bedeutende Rolle. Zur Nachbildung solcher Verhältnisse wurden die oben beschriebenen Lösungen vier Monate lang nach Abschluss der Experimente (Abschaltung der Homogenisierung nach 70 Tagen) stehen gelassen. Tabelle 5 stellt die Ergebnisse ausgewählter Elemente einer ICP-MS-Analyse zusammen und vergleicht die Urankonzentration mit den Werten am Ende des belüfteten Versuchs (70 Tage). < Tabelle 5> Es zeigt sich, dass die Urankonzentration in allen Fällen um mehr als 65 % gesenkt wurde, jedoch noch deutlich über dem Grenzwert der US EPA bleibt (30 µg/L). Im Referenzsystem (Tabelle 5) sind die Pb und Ba Konzentrationen höher als in den Systemen mit Dolomit und Pyrit. Dieses Ergebnis weist darauf hin, dass Sorption und Mitfällung sich senkend auf die Blei- und Bariumkonzentration ausgewirkt haben. Die Tatsache, dass die 14 Konzentrationen mehrerer Elemente in den Lösungen am Ende der vier monatigen Stillegung größer als die des Leitungswassers ist, deutet darauf hin, dass die Durchführung solcher Versuche unter Berücksichtigung mehrerer Zielschadstoffe von besonderer Bedeutung für die Untersuchung von geochemischen Prozessen im Labormaßstab sein kann. Der Einfluss der Gesteinsbestandteile auf einzelne Zielschadstoffe kann bei der Auswertung derartiger Versuche besser diskutiert werden. Tabelle 5 zeigt z.B., dass Elemente wie Co, Zn, Rb und Sr einer höherer Konzentration im System mit Dolomit aufweisen als im System mit Pyrit und im Referenzsystem. Für das Element Barium ist die Konzentration im Referenzsystem höher. Bedeutung für die Umweltforschung Diese Arbeit hat gezeigt, dass der Einsatz von natürlichen bzw. naturnahen Wässer Vorteile bei der Untersuchung des Einflusses von HCO 3-Ionen auf die Uranfreisetzung aus natürlichen Materialien haben kann. Bei allen eingesetzten Wassertypen wurden vom Hersteller eine Vorenteisenung vorgenommen, deshalb wäre es viel sinnvoller zusätzlich natürliche Wässer unterschiedlichen Zusammensetzungen (Fe, Ca, PO 4...) einzusetzen, um unter naturnahen Bedingungen deren Einfluß zu charakterisieren. Der Einsatz von Zusatzstoffen (z.B. Apatit), die in der Lage sind, in-situ reaktive Spezies zu produzieren, ist ebenfalls ein weiterzuführendes Experiment. Die parallele Charakterisierung solchen komplexen Systemen auf die Konzentrationsänderung von mehreren Elementen kann zum besseren Verständnis mancher geochemischen Prozesse verhelfen. Schlussfolgerung Die erzielten Ergebnisse ergänzen bisherige Erkenntnisse, wonach Uranfreisetzung bzw. transport im Untergrund und in uranhaltigem Haufwerk hauptsächlich durch Sorption von Uran an Fe/Mn/Ti-Oxiden, Tonmineralen und kohlenstoffhaltigen Verbindungen inhibiert wird. Diese Ergebnisse lassen sich wie folgt zusammenfassen: 15 • Die Uranmobilisierung aus einem natürlichen Gesteinsmaterial im pH-Bereich 7 bis 9 hängt stark vom Carbonatgehalt ab. Bei einer pH-Erhöhung auf Werte über 10 spielt die Uranmitfällung (z.B. als Uranat) eine größere Rolle. Die Folge ist eine geringere Mobilisierung in 0.1 M Na 2CO 3 als im CO 2-reichen Mineralwasser (MW). • Die Präsenz von Zusätzen (Carbonatminerale und Pyrit) verzögert die Uranfreisetzung. Während die Verzögerung in Versuchen mit Dolomit sich auf die Anfangsphase beschränkt, bewirkt die Pyritzugabe eine Freisetzungsverzögerung über Wochen hinaus. Diese Beobachtung ist auf die Entstehung einer begrenzten Menge hoch sorptiver Fe(III)-Species bzw. auf die Mitfällung von Uran zurückzuführen. • Als Mobilisierungsmittel für die Untersuchung der Stärke der Festlegung von Uran in natürlichen Proben können definierte Mineralwassersorten anstelle technischer Lösungen wie Na2CO3 eingesetzt werden, da erhöhte Carbonatkonzentrationen im natürlichen pH-Bereich erzielt werden. Somit werden natürliche Verhältnisse besser nachgebildet und die Uranmitfällung als Na-Uranate deutlich verhindert. • Für die belüfteten Standversuche im Leitungswasser wurde unter den gegebenen experimentellen Bedingungen nach 70 Tagen eine Gleichgewichtsurankonzentration von 8 mg/L erzielt, die ermittelte kinetische Konstante der Uranauflösung betrug 0,78 ± 0,11 µg.h1 .m-2 (NOUBACTEP ET AL. 2004). • Der gleichzeitige Einsatz von U-Gestein und Zusatzstoffen ist für die Charakterisierung geochemischen Prozesse im Labormassstab von besonderer Bedeutung. Danksagung Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (Vorhaben: DFG-Sa 501/151) finanziert. Die Autoren danken Herrn Dipl.-Min. Holger Dienemann vom Institut für Allgemeine Ökologie und Umweltschutz der Technischer Universität Dresden für die 16 sorgfältige Durchsicht des Manuskripts. Das Manuskript profitierte deutlich von wertvollen Hinweisen zweier anonymer Gutachter. Die ICP-MS Analyse in Tabelle 5 wurden freundlichst von Herrn Dr. Klaus Simon von der Abteilung Geochemie im Geowissenschaftlichen Zentrum der Universität Göttingen durchgeführt. Literaturverzeichnis Baas-Becking, L.G.M., Kaplan, I.R., Moore, D. (1960): Limits of the natural environments in terms of pH and oxidation-reduction potentials. – J. Geol., 68: 243–284. Bain, J.G., Mayer, K.U., Blowes, D.W., Frind, E. O., Molson, J.W.H., Kahnt, R., Jenk, U. (2001): Modelling the closure-related geochemical evolution of groundwater at a former uranium mine. J. Cont. Hydrol., 52: 109-135. Bayer, P., Finkel, M., Teutsch, G. (2004): Kombinierte „Pump-and-treat“-Barrieren-Systeme, Teil I: Minimierung der Grundwasserentnahmerate durch hydraulische Zusatzmaßnahmen. – Grundwasser 9: 173–180. Bernhard, G., Geipel, G., Brendler, V., Nitsche, H. (1996): Speciation of uranium in seepage waters from a mine tailing pile studied by time-resolved laser-induced fluorescence spectroscopy (TRLFS). – Radiochim. Acta, 74: 87–91. Braithwaite, A., Livens, F.R., Richardson, S., Howe, M.T., Goulding, K.W.T. (1997): Kinetically controlled release of uranium from soils. – Eur. J. Soil Sci., 48: 661–673. Brunauer, S., Emmett, P., Teller, E. (1938): Adsorption of gases in multimolecular layers. – J. Am. Chem. Soc., 60: 309–319. Buck, R.P., Rondinini, S., Covington, A.K., Baucke, F.G.K., Brett, C.M.A., Camoes, M.F., Milton, M.J.T., Mussini, T., Naumann, R., Pratt, K.W., Spitzer, P., Wilson, G.S. (2002): Measurement of pH. Definition, standards, and procedures (IUPAC Recommendations 2002). – Pure Appl. Chem., 74: 2169–2200. 17 Catchpole, G., Kirchner, G. (1995): Restoration of groundwater contaminated by alkaline insitu leach uranium mining. in: Uranium Mining and Hydrogeology (B Merkel, S. Hurst, E.P. Löhnert & W. Struckmeier eds) GeoCongress I Verlag Sven von Loga Köln/FRG 1: 81–89. Chapman, A.N., McKinley, I.G., Franca, P.E., Shea, E.M., Smellie, A.T. (1992): The Poços de Caldas project: an introduction and summary of its implications for radioactive waste disposal. – J. Geochem. Explor., 45: 1–24. Clark, D.L., Hobart, D.E., Neu, M.P. (1995): Actinide carbonate complexes and their importance in actinide environmental chemistry. – Chem. Rev., 95: 25–48. Duff, M.C., Amrheim, C., Bertsch, P., Hunter, B.D (1997): The chemistry of uranium in evaporation pond sediment in the San Joaquin Valley, California, USA, using X-ray fluorescence and XANES techniques. – Geochim. Cosmochim. Acta, 61: 73–81. Edel, H.-G., Voigt, T. (2001): Aktive und Passive Grundwassersanierung - ein Verfahrensund Kostenvergleich. TerraTech, 1: 40–44. Fredrickson, J.K., Zachara, J.M., Kennedy, D.W., Liu, C. Duff, M.C., Gorby, A.Y., Li, W.S.M, Krupkas S.-M. (2000): Reduction of U(VI) in Goethite (a-FeOOH) suspensions by a dissimilatory metal-reducing bacterium. – Geochim. Cosmochim. Acta, 64: 3085–3098. Gatzweiler, R., Mager, D. (1995): Altlasten des Uranbergbaus. Der Sanierungsfall WISMUT. – Geowissenschaften 11: 164–172. Helling, C. (1999): Hydrochemische Untersuchungen an Müllüberlagerten Urantailings unter dem Aspekt einer effektiven Langzeitüberwachung. Wiss. Mitt. Institut für Geologie der TU Bergakademie Freiberg, Band 9, 124 S. ISSN 1433–1284. Ho, C.H., Miller, N.H. (1986): Adsorption of uranyl species from bicarbonate solution onto hematite particles. – J. Colloid Interf. Sci., 110: 165–171. Hurst, S., Schneider, P., Meinrath, G. (2002): Remediating 700 years of Mining in Saxony: A Heritage from Ore Mining. – Mine Water and the Environment, 21: 3–6. 18 Ivanovich, M., Harmon, R.S. (1992): Uranium Series Disequilibrium. Applications To Earth, Marine And Environmental Sciences. 2nd Ed. Clarendon Press, Oxford. 910 S. Junghans, M., Helling, C. (1998): Historical Mining, uranium tailings and waste disposal at one site: Can it be managed? A hydrogeological analysis. In Tailings and Mine Waste '98, Balkema, Rotterdam:, 117–126. Kaplan, D.I., Serkiz, S.M. (2001): Quantification of thorium and uranium sorption to contaminated sediments. – J. Radioanal. Nucl. Chem., 248: 529–535. Kirchheimer, F. (1959a): Über radioaktive und uranhaltige Thermalsedimente, insbesondere von Baden-Baden. – Abh. Geol. Landesaamt Baden-Württ., 3: 67 S. Kirchheimer, F. (1959b): Die Radioaktivität der Phonolite des Hegaus und das Vorkommen der Uran-Opale. – Hegau, 8: 207–218. Köber, R., Ebert, M., Schäfer, D., Dahmke, A. (2001): Kombination von Fe° und Aktivkohle in Reaktionswänden zur Sanierung komplexer Mischkontaminationen im Grundwasser. – Altlasten Spektrum, 2: 91–95. Langmuir, D. (1978): Uranium solution-mineral equilibria at low temperatures with application to sedimentary ore deposits. – Geochim. Cosmochim. Acta, 42: 547–569. Langmuir, D. (1997): Aqueous Environmental Geochemistry. Prentice Hall, 600 S. Liu, C, Zachara, J.M., Qafoku, O., McKinley, J.P., Heald, S.M. and Wang, Z. (2004): Dissolution of uranyl microprecipitates in subsurface sediments at Hanford Site, USA. Geochim. Cosmochim. Acta, 68: 4519–4537. Mackay, M.D., Cherry, J.A. (1989): Groundwater contamination: Pump-and-treat remediation. – Environ. Sci. Technol., 23: 630–636. McMurty, D.C, Elton, R.O. (1985): New approach to in-situ treatment of contaminated groundwaters. – Environ. Progr., 4/3: 168–170. 19 Meinrath, A., Schneider, P., Meinrath, G. (2003): Uranium ores and depleted uranium in the environment - with a reference to uranium in the biosphere from the Erzgebirge/Sachsen, Germany. – J. Environ. Radioact., 64: 175–193. Meinrath, G., Volke, P., Helling, C., Dudel, E.G., Merkel, B.J. (1999): Determination and Interpretation of environmental water samples contaminated by uranium mining activities. – Fresenius J. Anal. Chem., 364: 1191–1202. Merkel, B. (2002): Kinetics of Uranium Removal from Water. In “Advanced groundwater remediation” Simon, Meggyes & McDonald (Eds), Thomas Telford, ISBN 0 7277 3121 1; 195–209. Morrison, J.S, Metzler, R.D., Carpenter, E.C. (2001): Uranium precipitation in a permeable reactive barrier by progressive irreversible dissolution of zerovalent iron. – Environ. Sci. Technol., 35: 385–390. Morrison, J.S., Sprangler, R.R. (1993): Chemical barriers for controlling groundwater contamination. – Environ. Progr., 12: 175–181. Neitzel, P.L.; Schneider, P., Hurst, S. (2000): Feldversuche zur In-situ Entfernung von Uran(NAT.) und Ra-226 aus Berge- und Fluttungswässern. Freiberger Forschungshefte C482 Geoingenieurwesen (TU Bergakademie Freiberg), 196–206. Noubactep, C. (2003): Untersuchungen zur passiven in-situ-Immobilisierung von U(VI) aus Wasser. Dissertation, TU Bergakademie Freiberg, Wiss. Mitt. Institut für Geologie der TU Bergakademie Freiberg, Band 21, 140 pp, ISSN1433-1284. Noubactep, C. Meinrath, G., Volke, P., Peter, H.-J., Dietrich, P., Merkel, B. (2002): Mechanism of uranium fixation by zero valent iron: the importance of co-precipitation. In Uranium in the Aquatic Environment. Merkel, Planer-Friedrich & Wolkersdorfer (Eds); Springer Verlag, Berlin: 577–586. Noubactep, C., Meinrath, G., Dietrich, P., Merkel, B. (2003) Mitigating Uranium in Ground Water: Prospects and Limitations. – Environ. Sci. Technol. 37: 4304–4308. 20 Noubactep, C., Sonnefeld, J., Sauter, M. (2004): Investigation of some Aspects the Uranium Release from a Natural Rock under oxic Natural near Conditions. – Submitted to Environ. Sci. Technol. Nyer, E.K., Kidd, D.F., Palmer, P.L., Crossman, T.L., Fam, S., Johns, II F.J., Boettcher, G. S., Suthersan, S.S. (1996): In Situ Treatment Technology. CRC Press, Boca Raton, 329 pp. Ohlendorf, F. (1999): Entstehung der radiologischen Altlasten des Bergbaus. In Bundesamt für Strahlenschutz & Sächsisches Staatministerium für Umwelt und Landwirtschaft (Hrsg.): Leitfaden Uranbergbau Sanierung Anlage 2. Peters, C. W. (1978): Exploration and Mining Geology. John Wiley & Sons, New York, 696 pp. Posey-Dowty, J., Axtmann, E., Crerar, D., Borcsik, M., Ronk, A., Woods, W. (1987): Dissolution rate of uraninite and uranium roll-front ores. – Econ. Geol., 82: 184–194. Read, D. (1992): Geochemical modelling of uranium redistribution in the Osamu Utsumi mine, Pocos de Caldas. – J. Geochem. Explor., 45: 503–520. Read, D., Lawless, R.J., Sims, R.J., Butter, K.R. (1993): The migration of uranium into peat rich soils at Broubster, Caithness, Scottland, U.K. – J. Cont. Hydrol., 14: 277–289. Reiche, P. (1950): A survey of weathering processes and products. University of New Mexico Press; Albuquerque, 95 pp. Reynolds, R.L., Goldhaber, M.B. (1978). Origin of a south Texas roll-type uranium deposit: I. Alteration of iron-titanium oxide minerals. Econ. Geol., 73: 1677– 1689. Romero, L., Neretnieks, I., Moreno, L. (1992): Movement of the redox front at the Osamu Utsumi uranium mine, Poços de Caldas, Brazil. – J. Geochem. Explor., 45: 471– 501. Schneider, P., Neitzel, P.L, Osenbrück, K., Hurst, S. (2000): Methoden zur Behandlung von Flutungswässer im Bergbau. Wasser und Abfall, 12: 20–24. 21 Smirnov, I.P., Matveev, A.A. (2001): Scientific Principles, Technology, and Equipment for Hydrometallurgical Processing of Uranium and Complex Ores. Atomic Energy, 91: 815– 823. U.S. EPA (2002): Field applications of in situ remediation technologies: permeable reactive barriers. Report prepared by Environment Management Support, Inc. under contract 68W-00-084, Work Assignment 006 with the U.S. EPA; available at: http://www.rtdf.org/. Williamson, M.A., J.D. Rimstidt, J.D. (1994): The kinetics and electrochemical rate- determining step of aqueous pyrite oxidation. – Geochim. Cosmochim. Acta, 58: 5443– 5454. Zanger, H. (2002): Heilwasser ... das natürlichste aller Naturheilmittel. Ralser & Heller, Erlangen. 307 S. 22 Tab 1: Eingesetzte Partikelgrößen der beiden uranhaltigen Gesteine (U-Gesteine) und deren spezifischen Oberfläche (S i , m2/g). S2 stellt die Oberfläche des Gesteins mit ca. 1.2 % U dar; (n.b. = nicht bestimmt). Korngröße 0.063 - 0.125 0.250 - 0.315 0.315 - 0.630 0.630 - 1.0 (mm) S1 (m2/g) 4.66 3.53 n.b. n.b. S2 (m2/g) 2.03 0.64 2.06 1.81 Code d1 d2 d3 d4 zweiten U- 23 Tab. 2: HCO 3-Gehalt und simulierte Bedingungen der eingesetzten Wässer (n.b. = nicht bestimmt). MW ist ein kommerzielles Mineralwasser mit 99 mg/L Ca. Wasser Code [HCO 3-] Sim. Bedingungen Beispiel (mg/L) (Reagent) Deionisat DW n.b. HCO3-armes Wasser Regenwasser Leitung LW 89 gängiges Grundwasser Sickerndes RegenW. Mineral MW 1854 HCO3-reiches Wasser Moorgrundwasser 24 Tab. 3: Absolute (PU) und relative (Prel) prozentuale Änderung der Uranfreisetzung in verschiedenen Lösungen als Funktion der Gesteinspartikelgröße. Die maximale Laugungsrate (100%) wurde in jedem Fall der kleinsten Partikelgröße (d1) zugewiesen. Diese Versuche wurden mit 40 g/L U-Gestein in Standversuchen für zwei Wochen durchgeführt. Lösung und prozentuale Laugungsrate (%) LW Na2CO3 H2SO 4 Prel PU Prel PU Prel PU d1 100 13 100 17 100 43 d2 81 10 53 9 68 29 d3 78 10 40 7 70 30 d4 61 8 28 5 47 20 25 Tab. 4: Änderung des pH-Wertes und der Uranlaugungsrate als Funktion der Laugungslösung für zwei Wochen. pHi = Anfangs-pH; pH f = End-pH und P U = perzentuale Uranlaugungsrate (%). [Si] ist die Siliziumlaugungsrate von einer reinen SiO2-Phase für 72 Stunden in der jeweiligen Lösung. Reagent pHi pHf ∆pH [U] PU [Si] (µg/L) (%) (mM/L) DW 5,86 7,82 1,96 583 0,3 0,12 TW 8,43 7,77 -0,66 3854 2,0 2,09 Na2CO3 11,47 11,20 -0,27 4634 2,2 10,15 MW 6,87 6,92 0,05 10028 5,0 - H2SO 4 1,09 1,24 0,15 51843 23,9 - 26 Tab. 5: Ergebnisse der ICP-MS Analyse der Proben nach vier Monaten Standzeit nach Ende des belüfteten Experiments (70 Tagen). PU (%) ist die prozentuale wässrigen Urankonzentration nach der Standzeit bezogen auf die Konzentration nach 70 Tagen. Alle Angaben in µg/L, n.n. = nicht nachweisbar. Die Zahlen im Klammern (Spalte Element) geben das verwendete Isotop der Messung an. Die Konzentrationen sind aufgrund der Isotopenhäufigkeit errechnet worden. Element LW Referenz Dolomit Pyrit Co (59) 0,76 6,92 6,72 7,46 Ni (60) 6,19 39 n.n. n.n. Cu (63) 2,12 n.n. n.n. n.n. Zn (66) 21 61 48,3 78 Rb (85) 1,94 29 n.n. 33 Sr (88) - 960 932 1123 Sb (121) 0,17 6,54 6,61 7,05 Ba (137) 46 90 80 71,57 Pb (208) 0,46 14,5 8,28 n.n. U (238) 0,97 2918 3624 2765 UEnde 0,97 8316 11770 8280 P U (%) 100 35,1 30,8 33,4 27 12000 Uran / [µg/L] 9000 11 % Gestein: 8 g/L Versuchsdauer: 14 Tage Partikelgröße: d2 69 % 19.3 42 % 6000 8.9 7.4 3000 11 % 0 1.1 DW LW Na2CO3 MW Laugungslösung Abb. 1: Urankonzentration als Funktion der Laugungslösung für eine Gesteinpartikelgröße von 0.250 - 0.315 mm (d2) und eine Versuchsdauer von 14 Tagen. Das Experiment wurde dreifach durchgeführt, Die Fehlerbalken geben die Standardabweichungen (%) an. Die Werte in den Balken geben die prozentualen relativen Laugungsrate an, bezogen auf die Laugungsrate in H 2SO4 (0.1 M). 28 7500 PO2 = 20 % PO2 = 2 % Gestein: 10 g/L Uran / [µg/L] 6000 4500 3000 1500 0 0 150 300 450 600 750 Zeit / [Stunden] Abb. 2: Vergleich der zeitlichen Änderung der Urankonzentration an der Luft (P O2 = 20 %; PCO2 = 0,035 %) und in einer Glove-Box (PO2 = 2 %). 29 12500 Referenz (Gestein) Gestein + Dolomit Gestein + Pyrit Uran / [µg/L] 10000 7500 5000 PCO2 = 0.035 % [Gestein] = 10 g/L [Zuschlagstoff] = 15 g/L 2500 0 0 15 30 45 60 75 Zeit / [Tage] Abb. 3: Zeitlicher Verlauf der Urankonzentrationsänderung als Funktion der Zusatzstoffe in belüfteten Batchversuchen für 70 Tage. P CO2 ist der Partialdruck von CO 2.
https://openalex.org/W3119633061	https://journal.ldubgd.edu.ua/index.php/Visnuk/article/download/2012/1926	Ukrainian	null	DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM»	Vìsnik Lʹvìvsʹkogo deržavnogo unìversitetu bezpeki žittêdìâlʹnostì	2,020	cc-by	7,922	DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Introduction. Formalized research of transport systems, improvement of terminology and classification tools is the way to gradually deepen and fully organize knowledge about transport in its broad sense, which should become the basis for finding sufficient means to increase economic activity. But, the problem is that the concept of “transport system” has not been clearly defined and is still contradictable and debatable. Purpose. The research aims to critically compare the concepts of “transport” and “transport system”, to provide a clear definition and explanation of the term “transport system”. Methods. The research has based on general principles of systems thinking and analysis. Systematized defined motivated flow of things or people that create cargo or passengers on the vehicle, as well as a logistics structure that provides transportation processes. The boundaries of economic activity defined the borders of the transport system. Methods. The research has based on general principles of systems thinking and analysis. Systematized defined motivated flow of things or people that create cargo or passengers on the vehicle, as well as a logistics structure that provides transportation processes. The boundaries of economic activity defined the borders of the transport system. Results. Studies on the development of transport quite often interpret “transport” as an infrastructure sector of the economy. Along with such terms as “transport”, “transport industry” and “transport system”, which used as synonyms, researchers mention “transport complex” and “transport infrastructure”. Of course, there should be a scientific rationale for using different terms for defining the same concept. Results. Studies on the development of transport quite often interpret “transport” as an infrastructure sector of the economy. Along with such terms as “transport”, “transport industry” and “transport system”, which used as synonyms, researchers mention “transport complex” and “transport infrastructure”. Of course, there should be a scientific rationale for using different terms for defining the same concept. Conclusions. The researchers concluded that the concept of transportation system expressed by different language means and by using different wording as it is a reflection of the researcher’s description of objects, links between the targets and the environment through the prism of the task to find the most effective ways of moving/ transporting things and people towards each other. In additional materials related to the field of the study and the particular task to be com- pleted or problem to be solved, we should provide all necessary clarifications and explanations. ОЗНАЧУВАНІСТЬ І ЗМІСТ ПОНЯТТЯ ТРАНСПОРТНОЇ СИСТЕМИ Проблема. Формалізоване дослідження транспортних систем, удосконалення термінології та кла- сифікаційного інструментарію — це шлях до планомірного поглиблення і цілковитого упорядкування знань про транспорт в широкому системному сенсі, що має стати основою для пошуку надійних засобів підвищення еко- номічної активності людства. Але на цьому шляху існує проблема: досі немає однозначного й несуперечливого тлумачення поняття «транспортна система». Мета дослідження — критично зіставити поняття «транспорт» і «транспортна система», намагаючись виснувати якнайточніше формальне означення чи змістовно якнайточніший наратив поняття «транспортна система». Мета дослідження — критично зіставити поняття «транспорт» і «транспортна система», намагаючись виснувати якнайточніше формальне означення чи змістовно якнайточніший наратив поняття «транспортна система». Методи дослідження. Дослідження спирається на загальні принципи системного мислення й аналізу. Си- стемотворними визнано мотивований потік речей чи людей, що на облавку транспортного засобу стають ванта- жами чи пасажирами, а також логістичну надбудову, що обслуговує процеси транспортування. Межі транспорт- ї ф і бі Методи дослідження. Дослідження спирається на загальні принципи системного мислення й аналізу. Си- стемотворними визнано мотивований потік речей чи людей, що на облавку транспортного засобу стають ванта- жами чи пасажирами, а також логістичну надбудову, що обслуговує процеси транспортування. Межі транспорт- ної системи окреслено межами сфери економічного обігу. Результати дослідження. З’ясовано, що у дослідженнях, присвячених питанням розвитку транспорту, його (транспорт) доволі часто трактують як інфраструктурну галузь народного господарства, і поряд з поняттями «транспорт», «транспортна галузь», «транспортна система», що їх часто беззастережно вважають синонімами, використовують ще й поняття «транспортний комплекс», «транспортна інфраструктура». Звісно, не може наука так без раціонального сенсу оперувати різними термінами на позначення одного і того ж. Висновки. Обґрунтовано, що транспортну систему доцільно загалом трактувати як цілісне відображення засобами мови спостерігача-дослідника множини об’єктів у всій повноті їх зв’язків між собою і з довкіллям в сенсах розв’язуваної спостерігачем задачі/проблеми забезпечення ефективної мобільності цінностей і людей назустріч одне одному. А всякі необхідні уточнення й доповнення є сенс відображати в додаткових наративах, відповідних конкретним розв’язуваним транспортним задачам/проблемам. Ключові слова: транспорт, транспортна система, зміст, означення, наратив. ТРАНСПОРТНІ ТЕХНОЛОГІЇ Вісник ЛДУБЖД УБЖД versity of Life Safety ІSSN 2078-4643 (print), ІSSN 2708-1389 (online) https://journal.ldubgd.edu.ua/index.php/Visnuk/ УДК 338.47+656.078 DOI: 10.32447/20784643.22.2020.09 ТРАНСПОРТНІ ТЕХНОЛОГІЇ УБЖД versity of Life Safety ІSSN 2078-4643 (print), ІSSN 2708-1389 (online) https://journal.ldubgd.edu.ua/index.php/Visnuk/ УДК 338.47+656.078 DOI: 10.32447/20784643.22.2020.09 ТРАНСПОРТНІ ТЕХНОЛОГІЇ ТРАНСПОРТНІ ТЕХНОЛОГІЇ Bulletin of Lviv State University of Life Safety П.М. Гащук, Ю.С. Тимошенко Львівський державний університет безпеки життєдіяльності П.М. Гащук, Ю.С. Тимошенко Львівський державний університет безпеки життєдіяльності DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Keywords: transport, transport system, content, definition, description. Вісник ЛДУБЖД, №22, 2020 66 1980…1990-х роках (і навіть тепер про нього чутно)? В [8] цей утямок означено як «сукупність шляхів сполучення, перевізних засобів, технічних пристроїв і механізмів, засобів управління та зв’язку, облаштувань усіх видів транспорту, об’єд- наних системою технологічних, технічних, інфор- маційних, правових і економічних відносин, що за- безпечують задоволення потреб народного госпо- дарства в перевезенні вантажів і пасажирів». Але чому «єдина транспортна система» відповідно до наведеного означення не є просто «транспортною системою»? Може в самому терміні «система» насправді закладена «єдиність» (чи краще — «єдність»). А як співвідносити утямок «транс- портна система» і утямок «транспортно-техно- логічна система», яким послуговуються, приміром, у процесі дослідження ефективності інтермодаль- них контейнерних перевезень [9]? Мотивація. Коли який-небудь термін набу- ває статусу наукового, то з’являється ілюзія, що ми вже десь поруч істини. Таку ілюзію навіюють, зо- крема, широко вживані в теорії транспортних про- цесів (і не тільки там) терміни «транспорт» і «транс- портна система». Звісно, досконалим можна вва- жати термін, настільки прозорий, що крізь нього легко бачити те, що він номінує. Однак саме терміни «транспорт» і «транспортна система», вияв- ляється, аж ніяк до прозорих не належать. Відтак важко окреслити, що ж позначають і терміни «національна транспортна система» чи «світова транспортна система» [1], якими доводиться послу- говуватись у наш час нестримної глобалізації. У дослідженнях, присвячених питанням ро- звитку транспорту, його (транспорт) доволі часто трактують як інфраструктурну галузь народного господарства, і поряд з утямками (вважатимемо далі незвичний термін «утямок» синонімом сумнівно українського терміну «поняття») «транспорт», «транспортна галузь», «транспортна система», що їх часто беззастережно вважають синонімами, вико- ристовують ще й утямки «транспортний комплекс» чи/та «транспортна інфраструктура». Звісно, не може наука розмовляти так «неохайно» і не може так без сенсу розтринькувати терміни. Складається враження, що ніби й здога- дуємося що таке транспорт і транспортна система, але якщо спробувати точно означити чи описати ці утямки, то виникають немалі труднощі. Здава- лося б транспорт — це і є система (або й система транспортних систем), але коли треба означити власне транспортну систему як таку, то транспорт (повністю чи частинно) ніби має правити за її структурний елемент (об’єкт). Формалізоване до- слідження структурованості транспортних си- стем, удосконалення термінології та кла- сифікаційного інструментарію — це шлях до пла- номірного поглиблення і цілковитого упорядку- вання знань про транспорт в широкому систем- ному сенсі, що має стати основою для пошуку надійних засобів підвищення ефективності госпо- дарської активності людства. Bulletin of Lviv State University of Life Safety, №22, 2020 DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Поняття «система», на відміну від понять «об’єкт» і «річ», відображає не щось окремішнє й неподільне, а су- перечливу єдність «багато чого» та «чогось єдиного». І аби яка-небудь сукупність стала си- стемою, має з’явитися системотворний елемент (чинник, компонент, субстрат). Вважатимемо си- стемотворними мотивований потік речей чи лю- дей, що на облавку транспортного засобу стають вантажами чи пасажирами, а поряд з цим також і логістичну надбудову. Системотворний чинник, звісно, не завжди може визнаватись складовим елементом системи. До прикладу, автомобіль — технічна система, а потоки споживаних енерго- носіїв — його системотворні чинники (завдяки їм нагромадження вузлів і агрегатів оживає як си- стема). А водій надає системі цілеспрямованості. чогось; інша річ — бачимо ми там, куди спря- мували пізнавальний погляд, систему чи не ба- чимо, хочемо бачити чи байдуже. Отже, не запе- речуючи об’єктивність як таку, все-таки можна дозволити собі бачити будь-де хоч-яку систему — «по-різному», зокрема й «по-своєму». Поняття «система», на відміну від понять «об’єкт» і «річ», відображає не щось окремішнє й неподільне, а су- перечливу єдність «багато чого» та «чогось єдиного». І аби яка-небудь сукупність стала си- стемою, має з’явитися системотворний елемент (чинник, компонент, субстрат). Вважатимемо си- стемотворними мотивований потік речей чи лю- дей, що на облавку транспортного засобу стають вантажами чи пасажирами, а поряд з цим також і логістичну надбудову. Системотворний чинник, звісно, не завжди може визнаватись складовим елементом системи. До прикладу, автомобіль — технічна система, а потоки споживаних енерго- носіїв — його системотворні чинники (завдяки їм нагромадження вузлів і агрегатів оживає як си- стема). А водій надає системі цілеспрямованості. Хай P — певна властивість, R — деяке відношення (деякий стосунок; для Берталанфі — це зв’язок), M — деяка множина предметів. Якщо на множині предметів M простежується яке-небудь відношення R , то це ще не дає підстав вважати, що M є системою. Предмети з M ра- зом будуть визнані системою, якщо відстежувати- меться відношення, яке цікавить саме нас, тобто відношення з певною (заданою) властивістю P . Тож ніби відразу можна стверджувати, що еле- ментом-предметом транспортної системи є транс- порт. Але, з іншого боку, транспорт можна тлу- мачити як щось принципово загальніше і об’ємніше за транспортну систему. А щось за- гальніше й об’ємніше позначають часто як транс- портний комплекс. Та загалом доведеться визнати, що в широкому сенсі транспорт — це все-таки те, що будучи залученим в систему, пе- ретворює її на транспортну систему. Транспорт існує сам по собі як щось безсистемне, і йому не слід надавати різних вузьких значень. DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Часом транспортну систему світу трактують як сукупність внутрішньоконтинентальних та між- континентальних транспортних систем, що залуча- ють залізничний, автомобільний, трубопровідний, річковий, морський та повітряний різновиди тран- спорту [2]. Тут ненароком поняття «сукупність» ототожнено з поняттям «система», а поняття «тра- нспорт» ненароком стало засобом означення тран- спортної системи — тож не збагнути чи транспорт є частиною транспортної системи, чи все-таки на- впаки: транспорт — сфера виробництва, що послу- говується транспортною системою. Мета дослідження — зіставити змістом утямки «транспорт» і «транспортна система», сподіваючись виснувати більш точне формальне означення чи більш точний змістовний наратив утямку «транспортна система». Цікаво також, що мав би номінувати термін «єдина транспортна си- стема»? А чи взагалі він потрібний? В транспортній системі інколи бачать просто територіальне поєднання різних видів транспорту, що спільно взаємодіючи, якнайповніше «задоволь- няють потреби народного господарства та насе- лення в перевезеннях вантажів та пасажирів» [3]. Транспортну систему світу також розглядають як сукупність (!) усіх видів транспорту та ланок транспортного процесу на всіх рівнях: національ- ному, міжнародному, міжконтинентальному, світовому [4]. Транспортну систему означують ще як поєднання усіх видів транспорту та засобів (!), що супроводжують його функціювання [5]. А от в [6] задекларовано таке означення: «всі шляхи спо- лучення, транспортні підприємства і транспортні засоби» сукупно «утворюють світову транспортну систему, у рамках якої взаємодіють окремі види транспорту, країни та регіони». Утямок (поняття) системи. Доводиться ро- зрізняти два розуміння системності — залежно від визнання того, властива цілісність геть усім-усім об’єктам, чи не завжди і не повсюдно. Одні вважа- ють, що системність притаманна усім об’єктам, по- заяк вона властива природній і соціальній дійс- ності, позаяк вона об’єктивна. Інші притриму- ються думки, що не всі сукупності існують як си- стеми, бо ж об’єктивно співіснують і неорганізо- вані сукупності, бо ж несистемним є хаос. В цьому другому випадку системність — не об’єктивна вла- стивість Світу, а лише спосіб його бачення. Гаразд, цілком можна погодитись: усе в Світі — системне, в Світі нема нічого окремішньо зайвого чи несуттєвого, все виникало чомусь і для Як не згадати утямок «єдина транспортна си- стема» [7, 8], яким нібито дуже плідно оперували у Bulletin of Lviv State University of Life Safety, №22, 2020 67 чогось; інша річ — бачимо ми там, куди спря- мували пізнавальний погляд, систему чи не ба- чимо, хочемо бачити чи байдуже. Отже, не запе- речуючи об’єктивність як таку, все-таки можна дозволити собі бачити будь-де хоч-яку систему — «по-різному», зокрема й «по-своєму». DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Суспільне виробництво й транспорт. Транспорт — активний учасник процесу опе- рування обмеженими ресурсами в системі народ- ної господарки — домашньої, національної, світо- вої. Аби унаочнити побудову народної госпо- дарки (економіки), покладаються на спрощені мо- дельні уявлення. Зокрема часто модель економіки подають [12, 13] у формі діаграми кругообігу по- токів благ і цінностей, рис. 1. Основним припу- щенням в такому разі є те, що народна господарка звідна до взаємодії двох суб’єктів економічної діяльності: підприємств (сфера виробництва) та домашніх (сімейних) господарств (сфера спожи- вання). Безпосереднє дотикання інтересів цих суб’єктів відбувається у сфері обігу, яку форму- ють ринок продуктів виробництва, ринок чин- ників виробництва та держава-уряд. Важливим учасником сфери обігу зазвичай є й ринок фінансів [14]. Звісно, реально кругообіг є склад- нішим — з додатковими зв’язками: приміром, уряд може ставати позичальником у разі бюджет- ного дефіциту і тому мати стосунки з ринком фінансів; впливовим може виявитися іноземний сектор економіки тощо. Тож скоріш за все існує дещо таке, що по- трібно називати транспортною системою і що справді є транспортною системою. Не дарма ж по- всюдно активно культивують утямок (поняття) «транспортна система», хоча часто й без нама- гання переконливо розкрити нам його зміст. А ще розрізняють транспортно-технологічні та транс- портно-логістичні системи. Але інколи взагалі не переймаються означенням чи наративом транс- порту і транспортної чи похідної від неї системи, незважаючи на те, що досліджують власне транс- портні операції [10]. В цьому випадку виявилося достатнім вирізнити ключові оптимізаційні за- дачі, що їх доводиться розв’язувати стосовно транспорту й транспортної системи. Часом, транспорт — це однозначно галузь економіки [11], що також можна вважати цілком слушним в усталеному дискурсі. В [7], приміром, задекларо- вано: «Транспорт є сукупність засобів переве- зення, шляхів сполучення, засобів керування і зв’язку, а також різних технічних пристроїв, ме- ханізмів і споруд, що забезпечують його роботу». Але «сукупність» не тотожна ні «системі», ні «елементу», за допомогою якого вибудовується цілісність. Виглядає так, що транспорт і транс- портна система — ці цілком різні речі. Узагальнено утямок системи можна тлу- мачити так (У. Росс Ешбі, У. Черчмен, Р. Акофф). Вісник ЛДУБЖД, №22, 2020 Вісник ЛДУБЖД, №22, 2020 68 Рисунок 1 – Діаграма кругообігу продуктів/послуг і доходів/витрат в народній господарці Рисунок 1 – Діаграма кругообігу продуктів/послуг і доходів/витрат в народній господарці Усе, що залучається у виробництво, назива- ють чинниками виробництва або економічними (господарськими) ресурсами [12, 15, 16]. Bulletin of Lviv State University of Life Safety, №22, 2020 DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Ресурси поділяють на матеріяльні, до яких відносять землю (ґрунт та інші природні блага) і капітал (головне майно чи засоби виробництва — будівлі, вер- стати…), та людські, до яких відносять працю (ро- бочу потенцію, робочу силу) і ще окремо — підприємливість (підприємницький хист та підприємницьку наснагу). Чинники виробництва «перетікають» від домашніх господарств до підприємств, а товари й послуги — від підприємств до домашніх господарств. Проти- лежно спрямовані потоки грошових засобів. Під чистими слід розуміти податки, від яких відняли трансферні платежі домогосподарствам та субсидії підприємствам з боку держави (уряду). Зауважмо, як фірми, так і домашні господарства є як продав- цями, так і покупцями, а тому всі активно оперу- ють потоками фінансів. портні засоби — мобільні машини (чи напірні си- стеми, якщо мова, приміром, про нафто(газо)гони), системи зв’язку, платіжні системи абощо. В си- стему матеріяльних потоків потрапляють людські ресурси, а отже поряд з вантажними транспорт- ними засобами в народну господарку залучені та- кож і пасажирські. Транспортна активність прони- зує сферу обігу, продуктивно сполучаючи сфери виробництва й споживання. Повчальним можна вважати історичний факт прояву активності: напередодні Першої світової війни найбільша на той час Британська імперія володіла майже чвертю світового сухо- долу і панувала над такою ж часткою світової людності; а стало це можливим завдяки цілкови- тому контролю над світовими морськими шля- хами та над міжнародною телеграфною мережею. Переконливою є також сентенція: «Три речі роблять націю великою і заможною: родючий ґрунт, діяльна промисловість, легкість пересу- вання людей і товарів» (Френсіс Бекон). Провідниками усіх потоків є інфраструк- турні утворення, а носіями цих потоків є транс- 69 Bulletin of Lviv State University of Life Safety, №22, 2020 стемним утворенням сутнісно і тому повинен вив- чатись не інакше як системно. Очевидно, що і сфера матеріяльного вироб- ництва, і сфера матеріяльного споживання техно- логічно послуговується кожна своїм транспортом. Понад те: чи сфера виробництва, чи сфера спожи- вання без транспорту, який доречно називати про- мисловим чи технологічним, у наш час обов’яз- ково виродиться, перестане існувати як така. Тож технологічний транспорт є підстави трактувати або як самостійну систему, підпорядковану чи ви- робничій, чи споживчій сфері, або ж вважати окре- мим елементом такої собі чи виробничої, чи спо- живчої системи. Тобто його нема жодного сенсу без застережень відносити до транспортної си- стеми у загальному сенсі. Звісно, загальна теорія систем «дозволяє», як зазначалося, кожному до- сліднику бачити «свою» систему. Але для того, аби технологічний транспорт бачити належним загаль- ній транспортній системі, потрібні особливі неоче- видні підстави. DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Тож, аби вирізнити справді транс- портну систему, треба звернути увагу на засоби ак- тивації сфери обігу, де власне здійснюється транс- портне виробництво (!). Але термін «транспорт» має надто багато зна- чень [8, 19], і тому ним оперувати вкрай непросто. А наведене означення є надто загальним і уникає прямої вказівки на транспорт саме як на систему. Транспорт чи якусь його частину можна було б без- застережно вважати транспортною системою, якщо б його чи її вдалося якось несуперечливо виокрем- ити як дещо цілісне й цілком особливе. Теорія транспортних систем як наукова дисципліна, що розробляє методологічні прин- ципи дослідження такого штибу систем, покли- кана дати єдине загальнонаукове означення ос- новних робочих понять, включаючи й поняття власне транспортної системи. Проте, незважаючи на виняткову важливість цього поняття, досі не винайдене єдине загально визнане його озна- чення. Транспортна система постійно ніби виско- взує з-під пізнавального інструменту дослідника. У декого поняття «транспортний комплекс» відображає сукупність декількох галузей еконо- міки, призначених для задоволення потреб суспі- льства у перевезеннях вантажів та пасажирів. Він ніби об’єднує: рухомий склад, що здійснює про- цес переміщення; транспортну інфраструктуру; транспортне машинобудування; транспортне бу- дівництво; логістику перевезень. Тож здається, що транспортний комплекс охоплює галузі, які за- безпечують функціювання транспорту, але при цьому можуть залишатись не пов’язаними між со- бою ні технологічно, ні організаційно. Тож цілком логічно транспорт ділити на дві групи [17]: 1) внутрішній транспорт чи виробни- чої сфери, чи сфери споживання — промисловий (технологічний), що задовольняє технологічні по- треби, та 2) транспорт зовнішній, що обслуговує сферу обігу. Щоправда, інколи, окремо вирізняючи магістральний транспорт, власне про- мисловий транспорт при цьому поділяють на внутрішній та зовнішній [8, 18]. До прикладу, внутрішній промисловий транспорт, як вважають, має переймати на себе цехові та міжцехові пере- везення всередині підприємства, а зовнішній про- мисловий транспорт натомість покликаний пере- давати потік продукції з виробництва на магістральний транспорт, чи/та доправляти сиро- вину з магістрального транспорту на підприєм- ство. Подібно, потрібно було б розрізняти внутрішній та зовнішній промислові (техно- логічні) транспорти також і сфери споживання. Поняття «транспортна система» і «транспо- ртний комплекс» трактують і як синоніми, і як іє- рархічну підпорядкованість штибу «система — підсистема». DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Приміром, існує твердження, що «… транспортний комплекс — це цілеспрямована пі- дсистема єдиної транспортної системи країни, яка сформована на основі інтеграції всіх універсаль- них засобів виробництва транспортної продукції з урахуванням спільної мети, взаємного допов- нення, взаємозамінності та єдиного керівництва їх функціонуванням та розвитком, яка забезпечує повне, своєчасне та якісне задоволення транспор- тних потреб суспільства з мінімально можливими сукупними суспільними витратами для даного рі- вня розвитку виробничих сил країни». Ознаки системності й означуваність транс- порту й транспортної системи. Транспорт — ви- сокого рівня організаційно-економічна і техніко- технологічна сфера людської діяльності, що, однак, є складовою частиною сфери діяльності ще вищого рівня — народної господарки (національної, а відтак і світової). Тому дослідження закономірно- стей розвитку і принципів ефективної експлуатації транспорту часто не може бути належно плідним, якщо в процесі цього не виходити за рамки транс- порту, якщо не розглядати проблеми транспорту з позицій народного господарства. Існують об’єкти, дослідження яких можна провадити в методологіч- ному сенсі цілком по-різному, зокрема й спираю- чись на системні засади. Натомість транспорт є си- За давньокитайським розумінням: пра- вильно назвати — значить правильно зрозуміти. А зворотно, мабуть, теж правильно: зрозумівши, легко назвати. Термінологічна точність — надзвичайно важливий аспект розуміння. Задля цього не можна абияк сіяти терміни. Під транспортом, як здається, доречно ро- зуміти все, що хоч якось пов’язане з транспортуван- ням, але наполягати на його системності формальних підстав не дуже багато, рис. 2. Транспорт — умовно Вісник ЛДУБЖД, №22, 2020 70 розпізнаваний сектор господарювання, покликаний забезпечувати нормальне функціювання і розвиток усього народного господарства (національної еко- номіки). Він поєднує, зокрема, транспортобудівну (приміром, автомобілебудівну) і транспортно- будівельну індустрії, транспортну мережу, основне транспортне, вторинне й допоміжне виробництва. Функцією транспортобудівної і транспортно- будівельної індустрії є виготовлення найрізно- манітніших (відповідно до унормованого типажу) транспортних засобів, будівництво шляхопроводів і пов’язаних з ними споруд... Але автомобілебуду- вання й дорожнє будівництво навряд чи можна без- посередньо пов’язувати з процесом транспорту- вання. Натомість інфраструктура інституційна, соціальна, логістична самі по собі є самостійними системами і повністю в транспорт не вбудовані. мування в належному стані рухомого складу (ут- римування рухомого парку на належному рівні готовності і якісне його зберігання). Транспортна інфраструктура покликана створювати і утри- мувати на прийнятному рівні умови для пересу- вання транспортних засобів. Основне призна- чення допоміжного виробництва (належного до транспортного виробництва та технічної інфра- структури, але не позначеного на рис. 2) — відновлювати рухомий склад та шляхи сполу- чення. Але транспортний комплекс також нема твердих підстав назвати транспортною системою. Л і Логічним виглядає означення: транспортна система (англ. Bulletin of Lviv State University of Life Safety, №22, 2020 DEFINITION AND CONTENT OF THE CONCEPT «TRANSPORT SYSTEM» Transport System) — це цілісно окреслене поєднання транспортних засобів, транспортних підприємств, транспортної інфра- структури, транспортної управлінської надбу- Рисунок 2 – Структурування автомобільного транспорту Рисунок 2 – Структурування автомобільного транспорту Все решта можна умовно назвати хіба що транспортною структурою, бо знову-таки нема підстав його ототожнювати з транспортною си- стемою. Певна структурованість далеко ще не означає системність. До того ж, капітальний ре- монт транспортних засобів до транспортного про- цесу безпосередньо ніяк не дотичний. дови... Але під таке означення краще підпадає транспортний комплекс. дови... Але під таке означення краще підпадає транспортний комплекс. За транспортні засоби зазвичай правлять: конвеєри, трубопроводи, ескалатори, ліфти, еле- ватори, вантажопідіймальні крани, канатні ви- тяги, фунікулери, плоти, судна, баржі, рухомі хідники (тротуари), автонавантажувачі, електро- кари, велосипеди, мотоцикли, автомобілі, авто- буси, тролейбуси, трамваї, поїзди, аеростати, літаки, вертольоти, гвинтокрили, ракети тощо. Але чи всі вони належні транспортній системі? А чи належні транспортній системі транспортні підприємства, зрештою? Тож залишається звернути увагу суто на те, що є сенс назвати транспортним комплексом, див. рис. 2. Комплекс (від лат. complexus — поєдна- ння, зв’язок) — сукупність предметів чи явищ, що становлять в певному сенсі єдине ціле (що пов’язані загальним задумом та сповідують спільну ціль). Це ніби система, але й не система в потрібному нам сенсі. Інфраструктура поєднує в собі перш за все транспортну мережу (дороги, залізничні колії, по- вітряні коридори, канали, трубопроводи, мости, тунелі, водні шляхи тощо), а також транспортні Функція транспортного виробництва — безпосереднє здійснення перевезень і підтри- 71 Bulletin of Lviv State University of Life Safety, №22, 2020 вузли й термінали, де проводиться переванта- ження вантажу або відбувається пересадка паса- жирів з одного виду транспорту на інший (аеро- порти, залізничні станції, автобусні зупинки, порти тощо). виготовлені на автомобілебудівному заводі, не належать транспортній системі, оскільки є тільки виробами (товаром) і не перебрали на себе жод- них транспортувальних функцій. Тож автомобіле- будівний завод належить машинобудівній галузі, але не транспортній системі; як і підприємства, що займаються будівництвом доріг і транспорт- них споруд. Управлінська надбудова — це зазвичай логістична система. Свого часу відома логістична ор- ганізація — Американське товариство інженерів- логістів запропонувала приблизно таке означення логістики: логістика — це мистецтво й наука ке- рування, техніка та технічні прийоми й методи пла- нування постачання та застосування засобів пе- реміщення для реалізації операцій заради досягнення поставленої мети. Логістична діяльність зазвичай охоплює транспортування, складування, обробку вантажів, захисне пакування, контроль і облік усіля- ких запасів, вибір місця розміщення виробництв і комірних центрів, формування замовлень на вироб- ництво продукції, прогнозування попиту, маркетинг і обслуговування споживачів. Тож логістична си- стема насправді підпорядковує собі транспортну си- стему (як водій підпорядковує автомобіль), а не є її складовою частиною, її елементом. Вона, зрештою, може трактуватись як елемент транспортно- логістичної чи логістично-транспортної системи. ру Зрештою, дати цілковито строге формальне означення поняття транспортної системи, мабуть, просто неможливо — власне настільки особливим є цей об’єкт пізнання. дови... Але під таке означення краще підпадає транспортний комплекс. Можна, приміром, спробу- вати опертися на таке загальне означення: си- стема — це цілісне відображення засобами мови спостерігача/дослідника цілісно вираженої мно- жини об’єктів (елементів) і їх зв’язків/відношень (стосунків) між собою і з оточенням/довкіллям в сенсах розв’язуваної спостерігачем/дослідником задачі/проблеми. Означення загалом хороше, але щоб у ньому побачити транспортну систему, треба залучити ще низку конкретніших і кон- структивніших понять, пов’язаних саме з транс- портом, а краще з транспортуванням. Але все ж не зникає проблема: скільки спостерігачів/до- слідників — стільки …? Якось не комільфо? Однозначного означення також немає й по- няття «транспортна інфраструктура». Дискусії то- чаться навіть навколо того, включати чи не вклю- чати в поняття «транспортна інфраструктура» ру- хомий склад, підприємства, що здійснюють пере- везення людей та вантажів, постачальницькі структури, галузеві навчальні заклади тощо. Ви- глядає радше так, що транспортна інфраструктура є складовою частиною єдиної транспортної си- стеми та покликана забезпечити належні умови її функціонування, але не включає в себе рухомий склад, підприємства, що здійснюють перевезення вантажів і пасажирів, та підприємства, що забез- печують роботу транспорту. Транспортна інфраструктура сприяє доціль- ній організації економічного простору. Під транс- портною інфраструктурою розуміють сукупність галузей і сфер діяльності, завдання яких зводиться до перенесення товарів від виробників до спожива- чів. А ще транспортну інфраструктуру розгляда- ють як комплекс підприємств, установ, організа- цій, покликаних обслуговувати процеси перемі- щення товарів. Або ж транспортну інфраструктуру означують як сукупність технічних засобів й уста- тковання, що забезпечують реалізацію транспорт- них процесів. Тобто суб’єктивно під транспортною інфраструктурою розуміють дуже різне. Виглядає так ніби транспортна система про- являє себе через транспортні засоби: де транспор- тні засоби — там ніби й транспортна система. З тих самих позицій можна говорити й про еле- менти транспортної інфраструктури. Але насправді транспортні засоби і відповідні еле- менти транспортної інфраструктури залучені в найрізноманітніші галузі матеріяльного вироб- ництва, а також у сферу споживання, та підпоряд- ковані там особливим системним цілям. Тобто вони є елементами різних систем зі сфери ма- теріяльного виробництва і сфери споживання, звідки вилучати їх в окрему транспортну систему нема жодного резону. Приміром, технологічний (промисловий) транспорт транспортній системі в загальному сенсі принципово не може належати, як і елементи технологічно-транспортної інфра- структури не можуть належати власне транспорт- ній інфраструктурі. Автомобілі, до слова, щойно у р у р р у «Інфраструктура» — в перекладі з латини «ос- нова», «фундамент». дови... Але під таке означення краще підпадає транспортний комплекс. Інфраструктурою є сенс вва- жати сукупний складник продуктивних сил, що поєднує в собі допоміжні й додаткові галузі вироб- ництва чи різновиди діяльності, які обслуговують безпосередньо основне виробництво (виробнича ін- фраструктура), а також галузі невиробничої сфери, що опосередковано пов’язані з процесом вироб- ництва і виконують різноманітні функції обслуго- вування всього процесу господарювання. Інфра- структура — це загалом системне поєднання ор- ганізаційно-економічних, соціальних та юридичних умов, а також споруд, будівель, систем і служб, необхідних для функціонування економіки, для здійснення матеріяльного виробництва, для забез- печення повсякденної життєдіяльності населення. Вісник ЛДУБЖД, №22, 2020 72 розмежовані сферою обігу, де транспорт власне і має втілювати корисні процеси. Транспорт цілісно вмонтований за посеред- ництва логістики в народне господарство. А от ін- фраструктуру народного господарства загалом складають три її різновиди: технічна (власне ви- робнича), інституційна і соціальна. Технічна ін- фраструктура передбачає наявність і організоване функціонування транспорту, доріг, споруд, ко- мор, терміналів тощо. Інституційна інфраструк- тура — це супутня діяльність банків, митниці, ор- ганів сертифікації тощо. Соціальна інфраструк- тура охоплює персонал, зайнятий в процесі ве- дення народного господарства. Ті самі різновиди інфраструктури, звісно, мають логістика і транс- порт, забезпечуючи впорядкований і взаємообу- мовлений рух матеріяльних, фінансових та інфор- маційних потоків. Щоправда, деякі елементи ін- фраструктури є належними все-таки народному господарству, хоча й обслуговують логістику й транспорт. Наприклад, операції страхування здійснюють спеціалізовані страхові компанії, що є водночас і самостійними суб’єктами страхового сектору (регістру), і частиною інституційної логістичної та транспортної інфраструктури. Тож транспорт є складовою частиною і рушієм (активатором) сфери обігу, системою, що забезпечує продуктивний зв’язок між сферою ма- теріяльного виробництва і сферою споживання. А от сфера матеріяльного виробництва і сфера спо- живання правлять за довкілля (середовище), посе- ред якого (у якому) функціонує транспортна си- стема. Коли хочуть це наочно підкреслити, то вда- ються до наведеної на рис. 3 схеми. Рисунок 3 — Схема ідентифікації транспортної системи Транспортна система — транспорт сфери обігу?!. Теорія — це не безплідне розумування «кабінетних вчених», як декому, хто гордий з себе практик, здається. Насправді усілякі «факти» без теорії беззвучні чи ніяк не хочуть розмовляти. Ви- датному економісту Джону Мейнардові Кейнсу (1883—1946) належать слова: «Люди-практики, що вважають себе цілком не підвладними інтеле- ктуальним впливам, зазвичай є рабами якого-не- будь економіста минулого». Рисунок 3 — Схема ідентифікації транспортної системи Але разом з тим, транспорт залучено ще й безпосередньо у сферу матеріяльного (промисло- вого й аграрного) виробництва. Bulletin of Lviv State University of Life Safety, №22, 2020 дови... Але під таке означення краще підпадає транспортний комплекс. Там транспорт — технологічний (внутрішньовиробничий), покли- каний переміщати предмети й засоби праці, а та- кож робочу силу, не проникаючи у сферу обігу. Він як технологічний (внутрішньовиробничий) також залучений і у сферу споживання, де покли- каний переміщати готовий продукт виробництва безпосередньо до осередку проміжного чи кінце- вого привласнення. Без сумніву, системно транспорт — це цілком самостійна галузь матеріяльного вироб- ництва, важлива сфера вкладення продуктивного капіталу. Відтак транспортна система мусить мати якісь визначальні межі. Зазвичай, якщо вка- зати хоч якусь частину межі, то легше вибудувати цю межу повністю — подібно до того, що якщо один корінь 1x заданого квадратного тричлена 2 ax bx c   визначено точно, то й другий корінь стає відомим: Тож транспорт сфери обігу за всіма озна- ками функціює як елемент галузі матеріяльного виробництва, як елемент самостійної транспорт- ної системи. А от технологічний (внутрішньови- робничий) транспорт є елементом системи про- мислового, будівельного, сільськогосподарсь- кого… виробництва і отже не має підстав вважа- тися самостійною галуззю народного господар- ства та й не може бути долучений до транспортної системи. Відтак системно транспорт — це скла- дова частина сфери обігу, що активує пе- реміщення продукту із сфери безпосереднього ви- робництва у сферу безпосереднього споживання. 2 1 b x x a   чи 2 1 1 c x x a  . Межі транспортної системи зумовлені об’єктивними законами, що панують в сфері сус- пільного виробництва: транспортна проми- словість, з одного боку, продовжує-довершує процеси виробництва в кожній іншій галузі про- мисловості, а з другого боку, вона живить сферу споживання продуктами виробництва. Отже транспортна система межує як зі сферою ма- теріяльного виробництва, так і зі сферою ма- теріяльного споживання. Але ці сфери об’єктивно Продукт, звісно, може бути проміжним, тобто таким, що править за сирець чи заготовку 73 Bulletin of Lviv State University of Life Safety, №22, 2020 і споживанням, між обігом матеріяльного і обігом не цілком матеріяльного. Тому все, що стосується вантажного транспорту можна, не торкаючись дещиць, переносити й на пасажирський. для виготовлення складнішого і кориснішого про- дукту. Приміром, продукт видобувної промисло- вості багатократно через сферу обігу потрапля- тиме у різні виробничі системи, рис. 4, перш ніж остаточно заляже чимсь корисним у такому складному завершеному продукті, яким є, ска- жімо, яка-небудь робоча машина. Він може бути і кінцевим-завершеним, цілком придатним до ви- користання (до споживання). дови... Але під таке означення краще підпадає транспортний комплекс. Рисунок 5 – Схема взаємодії сфер обігу та споживання Рисунок 4 — Схема перетину сфери виробництва Рисунок 5 – Схема взаємодії сфер обігу та споживання Основний (потрібен і додатковий) транс- порт зі сфери обігу є підстави тлумачити як магістральний. На рис. 3 прямі 1 1 , 2 2  , …, N N  якраз і позначають напрями активності різних видів магістрального транспорту. Але будь-який транспорт, що перебирає на себе функції магістрального, зазвичай не здатен, а ча- сто й не повинен, здійснювати транспортування за схемою «від порогу до порогу» (англ. «door-to- door» — від дверей до дверей). Тож в межах сфери обігу доводиться вдаватись до послуг додатко- вого транспорту, який є сенс умовно назвати ло- кальним (на противагу магістральному), див. рис. 3. Цей різновид транспорту покликаний на відносно коротких плечах доправляти вантаж (продукт) безпосередньо зі сфери виробництва до магістрального транспорту та від магістрального транспорту — безпосередньо у сферу споживання (проміжного чи кінцевого). Рисунок 4 — Схема перетину сфери виробництва Але й кінцевий продукт крізь сферу обігу може рухатись по-різному. Приміром, зернозби- ральний комбайн (фізично минаючи сферу спожи- вання) потрапить насправді відразу у сферу вироб- ництва (сільськогосподарського), відповідно до наведеної на рис. 3 схеми. Сільськогосподарське виробництво в такому разі має статус споживача. Але при цьому в сфері власне споживання мають залишитися сліди супутніх нематеріяльних по- токів. Та багато різних продуктів матеріяльного виробництва остаточно зависають у домашніх гос- подарствах, рис. 5: на схемі відображено, що пе- реміщення уособленого кінцевого продукту здійснюється в 1, i M  різних напрямках. До- машні господарства (домоволодіння) — це осе- редки безпосереднього споживання, до яких про- дукти доправляються або системним (таксі, марш- рутні автобуси, вантажівки…), або позасистемним (випадковим чи індивідуальним) транспортом (або навіть й без використання транспорту). Достачання вантажів (продукції) зі сфери виробництва до магістрального транспорту та від магістрального транспорту у сферу споживання здійснюють під’їзними шляхами до промислових підприємств, будівельних і торгових організацій, що мають бути належно досконалими. Таке транспортування — це виробничий процес сфери обігу незалежно від того, кому підпорядкований застосовуваний транспорт чи хто є його розпоряд- ником. Тож цей транспорт поряд з магістральним має належати транспортній системі як такій. Звісно, існують визнані осередки, де проду- кують культурні цінності і куди тягнуться спраглі духовних емоцій люди. Ці люди набувають ста- тусу пасажирів як у напрямі до храмів культури з бажанням духовно збагатитись, так і в напрямі до своїх домоволодінь з багажем набутих емоцій (і не тільки). дови... Але під таке означення краще підпадає транспортний комплекс. Тобто існує певна аналогія між ма- теріяльним і духовним (та іншим) продукуванням Зв’язок між локальним транспортом та магістральним здійснюється через спеціально улаштовані пункти, які називатимемо терміналами. Вісник ЛДУБЖД, №22, 2020 74 Термінали розташовують на залізничних станціях, у річкових і морських портах, в аеропортах тощо. підстав пропонували узагальнити до рівня си- стеми транспортування не тільки вантажу, а й ін- формації та енергії. Та все ж існує певна логіка на- ративного означування. Відтак модель транспортної системи стає можливим відобразити як цілісне поєднання в межах сфери обігу комунікацій (інциденцій), створюваних засобами й ресурсами магістраль- них різновидів транспорту разом з долученими до них через термінали засобами й ресурсами різних видів локального транспорту (див. рис. 3). Ло- кальний транспорт через термінали власне наво- дить зв’язок основних комунікацій з довкіллям. Отже транспортну систему загалом складають три цілком різнорідні об’єкти-елементи: система, що оперує локальним транспортом; магістральна транспортна система; термінальна система. Концепцію інтегрованої транспортної си- стеми розвивають у напрямі створення мультимо- дальних коридорів і регіональних транспортно- логістичних систем для освоєння важливих вантаж- них і пасажирських потоків на основі регіональної транспортної інфраструктури, що поєднує шляхи сполучення, транспортні підприємства, оптові тор- гові бази, комірні ємності (простори), а також то- варні біржі, банки, телекомунікаційні системи й ін- формаційні центри. Транспортний вузол в такому разі слугує ніби потужним терміналом на стику різ- них магістральних транспортів, рис. 6. І його обслу- говує власний технологічний транспорт. Під системним елементом «термінал» до- речно розуміти комплекс технічних засобів і облаштувань та сукупність відносин, що забезпе- чують ефективну взаємодію різних видів транс- порту і уможливлюють досконалу без жодних втрат передачу матеріяльного потоку з локального транспорту на магістральний і зворотно. В терміналах взаємодія транспортів може відбува- тись як з перевалюванням вантажів, так і без нього. Схему, що наведена на рис. 3, слід розуміти так, що в терміналах вантажі (пасажири) можуть передава- тись не тільки з магістрального транспорту на ло- кальний, але й з одного магістрального на інший. Рисунок 6 – Схема транспортування в транспортному коридорі Завдяки термінальній системі стає можли- вим сформувати транспортні вузли. Натомість магістральна транспортна система (разом з термінальною) дає змогу сформувати транспортні коридори. Рисунок 6 – Схема транспортування в транспортному коридорі Саме ефективні системні зв’язки і відно- сини є основою для існування окремих видів транспорту в системі. Чим повнішим є комплекс цих зв’язків і відносин і чим дієвіше вони себе проявляють, тим життєздатнішою стає транс- портна система і тим вищою буде ефективність її функціювання. Bulletin of Lviv State University of Life Safety, №22, 2020 дови... Але під таке означення краще підпадає транспортний комплекс. Усталені зв’язки й відносини відображають — усі по-своєму — організаційно- економічну природу народного господарства за- галом і транспортного виробництва зокрема. Вони ж характеризують рівень, глибину єдності транспортної системи. Тут доречно наголосити на аналогії, приміром, з моделями електричних або гідравлічних систем, щоправда, тільки в рамках адекватних проявів. Наприклад, електрична мережа відрізняється від усіх неелектричних тим, що оточена створюваним нею ж динамічним електричним полем, що простя- гається у нескінченність усіма напрямками. Тобто електрика існує не тільки у видимих провідниках, але й в невидимому просторі навколо них. Відтак електричну мережу розглядають як поєднання «мертвої» і «живої» структур. «Мертву» структуру (аналог — транспортна інфраструктура) описують тензором імпедансу, а «живу» (аналог — транспорт і вантаж чи пасажири) — двома електричними па- раметрами, один з яких, умовно кажучи, характери- зує електрику в провіднику, а другий — поза ним. Але навіть інженери-електрики часто трактують пе- ресилання електричного струму так, ніби він пере- буває в транспортному опакуванні — все залежить від контексту. Висновки. Нема жодного сенсу зазіхати на право бачити суто «свою» (цілком «по-своєму») транспортну систему. Розуміння будь-якої систем- ності визріває унаслідок вмотивованого поєднання в когнітивному акті «пізнаваного об’єкта», «суб’єкта — спостерігача/дослідника», «середо- вища/оточення/довкілля», «мови опису/відобра- ження», якою послуговуються. А таке поєднання ніколи не буде однозначним. Транспорт — це все те і всі ті, що і хто при- четні до здійснення переміщення будь-чого і будь-кого в життєвому просторі на догоду вироб- нику й споживачу цінностей, на догоду суспільс- тву. Утямки «транспорт» і «транспортна система» До прикладу, колись [20] транспортну си- стему, характеризовану трьома атрибутивами — «вантаж», «швидкість», «потужність», не без Bulletin of Lviv State University of Life Safety, №22, 2020 75 — не тотожні. Транспорт подільний на частинки, кожна з яких може слугувати цеглинкою у будові транспортної системи або ж цілком становити си- стему. Транспорт — це засіб, а транспортна сис- тема — це форма/концепція/технологія реалізації мобільності цінностей і людей назустріч одне од- ному за участі власне транспорту. загальному сенсі принципово не може належати, як і елементи технологічно-транспортної інфра- структури не можуть належати власне транспорт- ній інфраструктурі. Технологічний транспорт є підстави трак- тувати або як самостійну систему, підпорядко- вану чи виробничій, чи споживчій сфері (чи вва- жати окремим елементом такої собі чи виробни- чої (промислової), чи споживчої (торгівельної) системи). Тож у цілісному єднанні тільки транс- порт сфери обігу виражає себе як транспортну си- стему в широкому розумінні. Саме ця транс- портна система займається властиво транспорт- ним (!) виробництвом. Література: 1. Захарова О. В. Теоретичні підходи до визначення сутності та властивостей світової транспортної системи // Економіка: Вісник Маріу- польського державного університету, 2011, Вип. 1. — С. 21—25. Перевезення можна ділити на три різно- види: 1) перевезення в рамках особистих інтересів (задля особистого задоволення чи особистої ко- ристі) — транспортно-експедиційне обслуго- вування населення; 2) перевезення в інтересах за- доволення внутрішніх потреб якогось підприєм- ства — технологічні перевезення; 3) перевезення в сфері обігу — транспортне виробництво. Якщо б не було транспорту і транспортної системи, до- велося б повсюдно розосереджено розгортати ви- робництво потрібних усім речей. А так — власне транспортна система усуває ці клопоти, сама стає ніби виробництвом. 2. Рибчук А. В. Транспортні системи світу — важливий елемент глобальної виробничої ін- фраструктури // Актуальні проблеми економіки. — 2004. — № 7. — С. 99—104. 2. Рибчук А. В. Транспортні системи світу — важливий елемент глобальної виробничої ін- фраструктури // Актуальні проблеми економіки. — 2004. — № 7. — С. 99—104. 3. Яцківський Л. Ю., Зеркалов Д. В. Загаль- ний курс транспорту. — Київ: Арістей, 2007. — 504 с. 3. Яцківський Л. Ю., Зеркалов Д. В. Загаль- ний курс транспорту. — Київ: Арістей, 2007. — 504 с. 4. Ломакин В. К. Мировая экономика. — Москва: Юнити, 2004. — 735 с. 4. Ломакин В. К. Мировая экономика. — Москва: Юнити, 2004. — 735 с. 5. Хахлюк А. Для забезпечення транзитних потоків. Транспортна інфраструктура держави: регіональний аспект // Політика і час. — 2004. — № 2. — С. 71—81. Немає потреби оперувати утямком «єдина транспортна система». Термін «єдина» нічого особливого не характеризує стосовно системи, яка сама по собі є цілісне утворення, немислиме без гармонійного поєднання усіх наявних різно- видів транспорту. 6. Орловский П. Н., Скворцов Г. П. Систем- ный анализ транспортных узлов. — Кыйив: Ос- нова, 2007. — 596 с. Транспортні засоби і відповідні елементи транспортної інфраструктури залучені в найрізноманітніші галузі матеріяльного вироб- ництва, а також у сферу споживання, та підпоряд- ковані там особливим системним цілям. Тобто вони є елементами різних систем зі сфери ма- теріяльного виробництва і сфери споживання, звідки вилучати їх в окрему транспортну систему нема жодного резону. Приміром, технологічний (промисловий) транспорт транспортній системі в 7. Афанасьев Л. Л., Островский Н. Б., Цу- керберг С. М. Единая транспортная система и ав- томобильные перевозки. — Москва: Транспорт, 1984. — 336 с. 8. Аксенов И. Я. Единая транспортная си- стема. — Москва: Высшая школа, 1991. — 383 с. 9. Савельєва І. В., Дрожжин О. Л. дови... Але під таке означення краще підпадає транспортний комплекс. Для транспорту важливою класифікацій- ною ознакою є його місце в матеріяльному виро- бництві: в сфері виробництва (споживання) — це технологічний транспорт, що є складовою части- ною виробничого процесу; в сфері обігу — це еко- номічний (господарчий) транспорт, що наводить транспортно-економічні зв’язки між підприємст- вами і районами країни, але залишається самос- тійною галуззю виробництва. Тож саме еко- номічний (господарчий) транспорт належить до (є складовою частиною) транспортної системи. В транспортній системі доречно розпізна- вати магістральний транспорт і локальний транс- порт, що гармонійно взаємодіють у сфері обігу. Локальний транспорт тяжіє до певних осередків: чи до сфери виробництва, чи до сфери спожи- вання (тому, мабуть, його деколи називають зовнішнім технологічним). Звісно, радіус дії ло- кального транспорту в окремих випадках може виявитися таким, що до послуг магістрального транспорту вдаватись не буде сенсу — він пере- бере на себе функції магістрального. Утямок «транспорт» уживають в певному усталеному але доволі «розмитому» дискурсі. І однозначно означити його радше не вдасться. Але є підстави стверджувати: транспортна система — це цілісне відображення засобами мови спо- стерігача множини об’єктів у їх зв’язках між со- бою і з довкіллям в сенсах розв’язуваної спо- стерігачем задачі/проблеми, пов’язаної з забезпе- ченням мобільності цінностей і людей назустріч одне одному саме за допомогою транспорту. Література: Контейнерна транспортно-технологічна система як засіб реалізації Вісник ЛДУБЖД, №22, 2020 76 інтермодального перевезення // Вісник ЖДТУ: Тех- нічні науки. 2014. № 1 (68). — С. 12—16. 5. Khakhliuk A. (2004). Transits Streams Providing. The State Transport Infrastructure: Re- gional Aspect. Polinyka i chas, Issue 2, 71—81. (in Ukraine). 10. Кунда Н. Т. Дослідження операцій у транспортних системах. — Київ: Видавничий дім «Слово», 2008. — 400 с. 6. Orlovskyi, P. N., & Skvortsov, H. P. (2007). Systems analysis of transport knots. Kyiv: Osnova. (in Russian). 11. Савенко В. Я., Гайдукевич В. А. Транс- порт і шляхи сполучення. — Київ: Арістей, 2007. — 256 с. 7. Afanasiev, L. L., Ostrovskii, N. B., & Tsu- kerberg, S. M. (1984). Single transporting system and motorcar transportations. Moscow: Transport. (in Russian). 12. McConnell Campbell R., Brue Stanley L., Flynn Sean Masaki. Economics: Principles, Prob- lems, and Policies / 20th Edition — New York [etc.]: McGraw-Hill Education, 2014. — 890 p. (The McGraw-Hill series in economics). 8. Aksionov, I. Ya. (1991). Single transporting system. Moscow: Vysshaia shkola. (in Russian). 13. Mankiw N. Gregory. Principles of Eco- nomics / 6th Edition. The Dryden Press Harcourt Brace College Publishers, 2012. — 856 p. 9. Savelieva, I. V., & Drozhzhin, O. L. (2014). Container transporting-technological system as the mean of realization of інтермодального transporta- tion. Bulletin of ZhDTU: Engineering sciences. Issue 1 (68), 12—16. (in Ukraine). 14. Dolan Edwin G., Lindsay David E. Macro- economics. — Dryden press, 1994. — 464 p. 15. Samuelson Paul A., Nordhaus William D. h 10. Kunda, N. T. (2008). Operations analysis in the transport systems. Kyiv: Slovo. (in Ukraine). 15. Samuelson Paul A., Nordhaus William D. Economics / 19th Edition. — New York [etc.]: McGraw-Hill/Irwin, 2009. — 744 p. 16. Піндайк Роберт С., Рубінфелд Деніел Л. Мікроекономіка / Пер. з 77нгл.. А. Олійник, Р. С і й К ї О 1996 646 15. Samuelson Paul A., Nordhaus William D. Economics / 19th Edition. — New York [etc.]: McGraw-Hill/Irwin, 2009. — 744 p. 11. Savenko, V. Ya., & Haidukevych, V. A. (2007). Transport and ways of report. Kyiv: Aristei. (in Ukraine). McGraw-Hill/Irwin, 2009. — 744 p. 16. Піндайк Роберт С., Рубінфелд Деніел Л. Мі і / П 77 А О ій Р 12. McConnell, Campbell R., Brue, Stanley L., & Flynn, Sean Masaki. (2014). Economics: Princi- ples, Problems, and Policies (20th Edition). New York (etc.): McGraw-Hill Education (The McGraw-Hill se- ries in economics). Література: (in English). Скільський. — Київ: Основи, 1996. — 646 с. 17. Руссєв Г. В. Організація автомобільних перевезень. — Київ: Вища школа, 1971. — 256 с. 17. Руссєв Г. В. Організація автомобільних перевезень. — Київ: Вища школа, 1971. — 256 с. 18. Цветов Ю. М. Транспорт: системный подход (вопросы теории и практики). — Москва: Знание, 1980.— 64 с.— (Новое в жизни, науке, технике. Сер. «Транспорт»; № 12). 18. Цветов Ю. М. Транспорт: системный подход (вопросы теории и практики). — Москва: Знание, 1980.— 64 с.— (Новое в жизни, науке, технике. Сер. «Транспорт»; № 12). 13. Mankiw, N. Gregory. (2012). Principles of Economics (6th Edition). The Dryden Press Har- court Brace College Publishers. (in English). 19. Великий тлумачний словник сучасної української мови / Уклад. В. Бусел. — Київ; Ірпінь: ВТФ «Перун», 2001. — 1440 с. 19. Великий тлумачний словник сучасної української мови / Уклад. В. Бусел. — Київ; Ірпінь: ВТФ «Перун», 2001. — 1440 с. 14. Dolan, Edwin G., & Lindsay, David E. (1994). Macroeconomics. Dryden press. (in English). 20. Кузнецов П. Г. О возможности энерге- тического анализа основ организации обществен- ного производства. В кн.: Эффективность научно- технического творчества. — Москва: Наука, 1968. — С. 214—246. 20. Кузнецов П. Г. О возможности энерге- тического анализа основ организации обществен- ного производства. В кн.: Эффективность научно- технического творчества. — Москва: Наука, 1968. — С. 214—246. 15. Samuelson, Paul A., & Nordhaus, William 15. Samuelson, Paul A., & Nordhaus, William D. (2009). Economics (19th Edition). New York [etc.]: McGraw-Hill/Irwin. (in English). D. (2009). Economics (19th Edition). New York [etc.]: McGraw-Hill/Irwin. (in English). 16. Pindyck, Robert S., & Rubinfeld, Daniel L. (1992). Microeconomics. Kyiv: Osnovy. (in Ukraine). References: 17. Russiev H. V. (1971). Organization of mo- tor-car transportations. Kyiv: Vyshcha shkola. (in Ukraine). 1. Zakharova, O. V. (2011). The Theoretical Approaches to Definition of the World Transport Sys- tem and its Properties. Economics: Bulletin of the Ma- riupol state university, Issue 1, 21—25. (in Ukraine). 1. Zakharova, O. V. (2011). The Theoretical Approaches to Definition of the World Transport Sys- tem and its Properties. Economics: Bulletin of the Ma- riupol state university, Issue 1, 21—25. (in Ukraine). 18. Tsvetov Yu. M. (1980). Transport: Systems Approach (Questions of Theory and Practice). Mos- cow: Znaniie. — (New in life, science, technique. Se- ries «Transport»; Issue 12). (in Russian). 2. Rybchuk, А. V. (2004). The World Trans- porting Systems are an Important Element of Global Production Infrastructure. Actual Economy Problems, Issue 7, 99—104. (in Ukraine). 2. Rybchuk, А. V. (2004). The World Trans- porting Systems are an Important Element of Global Production Infrastructure. Actual Economy Problems, Issue 7, 99—104. (in Ukraine). 19. Busel, V. (2001). Large explanatory dic- tionary of modern Ukrainian. — Kyiv; Irpin: Perun. (in Ukraine). 3. Yatskivskyi, L. Yu., & Zerkalov, D. V. (2007). Flat Rate of Transport. Kyiv: Aristei. (in Ukraine). 20. Kuznietsov, P. G. (1968). About possibility of power analysis of bases of organization of public pro- duction. In Book: Efficiency of scientific and technical creation. Moscow: Nauka, 214—246. (in Russian). 4. Lomakin, V. K. (2004). World economics. Moscow: Yuniti. (in Russian). Bulletin of Lviv State University of Life Safety, №22, 2020 * Оглядова стаття 77 Bulletin of Lviv State University of Life Safety, №22, 2020
https://openalex.org/W4237345468	https://europepmc.org/articles/pmc4234829?pdf=render	English	null	Evolution of insulin sensitivity and its variability in out of hospital cardiac arrest (OHCA) patients treated with hypothermia	Critical care	2,014	cc-by	5,833	RESEARCH Open Access Open Access © 2014 Sah Pri et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Introduction: Therapeutic hypothermia (TH) is often used to treat out-of-hospital cardiac arrest (OHCA) patients who also often simultaneously receive insulin for stress-induced hyperglycaemia. However, the impact of TH on systemic metabolism and insulin resistance in critical illness is unknown. This study analyses the impact of TH on metabolism, including the evolution of insulin sensitivity (SI) and its variability, in patients with coma after OHCA. Methods: This study uses a clinically validated, model-based measure of SI. Insulin sensitivity was identified hourly using retrospective data from 200 post-cardiac arrest patients (8,522 hours) treated with TH, shortly after admission to the intensive care unit (ICU). Blood glucose and body temperature readings were taken every one to two hours. Data were divided into three periods: 1) cool (T <35°C); 2) an idle period of two hours as normothermia was re-established; and 3) warm (T >37°C). A maximum of 24 hours each for the cool and warm periods was considered. The impact of each condition on SI is analysed per cohort and per patient for both level and hour-to-hour variability, between periods and in six-hour blocks. Results: Cohort and per-patient median SI levels increase consistently by 35% to 70% and 26% to 59% (P <0.001) respectively from cool to warm. Conversely, cohort and per-patient SI variability decreased by 11.1% to 33.6% (P <0.001) for the first 12 hours of treatment. However, SI variability increases between the 18th and 30th hours over the cool to warm transition, before continuing to decrease afterward. Conclusions: OCHA patients treated with TH have significantly lower and more variable SI during the cool period, compared to the later warm period. As treatment continues, SI level rises, and variability decreases consistently except for a large, significant increase during the cool to warm transition. These results demonstrate increased resistance to insulin during mild induced hypothermia. Our study might have important implications for glycaemic control during targeted temperature management. can be highly insulin resistant and variable, particularly on the first two days of stay [14], as well as those who may particularly benefit from glycaemic control [4]. Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia Azurahisham Sah Pri1, J Geoffrey Chase1, Christopher G Pretty1, Geoffrey M Shaw2, Jean-Charles Preiser3, Jean-Louis Vincent3, Mauro Oddo4, Fabio S Taccone3, Sophie Penning5 and Thomas Desaive5 Correspondence: chris.pretty@canterbury.ac.nz 1Centre for Bio-Engineering, Department of Mechanical Engineering, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140, New Zealand Full list of author information is available at the end of the article Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Patients and data A retrospective analysis of glycaemic control data from 200 OHCA patients (8,522 hours) treated with TH, shortly after admission to intensive care. Data was ob- tained from intensive care units (ICUs) at Christchurch Hospital, New Zealand, at Erasme Hospital, Belgium, and CHUV-Lausanne Hospital, Switzerland. Patients from Christchurch Hospital (N = 20) were on the specialized relative insulin and nutrition titration (SPRINT) glycaemic control protocol [7], whereas the remaining 180 patients from Erasme (N = 99) and Lausanne (N = 81) hospitals were on local glycaemic control protocols and included in an institutional database (2008 to 2012). _G ¼ −pG:G tð Þ−SI tð Þ:G tð Þ: Q tð Þ 1 þ αGQ tð Þ þ P tð Þ þ EGP−CNS V G ð1Þ _I ¼ −nKI tð Þ− nLI tð Þ 1 þ αII tð Þ −nI I tð Þ−Q tð Þ ð Þ þ uex tð Þ V I þ 1−XL ð Þ uen tð Þ V I ð2Þ _Q ¼ nI I tð Þ−Q tð Þ ð Þ−nC Q tð Þ 1 þ αGQ tð Þ ð3Þ _G ¼ −pG:G tð Þ−SI tð Þ:G tð Þ: Q tð Þ 1 þ αGQ tð Þ þ P tð Þ þ EGP−CNS V G ð1Þ ð1Þ _I ¼ −nKI tð Þ− nLI tð Þ 1 þ αII tð Þ −nI I tð Þ−Q tð Þ ð Þ þ uex tð Þ V I þ 1−XL ð Þ uen tð Þ V I ð2Þ ð2Þ Blood glucose (BG) and temperature readings were taken one to two hourly. Data were divided into three periods: 1) cool (T <35°C); 2) an idle period of two hours as normothermia was restored; and 3) warm (T >37°C). A maximum of 24 contiguous hours and a minimum of 15 hours for each period were considered, ensuring a balance of contiguous data between periods. Overall demographics are shown in Table 1. _Q ¼ nI I tð Þ−Q tð Þ ð Þ−nC Q tð Þ 1 þ αGQ tð Þ ð3Þ ð3Þ Where G(t) represents the concentration of blood glu- cose (mmol/L). I(t) and Q(t) represent the plasma insulin and insulin interstitial concentrations (mU/L) respectively. Model parameters, rates and constants in this model were as fully defined in [21,24]. Glycaemic targets while treating OHCA patients in the three units were very similar and overlapped. The SPRINT protocol, used in the Christchurch Hospital ICU, targeted 4.0 to 7.0 mmol/L [7]. Introduction l Hyperglycaemia is prevalent in critical care [1-4] and increases the risks of further complications and mortality [1,4,5]. Glycaemic control has shown benefits in reducing mortality and morbidity [4,6,7]. However, many studies have found it difficult to reproduce these results [8-10] due in part to metabolic variability [11]. Out-of-hospital cardiac arrest (OHCA) patients often experience hypergly- caemia [12,13]. These patients belong to one group who Therapeutic hypothermia (TH) is often used with OHCA patients to protect against brain injury [15,16], which leads to a lowering of metabolic rate, reduces plasma insulin, in- duces insulin resistance and alters blood glucose homeo- stasis [17]. One of the adverse events associated with hypothermic therapy is a decrease in insulin sensitivity and endogenous insulin secretion [18]. However, this de- crease may not be observable in a cohort who is already highly insulin resistant and variable [14]. Hence, under- standing metabolic evolution and variability would enable * Correspondence: chris.pretty@canterbury.ac.nz 1Centre for Bio-Engineering, Department of Mechanical Engineering, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140, New Zealand Full list of author information is available at the end of the article Page 2 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Audit of the clinical data from SPRINT was given by the Upper South B Regional Ethics Committee and for the data study by Taccone et al. [17]. No approval was required as it was also a retrospective audit. safer and more accurate glycaemic control using insulin in this cohort. This study analyses the evolution of a clinically validated model-based measure of insulin sensitivity (SI) in OHCA patients to assess the impact of hypothermia therapy. Patients and data The protocol used in both the Erasme and Lausanne ICUs differed from SPRINT, but targeted 6.0 to 8.0 mmol/L [17]. Although two different protocols were used in the three units, the targets were very similar and within the relatively tight 4.0 to 8.0 mmol/L range. Model-based SI is identified hourly from patient data, producing an hourly piece-wise constant profile [25], capturing the whole-body glycaemic response to exogen- ous insulin and nutrition. The validity and independence of this patient-specific parameter have been validated using data from independent, clinically matched cohorts [19], in comparison to gold-standard insulin sensitivity tests [22] and in clinical glycaemic control [20,23]. Table 1 Demographic data and treatment information for both the cool and warm periods Variables Value Cool Warm Total patients, number (n) 200 Median age, years 61 [51, 72] Female gender, number (%) 40 (20.6%) ICU mortality, number (%) 85 (45.6%) Diabetes status, number (%) 26 (13.0%) Total treatment, hours (h) 4219 4303 Blood glucose, median (mmol/L) 7.6 [6.3,9.7] 6.8 [5.9,8.0] Insulin rate, median (U/hr) 3.4 [1.3,8.0] 3.5 [1.6,7.0] Glucose rate, median (g/hr) 2.7 [1.0,5.3] 5.4 [2.7,8.1] IQR: [interquartile range]. Table 1 Demographic data and treatment information for both the cool and warm periods Methods Model-based SI in this study is a patient-specific param- eter describing the overall whole-body effect of insulin. SI is identified for each hour, for each patient using a clinically validated glucose-insulin model [19-23]. The key model equations are defined: Analyses and metrics SI level and variability during the cool (T ≤35°C) and warm (T >35°C) periods are analysed on per-cohort and per-patient bases using six-hour blocks of data as per Table 2. SI level is compared between blocks as a cohort median and by per-patient median SI. Similarly, SI variabil- ity is calculated as the hour-to-hour percentage change in SI (Δ%SI) and is analysed per cohort for each block. %ΔSI ¼ SInþ1−SIn SIn 100 ð4Þ ð4Þ The use of percentage change, rather than absolute change, normalises the metric so patients with differing SI levels can be compared fairly. Page 3 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Table 2 Descriptions of six-hour blocks for data analysis Day Period Analysis Block Hour range 1 Cool 6-hour block 1 0 – 6 hours 2 6 – 12 hours 3 12 – 18 hours 4 18 – 24 hours Idle 2-hour period in between cool and warm 2 Warm 6-hour block 5 24 – 30 hours 6 30 – 36 hours 7 36 – 42 hours 8 42 – 48 hours Table 2 Descriptions of six-hour blocks for data analysis D P i d A l i Bl k H each six-hour block. Thus, a reduction in the IQR of Δ% SI over time would indicate a reduction in hour-to-hour variability for a given patient. SI level and variability are non-Gaussian and thus com- pared using non-parametric statistics and cumulative dis- tribution functions (CDFs). CDFs are particularly useful as they show the entire distribution that is often summarised as a median and IQR. The CDF for a given value of the independent variable (for example SI = x) describes the probability of observing a value less than or equal to x. All distributed data were compared using a Wilcoxon rank- sum test (Mann–Whitney U test), except for SI variability results. SI variability was compared using the Kolmogorov- Smirnov (KS) test as it has greater power to detect differ- ences in the shape of distributions when median values are similar. In all cases, P <0.05 is considered statistically significant. Bagshaw et al. [26] reported an association between both hypoglycaemia and BG variability with mortality during the first 24 hours of ICU stay. Analyses and metrics Thus, the acute evolution of SI over the first day using six-hour blocks was analysed as SI variability is a key contributor to BG variability. For the cohort analysis, SI and Δ%SI data from all patients was grouped into each appropriate time block. Median values for each time block were calculated for comparison to the previous block, thus capturing overall cohort changes over time in level and hour- to-hour variability. SI level analyses Figures 1 and 2 present the CDFs of hourly SI level by cohort and median SI per patient, respectively, using six-hour blocks. Table 3 presents the increase in median insulin sensitivity and corresponding P values between successive time blocks. For the per-patient analysis, the median value of SI and the interquartile range (IQR) of Δ% SI were calculated for each patient, for each time block. The IQR captures the width or degree of variability for a given patient within The results suggest that SI increases for the cohort and per patient are statistically significant for the first 36 hours (P <0.05) in both cases. Figure 1 Insulin sensitivity (SI) level distribution per cohort for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Figure 1 Insulin sensitivity (SI) level distribution per cohort for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Page 4 of 8 Page 4 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Figure 2 Insulin sensitivity (SI) level distribution per patient for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Figure 2 Insulin sensitivity (SI) level distribution per patient for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Results in Figure 2, Figure 3 and Table 3 are further reflected in Table 4, which shows that SI increases for a large proportion of patients between the six-hour blocks over the first 36 hours of ICU stay. Table 4 also shows that after 48 hours of treatment, only 86% of patients show rise in SI from the first six hours. Thus, while the general trend is obvious for increasing SI, it is not guar- anteed for all patients. Equally, these increases decelerate in terms of number of patients with increasing SI over time, going from left to right in the table. SI variability analyses I y y Figures 3 and 4 present the CDFs for changes in SI (%ΔSI) for six-hourly blocks per cohort and 50% range of SI variability per patient, respectively. Table 5 presents the reductions between successive blocks. Cohort and per-patient variability decreases for the first 24 hours. However, it increases across the cool to warm transition, indicating some potential stress across the cool to warm transition with negative reductions. The decreasing trend returns for all subsequent blocks. The results suggest that %ΔSI decreases per cohort and per patient are statistically significant (P <0.05) for the first 36 hours in both cases. SI level analyses Table 3 Increasing cohort and per-patient median SI during cool and warm periods as per six-hour blocks of data, where the P values compare successive six-hour blocks as shown in the first column for both the overall cohort and per-patient median values SI level Cohort analysis Per-patient analysis analysis % SI median increase P value % SI median increase P value (6-hr blocks) Block 1–2 (C) 35.1 <0.05 26.4 <0.05 (0–6 vs. 6–12 hr) Block 2–3 (C) 19.2 <0.05 31.1 <0.05 (6–12 vs. 12–18 hr) Block 3–4 (C) 31.8 <0.05 42.4 <0.05 (12–18 vs. 18–24 hr) Block 4–5 (C-W) 23.4 <0.05 18.3 <0.05 (18–24 vs. 24–30 hr) Block 5–6 (W) 23.9 <0.05 23.2 <0.05 (24–30 vs. 30–36 hr) Block 6–7 (W) 13.1 0.06 15.8 0.2 (30–36 vs. 36–42 hr) Block 7–8 (W) 4.4 0.4 3.2 0.5 (36–42 vs. 42–48 hr) P values are calculated using Wilcoxon rank-sum test. SI, insulin sensitivity metric (model-based). Table 3 Increasing cohort and per-patient median SI during cool and warm periods as per six-hour blocks of data, where the P values compare successive six-hour blocks as shown in the first column for both the overall cohort and per-patient median values Insulin sensitivity level The SI level results for both per-cohort and per-patient analysis suggest that OHCA patients undergoing TH treatment have significantly lower SI during the earlier Page 5 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Figure 3 Insulin sensitivity variability distribution (%ΔSI) per cohort for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Figure 3 Insulin sensitivity variability distribution (%ΔSI) per cohort for out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. However, the cool to warm transition at 24 hours shows an increase in variability likely due to the change of physiological conditions as body temperature increases from cool to warm between 18 and 36 hours. The lower decrease in SI variability after the 36th hour onward suggests that the patients’ metabolic condition has im- proved and become more stable. cool period on day 1 than the later warm period on day 2. Both results determine the general trend for overall in- creasing SI level for critically ill patients over time and are consistent with other ICU studies [14,27]. Further analysis shows that the increase in SI level during the first 36 hours are large and statistically significant for this cohort. The rapid increases in SI level for the first 36 hours is likely due to significant restart of human physiological systems and metabolic activities for these patients [13]. After 36 hours, the rapid SI increase abates as the patients’ metabolism improves and becomes more stable. Further analysis and comparison of SI variability between general ICU patients [14] and OHCA patients treated with TH shows that the main difference between them is the SI variability increase during the cool to warm transition period for the latter cohort. These SI variability results do not follow the same trend with other general ICU studies by Pretty et al. [14], and it is a unique finding for this cohort that could significantly impact glycaemic control and safety from hypoglycaemia. Insulin sensitivity variability Both per-cohort and per-patient analysis suggest that OHCA patients undergoing TH treatment have high initial variability that decreases over the first 36 hours. Table 4 Proportion of patients for whom median insulin sensitivity increases between the blocks indicated in the row and columns 6 -12 hr 12-18 hr 18-24 hr 24-30 hr 30-36 hr 36-42 hr 42-48 hr 0 – 6 hr 0.72 0.74 0.79 0.83 0.84 0.85 0.86 6 – 12 hr 0.66 0.72 0.74 0.76 0.82 0.82 12 – 18 hr 0.69 0.70 0.75 0.79 0.79 18 – 24 hr 0.66 0.65 0.70 0.72 24 – 30 hr 0.64 0.68 0.66 30 – 36 hr 0.58 0.61 36 – 42 hr 0.52 patients for whom median insulin sensitivity increases between the blocks indicated in the row Table 4 Proportion of patients for whom median insulin sensitivity increases between the blocks and columns 4 Proportion of patients for whom median insulin sensitivity increases between the blocks indicat l Page 6 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Figure 4 Per-patient 50% range of SI variability distribution of out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Figure 4 Per-patient 50% range of SI variability distribution of out-of-hospital cardiac arrest (OHCA) patients, treated with therapeutic hypothermia (TH) using six-hour blocks for both cool and warm periods. Implications for glycaemic control Clinically, these results have significant implications for managing glycaemia. Increased SI variability leads to increased variability in BG level for a given insulin inter- vention [11]. With low and variable insulin sensitivity, glycaemic levels might appear to remain unchanged and difficult to control effectively with exogenous insulin. This situation may result in increased glycaemic variabil- ity as well as an increased risk of hyperglycaemia and hypoglycaemia during the first 36 hours of treatment due to greater hour-to-hour SI variability with increased insulin resistance [17]. Thus, since glycaemic variability and hypoglycaemia are independent risk factors for the critically ill, it is important to understand and manage these patient-specific dynamics, especially those unique to a cohort, when implementing glycaemic control. This outcome is particularly important when OHCA patients transition from cool to warm. These results may also generalise to other areas where glycaemic control is applied to hypothermic patients, such as in the operating theatre. P values are calculated using the Kolmogorov-Smirnov test. SI, insulin sensitivity metric (model-based); IQR, interquartile range. Conclusions This study analyses the metabolic evolution of OHCA patients treated with TH. These analyses characterise the metabolic impact of TH treatment on the level and vari- ability of insulin sensitivity to inform control. Key messages Key messages increased variability would trade off a reduced risk of hypoglycaemia against increased hyperglycaemia. Ultim- ately, the preferred method for any unit may be influenced by practical considerations, such as clinical workload. OCHA patients treated with TH have significantly lower and highly variable SI during the first 24 hours of the cool period, compared to the later warm period in their ICU stay. Competing interests The authors declare that they have no competing interests. Abbreviations %ΔSI: hour-to-hour percentage changes in insulin sensitivity; BG: blood glucose; CDF: cumulative distribution function; ICU: intensive care unit; IQR: interquartile range; KS: Kolmogorov-Smirnov (test); OHCA: out-of-hospital cardiac arrest; SI: insulin sensitivity metric (model-based); SPRINT: specialized relative insulin and nutrition titration; TH: therapeutic hypothermia. Limitations There is an overall trend of increasing SI over the first 36 hours, both per-cohort and per-patient results. The parameters used in the glucose insulin system model are based on general ICU patients with normal body temperature conditions. Thus, the insulin sensitivity values derived during the cool period could be biased by model- ling errors or unmodelled effects. However, as noted previ- ously, the validity and independence of this patient-specific parameter has been validated using data from clinically matched cohorts and has been shown to correlate well in gold-standard insulin sensitivity tests. SI variability decreases consistently over time, except for a large, statistically significant increase during the cool to warm transition at 24 hours. This increase requires special consideration for glycaemic control as it increases risk of hypoglycaemia, BG variability and thus mortality. Insulin sensitivity variability is a key contributor to gly- caemic variability. Sechterberger et al. [28] showed an as- sociation between high glycaemic variability and mortality is not present in diabetic cohorts. Thus, a subgroup ana- lysis of diabetic OHCA patients in this study would be very interesting. However, only 26 of 200 (13%) OHCA patients in this study had previously diagnosed diabetes (Table 1), which is too few to enable a reliable analysis with these methods. Additionally, in this particular cohort, undiagnosed diabetes or impaired glucose regulation [29] may confound such a subgroup. Authors’ contributions ASP processed raw patient data from conventional spreadsheet into Matlab format, prepared statistical analysis and summarized results for interpretation. ASP, JGC, CGP, SP, and TD drafted the manuscript, performed analysis and interpretation of the data and critical revision of the manuscript for important intellectual content. GS provided clinical insight and supervised the acquisition of clinical data from Christchurch ICU patients. JCP, FT, JLV and MO provided clinical insight and supervised the acquisition of clinical data from Erasme and Lausanne ICU patients. All authors read and approved the final draft of the manuscript. Author details 1 1Centre for Bio-Engineering, Department of Mechanical Engineering, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140, New Zealand. 2Department of Intensive Care, Christchurch Hospital, Riccarton Avenue, Christchurch 8140, New Zealand. 3Department of Intensive Care, Erasme University Hospital (CUB), University of Brussels, Route de Lennik 808, 1070 Brussels, Belgium. 4Department of Intensive Care, Lausanne University Hospital (CHUV), Rue du Bugnon 46, 1011 Lausanne, Switzerland. 5Cardiovascular Research Center, Universite de Liege, Allée du 6 Août 17, B4000 Liege, Belgium. Two main conclusions are drawn as a result for these cohorts. i) SI level is much lower during TH and consistently increases over time, during both cool and warm periods. ii) Insulin sensitivity is more variable during the cool period and shows contrasting behaviour during the cool to warm transition period between 18 and 30 hours, which indicates that there are major changes in physiology and metabolic conditions between cool and warm as influenced by human body temperature. Otherwise, it decreases over time. Received: 25 May 2014 Accepted: 10 October 2014 Received: 25 May 2014 Accepted: 10 October 2014 Insulin sensitivity variability Table 5 Reductions in the interquartile range and median SI per patient range of hour-to-hour percentage SI change over time during cool and warm periods as per six-hour blocks of data, where the P values compare successive six-hour blocks as shown in the first column for both the overall cohort and per-patient median values SI variability Cohort analysis Per-patient analysis analysis % reduction of IQR P value % median decrease P value [6-hr blocks] Block 1–2 (C) 11.1 <0.05 33.6 <0.05 (0–6 vs. 6–12 hr) Block 2–3 (C) 20.7 <0.05 15.8 <0.05 (6–12 vs. 12–18 hr) Block 3–4 (C) 14.4 <0.05 22.6 <0.05 (12–18 vs. 18–24 hr) Block 4–5 (C-W) −19.7 <0.05 −14.9 <0.05 (18–24 vs. 24–30 hr) Block 5–6 (W) 23.1 <0.05 26.4 0.05 (24–30 vs. 30–36 hr) Block 6–7 (W) 4.6 <0.05 0.8 0.05 (30–36 vs. 36–42 hr) Block 7–8 (W) 13.0 0.08 17.1 0.06 (36–42 vs. 42–48 hr) P values are calculated using the Kolmogorov-Smirnov test. SI, insulin sensitivity metric (model-based); IQR, interquartile range. Table 5 Reductions in the interquartile range and median SI per patient range of hour-to-hour percentage SI change over time during cool and warm periods as per six-hour blocks of data, where the P values compare successive six-hour blocks as shown in the first column for both the overall cohort and per-patient median values There are several ways that this low and variable insulin sensitivity could be managed during glycaemic control. Re- ducing exogenous insulin doses, coupled with modulation of the glucose content of nutrition would diminish the im- pact of sudden changes of insulin sensitivity on glycaemic outcome. Equally, increased BG measurement frequency could improve control and reduce glycaemic variability. Accepting higher glycaemic targets during periods of Page 7 of 8 Page 7 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 References Brunkhorst FM, Engel C, Bloos F, Meier-Hellmann A, Ragaller M, Weiler N, Moerer O, Gruendling M, Oppert M, Grond S, Olthoff D, Jaschinski U, John S, Rossaint R, Welte T, Schaefer M, Kern P, Kuhnt E, Kiehntopf M, Hartog C, Natanson C, Loeffler M, Reinhart K, S German Competence Network: Intensive insulin therapy and pentastarch resuscitation in severe sepsis. N Engl J Med 2008, 358:125–139. 24. Pretty CG, Signal M, Fisk L, Penning S, Le Compte A, Shaw GM, Desaive T, Chase JG: Impact of sensor and measurement timing errors on model-based insulin sensitivity. Comput Methods Programs Biomed 2014, 114:e79–e86. 25. Hann CE, Chase JG, Lin J, Lotz T, Doran CV, Shaw GM: Integral-based parameter identification for long-term dynamic verification of a glucose- insulin system model. Comput Methods Programs Biomed 2005, 77:259–270 9. Finfer S, Chittock DR, Su SY, Blair D, Foster D, Dhingra V, Bellomo R, Cook D, Dodek P, Henderson WR, Hebert PC, Heritier S, Heyland DK, McArthur C, McDonald E, Mitchell I, Myburgh JA, Norton R, Potter R, Robinson BG, Ronco JJ: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283–1297. 26. Bagshaw SM, Egi M, George C, Bellomo R, Australia New Zealand Intensive Care Society Database Management C: Early blood glucose control and mortality in critically ill patients in Australia. Crit Care Med 2009, 37:463–470. 27. Langouche L, Vander Perre S, Wouters PJ, D'Hoore A, Hansen TK, Van den Berghe G: Effect of intensive insulin therapy on insulin sensitivity in the critically ill. J Clin Endocrinol Metab 2007, 92:3890–3897. 10. Preiser JC, Devos P, Ruiz-Santana S, Melot C, Annane D, Groeneveld J, Iapichino G, Leverve X, Nitenberg G, Singer P, Wernerman J, Joannidis M, Stecher A, Chiolero R: A prospective randomised multi-centre controlled trial on tight glucose control by intensive insulin therapy in adult intensive care units: the Glucontrol study. Intensive Care Med 2009, 35:1738–1748. y 28. Sechterberger MK, Bosman RJ, Oudemans-van Straaten HM, Siegelaar SE, Hermanides J, Hoekstra JB, De Vries JH: The effect of diabetes mellitus on the association between measures of glycaemic control and ICU mortality: a retrospective cohort study. Crit Care 2013, 17:R52. 11. References Chase JG, Le Compte AJ, Suhaimi F, Shaw GM, Lynn A, Lin J, Pretty CG, Razak N, Parente JD, Hann CE, Preiser JC, Desaive T: Tight glycemic control in critical care–the leading role of insulin sensitivity and patient variability: a review and model-based analysis. Comput Methods Programs Biomed 2011, 102:156–171. 29. Bartnik M, Ryden L, Ferrari R, Malmberg K, Pyorala K, Simoons M, Standl E, Soler-Soler J, Ohrvik J, Euro Heart Survey I: The prevalence of abnormal glucose regulation in patients with coronary artery disease across Europe. The Euro Heart Survey on diabetes and the heart. Eur Heart J 2004, 25:1880–1890. 29. Bartnik M, Ryden L, Ferrari R, Malmberg K, Pyorala K, Simoons M, Standl E, Soler-Soler J, Ohrvik J, Euro Heart Survey I: The prevalence of abnormal glucose regulation in patients with coronary artery disease across Europe. The Euro Heart Survey on diabetes and the heart. Eur Heart J 2004, 25:1880–1890. 12. Taylor NA, Griffiths RF, Cotter JD: Epidemiology of hypothermia: fatalities and hospitalisations in New Zealand. Aust N Z J Med 1994, 24:705–710. 13. Neumar RW, Nolan JP, Adrie C, Aibiki M, Berg RA, Bottiger BW, Callaway C, Clark RS, Geocadin RG, Jauch EC, Kern KB, Laurent I, Longstreth WT, Merchant MR Jr, Morley P, Morrison LJ, Nadkarni V, Peberdy MA, Rivers EP, Rodriguez-Nunez A, Sellke FW, Spaulding C, Sunde K, Vanden Hoek T: Post-cardiac arrest syndrome: epidemiology, pathophysiology, treatment, and prognostication. A consensus statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian and New Zealand Council on Resuscitation, European Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, Resuscitation Council of Asia, and the Resuscitation Council of Southern Africa); the American Heart Association Emergency Cardiovascular Care Committee; the Council on Cardiovascular Surgery and Anesthesia; the Council on Cardiopulmonary, Perioperative, and Critical Care; the Council on Clinical Cardiology; and the Stroke Council. Circulation 2008, 118:2452–2483. doi:10.1186/s13054-014-0586-x Cite this article as: Sah Pri et al.: Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia. Critical Care 2014 18:586. doi:10.1186/s13054-014-0586-x Cite this article as: Sah Pri et al.: Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia. Critical Care 2014 18:586. 14. References 1. Capes SE, Hunt D, Malmberg K, Gerstein HC: Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: a systematic overview. Lancet 2000, 355:773–778. y 2. McCowen KC, Malhotra A, Bistrian BR: Stress-induced hyperglycemia. Crit Care Clin 2001, 17:107–124. 3. Mizock BA: Alterations in fuel metabolism in critical illness: hyperglycaemia. Best Pract Res Clin Endocrinol Metab 2001, 15:533–5 Finally, this study shows the need for patient-specific glycaemic management to ensure good control and safety during treatment. These results have significant potential clinical impact on the metabolic treatment of these patients, and changes in clinical therapy are re- quired to safely treat patients as they transition from cool to warm. hyperglycaemia. Best Pract Res Clin Endocrinol Metab 2001, 15:533 551. 4. van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P, Bouillon R: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359–1367. 5. Krinsley JS: Association between hyperglycemia and increased hospital mortality in a heterogeneous population of critically ill patients. Mayo Clin Proc 2003, 78:1471–1478. 6. Krinsley JS: Effect of an intensive glucose management protocol on the mortality of critically ill adult patients. Mayo Clin Proc 2004, 79:992–1000. Page 8 of 8 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 Sah Pri et al. Critical Care 2014, 18:586 http://ccforum.com/content/18/6/586 7. Chase JG, Shaw G, Le Compte A, Lonergan T, Willacy M, Wong XW, Lin J, Lotz T, Lee D, Hann C: Implementation and evaluation of the SPRINT protocol for tight glycaemic control in critically ill patients: a clinical practice change. Crit Care 2008, 12:R49. 7. Chase JG, Shaw G, Le Compte A, Lonergan T, Willacy M, Wong XW, Lin J, Lotz T, Lee D, Hann C: Implementation and evaluation of the SPRINT protocol for tight glycaemic control in critically ill patients: a clinical practice change. Crit Care 2008, 12:R49. secretion test–a novel measure of insulin sensitivity. Metabolism 2011, 60:1748–1756. 23. Fisk LM, Le Compte AJ, Shaw GM, Penning S, Desaive T, Chase JG: STAR development and protocol comparison. IEEE Trans Biomed Eng 2012, 59:3357–3364. 8. doi:10.1186/s13054-014-0586-x Cite this article as: Sah Pri et al.: Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia. Critical Care 2014 18:586. References Pretty CG, Le Compte AJ, Chase JG, Shaw GM, Preiser JC, Penning S, Desaive T: Variability of insulin sensitivity during the first 4 days of critical illness: implications for tight glycemic control. Ann Intensive Care 2012, 2:17. 15. Eisenburger P, Sterz F, Holzer M, Zeiner A, Scheinecker W, Havel C, Losert H: Therapeutic hypothermia after cardiac arrest. Curr Opin Crit Care 2001, 7:184–188. 16. Lee R, Asare K: Therapeutic hypothermia for out-of-hospital cardiac arrest. Am J Health Syst Pharm 2010, 67:1229–1237. 17. Cueni-Villoz N, Devigili A, Delodder F, Cianferoni S, Feihl F, Rossetti AO, Eggimann P, Vincent JL, Taccone FS, Oddo M: Increased blood glucose variability during therapeutic hypothermia and outcome after cardiac arrest. Crit Care Med 2011, 39:2225–2231. 18. Hayashi N: Management of pitfalls for the successful clinical use of hypothermia treatment. J Neurotrauma 2009, 26:445–453. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 19. Chase JG, Suhaimi F, Penning S, Preiser JC, Le Compte AJ, Lin J, Pretty CG, Shaw GM, Moorhead KT, Desaive T: Validation of a model-based virtual trials method for tight glycemic control in intensive care. Biomed Eng Online 2010, 9:84. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 20. Evans A, Shaw GM, Le Compte A, Tan CS, Ward L, Steel J, Pretty CG, Pfeifer L, Penning S, Suhaimi F, Signal M, Desaive T, Chase JG: Pilot proof of concept clinical trials of Stochastic Targeted (STAR) glycemic control. Ann Intensive Care 2011, 1:38. References • Convenient online submission 21. Lin J, Razak NN, Pretty CG, Le Compte A, Docherty P, Parente JD, Shaw GM, Hann CE, Geoffrey Chase J: A physiological Intensive Control Insulin- Nutrition-Glucose (ICING) model validated in critically ill patients. Comput Methods Programs Biomed 2011, 102:192–205. 22. McAuley KA, Berkeley JE, Docherty PD, Lotz TF, Te Morenga LA, Shaw GM, Williams SM, Chase JG, Mann JI: The dynamic insulin sensitivity and
https://openalex.org/W3020933413	https://europepmc.org/articles/pmc7255551?pdf=render	English	null	COVID-19 Pandemic: A Neurological Perspective	Curēus	2,020	cc-by	5,464	Open Access Review Article Open Access Review Article Abstract Even though severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been observed to principally affect the respiratory system, neurological involvements have already been reported in some published work. We have reviewed original articles, case reports, and existing open-source data-sets to delineate the spectrum of neurological disorders potentially observed in SARS-CoV-2 positive cases. Neurological involvement in coronavirus disease 2019 (COVID- 19) corresponds to three situations: (a) neurological manifestations of viral infection, (b) post- infective neurological complications, and (c) infection in patients with neurological co- morbidity. Neurological manifestations can further be subdivided into the central nervous system (headache, dizziness, alteration of the sensorium, ataxia encephalitis, stroke, and seizures) and peripheral nervous system (skeletal muscle injury and peripheral nerve involvement including hyposmia and hypogeusia) symptomatology. Post-infective neurological complications include demyelinating conditions. Reduced mobility and dementia as co- morbidities may predispose a patient to have a viral infection. It is concluded that the pandemic of COVID-19 presents for a neurologist some unique challenges. We observe that SARS-CoV-2 may have various neurological manifestations and in many cases, neurological features may precede typical respiratory symptoms. Categories: Neurology, Infectious Disease Keywords: covid-19, neurological manifestations, viral infection COVID-19 Pandemic: A Neurological Perspective Durjoy Lahiri , Alfredo Ardila 1 2 1. Neurology, Institute of Post Graduate Medical Education & Research, Kolkata, IND 2. Neuropsychology, I.M. Sechenov First Moscow State Medical University, Moscow, RUS Corresponding author: Alfredo Ardila, ardilaalfredo@gmail.com Introduction And Background Coronavirus disease 2019 (COVID-19) has been declared a pandemic on the 11th of March, 2020 by the World Health Organization. The epicenter of this pandemic has shifted in quick succession from China to Europe to the United States of America in a matter of weeks. Since the middle of March 2020, South-east Asia has also seen a rise in the number of affected persons and it remains to be seen if there will be another twist in the story of this pandemic. One of the essential weapons to fight a pandemic of this stature is to gather as much knowledge as possible about the transmission dynamics and clinical manifestations while the quest for an effective vaccine keeps continuing. Received 04/14/2020 Review began 04/20/2020 Review ended 04/21/2020 Published 04/29/2020 DOI: 10.7759/cureus.7889 COVID-19 Pandemic: A Neurological Perspective Lahiri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received 04/14/2020 Review began 04/20/2020 Review ended 04/21/2020 Published 04/29/2020 Mechanism of neuro-invasion The putative mechanisms described to explain the neuro-invasion by a coronavirus (and one similar another RNA virus, influenza A) are hematogenous spread and retrograde axonal transport [1-4]. However, in light of contemporary evidence, some of the other possible routes of neuroinvasion by the SARS-CoV-2 deserve to be mentioned. Direct viral invasion of the brain leading to clinical encephalitis has been suspected after the treatment team of Beijing Ditan Hospital confirmed the presence of SARS-CoV-2 in the cerebrospinal fluid (CSF) of patients with COVID-19 by genome sequencing [5]. COVID-19 is widely known to cause respiratory insufficiency, and therefore, hypoxia needs to be considered among the major putative mechanisms of brain injury [6]. Cytokine storm, which is a well known immune reaction of this particular viral infection, may lead to inflammation and injury of the central nervous system (CNS) tissue. This idea is further supported by the observation that interleukin (IL)-6, an important member of the cytokine storm, is positively correlated with the severity of COVID-19 symptoms [7]. The affinity of the viral particle towards angiotensin-converting enzyme-2 (ACE- 2), a cardio-cerebral vascular protection factor, has been in discussion in recent papers [8]. The expression of ACE-2 in the nervous system and skeletal muscles can indeed explain some of the neurological features reported so far. It has been postulated that the viral attachment to the ACE-2 at the level of the blood-brain barrier may jeopardize the protective mechanism surrounding the encephalon, giving way to viral encephalitis. In a similar vein, spinal cord membranes expressing ACE-2 can culminate into myelitis-like features following SARS-CoV-2 infection. Concern has also been raised that the viral particles binding to ACE-2 in cerebral blood vessels may actually raise the luminal pressure of those vessels leading to intracerebral hemorrhage [9]. Thus there are multiple mechanisms elaborated so far in the available literature that can explain the observed as well as anticipated neurological features of this ailment. Classification and features of neurological involvement Early data on COVID-19 suggest neurological involvement in a variable percentage of cases with particular expression in more severe patients [10]. Neurological involvement in COVID-19 can be discussed in three sections: (1) neurological features of viral infection, (2) post-infective neurological complications, (3) infection in patients with neurological co-morbidity. Another issue that deserves to be mentioned is the precautions to be taken for neurologically ill patients who require immunosuppressive agents. The latter group mostly consists of multiple sclerosis, myasthenia gravis, and autoimmune encephalitis. © Copyright 2020 Even though severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been observed to mainly affect the respiratory system, neurological involvements have already been reported in some published work. Several medical news bulletins, blogs, and articles across the globe have also raised concerns about brain invasion by this particular strain of coronavirus. This is actually not surprising given our previous experience of neuro-invasion by severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome- related coronavirus (MERS-CoV). The body of literature on neurological aspects of SARS-CoV-2 Lahiri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. How to cite this article Lahiri D, Ardila A (April 29, 2020) COVID-19 Pandemic: A Neurological Perspective. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 is small but growing. However, we believe, an organized summary of the available information at this point would be indispensable to neurologists across the globe. Being well informed about the neurological presentations would not only help them have a high index of clinical suspicion but also take necessary precautions. Therefore, a brief literature review along with a critical appraisal of the evidence gathered so far is presented here from the perspective of a neurologist. In the present paper, we have reviewed the recently published or pre-print original articles, case reports, and existing open-source data-sets in order to delineate the spectrum of neurological disorders in SARS-CoV-2 positive cases. 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 Therefore, neurologist needs to pay attention and analyze even a simple symptom such as headache in a known COVID-19 case, especially if the headache is out of proportion to rise in temperature and is associated with certain other features such as vomiting and altered sensorium. Another noteworthy observation is the occurrence of cerebrovascular events (CVE) associated with this ailment. The initial retrospective case series study from Wuhan, China, reported that 5.7% (5/76) of the cases showing neurological involvement could be attributed to acute CVE [10]. Notably, four cases had an ischemic stroke, while cerebral hemorrhage was found in a single patient who died later on. Another recent paper from the same center, which analyzed 221 participants, elaborates that 5.88% (13/221) cases had some sort of new-onset CVE [16]. The majority of them presented acute ischemic stroke (11 patients), while hemorrhagic stroke (one patient) and cerebral venous sinus thrombosis (one patient) were also found but uncommonly. Therefore, thrombotic manifestations were way more frequent than hemorrhage. This observation may possibly be linked to the finding that patients with CVE were more likely to have enhanced inflammatory response as reflected in their C-reactive protein (CRP) and D- dimer levels. It is within the realm of possibilities that the viral infection may have given way to an inflammatory storm that ultimately culminated in accelerated thrombosis. The term "accelerated thrombosis" seems more meaningful if seen in the context of another finding in this particular retrospective study. Patients with CVE were not only significantly older than those without CVE but also were more likely to have pre-existent vascular risk factors. This observation is particularly relevant in the Indian scenario because vascular risk factors are common in the population, and so is the incidence of stroke. Moreover, any viral infection in a stroke patient not only delays recovery but also may actually worsen the neurological deficit, the latter being sometimes attributed to hemorrhagic transformation. The mortality rate was indeed found to be higher in COVID-19 stroke patients, with 38% indicating a worse prognosis in this group of patients. The above discussion brings out the two-way relationship between COVID-19 and stroke, which a neurologist has to bear in mind while attending patients in these difficult circumstances. The issue becomes more complicated when one considers treating a patient with ischemic stroke in the background of coronavirus infection. Neurological features of viral invasion Neurological manifestations of viral infection reported so far can further be subdivided into CNS and peripheral nervous system (PNS) features. CNS features include headache, dizziness, ataxia, alteration of sensorium, encephalitis, stroke, and seizures, while PNS features mostly 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 2 of 9 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 refer to skeletal muscle injury and peripheral nerve involvement in the form of hyposmia and hypogeusia. Headache can be a symptom of viral infection and usually remains associated with fever. Studies have reported an incidence of headaches ranging from 6 to 13% in COVID-19 cases [10- 13]. However, concerns have already been raised in recent correspondence if this particular symptom is a manifestation of viral meningitis or, for that matter, encephalitis, which may reveal itself subsequently in the form of drowsiness and seizures. Japanese colleagues shared their recent experience in dealing with a young male patient without any contact or travel history who presented features of meningitis before being diagnosed with the infection of SARS-CoV-2 [14]. The issue becomes more substantial as one considers the report of virus detection in CSF of a COVID-19 patient. Another very recent report describes the occurrence of acute hemorrhagic necrotizing encephalopathy in COVID-19 patient [15]. In this particular case, however, the CSF examination for SARS-CoV-2 was not carried out, although bacterial culture and tests for other viruses were essentially negative. Therefore, neurologist needs to pay attention and analyze even a simple symptom such as headache in a known COVID-19 case, especially if the headache is out of proportion to rise in temperature and is associated with certain other features such as vomiting and altered sensorium. Headache can be a symptom of viral infection and usually remains associated with fever. Studies have reported an incidence of headaches ranging from 6 to 13% in COVID-19 cases [10- 13]. However, concerns have already been raised in recent correspondence if this particular symptom is a manifestation of viral meningitis or, for that matter, encephalitis, which may reveal itself subsequently in the form of drowsiness and seizures. Japanese colleagues shared their recent experience in dealing with a young male patient without any contact or travel history who presented features of meningitis before being diagnosed with the infection of SARS-CoV-2 [14]. The issue becomes more substantial as one considers the report of virus detection in CSF of a COVID-19 patient. Another very recent report describes the occurrence of acute hemorrhagic necrotizing encephalopathy in COVID-19 patient [15]. In this particular case, however, the CSF examination for SARS-CoV-2 was not carried out, although bacterial culture and tests for other viruses were essentially negative. (addressed to their US colleagues) mentions the importance of recognizing poorly defined neurological features in confirmed as well as presumptive cases of COVID-19. Among these symptoms, altered sensorium indeed has drawn the attention of the neurologists mostly because it delays diagnostic procedures as the virus is widely known to be a respiratory pathogen. A detailed look at the description of a single case of encephalopathy in association with COVID-19 reveals that an altered level of consciousness may precede typical respiratory symptoms by days [17]. Given the transmission dynamics of the infection, this information is extremely important as health care providers at the emergency department may get inadvertent exposure while handling patients with altered sensorium. In a consecutive series of COVID-19 related acute respiratory distress syndrome (ARDS) patients (n=58) reported from France, altered consciousness, including agitation and confusion, has been documented in more than two-thirds of the cases. Additionally, 67% of the recruited patients in this study had prominent cortico-spinal signs [18]. In this context, telephonic conversation with the physician in charge of an infectious disease hospital in Kolkata (India) reveals that headache is more of a generalized symptom in both old and young patients, while delirium is a frequent presentation among the elderly. The hospital presently is catering to more than 60 confirmed cases of COVID-19, while around 250 presumptive cases are in isolation. In the Wuhan study, ataxia was found in only one patient, although a detailed description or anatomical substrate of this particular symptom was not available in this paper [10]. We at our center recently encountered a 72-year-old man presenting with acute onset cerebellar ataxia followed by encephalopathy, who was finally diagnosed with SARS-CoV-2 induced pneumonia. Notably, in our patient neurological manifestation preceded respiratory features by days. The association of seizure and COVID-19 can be multi-faceted. An early report suggested a very low incidence (0.5%) of seizure disorder [10]. However, as numbers start growing across the globe, this issue is anticipated to become a non-negligible one. Firstly, a seizure may be a manifestation of viral invasion into the CNS. Secondly, this particular infection is known to cause fatal pneumonia that gives way to severe hypoxemia, which may result in brain injury and seizures thereof. Metabolic perturbations and septic encephalopathy are some of the other concerns that need to be taken care of while attending a patient with seizure. The application of antiplatelets and anticoagulants is tricky because the virus is known to cause prominent respiratory involvement, and according to some authors, the involvement of the nervous system may be partly responsible for respiratory impairment. Impaired consciousness has so far been reported in 7.5% hospitalized patients of COVID-19 [10]. Severely affected patients are more likely to present impaired consciousness. There can be multiple underlying reasons for a patient with COVID-19 to present with altered sensorium, which include viral encephalitis, metabolic perturbation, infectious toxic encephalopathy, seizures with post-ictal confusion, and stroke (either strategic area involvement or large lesion with edema). A recent document, (published in Neurology Today Online) by Italian neurologists 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 3 of 9 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 Post-infective neurological complications With more number of patients recovering from the SARS-CoV-2 infection, it is imperative that post-infective complications would draw attention with time. CNS demyelinations have been documented previously following coronavirus infection [20]. An early report of Guillain-Barre syndrome (GBS) is available from China, although there is a concern regarding the causality in this particular case [21]. The patient developed typical symptoms of SARS-CoV-2 infection after seven days of hospitalization for GBS. Retrospective analysis, however, supports that she might have been harboring the infection since the beginning, as reflected in her blood counts (lymphocytopenia and thrombocytopenia). The authors duly speculate that the initial symptoms may have been too mild to be detected in this patient (as fever is present in less than half of the cases during the initial phase). A very recent correspondence describes five cases of GBS collected from three hospitals of northern Italy among 100 to 1200 cases of SARS-CoV-2 infection over three weeks [21]. Three of these cases fit criteria for the axonal variant of GBS, while the remaining two had prolonged distal latencies suggesting demyelinating neuropathy. Although each of these cases had a usual latency of five to ten days before the onset of neurological symptoms, one of the patients, similar to the previous one reported from China, was found negative on viral reverse transcription-polymerase chain reaction (RT-PCR) at the outset only to be detected positive subsequently. This observation is ominous because of two reasons: (1) there is a chance of inadvertent exposure to the infectious virus in the neurology ward both for attending health care staffs and other patients; (2) GBS is a disorder known to rapidly affect respiratory muscles particularly if bulbar involvement sets in which can cause sudden poorly explained worsening of a patient's status if the diagnosis of COVID-19 has not already been established. Another report from China describes a case of acute myelitis, possibly affecting the cervical spinal cord, as evidenced by the clinical features, in a known patient of SARS-CoV-2 infection [22]. In this particular description also the neurological symptoms were co-incident with the febrile period of the illness pointing towards para-infectious demyelination rather than post- infective complication in true sense. Lymphocytopenia accompanied by raised markers of inflammation (CRP and procalcitonin) was documented by the treating physicians. The patient received anti-viral therapy along with immune-suppressive and recovered from his limb weakness. Further studies will be required to elucidate the mechanisms underlying skeletal muscle injury in COVID-19. It has been reported that hypogeusia, as well as hyposmia, are fairly consistent symptoms of SARS-CoV-2 infection [10, 19]. Hypoplasia has also been reported but infrequently. These are all categorized as manifestations of peripheral nerve involvement, while another reported feature is neuralgia. However, noteworthy is that the olfactory nerve is considered part of the CNS, and hyposmia may actually be a reflection of olfactory bulb involvement rather than peripheral neuropathy. Similarly, hypoplasia, if due to optic neuropathy, may reflect CNS manifestation because optic nerve, as per classical teaching, is an extension of the brain. Post-infective neurological complications Although the literature is scarce at this point in time, the idea that SARS-CoV-2 can cause para-/post-infective complications affecting the neuroaxis at different levels seems realistic, and supposedly patients with the inflammatory storm will be more likely to manifest this. Additionally, since about 10% of hospitalized patients need assistance in intensive care wards, neurological monitoring must also be aimed at verifying the onset of the so-called "critical illness neuro-myopathy" type PNS problems. These issues are known to delay weaning from ventilation and pose a significant burden on the health care delivery system. The latter causes belong to the so-called group of "acute symptomatic seizure". Thirdly, known epileptic patients with COVID-19 may experience increased frequency and severity of seizures, particularly because of threshold lowering that remains associated with fever. A patient (mentioned above) with the encephalopathic presentation of COVID-19 had underlying gliosis in the right temporal region resulting from an old embolic stroke. He received prophylactic antiepileptic given the probability of subclinical seizures in the background of a structural brain lesion [17]. Seizures supposedly would complicate the clinical situation by the agency of skeletal muscle injury, which is already a well-known manifestation of the disease. The fact that neurological complications are more frequent in severely ill patients and also cardiovascular risk factors are predictors of severity, the drug interaction potential of several antiepileptic drugs would deserve attention in such clinical situations. Therefore from a neurologist's perspective, seizure in a COVID-19 patient will have some important implications both from diagnostic as well as therapeutic perspectives. Symptoms of skeletal muscle damage often associated with liver and kidney involvement have already been noted. The reported incidence is 10.7%, and like other neurological features, this one is also associated with a severe form of the illness [10]. It can be speculated that patients with pre-existing renal or hepatic impairment will be highly vulnerable to develop features of multi-organ failure in the backdrop of skeletal muscle injury. Muscle enzymes, including creatine kinase and lactate dehydrogenase (LDH), are seen to be highly elevated in the symptomatic patients - an observation that confirms muscle membrane damage. The exact mechanism of muscle damage, however, has not been established. Possibilities include viral muscle invasion through ACE-2 receptor tropism and immune-mediated muscle fiber damage. 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 4 of 9 The data so far on COVID-19 reveals that old age, as well as immune-dysregulated subjects, are more likely not only to acquire the infection but also to manifest increased severity. Therefore, similar to the oncology ward, patients in the neurology ward admitted receiving cyclic immunosuppressive drugs need attention. Appropriate caution has to be practiced while dealing with such cases both from the part of the neurologist as well as the patient's relatives. A recent article on this topic recommends that the benefits of continuing immunotherapy in patients with multiple sclerosis (MS) and related Contemporary neurology has seen wide use of immune-suppressive agents to combat several disorders of both CNS and PNS. The prototypical among these disorders is multiple sclerosis that calls for long term immunosuppression. The data so far on COVID-19 reveals that old age, as well as immune-dysregulated subjects, are more likely not only to acquire the infection but also to manifest increased severity. Therefore, similar to the oncology ward, patients in the neurology ward admitted receiving cyclic immunosuppressive drugs need attention. A i i h b i d hil d li i h h b h f h f h Patients with neurological co-morbidity During this hour of the pandemic, another group of patients will deserve the attention of 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 5 of 9 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 neurologists. This group comprises of neurologically ill patients who have already been in follow-up for some time. The need for different groups of patients will vary according to the nature of their illness. Data is insufficient at this point in time to conclude if chronic neurology patients are more predisposed to acquire infection. However, patients with reduced mobility and those on immune-suppression therapy may be anticipated to be more susceptible to infection. Recent correspondence has already discussed the challenges that will be faced by dementia patients during this period of social distancing and home isolation [23]. Obviously the crisis will be more for those subjects who depend on others for daily life activities. The situation will be similar for patients with prominent motor problems. Due to their restricted mobility, social distancing is supposed to affect them for worse. Reduced mobility and dementia may also predispose a patient to have a viral infection. So once again, the association becomes two-way. Contemporary neurology has seen wide use of immune-suppressive agents to combat several disorders of both CNS and PNS. The prototypical among these disorders is multiple sclerosis that calls for long term immunosuppression. The data so far on COVID-19 reveals that old age, as well as immune-dysregulated subjects, are more likely not only to acquire the infection but also to manifest increased severity. Therefore, similar to the oncology ward, patients in the neurology ward admitted receiving cyclic immunosuppressive drugs need attention. Appropriate caution has to be practiced while dealing with such cases both from the part of the neurologist as well as the patient's relatives. A recent article on this topic recommends that the benefits of continuing immunotherapy in patients with multiple sclerosis (MS) and related disorders may outweigh the risks of medication withdrawal in the apprehension of COVID-19. This is particularly because most infections, as in the general population, are anticipated to be mild an2d self-limiting. However, the authors emphasize the need for individualized decision- making in such circumstances because one size" may not fit all, and some of the patients may land up in severe infection leading to discontinuation of therapy [24]. Contemporary neurology has seen wide use of immune-suppressive agents to combat several disorders of both CNS and PNS. The prototypical among these disorders is multiple sclerosis that calls for long term immunosuppression. Acknowledgements We are sincerely thankful to Stefano F. Cappa (Professor of Neurology, University School for Advanced Studies, IUSS-Pavia) and Yutaka Tanaka (Professor of Neurology, Tanaka Clinic, Nara, Japan) for their valuable inputs in preparing the present version of the manuscript. Long term impact With the growing number in recovery from SARS-CoV-2 infection, rehabilitation issue is supposed to become a crucial one. Personal communication with neurologists working in the field in Italy reveals that there is early evidence for the need for rehabilitation, including neurological aspects, in clinically recovered patients. It can be assumed that the psycho-social effects of long term social distancing and home isolation will require adequate psychological rehabilitation measures as the pandemic will start waning off. Since the onset of the SARS-CoV-2 outbreak, if there is one pulmonary manifestation that has received maximum focus, it is ARDS. Evidence suggests a significant percentage of ARDS survivors may suffer long-term cognitive impairment [25]. Several factors, including mechanical ventilation, have been observed to cause a decline in higher brain functions following ARDS. Acute injury to the blood-brain barrier has been implicated as the underlying mechanism for cognitive impairment following ARDS. The effect of such injury may be amplified if there is a pre-existing cognitive impairment that corresponds to chronic blood- brain barrier damage. Patients with brain injury, on the other hand, have been found to develop neurogenic pulmonary edema. Therefore, it is postulated that the so-called brain-lung axis works both ways. The above observations are particularly relevant in the present circumstances, given the need for mechanical ventilation in the majority of the severely affected COVID-19 patients. As the pandemic continues to unfold, the number of people getting off mechanical ventilation will rise, and long-term cognitive outcomes will come into view. It can be anticipated that not only we shall witness cognitive decline lasting for months in this group of patients, but also some of them may progress to premature onset of dementia. 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 6 of 9 Disclosures Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Over time, the need to raise awareness among the neurologists is being increasingly recognized. The case record protocol for "First Few Cases (FFX) and their close contacts" published by WHO has a separate column to mention the neurological findings in addition to respiratory signs and symptoms [26]. This underlines the importance of recognizing the neurological manifestation of the illness. A recently published guideline from China lists some important precautionary measures that need to adapt by the neurologists to combat the pandemic [27]. The bottom line is to have a high index of clinical suspicion, particularly when working in an endemic zone. This will not only help early initiation of therapy but also will halt the chain of transmission. 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 Conclusions The pandemic of COVID-19 presents for a neurologist some unique challenges. We observe that SARS-CoV-2 may have various neurological manifestations, and in many cases, neurological features may precede typical respiratory symptoms. Holistic knowledge of the spectrum of neurological consequences of COVID-19 is important to get a hold on the spread of the virus. The most vital clinical information which we gather is that impaired consciousness may be a presenting feature of COVID-19, and therefore, a high index of suspicion for such patients will be the key to prevent or, at least, lessen exposure to health care providers and other patients. With the gradual subsidence of the outbreak, it can be anticipated that several post- infectious complications will surface up while rehabilitation measures will also deserve attention. Adequate caution has to be practiced while managing chronic neurology patients, particularly those requiring immune-modulator therapy since formulated guidelines are lacking at this point. The above summary of the neurological manifestations of COVID-19 will help neurologists have a basic preparation, which is of utmost importance to prevent horizontal infections. References 1. Desforges M, LeCoupanec A, Dubeau P, et al.: Human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system?. Viruses. 2020, 12:14. 10.3390/v12010014 2. Bohmwald K, Galvez N, Ríos M, Kalergis AM: Neurologic alterations due to respiratory virus infections. Front Cell Neurosci. 2018, 12:386. 10.3389/fncel.2018.00386 3. Mori I: Transolfactory neuroinvasion by viruses threatens the human brain . Acta Virol. 2015, 59:338-49. 10.4149/av_2015_04_338 4. Gu J, Gong E, Zhang B, et al.: Multiple organ infection and the pathogenesis of SARS . J Exp Med. 2005, 202:415-424. 10.1084/jem.20050828 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 7 of 9 5. Cai JH, Wang XS, Ge YL, et al.: First case of 2019 novel coronavirus infection in children in Shanghai (in Chinese). Chin J Pediatr. 2020, 58:E002. 10.3760/cma.j.issn.0578-1310.2020.0002 6. Guo Y, Cao Q, Hong Z, et al.: The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res. 2020, 7:11. 10.1186/s40779-020-00240-0 7. Wan SX, Yi QJ, Fan SB, et al.: Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP). MedRxiv. 2020, 10.1101/2020.02.10.20021832 8. Baig A, Khaleeq A, Ali U, et al.: Evidence of the COVID-19 virus targeting the CNS: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms. ACS Chem Neurosci. 2020, 11:995-998. 10.1021/acschemneuro.0c00122 9. Wu Y, Xu X, Chen Z, et al.: Nervous system involvement after infection with COVID-19 and other coronaviruses. Brain Behav Immun. 2020, in press: 10.1016/j.bbi.2020.03.031 10. Mao L, Wang MD, Chen SH, et al.: Neurological manifestations of hospitalized patients with COVID-19 in Wuhan, China: a retrospective case series study. Lancet Neurol. 2020, preprint:10.2139/ssrn.3544840 11. Guan WJ, Ni ZY, Hu Y, et al.: Clinical characteristics of coronavirus disease 2019 in China . N Engl J Med. 2020, 1-13. 10.1056/NEJMoa2002032 12. Chen N, Zhou M, Dong X, et al.: Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020, 395:507-513. 10.1016/S0140-6736(20)30211-7 13. Wang D, Hu B, Hu C, et al.: Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus‑infected pneumonia in Wuhan, China. JAMA. 2020, 323:1061-1069. 10.1001/jama.2020.1585 14. Poyiadji N, Shahin G, Noujaim D, Stone M, Patel S, Griffith B: COVID-19 associated acute hemorrhagic necrotizing encephalopathy: CT and MRI features. Radiology. 2020, 10.1148/radiol.2020201187 15. Moriguchi T, Harii N, Goto J, et al.: A first case of meningitis/encephalitis associated with SARS-Coronavirus-2. Int J Infect Dis. 2020, 94:55-58. 10.1016/j.ijid.2020.03.062 16. Filatov A, Sharma P, Hindi F, Espinosa PS: Neurological complications of coronavirus disease 16. Filatov A, Sharma P, Hindi F, Espinosa PS: Neurological complications of coronavirus disease (COVID-19). Cureus. 2020, 12:e7352. 10.7759/cureus.7352 16. Filatov A, Sharma P, Hindi F, Espinosa PS: Neurological com (COVID-19). Cureus. 2020, 12:e7352. 10.7759/cureus.7352 16. Filatov A, Sharma P, Hindi F, Espinosa PS: Neurological com (COVID-19). Cureus. 2020, 12:e7352. 10.7759/cureus.7352 17. Li Y, Wang T, Zhou N, et al.: Acute cerebrovascular disease following COVID- 19: a single centre, retrospective, observational study. Lancet. 2020, preprint:10.2139/ssrn.3550025 18. 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 Helms J, Kremer S, Merdji H, et al.: Neurologic features in severe SARS-CoV-2 infection . N Engl J Med. 2020, 10.1056/NEJMc2008597 19. Nath A: Neurologic complications of coronavirus infections . Neurology. 2020, 10.1212/WNL.0000000000009455 20. Yeh E, Collins A, Cohen M, Duffner PK, Faden H: Detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis. Pediatrics. 2004, 113:73-76. 10.1542/peds.113.1.e73 21. Zhao H, Shen D, Zhou H, Liu J, Chen S: Guillain-Barré syndrome associated with SARS-CoV-2 infection: causality or coincidence?. Lancet Neurol. 2020, 19:383-384. 10.1016/S1474- 4422(20)30109-5 22. Zhao K, Huang J, Dai D, Feng Y, Liu L, Nie S: Acute myelitis after SARS-CoV-2 infection: a case report. MedRxiv. 2020, 10.1101/2020.03.16.20035105 23. Wang H, Li T, Barbarino P, et al.: Dementia care during COVID-19. Lancet. 2020, 395:1190- 1191. 10.1016/S0140-6736(20)30755-8 24. Brownlee W, Bourdette D, Broadley S, Killestein J, Ciccarelli O: Treating multiple sclerosis and neuromyelitis optica spectrum disorder during the COVID-19 pandemic. Neurology. 2020, 10.1212/WNL.0000000000009507 10.1212/WNL.0000000000009507 25. Sasannejad C, Ely EW, Lahiri S: Long-term cognitive impairment after acute respiratory distress syndrome: a review of clinical impact and pathophysiological mechanisms. Crit Care. 2019, 23:352. 10.1186/s13054-019-2626-z 26. Stoecklin S, Rolland P, Silue Y, et al.: First cases of coronavirus disease 2019 (COVID-19) in France: surveillance, investigations and control measures, January 2020. Eurosurveillance. 2020, 25:10.2807/1560-7917.ES.2020.25.6.2000094 27. Jin H, Hong C, Chen S, et al.: Consensus for prevention and management of coronavirus disease 2019 (COVID-19) for neurologists. Stroke Vasc Neurol. 2020, 10.1136/svn-2020- 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 8 of 9 2020 Lahiri et al. Cureus 12(4): e7889. DOI 10.7759/cureus.7889 000382 9 of 9
https://openalex.org/W2093092324	https://europepmc.org/articles/pmc3797082?pdf=render	English	null	Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding	PloS one	2,013	cc-by	9,391	María Eugenia Alzugaray1, Mariana Laura Adami1,2, Luis Anibal Diambra3, Salvador Hernandez-Martinez4, Cristina Damborenea2, Fernando Gabriel Noriega5, Jorge Rafael Ronderos1,3* 1 Cátedra Histología y Embriología Animal, Facultad de Ciencias Naturales y Museo; Universidad Nacional de la Plata (FCNyM -UNLP), La Plata, Argentina, 2 División Zoología Invertebrados, Facultad de Ciencias Naturales y Museo; Universidad Nacional de la Plata (FCNyM-UNLP), La Plata, Argentina, 3 Centro Regional de Estudios Genómicos, Universidad Nacional de la Plata (CREG-UNLP), La Plata, Argentina, 4 Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública (CISEI-INSP), Cuernavaca, Mexico, 5 Department of Biological Sciences, Florida International University, Miami, Florida, United States of America Abstract Background: Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms. Methods: A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. Conclusions: AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion. Significance: Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion. Citation: Alzugaray ME, Adami ML, Diambra LA, Hernandez-Martinez S, Damborenea C, et al. (2013) Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding . PLoS ONE 8(10): e77520. doi:10.1371/journal.pone.0077520 Received March 27, 2013; Accepted September 3, 2013; Published October 15, 2013 Received March 27, 2013; Accepted September 3, 2013; Published October 15, 2013 garay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permi ution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2013 Alzugaray et al. This is an open-access article distributed under the terms of the Creative Commons Attribu unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financed with founds from the PICT 01287 (SECyT – Argentina) and and FCNyM-UNLP(N673) Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding María Eugenia Alzugaray1, Mariana Laura Adami1,2, Luis Anibal Diambra3, Salvador Cristina Damborenea2, Fernando Gabriel Noriega5, Jorge Rafael Ronderos1,3* María Eugenia Alzugaray1, Mariana Laura Adami1,2, Luis Anibal Diambra3, Salvador Hernandez-Martinez4, Cristina Damborenea2, Fernando Gabriel Noriega5, Jorge Rafael Ronderos1,3* October 2013 \| Volume 8 \| Issue 10 \| e77520 Introduction antibodies has been shown in the phylum Placozoa, which is probably the most ancestral group among metazoa - having no organs or specialized tissues- [2]. Non-peptidergic messengers such as epinephrine and norepinephrin have been found in Porifera [3,4] and Cnidaria [5]. In addition, serotonin has been reported in Hydrozoa, where it seems to be involved in the onset of metamorphosis [6]. Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Molecules involved in cell-cell recognition, as well as intercellular messengers, appeared early on in evolution. In fact, cellular messengers are not only present in multicellular organisms, but are also present in unicellular eukaryotes such as the insulin-like peptides of Tetrahymena pyriformis, Neurospora crassa and Aspergillus fumigate [1]. The expression of immunoreactivity against RFamide neuropeptide Cnidaria is the most ancient group of animals with differentiated tissues, represented by close to 10,000 existing aquatic species. They are located near the root of metazoan evolution and are likely the first group of animals exhibiting a nervous system [7]. In fact, two types of nerve cells have been PLOS ONE \| www.plosone.org 1 October 2013 \| Volume 8 \| Issue 10 \| e77520 1 Allatotropin: an ancestral myotropic neuropeptide (Tribolium castaneum) [50] and Lepidoptera (M. sexta) [51]. Interestingly, the detection of AT in several groups of Protostomata but apparently not in Deuterostomata suggests that this peptide could be a synapomorphic feature of Protostomata. Indeed, the presence of AT in organisms that do not undergo metamorphosis suggests that it was originally involved in myotropic activities, the induction of the synthesis of JHs being a secondary function [47]. As a first approach to understand the evolutionary origin of this pleiotropic peptide, we decided to investigate the expression of AT-like peptides in Hydra plagiodesmica (Hydroazoa: Cnidaria). A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. Immunochemical, physiological and bioinformatics evidence suggest that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion. Animals Hydra plagiodesmica hydroids were obtained from a colony maintained in dechlorinated water at 20±2°C with a 12:12 hour light/dark period. Animals were fed Artemia salina until they were processed. For physiological experiments, specimens were starved during the 72 hours prior to the beginning of the experiment. All the experiments included groups of 6 individuals for each treatment. Each specimen was kept isolated throughout the experiment. Introduction described, corresponding to sensory and motor neurons [8]. The presence of several peptidic cell messengers have been described, suggesting that peptides were the first type of molecules acting as neurotransmitters or neurohormones in the course of evolution [7]. Peptides are pleiotropic molecules with a diverse range of functions. In Hydrozoa neuropeptides induce metamorphosis [6,9] as well as differentiation of neural stem cells to neurons [10]. Neuropeptides in cnidarians are also associated with regenerative processes [11-13] (for review see also 14) and myoregulatory functions with both excitatory and inhibitory activities [7,14-18]. Nervous and endocrine systems emerged relatively early on in the history of animal evolution. Molecules, either acting as messengers with similar functions or having evolved new functions, are present in both ancestral and derived groups. For instance, peptides sharing sequence similarity with vasopressin and endothelin, two peptides originally described in vertebrates as potent vasoconstrictors, have been also found in Hydrozoa associated with nerve cell differentiation [19], developmental processes and muscle contraction [20]. Allatotropin (AT) is a peptide originally isolated from the nervous system of the lepidopteran Manduca sexta because of its ability to stimulate the secretion of juvenile hormone by the corpora allata [21]. The presence of AT was later described in numerous hemimetabolous and holometabolous insect species [22-28]. AT has pleiotropic functions; it inhibits ion transport in the midgut of M. sexta larvae [29], as well as it controls the release of digestive enzymes in the midgut of Spodoptera frugiperda [30]. In addition, the myoregulatory and cardioacceleratory role of AT has been demonstrated in various insect species [31-39], where it is secreted not only by the nervous system, but also by endocrine epithelial cells, acting in a paracrine and endocrine manner [36-38,40,41]. Finally, it has been also proposed that AT is involved in the control of circadian rhythms [40,42,43]. Immunohistochemistry and phalloidin labeling Allatotropin related peptides have been reported in other phyla beyond Arthropoda. In fact, genes codifying peptides sharing similarity with AT have been reported in the limpet Lottia gigantea (Gastropoda: Mollusca) and in the worms Capitella teleta (Polychaeta: Annelida) and Helobdella robusta (Hirudinea: Annelida) [44,45]. Recently, we have demonstrated the expression of an AT-like peptide in three species of free living flatworms (Platyhelminthes) from different groups of turbellaria (Catenulida, Macrostomorpha and Rhabdocoela), where the peptide is present in neurons morphologically and functionally associated with muscle tissues in the reproductive and digestive systems [46,47]. In fact, in the flatworm Mesostoma ehrenbergii (Rhabdocoela: Platyhelminthes), the exogenous administration of AT (10-14 and 10-12 M) induced muscular contractions, particularly in the pharynx, suggesting that it acts as a myoregulator [47]. Hydroids were fixed in formaldehyde-phosphate buffered saline (PBS) (4%) at 4 °C for 12 h. They were then washed in PBS-Tween (0.05%) (PBS-T), permeabilized in Triton X-100 (1%) (24 h at 4 °C) and blocked with 3% bovine serum albumin (BSA) for 2 h. Materials were then incubated overnight at 4°C with a polyclonal antiserum developed against allatotropin of the mosquito Aedes aegypti (1/500 in 3% BSA diluted in PBS- T). The antibody recognizes A. aegypti AT with the following sequence of amino-acids: APFRNSEMMTARGF [28]. The specificity of antibody binding was previously demonstrated by preadsorption with the cognate peptide, both in insect species [36,37,52] as well as in other invertebrate groups [46,47]. Hydroids were then incubated with a FITC-labeled goat anti- rabbit secondary antibody (Santa Cruz Biotechnology) (1/1000 in blocking-buffer) for 24 h at 4°C. To visualize the arrangement of the epithelial-muscular cell fibers, samples were co-incubated with a rhodamine-phalloidin solution (Sigma–Aldrich) (1/1000) [46,47]. After every step, hydroids were washed (3 times x 20 min) with PBS-T (0.05%). As controls, the incubation with the primary antibody was replaced with PBS. Specimens were mounted with Vectashield mounting The AT receptor was originally characterized in the silk moth Bombyx mori (Lepidoptera: Insecta) as a seven transmembrane domain protein pertaining to the family of the vertebrate orexin receptors [48]. Recently, the receptor was characterized in three additional holometabolous insect species from different orders - Diptera (Aedes aegypti) [49]; Coleoptera PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 2 Allatotropin: an ancestral myotropic neuropeptide medium (Vector Laboratories, Burlingame, CA) and analyzed with a Laser Scan Confocal Microscope Zeiss LSM 510 Meta. Immunohistochemistry and phalloidin labeling extruded was recorded. Data are expressed as percentage of the control. Identification of Allatotropin receptor sequences. A protein BLAST search was performed in GenBank using the sequence of Manduca sexta AT- receptor. A similar search was performed within the draft of the Hydra magnipapillata genome (http://hydrazome.metazome.net/cgi-bin/gbrowse/hydra/). Identification of Allatotropin receptor sequences. A protein BLAST search was performed in GenBank using the sequence of Manduca sexta AT- receptor. A similar search was performed within the draft of the Hydra magnipapillata genome (http://hydrazome.metazome.net/cgi-bin/gbrowse/hydra/). Sequences sharing a minimum of 27% identity with an e-value equal to or less than 1e-23 were then blasted against insect species. Only those sequences that presented all the complete transmembrane domains and shared more similitude between them than with the sequence of the Drosophila melanogaster fmrf-receptor (selected as outgroup) were included. A total of 22 sequences pertaining to Cnidaria, Mollusca, Arthropoda, and Nematoda were selected (Table 1). These sequences were aligned using the Clustal Wallis algorithm (http:// www.ebi.ac.uk/Tools/msa/clustalw2/) and further analyzed by the JalView 2.7 [53]. A phylogram was constructed using a Neighbor Joining method using the Blocks of Amino Acid Substitution Matrix (BLOSUM62) [54]. Qdot-AT binding to tissues Differences between treatments were analyzed by two sample t-test between percents analysis. Only differences equal or less than 0.05 were considered significant. Hydroids were incubated in a solution containing Qdot-AT conjugates for 30 mins, fixed for 12h at 4 °C in formaldehyde- phosphate buffered saline (PBS) and examined under microscopic at 543 nm. A similar sample was incubated, fixed and processed for AT-antibody immunohistochemistry as described above; allowing for the analysis of the coexistence of cell populations being labeled with the AT-antibody and binding Qdot-AT conjugates. As a control for the Qdot labeling, a third group of individuals was incubated in a solution containing non- conjugated Qdot nanocristals. To further test the specificity of Qdot-AT recognition of the population cells, another group of individuals was incubated with Qdot-AST-C conjugates and processed for microscopy as described above. Quantum dot-biotinylated peptide conjugates Quantum dot nanocrystals (Qdot) conjugated to streptavidin (1 µM) were purchased from Quantum Dot Corporation (Hayward, CA). Their peak of emission is at 605 nm (red). Biotinylated A. aegypti AT and Allatostatin-C (AST-C) were custom synthesized (Biopeptide, San Diego, CA). Each Qdot crystal is conjugated to 5-10 streptavidin molecules, with a total of 20-40 binding sites for biotin. Each biotinylated neuropeptide stock was dissolved at 0.92 mM in 100% DMSO (Dimethylsulfoxide, Sigma) and stored at -70 °C. An excess of neuropeptides (4.3 µl) was added to 10 µl of QDs, along with 58.7 µl of incubation buffer (2% BSA in 50 mM borate buffer pH 8.3 and 0.05% NaN3). The mixture was incubated for 1.5 h at RT. To remove the excess of free peptide, Qdot-peptide conjugates were washed five times with 0.5 ml of PBS in a Microcon-100 concentrator (Amicon Bioseparations, Bedford, MA, USA) at 10,000 rpm 5 min. The Qdot-peptide conjugates were recovered in 100 µl of PBS and stored at 4 °C. Myotropic assays To analyze the myoregulatory activity of the AT-like peptide in H. plagiodesmica, hydroids were treated with different doses of synthetic A. aegypti allatotropin. Six sets of six hydroids were starved for 72 hours and were individually placed in fresh water and acclimated for 10 to 15 min. Once the experimental specimens were acclimated, the solution was replaced with water containing AT at concentrations ranged from 10-16 to 10-8 M. A control group did not receive peptide. The experimental specimens were examined individually under a binocular microscope and the state of the hypostome (hypostome completely extruded or not) assessed at 3, 5, and 15 min. In a second series of experiments, we tested the ability of the A. aegypti AT-antiserum to block the hypostome extrusion. Similar groups of starved hydroids were incubated with Artemia salina in a solution containing anti-AT (1/100; 1/500; 1/1000; 1/5000 and 1/10000). Control groups did not received antiserum. The experimental specimens were examined individually under a binocular microscope, the position of the hypostome was assessed at 3, 5, 15, 30 and 45 min and the number of individuals showing the hypostome completely AT-like expression in Hydra plagiodesmica Figure 1 shows a generic scheme of a hydroid, with detailed views of the cellular organization at different regions of the body. The immunohistochemical analysis showed the existence of several AT-immunoreactive cell populations distributed throughout the hydroid body. The absence of immunoreactivity in control suggests that the labeling is specifically originated by recognition of an AT-like peptide (Figure 2A and B). The simultaneous use of the AT-antibody and phalloidin labeling demonstrated the presence of AT-like immunoreactive nerve cells that were anatomically related with the epithelial-muscular cells (phalloidin-labeled) present in the stalk (Figure 2C). A magnified view showed that allatotropic- like cells presented cytoplasmic projections running between epithelial-muscular cells that were circularly distributed (Figure 2D). The images in Figure 3A and B represent a 3D confocal reconstruction of the stalk’s cells; showing a detailed view of the morphological relationship between epithelial-muscular and nerve cell populations. Moreover, the close localization of cells producing the peptide (nerve cells) and cells containing f-actin filaments (epithelial-muscular cells) was shown by the co- localization of both signals in the same area (Figure 3B and C). The presence of a similar allatotropic-like cell population was detected in the pedal disc (Figure 3D and E). AT-Qdot labeling When both channels (immunohistochemistry label in green and Qdot label in red) were analyzed separately we could observe that motor nerve Allatotropin-like peptide myotropic activities regulating feeding Hydroids responded to the presence of food by extruding the hypostome to capture and ingest the prey. To analyze the response of the hypostome epithelial-muscular cells to AT, we studied the dose-dependent effect of AT on groups of 72 hours starved hydroids. Our results showed that the hypostome reacts to AT in a dose-response manner (Figure 6C). The role of AT as stimulant of hypostome activity was confirmed by an independent set of experiments where AT- antiserum was utilized to block the response of the hypostome to the presence of food. The control group, which did not receive treatment with the AT-antiserum, showed the normal extruding behavior of the hypostome; while groups treated with different concentrations of the AT-antiserum showed a dose- response inhibition of hypostome activity, suggesting that the movement of the hypostome was specifically blocked by the antiserum (Figure 6C). AT-Qdot labeling The microscopic analysis showed that after 30 min of incubation AT-Qdot conjugates were bound to H. plagiodesmica cell populations expressing putative AT PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 3 Allatotropin: an ancestral myotropic neuropeptide Table 1. Allatotropin receptors identified in Protostomata. Table 1. Allatotropin receptors identified in Protostomata. Name Order/Class Phylum Score e-value % identity Accession A. aegypti Diptera/Insecta Arthropoda 434 4e-146 53 AEN03789.1 T. castaneum Coleoptera/Insecta Arthropoda 417 2e-141 54 XP_973738.2 M. sexta Lepidoptera/Insecta Arthropoda 942 0.0 100 ADX66344.1 D. plexippus Lepidoptera/Insecta Arthropoda 604 0.0 74 ADX66344.1 B. mory Lepidoptera/Insecta Arthropoda 585 0.0 73 BAG68415.1 N. vitripennis Hymenoptera/Insecta Arthropoda 412 5e-140 55 XP_001604582.2 B. terrestris Hymenoptera/Insecta Arthropoda 412 2e-140 58 XP_003402490.1 B. impatients Hymenoptera/Insecta Arthropoda 414 2e-142 58 XP_003486747.1 A. florea Hymenoptera/Insecta Arthropoda 414 7e-141 52 XP_003690070.1 M. rotundata Hymenoptera/Insecta Arthropoda 376 2e-142 60 ADX66344.1 H. saltator Hymenoptera/Insecta Arthropoda 414 5e-126 54 EFN76143.1 S. gregaria Orthoptera/Insecta Arthropoda 407 4e-141 56 AEX08666.1 R. prolixus Hemiptera/Insecta Arthropoda 389 6e-135 63 Pending C. elegans Rhabditida/Secementea Nematoda 146 8e-39 33 NP_493283.3 C. brenneri Rhabditida/Secementea Nematoda 145 2e-38 35 EGT31251.1 C. briggsae Rhabditida/Secementea Nematoda 132 8e-34 35 XP_002646062.1 C. teleta Polychaeta/Capitellida Annelida 324 9e-105 47 ELU16138.1 C. gigas Ostreoidea/Bivalvia Mollusca 279 6e-89 46 EKC31663.1 H. magnipapillata Anthomedusae/Hydrozoa Cnidaria 127 7e-33 30 XP_002163508.1 H. magnipapillata Anthomedusae/Hydrozoa Cnidaria 114 3e-28 28 XP_004205906.1 H. magnipapillata Anthomedusae/Hydrozoa Cnidaria 100 4e-23 27 XP_002159553.1 T. adherens ----------------------------------- Placozoa 124 4e-31 27 XP_002117931.1 The table includes the score, e-value, percentage of identity and accession numbers. doi: 10.1371/journal.pone.0077520.t001 doi: 10.1371/journal.pone.0077520.t001 cells and their projections were intercalated running along spaces among epithelial-muscular cells (Figure 5C and D). receptors. The incubation of hydroids in a solution containing unconjugated Qdot nanocrystals did not generate a signal, suggesting that the binding to cells is caused by the recognition of the AT portion of the conjugated molecules. The distribution of the cell populations that bound the AT-Qdot conjugates were identical to those detected in the pedal disc and stalk using phalloidin, suggesting the mioepithelial nature of these cells (Figures 2C, 3C, D, 4A, 4B and 5A) and also showing a spatial coincidence with AT-immunolabeled cells. Notably, the distribution of immunolabeled populations of cells at the pedal disk and on the stalk were different (Figure 3), forming clusters in the pedal disk (Figure 4A and B) and groups of cells circularly orientated in the stalk (Figure 4C and D). AT-Qdot labeling Furthermore, when hydroids were incubated with AST-C-Qdot conjugates, the distribution of the labeled cells was clearly different from those labeled with AT-Qdots, suggesting that Hydra’s cells recognized specifically different peptides (Figure 4E and F). When hydroids were processed to detect both AT immunoreactivity and AT-Qdots binding, the microscopic analysis showed a clear concordance between the stalk cells producing the peptide and the population of cells recognizing the AT-Qdot conjugates (Figure 5A and B); indeed, in a magnified view, the motor nerve cells type morphology of the AT-like cells is evident (inset in Figure 5B). AT-Qdot conjugates were localized inside cytoplasmic vesicles, suggesting that the complex was internalized probably by means of AT receptor recognition (Figure 4D). When both channels (immunohistochemistry label in green and Qdot label in red) were analyzed separately we could observe that motor nerve receptors. The incubation of hydroids in a solution containing unconjugated Qdot nanocrystals did not generate a signal, suggesting that the binding to cells is caused by the recognition of the AT portion of the conjugated molecules. The distribution of the cell populations that bound the AT-Qdot conjugates were identical to those detected in the pedal disc and stalk using phalloidin, suggesting the mioepithelial nature of these cells (Figures 2C, 3C, D, 4A, 4B and 5A) and also showing a spatial coincidence with AT-immunolabeled cells. Notably, the distribution of immunolabeled populations of cells at the pedal disk and on the stalk were different (Figure 3), forming clusters in the pedal disk (Figure 4A and B) and groups of cells circularly orientated in the stalk (Figure 4C and D). Furthermore, when hydroids were incubated with AST-C-Qdot conjugates, the distribution of the labeled cells was clearly different from those labeled with AT-Qdots, suggesting that Hydra’s cells recognized specifically different peptides (Figure 4E and F). When hydroids were processed to detect both AT immunoreactivity and AT-Qdots binding, the microscopic analysis showed a clear concordance between the stalk cells producing the peptide and the population of cells recognizing the AT-Qdot conjugates (Figure 5A and B); indeed, in a magnified view, the motor nerve cells type morphology of the AT-like cells is evident (inset in Figure 5B). AT-Qdot conjugates were localized inside cytoplasmic vesicles, suggesting that the complex was internalized probably by means of AT receptor recognition (Figure 4D). In silico search for allatotropin receptors in invertebrates We identified a total of 22 sequences corresponding to Arthropoda, Cnidaria, Nematoda, Placozoa and Mollusca that shared significant similarity with the M. sexta AT receptor (Table 1). The alignment of all these sequences is shown in PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 4 Allatotropin: an ancestral myotropic neuropeptide Figure 1. Schematic representation of Hydra sp. Cross sections of two regions of the hydroid body. A: hypostome. B: pedal disk. C: detail of the nerve cells forming a net on the base of the epidermis and gastrodermis. cm: circular muscular layer formed by the contractile extensions of the nutritive muscle cells; cn: cnidocito; eg: epithelial gland cell; ep: epithelial-muscular cell; g: mucous and enzymatic gland cell; i: interstitial cell; lm: longitudinal muscle layer formed by the contractile extensions of the epithelial- muscular cells; n: nerve cell and ns: neurosensory cell. doi: 10.1371/journal.pone.0077520.g001 Figure 1. Schematic representation of Hydra sp. Cross sections of two regions of the hydroid body. A: hypostome. B: pedal disk. C: detail of the nerve cells forming a net on the base of the epidermis and gastrodermis. cm: circular muscular layer formed by the contractile extensions of the nutritive muscle cells; cn: cnidocito; eg: epithelial gland cell; ep: epithelial-muscular cell; g: mucous and enzymatic gland cell; i: interstitial cell; lm: longitudinal muscle layer formed by the contractile extensions of the epithelial- muscular cells; n: nerve cell and ns: neurosensory cell. doi: 10.1371/journal.pone.0077520.g001 and Annelida species, conforming all together the bilateria phyla represented in the tree. Non-bilateria phyla (i.e. Cnidaria and Placozoa) appeared in the base of the other groups. Regarding cnidarians, they included three sequences of H. magnipapillata sharing 30, 28 and 27 % of identity (access numbers: XP_002163508.1; XP_004205906.1; Figure S1. A phylogenetic analysis showed that all the holometabola insect sequences (orders Hymenoptera, Lepidoptera, Coleoptera and Diptera) cluster together on a branch in close proximity with the only two hemimetabolous species identified (orders Hemiptera and Orthoptera). The closer group to the Arthropoda was composed by three species of the genus Caenorhabditis sp. (Nematoda). The Ecdysozoa group (Arthropoda and Nematoda) were close to the Mollusca Figure S1. A phylogenetic analysis showed that all the holometabola insect sequences (orders Hymenoptera, Lepidoptera, Coleoptera and Diptera) cluster together on a branch in close proximity with the only two hemimetabolous species identified (orders Hemiptera and Orthoptera). common ancestor with the rest of the metazoan groups included in the analysis (Figure 7). In silico search for allatotropin receptors in invertebrates B: confocal 3D reconstruction of a cross-section of the hydroid body wall showing the anatomical relation between AT-like motor nerve cells and epithelial-muscular cells, as well as the colocalization of the cells producing the peptide, with contractile cells. C: Schematic representation of B including sensory nerve cells (blue) (modified from [69]). D and E: Panoramic (D) and detailed view (E) of an hydroid showing the presence of allatotropic-like cells in the pedal disc. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red). doi: 10.1371/journal.pone.0077520.g003 Figure 3. AT-like immunoreactivity. A: confocal 3D reconstruction showing AT-like nerve cells in a net-like arrangement and f- actin filaments B: confocal 3D reconstruction of a cross-section of the hydroid body wall showing the anatomical relation between Figure 3. AT-like immunoreactivity. A: confocal 3D reconstruction showing AT-like nerve cells in a net-like arrangement and f- actin filaments. B: confocal 3D reconstruction of a cross-section of the hydroid body wall showing the anatomical relation between AT-like motor nerve cells and epithelial-muscular cells, as well as the colocalization of the cells producing the peptide, with contractile cells. C: Schematic representation of B including sensory nerve cells (blue) (modified from [69]). D and E: Panoramic (D) and detailed view (E) of an hydroid showing the presence of allatotropic-like cells in the pedal disc. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red). doi: 10.1371/journal.pone.0077520.g003 In silico search for allatotropin receptors in invertebrates The closer group to the Arthropoda was composed by three species of the genus Caenorhabditis sp. (Nematoda). The Ecdysozoa group (Arthropoda and Nematoda) were close to the Mollusca XP_002159553.1). Beyond the identity, further analysis of the sequences showed similarity indices of 50, 50 and 49 % XP_002159553.1). Beyond the identity, further analysis of the sequences showed similarity indices of 50, 50 and 49 % PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 PLOS ONE \| www.plosone.org 5 Allatotropin: an ancestral myotropic neuropeptide Figure 2. AT-like immunoreactivity. A: Panoramic view of a specimen labeled with anti-AT antiserum showing the presence of immunoreactive material in different parts of the body. B: Similar view in which the primary antibody incubation was replaced for saline solution. C: Similar view as in A showing the co-existence of f-actin filaments and immunoreactive material D: A magnified view showing the spatial relationship between AT-like nerve cells and epithelial-muscular cells. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red). doi: 10.1371/journal.pone.0077520.g002 Figure 2. AT-like immunoreactivity. A: Panoramic view of a specimen labeled with anti-AT antiserum showing the presence of immunoreactive material in different parts of the body. B: Similar view in which the primary antibody incubation was replaced for saline solution. C: Similar view as in A showing the co-existence of f-actin filaments and immunoreactive material D: A magnified view showing the spatial relationship between AT-like nerve cells and epithelial-muscular cells. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red). doi: 10.1371/journal.pone.0077520.g002 common ancestor with the rest of the metazoan groups included in the analysis (Figure 7). common ancestor with the rest of the metazoan groups included in the analysis (Figure 7). respectively, suggesting a high conservation of the whole protein, as well as of the transmembrane domains, where the indices were higher (data not shown). Finally, T. adhaerens that showed 27% of identity and 47% similarity, shares a October 2013 \| Volume 8 \| Issue 10 \| e77520 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Allatotropin: an ancestral myotropic neuropeptide Figure 3. AT-like immunoreactivity. A: confocal 3D reconstruction showing AT-like nerve cells in a net-like arrangement and f- actin filaments. Discussion intercalated between epithelial-muscular cells, suggested the existence of anatomical and functional relationships between these two cell populations and advocates for myoregulatory functions of AT-like molecules in Hydra sp. Our results provided evidence for the existence of an AT-like peptide in hydroids of H. plagiodesmica (Cnidaria: Hydrozoa). Labeling with AT-specific antibodies revealed immunoreactivity in cells located in the stalk that clearly resembled nerve cells. The spatial distribution of these allatotropic-like cells, Qdot technology has been previously used in Hydra vulgaris to demonstrate GSH-mediated, highly specific, binding of Qdot- GSH conjugates to the gastric region; which indicated the PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 7 Allatotropin: an ancestral myotropic neuropeptide Figure 4. Qdot-AT and AST-C labeling and AT- immunoreactivity. A: Pedal disc view showing the distribution of cells labeled with Qdot-AT () B: Magnified view showing the cells in clusters C: View of the body wall of a hydroid showing AT recognizing epithelial-muscular cells at the stalk, as well as the distribution of the cells in a circular arrangement D: Magnified view of the epithelial-muscular circular cells at the stalk. Note the cells in two circular rows, as well as the presence of endocytic vesicles (arrow). E: View of the body wall of a hydroid showing AST-C recognizing epithelial- muscular cells at the stalk. Note that the distribution of the labeled cells is different to the labeling corresponding to the AT peptide. F: Magnified view of the same epithelial-muscular circular cells, also showing endocytic vesicles. doi: 10.1371/journal.pone.0077520.g004 receptors that specifically recognized the peptides in H. plagiodesmica. The combined treatment of hydroids with AT-Qdots and AT- antibody revealed the coincidence of both signals in the mouth region of the hypostome, as well as in the wall of the coelenteron. The distribution of AT-immunoreactive cells, along with the dose-response stimulation of feeding by AT, suggests that the AT-like peptide could regulate myotropic activities critical for the ingestion and digestion of meals. The cnidarians are the simplest organisms in which movements involved in feeding behavior are governed by a neuromuscular system [56]. The body column of the hydra during feeding undergoes a complex series of movements resembling those performed by organisms with a fully developed digestive system [57]. Comparison of the movements to those in mammals showed similarities in appearance to esophageal reflex, segmentation movement, and defecation reflex. October 2013 \| Volume 8 \| Issue 10 \| e77520 Discussion When nerve cells were eliminated, polyps showed only a weak segmentation movement, demonstrating that the diffuse nerve net in the body column of Hydra sp. primarily regulates the movements just as the netlike enteric nervous system does in mammals [57]. Acetylcholine and serotonin, which are regulating similar movements in the mammalian enteric system, were not involved in Hydra sp. [55]. Starved hydroids of H. plagiodesmica showed similar movements when they were exposed to A. salina or stimulated by the treatment with the synthetic AT. These feeding movements were inhibited in a dose-dependent fashion when individuals were treated with anti-AT antiserum. In fact, regurgitation, a movement which normally occurs between 6-9 hours after food ingestion [57], was evident in several hydroids around 30-40 min after treatment with AT, suggesting again that the AT-like peptide is involved in the process of ingestion. Figure 4. Qdot-AT and AST-C labeling and AT- immunoreactivity. A: Pedal disc view showing the distribution of cells labeled with Qdot-AT () B: Magnified view showing the cells in clusters C: View of the body wall of a hydroid showing AT recognizing epithelial-muscular cells at the stalk, as well as the distribution of the cells in a circular arrangement D: Magnified view of the epithelial-muscular circular cells at the stalk. Note the cells in two circular rows, as well as the presence of endocytic vesicles (arrow). E: View of the body wall of a hydroid showing AST-C recognizing epithelial- muscular cells at the stalk. Note that the distribution of the labeled cells is different to the labeling corresponding to the AT peptide. F: Magnified view of the same epithelial-muscular circular cells, also showing endocytic vesicles. doi: 10.1371/journal.pone.0077520.g004 Figure 4. Qdot-AT and AST-C labeling and AT- immunoreactivity. A: Pedal disc view showing the distribution of cells labeled with Qdot-AT () B: Magnified view showing the cells in clusters C: View of the body wall of a hydroid showing AT recognizing epithelial-muscular cells at the stalk, as well as the distribution of the cells in a circular arrangement D: Magnified view of the epithelial-muscular circular cells at the stalk. Note the cells in two circular rows, as well as the presence of endocytic vesicles (arrow). E: View of the body wall of a hydroid showing AST-C recognizing epithelial- muscular cells at the stalk. Note that the distribution of the labeled cells is different to the labeling corresponding to the AT peptide. Discussion F: Magnified view of the same epithelial-muscular circular cells, also showing endocytic vesicles. doi: 10.1371/journal.pone.0077520.g004 p g The physiological responses induced by AT, as well as the specific interactions of cell populations with Qdot-AT conjugates, suggested the presence of an AT-like receptor in H. plagiodesmica. Our search for orthologues of AT-receptors in invertebrates detected 22 sequences from several phyla including Placozoa, which is probably the most ancient group of Metazoa. The distance tree representing the relationships between sequences of the AT receptor family was coherent with recognized phylogenetic relationships between species. Receptors from insect species (Arthropoda) were closely related with those from Nematoda; these two groups together with another six phyla share a common ancestor, forming the monophyletic group of the Ecdysozoa [58,59]. Receptors from Mollusca and Annelida, two groups in the Lophotrochozoa, are related. Finally, the sequences pertaining to Cnidaria and Placozoa are well resolved as independent groups appearing as ancestors of the bilateria phyla. The evolutionary position of the Placozoa phylum is still controversial but modern molecular and genetic studies support its position as a basal lower metazoan phylum [60,61]. This is consistent with the position of the AT-receptor in this tree, and suggests that AT-receptors already appeared in the more primitive group of Metazoa. presence of GSH binding proteins in the endodermal cell layer [55]. Our results showed that the cells that bound the AT-Qdots conjugates were localized in the same stalk and pedal disc regions in which AT-like cells were present; suggesting again the existence of a functional relationship between the nerve cells producing the peptide and the targeted epithelial-muscular cells. The two different nanocrystal conjugates (AT and AST-C) were internalized by different target cells and detected inside endocytic vesicles, strongly suggesting the existence of Peptidic messengers play fundamental roles acting both as local neuromodulators, neurohormones and also in an endocrine and paracrine mode; being secreted not only by October 2013 \| Volume 8 \| Issue 10 \| e77520 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Allatotropin: an ancestral myotropic neuropeptide gure 5. Qdot-AT labeling and AT-immunoreactivity. A: Panoramic view at the bottom of the stalk showing the pedal disc ydroid. B: Magnified view of the stalk suggesting. a morpho-functional relationship between both cell populations. Inset: det ew of a nerve cell. Note that the morphology and spatial orientation of the cell and its projection clearly resembles the motor n the nerve cells. Discussion plagiodesmica after the ingestion of an A : confocal 3D reconstruction of an hydroid after double labeling with AT-antiserum (green) and nanocristals (red). N lization of both markers at the level of the hypostome (arrows) and gastroenteron (arrow heads). C: Physiologica strating the AT involvement in feeding movements. Black columns: Hypostome response of 72 h starved hydr ent with different doses of AT. Grey columns: Hypostome response of 72 h starved hydroids challenged with fo ed to different doses of AT-antiserum. The results are expressed as percentage of individuals that completely extrud ome. (): Significant differences between hydroids treated with AT or AT-antiserum and controls. 371/journal.pone.0077520.g006 Figure 6. Myotropic activity of AT-like peptide and feeding. A: Specimen of H. plagiodesmica after the ingestion of an A. salina egg. B: confocal 3D reconstruction of an hydroid after double labeling with AT-antiserum (green) and nanocristals (red). Note the colocalization of both markers at the level of the hypostome (arrows) and gastroenteron (arrow heads). C: Physiological assay demonstrating the AT involvement in feeding movements. Black columns: Hypostome response of 72 h starved hydroids to treatment with different doses of AT. Grey columns: Hypostome response of 72 h starved hydroids challenged with food and exposed to different doses of AT-antiserum. The results are expressed as percentage of individuals that completely extruded the hypostome. (): Significant differences between hydroids treated with AT or AT-antiserum and controls. doi: 10.1371/journal.pone.0077520.g006 Figure 6. Myotropic activity of AT-like peptide and feeding. A: Specimen of H. plagiodesmica after the ingestion of an A. salina egg. B: confocal 3D reconstruction of an hydroid after double labeling with AT-antiserum (green) and nanocristals (red). Note the colocalization of both markers at the level of the hypostome (arrows) and gastroenteron (arrow heads). C: Physiological assay demonstrating the AT involvement in feeding movements. Black columns: Hypostome response of 72 h starved hydroids to treatment with different doses of AT. Grey columns: Hypostome response of 72 h starved hydroids challenged with food and exposed to different doses of AT-antiserum. The results are expressed as percentage of individuals that completely extruded the hypostome. (): Significant differences between hydroids treated with AT or AT-antiserum and controls. doi: 10.1371/journal.pone.0077520.g006 Figure 6. Myotropic activity of AT-like peptide and feeding. A: Specimen of H. plagiodesmica after the ingestion of an A. salina egg. B: confocal 3D reconstruction of an hydroid after double labeling with AT-antiserum (green) and nanocristals (red). Discussion C and D: show both channels independently. Note that the position of nerve cells (C), clearly corresponding on-labeled spaces in (D) (open circles), showing that nerve cells are intercalated between epithelial-muscular cells recognizin eptide and reinforcing the morpho functional relationship between these two populations. Epithelial-muscular cells recognizing dots conjugates (red) and AT-like motornerve cells (green). Colocalization of markers (yellowish green). 10 1371/j l 0077520 005 Figure 5. Qdot-AT labeling and AT-immunoreactivity. A: Panoramic view at the bottom of the stalk showing the pedal disc of a hydroid. B: Magnified view of the stalk suggesting. a morpho-functional relationship between both cell populations. Inset: detailed view of a nerve cell. Note that the morphology and spatial orientation of the cell and its projection clearly resembles the motor nature of the nerve cells. C and D: show both channels independently. Note that the position of nerve cells (C), clearly corresponding with non-labeled spaces in (D) (open circles), showing that nerve cells are intercalated between epithelial-muscular cells recognizing AT peptide and reinforcing the morpho functional relationship between these two populations. Epithelial-muscular cells recognizing AT- Qdots conjugates (red) and AT-like motornerve cells (green). Colocalization of markers (yellowish green). doi: 10.1371/journal.pone.0077520.g005 On the other hand, allatotropins constitute a family of peptides originally characterized based on its ability to activate JH synthesis [21], but later demonstrated to be pleiotropic, inhibiting ion transport [29], inducing the secretion of gut enzymes [30], as well as been involved in circadian rhythms control [40-43]. Due to the difficulties of analyzing highly diverse and repetitive pro-peptides, the relationships among different families of peptides could be elusive. In fact, an orthologous relationship between allatostatin-A and galanin neurons, but also by epithelial cells. They likely comprise the most ancient group of neurotransmitters [7]. In this context, the Allatotropin/Orexin family of receptors would constitute a group of proteins broadly distributed from Placozoa to Chordata. The peptides associated with these receptors (allatotropins and orexins) are not chemically or functionally related. Orexins (also called hypocretins) are 28 to 33-amino-acid peptides mainly involved in the control of feeding, sleep-wakefulness, neuroendocrine homeostasis and autonomic regulation [62,63]. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77520 9 Allatotropin: an ancestral myotropic neuropeptide 6. Myotropic activity of AT-like peptide and feeding. A: Specimen of H. Discussion Note the colocalization of both markers at the level of the hypostome (arrows) and gastroenteron (arrow heads). C: Physiological assay demonstrating the AT involvement in feeding movements. Black columns: Hypostome response of 72 h starved hydroids to treatment with different doses of AT. Grey columns: Hypostome response of 72 h starved hydroids challenged with food and exposed to different doses of AT-antiserum. The results are expressed as percentage of individuals that completely extruded the hypostome. (*): Significant differences between hydroids treated with AT or AT-antiserum and controls. doi: 10.1371/journal.pone.0077520.g006 October 2013 \| Volume 8 \| Issue 10 \| e77520 10 PLOS ONE \| www.plosone.org Allatotropin: an ancestral myotropic neuropeptide Figure 7. Phylogram of AT-receptor. Species from the same phylum are labeled by a similar gray tone blocks. The tree is enrooted using the D. melanogaster FMFRamide receptor [50]. doi: 10.1371/journal.pone.0077520.g007 Figure 7. Phylogram of AT-receptor. Species from the same phylum are labeled by a similar gray tone blocks. The tree is enrooted using the D. melanogaster FMFRamide receptor [50]. doi: 10.1371/journal.pone.0077520.g007 the gut has been described in several insect species [23,31,32,34-39], as well as its cardioacceleratory effects [33,34,38,39]; suggesting that the ancient myoregulatory function has been maintained along the evolutionary process. Interestingly, it was recently shown that orexins also could have myoregulatory functions on the smooth muscle of the mouse duodenum [67], supporting the idea that this was the primitive function of these peptides and their receptors [7]. which is not evident at this level is supported by the orthology of their receptors and similar roles [64]. Our search of Allatotropin peptide or allatotropin precursor in H. magnipapillata genome did not show the presence of orthologous peptides, but demonstrated the presence of sequences corresponding to G-protein coupled receptors sharing 30% of identity and 50% of similarity with M. sexta. Furthermore, a search for orexin homologous did not reveal any significant match in Protostomata and the same was true when we searched for allatotropin related peptides in Deuterostomata; suggesting that even if both families of peptides share related receptors, the ligands evolved independently in these two groups. In fact the possibility that allatotropin/orexin and their receptors are orthologous representing an ancient bilaterian family was recently suggested [64]. G-protein coupled receptors are members of the largest, ubiquitous and most versatile family of cell membrane receptors. These types of receptors are also present in unicellular eukaryotes such as yeast [68]. Discussion The presence of AT- like receptors in Placozoa, probably the most ancient group of Metazoa, suggests that AT-receptors are a group of ancestral seven-transmembrane receptor originally involved in activities associated with feeding and digestion. As these activities are common and basic processes in any multicellular organism, we would like to suggest that AT and its receptors are in the base of the evolutionary origin of the message-receiver molecular systems based on G-protein receptors. The original function of AT as a myoactive regulator has been already previously proposed [65]. The presence of genes coding for AT-like peptides in Annelida and Mollusca [44,45], as well as the existence of cells producing AT-like peptides in Platyhelminthes has also been recently proposed [46,47]. Cnidarians are considered basal to the bilateral animals, being the first group which exhibits a neuronal tissue, which could be seen as the starting point in the evolution of the central nervous system [66]. Our results suggest that an AT-like peptide is involved in the feeding process in H. plagiodesmica; and its activity could be mediated by receptors members of the Allatotropin/orexin family. The myoregulatory activity of AT on References 20. Zhang J, Leontovich A, Sarras MP Jr (2001) Molecular and functional evidence for early divergence of an endothelin-like system during metazoan evolution: analysis of the Cnidarian, hydra. Development 128: 1607-1615. PubMed: 11290299. 1. Le Roith D, Shiloach J, Roth J, Lesniak MA (1980) Evolutionary origins of vertebrate hormones: Substances similar to mammalian insulins are native to unicellular eukaryotes. Proc Natl Acad Sci U_S_A 77: 6184-6188. doi:10.1073/pnas.77.10.6184. PubMed: 6449704. 2. Schuchert P (1993) Trichoplax adhaerens (Phylum Placozoa) has cells that react with antibodies against the neuropeptide RFamide. Acta Zool 74: 115-117. doi:10.1111/j.1463-6395.1993.tb01227.x. 21. Kataoka H, Toschi A, Li JP, Carney RL et al. (1989) Identification of an allatotropin from adult Manduca sexta. Science 243: 1481-1483. doi: 10.1126/science.243.4897.1481. PubMed: 17839751. 3. Lentz TL (1966) Histochemical localization of neurohumors in a sponge. J Exp Zool 162: 171-180. doi:10.1002/jez.1401620204. 22. Abdel-latief M, Meyering-Vos M, Hoffmann KH (2003) Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides. Insect Biochem Mol Biol 33: 467-476. doi: 10.1016/S0965-1748(03)00005-5. PubMed: 12706626. 4. Weyrer S, Rutzler K, Rieger R (1999) Serotonin in Porifera? Evidence from developing Tedania ignis, the caribbean fire sponge (Demospongiae) Mem Queensl. Museon 44: 659-665. ( p g ) 5. Kass-Simon G, Pierobon P (2007) Cnidarian chemical neurotransmission, an updated overview. Comp Biochem Physiol A Mol Integr Physiol 146: 9-25. doi:10.1016/j.cbpa.2006.09.008. PubMed: 17101286. 5. Kass-Simon G, Pierobon P (2007) Cnidarian chemical neurotransmission, an updated overview. Comp Biochem Physiol A Mol Integr Physiol 146: 9-25. doi:10.1016/j.cbpa.2006.09.008. PubMed: 17101286. ( ) 23. Paemen L, Tips A, Schoofs L, Proost P et al. (1991) Lom-AG- Myotropin: A novel myotropic peptide from the male accesory glands of Locusta migratoria. Peptides 12: 7-10. doi: 10.1016/0196-9781(91)90158-L. PubMed: 2052501. 6. Zega G, Pennati R, Fanzago A, De Bernardi F (2007) Serotonin involvement in the metamorphosis of the hydroid Eudendrium racemosum. Int J Dev Biol 51: 307-313. doi:10.1387/ijdb.062195gz. PubMed: 17554682. ( ) 24. Park C, Hwang JS, Kang SW, Lee BH (2002) Molecular characterization of a cDNA from the silk moth Bombyx mori encoding Manduca sexta allatotropin peptide. Zool Sci 19: 287-292. doi:10.2108/ zsj.19.287. PubMed: 12125926. 7. Grimmelikhuijzen CJP, Williamson M, Hansen GN (2002) Neuropeptides in cnidarians. Can J Zool 80: 1690-1702. doi:10.1139/ z02-137. 25. Scheng Z, Ma L, Cao MX, Li S, Jiang RJ (2007) Biochemical and molecular characterization of allatotropin and allatostatin from the Eri silkworm, Samia cynthia ricini. References Insect Mol Biol 37: 90-96. doi:10.1016/ j.ibmb.2006.09.007. 8. Grimmelikhuijzen CJP, Graff D, Koizumi O, Westfall J, McFarlane ID (1991) Neuropeptides in coelenterates: a review. Hydrobiologia 216/217: 555-563. doi:10.1007/BF00026513. 26. Truesdell PF, Koladich PM, Kataoka H, Kojima K, Suzuki A et al. (2000) Molecular characterization of a cDNA from the true armyworm Pseudaletia unipuncta encoding Manduca sexta allatotropin peptide. Insect Biochem Mol Biol 30: 691-702. doi:10.1016/ S0965-1748(00)00040-0. PubMed: 10876112. 9. Leitz T, Morand K, Mann M (1994) Metamorphosin A: A novel peptide controlling development of the lower metazoan Hydractinia echinata (Coelenterata, Hydrozoa). Dev Biol 163: 440-446. doi:10.1006/dbio. 1994.1160. PubMed: 7911112. 27. Veenstra JA, Costes L (1999) Isolation and characterization of a peptide and its cDNA from the mosquito Aedes aegypti related to Manduca sexta allatotropin. Peptides 20: 1145-1151. doi:10.1016/ S0196-9781(99)00117-5. PubMed: 10573285. 10. Takahashi T, Koizumi O, Ariura Y, Romanovitch A, Bosch TCG et al. (2000) A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra. Development 127: 997-1005. PubMed: 10662639. 28. Li Y, Unnithan GC, Veenstra JA, Feyereisen R, Noriega FG (2003) Stimulation of JH biosynthesis by the corpora allata of adult female Aedes aegypti in vitro: effect of farnesoic acid and Aedes allatotropin. J Exp Biol 206: 1825-1832. doi:10.1242/jeb.00371. PubMed: 12728004. 11. Schaller HC, Bodenmüller H (1981) Isolation and amino acid sequence of a morphogenetic peptide from hydra. Proc Natl Acad Sci U_S_A 78: 7000-7004. doi:10.1073/pnas.78.11.7000. PubMed: 16593123. 12. Hoffmeister SAH (1996) Isolation and characterization of two new morphogenetically active peptides from Hydra vulgaris. Development 122: 1941-1948. PubMed: 8674432. 29. Lee KY, Horodyski FM, Chamberlin ME (1998) Inhibition of midgut ion transport by allatotropin (Mas-At) and Manduca FRLFamides in the tobacco hornworm Manduca sexta. J Exp Biol 201: 3067-3074. PubMed: 9787126. 13. Hoffmeister-Ullerich SAH (2001) The foot formation stimulating peptide pedibin is also involved in patterning of the head in hydra. Mech Dev 106: 37-45. doi:10.1016/S0925-4773(01)00401-4. PubMed: 11472833. 30. Lwalaba D, Hoffmann KH, Woodring J (2009) Control of the release of digestive enzymes in the larvae of the fall armyworm, Spodoptera frugiperda. Arch Insect Biochem Physiol 73: 14-29. PubMed: 19771560. ( ) 14. Takahashi T, Fujisawa T (2009) Important roles for epithelial cell peptides in hydra development. Bioessays 31: 610-619. doi:10.1002/ bies.200800163. PubMed: 19382229. 31. Duve H, East PD, Thorpe A (1999) Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion. J Comp Neurol 413: 405-416. doi:10.1002/ (SICI)1096-9861(19991025)413:3. PubMed: 10502248. 15. (TIF) (TIF) Author Contributions Conceived and designed the experiments: JRR CD. Performed the experiments: MEA MLA. Analyzed the data: MEA MLA LAD JRR CD. Contributed reagents/materials/analysis tools: SHM FGN CD JRR LAD. Wrote the manuscript: JRR. Critically revised the manuscript: FGN LAD CD SHM. Acknowledgements We thank Mario Perez for his comments on the manuscript. LAD, MEA and CD are researchers of CONICET (Argentina) Supporting Information Figure S1. Alignment of 22 sequences corresponding to Arthropoda, Cnidaria, Nematoda, Placozoa and Mollusca that shared significant similarity with the M. sexta AT receptor. October 2013 \| Volume 8 \| Issue 10 \| e77520 PLOS ONE \| www.plosone.org 11 Allatotropin: an ancestral myotropic neuropeptide Allatotropin: an ancestral myotropic neuropeptide Allatotropin: an ancestral myotropic neuropeptide PubMed: 18192181. y 42. Homberg U, Reischig T, Stengl M (2003) Neural organization of the circadian system of the Cockroach Leucophaea maderae. Chronobiol Int 20: 577-591. doi:10.1081/CBI-120022412. PubMed: 12916714. 59. Aguinaldo AMA, Turbeville JM, Linford LS, Rivera MC, Garey JR et al. (1997) Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387: 489–493. doi:10.1038/387489a0. PubMed: 9168109. 43. Petri B, Homberg U, Loesel R, Stengl M (2002) Evidence for a role of GABA and Mass-allatotropin in photic entrainment of the circadian clock of the cockroach Leucophaea maderae. J Exp Biol 205: 1459-1469. PubMed: 11976357. 60. Dellaporta SL, Xu A, Sagasser S, Jakob W, Moreno MA et al. (2006) Mitochondrial genome of Trichoplax adhaerens supports Placozoa as the basal lower metazoan phylum. Proc Natl Acad Sci U_S_A 103: 8751-8756. doi:10.1073/pnas.0602076103. PubMed: 16731622. 44. Veenstra JA (2010) Neurohormones and neuropeptides encoded by the genome of Lottia gigantea, with reference to other mollusks and insects. Gen Comp Endocrinol 167: 86-103. doi:10.1016/j.ygcen. 2010.02.010. PubMed: 20171220. p 61. Ender A, Schierwater B (2003) Placozoa are not derived cnidarians: Evidence from molecular morphology. Mol Biol Evol 20: 130-134. doi: 10.1093/molbev/msg018. PubMed: 12519915. 45. Veenstra JA (2011) Neuropeptide evolution: Neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta. Gen Comp Endocrinol 171: 160-175. doi:10.1016/ j.ygcen.2011.01.005. PubMed: 21241702. 62. de Lecea L, Kilduff TS, Peyron C, Gao XB, Foye PE et al. (1998) The hypocretins: Hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U_S_A 95: 322-327. doi:10.1073/pnas. 95.1.322. PubMed: 9419374. 46. Adami ML, Damborenea C, Ronderos JR (2011) Expression of a neuropeptide similar to allatotropin in free living Turbellaria (Platyhelminthes). Tissue Cell 43: 377-383. doi:10.1016/j.tice. 2011.07.005. PubMed: 21937067. 63. Kukkonen JP (2013) Physiology of the orexinergic/hypocretinergic system: a revisit in 2012. Am J Physiol Cell Physiol 304: C2-C32. doi: 10.1152/ajpcell.00227.2012. PubMed: 23034387. 47. Adami ML, Damborenea C, Ronderos JR (2012) An allatotropin-like neuropeptide in Mesostoma ehrenbergii (Rhabdocoela, Platyhelminthes). Zoomorphology 131: 1-9. doi:10.1007/ s00435-012-0146-3. j 64. Jékely G (2013) Global view of the evolution and diversity of metazoan neuropeptide signaling. Proc Natl Acad Sci U_S_A 110: 8702-8707. doi:10.1073/pnas.1221833110. PubMed: 23637342. 48. Yamanaka N, Yamamoto S, Zitnan D, Watanabe K, Kawada T et al. (2008) Neuropeptide receptor transcriptome reveals unidentified neuroendocrine pathways. PLOS ONE 3(8): e3048. doi:10.1371/ journal.pone.0003048. PubMed: 18725956. 65. Elekonich MM, Horodyski FM (2003) Insect Allatotropins belong to a family of structurally-related myoactive peptides present in several invertebrate phyla. Allatotropin: an ancestral myotropic neuropeptide Allatotropin: an ancestral myotropic neuropeptide doi: serotonin. J Comp Neurol 428: 159-173. doi: 10.1002/1096-9861(20001204)428:1. PubMed: 11058230. 52. Hernández-Martínez S, Li Y, Lanz-Mendoza H, Rodríguez MH, Noriega FG (2005) Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegyti and Anopheles albimanus. Cell Tissue Res 321: 105-113. doi:10.1007/s00441-005-1133-5. PubMed: 15909164. 36. Santini MS, Ronderos JR (2007) Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated to diuresis in the Chagas disease vector Triatoma infestans (Klüg). J Exp Biol 210: 1986-1991. doi:10.1242/jeb.004291. PubMed: 17515423. 53. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) JalView version 2 – a multiple sequence alignement editor and analysis workbench. Bioinformatics. doi:10.1093/bioinformatics/btp033. 37. Santini MS, Ronderos JR (2009) Allatotropin-like peptide in Malpighian tubules: Insect renal tubules as an autonomous endocrine organ. Gen Comp Endocrinol 160: 243-249. doi:10.1016/j.ygcen.2008.12.002. PubMed: 19118556. p 54. Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U_S_A 89: 10915-10919. doi: 10.1073/pnas.89.22.10915. PubMed: 1438297. 38. Sterkel M, Riccillo FL, Ronderos JR (2010) Cardioacceleratory and myostimulatory activity of allatotropin in Triatoma infestans (Klüg). Comp Biochem Physiol A Mol Integr Physiol 155: 371-377. doi: 10.1016/j.cbpa.2009.12.002. PubMed: 19995615. 55. Tortiglione C, Quarta A, Tino A, Manna L (2007) Synthesis and Biological Assay of GSH Functionalized Fluorescent Quantum Dots for Staining Hydra vulgaris. Bioconjug Chem 18: 829-835. doi:10.1021/ bc060355t. PubMed: 17441682. 39. Veenstra JA, Lehman HK, Davis NT (1994) Allatotropin is a cardioacceleratory peptide in Manduca sexta. J Exp Biol 188: 347-354. PubMed: 7964382. 56. Miljkovic-Licina M, Gauchat D, Galliot B (2004) Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system. Biosystems 76: 75-87. doi:10.1016/ j.biosystems.2004.05.030. PubMed: 15351132. 40. Riccillo FL, Ronderos JR (2010) Allatotropin expression during the development of the fourth instar larvae of the kissing-bug Triatoma infestans (Klüg). Tissue Cell 42: 355-359. doi:10.1016/j.tice. 2010.07.011. PubMed: 20817237. 57. Shimizu H, Koizumi O, Fujisawa T (2004) Three digestive movements in Hydra regulated by the diffuse nerve net in the body column. J Comp Physiol Neuroethol Sens Neural Behav Physiol 190: 623-630. PubMed: 15168068. 41. Santini MS, Ronderos JR (2009) Daily variation of an allatotropin-like peptide in the Chagas disease vector Triatoma infestans (Klüg). Biol Rhythm Res 40: 299-306. doi:10.1080/09291010802214583. 58. Telford MJ, Bourlat SJ, Economou A, Papillon D, Rota-Stabelli O (2008) The evolution of the Ecdysozoa. Philos Trans R Soc Lond B Biol Sci 363: 1529- 1537. doi:10.1098/rstb.2007.2243. References McFarlane ID, Graff D, Grimmelikhuijzen CJP (1987) Excitatory actions of antho-rfamide, an anthozoan neuropeptide, on muscles and conducting systems in the sea anemone Calliactis parasitica. J Exp Biol 133: 157-168. 32. Duve H, Audsley N, Weaver RJ, Thorpe A (2000) Triple co-localisation of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut. Cell Tissue Res 300: 153-163. doi:10.1007/ s004410050056. PubMed: 10805084. 16. Takahashi T, Kobayakawa Y, Muneoka Y, Fujisawa Y, Mohri Y et al. (2003) Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps. Comp Biochem Physiol B Biochem Mol Biol 135: 309-324. doi:10.1016/ S1096-4959(03)00088-5. PubMed: 12798941. 33. Koladich PM, Cusson M, Bendena WG, Tobe SS, McNeil JN (2002) Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth Pseudaletia unipuncta. Peptides 23: 645-651. doi: 10.1016/S0196-9781(01)00658-1. PubMed: 11897383. 17. Takahashi T, Hayakawa E, Koizumi O, Fujisawa T (2008) Neuropeptides and their functions in hydra. Acta Biol Hung 59: 227-235. doi:10.1556/ABiol.59.2008.Suppl.32. PubMed: 18652396. 18. Yum S, Takahashi T, Koizumi O, Ariura Y, Kobayakawa Y et al. (1998) A Novel Neuropeptide, Hym-176, Induces Contraction of the Ectodermal Muscle in Hydra. Biochem Biophys Res Commun 248: 584-590. doi:10.1006/bbrc.1998.8831. PubMed: 9703970. 34. Matthews HJ, Audsley N, Weaver RJ (2007) Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea. J Insect Physiol 53: 75-83. doi:10.1016/ j.jinsphys.2006.10.007. PubMed: 17150228. 19. Morishita F, Nitagai Y, Furukawa Y, Matsushima O, Takahashi T et al. (2003) Identification of a vasopressin-like immunoreactive substance in hydra. Peptides 24: 17-26. doi:10.1016/S0196-9781(02)00272-3. PubMed: 12576081. 35. Rudwall AJ, Sliwowska J, Nässel DR (2000) Allatotropin-like neuropeptide in the cockroach abdominal nervous system: Myotropic actions, sexually dimorphic distribution and colocalization with 12 October 2013 \| Volume 8 \| Issue 10 \| e77520 PLOS ONE \| www.plosone.org Allatotropin: an ancestral myotropic neuropeptide Peptides 24: 1623-1632. doi:10.1016/j.peptides. 2003.08.011. PubMed: 14706542. 66. Holland ND (2003) Early central nervous system evolution: An era of skin brains? Nat Neurosci 4: 1-11. doi:10.1038/nrn1028. PubMed: 12894237. j 49. Nouzova M, Brockhoff A, Mayoral JG, Goodwin M et al. (2011) Functional characterization of an allatotropin receptor expressed in the corpora allata of mosquitoes. Peptides 34: 201-208. PubMed: 21839791. 67. Squecco R, Garella R, Luciani G (2011) Muscular effects of orexin A on the mouse duodenum: mechanical and electrophysiological studies. J Physiol 589: 5231-5246. PubMed: 21911618. 50. Vuerinckx K, Verlinden H, Lindemans M, Broeck JV, Huybrechts R (2011) Characterization of an allatotropin-like peptide receptor in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol 41: 815-822. doi:10.1016/j.ibmb.2011.06.003. PubMed: 21742031. y 68. Wang Y, Dohlman HG (2004) Pheromone signaling mechanisms in yeast: a prototypical sex machine. Science 306: 1508-1509. doi: 10.1126/science.1104568. PubMed: 15567849. 51. Horodyski FM, Verlinden H, Filkin N, Vandersmissen HP, Fleury C et al. (2011) Isolation and functional characterization of an allatotropin receptor from Manduca sexta. Insect Biochem Mol Biol 41: 804-814. doi:10.1016/j.ibmb.2011.06.002. PubMed: 21699978. 69. Mackie GO, Passano LM (1968) Epithelial conduction in Hydromedusae. J Gen Physiol 52: 600-621. doi:10.1085/jgp.52.3.600. PubMed: 438666219873635. PLOS ONE \| www.plosone.org 13 October 2013 \| Volume 8 \| Issue 10 \| e77520
https://openalex.org/W4221009339	https://www.researchsquare.com/article/rs-1403195/latest.pdf	English	null	Stressfulness of the Design Influences Consistency of Cognitive Measures and Their Correlation With Animal Personality Traits in Wild Mice (Mus Musculus)	Research Square (Research Square)	2,022	cc-by	11,106	Anja Guenther Max Planck Institute for Evolutionary Biology Max Planck Institute for Evolutionary Biology Stressfulness of the Design Influences Consistency of Cognitive Measures and Their Correlation With Animal Personality Traits in Wild Mice (Mus Musculus) Mathilde Delacoux (  mathilde.delacoux@gmail.com ) Max Planck Institute for Evolutionary Biology Research Article Keywords: Animal personality, cognitive repeatability, cognitive syndrome, individual differences, infrared thermography, Mus musculus, problem solving, spatial learning, stress Keywords: Animal personality, cognitive repeatability, cognitive syndrome thermography, Mus musculus, problem solving, spatial learning, stress Posted Date: March 11th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll i Page 1/23 Abstract Individual variation in cognition is being increasingly recognized as an important evolutionary force but contradictory results so far hamper a general understanding of consistency and association with other behaviors. Partly, this might be caused by external factors imposed by the design. Stress, for example, is known to influence cognition, with mild stress improving learning abilities, while strong or chronical stress impairs them. Also, there might be intraspecific variation in how stressful a given situation is perceived. We investigated two personality traits (stress coping and voluntary exploration), spatial learning with 2 mazes and problem-solving in low- and high-stress tests with a group of 30 female wild mice. For each test, perceived stress was assessed by measuring body temperature change with infrared thermography. None of the learning measures were significantly repeatable between the 2 stress conditions, indicating that the stress level impacts learning. We found correlations between learning and personality traits; however, they differed between the 2 stress conditions and between the cognitive tasks suggesting that different mechanisms underlie these processes. These findings could explain some of the contradicting findings in the literature and argue for very careful design of cognitive test setups to draw evolutionary implications. Introduction Since the past decade, we observe a growing interest in animal cognition in multiple biological disciplines. This young field aims to study the whole processes from the acquisition of information in the environment (perception), to the processing of that information (learning), its storage (memory) and later use (decision-making)1. Cognitive traits may influence the ecology and evolution of populations as some traits have been shown to be directly related to fitness2. The evolutionary potential relies on the heritability of a trait and it´s consistency over time and contexts3. Consistency in cognition is so far often neglected and has only been demonstrated recently. A meta-analysis from 4 shows both temporal (R = 0,18) and contextual consistency (R = 0,20 to 0,27) in various animal cognitive abilities4. Nevertheless, the demonstrated consistency in cognitive traits is lower than that found for other behavioral traits (R usually varying between 0,29 and 0,41)5–7. Whether this low consistency is an inherent biological phenomenon or caused by the low number of empirical studies and methodological heterogeneity, is not yet known. Contrasting results are also often reported in the literature regarding associations between cognition and personality traits, so-called “cognitive syndromes”, although such associations are predicted by several hypotheses8–10. Shared underlying risk-reward trade-offs are hypothesized to lead to predictable associations between personality and cognitive traits10. Through these trade-offs, personality and cognitive traits may even be integrated with life history11. The pace-of-life syndrome hypothesis states that many different biological traits (life history, physiology and behavior) could be correlated to form a “fast-slow” pace-of-life gradient. Fast individuals should express a more “proactive” behavior (bold, fast explorers, etc.) and should also be better learners in new cognitive tasks. On the contrary, slow individuals are expected to be more “reactive” (fearful, slow explorers, etc.) and perform better in a reversal learning Page 2/23 Page 2/23 task8,11,12. For example, an association between associative learning and exploration has been found in the black-capped chickadee13. Nevertheless, no such correlation was reported in a related species, the great tit14. On the other hand, exploration was positively correlated with social learning in great tits15. Also on great tits, Titulaer et al (2012) found a relation between exploration and reversal learning, but only in the most difficult test and in a sex-dependent way16. Introduction To achieve a better understanding of the ecological and evolutionary impact of individual differences in cognitive abilities, we therefore need a better understanding of the processes underlying consistency in cognition and correlations with personality traits, which is probably way more complex than we might think (for a full discussion, see17). One important step in this process is studying the effects of potential confounding factors on both consistency and cognitive syndromes18. An important factor which is known to influence cognitive performance is stress. However, the relations between stress and learning performance are complex. In Pavlovian conditioning, acute stress has been found to lead to a linear increase in learning abilities19. However, for some other learning abilities such as spatial learning, this relation has been found to resemble an inverted U-shape19,20. In these cognitive tasks, mild stress should improve learning abilities while strong or chronic stress would impair it21. Therefore, cognitive tasks that differ in the stress they induce are expected to influence learning performances differently22,23. In addition to that, all individuals do not cope the same way with stress. Some factors such as the age, sex21 and personality can be linked with how stressful a situation is perceived and how that situation is dealt with (coping styles)24. Across taxa, ample evidence shows associations between personality traits such as boldness, exploration, activity, sociability and stress measures such as glucocorticoid level and anxiety-like behaviors25–30. Assuming an inherent relationship between cognition and personality, these individual differences in sensibility to stress may influence the way that stress influences cognition. During a cognitive test, stress-sensitive individuals would be better learners in a low-stress situation, but they would be more easily overwhelmed by stress in a stressful task and consequently be the worse learners (and vice versa)20,31. Accordingly, we expect to find no consistency between cognitive performance in different stress conditions, and the strength and/or sign of correlations between personality and cognition to depend on how stressful the test situations are experienced and/or how they are dealt with. In this study, we want to investigate how the stress induced by the experimental design of both, cognitive and personality tests influence a) the consistency cognitive traits and b) the personality-cognition relationships. Therefore, we tested descendants of wild mice (Mus musculus domesticus) in multiple cognitive tasks. We used 2 mazes to measure spatial learning performances and 4 different problem- solving (PS) tasks. Animals and housing Thirty female wild mice (Mus musculus domesticus) ~ 6–7 months of age at the start of experiments were used in this study. They were kept in sister pairs, with food (Altromin 1324, Germany) and water ad libitum. Housing cages were standard type III (24 x 40 x 14 cm) laboratory cages littered with wood chips. Cages always included a shelter made of egg carton and nesting material such as toilet paper. In addition, mice received various physical enrichment changed every 2–4 weeks. The room temperature varied seasonally between 16°C and 24°C and the animals received artificial light in addition to natural light from a large window from 8:00 am to 4:30 pm. The home cage could be connected to the experimental setups with a tube through a hole made in the cage, thus, allowing the mouse to enter and leave voluntarily without handling. Except during experiments, the tube was closed by a sliding door. Behavioral and cognitive tests We measured stress coping using an open field test (OF) and voluntary exploration using a novel environment test (NE). Every individual was tested twice in order to estimate repeatability. Learning performance was assessed with two mazes and four problem-solving tasks, half of which have been designed stressful for the mice while the others were designed in a non-stressful way (see in the detailed descriptions). Half of the individuals started with the high-stress test and the other half with the low- stress test. Introduction Despite the high variability in consistency found in the literature these cognitive traits were generally found to be repeatable in mice both on a temporal and contextual point of view32. Half of our tests have been designed to be more stressful for the mice (1/2 mazes and 2/4 PS). The stress was Page 3/23 Page 3/23 directly induced by the test itself and was simulated as biologically relevant as possible. We also tested the mice with 2 commonly used personality tests. We chose the novel environment to investigate exploration and an open field to study stress coping, 2 personality traits that have already been linked to problem-solving and/or spatial learning abilities20,33. To determine the stress level of the different tests and individual differences in immediate stress response, infrared thermography has been used. The acute temperature increase of the eyes during a test situation such as the open field has been recently shown to be correlated with behavioral measures of stress in rodents and has been then proposed to be a reliable, non-invasive way to measure physiological stress34 (for a review on multiple endothermic taxa, see35). Personality For the OF, the individual is released in the center of an empty white box (60x60 cm). Its behavior is directly recorded for 5 minutes. The total distance covered, and the time spend in the central area (a square of 30x30 cm in the middle of the arena) are calculated by the software VideoMot2 (TSE systems). The test takes place in a brightly illuminated room that the individuals do not know, they are forced to enter the experimental setup, and have no place to hide, which makes this a more stressful situation. On the contrary, for the NE, the home cage with one animal inside is placed in a white box (60x60 cm) covered with bedding and containing objects the individual does not know. The mouse is then free to leave the cage and explore the novel environment through the opening in the cage wall. If it leaves the cage within 5 minutes after the test has started, the behavior is recorded for 5 additional minutes, if not, the test is stopped. The behavioral measures are the latency to leave the cage the first time, the time spend actively exploring the NE, and the number of times it is going back to its cage (i.e., the number of exploration trips). This test takes place in the room where the individuals are housed, the box is poorly illuminated, and the individuals have access to their own cages at any time of the test, which makes it as stress-free as possible for the mice. Physiological stress measure Before and after each test, the home cage was opened and put in a black box where multiple live traps were placed. To avoid any stress of handling, we waited until the focal mouse had entered one of the traps voluntarily. Since the mice were used to this method, this usually only took 1–3 minutes. The live traps were covered with a wire mesh. Then, 3 pictures were taken with an infrared thermography camera (FLIR T860) focusing on the eyes of the mouse from a distance of about 40 cm. The increase in Page 4/23 Page 4/23 temperature was measured as the difference between the average temperature of the eyes after the test minus the average temperature of the eyes before the test. temperature was measured as the difference between the average temperature of the eyes after the test minus the average temperature of the eyes before the test. Mazes Spatial learning performance was measured with two mazes, following a similar protocol. The mouse is placed in a small black box (15 x 10 cm) connected to one end of the maze and is free to leave this box in order to reach the reward to the other end (its own cage). Each individual has 5 trials (1 trial/day). The latency to enter the maze, the time spend in the maze and the number of mistakes is measured for each trial. The learning measures are defined as a) the difference between the initial number of errors and the least number of errors, b) the number of the trial with the least number of errors (i.e. how fast the individual learned to navigate the maze), c) the difference between the time spend during the first trial and this trial, and d) the number of errors at trial 3. For this last measure, trial 3 has been chosen for multiple reasons. It is the first trial for which the percentage of error is significantly below 50% (therefore different from random chance) and the first trial in which one individual could reach the end of the maze without any mistake. In addition to that, this trial is exactly at the halfway of the experiment and shows therefore the state of the learning of the different individuals in the middle of the learning process. The Low-Stress Maze (LSM) takes place in the room where the individuals are housed. The maze is dark gray, poorly illuminated, smaller (154cm long), with narrow corridors (5cm) and has a lid, thus, giving the animals the feeling of being in a narrow corridor. On the other hand, the second maze (High-Stress Maze; HSM) takes place in a room the animals do not know. The maze has white walls, wide corridors (15cm), is larger (540cm) and is brightly illuminated, making it more stressful. Problem-solving Problem-solving Page 5/23 Page 5/23 Four different tests have been used (2 low-stress and 2 high-stress). For the low-stress PS task, the mice are first habituated to the experimenter opening the cage and putting a plate containing a mealworm inside the cage during two consecutive days. Then, the easiest PS is conducted the following day and the hardest one, one day after. Mazes The easy PS task consists of pushing/lifting a dome (here after LSPS1 (Low-Stress Problem-Solving 1)) and in the hard PS task, the mouse has to slide a tab (LSPS2), in order to access the mealworm hidden below. Both tests take place in the mouse’s own cage and the animal could voluntarily decide to participate or stay hidden in its shelter. They are conducted as follows. The opened apparatus is put in the cage with a mealworm inside. After the mouse has eaten the mealworm, a second is put in and the apparatus is closed. The test ends when the mouse solves the problem-solving task (max 15 min). The behavior of the mouse is recorded for the whole test. Whether or not the problem has been solved in 15 min, the latency to solve and the time spend exploring the apparatus are the 3 learning measures. In addition to that, two other behaviors are measured: the latency to eat the first mealworm (motivation) and the latency to approach the apparatus (exploration). The two high-stress tests include escaping an unknown brightly illuminated box by pulling (1) a door (HSPS1 (High-Stress Problem-Solving 1)) or (2) a lever (HSPS2), in order to get back to its own cage. Half of the individuals were randomly selected to start with the door test (1) and the other half started with the lever test (2). The second test is done the following day. As for the low-stress PS, the individuals have 15 minutes to solve the PS task and the same learning measures are taken. In addition to this, one other behavior is measured (latency to approach the apparatus, exploration). All experimental setups are cleaned with alcohol between each test. Ethics Animal facilities are approved and controlled regularly for keeping of wild mice by the local veterinary office. All animal procedures are in accordance with European and German animal protection laws and were carried out under licence V244-19227/2019(43 − 4/19) granted by the MELUND (Ministerium für Energiewende, Landwirtschaft, Umwelt, Natur und Digitalisierung des Landes Schleswig-Holstein). The statistical analysis has been done with the software R version 4.1.1. The statistical analysis has been done with the software R version 4.1.1. First, we checked for each category of tests (personality, maze and problem-solving) whether there were differences in the temperature increase or in the behavior to verify the assumed difference in stressfulness induced by the tests. Linear mixed models (lmer() function from lme4 package) have been used to compare the temperature increase, with individual ID as random effect and with the test as fixed effect. For the mazes, we included in addition the interaction trialtest. For the behavior in the maze, it has been tested whether there was an effect of the test, the trial and their interaction on the latency to enter the maze with a glmm (glmer() function from lme4 package, Poisson distribution, individual ID as Page 6/23 Page 6/23 random effect). Model assumption of the normality of the residuals and heterodasticity were checked. However, in the case of the latency to approach the set-up in the PS task, model assumption were not fulfilled and we instead used a non-parametric test (aovperm() from permuco package, with ID as random effect and test as fixed effect). random effect). Model assumption of the normality of the residuals and heterodasticity were checked. However, in the case of the latency to approach the set-up in the PS task, model assumption were not fulfilled and we instead used a non-parametric test (aovperm() from permuco package, with ID as random effect and test as fixed effect). Repeatability has been assessed for the different behavioral measures in order to see which could be considered as personality traits, and for the different learning measures to test for consistency between the low- and high-stress conditions (i.e. contextual repeatability). The function rpt() from rptR package was used to test for the repeatability (with arguments family = “Poisson” for the traits that followed a Poisson distribution (Time spend in the center (OF), Latency to leave the trap (NE), Number of exploration sessions (NE), Difference between the time spend in the maze during the first and the best trial (HSM and LSM), Number of mistakes at trial 3 (HSM and LSM), Latency to solve (PS) and Time spend exploring the setup (PS)) or family = “Binary” for one learning measure in the PS tasks (solved or not)). The statistical analysis has been done with the software R version 4.1.1. Before checking for the presence of correlations between the temperature increase, personality traits and learning in the different conditions, we simplified the correlation table. First, only repeatable, i.e., personality traits have been considered, and the mean value across both trials for each individual was used for the calculation of correlations. Regarding the learning measures, they have all been kept for the mazes. For the PS tasks, only the learning measures from LSPS1 have been used for the low-stress condition, because too few animals solved the LSPS2 task (4/22). For the high-stress condition, the mean between the learning measures from both tasks was used since they were found to be repeatable. The correlations have then been calculated with the rcorr() function from the Hmisc package (with argument type="spearman" as some of the measures do not follow a normal distribution). In addition to the significant correlations, we also took biologically relevant correlations into consideration. We defined a biologically relevant correlation as any correlation whose coefficient is above 0,3 or below − 0,3. We chose this value following the meta-analysis from Bell et al. 5 estimating the mean repeatability of a behavior around 0,37, following the one from Cauchoix et al. 4 that calculated the repeatability of cognitive traits between 0,18 and 0,27, and the meta-analysis from Garamszegi et al. 36 that found a mean effect size for correlations between different personality traits between 0,074 and 0,566. In order to find significance with such a correlation, we would have needed a group size of 85 individuals (according to a power analysis with the function pwr.r.test() from the package pwr with arguments r = 0.3, sig.level = 0.05, power = 0.8). We therefore preferred to keep a smaller group size for ethical reasons but to still consider them. Differences between tests (perceived stressfulness of the tests situations) For all the categories of tests (personality, maze and problem-solving), the increase in temperature was significantly higher in the condition we assumed to be more stressful (personality (p = 0,034); maze (p = Page 7/23 Page 7/23 0,0011); PS: effect of the test (p = 0,0011), LSPS1 – HSPS2 (p = 0,048), LSPS2 – HSPS2 (p = 0,048); Fig. 1). All together, the mean increase in temperature in the low-stress condition was + 0,38 ± 0,84°C, while it was of + 0,71 ± 0,92°C in the high-stress condition. Trial or the interaction between the test and the trial were never significant. In the mazes, the effect of the interaction between test and trial on the latency to enter the maze was highly significant (p = 0,00041). The latency to enter the maze on the first trial was comparable in both mazes, but it was rapidly decreasing in the LSM, while it stayed constant in the HSM (Fig. 2a). For the PS tasks, the test has a significant effect on the latency to approach the experiment set-up (f = 3,4; p (parametric) = 0,021; p (permutation) = 0,012). The latency to approach the set-up was lower in the high- stress condition compared to the low-stress condition (Fig. 2b). b l In the mazes, the effect of the interaction between test and trial on the latency to enter the maze was highly significant (p = 0,00041). The latency to enter the maze on the first trial was comparable in both mazes, but it was rapidly decreasing in the LSM, while it stayed constant in the HSM (Fig. 2a). For the PS tasks, the test has a significant effect on the latency to approach the experiment set-up (f = 3,4; p (parametric) = 0,021; p (permutation) = 0,012). The latency to approach the set-up was lower in the high- stress condition compared to the low-stress condition (Fig. 2b). Learning For the mazes, contextual consistency wasn´t found for any of the learning measures between the high- and low-stress condition (Number of the best trial (p = 1), Δ number of mistakes between the first and the best trial (p = 0,246), Δ time spend in the maze between the first and the best trial (p = 0,255), number of mistakes at trial 3 (p = 1)). To investigate consistency in problem-solving performance, repeatability has first been estimated pooled across all PS tests (except LSPS2, which had too few solvers). In this situation, none of the learning measures were found to be repeatable (whether the task has been solved or not (p = 0,15), latency to solve the task (p = 0,5), time spend exploring the set-up (p = 0,45)). However, when repeatability was assessed by testing pairwise between and within conditions (Fig. 3), we observed that learning performance were generally consistent within the high-stress condition (HSPS1 – HSPS2: solved or not (R = 0,93; p = 0,00052), latency to solve (R = 0,51; p = 0,01), exploration time (R = 0,35; p = 0,083)). On the contrary, repeatability between high- and low-stress conditions was never significant. Temperature We wanted to know if the temperature increase is a consistent individual characteristic. Therefore, we tested it´s repeatability across the different tests and trials (with the test and the trial as explanatory factor). We found that the increase in temperature was indeed consistent, even if the repeatability estimate was very low (R = 0,070; p = 0,00072). However, the repeatability of the temperature increase was higher when only including the high stress conditions measurements in the analysis (R = 0,116; suppl. mat. S1). In addition to that, we also found that the basal temperature (temperature before the test) was consistent over time (R = 0,17; p = 1,1310− 10). Personality Page 8/23 For the OF test, only the distance covered was repeatable (R = 0,55; p = 0,00045 – time spend in the center: p = 0,24). For the NE test, the number of explorations was repeatable (R = 0,35; p = 0,045) and the For the OF test, only the distance covered was repeatable (R = 0,55; p = 0,00045 – time spend in the center: p = 0,24). For the NE test, the number of explorations was repeatable (R = 0,35; p = 0,045) and the Page 8/23 Page 8/23 latency to enter the novel environment tended to be repeatable (R = 0,29, p = 0,063) but not the time spend exploring (p = 0,116). Three personality measures were kept for the correlation analyses: the distance covered (OF), the number of explorations (NE) and the latency to enter the novel environment (NE). latency to enter the novel environment tended to be repeatable (R = 0,29, p = 0,063) but not the time spend exploring (p = 0,116). Three personality measures were kept for the correlation analyses: the distance covered (OF), the number of explorations (NE) and the latency to enter the novel environment (NE). C l ti Learning – personality/temperature The only clear correlation between learning performance and measures (behavior or stress level) in the personality tests is a positive correlation between learning in the HSM and the increase in temperature in the OF (average R across learning measures = 0,20 ± 0,23, with 2 significant positive correlations; Fig. 5). This indicates that individuals with a higher temperature increase in the OF were better or faster learners in the HSM. In addition to this, the correlations suggest two other associations between learning in the LSPS and the NE measures. The first one is a positive correlation between learning performance in the LSPS and a proactive behavior in the NE (average R = 0,15 ± 0,14, with one biologically relevant positive correlation; Fig. 5)). And the second one may be a positive correlation between learning in the LSPS and a steeper temperature increase in the NE (average R = 0,13 ± 0,23, with one significant positive correlation; Fig. 5)). It indicates that the best learners in a low-stress PS task are also the fastest explorers and/or the most sensitive to stress in a NE test, albeit these relations are weaker compared to correlations among high-stress situations. All the observed correlations are summarized on Fig. 6. Learning – learning Within cognitive tasks, the different measures used to assess learning performance were almost all positively correlated, although not always significantly (see Fig. 4 1st column). Correlations within high- stress correlated on average with R = 0,36 ± 0,29 while correlations within the low-stress situations correlated on average with R = 0,29 ± 0,30. We found no statistical difference in the strength of correlations with a paired t-test; t = 0,21; df = 7; p = 0,83. Across cognitive tasks within either high or low stress condition, the learning measures were also in general positively correlated in the high-stress condition (average R = 0,14 ± 0,27). On the other hand, learning measures between the maze and the PS task in the low stress conditions were not correlated (average R = 0,01 ± 0,18) (Fig. 4 2nd column). The strength of the correlations was significantly lower in the latter (paired t-test; t = 2,71; df = 11; p = 0,02). Across cognitive tasks of different stress conditions, the only positive correlation found is between learning in the problem-solving task in the high and low stress conditions (average R = 0,24 ± 0,18, with 1 significant and 3 biologically relevant positive correlations out of 9). No other correlations have been found between learning performance in the high- and low-stress situations (Fig. 4 3rd column). Discussion Page 9/23 With this study, we wanted to investigate whether the experimental design, particularly the stress it induces, would influence the consistency of learning performances in cognitive tasks and their associations with personality traits. We therefore designed both low- and high-stress versions for each test. Across all tests, we observed a significantly higher temperature increase during the tests that have been designed to be more stressful compared to the low-stress tests. Moreover, the difference in temperature increases with the duration of the tests in a high-stress test while it remains constant in a low-stress situation (suppl. mat. S3). In addition to that, some behaviors also indicated that the high- stress situations were really experienced as more stressful. In the mazes, the latency to enter decreased by the 2nd trial in the low-stress situation, indicating a quick habituation. On the contrary, in the high- stress condition, the latency to enter stays steady across all five trials. In the PS tasks, the latency to approach the setup seems higher in the low-stress condition, potentially indicating a higher motivation to get access back to their home cage in the HSPS. However, the different approach latencies could also indicate a difference in perception and/ or attention rather than indicating a difference in stress. All together, the physiological and behavioral results show that the tests that were designed more stressful were also perceived as more stressful by the mice. In addition to the measure of the stressfulness of the design, infrared thermography could be used as a measure of individual sensibility to stress. This method has been proposed to be a new non-invasive measure of stress in a couple of studies35. In mice, for example, differences in temperature increase have been linked with some personality and behavioral traits34,37. In this study, the temperature increase was consistent over quite a substantial time period (ca. 6 months) and across contexts, revealing consistent inter-individual differences in this physiological trait. We did not find consistency between learning performances in low- and high-stress conditions although in general, wild mice exhibit consistency in the problem-solving tasks we used38 and we found problem- solving to be consistent when only considering the high-stress situations. Furthermore, we found that the personality-cognition correlations differ between the high and low stress conditions. This shows that the design-induced stress can have an impact on both trait consistency and cognitive syndromes. Stress and cognition: absence of consistency In the meta-analysis from Cauchoix et al.4, cognition was generally found to be repeatable across time and contexts. Despite the high variability in consistency between species and cognitive tasks, problem- solving and spatial learning were always found to be repeatable in adult rodents (including Mus musculus) except some species of the genus Sciurus4,9,39. While problem-solving was also repeatable in Mus musculus (even of the same population as used in this study) and in Apodemus agrarius using the same problem-solving test setups as in our low-stress situation38,40, here, we could not find consistency across the high- and low stress situation. Page 10/23 Stress is already known to influence behavior in such a way that it impacts the repeatability of personality traits. For example, acclimation time has been shown to influence the repeatability of activity Page 10/23 Page 10/23 in guppies41. The methodology is then expected to also influence results in cognitive experiments. Different spatial learning testing set-ups have already been shown to induce different stress levels22 and influence memory retention and working memory42. Nevertheless, the influence of stress varies from one context to the other: while strong or chronic stress is expected to impair cognitive performances, mild fear could have the opposite effect21,43. Additionally, perceived stress will be different from one individual to the other, which will modulate the effect of stress on cognitive performance by displacing or changing the shape of the relations stress-cognition. For example, it has been suggested that the inverted U-shape of the cognitive abilities could be moved along the stress axis following the personality of an individual. This means that the “optimal stress” (i.e., the stress level at which learning performances are the highest) could vary from one individual to the other20. In such a situation, stress-sensitive individuals would be better learners in a low-stress situation but the worse learners in a more stressful task, and vice versa, explaining the lack of consistency found in this study. A couple of studies tend to support this statement20,31,44 while some other studies do not33,45,46, however, the lack of control for the stress level of the task in the latter makes it difficult to interpret the generality and importance of this phenomenon. in guppies41. The methodology is then expected to also influence results in cognitive experiments. Some factors other than stress are known to influence learning performance and might consequently decrease consistency if not accounted for in experimental designs. Underlying mechanisms For both the maze and PS, correlations between learning measures and personality were different in high- and low-stress conditions. Learning performances in the HSM were linked with individual sensitivity to stress in the OF, while we found no personality-learning correlations with the LSM. On the contrary, learning in the HSPS tasks wasn’t linked with any personality measure but learning in the LSPS task was linked with the behavior and the temperature increase in the NE. Correlations between different biological traits can result from shared underlying trade-offs10. For example, an active and bold individual is expected to explore a task faster, find cues easily and react to them more quickly than a shy individual. However, in this study, different correlations between personality and cognition are found following the stress level of the task, suggesting that these relations might be more complex than imagined17. As suggested for the lack of consistency, this could also be due to different underlying mechanisms. In an experiment on great tits (Parus major), Titulaer et al (2012) found personality-learning correlations only in the most difficult reversal learning task. They hypothesize that easy and hard tasks might be differently perceived for example by the attention that is paid to the cues. Correlations between different biological traits can result from shared underlying trade-offs10. For example, an active and bold individual is expected to explore a task faster, find cues easily and react to them more quickly than a shy individual. However, in this study, different correlations between personality and cognition are found following the stress level of the task, suggesting that these relations might be more complex than imagined17. As suggested for the lack of consistency, this could also be due to different underlying mechanisms. In an experiment on great tits (Parus major), Titulaer et al (2012) found personality-learning correlations only in the most difficult reversal learning task. They hypothesize that easy and hard tasks might be differently perceived for example by the attention that is paid to the cues. These context differences could then reveal mechanisms that underlie cognition16. Specifically, it is known that different neurobiological mechanisms are involved in low- and high-stress cognitive tests19. It has been shown in a spatial learning task that the memory-related protein ERK2 was activated during learning in the amygdala, a brain region linked to fear, in a high-stress situation, but not in a low-stress one50. Stress and cognition: absence of consistency For example, previous experience of a task has been shown to increase performance in a similar task47,48. However, this has been controlled for in this study by splitting our set of animals into 2 groups which started either with high- or low-stress tests and our results show that there is no influence of the sequence of the test on the different learning measures (see Suppl mat S5). Motivation is another important factor that can influence performance in a cognitive test47. We tested the effect of motivation in the LSPS tasks by measuring the latency to eat the first mealworm. Despite a trend of the latency to eat the mealworm to influence the latency to solve the PS, the 2 other learning measures were not influenced by motivation (see Suppl mat S5) in accordance with previous results on wild mice38. This result suggests that, at least for the LSPS task, motivation did not influence learning performances. Another factor explaining the lack of consistency is that some tasks that appear very similar to us could in fact rely on different mechanisms4. For example, an experiment of Troisi et al.49 showed that learning was not consistent in a spatial and a reversal learning task between 2 different spatial scales. They hypothesize that this could be due to differences in the cognitive mechanisms implied at different spatial scales (allo- vs egocentric navigation and cue use)49. This could contribute to the lack of consistency we observe between the high- and the low-stress maze, as one of the differences between them is their size. However, in their study, for both the spatial learning and the reversal learning tasks, the smaller scale test took place in the individual’s cage, while the larger scale tests happened in another room. We showed that such a difference in design leads to a different perceived stress for the animals, both, behaviorally and physiologically, while the stress level was not controlled for in the study of Troisi et al.49. Furthermore, a scale difference may only explain a lack of consistency in the mazes while all the problem-solving setups were of approximately the same size but still lacking in consistency. Nevertheless, this example shows Page 11/23 Page 11/23 that experimental setups and their intended effects on animals need to be designed very rigorously and several potential confounding factors need to be considered. Underlying mechanisms Similarly, stress-related hormones such as glucocorticoids can bind to their respective receptors in the brain and affect cognitive performances51. In rats, corticosterone blockade in a stressful situation decreased learning performances in a spatial learning task, while corticosterone enhancement in a low- stress situation increased them52. This could easily explain both the lack of consistency found between the HSM and the LSM and that learning performances in a high-stress situation such as the HSM are correlated with stress-sensitivity. On the other hand, exploration is often linked with cognitive performances8, and especially aspects of problem-solving33,53. This explains that learning in a low-stress condition such as the LSPS task was correlated with personality in the NE. The involvement of these different physiological reactions following the stress context indicates that the cognitive performances are strongly influenced by the design of the experiment, while personality remains relatively consistent, hence, leading to different personality-learning associations. In addition, correlations between cognitive measures appeared stronger in high-stress conditions compared to the low stress conditions. It is suggesting that some of the mechanisms involved in the stressful situation are common to multiple learning tasks, while the different cognitive tasks in the low-stress situation are more independent or more strongly influenced by external factors. These context differences could then reveal mechanisms that underlie cognition16. Specifically, it is known that different neurobiological mechanisms are involved in low- and high-stress cognitive tests19. It has been shown in a spatial learning task that the memory-related protein ERK2 was activated during learning in the amygdala, a brain region linked to fear, in a high-stress situation, but not in a low-stress one50. Similarly, stress-related hormones such as glucocorticoids can bind to their respective receptors in the brain and affect cognitive performances51. In rats, corticosterone blockade in a stressful situation decreased learning performances in a spatial learning task, while corticosterone enhancement in a low- stress situation increased them52. This could easily explain both the lack of consistency found between the HSM and the LSM and that learning performances in a high-stress situation such as the HSM are correlated with stress-sensitivity. On the other hand, exploration is often linked with cognitive performances8, and especially aspects of problem-solving33,53. This explains that learning in a low-stress condition such as the LSPS task was correlated with personality in the NE. Underlying mechanisms The involvement of these different physiological reactions following the stress context indicates that the cognitive performances are strongly influenced by the design of the experiment, while personality remains relatively consistent, hence, leading to different personality-learning associations. In addition, correlations between cognitive measures appeared stronger in high-stress conditions compared to the low stress conditions. It is suggesting that some of the mechanisms involved in the stressful situation are common to multiple learning tasks, while the different cognitive tasks in the low-stress situation are more independent or more strongly influenced by external factors. Additionally, the lack of consistency between the two stress conditions suggests that cognitive traits may be particularly flexible. On an evolutionary point of view, the flexibility of the underlying mechanisms of Page 12/23 Page 12/23 cognition can be adaptive, as the optimal cognitive phenotype can be different in different stress contexts. Indeed, multiple trade-offs are involved in the determination of cognitive abilities and the optimal outcome can be different following the situation. The first one is the trade-off costs-benefits of cognition4. Advantages provided by learning are sometimes counteracted by direct (time and energy demand) and indirect (correlations with other traits such as personality) costs54. Secondly, the speed- accuracy trade-off describes the compromise between the precision/accuracy during a task and the time necessary to perform it55. The stakes of low- and high-stress situation are intrinsically different, as the individual’s life is supposedly more at risk in a stressful situation. This can influence the energy allocation and the importance given to speed and/or accuracy in a cognitive task55,56. Acting distinctively on cognitive performances in low- and high-stress situation would allow for a better fine-tuning of these traits. However, we also find some differences between learning tasks. Cognitive syndromes are only present in the low-stress condition in the PS tasks, but we only find personality-learning correlations in the high- stress condition in the maze. In addition, while learning performances in the mazes are drastically different between the 2 stress conditions, it is much less strong in the PS tasks. Indeed, even if consistency could not be found between the learning measures from LSPS and HSPS, they still tended to be positively correlated. In the same way as consistency in cognition4, our results suggest that the effect of stress on cognitive performances and its strength are highly dependent on the cognitive task. Underlying mechanisms Different mechanisms are probably involved in the different learning tasks and, therefore, they are differentially mediated by the stress level of the test. More cognitive abilities should be tested in order to have a broader view on the influence of stress on cognitive performances. Acknowledgments We would like to thank Milan Jovicic for his dedication to helping people and taking care of the mice and Karem Lopez Hervas for her wise advice. We also want to acknowledge our 30 female Mus musculus domesticus for the few mysteries they helped solve, the many other questions they raised and without which this experiment wouldn’t have been possible. Data availability Raw data and R code is available: See attached files Competing interests The authors declare no competing interests. Author contributions M.D. and A.G. planned the study. M.D. collected the data. M.D. analyzed the data. M.D. and A.G. wrote the paper. Evolution of cognition For the two studied cognitive tasks, learning performances have been shown to be not consistent across different stress contexts and the correlations between the measured learning and personality traits were different. Controlling for these conditions would be very important to unravel when cognitive syndromes may affect fitness and what are their underlying mechanisms are. There are 3 important conditions for a trait to be subject to evolution2,57. The first one, the presence of inter-individual variation in a population, seems to be generally verified for cognition4. Secondly, the cognitive traits should be heritable. Even if not completely clear, multiple clues tend to indicate that at least some of these traits are transmitted to the next generation2,58. Finally, the inter-individual differences should lead to differences in fitness. A causality between cognition and fitness is hard to determine, but several correlations have been found2,4,54 suggesting that such a relationship exists. In addition to that, some studies found differences in cognition between populations or related species with different ecology that could be adaptive38,59. These results imply that cognitive traits probably under selection pressure. It also means that if cognition is correlated with some other traits, they won’t evolve in isolation8,57. Then, evolutionary changes in one of these traits would lead to concurrent changes in correlated traits even if these are not under direct selection60,61. The differences found in learning performances and their correlations with personality Page 13/23 Page 13/23 between stressful and non-stressful conditions shows the importance of taking stress into account when designing a cognitive experiment, as it could drastically bias and induce misleading conclusions on the underlying mechanisms and therefore also on the evolution of cognition. between stressful and non-stressful conditions shows the importance of taking stress into account when designing a cognitive experiment, as it could drastically bias and induce misleading conclusions on the underlying mechanisms and therefore also on the evolution of cognition. References 1. Shettleworth, S. J. Cognition, Evolution, and Behavior. (Oxford University Press, 2009). 2. Cauchoix, M. & Chaine, A. How Can We Study the Evolution of Animal Minds? Front. Psychol. 7, 358 (2016). 2. Cauchoix, M. & Chaine, A. How Can We Study the Evolution of Animal Minds? Front. Psychol. 7, 358 (2016). Endler, J. A. Natural Selection in the Wild. (Princeton University Press, 1986) 3. Endler, J. A. Natural Selection in the Wild. (Princeton University Press, 1986). Page 14/23 4. Cauchoix, M. et al. The repeatability of cognitive performance: a meta-analysis. Philos. Trans. R. Soc. B Biol. Sci. 373, 20170281 (2018). 5. Bell, A. M., Hankison, S. J. & Laskowski, K. L. The repeatability of behaviour: a meta-analysis. Anim. Behav. 77, 771–783 (2009). 6. Dochtermann, N. A., Schwab, T. & Sih, A. The contribution of additive genetic variation to personality variation: heritability of personality. Proc. R. Soc. B Biol. Sci. 282, 20142201 (2015). 7. Holtmann, B., Lagisz, M. & Nakagawa, S. Metabolic rates, and not hormone levels, are a likely mediator of between-individual differences in behaviour: a meta-analysis. Funct. Ecol. 31, 685–696 (2017). 8. Carere, C. & Locurto, C. Interaction between animal personality and animal cognition. Curr. Zool. 57, 491–498 (2011). 9. Guenther, A. & Brust, V. Individual consistency in multiple cognitive performance: behavioural versus cognitive syndromes. Anim. Behav. 130, 119–131 (2017). 10. Sih, A. & Del Giudice, M. Linking behavioural syndromes and cognition: a behavioural ecology perspective. Philos. Trans. R. Soc. B Biol. Sci. 367, 2762–2772 (2012). 11. Brust, V., Wuerz, Y. & Krüger, O. Behavioural Flexibility and Personality in Zebra Finches. Ethology 119, 559–569 (2013). 12. Bebus, S. E., Small, T. W., Jones, B. C., Elderbrock, E. K. & Schoech, S. J. Associative learning is inversely related to reversal learning and varies with nestling corticosterone exposure. Anim. Behav. 111, 251–260 (2016). 13. Guillette, L. M., Reddon, A. R., Hurd, P. L. & Sturdy, C. B. Exploration of a novel space is associated with individual differences in learning speed in black-capped chickadees, Poecile atricapillus. Behav. Processes 82, 265–270 (2009). 14. Groothuis, T. G. G. & Carere, C. Avian personalities: characterization and epigenesis. Neurosci. Biobehav. Rev. 29, 137–150 (2005). 15. Marchetti, C. & Drent, P. J. Individual differences in the use of social information in foraging by captive great tits. Anim. Behav. 60, 131–140 (2000). 16. Titulaer, M., van Oers, K. & Naguib, M. References Personality affects learning performance in difficult tasks in a sex-dependent way. Anim. Behav. 83, 723–730 (2012). 16. Titulaer, M., van Oers, K. & Naguib, M. Personality affects learning performance in difficult tasks in a sex-dependent way. Anim. Behav. 83, 723–730 (2012). 17. Griffin, A. S., Guillette, L. M. & Healy, S. D. Cognition and personality: an analysis of an emerging field. Trends Ecol. Evol. 30, 207–214 (2015). 17. Griffin, A. S., Guillette, L. M. & Healy, S. D. Cognition and personality: an analysis of an emerging field. Trends Ecol. Evol. 30, 207–214 (2015). 18. Thornton, A., Isden, J. & Madden, J. R. Toward wild psychometrics: linking individual cognitive differences to fitness. Behav. Ecol. 25, 1299–1301 (2014). 18. Thornton, A., Isden, J. & Madden, J. R. Toward wild psychometrics: linking individual cognitive differences to fitness. Behav. Ecol. 25, 1299–1301 (2014). 19. Sandi, C. & Pinelo-Nava, M. T. Stress and Memory: Behavioral Effects and Neurobiological Mechanisms. Neural Plast. 2007, 78970 (2007). 19. Sandi, C. & Pinelo-Nava, M. T. Stress and Memory: Behavioral Effects and Neurobiological Mechanisms. Neural Plast. 2007, 78970 (2007). 20. Salehi, B., Cordero, M. I. & Sandi, C. Learning under stress: The inverted-U-shape function revisited. Learn. Mem. 17, 522–530 (2010). 20. Salehi, B., Cordero, M. I. & Sandi, C. Learning under stress: The inverted-U-shape function revisited. Learn. Mem. 17, 522–530 (2010). Page 15/23 Page 15/23 21. Sandi, C. Stress and cognition. WIREs Cogn. Sci. 4, 245–261 (2013). 21. Sandi, C. Stress and cognition. WIREs Cogn. Sci. 4, 245–261 (2013). 22. Harrison, F. E., Hosseini, A. H. & McDonald, M. P. Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behav. Brain Res. 198, 247–251 (2009). 23. Hölscher, C. Stress impairs performance in spatial water maze learning tasks. Behav. Brain Res. 100, 225–235 (1999). 24. Koolhaas, J. M. et al. Coping styles in animals: current status in behavior and stress-physiology. Neurosci. Biobehav. Rev. 23, 925–935 (1999). 25. Clary, D. et al. Shyness–Boldness, but not Exploration, Predicts Glucocorticoid Stress Response in Richardson’s Ground Squirrels (Urocitellus richardsonii). Ethology 120, 1101–1109 (2014). 26. Cockrem, J. F. Stress, corticosterone responses and avian personalities. J. Ornithol. 148, 169–178 (2007). 27. Ferreira, V. H. B. et al. Personality traits modulate stress responses after enclosure change of captive capuchin monkeys (Sapajus libidinosus). Appl. Anim. Behav. Sci. 232, 105111 (2020). 28. Martins, C. I. M. et al. Linking Fearfulness and Coping Styles in Fish. References PLoS ONE 6, e28084 (2011). 29. Raynaud, J. & Schradin, C. Experimental increase of testosterone increases boldness and decreases anxiety in male African striped mouse helpers. Physiol. Behav. 129, 57–63 (2014). 30. Seltmann, M. W. et al. Stress responsiveness, age and body condition interactively affect flight initiation distance in breeding female eiders. Anim. Behav. 84, 889–896 (2012). 31. Brinks, V., van der Mark, M., de Kloet, R. & Oitzl, M. Emotion and Cognition in High and Low Stress Sensitive Mouse Strains: A Combined Neuroendocrine and Behavioral Study in BALB/c and C57BL/6J Mice. Front. Behav. Neurosci. 1, 8 (2007). 32. Cauchoix, M., Hermer, E., Chaine, A. S. & Morand-Ferron, J. Cognition in the field: comparison of reversal learning performance in captive and wild passerines. Sci. Rep. 7, 12945 (2017). 33. Medina-García, A., Jawor, J. M. & Wright, T. F. Cognition, personality, and stress in budgerigars, Melopsittacus undulatus. Behav. Ecol. 28, 1504–1516 (2017). 34. Lecorps, B., Rödel, H. G. & Féron, C. Assessment of anxiety in open field and elevated plus maze using infrared thermography. Physiol. Behav. 157, 209–216 (2016). 34. Lecorps, B., Rödel, H. G. & Féron, C. Assessment of anxiety in open field and elevated plus maze using infrared thermography. Physiol. Behav. 157, 209–216 (2016). 35. Travain, T. & Valsecchi, P. Infrared Thermography in the Study of Animals’ Emotional Responses: A Critical Review. Anim. Open Access J. MDPI 11, 2510 (2021). 36. Garamszegi, L. Z., Markó, G. & Herczeg, G. A meta-analysis of correlated behaviors with implications for behavioral syndromes: relationships between particular behavioral traits. Behav. Ecol. 24, 1068– 1080 (2013). 37. Duparcq, M. et al. Differences between fast and slow explorers in short-term tail temperature responses to handling in a rodent of wild origin. Behav. Brain Res. 376, 112194 (2019). 37. Duparcq, M. et al. Differences between fast and slow explorers in short-term tail temperature responses to handling in a rodent of wild origin. Behav. Brain Res. 376, 112194 (2019). 38. Vrbanec, L., Matijević, V. & Guenther, A. Enhanced problem-solving ability as an adaptation to urban environments in house mice. Proc. R. Soc. B Biol. Sci. 288, 20202504 (2021). 38. Vrbanec, L., Matijević, V. & Guenther, A. Enhanced problem-solving ability as an adaptation to urban environments in house mice. Proc. R. Soc. B Biol. Sci. 288, 20202504 (2021). Page 16/23 Page 16/23 39. Schuster, A. C., Carl, T. & Foerster, K. References Repeatability and consistency of individual behaviour in juvenile and adult Eurasian harvest mice. Sci. Nat. 104, 10 (2017). 40. Mazza, V. & Guenther, A. City mice and country mice: innovative problem solving in rural and urban noncommensal rodents. Anim. Behav. 172, 197–210 (2021). 41. O’Neill, S. J., Williamson, J. E., Tosetto, L. & Brown, C. Effects of acclimatisation on behavioural repeatability in two behaviour assays of the guppy Poecilia reticulata. Behav. Ecol. Sociobiol. 72, 166 (2018). 42. Llano Lopez, L., Hauser, J., Feldon, J., Gargiulo, P. A. & Yee, B. K. Evaluating spatial memory function in mice: A within-subjects comparison between the water maze test and its adaptation to dry land. Behav. Brain Res. 209, 85–92 (2010). 43. de Kloet, E. R., Oitzl, M. S. & Joëls, M. Stress and cognition: are corticosteroids good or bad guys? Trends Neurosci. 22, 422–426 (1999). 44. Radtke, E. L., Düsing, R., Kuhl, J., Tops, M. & Quirin, M. Personality, Stress, and Intuition: Emotion Regulation Abilities Moderate the Effect of Stress-Dependent Cortisol Increase on Coherence Judgments. Front. Psychol. 11, 339 (2020). 45. de Haas, E. N., Lee, C., Hernandez, C. E., Naguib, M. & Rodenburg, T. B. Individual differences in personality in laying hens are related to learning a colour cue association. Behav. Processes 134, 37–42 (2017). 46. Overall, K. L., Dunham, A. E., Scheifele, P. & Sonstrom Malowski, K. Fear of noises affects canine problem solving behavior and locomotion in standardized cognitive tests. Appl. Anim. Behav. Sci. 221, 104863 (2019). 47. Cooke, A. C., Davidson, G. L., van Oers, K. & Quinn, J. L. Motivation, accuracy and positive feedback through experience explain innovative problem solving and its repeatability. Anim. Behav. 174, 249– 261 (2021). 48. Shaw, R. C. Testing cognition in the wild: factors affecting performance and individual consistency in two measures of avian cognition. Behav. Processes 134, 31–36 (2017). 48. Shaw, R. C. Testing cognition in the wild: factors affecting performance and individual consistency in two measures of avian cognition. Behav. Processes 134, 31–36 (2017). 49. Troisi, C. et al. No evidence for cross-contextual consistency in spatial learning and behavioural flexibility in a passerine. Anim. Behav. Cogn. 8, 446–461 (2021). 49. Troisi, C. et al. No evidence for cross-contextual consistency in spatial learning and behavioural flexibility in a passerine. Anim. Behav. Cogn. 8, 446–461 (2021). 50. Akirav, I., Sandi, C. & Richter-Levin, G. References Differential activation of hippocampus and amygdala following spatial learning under stress. Eur. J. Neurosci. 14, 719–725 (2001). 50. Akirav, I., Sandi, C. & Richter-Levin, G. Differential activation of hippocampus and amygdala following spatial learning under stress. Eur. J. Neurosci. 14, 719–725 (2001). 51. von Dawans, B., Strojny, J. & Domes, G. The effects of acute stress and stress hormones on social cognition and behavior: Current state of research and future directions. Neurosci. Biobehav. Rev. 121, 75–88 (2021). 52. Akirav, I. et al. A Facilitative Role for Corticosterone in the Acquisition of a Spatial Task Under Moderate Stress. Learn. Mem. 11, 188–195 (2004). 52. Akirav, I. et al. A Facilitative Role for Corticosterone in the Acquisition of a Spatial Task Under Moderate Stress. Learn. Mem. 11, 188–195 (2004). 53. Griffin, A. S. & Guez, D. Innovation and problem solving: A review of common mechanisms. Behav. Processes 109, 121–134 (2014). 53. Griffin, A. S. & Guez, D. Innovation and problem solving: A review of common mechanisms. Behav. Processes 109, 121–134 (2014). Page 17/23 Page 17/23 54. Morand-Ferron, J., Cole, E. F. & Quinn, J. L. Studying the evolutionary ecology of cognition in the wild: a review of practical and conceptual challenges. Biol. Rev. 91, 367–389 (2016). 55. Chittka, L., Skorupski, P. & Raine, N. E. Speed–accuracy tradeoffs in animal decision making. Trends Ecol. Evol. 24, 400–407 (2009). 56. van Maanen, L. Is There Evidence for a Mixture of Processes in Speed-Accuracy Trade-Off Behavior? Top. Cogn. Sci. 8, 279–290 (2016). 57. Thornton, A. & Wilson, A. J. In search of the Darwinian Holy Trinity in cognitive evolution: a comment on Croston et al. Behav. Ecol. 26, 1460–1461 (2015). 58. Sorato, E., Zidar, J., Garnham, L., Wilson, A. & Løvlie, H. Heritabilities and co-variation among cognitive traits in red junglefowl. Philos. Trans. R. Soc. B Biol. Sci. 373, 20170285 (2018). 59. Henke-von der Malsburg, J., Kappeler, P. M. & Fichtel, C. Linking cognition to ecology in wild sympatric mouse lemur species. Proc. R. Soc. B Biol. Sci. 288, 20211728 (2021). 60. Louison, M. J., Hage, V. M., Stein, J. A. & Suski, C. D. Quick learning, quick capture: largemouth bass that rapidly learn an association task are more likely to be captured by recreational anglers. Behav. Ecol. Sociobiol. 73, 23 (2019). 61. Walsh, B. & Blows, M. W. Abundant Genetic Variation + Strong Selection = Multivariate Genetic Constraints: A Geometric View of Adaptation. Annu. References Rev. Ecol. Evol. Syst. 40, 41–59 (2009). Figures Page 18/23 Page 18/23 Figure 1 Increase in temperature related to physiological stress for each test. NE = novel environment, OF = open field, LSM = low-stress maze, HSM = high-stress maze, LSPS = low-stress problem-solving, HSPS = high- stress problem-solving. The high stress tests are represented in orange and the low-stress test in grey. The significant differences are shown by stars (: 0,01 < p < 0,05; : 0,005 < p < 0,01; *: p < 0,005). Figure 1 Figure 2 Differences in behavior between the high and low stress conditions in cognitive tests. a) Latency to enter the maze for each trial, b) latency to approach the PS set-up for each test. High-stress condition is represented by the orange color and low-stress condition by the grey color. Note: 22/290 values above 120 are not visible on fig. a) (of which 4 are from the LSM and 18 from HSM) and 4/108 above 120 on fig. b) (all from LSPS). Differences in behavior between the high and low stress conditions in cognitive tests. a) Latency to enter the maze for each trial, b) latency to approach the PS set-up for each test. High-stress condition is represented by the orange color and low-stress condition by the grey color. Note: 22/290 values above 120 are not visible on fig. a) (of which 4 are from the LSM and 18 from HSM) and 4/108 above 120 on fig. b) (all from LSPS). Figure 3 Repeatability estimates found between the learning measures of the different PS tasks: (a) whether the task has been solved or not (b) the latency to solve the task (c) the time spend exploring the set-up before solving. Solid arrows show a significant repeatability (: 0,01 < p < 0,05; : 0,005 < p < 0,01; : p < 0,005) and dotted arrows an almost significant repeatability (. : 0,05 < p < 0,1). The high stress tests are Figure 3 Figure 3 Figure 1 Increase in temperature related to physiological stress for each test. NE = novel environment, OF = open field, LSM = low-stress maze, HSM = high-stress maze, LSPS = low-stress problem-solving, HSPS = high- stress problem-solving. The high stress tests are represented in orange and the low-stress test in grey. The significant differences are shown by stars (: 0,01 < p < 0,05; : 0,005 < p < 0,01; : p < 0,005). Page 19/23 Page 19/23 Figure 3 Repeatability estimates found between the learning measures of the different PS tasks: (a) whether the task has been solved or not (b) the latency to solve the task (c) the time spend exploring the set-up before solving. Solid arrows show a significant repeatability (: 0,01 < p < 0,05; : 0,005 < p < 0,01; : p < 0,005) and dotted arrows an almost significant repeatability (. : 0,05 < p < 0,1). The high stress tests are Repeatability estimates found between the learning measures of the different PS tasks: (a) whether the task has been solved or not (b) the latency to solve the task (c) the time spend exploring the set-up before solving. Solid arrows show a significant repeatability (: 0,01 < p < 0,05; : 0,005 < p < 0,01; *: p < 0,005) and dotted arrows an almost significant repeatability (. : 0,05 < p < 0,1). The high stress tests are Page 20/23 Page 20/23 Page 20/23 represented in orange and the low-stress test in grey. Numbers above arrows show repeatability i represented in orange and the low-stress test in grey. Numbers above arrows show repeatability estimates. represented in orange and the low-stress test in grey. Numbers above arrows show repeatability estimates. estimates. Figure 4 Table of the correlations between the learning measures in the cognitive tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is statistically significant. Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors at trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Figure 4 Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors a trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS: ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Behavioral measure in the OF: d = distance. Behavioral measure in the NE: #ex = number of exploration sessions, le = latency to emerge in the NE. Stress measures: ΔT1 = Increase in temperature during trial 1, ΔT2 = Increase in temperature during trial 2. Figure 4 Table of the correlations between the learning measures in the cognitive tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is statistically significant. Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors at trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS: ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Page 21/23 Figure 5 Table of the correlations between learning measures in the cognitive tests and stress and behavioral measures from the personality tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is significant. Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors at trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS: ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Behavioral measure in the OF: d = distance. Behavioral measure in the NE: #ex = number of exploration sessions, le = latency to emerge in the NE. Stress measures: ΔT1 = Increase in temperature during trial 1, ΔT2 = Increase in temperature during trial 2. Figure 5 Table of the correlations between learning measures in the cognitive tests and stress and behavioral measures from the personality tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is significant. Figure 6 Summary of the observed correlations between learning measures and with personality traits. Positive correlations are shown in blue. Thick arrows represent stronger correlations and dashed arrows uncertain correlations. Figure 5 Table of the correlations between learning measures in the cognitive tests and stress and behavioral measures from the personality tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is significant. Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors at trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS: ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Behavioral measure in the OF: d = distance. Behavioral measure in the NE: #ex = number of exploration sessions, le = latency to emerge in the NE. Stress measures: ΔT1 = Increase in temperature during trial 1, ΔT2 = Increase in temperature during trial 2. Table of the correlations between learning measures in the cognitive tests and stress and behavioral measures from the personality tests. Positive coefficients are displayed in blue and negative coefficients in red. The cell is also colored in light blue or red when the correlation is biologically relevant (above 0,30 or below -0,30) and in bright blue or red when the correlation is significant. Learning measures of the maze: Δe = difference in the number of errors between the first and the best trial, e3 = number of errors at trial 3, ΔD = difference in the duration of the test between the first and the best trial, #t = number of the best trial. Learning measures of the PS: ex = time spend exploring the set-up before solving, ls = latency to solve, s = solved or not. Behavioral measure in the OF: d = distance. Behavioral measure in the NE: #ex = number of exploration sessions, le = latency to emerge in the NE. Stress measures: ΔT1 = Increase in temperature during trial 1, ΔT2 = Increase in temperature during trial 2. Page 22/23 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Curiosity.csv Page 23/23 Curiosity.csv DelacouxandGuentherStressCognitionSupplementarymaterial.docx DelacouxandGuentherStressCognitionstatisticsR.rmd IranBehaviors.csv IraniansALL.csv Learningmaze.csv MazeALL.csv Openfield.csv Probsol.csv Temperatures.csv DelacouxandGuentherStressCognitionSupplementarymaterial.docx DelacouxandGuentherStressCognitionstatisticsR.rmd IraniansALL.csv Learningmaze.csv Page 23/23 Page 23/23
https://openalex.org/W2993917445	https://aip.scitation.org/doi/pdf/10.1063/1.5129621	English	null	Energy transfer speed of polymer network and its scaling-law of elastic modulus—New insights	Journal of applied physics	2,019	cc-by	5,098	Articles You May Be Interested In 24 October 2024 05:16:25 24 October 2024 05:16:25 RESEARCH ARTICLE \| DECEMBER 02 2019 Energy transfer speed of polymer network and its scaling- law of elastic modulus—New insights RESEARCH ARTICLE \| DECEMBER 02 2019 RESEARCH ARTICLE \| DECEMBER 02 2019 Energy transfer speed of polymer network and its scaling law of elastic modulus—New insights Ziqian Li ; Zishun Liu Articles You May Be Interested In Energy transfer speed of polymer network and its scaling-law of elastic modulus—New insights Energy transfer speed of polymer network and its scaling-law of elastic modulus—New insights Cite as: J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 Submitted: 30 September 2019 · Accepted: 12 November 2019 · Published Online: 2 December 2019 ABSTRACT The elastic modulus E of a solvent-absorbable polymer network and its polymer-fraction f (or, conversely, water content) obey a scale-law E ≏fν. In particular, for a polymer network paired with a good solvent, ν = 2.3. We now provide a fresh look at this problem from the perspective of energy transfer speed deﬁned for a complex fractal network. Two core algorithms are designed, showing that the elastic modulus actually stands for the ability to transfer energy within a certain matter. Our algorithms can directly give the scaling exponent of ν ¼ 2:3. It is much easier to understand the scaling law E ≏fν from the perspective of energy transfer speed. © 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5129621 October 20 AFFILIATIONS International Center for Applied Mechanics, State Key Laboratory for Strength and Vibration of Mechanical Structures, School of Aerospace, Xi’an Jiaotong University, Xi’an 710049, China International Center for Applied Mechanics, State Key Laboratory for Strength and Vibration of Mecha School of Aerospace, Xi’an Jiaotong University, Xi’an 710049, China a)zishunliu@mail.xjtu.edu.cn INTRODUCTION simply adjusted for the elastic contributions of swollen elastomers, respectively. However, they did not explain the origin of ν ¼ 2:3 (they adopted another approximant value ν ¼ 2:25 instead of 2:3 in their papers). Okumura et al. added two parameters into the F-R model and analyzed their eﬀects.10 Xiang et al. built a new general constitutive model of soft elastomers from the microscopic perspec- tive of statistic mechanics, but it failed to explain ν ¼ 2:3 too.11 By far, only De Gennes provided a relatively good explanation of E ≏f2:3.12 De Gennes deemed that ν ¼ 2:3 may be caused by many factors, including (i) types of solvent (good solvent, poor solvent, or athermal solvent), (ii) deformation of chains, (iii) mean- square end-to-end distance of network strands in both polymer’s preparation state and ﬁnal state, and (iv) types of solvent concentra- tion (dilute, semidilute, or concentrated concentration).2 Although De Gennes’ theory is ingenious, the considered multifarious con- cepts may be confusing because its methodology is quite diﬀerent from the F-R model. Could this question be simpliﬁed for easier understanding? Our answer may be yes. 16:25 The physics of long ﬂexible chains was pioneered by Von Werner Kuhn, Paul J. Flory, and so forth. They constructed the basic theories summarized in Flory’s book.1 The Flory-Rehner theory (the F-R model) gives a rigorous mathematical derivation, showing that the elastic modulus E of a hydrophilic polymer network and its polymer fraction f follow a scale-law E ≏f1=3 for a Gaussian polymer chain (random walk conﬁguration).2–5 Polymer chains in reality are usually non-Gaussian chains [self-avoiding walk (SAW) conﬁguration]. Experimental results show their scaling-law relationship from the preparation state to dehydration state, paired with a good solvent, can be approximated by E ≏f2:3 instead.6,7 Consistent results are obtained when we conduct experiments on the polyacrylamide (PAAm) gel. This is confusing because the F-R model claims that it includes all main physical factors, including polymer molecules, solvent molecules as well as the interplay between them, and hence, its derivation should not have conﬂicted with the experimental results. The reason we conjecture is that the F-R model has defects in describing the complex three-dimensional cross-linked network conﬁguration as it ignores the conﬁguration diﬀerences between chains.2 Journal of Applied Physics ARTICLE scitation.org/journal/jap METHODOLOGY For a cross-linked network of the PAAm gel, many people depict it as a regular net much like a ﬁshing net.17–19 However, the PAAm network can become entangled and form messy coils and strands; i.e., it is more likely to have a self-similar (fractal) network conﬁguration rather than a regular net-structure. To capture this essential property, the best candidate is the “self- avoiding walk” (SAW) model proposed by Flory.1 In this paper, the CNG algorithm is designed for the three- dimensional discrete PBC space, as the 3D space is much closer to the reality than the 2D one. The design ideas in the 3D space (see Movie 3 in the supplementary material) are consistent with the case in the 2D space as shown previously. In the discrete 3D space, one set of spatial coordinates consists of three integers. Thus, the total number of positions (spatial coordinates) is constant for space with a determinate width w. For example, if the width of the space w is 10, the total number of particles that space can involve is 1000. Every particle, including monomers, cross-linkers, and water mole- cules, only occupies one position. Monomers are shown in blue, Physicists attempt to simplify polymer chains as much as pos- sible by considering them as threads, shoelaces, or necklaces made of beads on a string. For a cross-linked network of the PAAm gel, many people depict it as a regular net much like a ﬁshing net.17–19 However, the PAAm network can become entangled and form messy coils and strands; i.e., it is more likely to have a self-similar (fractal) network conﬁguration rather than a regular net-structure. To capture this essential property, the best candidate is the “self- avoiding walk” (SAW) model proposed by Flory.1 SAW is a path on a lattice that does not visit the same site more than once. One path of SAW can be used to approximate a single polymer chain (see Movie 1 in the supplementary material). In spite of the simple deﬁnition of SAW, most basic questions about this model are diﬃcult to resolve in a mathematically rigorous fashion. In fact, they are achieved by a variety of nonrigorous methods, primarily including computer simulations.20 Consequently, using a rigorous mathematical derivation to get the scaling-law of E ≏fν will lead to a dead end. We resort to computer simulations to mimic the network conﬁguration of the PAAm gel in this paper. METHODOLOGY We put up a new perspective from “complex network theory” (CN theory) and energy transfer (ET) speed to reconsider the scaling- law relationship of E ≏fν. Polymers, including rubbers and hydro- gels, are macromolecules and usually have their own network In the open literature, some trials to amend the F-R model to explain ν ¼ 2:3 instead of 1=3 have been carried out. Bastide et al.8 and Durning and Morman9 developed their scaling approaches J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019 J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 126, 215101-1 Journal of Applied Physics scitation.org/journal/jap ARTICLE of ultraviolet radiation catalysis and photoinitiators, the thermody- namic motion of molecules assembles them into a huge cross- linked network.21 This process can be imagined as playing the LEGO bricks game. Figure 1 illustrates an example of the growth process of the network in the 2D space. The corresponding visuali- zation process is shown in Movie 2 in the supplementary material, where red points are cross-linkers (MBAA), and blue parts are polymer chains (AAm). conﬁgurations. We wonder whether CN theory, an emerging interdis- ciplinary ﬁeld,13 could be used to analyze the properties of the polymer network. We infer that CN theory may be appropriate as it has successfully solved many similar scaling-law problems in diﬀerent ﬁelds, such as the World Wide Web, earthquakes, epidemic spreading as well as the social network.13–16 CN theory’s mathematical base has not been completely and systematically built yet, but computer simu- lations are helpful and reliable in this regard. Therefore, we plan to abstract a discrete network model from a cross-linked PAAm gel and use MATLAB (MathWorks) to analyze the relationship of E ≏fν. Two core algorithms are designed: “Cross-linked-Network-Growth algorithm” (CNG algorithm) and “Energy-Transfer algorithm” (ET algorithm), and the MATLAB codes are provided online (http://www. zsliu.net/index.php/publications). Simulating a full-scale gel network is impossible, and hence, we only plan to obtain a partial mesoscopic network. Since the macro- scopic world is almost inﬁnite compared with the size of monomer molecules, the periodic boundary condition (PBC) is adopted for equating the inﬁnite space. The 2D example that explains the rules of PBC is shown in Fig. 2. p p p Physicists attempt to simplify polymer chains as much as pos- sible by considering them as threads, shoelaces, or necklaces made of beads on a string. METHODOLOGY The PAAm gel consists of acrylamide (AAm) monomers and N, N-methylenebisacrylamide (MBAA) as cross-linkers. With the help FIG. 2. The schematic of the periodic boundary space (2D) and the illustration of the periodic boundary rule: When a molecular unit reaches the periodic boun- dary of the space, take the AAm (marked as Pr) on the right side; for example, it ends growth. It continues to grow synchronously from the new position (marked as Pl) on the opposite side of the space again. However, it should be regarded as a new chain. Similarly, the Pd monomer that touches the down-side will jump to the up-side and continually grow. 24 October 2024 05:16:25 24 October 2024 05:16:25 FIG. 1. The schematic of the growth process of a cross-linked PAAm network in the two-dimensional space. (a) Eight AAm monomers connected end to end, forming a short chain. The identity of the ninth position depends on the number ratio of AAm and MBAA. For example, if there are 40 000 AAm and 16 MBAA randomly wander in the space at present, then the ninth position has 99.96% probability to be AAm again or 0.04% probability to be an MBAA. (b) If the ninth position is an MBAA, it can connect three other AAm as new neighbors. (c) A huge cross-linked network will form in this way after a long time. This process can be abstracted as a SAW model. FIG. 1. The schematic of the growth process of a cross-linked PAAm network in the two-dimensional space. (a) Eight AAm monomers connected end to end, forming a short chain. The identity of the ninth position depends on the number ratio of AAm and MBAA. For example, if there are 40 000 AAm and 16 MBAA randomly wander in the space at present, then the ninth position has 99.96% probability to be AAm again or 0.04% probability to be an MBAA. (b) If the ninth position is an MBAA, it can connect three other AAm as new neighbors. (c) A huge cross-linked network will form in this way after a long time. This process can be abstracted as a SAW model. FIG. 1. The schematic of the growth process of a cross-linked PAAm network in the two-dimensional space. (a) Eight AAm monomers connected end to end, forming a short chain. The identity of the ninth position depends on the number ratio of AAm and MBAA. TABLE I. Polymer fraction and its corresponding space width. The precise value of the corresponding polymer fraction is f ¼ 10:10%. Table I lists the one-to-one correspondence of f and w. Figure 4 shows four network conﬁgurations with diﬀerent polymer fractions. csolid, s ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ G=ρ p , (1) (1) The network conﬁgurations generated by the CNG algorithm are static, and it is by far impossible to “stretch” them and get strain-stress curves to calculate their modulus. The diﬃculty lies in building a connection between the network conﬁgurations and where G is the shear modulus and ρ is the density. In other words, shear modulus and sound velocity obey a scaling relation G ≏c2 solid, s. In addition, sound is, in fact, a form of energy. Therefore, the concept of sound velocity here may be directly compared to the new concept of energy transfer speed V. The energy transfer speed V and material elastic modulus E might also have a scaling relation of FIG. 3. Schematic of one position (green hexagram) in the three-dimensional discrete space and its 26 neighbors (blue ﬁlled points at the front and hollow points at back), including 6 neighbors on faces, 12 neighbors on sides, and 8 neighbors at corners. The distance from the central position to a surface neighbor is 1. g g E ≏V2: (2) E ≏V2: (2) 0 05 6 (2) Now, we have found a bridge connecting the static network conﬁgurations generated by the CNG algorithm and elastic modulus. If we can design an “energy transfer (ET) algorithm” to calculate the time of energy spreading within a network, it is possi- ble to determine the corresponding energy transfer speed. To better understand the concept of energy transfer, it is easier to imagine it as a serious forest ﬁre ﬂaring up in this network space (see Movie 4 in the supplementary material). AAm and MBAA particles can be seen as trees, and the energy spreading among them is like a spreading ﬁre. The idea of determining the ﬁre spreading speed is to ignite some trees located on one edge of the forest and record the total time cost for spreading the whole forest. In the beginning, all particles on the one side are ignited. Then, the burning trees ignite their neighbors according to some designated rules (introduced later). TABLE I. Polymer fraction and its corresponding space width. TABLE I. Polymer fraction and its corresponding space width. cross-linkers are shown in red, and unoccupied positions represent water molecules. Every position has a total of 26 nearest neighbors around, as shown in Fig. 3. Rigorously speaking, the distances from these neighbors to the central seat are not identical, but the tiny diﬀerences in neighbor-to-center distances can be ignored. This approximation has a great advantage as it reduces the probability that a growing chain walks into the dead end, becoming a dangling chain. their modulus. It is gratifying that this problem may be solved by another novel perspective on the physical meaning of material modulus. The modulus of a material, by our conjecture, stands for its ability to resist disturbance or even deformation. High modulus represents that the interactions within the microstructure of the material are strong. More atoms or molecules can react together against external disturbances or stimulations at the same time. It also means that they can transfer external energy to their neighbors and spread energy to distant places faster. There is an example in our life that can help to understand this perspective. It is well known that diﬀerent materials can spread sound waves at diﬀerent speeds. At 20 °C, the speed of sound in air is about 343 m=s. Sound travels at 1493 m=s in water, at 5130 m=s in steel, and at 12 000 m=s in diamond. In acoustics, the sound velocity of shear waves (csolid, s) propagating in a homogeneous three-dimensional solid is given by All CNG algorithm input parameters only involve the number of cross-linkers nMBAA, the number of monomers nAAm, and the space width w. The polymer fraction f in the CNG algorithm is determined only by w and nAAm. The mass of MBAA is ignored as its mass proportion is negligible.6 Here, we present an example of how to calculate the w when f ¼ 10%. The mass fraction of AAm is 10% and water accounts for 90%. The molar masses of the water mol- ecule and AAm molecule are 18 g=mol and 71:08 g=mol, respectively. Thus, the molecular number ratio for water and AAm is about 35:54. If we take nMBAA ¼ 19 494 and nMBAA ¼ 9, the total positions shall be about Nspace ¼ 19 494 (1 þ 35:54) 712 311 ¼ w3. Then, w is 89:31, taking integer 89 as the ﬁnal result. METHODOLOGY For example, if there are 40 000 AAm and 16 MBAA randomly wander in the space at present, then the ninth position has 99.96% probability to be AAm again or 0.04% probability to be an MBAA. (b) If the ninth position is an MBAA, it can connect three other AAm as new neighbors. (c) A huge cross-linked network will form in this way after a long time. This process can be abstracted as a SAW model. FIG. 2. The schematic of the periodic boundary space (2D) and the illustration of the periodic boundary rule: When a molecular unit reaches the periodic boun- dary of the space, take the AAm (marked as Pr) on the right side; for example, it ends growth. It continues to grow synchronously from the new position (marked as Pl) on the opposite side of the space again. However, it should be regarded as a new chain. Similarly, the Pd monomer that touches the down-side will jump to the up-side and continually grow. J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019 126, 215101-2 126, 215101-2 Journal of Applied Physics ARTICLE scitation.org/journal/jap TABLE I. Polymer fraction and its corresponding space width. TABLE I. Polymer fraction and its corresponding space width. As the polymer fraction increases, the PAAm gel network becomes more con- densed. It looks more like a nonfractal construction when the polymer fraction reaches 70%. beginning [Fig. 5(a) t = 0], all particles on the right side are ignited (introduced a ﬁre source), and they are marked as green. Which side to choose as the source of ﬁre is an artiﬁcial choice. We also have tried the particles on the up-side and front-side as the ﬁre source, and the energy speed output is indeed diﬀerent for the same network conﬁguration because the network is not isotropic and homogeneous. Especially for the network conﬁguration with low f, the divergences among the left-side, up-side, and front-side are distinct. However, the ﬁnal statistic results are still very close to each other. In the following step [Fig. 5(a) t = 1], those already ignited trees at t = 0 are marked as black. These green points will ignite their neighbors in the next step (t = 2). In this way, trees are ignited gradually until there are no other new trees that can be ignited and the spreading ﬁre cannot sustain anymore. 9 nearest neighbors (P2–P10) belonging to three diﬀerent chains. P2 and P3 are the nearest neighbors sharing the same chain with P1. P2 has been already burnt at the previous time step, and P3 is still untouched. P4–P7 are on another chain, while P8 and P9 are on the third chain, where P4, P5, P8, and P9 have been burnt and the rest are still intact. For the next time step, P3 will deﬁnitely be ignited. Moreover, one more neighbor should also be ignited from P6, P7, and P10 randomly. This stipulation comes from a deduction that one AAm monomer forms only one hydrogen bond with another AAm through water molecules. It only happens when two chains are close enough, usually at a distance equivalent to the size of one or two water molecules.22,23 Hydrogen bonds are also able to trans- fer energy by acting as a virtual connection. The spreading ﬁre must obey some rules as well. A burning tree does not ignite all its neighbors at once. Figure 5(b) illustrates an easy-to-understand example explaining the energy transfer rules. In Fig. 5(b), there are ten points in the local space. The central green point (marked as P1) is on ﬁre at the present time step. It has TABLE I. Polymer fraction and its corresponding space width. At the end, after all ignitable trees are burnt, the energy transfer speed V is deﬁned as the number of “burnt trees” nb divided by the total number of time steps T. V is a dimensionless quantity, FIG. 3. Schematic of one position (green hexagram) in the three-dimensional discrete space and its 26 neighbors (blue ﬁlled points at the front and hollow points at back), including 6 neighbors on faces, 12 neighbors on sides, and 8 neighbors at corners. The distance from the central position to a surface neighbor is 1. V ¼ def nb=T: (3) (3) The details about the ET algorithm are shown in Fig. 5. Figure 5(a) shows the general process of energy transfer. At the J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019 126, 215101-3 126, 215101-3 Journal of Applied Physics scitation.org/journal/jap FIG. 4. Conﬁgurations of PAAm gel networks generated by the CNG algo- rithm for different polymer fractions: (a) f¼10wt:%, w¼89; (b) f¼20wt:%, w¼69; (c) f¼40wt:%, w¼51; and (d) f ¼ 70 wt: %, w ¼ 37. The messy blue lines represent polymer chains, and red dots are cross-linked points. For conﬁguration f ¼ 10%, macromo- lecular chains in the space are sparse that looks like a fractal construction. As the polymer fraction increases, the PAAm gel network becomes more con- densed. It looks more like a nonfractal construction when the polymer fraction reaches 70%. 24 October 2024 05:16:25 FIG. 4. Conﬁgurations of PAAm gel networks generated by the CNG algo- rithm for different polymer fractions: (a) f¼10wt:%, w¼89; (b) f¼20wt:%, w¼69; (c) f¼40wt:%, w¼51; and (d) f ¼ 70 wt: %, w ¼ 37. The messy blue lines represent polymer chains, and red dots are cross-linked points. For conﬁguration f ¼ 10%, macromo- lecular chains in the space are sparse that looks like a fractal construction. As the polymer fraction increases, the PAAm gel network becomes more con- densed. It looks more like a nonfractal construction when the polymer fraction reaches 70%. FIG. 4. Conﬁgurations of PAAm gel networks generated by the CNG algo- rithm for different polymer fractions: (a) f¼10wt:%, w¼89; (b) f¼20wt:%, w¼69; (c) f¼40wt:%, w¼51; and (d) f ¼ 70 wt: %, w ¼ 37. The messy blue lines represent polymer chains, and red dots are cross-linked points. For conﬁguration f ¼ 10%, macromo- lecular chains in the space are sparse that looks like a fractal construction. RESULTS Similarly, black points (P4, P5, P2, P8, and P9) are monomers that have already activated by energy (already burnt trees), and the central green point P1 is the current ﬁre source. Black points cannot be ignited anymore; only P3, P6, P7, and P10 are the candidates of being ignited by P1. completely, and it becomes a dense entity. Then, T is w=2, and nb is w3, resulting in Vlimit ¼ 2w2. Based on our earlier hypothesis [see Eq. (2)], we have the following power-law relationship: r 2024 05:16: 1:16 according to two diﬀerent ﬁtting methods. Besides, the theo- retical upper limit Vlimit in Fig. 6 is calculated from the case where f ¼ 1. In this situation, every position in the space is occupied by monomers or cross-linkers. Fractal network conﬁguration disappears E ≏V2 ≏f2τ: (4) 5 (4) FIG. 6. Discrete data of polymer fraction against energy transfer speed. The dis- crete data range from f ¼ 10% to f ¼ 70%. The data at f ¼ 1 are the theoret- ical upper limit. Two ﬁtting methods give the 1.08 and 1.16 power-law exponents. Taking the average of τ ¼ 1:08 and 1:16, the result is Taking the average of τ ¼ 1:08 and 1:16, the result is Taking the average of τ ¼ 1:08 and 1:16, the result is E ≏f2:24: (5) (5) It is a satisfying result where 2.24 is very close to our target value 2.3, and it reinforces our hypothesis in this paper to a quite extent. In fact, we also ran our models for diﬀerent scales, including 10 752 AAm plus 2 MBAA, 53 760 AAm plus 16 MBAA, and even 108 300 AAm plus 50 MBAA, obtaining similar results. It is a satisfying result where 2.24 is very close to our target value 2.3, and it reinforces our hypothesis in this paper to a quite extent. In fact, we also ran our models for diﬀerent scales, including 10 752 AAm plus 2 MBAA, 53 760 AAm plus 16 MBAA, and even 108 300 AAm plus 50 MBAA, obtaining similar results. RESULTS We run CNG and ET algorithms hundreds of times. The rela- tionship of V ≏fτ can then be ﬁtted by MATLAB built-in app “Curve Fitting.” The result is shown in Fig. 6, with τ ¼ 1:08 or J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019 126, 215101-4 Journal of Applied Physics scitation.org/journal/jap FIG. 5. (a) The illustrations of the energy transfer process. In every code loop, the ET algorithm ﬁrst shows the “molecular trees” (AAm) that are burning currently (marked as green points) and then calculate which neighbors will be ignited (highlighted as green in the next time step). Those already ignited trees at the previous time steps are marked as black, and they cannot ignite others and be ignited by others again. Besides, since this space is periodic, when the ﬁre reaches the periodic boundary, it will ignite the trees on the boundary and the counterpart trees on the opposite boundary. (b) The schematic diagram of energy transfer rules. There are three partial polymer chains (blue line: line 2-1-3, line 4-5-6-7, and line 8-9-10) within the neighbor area of the center point. Similarly, black points (P4, P5, P2, P8, and P9) are monomers that have already activated by energy (already burnt trees), and the central green point P1 is the current ﬁre source. Black points cannot be ignited anymore; only P3, P6, P7, and P10 are the candidates of being ignited by P1. 24 O FIG. 5. (a) The illustrations of the energy transfer process. In every code loop, the ET algorithm ﬁrst shows the “molecular trees” (AAm) that are burning currently (marked as green points) and then calculate which neighbors will be ignited (highlighted as green in the next time step). Those already ignited trees at the previous time steps are marked as black, and they cannot ignite others and be ignited by others again. Besides, since this space is periodic, when the ﬁre reaches the periodic boundary, it will ignite the trees on the boundary and the counterpart trees on the opposite boundary. (b) The schematic diagram of energy transfer rules. There are three partial polymer chains (blue line: line 2-1-3, line 4-5-6-7, and line 8-9-10) within the neighbor area of the center point. CONCLUDING REMARKS The scaling law between modulus and polymer fraction can be explained by complex network theory as the network conﬁguration of the PAAm gel can be abstracted into a self-avoiding walk model. We hereby reconsider the physical meaning of modulus to look for a bridge to connect our network model and elastic modulus. It is reasonable to think that the modulus of a material reﬂects its ability to transfer disturbance, deformation, or any form of energy. The number of particles participating in the dissipation of external energy within a certain amount of time determines the modulus. FIG. 6. Discrete data of polymer fraction against energy transfer speed. The dis- crete data range from f ¼ 10% to f ¼ 70%. The data at f ¼ 1 are the theoret- ical upper limit. Two ﬁtting methods give the 1.08 and 1.16 power-law exponents. J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019 126, 215101-5 Journal of Applied Physics scitation.org/journal/jap ARTICLE 3L. R. G. Treloar, The Physics of Rubber Elasticity, 3rd ed. (Oxford University Press, New York, 1975). Actually, this can be seen as a macroscopic reﬂection of microinter- plays. Higher modulus means that it can spread energy to every part of the material faster. Therefore, a new concept, energy trans- fer speed, is deﬁned in this paper, which can be used to explain modulus. From the perspective of energy transfer, an interesting bridge connecting material modulus and polymer fraction, E ≏V2 ≏f2τ, has been found. In order to ﬁnd out the exact value of τ, two algorithms are designed in this work, which are the cross- linked network-growth algorithm and energy transfer algorithm. Finally, a satisfying result of E ≏f2:24 is obtained. We hope our work can inspire others with a fresh look at hydrogels. Moreover, we will continue to handle the questions on the fracture behaviors of hydrogels based on this methodology. 4Z. Liu, W. Toh, and T. Y. Ng, Int. J. Appl. Mech. 7, 1530001 (2015) 5 5S. Zheng, Z. Li, and Z. Liu, Int. J. Appl. Mech. 137, 263 (2018). 6 6E. Zhang, R. Bai, X. P. Morelle, and Z. Suo, Soft Matter 14, 3563 (2018). 7 7K. Urayama, T. Kawamura, and S. Kohjiya, J. Chem. Phys. 105, 4833 (1996). 8J. Bastide, S. Candau, and L. Leibler, Macromolecules 14, 719 (1981). 9 9C. J. Durning and K. N. Morman, Jr., J. Chem. Phys. 98, 4275 (1993). CONCLUDING REMARKS 10 10D. Okumura, A. Kondo, and N. Ohno, J. Mech. Phys. Solids 90, 61 (2016). 11Y. Xiang, D. Zhong, P. Wang, G. Mao, H. Yu, and S. Qu, J. Mech. Phys. Solids 117, 110 (2018). 12P.-G. De Gennes, Scaling Concepts in Polymer Physics (Cornell University Press, New York, 1979). 13M. Newman, Networks (Oxford University Press, 2018). SUPPLEMENTARY MATERIAL y 14A.-L. Barabási and R. Albert, Science 286, 509 (1999). 15 14A.-L. Barabási and R. Albert, Science 286, 509 (1999). 15P. Bak, How Nature Works: The Science of Self-Organized Criticality (Springer Science & Business Media, 2013). See the supplementary material for Movies 1–4 mentioned in this paper. 16R. Pastor-Satorras and A. Vespignani, Phys. Rev. Lett. 86, 3200 (2001). 17X. Zhao, Soft Matter 10, 672 (2014). ACKNOWLEDGMENTS 18F. J. Vernerey, R. Brighenti, R. Long, and T. Shen, Macromolecules 51, 6609 (2018). We gratefully acknowledge the support from the National Natural Science Foundation of China (NNSFC) through Grant Nos. 11811530287 and 11572236. 19R. Bai, J. Yang, X. P. Morelle, C. Yang, and Z. Suo, ACS Macro Lett. 7, 312 (2018). 20N. Madras and G. Slade, The Self-Avoiding Walk (Springer Science & Business Media, 2013). REFERENCES 21T. Tanaka, Gels. Sci. Am. 244, 124 (1981). 22 21T. Tanaka, Gels. Sci. Am. 244, 124 (1981). 22 1P. J. Flory, Principles of Polymer Chemistry (Cornell University Press, New York, 1953). 2 22J. N. Israelachvili, P. M. Mcguiggan, and A. M. Homola, Science 240, 189 (1988). 23E. Bonaccurso, M. Kappl, and H.-J. Butt, Phys. Rev. Lett. 88, 076103 (2002). 24 Oc 2M. Rubinstein and R. H. Colby, Polymer Physics (Oxford University Press, New York, 2003), Vol. 23. 2M. Rubinstein and R. H. Colby, Polymer Physics (Oxford University Press, New York, 2003), Vol. 23. 126, 215101-6 126, 215101-6 J. Appl. Phys. 126, 215101 (2019); doi: 10.1063/1.5129621 © Author(s) 2019
https://openalex.org/W2052313781	https://www.scielo.br/j/ni/a/x9ZLtyDm4j3nY64vnqgXj5z/?lang=en&format=pdf	Spanish; Castilian	null	Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains	Neotropical ichthyology/Neotropical Ichthyology	2,011	cc-by	8,527	1Universidade de São Paulo, Museu de Zoologia, P. O. Box 42494, 04218-970 São Paulo, Brazil. alnferreira@gmail.com; oyakawa@usp.br; cezarnolasco@yahoo.com.br 2Instituto Nacional de Pesquisas da Amazônia, Coordenação de Pesquisas em Biodiversidade, P. O. 478, 69060-001 Manaus, Brazil. zuanon@inpa.gov.br Neotropical Ichthyology, 9(4): 767-775, 2011 Copyright © 2011 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 9(4): 767-775, 2011 Copyright © 2011 Sociedade Brasileira de Ictiologia Lebiasina yepezi, a new Lebiasininae (Characiformes: Lebiasinidae) from the Serra Parima-Tapirapecó mountains André L. Netto-Ferreira1, Osvaldo T. Oyakawa1, Jansen Zuanon2 and José C. Nolasco1 André L. Netto-Ferreira1, Osvaldo T. Oyakawa1, Jansen Zuanon2 and J Ferreira1, Osvaldo T. Oyakawa1, Jansen Zuanon2 and José C. Nolasco1 Lebiasina yepezi, a new Lebiasininae with a conspicuous color pattern, is described. The new species is endemic of the headwaters of the rio Negro, rio Branco, and rio Orinoco in the Serra Parima-Tapirapecó Mountains, at the border of Brazil and Venezuela. The new species is readily distinguished from all other Lebiasininae by the presence of four black longitudinal stripes on the trunk, and the triangular shaped dorsal surface of the mesethmoid, lacking lateral projections. The unusual color pattern is contrasted with those of other lebiasinin, as well as members of the pyrrhulinin genus Nannostomus. A close relationship between Lebiasina yepezi and the Gran Sabana (Venezuela) species is suggested based in color pattern features. The present contribution corresponds to a further refutation of the type locality of L. intermedia, as suggested on its description, since Lebiasinins, except L. bimaculata, L. boruca, and L. festae, do not occur in low land waters. Lebiasina yepezi, um novo Lebiasininae com um conspícuo padrão de colorido, é descrito. A nova espécie é endêmica das cabeceiras do rio Negro, rio Branco e rio Orinoco, na Serra Parima-Tapirapecó, ao longo da fronteira entre Brasil e Venezuela. A nova espécie é prontamente diferenciada das demais espécies pela presença de quatro faixas longitudinais escuras ao longo do corpo, e a superfície dorsal do mesetmoide com o formato triangular, não apresentando projeções laterais. O padrão de colorido incomum é comparado com o dos demais Lebiasininae e também com o dos Pyrrhulininae do gênero Nannostomus. Uma próxima relação é proposta entre Lebiasina yepezi e as espécies da Gran Sabana (Venezuela) com base em caracteres de colorido. A presente contribuição corresponde a mais uma refutação da localidade-tipo de L. intermedia conforme sugerida na descrição daquela espécie, uma vez que os lebiasiníneos, com exceção de L. bimaculata, L. boruca e L. festae, não ocorrem em rios de terras baixas. Key words: Distribution pattern, Gran Sabana, Guyana Shield, Ostariophysi, Systematics. Nannostomus or Pyrrhulina.-Géry, 1977: 588 [photography]. Lebiasina sp.-Géry & Zarske, 2002: 44-45 [comparison with Derhamia hoffmannorum Géry & Zarske]. Nannostomus or Pyrrhulina.-Géry, 1977: 588 [photography]. Lebiasina sp.-Géry & Zarske, 2002: 44-45 [comparison with Derhamia hoffmannorum Géry & Zarske]. y Collections in the upper courses of the rio Negro, rio Branco, and rio Orinoco draining from the Serra Parima- Tapirapecó, along the border of Brazil and Venezuela, yielded specimens of a conspicuously patterned new lebiasinin, which is described herein, and its relationships among other Lebiasininae are discussed. The new species is assigned to the genus Lebiasina following anterior studies questioning the validity of Piabucina (Eigenmann & Allen, 1942; Weitzman, 1964; Machado-Allison, 1974; Dahl, 1971; Géry, 1977; Taphorn, 1992; Géry & Zarske, 2002), or disregarding it (Ardila-Rodríguez, 1994, 1999, 2000, 2001, 2002, 2004, 2008a, 2008b, 2009, 2010), not withstanding the presence of an adipose fin in most species described. Holotype. MNRJ 39067, 152.9 mm SL, Brazil, Amazonas State, upper rio Negro drainage, rio Marari, tributary of rio Padauari, at basecamp between 700 and 1000 m elev., upstream to Missão Marari of Ianomami Village, Serra Tapirapecó, 1°12’26”N 64°47’18”W, 12 Apr to 20 May 2004, U. Caramaschi & D. F. Moraes. Paratypes. MNRJ 38917, 24, 23.7-123.5 mm SL (4, 97.5-123.5 mm SL), same data as holotype. Brazil. Roraima State, rio Branco drainage: INPA 15091, 2, 21.8-40.5 mm SL, MZUSP 62918, 3, 31.9-81.5 mm SL, MZUSP 81128 (1 c&s, 42.8 mm SL) Igarapé das Irmãs, rio Parima, tributary of rio Branco, 21 Sep 1998, Oncocercosis Team. INPA 15742 (1, 88.2 mm SL), rio Parima, tributary of rio Branco, Xitei-Xidea, 2°36’29”N 63°52’18”W, 17 Apr 1979, U. Barbosa (Oncocercosis Team). INPA 15743 (9/18, 14.6-22.6 mm SL), MZUSP 62917 (8, 12.0-38.2 mm SL), Irmãs Xiteí, 9 Dec 1997, Oncocercosis Team. INPA 20518 (1, 93.6 mm SL), MZUSP 81129, 1, 90.2 mm SL, rio Mucajaí, tributary of rio Branco, Yanomami’s indigenous area, Apr 2002, M. William. MPEG 1830, 1, 94.5 mm SL, Serra Parima, tributary of rio Uraricoera, 1200 m elev., Mar 1962, M. Hidasi [specimen examined by Géry, in Géry & Zarske, 2002: 45]. MZUSP 62916, 1, 96.5 mm SL, rio Parima, tributary of rio Branco, Xitei-Xidea, 2°36’29”N 63°52’18”W, 17 Apr 1979, U. Barbosa (Oncocercosis Team). USNM 306560, 3, 21.8-27.0 mm SL, río Parima, H. Axelrod, March 1963 [one specimen photographed and ilustrated in Géry, 1977: 588]. Venezuela. Amazonas State, rio Orinoco drainage: MAC-PAY 591 8, 37.1-134.9 mm SL (3, 90.6-134.9 mm SL), rio Niyayobä, Sierra Parima, 2°45’N 64°18’W, 17 Oct 1982, J. Fernández. Nannostomus or Pyrrhulina.-Géry, 1977: 588 [photography]. Lebiasina sp.-Géry & Zarske, 2002: 44-45 [comparison with Derhamia hoffmannorum Géry & Zarske]. MAC-PAY 619, 3, 27.3-40.0 mm SL, Cañito Jopajewä, tributary of left margin of río Padamo, upstream of Coshilowäteri, tributary of right margin of rio Orinoco, 2º54’N 65º17’W, 22 Oct 1982, J. Fernández. MBUCV 11000, 2, 114.3-145.8 mm SL (1, 145. 8 mm SL), Atabapo, Caño Herita, río Ocamo, tributary of right margin of upper rio Orinoco, 3°45’N 64°35’W, Nov 1975, J. Lizot. MHNLS 4160, 1, 106.7 mm SL, Mavaca, upper rio Orinoco, mouth of río Mavaca, 2º31’N 65º11’W, Nov 1981, J. Finkers. MHNLS 797, 2, 40.8-83.8 mm SL, Venezuela, Amazonas, Raudal Culebra 3º36’N 65º48’W, Dec 1951, A. Fernández-Yepez. Results high waters in the rio Amazonas, rio Orinoco and coastal drainages on the Atlantic and Pacific slopes of Colombia, Costa Rica, Ecuador, Guyana, Panama, Peru, and Venezuela. The only exception would be Lebiasina intermedia Meinken, presumably from Santarém in the lower Amazon (Géry, 1977; Weitzman & Weitzman, 2003). Introduction miniature Nannostomus anduzei Fernandez & Weitzman, about 1.6 mm SL, to species of Lebiasina, that reach up to 200.0 - 250.0 mm SL. The Lebiasinidae is a Neotropical characiform family consisting of seven genera distributed in two subfamilies: Lebiasininae (Lebiasina Valenciennes, plus Piabucina Valenciennes), and Pyrrhulininae (Pyrrhulina Valenciennes, Copeina Fowler, Copella Myers and Nannostomus Günther). The monotypic genus Derhamia Géry & Zarske was originally assigned to the Lebiasininae (Géry & Zarske, 2002), but it was not placed in either subfamilies by Weitzman & Weitzman (2003), and it is currently considered an incertae sedis genus in the Lebiasinidae (Nelson, 2006). The species belonging to the family are small- to medium-sized fishes ranging from the The greatest diversity of the family occurs in the Pyrrhulininae, which are distributed primarily in the Amazon and Orinoco basins, and coastal rivers of the Guyana Shield, occuring mainly in shallow waters of forest streams and small ponds along the lower parts of larger rivers. Pyrrhulina, the most widespread and speciose genus in the family, is the only one known for the Paraná-Paraguay system (including the rio Uruguay). Species of Lebiasininae, on the other hand, are usually found in the upper courses of streams with rocky or sandy bottoms and well oxygenated 767 A new Lebiasininae from the border of Brazil and Venezuela 768 Material and Methods C. Nolasco Fig. 1. Lebiasina yepezi: a) MNRJ 39067 holotype (152.9 mm SL), Brazil, Amazonas, rio Marari, tributary of rio Padauari, Serra Tapirapecó; b) MNRJ 38917, paratype (97.5 mm SL), same data as holotype. Fig. 1. Lebiasina yepezi: a) MNRJ 39067 holotype (152.9 mm SL), Brazil, Amazonas, rio Marari, tributary of rio Padauari, Serra Tapirapecó; b) MNRJ 38917, paratype (97.5 mm SL), same data as holotype. distinctly larger, teeth gradually decreasing in size posteriorly. Inner series with several minute conical teeth extending from the symphysis to coronoid process. Branchiostegal rays 4, 3 articulating with anterior-ceratohyal, and one with posterior- ceratohyal. yuruaniensis Ardila-Rodríguez) by the secondary stripe extending along scales of second and third longitudinal series (vs. stripe extending along scales of first and second longitudinal series), and caudal-fin blotch restricted to the caudal-fin median rays (vs. anterior margin of candal-fin blotch extending onto caudal peduncle). The new species also differ from its closest geographical relatives, L. yepezi differs from L. taphorni Ardila-Rodríguez, L. unitaeniata (Günther), L. uruyensis, and L. yuruaniensis Ardila-Rodríguez by the extent of the intermediate stripe (from infraorbitals to anal-fin origin; vs. intermediate stripe restricted to infraorbitals 2 to 4). Scales cycloid, circuli restricted to posterior border of scales, several radii (approx. 25) converging to center of scales and strongly anastomosed in the center, forming several cells. Lateral line longitudinal series with 26(1), 27(5), 28(4), or 29(1) scales, of which only 3(3), 4(1), 5(9), or 6(1) are perforated. Longitudinal rows of scales between dorsal and pelvic fins 7. Predorsal scales 12(7) or 13(7). First longitudinal row of scales usually reaching vertical through dorsal-fin terminus, but may extend beyond that point by one or two scales. Circumpeduncular scales 12. Description. Morphometric data of Lebiasina yepezi are presented in Table 1. Body compressed and elongate. Dorsal profile of body distinctly convex from upper lip to vertical through nares, becoming slightly convex from that point to vertical through pectoral-fin origin, then nearly straight from latter to dorsal-fin origin; and slightly concave from that point to dorsal caudal-fin procurrent rays. Ventral profile of head and trunk convex from lower lip to pelvic-fin origin; straight from that point to anal-fin origin, convex at anal fin base, and concave from anal fin terminus to caudal-fin procurrent rays. Mouth terminal. Premaxillary with single row of 9(1) or 11(1) tricuspid teeth decreasing in size posteriorly. Material and Methods Counts and measurements follow Fink & Weitzman (1974) and Menezes & Weitzman (1990). All measurements were made point-to-point on the left side of the specimens whenever possible, with dial calipers with a precision of 0.1 mm. Standard length is presented in mm, all other measurements are presented as proportions of standard length (SL), except for subunits of head, which are presented as proportions of head length (HL). Meristic data are given in the description, followed by the frequency of each count in parenthesis, and an asterisk indicates the value of the holotype. Counts of teeth, vertebrae, supraneurals, and procurrent caudal-fin rays were taken only from cleared and double-stained paratypes (c&s), prepared according to Taylor & van Dyke (1985), or x- rayed specimens. Vertebrae of the Weberian apparatus were included as four elements in the precaudal counts, and the fused PU1+U1 of the caudal region as a single element in the caudal counts. Pattern of circuli and radii was observed on scales sampled from the region between the lateral line series and the insertion of the dorsal fin. Color pattern nomenclature follows Weitzman (1966). Institutional abbreviations follow Ferraris (2007), with the addition of CAR (Colección Carlos A. Ardila Rodríguez, Barranquilla, Atlantico, Colombia), IAvH-P (Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Colleción de Pecas, Villa de Leyva, Boyacá, Colombia), MAC-PAY (Ministerio de Agricultura y Cría, Instituto Nacional de Investigaciones Agricolas (INIA), Estación Experimental Amazonas, Puerto Ayacucho, Amazonas, Venezuela), UCR (Universidad de Costa Rica, Escuela de Biología, Museo de Zoologia, Departamento de Biología, Collección de Ictiología, San Jose, Costa Rica) and UF (University of Florida, Florida Museum of Natural History, Gainesville, Florida, USA.). Abbreviations used in the text are: FRO - frontal; LAT - lateral ethmoid; MES - mesethmoid; NAS - nasal; PRE - premaxillary. Diagnosis. Lebiasina yepezi is readily distinguished from all other members of Lebiasininae by the presence of a primary, secondary, tertiary, and intermediate stripes on the sides of the trunk (Fig. 1; vs. primary and secondary stripes present in most Lebiasininae, except: L. astrigata (Regan), L. aureogutatta (Fowler), L. chocoensis Ardila-Rodríguez and L. multimaculata Boulenger, which present only the secondary stripe), and dorsal surface of the mesethmoid triangular shaped (Fig. 2), without lateral projections (vs. mesethmoid T-shaped). Lebiasina yepezi further differs from its congeners (except L. taphorni Ardila-Rodríguez, L. unitaeniata (Günther), L. uruyensis Fernandez-Yepez, and L. 769 A. L. Netto-Ferreira, O. T. Oyakawa, Jansen Zuanon & J. Material and Methods Holotype n Range Mean SD Standard length 152.9 22 12.0-152.9 80.7 - Percentage of SL Depth at dorsal-fin origin 20.9 12 18.6-23.2 21.3 1.4 Snout to anal-fin origin 73.5 12 73.5-79.3 76.0 1.8 Snout to pelvic-fin origin 50.2 12 49.8-53.9 51.8 1.4 Snout to dorsal-fin origin 53.5 12 53.5-57.0 54.8 1.2 Dorsal-fin origin to caudal-fin base 47.7 12 45.0-48.3 46.5 1.1 Dorsal-fin length 18.5 11 18.2-19.6 18.9 0.5 Dorsal-fin base 9.9 12 9.0-10.9 9.9 0.5 Adipose-fin to caudal peduncle 12.5 12 11.5-13.4 12.5 0.7 Caudal-peduncle Length 14.8 12 14.2-16.3 15.2 0.6 Caudal-peduncle Depth 9.9 12 8.7-10.6 9.5 0.7 Anal-fin length 19.9 12 18.1-21.3 19.7 1.0 Anal-fin base 10.7 12 9.3-12.3 10.5 1.1 Pelvic- to anal-fin origin 24.6 12 22.3-25.9 24.8 1.1 Pelvic-fin length 12.2 12 12.0-15.1 12.9 0.9 Pectoral to pelvic-fin origin 28.5 12 27.9-31.3 29.5 1.1 Pectoral-fin length 15.9 12 15.5-17.7 16.4 0.7 Snout to pectoral-fin origin 21.2 12 21.2-25.9 23.1 1.4 Bony head length 23.6 12 23.4-26.6 25.2 1.0 Percentage of HL Head width 13.0 12 12.7-14.6 13.4 0.6 Horizontal eye diameter 4.5 12 4.5-6.3 5.4 0.6 Distance snout tip to eye 6.7 12 6.7-8.1 7.6 0.4 Interorbital distance 9.1 12 9.1-9.7 9.3 0.2 Lower jaw length 10.3 12 10.1-12.2 11.2 0.7 Upper jaw length 5.1 12 5.1-10.3 7.0 2.1 Table 1. Morphometric and meristic data for Lebiasina yepezi. SD - standard deviation. Table 1. Morphometric and meristic data for Lebiasina yepezi. SD - standard deviation. p Trunk dark dorsally, becoming lighter ventrally from third longitudinal series of scales. Abdominal area yellowish, lacking pigmentation between the isthmus and anal-fin origin. Mid-dorsal series with a black longitudinal stripe from back of head to dorsal-fin origin. Humeral blotch inconspicuous, round, overlapped by primary stripe. Four dark longitudinal stripes on the trunk. Primary stripe conspicuous, originating posterior to eye, extending across infraorbitals four and five, opercle and along trunk, to terminate at rear of caudal peduncle. Stripe subdivided starting at vertical through dorsal-fin terminus, into 10-12 small round blotches, overlying scales of fourth longitudinal series. Secondary stripe inconspicuous, extending from supracleithrum to middle rays of upper caudal- fin lobe, over second and third longitudinal series of scales. Conspicuous intermediate stripe usually extending from mouth to vertical through anal-fin origin, onto scales of fifth longitudinal series, between primary and tertiary stripes. Material and Methods (MZUSP 81128, 42.8 mm SL), showing the usual T-shaped mesethmoid of Lebiasininae and the absence of lateral projections, respectively (FRO - frontal; LAT - lateral ethmoid; MES - mesethmoid; NAS - nasal; PRE - premaxillary). Fig. 2. Anterior portion of the skull in dorsal view of: a) Lebiasina astrigata (MEPN 4418, 94.5 mm SL); b) Lebiasina yepezi. (MZUSP 81128, 42.8 mm SL), showing the usual T-shaped mesethmoid of Lebiasininae and the absence of lateral projections, respectively (FRO - frontal; LAT - lateral ethmoid; MES - mesethmoid; NAS - nasal; PRE - premaxillary). ventral procurrent rays 8(1) or 9(1). Precaudal vertebrae 23(2); caudal vertebrae 19(2). specimens longer than 123.0 mm SL, in which it may be represented by patches of chromatophores. Anterior border of caudal-fin blotch not extending onto caudal peduncle. Pelvic and dorsal fins hyaline, base of pectoral-fin rays densely pigmented, forming a round dark blotch. Anal fin lightly pigmented, grayish, with distal border hyaline. Base of last two anal-fin branched rays densely pigmented, forming an oblong dark blotch. Caudal-fin rays densely pigmented. Color in alcohol. Background color yellowish. Head densely pigmented from upper lip to origin of scales on the mid- posterior portion of parietals. Dark brown pigmentation extending from that point to caudal-fin insertion, through mid- dorsal series of scales and immediately bordering scale rows. Infraorbital bones and opercular series with yellowish background color, with dark portions where primary, secondary and intermediate stripes extend across. Lower lip dark, densely pigmented. Ventral portion of head with minute scattered chromatophores. Table 1. Morphometric and meristic data for Lebiasina yepezi. SD - standard deviation. Material and Methods Posterior tip of maxillary reaching distinctly beyond vertical through anterior margin of orbit. Maxillary with 5(1) or 7(1) tricuspid teeth. Dentary with two series of teeth. Outer series with 12(1) or 18(1) pedunculate tricuspid teeth with central cusp Description. Morphometric data of Lebiasina yepezi are presented in Table 1. Body compressed and elongate. Dorsal profile of body distinctly convex from upper lip to vertical through nares, becoming slightly convex from that point to vertical through pectoral-fin origin, then nearly straight from latter to dorsal-fin origin; and slightly concave from that point to dorsal caudal-fin procurrent rays. Ventral profile of head and trunk convex from lower lip to pelvic-fin origin; straight from that point to anal-fin origin, convex at anal fin base, and concave from anal fin terminus to caudal-fin procurrent rays. Pectoral fin with i,13(1), 14(4), 15(5), or 16(2) rays. Pelvic fin i,7. Supraneurals 10(8), anterior to neural spine of centra 5 to 14(8). Dorsal fin ii,8(8) or 9(4) rays. First dorsal-fin pterygiophore inserted posterior to neural spine of centrum 14(8). Distal margin of extended dorsal fin rounded. Dorsal- fin origin closer to caudal-fin origin than to tip of snout. Base of last dorsal-fin ray anterior to vertical through anal- fin origin. Anal fin iii,8*(9) or 11(1), last ray adnate. Distal border of extended anal fin rounded. First anal-fin pterygiophore inserted posterior to hemal spine of centrum 24(8). Adipose fin present. Caudal fin furcate, with upper lobe distinctly longer than lower lobe; both lobes rounded. Principal caudal-fin rays i,9/8,i. Dorsal procurrent rays 9(2); Mouth terminal. Premaxillary with single row of 9(1) or 11(1) tricuspid teeth decreasing in size posteriorly. Posterior tip of maxillary reaching distinctly beyond vertical through anterior margin of orbit. Maxillary with 5(1) or 7(1) tricuspid teeth. Dentary with two series of teeth. Outer series with 12(1) or 18(1) pedunculate tricuspid teeth with central cusp A new Lebiasininae from the border of Brazil and Venezuela 770 Fig. 2. Anterior portion of the skull in dorsal view of: a) Lebiasina astrigata (MEPN 4418, 94.5 mm SL); b) Lebiasina yepe (MZUSP 81128, 42.8 mm SL), showing the usual T-shaped mesethmoid of Lebiasininae and the absence of lateral projection respectively (FRO - frontal; LAT - lateral ethmoid; MES - mesethmoid; NAS - nasal; PRE - premaxillary). Fig. 2. Anterior portion of the skull in dorsal view of: a) Lebiasina astrigata (MEPN 4418, 94.5 mm SL); b) Lebiasina yepezi. Material and Methods Tertiary stripe densely pigmented,except at proximal portion of caudal peduncle, extending from pectoral-fin origin to middle portion of caudal-fin lower lobe, onto scales of sixth longitudinal series. Caudal-fin blotch diffuse and rounded in juvenile specimens, located at basal portion of five median caudal-fin rays; caudal-fin blotch inconspicuous or absent in 771 A. L. Netto-Ferreira, O. T. Oyakawa, Jansen Zuanon & J. C. Nolasco Fig. 3. Anal-fin of Lebiasina yepezi evidencing the sexual dimorphism: a) Male (MNRJ 39067 - holotype) 152.9 mm SL; b) female (MNRJ 38917 - paratype) 97.5 mm SL. Arrow indicates breeding tubercles on the scales. Sexual Dimorphism. Mature males of Lebiasina yepezi present the most common pattern of sex dimorphism among species of the genus: in which the anal-fin rays and anal-fin base are distinctly longer and thicker than females (Fig. 3); hypertrophy of the inclinator muscles and the spiniform processes in which the muscles attach to the first lepidotrichia; scales of the fifth longitudinal series along the base of the anal fin modified (covered by a thin layer of a spongeous, apparently glandular, tissue); and breeding tubercles present on pectoral-, pelvic- and anal-fins rays, as well as on scales and sides of the head. Distribution. Specimens of Lebiasina yepezi are known from rio Parima (rio Mucajaí system) and rio Uraricoera in the rio Branco drainage, rio Amazonas system, Roraima State, Brazil, the rio Marari (rio Padauari system) in the rio Negro drainage, rio Amazonas system, Amazonas State, Brazil, and the upper rio Orinoco drainage, upstream of La Esmeralda, Amazonas State, Venezuela (Fig. 4). Etymology. The specific name honors Agustín Fernández Yépez, first collector of the new species described herein. Ecological notes. Lebiasina yepezi inhabits small and shaded forest streams, with fast-flowing clear water and a substrate consisting mainly of rocks and sand. Discussion In his study of the fish fauna near the río Yaracuy in Venezuela, Fernández-Yépez (1972: 13) mentions in passing a species yet to be described from the upper course of the Orinoco. Given that author had collected specimens of Lebiasina yepezi in 1951 (MHNLS 797), it is likely that he was referring to this species in his comment. Géry (1977: 588) was the first to depict the new species in a photo by Axelrod of a juvenile specimen collected at the Parima mountains in northern Brazil, which is currently cataloged as USNM 306560. In the occasion, Géry listed it as an unidentified characiform, “possibly near Nannostomus or Pyrrhulina”. Subsequently, Géry & Zarske (2002: 44-45) proposed a possible relationship between this species and Derhamia hoffmannorum, based on their conception of the relationships of the lebiasinid genera, but the authors did not provide evidence to such a hypothesis. In two phylogenetic studies based on morphologic data of the family Lebiasinidae, Netto-Ferreira (2006, 2010) refuted this hypothesis, proposing L. yepezi as being more closely related to the Lebiasinins from the Guyana Shield than Derhamia. Such close relationship between Lebiasina yepezi and its congeners from the Gran Sabana, Venezuela, L. taphorni, L. unitaeniata, L. uruyensis, and L. yuruaniensis (fig. 4) is supported by: first longitudinal series of scales short, ending near the dorsal-fin terminus; posteriorly displaced caudal blotch, not located on the caudal peduncle; secondary stripe passing onto the scales of the second and third longitudinal rows of scales; and the presence Fig. 3. Anal-fin of Lebiasina yepezi evidencing the sexual dimorphism: a) Male (MNRJ 39067 - holotype) 152.9 mm SL; b) female (MNRJ 38917 - paratype) 97.5 mm SL. Arrow indicates breeding tubercles on the scales. of the intermediate stripe, passing at least onto third infraorbital, usually represented by a horizontally elongate blotch (which in few specimens of L. uruyensis may form an inconspicuous stripe, passing onto the scales of the fifth longitudinal series in a way similar to that in L. yepezi). The presence of multiple dark longitudinal stripes along the body is an unusual color pattern among the Lebiasininae, albeit common among species of the Pyrrhulininae genus Nannostomus (three stripes present in N. limatus Weitzman, N. marginatus Eigenmann, N. marilynae Weitzman & Cobb, N. minimus Eigenmann, N. mortenthaleri Paepke & Arendt, N. rubrocaudatus Zarske, and N. trifasciatus Steindachner; A new Lebiasininae from the border of Brazil and Venezuela 772 Fig. 4. Discussion Map of northern portion of South America with the distribution of Guyana Shield species of Lebiasininae: Lebiasina yepezi (stars), L. unitaeniata (diamonds), L. uruyensis (squares), L. taphorni (triangle) and L. yuruaniensis (circles). White polygons represent type-localities. Fig. 4. Map of northern portion of South America with the distribution of Guyana Shield species of Lebiasininae: Lebiasina yepezi (stars), L. unitaeniata (diamonds), L. uruyensis (squares), L. taphorni (triangle) and L. yuruaniensis (circles). White polygons represent type-localities. four stripes in N. britskii Weitzman and N. eques Steindachner). Young specimens of Lebiasina yepezi can be distinguished from these species of Nannostomus with a somewhat comparable color pattern by the broader and slightly upturned mouth and the presence of tricuspidate teeth (vs. a narrow terminal mouth with spatulate multicuspid teeth in Nannostomus). upper portions of the rio Amazonas and the rio Orinoco systems represent further evidence that, with exception of Lebiasina bimaculata, L. boruca, and L. festae, species of Lebiasininae do not inhabit low land waters, living exclusively in fast flowing clear headwaters. Therefore, Meinken’s suggestion that the type locality of L. intermedia, “near Santarém” is most likely inaccurate as suggested by Géry (1977) and Weitzman & Weitzman (2003). So far, any recent collection yielded specimens comparable with that described by Meinken, and it is likely that specimens of L. intermedia are still awaiting to be rediscovered in either Guyana or Brazilian Shields. Lebiasina yepezi represents an additional species inhabiting both the rio Negro and rio Orinoco drainages. The continuous discovery of new species restricted to the headwaters of rivers draining from the Guyana Shield (Fernández-Yépez, 1967; Ardila-Rodríguez, 1999, 2000, 2004) suggests that the diversity of the Lebiasininae in that area still needs further investigation. Ongoing revisionary studies conducted by the senior author revealed three new lebiasinins occurring in the headwaters of the rio Mazaruni and the rio Potaro (a drainage exhaustively sampled by Eigenmann in the early 20th century) in the Guyana Shield. Comparative Material. Lebiasina astrigata: Colombia. CAR 187 (11, 70.6-138.4 mm SL), Nariño, creek tributary of Quebrada Indó, Quebrada Indún, 01°42’00”N 78°19’00”W. Ecuador. MEPN 4109 (4, 93.2-101.2 mm SL), Carchi, Quebrada Pailón, 00°28’59”N 77°55’01”W. MEPN 4130 (1, 56.9 mm SL), Carchi, Quebrada Negra, sítio San Marcos, 01°03’25”N 78°18’14”W. BMNH 1898.4.28:164- 6 Syntypes (3, 67.5-69.5 mm SL), Carchi/Imbabura, Paramba. BMNH1901.3.29:71 Syntype (1, 130.5 mm SL), Esmeraldas, San Javier, río Cachaví. BMNH 1902.7.29:59 Syntype (1,158.8 mm SL), Esmeraldas, río Sapayo. Discussion MCZ 54125 (3, 112.5-141.9 mm SL), Additionally, three new species have also been discovered in the headwaters of rio Xingu and rio Tapajós, in the Brazilian Shield, an area where lebiasinins were not expected to occur. The apparent peripheric distribution of these species in the A. L. Netto-Ferreira, O. T. Oyakawa, Jansen Zuanon & J. C. Nolasco 773 Esmeraldas, rios Cayapas, Hoja Blanca and San Miguel [de Cayapas], río Santiago drainage, 78°55’W 00°46’N. MEPN 4418 (2 c&s, 94.5- 103.6 mm SL; 21, 29.2-105.4 mm SL), Esmeraldas, creek of rio Mira, 80 m downstream of Casa Manuel Puy, 01°13’N 78°36’W. Lebiasina aureogutatta, Ecuador. ANSP 39103 Holotype (1, 127.6 mm SL), Guayas, tributary of río Chimbo, near Bucay, 02°10’00”S 79°06’00”W. ANSP 45474 (1, 80.5 mm SL), Mina [Guayas, río Minas, río Guayas drainage, 02°41’26”S 79°38’16”W. MCNG 19378 (1, 104.0 mm SL), Bolívar, río Chimbo, 2°05’56”S 79°41’47”W. MEPN 6232 (5, 79.4- 112.9 mm SL), Bolivar, río Chanquil, near San José del Tambo. FMNH 93122 (19/80, 38.1-141.6 mm SL), Los Rios, small pool at Centro Científico río Palenque, 00°35’00”S 79°22’00”W. MCZ 48728 (1, 57.1 mm SL), Pichincha, arroyo Guanecilla, río Blanco drainage, 00°13’S 79°09’W. MEPN 9469 (4, 73.6-142.9 mm SL), Pichincha, río Pachijal, affluent of río Guayllabamba, río Esmeraldas drainage, 0°12’10”N 78°43’16”W. CAS uncat. (ex-IU 13534) (32, 43.3-90.7 mm SL), Pichincha, río Mindo [río Guayllabamba drainage], in a farm west of Quito, 00°05’S 78°35’W. Lebiasina bimaculata, Peru. ROM 52209 (26, 23.0-61.4 mm SL), Ancash, río Sechin, 4 km east of Buena Vista, near Panamericana Highway, 9°29’03”S 78°18’15”W. MUSM 5188 (4, 35.2-46.1), Cajamarca, San Ignacio, tributary of río Tabaconas, 05°07’22”S 78°57’10”W. MZUSP 80083 (13, 34.0-47.9 mm SL), Cajamarca, Magdalena, río Jequetepeque, 7°15’30”S 78°38’47”W. LIRP 4379 (4, 43.0-49.1 mm SL), Cajamarca, Magdalena, río Jequetepeque, 07°13’35”S 78°50’00”W. MCZ 54123 (1, 121.3 mm SL), Lima, Chosica, río Rimac, 40 Km southeastern from Lima, 11°56’30”S 76°41’54”W. MNHN 141 Holotype (1, 76.0 mm SL), [Lima] río Rimac, near Lima, CAS 70420 (ex-IU 17587) (12, 34.4- 162.0 mm SL), La Libertad, laguna Hornito, mouth of río Jequetepeque, 07°19’41”S 79°35’26”W. Lebiasina boruca, Costa Rica. UCR 42-2 Paratypes (4, 114.2-121.4 mm SL), Alajuela, between La Marina and Águas Zarcas, 10°18’N 84°18’W. ANSP 140656 (3, 68.2-78.7 mm SL), Puntarenas, creek near the road Palmar Norte- Puerto Cortes, 9°00’N 83°30’W. LACM 2926 Paratype (1, 117.4 mm SL), Puntarenas, río Sonador, 570 m elev., 09°14’47.7”N 83°29’37.4”W. Discussion MUSM 5188 (4, 35.2-46.1), Cajamarca, San Ignacio, tributary of río Tabaconas, 05°07’22”S 78°57’10”W. MZUSP 80083 (13, 34.0-47.9 mm SL), Cajamarca, Magdalena, río Jequetepeque, 7°15’30”S 78°38’47”W. LIRP 4379 (4, 43.0-49.1 mm SL), Cajamarca, Magdalena, río Jequetepeque, 07°13’35”S 78°50’00”W. MCZ 54123 (1, 121.3 mm SL), Lima, Chosica, río Rimac, 40 Km southeastern from Lima, 11°56’30”S 76°41’54”W. MNHN 141 Holotype (1, 76.0 mm SL), [Lima] río Rimac, near Lima, CAS 70420 (ex-IU 17587) (12, 34.4- 162.0 mm SL), La Libertad, laguna Hornito, mouth of río Jequetepeque, 07°19’41”S 79°35’26”W. Lebiasina boruca, Costa Rica. UCR 42-2 Paratypes (4, 114.2-121.4 mm SL), Alajuela, between La Marina and Águas Zarcas, 10°18’N 84°18’W. ANSP 140656 (3, 68.2-78.7 mm SL), Puntarenas, creek near the road Palmar Norte- Puerto Cortes, 9°00’N 83°30’W. LACM 2926 Paratype (1, 117.4 mm SL), Puntarenas, río Sonador, 570 m elev., 09°14’47.7”N 83°29’37.4”W. LACM 4743 Paratypes (5/6, 17.6-24.4 mm SL), Puntarenas, río Convento, 09°14’19.3”N 83°29’19.1”W. LACM 9239 Holotype (1, 117.4 mm SL), Puntarenas, creek on the road to Golfito, Panamericana Highway, 08°37’11.23”N 83°03’36.7”W. UF 19537 (1, 97.0 mm SL), San Jose, río San Isidro, San Isidro del General, Panamericana Highway. USNM 194229 (2, 55.5-107.6 mm SL), San Jose, río Unión near San Isidro, Panamericana Highway, 09°22’00”N 83°41’17.2”W. Panama. ANSP 151023 (4, 77.9-92.7 mm SL), Chiriqui, creek 29 km south of Interamericana Highway, 08°19’9.6”N, 82°49’34.0”W. Lebiasina chocoensis, Colombia. ANSP 84172 (3, 64.0-112.0 mm SL), Chocó, Nuquí, upper río Jurubidá, aprox. 05°50’N 77°17’W, north of Baudó and east of Quibdó; CAR 185 Paratypes (5/6, 108.0-148.6 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. CAR 186 (12/18, 18.9-136.8 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. CAR 315 (ex-CAR 185) Holotype (1, 108.0 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. IAvH-P 573 (1, 76.8 mm SL), Chocó, río del Valle, 06°06’37"N 77°24’45”W, coastal drainage. ICNMHN 6872 Paratypes [not ICN 6772 as in Ardila-Rodríguez, 2010:05] (4, 65.6-107.1 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. Lebiasina colombia, Colombia. CAR 190 Holotype (1, 131.6 mm SL), Córdoba, Tierralta, Quebrada el Higuerón, tributary of río Sinú, 08°11’38.6”N 76°04’44.0”W. CAR 191 192 Paratypes (10, 59.7-138.0 mm SL), same data as holotype. CAR 408 (5/7, 94,4-125.6 mm SL), Córdoba, Quebrada la Corobá, tributary of Quebrada Tuis Tuis, 08°03’38.83”N 76°07’53.7”W. CAR 447 (1, 94,4-125.6 mm SL), Córdoba, río Manso, PNN [Parque Nacional Natural] Paramillo, 07°39’60”N 76°10’00”W. CAS uncat. Discussion (ex-SU 49514) (1, 59.8 mm SL), Córdoba, Crucito, upper río Sinú, 09°24’N 75°49’W. ICNMHN 5314 Paratype (1, 71.44 mm SL), Córdoba, río Sinú, near mouth to río Urrá, 07°56’21.4”N 76°11’58.1”W. ICNMHN 10692 (3, 79.4-115.4 mm SL), Córdoba, Tierralta, Quebrada el Higuerón, tributary of río Sinú, 08°11’38.6”N 76°04’44.0”W. Lebiasina elongata, Ecuador. BMNH 1880.12.8.123 Lectotype (1, 101.8 mm SL), Pastaza, Canelos [upper río Bobonaza, left bank tributary of río Pastaza, río Marañon drainage, 01°35’22.0”S 77°44’47.7”W. BMNH 1880.12.8.124 Paralectotype (1, 111.9 mm SL), same data as lectotype. MEPN 9131 (5, 83.2- 137.1 mm SL), Napo, río Pucuno, 300 m elev., 01°03’56”S 77°38’16”W. USNM 164062 (7, 67.8-108.7 mm SL), Napo río Misahuallí, in Misahuallí 01°02’00.3”S 77°39’52.0”W. USNM 258065 (3, 44.1-74.7 mm SL), Napo, Quebrada in Sarayacu, tributary of río Misahuallí, río Napo drainage, in the highway Baeza- Archidona, 1500 m elev., 00°39’18.9”S 77°46’55.2”W. Colombia. CAR 136 (9/10, 22.7-66.2 mm SL), Caquetá, Quebrada Pabarpaco, upper río Caquetá, 01°04’54”N 75°46’19”W. CAR 149 (2, 72.6- 117.7 mm SL), Putumayo, Orito, vereda el Líbano, upper río Putumayo, 775-850 m elev. , 00°40’53.8”N 77°02’07.2”W. Peru. MUSM 5666 (1, 118.2 mm SL), Amazonas, arroyo Condorcangui, río Comainas, downstream of PV22 basecamp, 04°01’00.4”S 78°24’7.9”W. Lebiasina erythrinoides, Colombia. IAvH-P 9209 (1, 27.5 mm SL), Casanare, Pore, río Pore, 100 m bellow the road Yopal-Pore, 360 m elev., 05°43’45”N 72°00’04”W. IAvH-P 9210 (3, 60.5-94.8 mm SL), Casanare, Yopal, Quebrada Agua Blanca, tributary of río Cravo Sur, 760 m elev., 05°28’17.6”N 72°27’04.5”W. CAR 205 (3, 33.9-69.5 mm SL), Norte de Santander, Pamplonita, Quebrada Mutiscua 7°17’59.4”N 72°44’58.6”W. Venezuela. MNHN 4614 Syntypes (2, 123.7-144.7 mm SL), [Zulia] Lake Maracaibo. MNRJ 14230 (7, 53.4-82.6 mm SL), Barinas, Quebrada tributary of río Qui, 2 km north of higway and upstream of Fazenda Qui, 8°04’30”N 70°56’55”W. MBUCV uncat (1, 46.9 mm SL), Barinas, río Masparro, between Boconoito and Barrancas, in front of Masparro, 08°47’40.7”N 70°04’46.8”W. INHS 28919 (3, 64.5- 127.3 mm SL), Carabobo, río Capa, río Urama basin, Caribbean sea drainage, 10°18’44.1”N 68°17’00”W. CAR 450 (ex-MHNLS 4336) (2, 130.0-153.4 mm SL), Yaracuy, Quebrada el Charcal, Finca Jaguar, Quebrada el Charal 10°31’22.8”N 68°57’45.9”W. INHS 34955 (2, 163.2-192.9 mm SL), Trujillo, creek tributary of Montatán, 09°12’46”N 70°40’08”W. UF 25457 (3, 71.7-129.2 mm SL), Zulia, Heras, bridge of río Frio, Panamerican highway, 08°58’08.0”N 71°24’58.4”W. Lebiasina festae, Colombia. CAR 143 (8, 80.9- 130.0), Antioquia, Vigia del Fuerte, 06°35’48.8”N 76°53’10.0”W. ANSP 84264 (2, 95.7-110.5 mm SL), Chocó, río Dagua, Buenaventura, 03°25’44.9”N 77°3’10.4”W. Discussion LACM 4743 Paratypes (5/6, 17.6-24.4 mm SL), Puntarenas, río Convento, 09°14’19.3”N 83°29’19.1”W. LACM 9239 Holotype (1, 117.4 mm SL), Puntarenas, creek on the road to Golfito, Panamericana Highway, 08°37’11.23”N 83°03’36.7”W. UF 19537 (1, 97.0 mm SL), San Jose, río San Isidro, San Isidro del General, Panamericana Highway. USNM 194229 (2, 55.5-107.6 mm SL), San Jose, río Unión near San Isidro, Panamericana Highway, 09°22’00”N 83°41’17.2”W. Panama. ANSP 151023 (4, 77.9-92.7 mm SL), Chiriqui, creek 29 km south of Interamericana Highway, 08°19’9.6”N, 82°49’34.0”W. Lebiasina chocoensis, Colombia. ANSP 84172 (3, 64.0-112.0 mm SL), Chocó, Nuquí, upper río Jurubidá, aprox. 05°50’N 77°17’W, north of Baudó and east of Quibdó; CAR 185 Paratypes (5/6, 108.0-148.6 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. CAR 186 (12/18, 18.9-136.8 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. CAR 315 (ex-CAR 185) Holotype (1, 108.0 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. IAvH-P 573 (1, 76.8 mm SL), Chocó, río del Valle, 06°06’37"N 77°24’45”W, coastal drainage. ICNMHN 6872 Paratypes [not ICN 6772 as in Ardila-Rodríguez, 2010:05] (4, 65.6-107.1 mm SL), Chocó, Quibdó, Quebrada affluent of río Tutunendo, 05°46’56”N 76°34’18”W. Lebiasina colombia, Colombia. CAR 190 Holotype Esmeraldas, rios Cayapas, Hoja Blanca and San Miguel [de Cayapas], río Santiago drainage, 78°55’W 00°46’N. MEPN 4418 (2 c&s, 94.5- 103.6 mm SL; 21, 29.2-105.4 mm SL), Esmeraldas, creek of rio Mira, 80 m downstream of Casa Manuel Puy, 01°13’N 78°36’W. Lebiasina aureogutatta, Ecuador. ANSP 39103 Holotype (1, 127.6 mm SL), Guayas, tributary of río Chimbo, near Bucay, 02°10’00”S 79°06’00”W. ANSP 45474 (1, 80.5 mm SL), Mina [Guayas, río Minas, río Guayas drainage, 02°41’26”S 79°38’16”W. MCNG 19378 (1, 104.0 mm SL), Bolívar, río Chimbo, 2°05’56”S 79°41’47”W. MEPN 6232 (5, 79.4- 112.9 mm SL), Bolivar, río Chanquil, near San José del Tambo. FMNH 93122 (19/80, 38.1-141.6 mm SL), Los Rios, small pool at Centro Científico río Palenque, 00°35’00”S 79°22’00”W. MCZ 48728 (1, 57.1 mm SL), Pichincha, arroyo Guanecilla, río Blanco drainage, 00°13’S 79°09’W. MEPN 9469 (4, 73.6-142.9 mm SL), Pichincha, río Pachijal, affluent of río Guayllabamba, río Esmeraldas drainage, 0°12’10”N 78°43’16”W. CAS uncat. (ex-IU 13534) (32, 43.3-90.7 mm SL), Pichincha, río Mindo [río Guayllabamba drainage], in a farm west of Quito, 00°05’S 78°35’W. Lebiasina bimaculata, Peru. ROM 52209 (26, 23.0-61.4 mm SL), Ancash, río Sechin, 4 km east of Buena Vista, near Panamericana Highway, 9°29’03”S 78°18’15”W. Discussion CAR 145 (12, 66.3-137.3 mm SL), Valle del Cauca, Alcalá, creek tributary of río Barbas, 200 m near the río La Vieja, 04°40’46.3”N 75°47’33.7”W. CAR 157 Paratypes (4, 64.1-119.8 mm SL), Valle del Cauca, Florida, Quebrada tributary of río Cañas, Vereda el Tambor, 1100 m elev., 03°18’50.7”N 76°14’26.8”W. CAR 265 Paratypes (13 [not 7 as in Ardila-Rodríguez, 2008b:20], 34.7- 114.8 mm SL), Valle del Cauca, tributary of río las Cañas, 200 m near the road between Florida and Miranda, 03°16’41.7”N 76°13’17.9”W. CAR 15-04-45 (1, 72.3 mm SL), Quindio, Quebrada el Broche, río Baragán drainage, in the road to Caicedonia, 04°20’07.2”N 75°42’02.8”W. Lebiasina panamensis, Panama. USNM 16676 Lectotype (1, 65.9 mm SL), creek open into the Atlantic Ocean, channel area. USNM 16677 Paralectotype (1, 65.3 mm SL), Canal Zone, río Frijoles, 09°08’24.9”N 79°45’43”W. USNM 78656 (2, 120.1-128.3 mm SL), Agua Clara, río Trinidad, a large tributary of the río Chagres west of the route to the Channel, 09°18’N 79°47’W. ANSP 126193 (4, 64.5-134.4 mm SL), 16 km de Cerro Azul, Pacific Ocean drainage, Cerro Azul, 09°10’00”N 79°25’00”W. INHS 68116 (1, 54.3 mm SL), Colón, río Frijolito, bacia do río Chagres, Parque Nacional da Soberania, 09°10’11.1”N 79°46’54.6”W. INHS 68151 (5, 52.8-87.3 mm SL), Colón, Soberania State Park, río Frijolito, río Chagres drainage, 09°10’11.1”N 79°46’54.6”W. MCZ 45820 (1, 118.3 mm SL), channel area, near Gamboa, tributary of río Chagres, 09°07’N 79°42’W. MCZ 80749 (11, 53.3-141.9 mm SL), creek in the Barro Colorado island channel area, 09°09’47.9”N 79°49’43.3”W. MNRJ 14541 (1, 139.1 mm SL; 1 c&s, 100.0 mm SL), Comarca de San Blas, creek Bulebgandi, tributary of río Azúcar, 09°24’53.1”N 78°38’20.0”W. Lebiasina taphorni, Venezuela. CAR 15-04-47 Holotype (ex-MCNG 19939) (1, 87.1 mm SL), Bolívar, Quebrada in the middle río Caura. CAR 15-04-48 Paratypes (ex-MCNG 19939) (6, 34.0-62.4 mm SL), Bolívar, Quebrada in the middle río Caura. CAR 15-04-15 (ex-MCNG 18817) (3, 106.6-123.7 mm SL), Bolívar, Cedeño, Guanajuja, creek upstream from a small fall, 05°09’00”N 64°09’00”W. MCNG 18814 (5, 61.3- 123.0 mm SL; 1 c&s, 67.9 mm SL, Bolívar, Cedeño, Guanajuja, creek upstream from a small fall, 05°09’00"N 64°09’00"W. Lebiasina unitaeniata, Venezuela. BMNH 1988.2.16.1 Holotype (1, 100.4 mm SL), Guyana [Venezuela, Bolívar?]. AMNH 199331 (6, 103.4- 131.8 Discussion CAR 477 (ex-MBUCV 17453) (5, 37.9- 69.9 mm SL), Bolívar, Gran Sabana, Quebrada Aropán, between the río Anavaic and río Kamoirán. MBUCV 15286 (1, 126.2 mm SL), Bolívar, río Kakopauri between Santa Elena and San Ignacio de Yuruaní. MCNG 18992 (4, 41.5-69.3 mm SL), Bolívar, estrada El Dorado, Gran Sabana, km 149, 05°39’16.9"N 61°26’12.1"W. MCNG 19006 (7, 46.7-68.9 mm SL), Bolívar, Gran Sabana, 22 km south of Whao-Whao. MCNG 48089 (3, 24.5-70.1 mm SL), Bolívar, Heres, near the Salto Catedral, in Carucú-Maru, 4,6 km west of Santa Elena, 04°34’29.8”N 61°12’58.8”W. elev., 05°45’N 61°43’W. CAR 485 (ex-MBUCV-V 22331) (7, 44.5- 98.4 mm SL), Bolívar, Gran Sabana, Quebrada Tarotá, río Apon Wao system. MZUSP 73090 (ex-MBUCV-V 29376) (10, 51.2-102.6 mm SL), Bolívar, Gran Sabana, Quebrada Tarotá, río Apon Wao system. USNM 163426 (5, 102.0-129.1 mm SL), Bolívar, Soropan-tepuí, 5600’ elev., 05°45’N 61°43’W. USNM 307215 (9, 50.6-110.4 mmSL), Bolívar, río Tarota, tributary of río Aponguao [=río Apon Wao]. Lebiasina uruyensis, Venezuela. AMNH 14263 (1, 82.1 mm SL), Bolívar, río Haicha, sopé do Auyán-tepui, 450 m elev., 05°44’41”N 62°27’37”W. AMNH 59048 (20/52, 41.7-127.9 mm SL), Cerro Guaiquinima, creek into the forest, near the basecamp, 05°49’00”N 63°40’00”W. AMNH 59049 (4, 63.0-111.9 mm SL), Bolívar, tributary of the left margin of río Carapo, tributary of the right margin of río Paragua, 450- 550 m elev., 05°39’N 63°40’W. CAR 15-04-14 (ex-MHNLS 787) (3, 87.4-90.4 mm SL), Bolívar, río Guayaraca, south of Auyan Tepuy, 980 m elev. 05°41’N 62°32’W. CAR 456 (ex-MHNLS 14458) (4, 64.4- 81.4 mm SL), Bolívar, río Wareipita, Canaima State Park, Wareipa, 06°00’32”N 62°46’52”W. MHNLS 789 Holotype (110.9 mm SL) and Paratypes (9, 96.6-118.0 mm SL), Bolívar, rio Guayaraca, ao sul de Auyán-tepui, 980 m elev., 05°41’N 62°32’W. Lebiasina yuruaniensis. Venezuela. AMNH 1536 (1, 70.7 mm SL), Bolívar, Erkin creek, río Arabopo, 04°47’N 60°52’W.AMNH 9662 (3, 82.3-118.5 mm SL), Bolívar, Arabupu, Roraima, 13 km southeast of Monte Roraima, 4200 m elev., 05°06’N 60°44’W, AUM 36649 (4, 27.7- 77.7 mm SL), Bolívar, tributary of río Waiparú, km 77, in Waipan- Tepuy, río Caroní drainage, 04°30’N 61°36’W. AUM 36651 (2, 80.1- 83.9 mm SL), Bolívar, Fundo Cantarrana, río Ikabaru, tributary of río Caroní, km 95, 04°24’N 61°42’W. CAR 457 (ex-MBUCV 17426) (6, 30.5-85.1 mm SL), Bolívar, Quebrada Cantarrana, 30 km north of Icabarú, Gran Sabana. CAR 477 (ex-MBUCV 17453) (5, 37.9- 69.9 mm SL), Bolívar, Gran Sabana, Quebrada Aropán, between the río Anavaic and río Kamoirán. Ardila-Rodríguez, C. A. 1994. Lebiasina floridablancaensis, una nueva especie de pez para Colombia (Teleostei: Characiformes: Lebiasinidae). Revista Unimetro, 10 (19): 1-8. Discussion MBUCV 15286 (1, 126.2 mm SL), Bolívar, río Kakopauri between Santa Elena and San Ignacio de Yuruaní. MCNG 18992 (4, 41.5-69.3 mm SL), Bolívar, estrada El Dorado, Gran Sabana, km 149, 05°39’16.9"N 61°26’12.1"W. MCNG 19006 (7, 46.7-68.9 mm SL), Bolívar, Gran Sabana, 22 km south of Whao-Whao. MCNG 48089 (3, 24.5-70.1 mm SL), Bolívar, Heres, near the Salto Catedral, in Carucú-Maru, 4,6 km west of Santa Elena, 04°34’29.8”N 61°12’58.8”W. Floridablanca Quebrada el Gaque, upper río Lebrija, 07°03’58.9”N 73°05’21.3”W. BMNH 1947.47.7.1.23-32 (9, 45.9-106.2 mm SL), Santander, Bucaramanga, 1000 m elev., río Magdalena drainage. CAR 15-04-23 (2, 105.1-117.9 mm SL), Santander, San Vicente de Chucuri Quebrada La Carbonera, tributary of río la Llana, 06°53’47.1”N 73°25’39.6”W. IAvH-P 4730 (2, 89.2- 106.3 mm SL), Santander, Girón, Quebrada el Palmar, tributary of río Del Oro, 07°04’18.5”N 73°10’32.5”W. CAR 260 (14, 131.2-46.0 mm SL), Santander, Lebrija, Vereda Lisboa, tributary of Sucio, río Lebrija drainage, 07°07’10.3”N 73°13’13.6”W. Lebiasina multimaculata, Colombia. BMNH 1910.7.11:167 Lectotype (1, 91.2 mm SL), Chocó, Condoto, río Condoto, 45 m elev., 06°06’00”N 76°42’00”W. CAR 15-04-34 (35, 21.6-48.7 mm SL), Chocó, Andagoya, Quebrada tributary of río San Juan, near the mouth río Condoto, 05°06’03.9”N 76°41’43.8”W. CAR 138 (5, 52.9-103.7 mm SL), Chocó, Opogodó, río Opogodó, 5°3’0”N 76°39’0”W. CAR 139 (13, 21.7-118.3 mm SL), Chocó, near the Tadó village, 05°15’25.2”N 76°33’12.9”W. CAR 451 (2, 110.9- 123.8 mm SL), Chocó, Quebrada Weguerál, río San Juan. Lebiasina ortegai, Colombia. CAR 445 (1, 55.8 mm SL), Cauca, Quebrada Gemagra, Popayán 1730 m elev., 02°27’03.5”N 76°37’12.1”W. CAR 145 (12, 66.3-137.3 mm SL), Valle del Cauca, Alcalá, creek tributary of río Barbas, 200 m near the río La Vieja, 04°40’46.3”N 75°47’33.7”W. CAR 157 Paratypes (4, 64.1-119.8 mm SL), Valle del Cauca, Florida, Quebrada tributary of río Cañas, Vereda el Tambor, 1100 m elev., 03°18’50.7”N 76°14’26.8”W. CAR 265 Paratypes (13 [not 7 as in Ardila-Rodríguez, 2008b:20], 34.7- 114.8 mm SL), Valle del Cauca, tributary of río las Cañas, 200 m near the road between Florida and Miranda, 03°16’41.7”N 76°13’17.9”W. CAR 15-04-45 (1, 72.3 mm SL), Quindio, Quebrada el Broche, río Baragán drainage, in the road to Caicedonia, 04°20’07.2”N 75°42’02.8”W. Lebiasina panamensis, Panama. USNM 16676 Lectotype (1, 65.9 mm SL), creek open into the Atlantic Ocean, channel area. USNM 16677 Paralectotype (1, 65.3 mm SL), Canal Zone, río Frijoles, 09°08’24.9”N 79°45’43”W. Acknowledgements We are grateful to R. P. Vari for valuable comments and suggestions on the manuscript, and E. Baena for the photos and drawings in Fig. 2. We thank U. Barbosa and L. Py- Daniel for the collection and donation of several specimens of the new species to INPA’s fish collection. Authors were financially supported by FAPESP (fellowship 06/04161-5 ALN-F), www.biotasp.org.br (project 00/04300-9 OTO, “Diversidade de peixes de riachos e cabeceiras da Bacia do rio Ribeira de Iguape no Estado de São Paulo”) and CNPq (productivity grant #307464-2009-1 JZ). Discussion CAR 137 (8/10, 30.9- 118.0 mm SL), Chocó, Andagoya, Quebrada tributary of río Condoto, 05°06’03.9”N 76°41’43.8”W. CAR 142 (9, 33.2-96.0), Chocó, Quebrada tributary of río Tutunendo, 05°47’04.4”N 76°34’6.5”W. CAR 454 (1, 127.2 mm SL), Chocó, Comunidade Munguidó. Panama: MZUT 1591 Holotype (1, 134.4 mm SL), Darién, laguna Della Pita. CAS 66982 (6, 50.5-73.9 mm SL), Darién, Yaviza, 08°10’18.4”N 77°41’10.3”W. NRM 35875 (3, 58.5-78.1 mm SL), Darién, Yaviza, tributary of río Tupisa, 08°11’59.9”N 77°41’17.3”W. USNM 78641 (1, 107.8 mm SL), Dárien, río Yape, 08°06’58.7”N, 77°34’45.4”W. Lebiasina floridablancaensis, Colombia CAR 15 04 01 Holotype (1 109 2 mm SL) Santander A new Lebiasininae from the border of Brazil and Venezuela 774 elev., 05°45’N 61°43’W. CAR 485 (ex-MBUCV-V 22331) (7, 44.5- 98.4 mm SL), Bolívar, Gran Sabana, Quebrada Tarotá, río Apon Wao system. MZUSP 73090 (ex-MBUCV-V 29376) (10, 51.2-102.6 mm SL), Bolívar, Gran Sabana, Quebrada Tarotá, río Apon Wao system. USNM 163426 (5, 102.0-129.1 mm SL), Bolívar, Soropan-tepuí, 5600’ elev., 05°45’N 61°43’W. USNM 307215 (9, 50.6-110.4 mmSL), Bolívar, río Tarota, tributary of río Aponguao [=río Apon Wao]. Lebiasina uruyensis, Venezuela. AMNH 14263 (1, 82.1 mm SL), Bolívar, río Haicha, sopé do Auyán-tepui, 450 m elev., 05°44’41”N 62°27’37”W. AMNH 59048 (20/52, 41.7-127.9 mm SL), Cerro Guaiquinima, creek into the forest, near the basecamp, 05°49’00”N 63°40’00”W. AMNH 59049 (4, 63.0-111.9 mm SL), Bolívar, tributary of the left margin of río Carapo, tributary of the right margin of río Paragua, 450- 550 m elev., 05°39’N 63°40’W. CAR 15-04-14 (ex-MHNLS 787) (3, 87.4-90.4 mm SL), Bolívar, río Guayaraca, south of Auyan Tepuy, 980 m elev. 05°41’N 62°32’W. CAR 456 (ex-MHNLS 14458) (4, 64.4- 81.4 mm SL), Bolívar, río Wareipita, Canaima State Park, Wareipa, 06°00’32”N 62°46’52”W. MHNLS 789 Holotype (110.9 mm SL) and Paratypes (9, 96.6-118.0 mm SL), Bolívar, rio Guayaraca, ao sul de Auyán-tepui, 980 m elev., 05°41’N 62°32’W. Lebiasina yuruaniensis. Venezuela. AMNH 1536 (1, 70.7 mm SL), Bolívar, Erkin creek, río Arabopo, 04°47’N 60°52’W.AMNH 9662 (3, 82.3-118.5 mm SL), Bolívar, Arabupu, Roraima, 13 km southeast of Monte Roraima, 4200 m elev., 05°06’N 60°44’W, AUM 36649 (4, 27.7- 77.7 mm SL), Bolívar, tributary of río Waiparú, km 77, in Waipan- Tepuy, río Caroní drainage, 04°30’N 61°36’W. AUM 36651 (2, 80.1- 83.9 mm SL), Bolívar, Fundo Cantarrana, río Ikabaru, tributary of río Caroní, km 95, 04°24’N 61°42’W. CAR 457 (ex-MBUCV 17426) (6, 30.5-85.1 mm SL), Bolívar, Quebrada Cantarrana, 30 km north of Icabarú, Gran Sabana. Discussion USNM 78656 (2, 120.1-128.3 mm SL), Agua Clara, río Trinidad, a large tributary of the río Chagres west of the route to the Channel, 09°18’N 79°47’W. ANSP 126193 (4, 64.5-134.4 mm SL), 16 km de Cerro Azul, Pacific Ocean drainage, Cerro Azul, 09°10’00”N 79°25’00”W. INHS 68116 (1, 54.3 mm SL), Colón, río Frijolito, bacia do río Chagres, Parque Nacional da Soberania, 09°10’11.1”N 79°46’54.6”W. INHS 68151 (5, 52.8-87.3 mm SL), Colón, Soberania State Park, río Frijolito, río Chagres drainage, 09°10’11.1”N 79°46’54.6”W. MCZ 45820 (1, 118.3 mm SL), channel area, near Gamboa, tributary of río Chagres, 09°07’N 79°42’W. MCZ 80749 (11, 53.3-141.9 mm SL), creek in the Barro Colorado island channel area, 09°09’47.9”N 79°49’43.3”W. MNRJ 14541 (1, 139.1 mm SL; 1 c&s, 100.0 mm SL), Comarca de San Blas, creek Bulebgandi, tributary of río Azúcar, 09°24’53.1”N 78°38’20.0”W. Lebiasina taphorni, Venezuela. CAR 15-04-47 Holotype (ex-MCNG 19939) (1, 87.1 mm SL), Bolívar, Quebrada in the middle río Caura. CAR 15-04-48 Paratypes (ex-MCNG 19939) (6, 34.0-62.4 mm SL), Bolívar, Quebrada in the middle río Caura. CAR 15-04-15 (ex-MCNG 18817) (3, 106.6-123.7 mm SL), Bolívar, Cedeño, Guanajuja, creek upstream from a small fall, 05°09’00”N 64°09’00”W. MCNG 18814 (5, 61.3- 123.0 mm SL; 1 c&s 67 9 mm SL Bolívar Cedeño Guanajuja creek upstream Floridablanca Quebrada el Gaque, upper río Lebrija, 07°03’58.9”N 73°05’21.3”W. BMNH 1947.47.7.1.23-32 (9, 45.9-106.2 mm SL), Santander, Bucaramanga, 1000 m elev., río Magdalena drainage. CAR 15-04-23 (2, 105.1-117.9 mm SL), Santander, San Vicente de Chucuri Quebrada La Carbonera, tributary of río la Llana, 06°53’47.1”N 73°25’39.6”W. IAvH-P 4730 (2, 89.2- 106.3 mm SL), Santander, Girón, Quebrada el Palmar, tributary of río Del Oro, 07°04’18.5”N 73°10’32.5”W. CAR 260 (14, 131.2-46.0 mm SL), Santander, Lebrija, Vereda Lisboa, tributary of Sucio, río Lebrija drainage, 07°07’10.3”N 73°13’13.6”W. Lebiasina multimaculata, Colombia. BMNH 1910.7.11:167 Lectotype (1, 91.2 mm SL), Chocó, Condoto, río Condoto, 45 m elev., 06°06’00”N 76°42’00”W. CAR 15-04-34 (35, 21.6-48.7 mm SL), Chocó, Andagoya, Quebrada tributary of río San Juan, near the mouth río Condoto, 05°06’03.9”N 76°41’43.8”W. CAR 138 (5, 52.9-103.7 mm SL), Chocó, Opogodó, río Opogodó, 5°3’0”N 76°39’0”W. CAR 139 (13, 21.7-118.3 mm SL), Chocó, near the Tadó village, 05°15’25.2”N 76°33’12.9”W. CAR 451 (2, 110.9- 123.8 mm SL), Chocó, Quebrada Weguerál, río San Juan. Lebiasina ortegai, Colombia. CAR 445 (1, 55.8 mm SL), Cauca, Quebrada Gemagra, Popayán 1730 m elev., 02°27’03.5”N 76°37’12.1”W. Literature Cited Literature Cited Ardila-Rodríguez, C. A. 1994. Lebiasina floridablancaensis, una nueva especie de pez para Colombia (Teleostei: Characiformes: Lebiasinidae). Revista Unimetro, 10 (19): 1-8. 775 A. L. Netto-Ferreira, O. T. Oyakawa, Jansen Zuanon & J. C. Nolasco Ferraris, C. J. 2007. Checklist of catfishes, recent and fossil (Osteichthyes: Siluriformes), and catalogue of siluriform primary types. Zootaxa, 1418: 1-628. Ardila-Rodríguez, C. A. 1999. Lebiasina provenzanoi, una nueva especie de pez para Venezuela (Teleostei: Characiformes: Lebiasinidae). Revista Unimetro, 13 (5 & 6): 1-10. Fink, W. L. & Weitzman, S. H. 1974. The so-called cheirodontin fishes of Central America with description of two new species (Pisces, Characidae). Smithsonian Contributions to Zoology, 172: 1-46. Ardila-Rodríguez, C. A. 2000. Lebiasina yuruaniensis, una nueva especie de pez para Venezuela (Teleostei: Characiformes: Lebiasinidae). Revista Unimetro, Separata Especial N°s. 2, 3 (25 & 26): 1-16. Géry, J. 1977. Characoids of the World. Tropical Fish Hobbyist Publications, Neptune City, 672p. Ardila Rodríguez, C. A. 2001. Lebiasina chucuriensis una nueva especie de pez para Colombia (Teleostei: Characiformes: Lebiasinidae). Revista Unimetro, 13 (27-28): 1-20. Géry, J. & Zarske A. 2002. Derhamia hoffmannorum gen. et sp. n - a new pencil fish (Teleostei, Characiformes, Lebiasinidae), endemic from the Mazaruni River in Guyana. Zoologische Abhandlungen; Staatliches Museum für Tierkunde in Dresden, 52: 35-47. Ardila-Rodríguez, C. A. 2002. Lebiasina nariñensis, una nueva especie de pez para Colombia (Teleostei: Characiformes, Lebiasinidae). Dahlia, 5: 11-18. Ardila-Rodríguez, C. A. 2004. Lebiasina taphorni (Pisces: Characiformes, Lebiasinidae), una nueva especie. Dahlia, 7: 57-65. Machado-Allison, A. 1974. Etapas del desarrollo del pez Piabucina pleurotaenia Regan 1903 (Characiformes: Lebiasinidae). Acta Biológica Venezoelana, 8: 579-622. Ardila-Rodríguez, C. A. 2008a. Lebiasina ortegai (Characiformes: Lebiasinidae), nueva especie, sistema del río Cauca, Colombia. Dahlia, 10: 17-25. Menezes, N. A. & S. H. Weitzman. 1990. Two new species of Mimagoniates (Teleostei: Characidae: Glandulocaudinae), their phylogeny and biogeography and a key to the gladulocaudin fishes of Brazil and Paraguay. Proceedings of the Biological Society of Washington, 102: 380-426. Ardila-Rodríguez, C. A. 2008b. Lebiasina colombia (Characiformes: Lebiasinidae), nueva especie cuenca del río Sinú, Colombia. Dahlia, 10: 27-32. Nelson, J. S. 2006. Fishes of the World. John Wiley and Sons, Inc. New York, 601p. Ardila-Rodríguez, C. A. 2009. Lebiasina colombia (Ardila Rodriguez, 2008). Peces del Alto río Sinu, 1: 1-20. Netto-Ferreira, A. L. 2006. Relações filogenéticas dos gêneros de Lebiasinidae (Ostariophysi, Characiformes). Unpublished M.Sc. Dissertation, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 374p. Ardila-Rodríguez, C. A. 2010. Literature Cited Lebiasina chocoensis, una nueva especie de pez para Colombia (Teleostei: Characiformes: Lebiasinidae, Lebiasininae). Peces del Departamento del Chocó, 1: 1-20. Dahl, G. 1971. Los Peces del norte de Colombia. Instituto de Desarrollo de Los Recursos Naturales Renovables (INDERENA), Bogotá, 391p. Netto-Ferreira, A. L. 2010. Revisão taxonômica e relações interespecíficas de Lebiasininae (Ostariophysi: Characiformes: Lebiasinidae). Unpublished Ph.D. Dissertation, Universidade de São Paulo, São Paulo, Brazil, 443p. Eigenmann, C. H. 1910. Catalogue of freshwaters fishes of tropical and south temperate America. Reports of Princeton University Expedition Patagonia, 1896-1899. Princeton, N. J., Princeton University, 3: 375-511. Taphorn, D. 1992. Characiform fishes of the Apure River drainage, Venezuela. BioLlania. UNELLEZ, Guanare, Venezuela, 537p. Eigenmann, C. H. & W. R. Allen. 1942. Fishes of western South America. I. The Intercordilleran and Amazonian lowlands of Peru. II. The high pampas of Peru, Bolivia and northern Chile. University of Kentucky, Lexington, 494p. Taylor, W. R. & G. C. van Dyke. 1985. Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium, 9: 107-119. Weitzman, S. H. 1964. Osteology and relationships of South American characid fishes of subfamilies Lebiasinidae and Erythrinidae with special references to subtribe Nannostomina. Proceedings of the United States National Museum, 116: 127-170. Fernández-Yépez, A. 1967. Primera contribución al conocimiento de los peces, con descripción de dos especies y una subespecie nuevas. Resultados zoológicos de la expedición de la Universidad Central de Venezuela. Acta Biologica Venezuelica, 5: 159-177. Weitzman, S. H. 1966. Review of South American characid fishes of subtribe Nannostomina. Proceedings of the United States National Museum, 119: 1-56. Fernández-Yépez, A. 1972. Analisis Ictiológico del Complejo Hidrográfico (04) “río Yaracuy”. Dirección de Obras Hidraúlicas, Ministerio de Obras Públicas, Republica de Venezuela, 25p. Weitzman, M. & S. H. Weitzman. 2003. Family Lebiasinidae. Pp. 241-250. In: Reis, R. E.; S. O. Kullander & C. J. Ferraris, Jr. (eds.). Check List of the freshwater fishes of South and Central America, Porto Alegre, Edipucrs, 729p. Submitted August 19, 2011 Accepted November 28, 2011 Published December 26, 2011 Submitted August 19, 2011 Accepted November 28, 2011 Published December 26, 2011
https://openalex.org/W1985522974	http://wrap.warwick.ac.uk/539/1/WRAP_Kumar_Serum_leptin.pdf	English	null	Serum leptin and its relation to anthropometric measures of obesity in pre-diabetic Saudis	Cardiovascular diabetology	2,007	cc-by	5,694	University of Warwick institutional repository: http://go.warwick.ac.uk/wrap This paper is made available online in accordance with publisher policies. Please scroll down to view the document itself. Please refer to the repository record for this item and our policy information available from the repository home page for further information. To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription. Author(s): Nasser M Al-Daghri, Omar S Al-Attas, Khalid Al-Rubeaan, Mehad Mohieldin, Mohammad Al-Katari, Alan F Jones and Sudhesh Kumar Article Title: Serum leptin and its relation to anthropometric measures of obesity in pre-diabetic Saudis Year of publication: 2007 Link to published version: http://dx.doi.org/10.1186/1475-2840-6-18 Publisher statement: None To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription. Access to the published version may require a subscription. Author(s): Nasser M Al-Daghri, Omar S Al-Attas, Khalid Al-Rubeaan, Mehad Mohieldin, Mohammad Al-Katari, Alan F Jones and Sudhesh Kumar Article Title: Serum leptin and its relation to anthropometric measures of obesity in pre-diabetic Saudis Year of publication: 2007 Link to published version: http://dx.doi.org/10.1186/1475-2840-6-18 Publisher statement: None BioMed Central Published: 7 July 2007 Received: 2 May 2007 Accepted: 7 July 2007 Cardiovascular Diabetology 2007, 6:18 doi:10.1186/1475-2840-6-18 This article is available from: http://www.cardiab.com/content/6/1/18 © 2007 Al-Daghri et al; licensee BioMed Central Ltd. © 2007 Al Daghri et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 2 May 2007 Accepted: 7 July 2007 Published: 7 July 2007 Abstract Background: Little information is available on leptin concentrations in individuals with IGT. This study aims to determine and correlate leptin levels to anthropometric measures of obesity in pre- diabetic, (IFG and IGT), type 2 diabetic and normoglycaemic Saudis. Methods: 308 adult Saudis (healthy controls n = 80; pre-diabetes n = 86; Type 2 diabetes n = 142) participated. Anthropometric parameters were measured and fasting blood samples taken. Serum insulin was analysed, using a solid phase enzyme amplified sensitivity immunoassay and also leptin concentrations, using radio-immunoassay. The remaining blood parameters were determined using standard laboratory procedures. Results: Leptin levels of diabetic and pre-diabetic men were higher than in normoglycaemic men (12.4 [3.2–72] vs 3.9 [0.8–20.0] ng/mL, (median [interquartile range], p = 0.0001). In females, leptin levels were significantly higher in pre-diabetic subjects (14.09 [2.8–44.4] ng/mL) than in normoglycaemic subjects (10.2 [0.25–34.8] ng/mL) (p = 0.046). After adjustment for BMI and gender, hip circumference was associated with log leptin (p = 0.006 with R2 = 0.086) among all subjects. Conclusion: Leptin is associated with measures of adiposity, hip circumference in particular, in the non-diabetic state among Saudi subjects. The higher leptin level among diabetics and pre-diabetics is not related to differences in anthropometric measures of obesity. cultural changes over the past thirty years. Approximately 60% of the population are urbanised and have adopted a 'Westernised' lifestyle in terms of diet and physical activity Op Original investigation Serum leptin and its relation to anthropometric measures of obesity in pre-diabetic Saudis Address: 1Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia, 2Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia, 3Medicine Department, College of Medicine, King Saud University, Riyadh, Saudi Arabia, 4Diabetic Center, KAUH, Riyadh, Saudi Arabia, 5Diabetic Center, KAUH, Riyadh, Saudi Arabia, 6Clinical Biochemistry, Birmingham Heartlands Hospital, Birmingham, B9 5SS, UK and 7Warwick Medical School, Diabetes & Metabolism Unit, University of Warwick, Coventry, CV4 7AL, UK Email: Nasser M Al-Daghri* - aldaghri2000@hotmail.com; Omar S Al-Attas - omrattas@ksu.edu.sa; Khalid Al-Rubeaan - krubeaan@ksu.edu.sa; Mehad Mohieldin - mohieldin@ksu.edu.sa; Mohammad Al-Katari - malkatari@ksu.edu.sa; Alan F Jones - afjones@doctors.org.uk; Sudhesh Kumar - Sudhesh.Kumar@warwick.ac.uk * Corresponding author Cardiovascular Diabetology Open Access Methods Subjects In the present study, subjects were selected from the roster of adult, ambulant, non-pregnant patients attending the Diabetes Center at the King Abdulaziz University Hospital in Riyadh, Saudi Arabia. Ethical approval was obtained from the local institution's review committee and consent was obtained from all participants. For the purposes of this study, subjects were classified into categories of abnormal glucose homeostasis if they had a single abnor- mal FPG. 142 patients had type-2 diabetes (FPG > 7.1 mmol/L) (97 males and 45 females). All patients with dia- betes were treated with a low-carbohydrate diet, with or without oral antidiabetetic agents, mainly glibenclamide and metformin. 86 pre-diabetic subjects (49 males and 37 females) were defined on the basis of modified ADA crite- ria [24]. Pre-diabetes was diagnosed when the fasting plasma glucose was between 5.6 mmol/L – 7.0 mmol/L (100–126 mg/dL). 80 Saudi individuals (45 males and 35 females), with no prior history of CHD or diabetes, were recruited as controls (no diabetes). Increased risk of CHD in subjects with IGT is supported by data from the Honolulu [8], Chicago [9] and Islington [10] heart studies, which demonstrated that mortality from CHD increased gradually as glucose tolerance deteri- orated, with no apparent threshold value. In 1997, the American Diabetes Association (ADA) defined pre-diabe- tes as fasting plasma glucose (FPG) 6.1–7.0 mmol/L (110–126 mg/dl) [11]. The choice of the 6.1 mmol/L threshold was based partly on the increased risk of devel- oping both microvascular and macrovascular complica- tions above this level [11]. However, it has now been shown that the fasting glycaemia threshold of 6.1 mmol/ L does not create a category of glucose homeostasis equiv- alent to that of IGT, either for the subsequent develop- ment of diabetes or CHD [12]. Diabetes increases the risk of developing cardiovascular disease by 2–3 times and increases by as much as 50% the risks of non-cardiovascular mortality associated with this condition [13-15]. This high risk is not completely explained by the traditional risk factors [16,17]. Pre-dia- betes is also associated with cardiovascular diseases (CVD) [18,19], but it is unclear if it is an independent risk factor, because it commonly co-exists with other cardio- vascular risk factors present in the metabolic syndrome. The overall prevalence of type 2 diabetes in Saudi adults is 23.7% [20] while the overall prevalence of CHD is 5.5% [5]. Background Background Saudi Arabia, a Middle Eastern country with a population of 22 million, has undergone significant economic and Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) http://www.cardiab.com/content/6/1/18 Cardiovascular Diabetology 2007, 6:18 with those in patients who have pre-diabetes and to corre- late it with anthropometric measures of obesity. [1]. Obesity and associated components of the metabolic syndrome have become increasingly common in the Saudi population, which may partially explain the increased incidence of coronary heart disease (CHD) [2- 7]. In the USA, 17.1% of overweight adults aged 45–74 years were found to have impaired glucose tolerance (IGT); 11.9% had impaired fasting glucose (IFG); 22.6% had prediabetes; and 5.6% had both IGT and IFG [8]. Laboratory methods Blood samples were collected after a 12-hour fast for the determination of glucose, insulin, total cholesterol, HDL, triglycerides, apolipoprotein A1 (apo A1), apolipoprotein A2 (apo A2) and leptin. All samples were stored at -70°C prior to analysis by using routine laboratory methods, except for insulin and leptin assay. Insulin was analyzed by a solid phase enzyme amplified sensitivity immu- noassay (Medgenix INS-ELISA, Biosource, Belgium). Lep- tin concentrations were measured by radio-immunoassay (Linco Research, St. Charles, MO). Homeostasis model assessment-insulin resistance (HOMA-IR) and β-cell func- tion index were derived using the HOMA equation [25]. In human beings, serum leptin concentration is directly proportional to body fat mass, but it is leptin resistance and not leptin deficiency per se which is regarded as a pathogenic mechanism in human obesity. Leptin concen- trations vary widely among individuals with similar fat mass, indicating other possible factors for its determina- tion [21,22]. Leptin may be a marker of risk of CHD, at least in males, and contributes to the CHD risk profile in subjects with insulin resistance [23]. Not much informa- tion is available on leptin concentrations in individuals with IGT and to our knowledge there has been no study of leptin in pre-diabetics within the region. Methods Subjects However, the percentage of Saudi subjects who had an FPG in the impaired fasting glucose range (6.1–7.0 mmol/ L) was 14.1% [7]. Anthropometric parameters All subjects underwent a full physical examination and completed a general questionnaire. Height was measured to the nearest 0.5 cm and weight to the nearest 0.1 kg. Waist circumference (cm) was measured at the horizontal circumference midway between the lowest rib margin and the iliac crest and the hip circumference (cm) was meas- ured at the maximum circumference over the buttocks. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Groups were matched for BMI. Results bl Table 1 presents the clinical and metabolic characteristics of the male subjects. It is evident that those in the diabetes group were significantly older (p < 0.0001), with higher fasting glucose and leptin levels (p < 0.0001, 0.012 respec- tively) than those in the pre-diabetes and control groups. At the same time, the pre-diabetes group had significantly elevated systolic blood pressure (p = 0.046) and fasting glucose levels (p < 0.0001), compared to the controls. Table 2 highlights the clinical and metabolic parameters in the females. In the diabetes and pre-diabetes group, FPG and LDL were significantly higher than in the control. Total cholesterol was also significantly higher in women with diabetes (p = 0.03). Data were presented according to gender, since it is already an established fact that leptin levels are signifi- cantly higher in women than in men. There are several possible explanations for the difference. One is that females have more adipose tissue than males, but a grow- ing literature indicates that estrogen, especially at higher levels, will stimulate the production of leptin, whereas androgens will suppress the levels of leptin [37]. In this cross-sectional study, we found positive correlations between leptin and hip circumference in pre-diabetic patients. This is consistent with a previous study, which also showed that this group is at high risk of developing Serum leptin was significantly higher in the men with dia- betes than in their non-diabetic counterparts (12.4 [3.2– 72]; p = 0.0001). In the female patients, serum leptin was higher in those with pre-diabetes (14.09 [2.8–44.4]) than in the controls. Table 1: Clinical characteristics and metabolic parameters of male subjects with type-2 diabetes, pre-diabetes and no diabetes No Diabetes Pre-diabetes Type-2 Diabetes N 45 49 97 Age (years) 45.7(12.6) 50.1(11.6) 54.5(10.7)f Systolic blood pressure (mm Hg) 123.1(16.9) 132.8(25.1)a 130.7(17.5) Diastolic blood pressure (mm Hg) 79.9(9.1) 85.1(11.4) 82.9(10.5) BMI (Kg/m2) 29.2(7.3) 28.5(4.3) 27.3(4.1) Waist (cm) 93.7(18.2) 98.6(10.1) 96.4(15.1) Hips (cm) 95.9(15.7) 100.5(13.9) 99.8(14.4) Fasting Glucose (mmol/L) 4.8(0.6) 6.1(0.4)f 13.7(5.0)f Cholesterol (mmol/L) 5.2(1.6) 5.6(1.4) 6.5(1.9)f HDL (mmol/L) 0.94(0.3) 1.2(0.4) 0.9(0.4) Triglyceride (mmol/L) 1.8(0.6–6.7) 1.9(0.7–6.3) 2.5(1.6–15) LDL (mmol/L) 3.3(1.1) 3.9(1.6) 4.2(1.8)b Insulin μmol/L * 12.6(2–99) 12.3 (2–36) 16.2(2–70) Leptin ng/mL * 3.9(0.8–20) 7.6(1.2–72)d 12.4(3.2–72)c APO A1 mg/dL 0.9(0.3) 0.9(0.5) 0.9(0.3) APO A2 mg/dL 0.4(0.1) 0.4(0.1) 0.3(0.1) HOMA-IR * 3.3(0.3–23.8) 3.5(0.5–10.2) 9.6(1.1–49.7)e Data are presented as means (SD) or as medians (interquartile range) for insulin, leptin and HOMA-IR data. Discussion Insulin resistance is the common factor in a range of risk factors for atherosclerosis, specifically hypertension, dysl- ipidemia and abnormal glucose metabolism. As has been shown in other populations [26-29], the pre-diabetic males had a significantly higher systolic blood pressure and fasting plasma glucose than the control subjects had. Many [30,31], but not all studies [32,33] of the general population have shown a positive association of indices of insulin resistance with arterial wall changes, CHD and CVD. The clustering of risk factors increases the risk of atherosclerosis [34-36]. Statistical Analysis Data were analysed using the Statistical Package for the Social Sciences (SPSS) version 10 (SPSS, Evanston, IL, USA) for Windows. Biochemical parameters not normally This study aims to determine and compare the serum lep- tin concentrations of diabetic and non-diabetic patients Page 2 of 6 (page number not for citation purposes) Cardiovascular Diabetology 2007, 6:18 http://www.cardiab.com/content/6/1/18 distributed were analyzed after being logarithmically transformed. Students' unpaired t-test and one-way anal- ysis of variance (ANOVA) were used to compare the results of the different groups. Simple and partial correla- tion coefficients between the variables were determined and multiple regression analysis was performed to deter- mine the relationships between the variables of interest. Data were expressed as mean (SD) or median (range); sta- tistical significance was set at p < 0.05. Stepwise linear regression analysis revealed a positive cor- relation between log leptin and hip circumference and BMI (R = 0.31, p = 0.002; R = 0.26, p = 0.006 respectively). Even after adjustment for BMI, there was a positive associ- ation between log leptin and hip circumference with an R2 = 0.086 and p value of 0.006 (see Figure 1). The rest of the findings were non-contributory. Results bl The P-values shown are comparisons of metabolic characteristics for case control versus either prediabetic patients or those with T2DM: a) 0.046; b) p = 0.03; c) p = 0.012; d) p = 0.004; e) p = 0.001; f) p < 0.0001. stics and metabolic parameters of male subjects with type-2 diabetes, pre-diabetes and no diabetes Table 1: Clinical characteristics and metabolic parameters of male subjects with type-2 diabetes, pre-diab Data are presented as means (SD) or as medians (interquartile range) for insulin, leptin and HOMA-IR data. The P-values shown are comparisons of metabolic characteristics for case control versus either prediabetic patients or those with T2DM: a) 0.046; b) p = 0.03; c) p = 0.012; d) p = 0.004; e) p = 0.001; f) p < 0.0001. Page 3 of 6 (page number not for citation purposes) Page 3 of 6 The association of leptin to waist circumference (a surrogate for upper body obesity) and hip circumference confirms findings in Mexican American populations [49,50]. After adjustment for BMI, serum lep- tin concentrations in pre-diabetic men were independent of waist circumference, but in women they were associ- ated with hip circumference. Hip circumference is a proxy measure of peripheral fat in serum leptin concentrations, aside from the fact that women have a significantly larger volume of subcutaneous fat than do men. In the control subjects, leptin concentrations were directly and signifi- cantly related to subcutaneous fat, with a strong inverse relationship to waist-hip ratio. When the results were con- sidered according to gender, it was found that 33.1% of the male Saudis with waist circumference >102 cm, had diabetes. In female subjects, 27% of those with a high waist circumference ≥88 cm had diabetes, compared to only 13.4% of those with a normal waist circumference [20]. Leptin is related to waist and hip circumference and is directly proportional to body fat. However, in those with diabetes, this relationship is lost, reflecting the effect of other factors, including hyperinsulinaemia and the acti- vation of sub-clinical inflammation. lated to have closer associations with the biomarkers of CHD than BMI has [48]. The association of leptin to waist circumference (a surrogate for upper body obesity) and hip circumference confirms findings in Mexican American populations [49,50]. After adjustment for BMI, serum lep- tin concentrations in pre-diabetic men were independent of waist circumference, but in women they were associ- ated with hip circumference. Hip circumference is a proxy measure of peripheral fat in serum leptin concentrations, aside from the fact that women have a significantly larger volume of subcutaneous fat than do men. In the control subjects, leptin concentrations were directly and signifi- cantly related to subcutaneous fat, with a strong inverse relationship to waist-hip ratio. When the results were con- sidered according to gender, it was found that 33.1% of the male Saudis with waist circumference >102 cm, had diabetes. In female subjects, 27% of those with a high waist circumference ≥88 cm had diabetes, compared to only 13.4% of those with a normal waist circumference [20]. Leptin is related to waist and hip circumference and is directly proportional to body fat. However, in those with diabetes, this relationship is lost, reflecting the effect of other factors, including hyperinsulinaemia and the acti- vation of sub-clinical inflammation. Page 3 of 6 Serum leptin concentration may contribute to the risk of CVD by altering lipid metabolism and contributing to hypertension via the activation of the sympathetic nerv- ous system and increasing renal sodium re-absorption [42,43]. However, in our study there was no correlation between leptin and blood pressure. In another study, lep- tin signaling directly promoted atherosclerosis and may therefore represent a therapeutic target for the prevention of atherosclerosis [44]. Lichnovska and her colleagues in their recent report mentioned the significant role of serum leptin in the progression of insulin resistance, but this was not confirmed in the present study [45]. The difference can be due to the fact that the earlier study considered eld- erly and hyperlipemic patients, while the present study focused on pre-diabetic, diabetic and non-diabetic adults. Our results, however, confirm the results of Adamia et al., which reveal no correlation between leptin and insulin resistance [46]. Page 3 of 6 http://www.cardiab.com/content/6/1/18 Cardiovascular Diabetology 2007, 6:18 Table 2: Clinical characteristics and metabolic parameters of female subjects with type-2 diabetes, pre-diabetes and healthy control subjects No diabetes Pre-diabetes Type-2 diabetes N 35 37 45 Age (years) 43.6(11.3) 43.3(11.7) 49.0(12.6)d Systolic blood pressure (mm Hg) 122.8(23.1) 127.3(24.2) 123.6(18.6) Diastolic blood pressure (mm Hg) 79.4(16.5) 79.8(8.03) 80.7(10.3) BMI (kg/m2) 30.4(6.4) 32.5(8.4) 32.5(10.3) Waist (cm) 90.5(12.5) 91.8(13.5) 96.2(17.9) Hips (cm) 105.3(14.1) 104.8(17.9) 106.7(20.8) Fasting Glucose (mmol/L) 4.8(0.9) 6.1(0.4)b 14.1(5.5)b Cholesterol (mmol/L) 5.2(1.3) 5.3(1.1) 6.6(1.9)b HDL (mmol/L) 1.1(0.4) 0.9(0.4) 1.1(0.3) Triglyceride (mmol/L) 1.9(1.9) 1.6(0.9) 2.6(1.4) LDL (mmol/L) 3.5(1.4) 3.6(1.1)d 4.4(1.5)c Insulin (μmol/L)* 15.2(2–62) 13.5(6–26) 14.04(1–52) Leptin ng/mL * 10.2(0.25–34.8) 14.09(2.8–44.4)a 13.3(3.6–49.1) APO A1 mg/dL 0.8(0.4) 0.9(0.4) 1.2(2.5) APO A11 mg/dL 0.4(0.1) 0.4(0.1) 0.4(0.2) HOMA IR * 3.1(0.3–12.5) 3.6(1.5–7.3) 7.8(0.6–28.4)b Data are presented as means (SD) or as medians (interquartile range) for insulin, leptin and HOMA-IR data. The P-values shown are comparisons of metabolic characteristics for case control versus either prediabetics or patients with T2DM: a) 0.046; b) p = 0.03; c) p = 0.004; d) p < 0.001 Table 2: Clinical characteristics and metabolic parameters of female subjects with type-2 diabetes, pre-diabetes and healthy control subjects istics and metabolic parameters of female subjects with type-2 diabetes, pre-diabetes and healthy control Table 2: Clinical characteristics and metabolic parameters of female subjects with type-2 diabetes, pre-diabetes and healthy control subjects e 2: Clinical characteristics and metabolic parameters of female subjects with type-2 diabetes, pre-diabetes t Data are presented as means (SD) or as medians (interquartile range) for insulin, leptin and HOMA-IR data. The P-values shown are comparisons of metabolic characteristics for case control versus either prediabetics or patients with T2DM: a) 0.046; b) p = 0.03; c) p = 0.004; d) p < 0.001 *Data are presented as means (SD) or as medians (interquartile range) for insulin, leptin and HOMA-IR data. The P-valu of metabolic characteristics for case control versus either prediabetics or patients with T2DM: a) 0.046; b) p = 0.03; c) CHD [38]. The positive association with hip circumfer- ence remained significant after adjusting for gender and BMI, which confirmed the results in previous studies for healthy men and women [39,40]. With impaired glucose tolerance, small changes in circulating insulin also alter insulin levels [41]. lated to have closer associations with the biomarkers of CHD than BMI has [48]. Conclusion A family history of diabetes was found to have a negative association with leptin in pre-diabetic patients; this con- firms a previous study which reported that healthy lean non-diabetic Asian Indians are more insulin resistant than other ethnic groups, despite similarities in their living environment [47]. Waist circumference has been postu- The significant correlation of leptin to selected anthropo- metric measurements of obesity is confirmed in non-dia- betic Saudi Subjects. In those with diabetes, this relationship is lost, reflecting the effect of other factors, including hyperinsulinaemia and the activation of sub- clinical inflammation. Page 4 of 6 (page number not for citation purposes) Cardiovascular Diabetology 2007, 6:18 http://www.cardiab.com/content/6/1/18 http://www.cardiab.com/content/6/1/18 L l ti ( / l) Hi i f ( ) Fi 1 -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 2.50 0 20 40 60 80 100 120 140 160 Hips (cm) log leptin (ng/ml) Log leptin (ng/ml) vs. Hip circumference (cm) Figure 1 Log leptin (ng/ml) vs. Hip circumference (cm). The correlation of log leptin (ng/mL) with hip circumference (cm) using univariate analysis across all Saudi subjects, case con- trols, T2DM and pre-diabetic subjects. Blue denotes the actual values with the line of best fit shown in square (R2 0.086 p-value = 0.006). -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 2.50 0 20 40 60 80 100 120 140 160 Hips (cm) log leptin (ng/ml) Log leptin (ng/ml) vs. Hip circumference (cm) Figure 1 Log leptin (ng/ml) vs. Hip circumference (cm). The correlation of log leptin (ng/mL) with hip circumference (cm) using univariate analysis across all Saudi subjects, case con- trols, T2DM and pre-diabetic subjects. Blue denotes the actual values with the line of best fit shown in square (R2 0.086 p-value = 0.006). -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 2.50 0 20 40 60 80 100 120 140 160 Hips (cm) log leptin (ng/ml) 9. Lowe LP, Liu K, Greenland P, Metzger BE, Dyer AR, Stamler J: Dia- betes, asymptomatic hyperglycemia, and 22-year mortality in black and white men. The Chicago Heart Association Detection Project in Industry Study. Diabetes Care 1997, 20(2):163-169. ( ) 10. Jackson CA, Yudkin JS, Forrest RD: A comparison of the relation- ships of the glucose tolerance test and the glycated haemo- globin assay with diabetic vascular disease in the community. The Islington Diabetes Survey. Diabetes Res Clin Pract 1992, 17(2):111-123. ( ) 11. Acknowledgements J ( ) 21. Lonnqvist F, Arner P, Nordfors L, Schalling M: Overexpression of the obese (ob) gene in adipose tissue of human obese sub- jects. Nat Med 1995, 1(9):950-953. The authors extend their sincerest appreciation to Dr. Shaun Louie Sabico for his writing and editing assistance. j ( ) 22. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, et al.: Serum immunoreactive-leptin concentrations in normal- weight and obese humans. N Engl J Med 1996, 334(5):292-295. Competing interests The author(s) declare that they have no competing inter- ests. 18. Barzilay JI, Spiekerman CF, Kuller LH, Burke GL, Bittner V, Gottdi- ener JS, Brancati FL, Orchard TJ, O'Leary DH, Savage PJ, Cardiovas- cular Health Study: Prevalence of clinical and isolated subclinical cardiovascular disease in older adults with glu- cose disorders: the Cardiovascular Health Study. Diabetes Care 2001, 24(7):1233-1239. Authors' contributions NA for the concept and design as well as statistical analy- sis; OA for the drafting and revising of the manuscript; KA and AJ for the acquisition and interpretation of data; MM and MK for the screening and collection of data; KS for the concept and final revision of the manuscript. All the authors have read and approved the final version. ( ) 19. Fisman EZ, Motro M, Tenenbaum A, Boyko V, Mandelzweig L, Behar S: Impaired fasting glucose concentrations in nondiabetic patients with ischemic heart disease: a marker for a worse prognosis. Am Heart J 2001, 141(3):485-490. p g J ( ) 20. Al-Nozha MM, Al-Maatouq MA, Al-Mazrou YY, Al-Harthi SS, Arafah MR, Khalil MZ, Al-Khadra A, Al-Marzouki K, Nouh MS, Abdullah M, Attas O, Al-Shahid MS, Al-Mobeireek A: Diabetes mellitus in Saudi Arabia. Saudi Med J 2004, 25(11):1603-1610. Conclusion [No authors listed]: Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes care 1997, 20(7):1183-1197. ( ) 12. Meigs JB, Muller DC, Nathan DM, Blake DR, Andres R, Baltimore Longitudinal Study of Aging: The natural history of progression from normal glucose tolerance to type 2 diabetes in the Bal- timore Longitudinal study of aging. Diabetes 2003, 52(6):1475-1484. ( ) 13. Kannel WB, McGee DL: Diabetes and cardiovascular disease: The Framingham Study. JAMA 1979, 241(19):2035-2038. Log leptin Figure 1 g y J ( ) 14. Gu K, Cowie CC, Harris MI: Mortality in adults with and without diabetes in a national cohort of the US population, 1971– 1993. Diabetes Care 1998, 21(7):1138-1145. g p ( g ) p ( ) g Log leptin (ng/ml) vs. Hip circumference (cm). The correlation of log leptin (ng/mL) with hip circumference (cm) using univariate analysis across all Saudi subjects, case con- trols, T2DM and pre-diabetic subjects. Blue denotes the actual values with the line of best fit shown in square (R2 0.086 p-value = 0.006). 15. Woodward M, Zhang X, Barzi F, Pan W, Ueshima H, Rodgers A, Mac- Mahon S, Asia Pacific Cohort Studies Collaboration: The effects of diabetes on the risks of major cardiovascular diseases and death in the Asia-Pacific region. Diabetes Care 2003, 26(2):360-366. ( ) 16. Kannel WB: Lipids, diabetes, and coronary heart disease: insights from the Framingham Study. Am Heart J 1985, 110(5):1100-1107. ( ) 17. Stamler J, Vaccaro O, Neaton JD, Wentworth D: Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Dia- betes Care 1993, 16(2):434-444. References 1. al-Nuaim AR, al-Rubeaan K, al-Mazrou Y, al-Attas O, al-Daghari N, Khoja T: High prevalence of overweight and obesity in Saudi Arabia. Int J Obes Relat Metab Disord 1996, 20(11):547-552. 23. Al-Daghri N, Al-Rubean K, Bartlett WA, Al-Attas O, Jones AF, Kumar S: Serum leptin is elevated in Saudi Arabian patients with metabolic syndrome and coronary artery disease. Diabet Med 2003, 20(10):832-837. J ( ) 2. al-Nuaim AR, al-Rubeaan K, al-Mazrou Y, al-Attas O, al-Daghari N: Prevalence of hypercholesterolemia in Saudi Arabia, epide- miological study. Int J Cardiol 1996, 54(1):41-49. ( ) 24. American Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care 2004, 27(Suppl 1):S5-S10. g y J ( ) 3. al-Shammari SA, Ali M, al-Shammari A, al-Maatouq M, Tennier A, Armstrong K: Blood lipid concentrations and other cardiovas- cular risk factors among Saudis. Fam Pract 1994, 11(2):153-158. 4 Al Dawood KM: Pattern of smoking among parents of school g y J ( ) 3. al-Shammari SA, Ali M, al-Shammari A, al-Maatouq M, Tennier A, Armstrong K: Blood lipid concentrations and other cardiovas- cular risk factors among Saudis. Fam Pract 1994, 11(2):153-158. 4. Al-Dawood KM: Pattern of smoking among parents of school- boys. Saudi Med J 2000, 21(8):735-739. 25. Matthews DR, Rudenski AS, Naylor BA, Treacher DF, Turner RC: Homeostasis model assessment: insulin resistance and beta- cell function from fasting plasma glucose and insulin concen- trations in man. Diabetologia 1985, 28(7):412-419. y J ( ) 5. Al-Nozha MM, Arafah MR, Al-Mazrou YY, AL-Maatouq MA, Khan NB, Khalil MZ, Al-Khadra AH, Al-Marzouki K, Abdullah MA, Al-Harthi SS, Al-Shahid MS, Nouh MS, Al-Mobeireek A: Coronary Artery Dis- ease in Saudi Arabia. Saudi Med J 2004, 25(9):1165-1171. g ( ) 26. Haffner SM, Stern MP, Hazuda HP, Mitchell BD, Patterson JK: Cardi- ovascular risk factors in confirmed prediabetic individuals. Does the clock for coronary heart disease start ticking before the onset of clinical diabetes? JAMA 1990, 263(21):2893-2898. J ( ) 6. Al-Turki YA: Overview of chronic diseases in the Kingdom of Saudi Arabia. Saudi Med J 2000, 21(5):499-500. 27. Medalie JH, Papier CM, Goldbourt U, Herman JB: Major factors in the development of diabetes mellitus in 10,000 men. Arch Intern Med 1975, 135(6):811-817. J ( ) 7. Al-Nuaim AR: Prevalence of glucose intolerance in urban and rural communities in Saudi Arabia. Diabet Med 1997, 14(7):595-602. J ( ) 7. References Al-Nuaim AR: Prevalence of glucose intolerance in urban and rural communities in Saudi Arabia. Diabet Med 1997, 14(7):595-602. 28. 28. McPhillips JB, Barrett-Connor E, Wingard DL: Cardiovascular dis- ease risk factors prior to the diagnosis of impaired glucose ( ) 8. Baron AD: Impaired glucose tolerance as a disease. Am J Cardiol 2001, 88(6A):16H-19H. Page 5 of 6 (page number not for citation purposes) Page 5 of 6 (page number not for citation purposes) Cardiovascular Diabetology 2007, 6:18 http://www.cardiab.com/content/6/1/18 http://www.cardiab.com/content/6/1/18 48. Lofgren I, Herron K, Zern T, West K, Patalay M, Shachter NS, Koo SI, Fernandez ML: Waist circumference is a better predictor than body mass index of coronary heart disease risk in over- weight premenopausal women. J Nutr 2004, 134(5):1071-1076. tolerance and non-insulin-dependent diabetes mellitus in a community of older adults. Am J Epidemiol 1990, 131(3):443-453. tolerance and non-insulin-dependent diabetes mellitus in a community of older adults. Am J Epidemiol 1990, 131(3):443-453. 29. Weyer C, Bogardus C, Mott DM, Pratley RE: The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 1999, 104(6):787-794. 49. g p p J ( ) 49. Haffner SM, Gingerich RL, Miettinen H, Stern MP: Leptin concen- trations in relation to overall adiposity and regional body fat distribution in Mexican Americans. Int J Obes Relat Metab Disord 1996, 20(10):904-908. ( ) 30. Pyorala M, Miettinen H, Laakso M, Pyorala K: Hyperinsulinemia and the risk of stroke in healthy middle-aged men: the 22- year follow-up results of the Helsinki Policemen Study. Stroke 1998, 29(9):1860-1866. ( ) 50. Kafulafula G, Moodley J: Leptin levels in the obese African par- turient. J Obstet Gynaecol 2001, 21(3):228-231. ( ) 31. Jarrett RJ: Why is insulin not a risk factor for coronary heart disease? Diabetologia 1994, 37(9):945-947. 32. Niskanen L, Rauramaa R, Miettinen H, Haffner SM, Mercuri M, Uusitupa M: Carotid artery intima – media thickness in elderly patients with NIDDM and in nondiabetic subjects. Stroke 1996, 27(11):1986-1992. ( ) 33. Welin L, Eriksson H, Larsson B, Ohlson LO, Svardsudd K, Tibblin G: Hyperinsulinaemia is not a major coronary risk factor in eld- erly men: The study of men born in 1913. Diabetologia 1992, 35(8):766-770. ( ) 34. DeFronzo RA, Ferrannini E: Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia and atherosclerotic cardiovascular disease. Dia- betes 1991, 14(3):173-194. 35. http://www.cardiab.com/content/6/1/18 48. Lofgren I, Herron K, Zern T, West K, Patalay M, Shachter NS, Koo SI, Fernandez ML: Waist circumference is a better predictor than body mass index of coronary heart disease risk in over- weight premenopausal women. J Nutr 2004, 134(5):1071-1076. 49. Haffner SM, Gingerich RL, Miettinen H, Stern MP: Leptin concen- trations in relation to overall adiposity and regional body fat distribution in Mexican Americans. Int J Obes Relat Metab Disord 1996, 20(10):904-908. 50 Kafulafula G, Moodley J: Leptin levels in the obese African par- ( ) 50. Kafulafula G, Moodley J: Leptin levels in the obese African par- turient. J Obstet Gynaecol 2001, 21(3):228-231. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 44. Reilly MP, Iqbal N, Schutta M, Wolfe ML, Scally M, Localio AR, Rader DJ, Kimmel SE: Plasma leptin levels are associated with coro- nary atherosclerosis in type 2 diabetes. J Clin Endocrinol Metab 2004, 89(8):3872-3878. ( ) 45. Lichnovska R, Gwozdziewiczova A, Hrebicek J: Gender differences in factors influencing insulin resistance in elderly hyperli- pemic non-diabetic subjects. Cardiovasc Diabetol 2002, 1:4. 46. Adamia N, Virsaladze D, Charkviani N, Skhirtladze M, Khutsishvili M: Effect of metformin therapy on plasma adiponectin and lep- tin levels in obese and insulin resistant postmenopausal females with type 2 diabetes. Georgian Med News 2007:52-55. yp g 47. Liew CF, Seah ES, Yeo KP, Lee KO, Wise SD: Lean, nondiabetic Asian Indians have decreased insulin sensitivity and insulin clearance, and raised leptin compared to Caucasians and Chinese subjects. Int J Obes Relat Metab Disord 2003, 27(7):784-789. References Reaven GM, Laws A: Insulin resistance, compensatory hyperin- sulinaemia, and coronary heart disease. Diabetologia 1994, 37(9):948-952. ( ) 36. Fujioka S, Matsuzawa Y, Tokunaga K, Tarui S: Contribution of intra-abdominal fat accumulation to the impairment of glu- cose and lipid metabolism in human obesity. Metabolism 1987, 36(1):54-59. ( ) 37. Castracane VD, Henson MC: When did leptin become a repro- ductive hormone? Semin Reprod Med 2002, 20(2):89-92. p ( ) 38. Alexander CM, Landsman PB, Teutsch SM: Diabetes mellitus, impaired fasting glucose, atherosclerotic risk factors, and prevalence of coronary heart disease. Am J Cardiol 2000, 86(9):897-902. ( ) 39. Zhong N, Wu XP, Xu ZR, Wang AH, Luo XH, Cao XZ, Xie H, Shan PF, Liao EY: Relationship of serum leptin with age, body weight, body mass index, and bone mineral density in healthy mainland Chinese women. Clin Chim Acta 2005, 351(1– 2):161-168. ) 40. Maugeri D, Bonanno MR, Speciale S, Santangelo A, Lentini A, Russo MS, Calanna A, Malaguarnera M, Motta M, Testai' M, Panebianco P: The leptin, a new hormone of adipose tissue: clinical findings and perspectives in geriatrics. Arch Gerontol Geriatr 2002, 34(1):47-54. ( ) 41. Leonhardt W, Horn R, Brabant G, Breidert M, Temelkova-Kurkt- schiev T, Fucker K, Hanefeld M: Relation of free and specifically bound leptin to insulin secretion in patients with impaired glucose tolerance (IGT). Exp Clin Endocrinol Diabetes 1997, 107(1):46-52. ( ) 42. Asakawa H, Tokunaga K, Kawakami F: Relationship of leptin level with metabolic disorders and hypertension in Japanese type 2 diabetes mellitus patients. J Diabetes Complications 2001, 15(2):57-62. 43. Couillard C, Mauriege P, Prud'homme D, Nadeau A, Tremblay A, Bouchard C, Despres JP: Plasma leptin concentrations: gender differences and associations with metabolic risk factors for cardiovascular disease. Diabetologia 1997, 40(10):1178-1184.
https://openalex.org/W2084023097	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0082984&type=printable	English	null	Rural Clinician Scarcity and Job Preferences of Doctors and Nurses in India: A Discrete Choice Experiment	PloS one	2,013	cc-by	8,007	Abstract The funders (World Bank) participated in the study design, decision to publish, and preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: The study was funded by a grant from the World Bank. Two of the authors were members of the World Bank at the time of this study. The funders (World Bank) participated in the study design, decision to publish, and preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kd.rao@phfi.org Krishna D. Rao1, Mandy Ryan2, Zubin Shroff3, Marko Vujicic4, Sudha Ramani5, Pet 1 Public Health Foundation of India, New Delhi, India, 2 Health Economics Research Unit, Institute of Applied Health Sciences, University of Aberdeen, Aberdeen, Scotland, 3 Department of Global Health and Population, Harvard School of Public Health, Boston, Massachusetts, United States of America, 4 American Dental Association, Chicago, Illinois, United States of America, 5 Indian Institute of Public Health (Hyderabad), Hyderabad, India, 6 Department of Global Health and Population, Harvard School of Public Health, Boston, Massachusetts, United States of America December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org Abstract The scarcity of rural doctors has undermined the ability of health systems in low and middle-income countries like India to provide quality services to rural populations. This study examines job preferences of doctors and nurses to inform what works in terms of rural recruitment strategies. Job acceptance of different strategies was compared to identify policy options for increasing the availability of clinical providers in rural areas. In 2010 a Discrete Choice Experiment was conducted in India. The study sample included final year medical and nursing students, and in-service doctors and nurses serving at Primary Health Centers. Eight job attributes were identified and a D-efficient fractional factorial design was used to construct pairs of job choices. Respondent acceptance of job choices was analyzed using multi-level logistic regression. Location mattered; jobs in areas offering urban amenities had a high likelihood of being accepted. Higher salary had small effect on doctor, but large effect on nurse, acceptance of rural jobs. At five times current salary levels, 13% (31%) of medical students (doctors) were willing to accept rural jobs. At half this level, 61% (52%) of nursing students (nurses) accepted a rural job. The strategy of reserving seats for specialist training in exchange for rural service had a large effect on job acceptance among doctors, nurses and nursing students. For doctors and nurses, properly staffed and equipped health facilities, and housing had small effects on job acceptance. Rural upbringing was not associated with rural job acceptance. Incentivizing doctors for rural service is expensive. A broader strategy of substantial salary increases with improved living, working environment, and education incentives is necessary. For both doctors and nurses, the usual strategies of moderate salary increases, good facility infrastructure, and housing will not be effective. Non-physician clinicians like nurse- practitioners offer an affordable alternative for delivering rural health care. Editor: Waldemar A. Carlo, University of Alabama at Birmingham, United States of America Received July 25, 2013; Accepted October 29, 2013; Published December 20, 2013 Copyright: 2013 Rao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by a grant from the World Bank. Two of the authors were members of the World Bank at the time of this study. Introduction involves deploying non-physician clinicians (e.g. nurse-practitioner or medial assistant) to perform many of the functions of a doctor [8]. Studies have shown that non-physician clinicians can perform comparably with doctors in primary care settings [9–13]. Health systems in low and middle-income countries (LMIC) like India struggle to provide quality clinical services to rural populations. An important constraint facing these countries is the scarcity of physicians in rural areas. In India, for example, almost 60% of health workers reside in urban areas even though 74% of the country’s population is rural [1]. This rural scarcity is particularly stark when compared to the urban availability of clinical care providers. In rural (urban) India there are 1.2(11.3) physicians and 0.7(4.3) nurses and midwifes per 10,000 population [1]. Indian strategies to improve rural recruitment have been born out of an administrative response, rather than, from a systematic understanding of health worker preferences [14]. This has prevented policy makers from taking a comprehensive view of the rural recruitment problem. Moreover, strategies that have been attempted like higher salaries for rural postings, admission to specialist training after some years in rural service, and housing have been offered as singleton incentives (typically only salary increases) [15–16]. While task shifting for primary care has been attempted on a limited scale, concerns about quality of care have contributed to restricting further expansion [17]. In general, little evidence exists on the effectiveness of individual rural retention strategies or how they can be improved. Importantly, current strategies mostly focus on doctors with little information on nurses. Such approaches based on a narrow view of the rural recruitment problem have limited the range of strategies that have been attempted. Efforts to reduce the scarcity of rural clinicians in LMICs have broadly focused on three strategies. One is to make rural service compulsory; however, this has generally been unpopular and unsuccessful [2]. A second strategy is to make rural service more attractive by offering monetary and non-monetary incentives. Several studies have shown that job choices are driven by salary, as well as, non-monetary job characteristics such as living conditions, educational opportunities for children, opportunities for training, and future career prospects [3–7]. A third strategy of task shifting December 2013 \| Volume 8 \| Issue 12 \| e82984 1 PLOS ONE \| www.plosone.org Rural Clinician Scarcity In India Table 1. Attributes and levels [Reference category for each attribute highlighted in italics]. Table 1. Introduction Attributes and levels [Reference category for each attribute highlighted in italics]. Attribute Levels 1 Type of health center 1. Clinic 2. Small hospital (20–30 beds) 3. Large hospital (50–100 beds) 2 Area 1. Located in a poorly connected place with bad education facility for children and poor housing provided 2. Located in a poorly connected place with bad education facility for children but good housing provided 3. Located in a well-connected place, having good education facilities for children but poor quality housing provided 4. Located in a well-connected place, having good education facilities for children and good quality housing provided 3 Health center infrastructure 1. Building in poor condition, inadequate equipment, and frequent shortages of supplies and drugs 2. Well maintained building, adequately equipped with few shortages of supplies and drugs. 4 Staff 1. Few staff and heavy workload 2. Fully staffed and moderate workload 5 Salary (including allowances, Rs/month) 1. Doctors: 30,000 45,000 65,000 80,000 2. Nurses: 10,000 15,000 25,000 30,000 6 Change in location to city/town 1. Uncertain 2. On completion of 3 years 7 Professional development 1. Short duration training courses for skill development 2. Easier admission to PG after 3 years of service in same job through reservation/quota. 8 Job location 1. The job is not located in your native area 2. The job is located in your native area Source: Study data. doi:10.1371/journal.pone.0082984.t001 This study aims to better understand ‘what works’ in terms of strategies for increasing the presence of qualified clinical care providers in rural areas of India. This is approached from the perspective of job preferences of both trainee and in-service doctors and nurses. This study has the following specific aims. First, to identify effective rural recruitment strategies based on health worker job preferences. Second, to compare how the effectiveness of these strategies differs between doctors and nurses. Third, to outline policy options for increasing the availability of clinical care providers in rural areas. The study focused on final year medical and nursing students, as well as, doctors and nurses serving at Primary Health Centers (PHC) and sub-district hospitals. The focus on final year students is because they will soon enter the job market, and on doctors and nurses is because they both provide clinical care at PHCs. The two Indian states where this study was conducted represent diversity both in terms of geography and in their capacity to produce doctors and nurses. Introduction The two Indian states where this study was conducted represent diversity both in terms of geography and in their capacity to produce doctors and nurses. To elicit health worker preferences for different job attributes we use the Discrete Choice Experiment (DCE) methodology. The DCE method has been used extensively in the health economics field [3–7,18–21]. To the best of our knowledge, this is the first DCE to be conducted in India for this purpose. Importantly, this study is one of the few that compares the stated job preferences of doctors and nurses for rural recruitment in the same setting. Introduction To elicit health worker preferences for different job attributes we use the Discrete Choice Experiment (DCE) methodology. The DCE method has been used extensively in the health economics field [3–7,18–21]. To the best of our knowledge, this is the first DCE to be conducted in India for this purpose. Importantly, this study is one of the few that compares the stated job preferences of doctors and nurses for rural recruitment in the same setting. particular attributes of a job by asking them to state their preferred choice over hypothetical job alternatives. The DCE methodology has its theoretical foundations in the random utility framework [21]. A respondent (i.e. health worker) is assumed to choose among several alternative jobs. He or she will choose the job that produces the highest utility. The random utility framework assumes that the utility of a given job has two components - deterministic and random [21]. The deterministic component is a function of observable job attributes (e.g. pay, working conditions, location), each of which has a ‘ utility weight’ (see equation 1). The random component is determined by unobserved job attributes in addition to individual-level preference variation. The utility derived from a job is not directly observed, implying that the utility weights of the job attributes cannot be directly estimated. In the DCE methodology, when respondents choose their preferred job from a set of alternative jobs, the probability of choosing a job can be estimated (see equation 2). And after making certain assumptions, the job attribute utility weights can be estimated using standard econometric techniques [21]. It should be noted that an underlying assumption of these models is that individuals have a complete ranking of job opportunities that is determined by their preferences for the varying job attributes. y g p First, to identify effective rural recruitment strategies based on health worker job preferences. Second, to compare how the effectiveness of these strategies differs between doctors and nurses. Third, to outline policy options for increasing the availability of clinical care providers in rural areas. The study focused on final year medical and nursing students, as well as, doctors and nurses serving at Primary Health Centers (PHC) and sub-district hospitals. The focus on final year students is because they will soon enter the job market, and on doctors and nurses is because they both provide clinical care at PHCs. Statistical Analysis Respondents were presented with a set of 16 choice sets, each choice set containing a pair of jobs that had the same attributes but not all at the same level. The survey used a two-stage response design in which the respondent first made a choice between the presented job pairs by responding to the question ‘‘Which of the two jobs do you prefer’’. In the second stage, medical and nursing students were first asked ‘‘Will you choose this job if it was offered to you’’, and in- service doctors and nurses were asked ‘‘Will you choose this job over your current job’’. The second stage serves as a ‘opt-out’ option and offers respondents the opportunity to reject the forced choice made in the first stage. Forcing choice to be confined to the first stage can potentially bias respondent preferences for job attributes [21]. Data collected from the field survey was cleaned and double entered into a CSPro version 4.1 (US Census Bureau) database. All analysis was done in Stata 12 [24]. All analysis was stratified by students, in-service doctors, and nurses. Analysis of the information collected included presenting summary statistics of respondent characteristics. Individuals who ‘‘failed’’ both dominance tests were dropped from the analysis. In the DCE, each respondent stated their choice between 16 pairs of job choices. Further, respondents were also selected from a common school or district. Therefore the study data was hierarchically structured with the possibility of data being correlated within each level of hierarchy – there could be correlation in the job choice responses belonging to the same individual (intra-respondent), as well as, correlation among responses of individuals in the same school or district (intra- location). The outcome of interest is if the respondent accepted the job that he/she selected in the first stage. The binary response to the following questions were modeled: for medical and nursing students -‘‘Will you choose this job if it was offered to you’’, and in-service doctors and nurses - ‘‘Will you choose this job over your current job’’. Multi-level logistic regression was used to model the binary responses related to job acceptance [25–27]. Models were run separately for medical students, nursing students, doctors and nurses. Because students were sampled within schools and in- service respondents from the same district, we also ran three-level models in which school/district was the third level to accommo- date the intra-school or intra-district correlation. Sample Selection The target sample (size) was final year undergraduate medical students (150), final year GNM nursing students (150), in-service doctors (150) and nurses (150) working at PHCs. doctors (150) and nurses (150) working at PHCs. The utility or satisfaction derived from job defined by choice-set i and by person j is represented by a continuous variable, y ij [28]. In the two level random effects linear regression framework shown below, where choice-sets are at level 1 and individuals at level 2, we can model y ij as: Medical and nursing schools and students. The student sample was from the state of Andhra Pradesh. The selection of medical and nursing students was a two-step process- first; medical/nursing schools were purposively selected, followed by a convenience sampling of final year medical and nursing students. Medical and nursing schools and students. The student sample was from the state of Andhra Pradesh. The selection of medical and nursing students was a two-step process- first; medical/nursing schools were purposively selected, followed by a convenience sampling of final year medical and nursing students. One medical and nursing school was selected from each of the three regions of the state in such a manner that the aggregate sample of colleges had representation from public and private colleges, urban and rural locations and a range of academic reputations. A total of 4 (3 public and 1 private) medical and 4 nursing (2 public and 2 private) schools were selected. Within each school, students in their final year – fourth year MBBS students and second year General Nursing and Midwifery (GNM) students- were invited to participate in the study by the school administra- tion. The majority of students in the class accepted and participated. No information on non-participants was collected. Among medical students willing to participate, an equal number of male and female students were administered the questionnaire. Informed written consent was obtained from all respondents. y ij~b0zb1Xijzb2ZijzeijzU0j ð1Þ ð1Þ where, b0 is the constant term, Xij is a vector of job attribute-levels (see Table 1) and b1 is the vector of regression coefficients associated with these attribute levels. Zij is a vector of individual- level characteristics (see Table 2), and b2 is the vector of regression coefficients associated with these characteristics. Methods p A qualitative study was conducted between January and July 2010 in the two study states to identify what job attributes were important for trainee and in-service doctors and nurses [22]. A total of 80 in-depth interviews were conducted with medical students, nursing students, and doctors and nurses in primary health centers. A diverse set of job attributes was elicited and Ethical approval was obtained from the Ethical Review Committee of the Public Health Foundation of India. The Discrete Choice Experiment (DCE) method is a quantita- tive technique that aims to elicit stated preferences of individuals [19]. This technique helps to uncover how individuals’ value December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org 2 Rural Clinician Scarcity In India candidates from the selected districts who were working in PHCs were invited to participate in the study. The majority of nurses and doctors in the district accepted and participated. No information on non-participants was collected. In Uttarkhand, a listing was made of the number of sanctioned posts for doctors and nurses at PHCs in each district of the state. Nurses posted at sub-district hospitals were also included in the sample. Six districts with the largest number of sanctioned posts for Medical Officers were selected from the two regions of the state. All eligible candidates from the selected districts were invited to participate in the study. Informed written consent was obtained from all respondents. clustered based on the frequency with which attributes were cited in health worker interviews and from policy maker ratings on the ‘actionablility’ of the attribute. Table 1 describes the final set of eight job attributes and their levels. Given the number of attributes and their associated levels, a total of 1,536 (2531*42) unique jobs can be derived from different combinations of these attributes and levels. To limit the number of job choice sets to 16 (which is generally the convention for DCE studies), a statistically efficient (D-efficient) fractional factorial design was used. This ensures minimum variation around the parameter estimates by minimizing the estimated standard errors. SAS software was used to generate the design [23]. Statistical Analysis If these correlations were found to be significant then school or district level random effects were also included in the model. Four additional choice sets were inserted. Two of these were dominance tests, in which one job was superior to the other in terms of all attributes, and expected to be chosen by a rational respondent. For the attributes ‘‘place of work’’ and ‘‘location of job’’, no level was consistently considered to be dominant over the other, and hence, for the rationality test, the level of these two attributes was kept same in job 1 and job 2. Another two choice sets were added as ‘hold outs’, responses to which enabled evaluation of the model’s predictive accuracy. The questionnaire was administered in English to medical students and doctors and in the local language to nurses and nursing students. December 2013 \| Volume 8 \| Issue 12 \| e82984 Sample Selection The mean duration of government service was similar for the sample of doctors and nurses. Note: Figures in parentheses are standard deviations. N/a = not applicable. Source: Study data. doi:10.1371/journal.pone.0082984.t002 The predictive accuracy of the model for medical students (80%), nursing students (72%), doctors (78%), and nurses (74%) indicated good model fit. The out-of-sample prediction using the two hold-out choice sets yielded the following proportions of responses correctly predicted– medical students (87%, 65%), nursing students (74%, 60%), doctors (90%, 56%), and nurses (77%, 70%). The statistical significance of the intra-cluster coefficient, rho, confirms the appropriateness of introducing the two or three-level random intercept model (see Table S1). Note that three-levels were included in the model only when the intra- school/district correlations were found to be significant. that yij = 1, as follows: log it(pij)~b0zb1Xijzb2ZijzU0j ð2Þ ð2Þ where, eb1 is the odds ratio associated with each attribute-level of the job. This odds ratio can be interpreted as how much more likely (if .1) or less likely (if ,1) a job will be accepted when that attribute-level is present, compared to when it is not present, with other attribute-levels and covariates being at their reference categories. The vector of regression coefficients, eb2 is the odds ratio associated with each individual characteristic. These odds ratios are interpreted as how much more or less likely a job will be accepted if that individual characteristic was present, compared to when it was not present, with other attribute-levels and covariates being at their reference categories. where, eb1 is the odds ratio associated with each attribute-level of the job. This odds ratio can be interpreted as how much more likely (if .1) or less likely (if ,1) a job will be accepted when that attribute-level is present, compared to when it is not present, with other attribute-levels and covariates being at their reference categories. The vector of regression coefficients, eb2 is the odds ratio associated with each individual characteristic. These odds ratios are interpreted as how much more or less likely a job will be accepted if that individual characteristic was present, compared to when it was not present, with other attribute-levels and covariates being at their reference categories. g Figure 1 presents the results from the multi-level logistic regression models. The plotted regression coefficients are in the form of odds-ratios and their 95% confidence intervals (Table S1). Sample Selection An attribute/characteristic is positively and significantly associated with accepting a job if the 95% confidence interval of the odds- ratio is greater than 1 i.e. above the vertical dashed line. The odds ratios represent the likelihood of accepting a job when that particular job attribute or individual characteristic is present in a reference case job. The reference case refers to a job in a clinic, located in an area with poor connectivity, poor education facilities for children, and poor housing; the clinic has poor infrastructure; it is poorly staffed and workload is high; transfer is uncertain; there is no reservation for in-service staff for higher education; job is not located in the respondent’s native area; and salary is entry level (Rs 30,000 for doctors and Rs 10,000 for nurses). In short, the reference case refers to a government job at a typical PHC located in a remote area with salary levels around what new recruits receive. The results from the logistic regression can be used to estimate the probability (pij) of accepting a job: pij~ eb0zb1Xijzb2ZijzU0j 1zeb0zb1Xijzb2ZijzU0j ð3Þ pij~ eb0zb1Xijzb2ZijzU0j 1zeb0zb1Xijzb2ZijzU0j ð3Þ ð3Þ Equation (3) can be used to estimate the percentage of respondents who would take up a job for different combinations of job attribute-levels and individual characteristics. Wage supply elasticities were calculated by estimating the percentage change in the proportion of respondents willing to accept a job relative to the percentage change in salary. Among medical students and doctors, the likelihood of accepting a job, was significantly and positively (95% CI of odds-ratio. 1) associated with the following attributes - area (good education facilities for children and connectivity), good health facility infrastructure, adequate staff availability, guaranteed transfer after three years of service, reservation for higher education, posting in one’s native area, and salary. Job attributes that were not significantly associated were – a job in a small or big hospital, only having good housing in an area, and guaranteed transfer after three years (for doctors). For both medical students and in-service doctors a job with 20% higher salary was 1.23 (1.20–1.25) times more likely to be accepted, compared to a job with reference salary and other attribute levels. Notably, among all the job attributes, jobs with higher education training opportuni- ties had the highest likelihood of being accepted. Sample Selection eij is the choice- set level error term, U0j is the individual level random effects term and is assumed to be normally distributed with a mean of zero and a between-respondent level variance of s2 u. We however only observe the binary variable, yij, such that yij = 1 if y ij .0, and yij = 0 if y ij#0. In the DCE context, yij = 1 if person j accepts the job he/she selected in choice-set i. Otherwise, yij = 0. Conditional on U0j and assuming that eij follows a standard logistic distribution [28], we model pij the probability In-service doctors and nurses. To select in-service doctors and nurses employed at PHCs in Andhra Pradesh, one district from each of the state’s three regions was randomly selected. All December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e82984 3 Rural Clinician Scarcity In India to 457 doctors and nurses. After retaining those observations in the sample that met the sample eligibility requirements, and dropping four respondents that failed both the dominance tests the final sample size was 221 doctors and 232 nurses. Table 2. Sample description. Students In-service Medical Nursing Doctors Nurses Age (years) 21.90 20.38 35.98 32.43 (1.20) (1.62) (7.41) (7.38) Male (%) 50 5 74 3 Rural upbringing (%) 12 73 28 51 Private school (%) 39 45 26 29 Years of service N/a N/a 5.78 6.61 (4.55) (6.60) Sample size 161 132 221 232 Note: Figures in parentheses are standard deviations. N/a = not applicable. Source: Study data. d i 10 1371/j l 0082984 t002 About half the medical students who participated were male with a mean age of about 22 years (Table 2). Nursing students were overwhelmingly female and were slightly younger. Only 12 percent of medical students had had grown up in a rural area compared to the majority of nursing students. Most of the medical school students and about half the nursing students were studying at government institutions. The majority of doctors were male, had grown up in an urban area, attended public medical colleges, and had served for five years on average as Medical Officers. The overwhelming majority of nurses were female. Nurses tended to be younger than doctors, more than half of them had rural upbringing, and most of them had trained in government colleges. Sample Selection For instance, its effect size for medical students (OR = 5.12(4.36–6.01)) and doctors The model’s predictive accuracy was measured by the area under the Receiver Operating Characteristic (ROC) curve, which gives an estimate of the percent of outcomes correctly predicted by the model [29]. The in-sample prediction is based on the range of data used to populate the model. The out-of-sample prediction was made with the help of the two holdout choice sets, which were not used in estimating the regression equations. Discussion For instance, 6.7% more doctors would take up a job if it possessed the best area attribute, and around 9.0% (2.8% for medical students) more would accept a rural job if offered specialist training in exchange for three years of rural service. Health facilities with good infrastructure would attract 3% more in-service doctors over baseline levels. In India’s context, reservation of specialist seats in exchange for a few years of rural service is a powerful strategy to attract and retain doctors (and nurses, and nursing students) to rural posts. Several states currently offer this scheme to their in-service doctors. In our study, this strategy had a large effect on the likelihood of selecting a rural job. For doctors, its effect on accepting a rural job was almost as powerful as a job in the ‘best’ area (i.e. having good connectivity, education facilities for children, and housing). This is expected since the pressing ambition of Indian medical graduates is to become a specialist, and the competition for specialist seats is intense due to the small number of seasts avaialble in comparison to the number of medical graduates. However, there is a larger question about how well qualified and motivated medical graduates are to serve as rural doctors. Job attributes had a larger effect on nursing students and nurse (Figure 3) uptake of rural jobs over baseline levels, compared to medical students and doctors. In the presence of the best area attribute, 15% (8%) more nursing students (nurses) would opt for rural service. For both nursing students and nurses, jobs in health facilities that had good infrastructure, and the presence of higher education opportunities (e.g. BSc nursing) has relatively large effects on increasing the percentage of respondents opting for rural jobs. For doctors and nurses, individual characteristics had small effects on increasing the percent of respondents opting for a rural job, over baseline levels. Figure 4 shows the labor supply curves for government jobs located in a typical rural area i.e. the reference case situation. Overall, for every salary level, a considerably higher proportion of nursing students and nurses were willing to accept a rural job compared to medical students and doctors. A doubling of salary from base levels increases the percentage of medical students (0.73% to 2%) and doctors (2% to 5%) available for rural service. Discussion The DCE reported in this study highlighted several strategies available in LMICs like India for increasing the availability of clinical care providers in rural areas. Expectedly, location matters; for both trainee and in-service doctors and nurses, jobs located in an area with the ‘best’ attributes had one of the highest likelihoods of being accepted, and this declined as the area attributes became less desirable. This has several implications. For one, it highlights the importance of creating a good living environment for health workers in the area where they are posted. Since amenities like connectivity and children’s education are such important drivers of where health workers would like to work, effort should be made in providing such facilities where rural posts are located. The responsibility for creating such an environment will require an integrated approach that necessarily goes beyond the health ministry and include the education and rural development sectors in government. However, the cost of providing such an environment may ultimately be unaffordable to countries like India. Secondly, state health departments can expect greater success in posting doctors and nurses in rural posts that are close to urban settlements since the amenities of urban life are within reach. The greater concentration of doctors and nurses in semi- urban areas is testimony to this. However, for postings that are remote, a broader strategy of improving living environments is required. So, for instance, the common strategy of only providing higher salary or good housing will have limited success in making rural posts attractive. Figure 2 (medical students and doctors) and Figure 3 (nursing students and nurses) shows the change in the percentage of respondents, over baseline (described earlier), who would accept a rural job in the presence of specific job attributes and individual characteristics (see Equation 3). Among medical students, the presence of different job attributes did not substantially increase the percentage of respondents opting for a rural job over baseline levels. For instance, in the presence of the best area attribute, 3% more medical students accepted a rural job, over baseline levels. For a job with reference case conditions and salary, less than 1.0% of medical students stated acceptance. In contrast, in-service doctors were more influenced by job attributes for taking up rural posts. Rural Clinician Scarcity In India Rural Clinician Scarcity In India Among nursing students and nurses, the job attributes significantly associated with the likelihood of accepting a job included – job in a small or large hospital (as opposed to a health center), good area characteristics, good health facility infrastruc- ture, adequate staff, reservation for higher education, posting in one’s native area (for nurses), and salary. Job attributes not significantly associated with job acceptance were- the availability of good housing only, guaranteed transfer after three years of service, and posting in one’s native area (for nursing students). Good facility infrastructure had the second biggest effect size after the best area attribute. For both nursing students and nurses a job with 50% higher salary was 1.47 (1.41–1.54) and 1.56(1.50–1.63) times more likely to be accepted, respectively, compared to a job with reference salary and other attribute levels. The likelihood of accepting a job diminishes as the area becomes progressively ‘rural’. None of the individual attributes (rural upbringing, private schooling, being male) were significantly associated with the likelihood of accepting a job. However, for nurses, being older significantly reduced the likelihood of accepting a job. percentage increase in salary. For nursing students and nurses, supply was elastic at lower salary levels and became inelastic towards the upper end of the salary range. Further, the supply of nursing students and nurses is more elastic, relative to medical students and doctors, at lower salary levels. December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org Results Of the 308 medical and nursing students who answered the questionnaire, 15 respondents who failed both the dominance (i.e. did not choose the job with better attribute levels) tests were dropped, reducing the sample size to 293 (161 medical and 132 nursing) students. The in-service questionnaire was administered December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e82984 4 Rural Clinician Scarcity In India (OR = 5.41(4.76–6.16)) was greater than that of a job possessing h b ib h lik lih d f i j b (OR = 2.18(1.76–2.17 d i f ili i Figure 1. Determinants of job acceptance among trainee and in-service doctors and nu doi:10.1371/journal.pone.0082984.g001 Figure 1. Determinants of job acceptance among trainee and in-service doctors and nurses. doi:10.1371/journal.pone.0082984.g001 (OR = 5.41(4.76–6.16)) was greater than that of a job possessing the best area attribute. The likelihood of accepting a job diminished as the area became progressively ‘rural’. For example, medical students were five times more likely (OR = 5.00(4.07– 6.05)) to accept a job in the ‘best’ area (i.e. good education facilities for children, connectivity, housing) or were twice as likely (OR = 5.41(4.76–6.16)) was greater than that of a job possessing the best area attribute. The likelihood of accepting a job diminished as the area became progressively ‘rural’. For example, medical students were five times more likely (OR = 5.00(4.07– 6.05)) to accept a job in the ‘best’ area (i.e. good education facilities for children, connectivity, housing) or were twice as likely (OR = 2.18(1.76–2.17)) to accept a job in an area with only good education facilities and connectivity, compared to an area without these attributes. In general, none of the individual characteristics – being male (for medical students), rural upbringing, private medical school education – was significantly associated with the likelihood of accepting a job. December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Discussion In contrast, a doubling of salary has a greater effect on increases in the percentage of nursing students (7% to 14%) and nurses (3% to 7%) accepting rural jobs. At the high salary level of Rs. 100,000 per month, 13% (31%) of medical students (doctors) accepted a rural job. At half that salary level, 61% (52%) of nursing students (nurses) accepted a rural job. For both trainee and in-service doctors and nurses, job attributes such as moderate salary increases, staffing health facilities adequately so that workloads are not overbearing, and good facility infrastructure, while important, did not have large effects on the likelihood of a rural job being accepted. In general, such singleton attributes are not very effective in increasing rural recruitment. However, when combined, they can have an additive effect- incentive ‘packages’ based on these attributes will be more effective in increasing rural recruitment than singleton incentives. This again reinforces the importance of taking a broader approach to designing strategies for increasing rural recruitment. This study also highlights the relatively limited role that certain respondent characteristics play in uptake of rural jobs in India. Those who were brought up in rural areas were no more likely to accept rural jobs than those with an urban background. This Overall, the wage elasticity of supply of medical students and doctors was elastic over the salary rage. That is, the percentage increase in supply of health workers was greater than the PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e82984 6 Rural Clinician Scarcity In India Figure 2. Percentage change (over base) in number of medical students and doctors willing to accept a rural job in the presence of specific job attributes and individual characteristics (base: salary Rs.30,000/month). doi:10.1371/journal.pone.0082984.g002 Figure 2. Percentage change (over base) in number of medical students and doctors willing to accept a rural job in the presence of specific job attributes and individual characteristics (base: salary Rs.30,000/month). doi:10.1371/journal.pone.0082984.g002 Figure 3. Percentage change (over base) in number of nursing students and nurses willing to accept a rural job in the presence of specific job attributes and individual characteristics (base: salary Rs.10,000/month). doi:10.1371/journal.pone.0082984.g003 igure 3. Percentage change (over base) in number of nursing students and nurses willing to accept a rural job in the presence of pecific job attributes and individual characteristics (base: salary Rs.10,000/month). Figure 3. Discussion Percentage change (over base) in number of nursing students and nurses willing to accept a rural job in the presence of specific job attributes and individual characteristics (base: salary Rs.10,000/month). doi:10.1371/journal.pone.0082984.g003 December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org 7 7 Rural Clinician Scarcity In India Figure 4. Supply of trainee and in-service doctors and nurses. doi:10.1371/journal.pone.0082984.g004 non-physician clinician cadres, coupled with their greater propensity for rural service, makes them both a viable and affordable option for strengthening rural health services. suggests that a strategy of purposively recruiting students from Indian rural areas into medical and nursing schools may not be effective in increasing rural recruitment, and is somewhat at odds with previous research in other countries [13]. The findings do not provide any evidence that those educated at government medical colleges, where education is highly subsidized by the government, are more likely to accept a rural job than those educated at (more expensive) private medical colleges. suggests that a strategy of purposively recruiting students from Indian rural areas into medical and nursing schools may not be effective in increasing rural recruitment, and is somewhat at odds with previous research in other countries [13]. The findings do not provide any evidence that those educated at government medical colleges, where education is highly subsidized by the government, are more likely to accept a rural job than those educated at (more expensive) private medical colleges. This study has several notable limitations. Because the DCE method relies on stated preferences, how health workers might behave (i.e. revealed preference) when actually faced with these incentives, can be different [22,30]. Secondly, in the DCE health workers made a choice between hypothetical jobs. While every effort was made to make these job descriptions as realistic as possible in terms of their attributes and levels, nevertheless, they are limited in their realism. Third, respondents could be making job choices based on a few select attributes (e.g. salary) rather than assessing all the attributes. In this case preferences for select attribute-levels will be incorrectly interpreted as preference for all the attribute-levels that define that job. Fourth, one also needs to be cautious in interpreting the findings emerging from this DCE. Since the DCE focused on specific cadres (nurses and doctors) the findings are applicable to them. December 2013 \| Volume 8 \| Issue 12 \| e82984 References 1. Rao KD, Bhatnagar A, Berman P (2012). So many, yet few: Human resources for health in India. Human Resources for Health, 10(1), 19. doi:10.1186/1478- 4491-10-19. 15. Shroff Z, Murthy S, Rao KD (2013). Attracting Doctors to Rural Areas: A Case Study of the Post-Graduate Reservation Scheme in Andhra Pradesh. Indian Journal of Community Medicine, 38(1), 27–32. doi:10.4103/0970-0218.106624. 15. Shroff Z, Murthy S, Rao KD (2013). Attracting Doctors to Rural Areas: A Case Study of the Post-Graduate Reservation Scheme in Andhra Pradesh. Indian Journal of Community Medicine, 38(1), 27–32. doi:10.4103/0970-0218.106624. 2. Kalantri SP (2007). Getting doctors to the villages: will compulsion work? Indian Journal of Medical Ethics, 4(4), 152–153. 16. Sundararaman T, Gupta G (2011). Indian approaches to retaining skilled health workers in rural areas. Bulletin of the World Health Organization, 89(1), 73–77. doi:10.2471/BLT.09.070862. 16. Sundararaman T, Gupta G (2011). Indian approaches to retaining skilled health workers in rural areas. Bulletin of the World Health Organization, 89(1), 73–77. doi:10.2471/BLT.09.070862. 3. Blaauw D, Pagaiya E, Tangcharoensathein N, Mullei V, Mudhune K, et al. (2010). Policy interventions that attract nurses to rural areas: a multicountry discrete choice experiment. Bulletin of the World Health Organization, 88(5), 350–356. doi:10.2471/BLT.09.072918. 17. Rao KD, Sundararaman T, Bhatnagar A, Gupta G, Kokho P, et al. (2013). Which Doctor for Primary Health Care? Quality of Care and Non-Physician Clinicians in India. Social Science & Medicine. doi:10.1016/j.socs- cimed.2013.02.018. 4. Kruk ME, Johnson JC, Gyakobo M, Agyei-Baffour P, Asabir K, et al. (2010). Rural practice preferences among medical students in Ghana: a discrete choice experiment. Bulletin of the World Health Organization, 88(5), 333–341. doi:10.2471/BLT.09.072892. 18. Lancsar J, Louviere E (2008). Conducting discrete choice experiments to inform healthcare decision making: a user’s guide. Pharmacoeconomics, 26(8), 661–677. 19. Mangham L, Hanson K, McPake B (2009). How to do (or not to do)… Designing a discrete choice experiment for application in a low-income country. Health Policy and Planning, 24(2), 151–158. doi:10.1093/heapol/czn047. 5. WHO (2010). Increasing access to health workers in remote and rural areas through improved retention: global policy recommendations. World Health Organization, Geneva. 20. Miranda JJ, Diez-Canseco F, Lema C, Lescano AG, Lagarde M, et al. (2012). Stated preferences of doctors for choosing a job in rural areas of Peru: a discrete choice experiment. PLoS One. 2012;7(12): e50567. doi:10.1371/journal.- pone.0050567. g 6. Kolstad J (2011). How to Make Rural Jobs More Attractive to Health Workers. Table S1 Multi-level Logistic Regression Results of Job Acceptance on Job and Individuals Characteristics. (DOCX) Table S1 Multi-level Logistic Regression Results of Job Acceptance on Job and Individuals Characteristics. (DOCX) References Findings from A Discrete Choice Experiment in Tanzania. Health Economics, (211 (January 2010)), 196–211. doi:10.1002/hec.1581. ( (J y )) 7. Vujicic M, Alfano M, Shengelia B, Witter S (2010). Attracting Doctors and Medical Students to Rural Vietnam: Insights from a Discrete Choice Experiment. Health, Nutrition and Population Discussion Paper. Human Development Network, World Bank. 21. WHO (2012). ‘‘How to conduct a Discrete Choice Experiment for Health Workforce Recruitment and Retention in Remote and Rural Areas: A User Guide with case Studies.’’ World Health Organization, Geneva. 22. Rao KD, Ramani S, Khandpur N, Murthy S, Hazarika I, et al. (2010). Health Worker Attitudes Towards Rural Service In India: Results From Qualitative Research. HNP Discussion Paper, The World Bank, Washington DC. 8. Fulton BD, Scheffler RM, Sparkes SP, Auh EY, Vujicic M, et al. (2011). Health workforce skill mix and task shifting in low income countries: a review of recent evidence. Human resources for health, 9(1), 1. doi:10.1186/1478-4491-9-1. 23. Kuhfeld W (2010). Marketing Research Methods in SAS, Version 9.2. Cary, NC, USA: SAS Institute. 9. Abegunde DO, Shengelia B, Luyten A, Cameron A, Celletti F, et al. (2007). Can non-physician health-care workers assess and manage cardiovascular risk in primary care? Bulletin of the World Health Organization, 85(6), 432–40. 24. Stata Corp (2011). Stata Statistical Software: Release 12. College Station, Texas. 25. Cheng J, Pullenayegum E, Marshall D, Marshall JK, Thabane L (2012). An empirical comparison of methods for analyzing correlated data from a discrete choice survey to elicit patient preference for colorectal cancer screening. BMC medical research methodology, (12(15)). doi:10.1186/1471-2288-12-15. 10. Huicho L, Scherpbier RW, Nkowane AM, Victora CG (2008). How much does quality of child care vary between health workers with differing durations of training? An observational multicountry study. Lancet, 372(9642), 910–6. doi:10.1016/S0140-6736(08)61401-4. medical research methodology, (12(15)). doi:10.1186/1471-2288- 26. Opuni M, Bishai D, Gray GE, McIntyre JA, Martinson NA (2010). Preferences for characteristics of antiretroviral therapy provision in Johannesburg, South Africa: results of a conjoint analysis. AIDS and Behavior, 14(4), 807–815. doi:10.1007/s10461-009-9584-4. ( ) 11. McCord C, Mbaruku G, Pereira C, Nzabuhakwa C, Bergstrom S (2009). The quality of emergency obstetrical surgery by assistant medical officers in Tanzanian district hospitals. Health affairs (Project Hope), 28(5), w876–85. doi:10.1377/hlthaff.28.5.w876. 27. Wooldridge J (2002). Econometric Analysis of Cross Section and Panel Data. Cambridge, MA: MIT Press. 12. Mullan F, Frehywot S (2007). Non-physician clinicians in 47 sub-Saharan African countries. Lancet, 370(9605), 2158–2163. doi:10.1016/S0140- 6736(07)60785-5. Discussion However, in a health system context where health workers operate in teams and not in isolation, it is usually difficult to change incentives (e.g. higher salary) for only some cadre without others also demanding improved compensation. This can rapidly become an expensive proposition for government. Moreover, certain strategies, such as reserving seats for specialist training, might not be feasible in certain contexts. Incentivizing doctors to serve in rural areas is challenging and expensive. The supply of both student and in-service doctors for rural posts was not responsive to increases in salary, particularly at lower salary levels. The high salary levels at which a respectable proportion of medical students and doctors were willing to accept rural job is unaffordable for countries with limited health resources. In contrast, nursing students and nurses are more willing to accept a rural job, as well as, with greater enthusiasm in the presence of favorable job attributes. Moreover, the supply of nursing students and nurses is much more responsive to increases in salaries, particularly at lower levels, relative to medical students and doctors. The nurses in this study were not trained to take on the clinical functions of doctors. However, their cadre offers the potential of becoming providers of basic clinical care. Several countries in Africa and Asia, as well as two states within India, have used nurse-practitioners or Medial Assistants to successfully staff primary care facilities [11,16]. This potential of nurses or other December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org 8 Rural Clinician Scarcity In India For countries like India that have limited health resources, creating a health system capable of providing reliable and quality clinical services in rural areas is an especially difficult challenge. Strategies that offer ‘packages of incentives’ that address both the professional and personal needs of doctors can improve their rural recruitment to a certain extent. Elements of this package should include, substantial salary increases, improvements to the living environment, provision of good children’s education, and where possible, reservation of seats for specialist training. However, pursuing this route to its full extent might ultimately be unaffordable and requires health departments to engage with other government departments (e.g. education, roads). The experience of several countries that have adopted non-physician clinicians as an alternative way of providing basic health services to rural populations offers important lessons for India’s own ambitions for achieving universal health coverage. Author Contributions Conceived and designed the experiments: KDR MR ZS MV SR PB. Performed the experiments: KDR ZS SR. Analyzed the data: KDR MR ZS SR. Contributed reagents/materials/analysis tools: KDR MR MV PB. Wrote the paper: KDR MR ZS MV SR PB. Conceived and designed the experiments: KDR MR ZS MV SR PB. Performed the experiments: KDR ZS SR. Analyzed the data: KDR MR ZS SR. Contributed reagents/materials/analysis tools: KDR MR MV PB. Wrote the paper: KDR MR ZS MV SR PB. References 28. Guo G, Zhao H (2000). Multilevel Modeling For Binary Data. Annual Review of Sociology, 26: 441–62. d ( ) 13. Wilhelm TJ, Thawe IK, Mwatibu B, Mothes H, Post S (2011). Efficacy of major general surgery performed by non-physician clinicians at a central hospital in Malawi. Tropical doctor, 41(2), 71–5. doi:10.1258/td.2010.100272. 29. Hosmer DW, Lemeshow WD (2000). Applied Logistic Regression. 2nd Edition, New York: John Wiley & Sons. 30. Lagarde M, Blaauw D (2009). A review of the application and contribution of discrete choice experiments to inform human resources policy interventions. Human Resources for Health. 7: 62. 14. Dolea C, Stormont L, Braichet JM (2010). Evaluated strategies to increase attraction and retention of health workers in remote and rural areas. Bulletin of the World Health Organization, 88(5), 379–85. doi:10.2471/BLT.09.070607. December 2013 \| Volume 8 \| Issue 12 \| e82984 PLOS ONE \| www.plosone.org 9
https://openalex.org/W2030458387	https://europepmc.org/articles/pmc3480047?pdf=render	English	null	Moving to malaria elimination in Cape Verde	Malaria journal	2,012	cc-by	847	Background This case study examines the history of malaria in Cape Verde and presents details of the interventions implemen- ted over the last 60 years with the aim of disease elimina- tion. Trends in the malaria situation and programme policies and operations over time were evaluated and les- sons captured to assist other countries in making well- informed decisions regarding malaria elimination. The case study is a part of a joint series of malaria elimination case studies conducted by the WHO Global Malaria Pro- gramme and the University of California, San Francisco, Global Health Group. Low level malaria transmission continued on Santiago and, in 2003, reappeared on the island of Boa Vista. The annual parasite incidence for the whole archipelago has remained below 0.3 per 1000 population, exceeding 0.5 per 1000 population on Santiago Island only once, in 2000. Over the last 20 years, activities have been restricted to passive case finding and investigation with case-based surveillance on Santiago, and to early detection of imported cases elsewhere. Unfortunately, in 2006, an unexpected 8 malaria deaths occurred. Results Before the 1950s, Cape Verde was meso-endemic (Plasmo- dium falciparum, P.vivax and P.malaraie were detected) with annual incidence rates around 100 per 1000 popula- tion. The first malaria elimination campaign was initiated in 1953. Using IRS with DDT as a main strategy, and apply- ing additionally antilarval activities and active case finding, malaria transmission was interrupted in 1967; and in 1969 control interventions were stopped. The unique vector Anopheles arabiensis had been eradicated from all islands but Santiago. Moving to malaria elimination in Cape Verde Julio Monteiro Rodriguez1, Jean Olivier Guintran2*, Carolina Gomes3, Socé Fall4, Aafje Rietveld2, Richard Cibulskis2, Robert D Newman2, Rossitza Kurdova-Mintcheva2 From Challenges in malaria research Basel, Switzerland. 10-12 October 2012 5 years and transmission was again interrupted (1983- 1986). A new epidemic occurred in 1987-88 in Santiago Island and localised IRS operations were reactivated for 2 years; the country has not been able to invest sufficient resources to operate a new elimination plan. © 2012 Rodriguez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Materials and methods A review of published literature in English, French and Portuguese, of relevant documents and guidelines of the Ministry of Health, documents from WHO archives, and archives from the “Instituto de Medicina Tropical” (IMT) in Lisbon was conducted. Epidemiological, programma- tic, demographic, social, and other relevant data was extracted, analysed and evaluated. p Formulated in 2007, the National Health Policy sets out the strategy for durably eliminate malaria by 2020. The Government developed a National Strategic Plan 2009- 2013 with an integrated approach and the following main strategies: drug policy change to artemisinin-based combi- nation therapy (ACT) for P. falciparum; case detection among all febrile patients with positive travel history; full reporting of microscopically confirmed cases; case and foci investigation; vector control – including focal larval con- trol where appropriate, and IRS. National funding for malaria has been increased; in 2011, a 5-year Global Fund grant was secured to support the programme transition to elimination. 2Global Malaria Programme, World Health Organization, Geneva, Switzerland Full list of author information is available at the end of the article © 2012 Rodriguez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rodriguez et al. Malaria Journal 2012, 11(Suppl 1):O9 http://www.malariajournal.com/content/11/S1/O9 Conclusions In Cape Verde, malaria transmission has already been interrupted twice within the last 50 years, confirming that elimination is technically feasible; the challenge is now sustainability. The importance of preventing malaria rein- troduction should be fully taken into account if elimina- tion is to be achieved. In 1973, transmission reappeared in Santiago, culminat- ing in a major epidemic with 844 cases and 13 deaths in 1978. Widespread IRS operations were resumed for 2Global Malaria Programme, World Health Organization, Geneva, Switzerland Full list of author information is available at the end of the article Page 2 of 2 Page 2 of 2 Rodriguez et al. Malaria Journal 2012, 11(Suppl 1):O9 http://www.malariajournal.com/content/11/S1/O9 Author details 1 1Ministry of Health, Praia, Cape Verde. 2Global Malaria Programme, World Health Organization, Geneva, Switzerland. 3WHO country Office, Praia, Cape Verde. 4WHO Regional Office for Africa, Brazzaville, Congo. Published: 15 October 2012 doi:10.1186/1475-2875-11-S1-O9 Cite this article as: Rodriguez et al.: Moving to malaria elimination in Cape Verde. Malaria Journal 2012 11(Suppl 1):O9. doi:10.1186/1475-2875-11-S1-O9 Cite this article as: Rodriguez et al.: Moving to malaria elimination in Cape Verde. Malaria Journal 2012 11(Suppl 1):O9. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4242325818	https://zenodo.org/records/1509595/files/article.pdf	Latin	null	1 Thessalonicher 5, 1—10.	Zeitschrift für die neutestamentliche Wissenschaft und die Kunde der älteren Kirche	1,916	public-domain	3,923	G. F rster, l Thessalonicher 5, 1—10. G. F rster, l Thessalonicher 5, 1—10. 169 Von Dr. jur. Gerh. F rster in Freiberg Sa, l Thess 5, l—10 handelt von der Parusie Christi. Nachdem Paulus in v. l darauf hingewiesen hat, da er nicht n tig habe, ber das allen bekannte Thema π€ρι των χρόνων ( ber die Zeit bis zur Wiederkehr) και των καιρών ( ber die Zeit des Eintritts der Wiederkehr) zu schreiben, hei t es in v. 2: αυτοί γαρ ακριβώς οϊδατε, δτι (ή) ήμερα κυρίου ως κλέπτης εν νυκτι ούτως έρχεται. η ύ Da Paulus der Gemeinde nichts Neues sagt, .bringt er selbst durch die Worte αυτοί γαρ ακριβώς ρϊδατε zum Ausdruck. Wernle, Anf nge2, 152, und von Dobsch tz in Meyers Kommentar 7. Aufl. 1909 nehmen daher an, da es sich um ein Herrenwort handelt, und W. Br ckner, Entstehung der Paulinischen Chronologie S. 179, vermutet sogar, es mit einem vorchristlichen apokalyptischen Worte zu tun zu haben. Weder das eine noch das andere ist der Fall. Die Bedeutung der Worte ist, je nachdem man von der mosaischen oder griechischen Anschau- ungsweise ausgeht, verschieden. Nach der Vorstellung des mosaischen Rechtes ist das dem Diebe in der Nacht — zu denken ist an einen Einbrecher — wesentliche Merkmal, da er sich nicht scheut, von der Gewalt gegen die Sache zur Gewalt gegen die Person berzu- gehen. Ex. 22, 1. u. 2, Hos. 4,2; Jer 2, 34; Joseph. Antiq. IV 8, 27; Mischna Sanh.VIIIo. Der Einbrecher in der Nacht bedroht nicht nur das Besitztum, sondern auch das Leben. Von der Anschauungsweise des mosaischen Rechtes, wie des ihm hierin gleichartigen r mischen Rechtes (zu vgl. das Zw lftafelgesetz: Cic. pro T ll. 20, 47; 21,50; Dig. 47, 17, L 1; Der Verf., Zeitschr. f. Assyriologie XXVIII S. 337 f.), w rden die Worte des Apostels dahin zu verstehen sein: Der Tag des Herrn kommt mit der Gewalt eines n chtlichen Einbrechers, er kommt mit vernichtender Gewalt. Anders die Auffassung des griechischen Rechtes., Nach ihr ist das dem Diebe wesentliche Merkmal die Heimlichkeit, mit der er zu Werke geht: Plat. leg.IX7 (864); VIII11 (846); 1X12 (874); res publ.IXS G· F rster, l Thessalonicher 5, 1—10. 170 (574); Xenoph. Oicon. 14,5; 20,15; mem. Socr. I 2,62 u. a.; Lipsius, Das Attische Recht und Rechtsverfahren, Leipzig, Reisland, Bd. 2. S. 442. Die Heimlichkeit findet ihren sch rfsten Ausdruck in der zur Nachtzeit begangenen Tat. Horn. II. Ill 10 u. 11; Demosth. Timocr. 113 f (736). Von Dr. jur. Gerh. F rster in Freiberg Sa, Vom Gesichtspunkte des griechischen Rechtes bedeuten die Worte: Der Dieb in der Nacht macht sich, um verborgen zu bleiben, das Dunkel der Nacht zunutze. Wer heimlich kommt, kommt unerwartet. Un- erwartet ist die Wiederkehr des Herrn. Die Worte lassen sich als Herrenwort nur vom Gesichtspunkte des mosaischen Rechtes beurteilen. W rden wir ein Herrenwort vor uns haben, so w rde es Paulus, wenn er nicht Christus mi verstanden h tte, in der Bedeutung gebraucht haben, die der Vorstellung des mosaischen Rechtes entspricht. Paulus hat aber die Worte nicht vom Gesichtspunkte des mosaischen, sondern des griechischen Rechtes ge- braucht. Wie v. l zeigt, spricht er ber die Zeit der Wiederkehr Jesu. Er will darauf hinweisen, da der Tag heimlich, also unerwartet, zu einer nicht voraussehbaren Zeit kommt. Um dies zu verdeutlichen, nicht um die ..Gewalt zu schildern, mit der der Herr kommt, vergleicht er sein Kommen mit dem des Diebes in der Nacht. Wenn Paulus sagt: αυτοί γαρ άκριβακ οϊδατε, will er nicht auf ein Herrenwort, son- dern auf das der Thessalonichergemeinde bekannte, unter dem Einfl sse griechischer Rechtsanschauung entstandene Gleichnis vom Dieb.Bezug nehmen (Mcl3,33f., Mt24,42f.; Lcl2,39f.). Wie der Dieb in der Nacht unerwartet kommt, so kommt auch der Tag des Herrn uner- wartet. Da es sich hierbei um ein von Paulus selbst geschaffenes Gleichnis handelt, l t sich nicht mit Bestimmtheit sagen, ist aber wahrscheinlich. Dem inneren Zusammenhange der Worte des Apostels in v. 2 mit dem Gleichnisse vom Diebe entspricht es, da Paulus seine -Ausf hrungen ber die Wiederkehr Christi in v. 6 mit einem Hinweis auf die durch das Gleichnis vom Dieb veranschaulichten sittlichen Pflichten abschlie t: δρα o v μη καθεύδιυμβν Obc οί λοιποί, άλλα γρηγο- ρώμεν και νήφωμεν. Wir sollen wachen in der Zeit bis zur Wieder- kehr des Herrn (περί των χρόνων), uns h ten vor den Versuchungen der Nacht, die heimlich wie ein Dieb an uns herantreten. Wir sollen aber auch bereit sein auf die Wiederkehr des Herrn (περί των καιρών), damit uns der Herr nicht unvorbereitet trifft. Denn der Herr kommt als Richter der Welt unerwartet, wie das Licht des Tages. Der Zweck des Wachens ist, zu verhindern, da nicht der Dieb kommt, der Grund des Wachens, bereit zu sein auf die Wiederkunft des Herrn. Der Vergleichspunkt ist die Heimlichkeit, mit der der Dieb in der Nacht Brought to you by \| Simon Fraser University Authenticated Download Date \| 5/30/15 11:41 AM G. Von Dr. jur. Gerh. F rster in Freiberg Sa, F rster, l Thessalonicher 5, 1—10. 171 und der Herr am Tage des Gerichts kommen. Das Gemeinsame wird durch den Gegensatz der in Vergleich gestellten Begriffe ge- steigert. Auf der einen Seite der Dieb als das Urbild eines Ver- brechers, auf der ndern Seite der Herr als der Richter ber den Verbrecher. Nacht und Tag. Das B se und die sittliche Vergeltung des B sen. Im Anschlu an diese Gedanken gibt Paulus in v. 3 und 4 ein in k hnen Strichen entworfenes Bild. Die Verse sind in verschiedenen Lesarten erhalten. Von Bedeutung ist die doppelte Lesart in v. 4: κλέπτας auf der einen und κλέπτης auf der ndern Seite. Lesart B. KDFG al. AB boh. v. 3. όταν δε λέγυυαν Ειρήνη και άςφάλεια, τότε αιφνίδιος έφίςταται αύτοΐς όλεθρος ώςπερ ή ώδιν τη εν γαςτρί έχούςη, και ου μη έκφύγωςιν. γαρ statt δε KLP al. om. AK* FG al. Iren. Tert. Cypr. όταν δε λέγυυαν Ειρήνη και άςφάλεια, τότε αιφνίδιος έφίςταται αύτοΐς όλεθρος ώςπερ ή ώδιν τη εν γαςτρί έχούςη, και ου μη έκφύγωςιν. γαρ statt δε KLP al. om. AK* FG al. Iren. Tert. Cypr. ύμεΐς be, αδελφοί, ουκ έςτέ εν ςκότει, ίνα ή ήμερα υμάς ως κλέπ- τας καταλάβη. . ύμεΐς δε, αδελφοί, ουκ έςτέ εν ςκότει, ίνα ή ήμερα υμάς ως κλέπ- της καταλάβη. KDFG al. Lesart A. V. 3 und 4 sind nicht Nachs tze zu v. 2. Sie wollen nicht v. 2, sondern wie dieser Vers v. l begr nden. Auf die Frage des Apostels, warum er es nicht n tig habe, ber die Zeit der Wiederkehr Christi zu schreiben, gibt er die Antwort: Weil die Thessalonicher nicht, wie die in v. 3 Geschilderten, in der Finsternis leben. Unter Finsternis ver- steht er die dem Bereiche Gottes entgegengesetzte Macht des B sen ή έΞουςία του ςκότους. So bei Lc 22,53 und Kol l, 13; 2 Kor 6,14:... ή τις κοινωνία φιυτι προς ςκότος. In der Finsternis herrschen die b sen Geister der Welt, Eph 6,12. In ihr leben die S nder, Eph5,8. Der Bedeutung des Wortes entspricht es, da mit ihm bestimmte dem Leben entnommene Vorstellungen verbunden werden. Die Nacht ist die Zeit des Schlafes (!Thess5,7), der Schlaf als Sinnbild der Schw che und des Todes gedacht (Rom 13,11; l Kor 11,30; 15,20). Sie ist aber auch die Zeit ppiger Gelage (Rom 13,13; IThessS,?) und b ser Werke (Rom 13,12; Eph 5,11). Alles, was schandbar ist, verbirgt sich im Dunkel der Nacht (2 Kor 4,2; Eph 5,12). In ihr ge- schieht Verrat (l Kor 11, 23; Lc 22, 53; Joh 13,30) und unter ihrem Sch tze arbeitet der Dieb (Mcl3,33f.; Mt24,42f.; Lei2,39f.). G· F rster, l Thessalonicher 5, 1—10. 172 Als Kinder der Nacht f hrt der Apostel in v. 3 eine Diebesbande oder Schlafende, Schwelgende und Trunkene vor Augen. Sie berf llt in ihrer Sorglosigkeit das Verderben. Auf das doppelte Bild, von dem sich Lc21,34 und 35 eine Nachbildung findet, nimmt Paulus im fol- genden Bezug, auf das Bild von den beim n chtlichen Einbruch be- troffenen Dieben in v. 4 und 5 und auf das Bild von den Schlaftrun- kenen und den zum n chtlichen Mahle Vereinten in v. 6 und 7. Der, der die 'Diebesbande in ihrer Sorglosigkeit berrascht und er- greift, ist ή ημέρα κυρίου. Aus der Gegen berstellung der Worte ήμερα und CKOTOC in v. 4 und aus den Worten in v. 5: ύμεΐο υιοί φωτόο ecre και υιοί ήμέραο einerseits und ουκ έςμεν νυκτόο ουδέ οκότουο anderer- seits geht hervor, da ήμερα als das Licht des Tages im Gegensatz zur Finsternis der Nacht gebraucht wird. CH ήμερα κυρίου ist das Licht am Tage des Herrn. Der Tag macht eines jeden Werke offenbar (l Kor 3,13; Eph 5,13). Lesart A. Ι 91 S. 113: οι μεν έπ' αύτοφώρψ Aovrec, εάν μεν όμολογώα, παραχρήμα θανάτψ Ζημιουνται. Plat. leg. IX 12 (874Β), Xenoph. oecon. 14, 5. Der b sen Tat soll unmittelbar die Strafe folgen. Wer aus Bosheit und Feigheit sein Tun der ffentlichkeit entzieht, geht des regelrechten Strafverfahrens verlustig. Das Verfahren, das bei ihm Platz greift, ist ein abgek rztes. An Stelle der Ladung und Vorf h- rung durch die ffentlichen Organe tritt die Apagoge. Wer ein Ver- brechen entdeckt, ist berechtigt, als Ankl ger und Zeuge den T ter sofort vor Gericht zu bringen; er kann zur Durchf hrung dieser Auf- gabe zu jeder erforderlichen Ma nahme greifen. Sucht der T ter zu entfliehen, so kann er get tet werden. Der, der ihn ergreift, ist dann nicht nur Ankl ger und Zeuge, er ist zugleich Richter. Er f llt und vollstreckt das Urteil unmittelbar nach der Tat. Das Licht der Sonne, das das, was die Nacht verborgen hat, offenbart und den Tag zu der Zeit macht, zu der ber den Verbrecher der Nacht das Urteil ge- sprochen und vollstreckt wird, vollzieht bei Paulus die Apagoge. Es berrascht und· ergreift den Verbrecher und bringt ihn zu seiner so- fortigen Aburteilung. dem Apostel die Apagoge des griechischen Rechts vor Augen. Bei den aus κακουργία begangenen Verbrechen, insbesondere dem Dieb- stahle in der Nacht, kann der auf frischer Tat betroffene T ter (έπ' αυτοφώρω) sofort vor die Beh rde (ο! ένδεκα) abgef hrt und, wenn er gest ndig ist, abgeurteilt werden (Lipsius, Das att. Recht u. Rechtsverf., Bd. l S.78, B.2 S.317f.).· Demosth. Timocr. 113f. (735f.): o CoXuuv... νόμον €ΐ€ήν€γκ€ν, ei μεν TIC μεθ' ήμέραν υπέρ πεντήκοντα δραχμάο κλέπτοι, άπαγωγήν πρόο τούο έ'νδεκ' είναι, εί be TIC νύκτωρ ότιουν κλέπ- τοι, τούτον έΗεΐναι και άποκτεΐναι και πρώοαι διώκοντα και άπαγαγεΐν TOIC ένδεκ', εί βούλοιτο'^τώ δ'άλόντι ων αί άπαγωγαί eiciv, ουκ έγγυητόκ KaracrrjcavTi £κτιαν είναι των κλεμμάτιυν, άλλα θάνατον την ίημίαν. Aesch. Ι 91 S. 113: οι μεν έπ' αύτοφώρψ Aovrec, εάν μεν όμολογώα, παραχρήμα θανάτψ Ζημιουνται. Plat. leg. IX 12 (874Β), Xenoph. oecon. 14, 5. Der b sen Tat soll unmittelbar die Strafe folgen. Wer aus Bosheit und Feigheit sein Tun der ffentlichkeit entzieht, geht des regelrechten Strafverfahrens verlustig. Das Verfahren, das bei ihm Platz greift, ist ein abgek rztes. An Stelle der Ladung und Vorf h- rung durch die ffentlichen Organe tritt die Apagoge. Lesart A. Er bringt an das Licht, was sich im Dunkel der Nacht verborgen h lt (l Kor 4,5). Der Tag ist aber auch die Zeit des ffentlichen Lebens. An ihm stehen die Gerichtsh fe offen und die H ter des Gesetzes wachen. Philo, de spec. leg. IV (2) 9 p. 337 Cohn: Μεθ' ήμέραν μέντοι άναπέπταται δικαστήρια και βουλευτήρια, έπλήθυνε δε των ουλληψομένων ή πολιό, ων οι μεν φύλακεο των νόμων κεχειροτόνηνται, οι δε άνευ χειροτονίαο, μιοοπονήριμ πάθει την υπέρ των ήδικημένυυν αύτοκέλευοτοι τάξιν αίρουνται, προο oOc τον κλέπτην · άκτέον. Am Tage kann man den Schuldigen zur Rechenschaft ziehen, es wird Gericht gehalten und die Strafe vollstreckt. Senec. de ira III, 19: quid tarn inauditum quam nocturnum supplicium? cum latrocinia tenebris abscondi soleant, animadversiones, quo notiores sunt, plus in exemplum emendationemque proficiant. Plat. Phaed. p. 61 e und 116e (vgl. dagegen Herod. IV 146 und Val Max. IV 6 ext. 3); Deut 2.1,23. Nach Mischna Sanh.IVl mu bei Kriminalprozessen am Tage verhandelt und Beschlu gefa t, und nach Sanh. VI l im unmittel- baren Anschlu daran das auf Steinigung lautende Urteil vollstreckt werden. Im Orient ist der Wechsel von Nacht und Tag ein j her. Wie die Finsternis der Nacht den Wanderer berrascht (Horn. 11.24,366: νύ£ θοή; Job 12,35), so berf llt (aiqpvibioc έφίαταται) das Licht des Tages den Dieb beim n chtlichen Handwerk. Es ergreift ihn (κατα- λαμβάνειν), richtet ihn (Rom 2,16) und straft ihn (Eph 5,13; Joh3,20). Wie den vom Tageslicht betroffenen Dieben soll es am j ngsten Tage denen ergehen, die noch im Dunkel der Nacht leben. g Bei den Worten ϊνα ή ήμερα ύμάκ be κλέιττας καταλάβη schwebt G. F rster, l Thessalon icher 5, l—10. 173 dem Apostel die Apagoge des griechischen Rechts vor Augen. Bei den aus κακουργία begangenen Verbrechen, insbesondere dem Dieb- stahle in der Nacht, kann der auf frischer Tat betroffene T ter (έπ' αυτοφώρω) sofort vor die Beh rde (ο! ένδεκα) abgef hrt und, wenn er gest ndig ist, abgeurteilt werden (Lipsius, Das att. Recht u. Rechtsverf., Bd. l S.78, B.2 S.317f.).· Demosth. Timocr. 113f. (735f.): o CoXuuv... νόμον €ΐ€ήν€γκ€ν, ei μεν TIC μεθ' ήμέραν υπέρ πεντήκοντα δραχμάο κλέπτοι, άπαγωγήν πρόο τούο έ'νδεκ' είναι, εί be TIC νύκτωρ ότιουν κλέπ- τοι, τούτον έΗεΐναι και άποκτεΐναι και πρώοαι διώκοντα και άπαγαγεΐν TOIC ένδεκ', εί βούλοιτο'^τώ δ'άλόντι ων αί άπαγωγαί eiciv, ουκ έγγυητόκ KaracrrjcavTi £κτιαν είναι των κλεμμάτιυν, άλλα θάνατον την ίημίαν. Aesch. Lesart A. Wer ein Ver- brechen entdeckt, ist berechtigt, als Ankl ger und Zeuge den T ter sofort vor Gericht zu bringen; er kann zur Durchf hrung dieser Auf- gabe zu jeder erforderlichen Ma nahme greifen. Sucht der T ter zu entfliehen, so kann er get tet werden. Der, der ihn ergreift, ist dann nicht nur Ankl ger und Zeuge, er ist zugleich Richter. Er f llt und vollstreckt das Urteil unmittelbar nach der Tat. Das Licht der Sonne, das das, was die Nacht verborgen hat, offenbart und den Tag zu der Zeit macht, zu der ber den Verbrecher der Nacht das Urteil ge- sprochen und vollstreckt wird, vollzieht bei Paulus die Apagoge. Es berrascht und· ergreift den Verbrecher und bringt ihn zu seiner so- fortigen Aburteilung. Hat der Apostel in v. 3 bis 5 die in v. l aufgestellte Behauptung erl utert, da er nicht n tig habe, ber die Zeit der Wiederkehr Christi zu schreiben, so gibt er in v. 6 f. als Ergebnis dieser Betrachtung die f r das Leben der Gl ubigen praktische Bedeutung der Lehre von der Parusie. Die, die nicht mehr in der Finsternis der Nacht leben, son- dern zu denen das Licht des Tages gekommen ist (Rom 13,12; l Job 2, 8), sollen aufwachen (Rom 13,11) und, wie vom Schlafe, so von den Toten auferstehen (Eph 5,14). Sie sollen ablassen von den Werken der Finsternis (Rom 13,12), ehrbar wandeln (Rom 13,13; Johl2,35) und n chtern sein. Sie sollen als B nder des Lichtes in der Welt der Finsternis scheinen (Phil 2,15). 174 G· F rster, l Thessalonicher 5, 1—10. Im Gegensatze zu v. 3, in dem die Gottlosen als eine im Dunkel der Nacht arbeitende Verbrecherbande, und zu v. 6 und 7, wo sie als zu einem ppigen Nachtgelage Vereinigte oder vom Schlafe Befallene geschildert werden, f hrt der Apostel in v. 8 die Gl ubigen als Krieger mit Panzer und Helm .vor Augen, die fest und voll Vertrauen Aus- schau halten nach dem Feind: Das von der Kirche festgehaltene Bild der militia Christi. Diese Krieger sind Kinder des Tages, die mit Waffen des Lichtes (Rom 13,12) gegen die b sen Geister, die in der Finsternis dieser Welt herrschen (Eph 6,12), k mpfen und ihre un- fruchtbaren Werke strafen (Eph 5,· 11). Lesart B. Ein anderes Bild gibt Lesart B. V. 3, hier nicht von v. l son- dern von v. 2 abh ngig, soll den Gedanken in v. 2 durch ein Bild erkl ren. Inwiefern wird der Tag des Herrn wie ein Dieb in der Nacht kommen? Er wird pl tzlich wie ber die Sorglosen in v. 3 kommen. Diese glauben, es sei Frieden. Sie vergessen die Gefahr vor n chtlichen Dieben. Da berrascht sie ein berfall. Sie k nnen nicht entfliehen und werden von dem als Dieb vorgestellten Tag des Herrn ergriffen. W hrend die Lesart A in v. 2, wie im Gleichnisse vom Dieb, das unerwartete Kommen des j ngsten Gerichteis mit dem heimlichen Nahen des Diebes vergleicht und gleichzeitig den Dieb in der Nacht als typisches Beispiel des Schlechten in Gegensatz zu Christus als den Richter in der Lichtf lle des Tages bringt, wird in der Lesart B nicht nur das Kommen des Herrn, sondern der Herr selbst mit dem Diebe in Vergleich gebracht. Der Dieb ist nicht mehr typisches Beispiel des Schlechten, sondern Bild der vernichtenden Gewalt des Herrn am Tage des Gerichts. Sowohl der Gegensatz des Diebes als Beispiel eines Verbrechers und des Herrn als Beispiel des Richters wie der Gegen- satz der Nacht als die Zeit des Verbrechens und des Tages als die Zeit des Gerichts schwindet. Das Bild selbst wird in seiner nat r- lichen Einfachheit zerst rt. Vom Tag (ήμερα) wird gesagt, da er die Pflichtvergessenen ergreift, wie ein Dieb, der doch gerade nicht am Tage zu Werke zu gehen pflegt. Ein Dieb soll mehrere ergreifen! Die das Bild abschlie enden Worte in v. 5: πάντ€€ γαρ ύμεις υιοί cpurroc dcre και υιοί ήμέραο werden zu einer gehaltlosen Redensart. Die Lesart A, die auf griechische Rechtsanschauung zur ckgeht, ist paulinisch, die Lesart B eine Umarbeitung unter dem Einfl sse des Brought to you by \| Simon Fraser University Authenticated Download Date \| 5/30/15 11:41 AM G. F rster, l Thessalonicher 5, 1—10. 175 mosaischen, vielleicht auch des mit ihm bereinstimmenden r mischen Rechtes. Wie in l Thess 5, 4 macht sich in den hiervon abh ngigen Stellen die mosaisch-r mische Auffassung geltend. g g In 2 Pt 3,10 hei t es: ή£ει δε ήμερα κυρίου ως κλέπτης . . . Der t. r. (nach C31 KLal.) f gt dem κλέπτης das εν νυκτί bei. Aus den der Stelle in v. Lesart B. 8 vorausgehenden Worten: δτι μία ήμερα παρά'κυρίψ ως χίλια ?τη και χίλια Ιτη ως ήμερα μία und aus der in v. 9 gegebenen Begr ndung des Verzugs der Wiederkunft Christi geht hervor, da der Petrusbrief einen Hinweis auf die Zeit der Wiederkunft geben will, da das Bild also auch hier nicht anders wie in l Thess 5, 2 verstanden werden kann. Wenn aber dann im unmittelbaren Anschlu an das Bild in v. 10 fortgefahren wird: εν rj οι ουρανοί ροιζηδόν παρελεύςονται, ςτοιχεΐα δε καυςούμενα λυθήςεται και γη και τα εν αυτή ?ργα εύρεθήςεται, so entspricht-diese Schilderung von dem allgemeinen Verderben und dem Untergange der irdischen Welt am Tage der Parusie nicht der hellenistischen, sondern der mosaisch-r mischen Vorstellung von der Gewalt, mit der der Dieb in der Nacht kommt. In hnlicher Weise vermischen sich beide Vorstellungen bei dem in der Offenbarung Johannis wiederkehrenden Bilde. Apc 3, 3 lautet: εάν ουν μη γρηγορήςης ή&υ ως κλέπτης, και ου μη γνψς, ποίαν ώραν ή£ιυ επί ςέ. Nach K u. a. sind auch hinter γρηγορήςης, ή£ιυ die Worte επί ςέ einzuf gen. Die Worte εάν ουν μη γρηγορήςης und και ου μη γνψς weisen auf den Ursprung des Bildes. Die Vorstellung von der Ge- walt des Diebes kommt dagegen in dem ή£ιυ επί ςέ zum Ausdruck. Sein Kommen bedroht nicht nur das Verm gen, sondern auch die Person des im Petrusbrief Angeredeten. Der Dieb kommt mit einer alles ver- nichtenden Gewalt. Apc 16,15 gibt eine sich an die Worte des vorausgehenden Verses ςυναγαγεΐν αυτούς είς τον πόλεμον της μεγάλης ημέρας του θεού του παντοκράτορος lose anschlie ende, urspr nglich nicht zum Texte ge- h rige Einschiebung. Es hei t: ιδού έρχομαι ως κλέπτης· μακάριος 6 γρήγορων και τηρών τα ιμάτια αυτού, ίνα μη γυμνός περιπατή και βλέ- πιυςιν την άςχημοςύνην αυτού. Wie in Apc 3, 3 wird auch hier mit dem Diebe nicht ή ήμερα κυρίου, sondern ό κύριος selbst verglichen. Ό κύριος ist aber als der παντοκράτωρ angef hrt. Dementsprechend ist bei den Worten ίνα μη γυμνός περιπατή και βλέπιυςιν την άςχη- μοςύνην αυτού nicht an einen heimlich Bestohlenen, sondern seines ganzen Hab und Gutes, ja seiner Kleider am Leibe Beraubten zu denken. — G· F rster, l Thessalon icher 5, 1—10. 176 Der Unterschied der Lesart B von der Lesart A liegt nicht nur auf dem Gebiete des Rechtes, sondern auch auf dem der Theologie. Lesart B. Wenn Paulus die Wiederkehr des Herrn mit den ersten Strahlen des erwachenden Tages vergleicht, so ist das nicht nur Ausflu poetischen Empfindens. Paulus geht mit dem Vergleiche zur ck auf bestimmte, der gesamten Kulturwelt der damaligen Zeit gel ufige Vorstellungen. Die Sonne ist durch die Fortschritte astronomischer Wissenschaft als das den gesamten gestirnten Himmel beherrschende Gestirn erkannt. Aristarch; Cic. Somn. Scip. c. 4: sol dux et princeps et moderator lu- minum reliquorum, mens mundi et temperatio. Tuscul. I 28, 68; Plin. HN II 6 § 12; Julian von Laodicea, Cat. codd. astr. I S. 136, Z. 1. Im Lichte der Sonne sieht man die siegreiche Kraft des Guten ber die d monischen M chte der Finsternis. Zu vgl. das von Windischmann bersetzte Opfergebet Mihir Yasht III 11; IV 13; XII 49, 50; XVI 65; XXVIII 112,113; Cumont-Gehrich, Die Mysterien des Mithra, Leipzig 1911; Apollon λύκειος έκάεργος έκηβόλοο, άκέαοο, αυτήρ usw. Das Licht der Sonne ist rein. Joseph. Bell. Jud. II 8, 5, 9. Die griechische Philosophie fa t es als das stets gegenw rtige Bild des unsichtbaren Wesens (numen praesens oder praesentissimum CIL. XIV 3567; VII, 481; cf. CIG 2635, 5), Philo, de opific. mundi (6) 25, (8) 31, (17) 53 (Cohn), als die sich in die Sinnenwelt umsetzende g ttliche Vernunft. In der durch den Planetenkult verbreiteten Lehre vom Aufstieg der Seele erhebt das dem Auge wahrnehmbare Licht der Gestirne ber alles Irdisch-Beschr nkte und f hrt zur Erkenntnis der die Welt be- herrschenden Gesetze. Plat, res publ. X 616; Pseudo-Arist., περί κόομου c.5f.; Philo, de Abrahamo, (15)70, de opific. mundi (17) 54; (18) 55; (23) 70; de spec. leg. III (34) 187 (Cohn); Cumont-Gehrich, Die orient. Religionen i. r m. Heidentum, Leipzig 1910, S. 191 f., S. 312 f. Paulus will durch den Vergleich des Herrn mit dem Lichte des Tages nicht philosophische oder mythologische Lehren in das Christen- tum hineintragen, sondern durch die allgemein mit dem Lichte ver- bundenen Vorstellungen das andeuten, was er ber die Art der Wieder- kehr Christi zu sagen wei . Christus kommt nicht als Herr des Volkes Israel, sondern als der Herr der gesamten Welt, er kommt nicht in k rperlicher Gestalt, sondern rein - geistig als die siegreiche Kraft des Guten ber das B se. Die Kirche erh lt sich nicht auf der H he paultnischer Lehre. Lesart B. Der Vergleich des Herrn mit dem Lichte der Sonne erscheint in einer Zeit des Kampfes gegen den Mithrasdienst bedenklich. J dische Vorstel- G. F rster, l Thessalonicher δ, l—10. 177 langen von einer sinnenf lligen Wiederkehr des Messias gewinnen die Oberhand. Ihnen tr gt die Lesart B Rechnung. Befreien wir die paulinische Stelle von der Verunstaltung einer j ngeren Zeit. Lesen wir ίνα ή ήμερα ύμαο ωό κλέπταο καταλάβη und nicht . . . tue κλέπτης καταλάβη. Die sich am Morgen erhebende Sonne sei uns ein Zeichen der Wiederkehr Christi am Tage des Ge- richts, ihr Licht ein sich t glich erneuerndes Sinnbild der siegenden Kraft des Guten und der die Welt beherrschenden ausgleichenden Ge- rechtigkeit,
https://openalex.org/W4232209212	https://www.qeios.com/read/AK2DL7/pdf	English	null	Natural Log Transformed Area Under the Curve From Dosing to Last Concentration	Definitions	2,020	cc-by	66	Natural Log Transformed Area Under the Curve From Dosing to Last Concentration National Cancer Institute Qeios ID: AK2DL7 · https://doi.org/10.32388/AK2DL7 Qeios · Definition, February 2, 2020 Source National Cancer Institute. Natural Log Transformed Area Under the Curve From Dosing to Last Concentration. NCI Thesaurus. Code C161414. The natural log transformed area under the curve (AUC) from the time of dosing to the last measurable concentration. 1/1
https://openalex.org/W3166111610	https://www.repository.cam.ac.uk/bitstream/1810/323387/2/cells-10-01365.pdf	English	null	Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis	Cells	2,021	cc-by	21,342	cells cells Citation: Agarwal, N.; Mak, C.; Bojanic, C.; To, K.; Khan, W. Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis. Cells 2021, 10, 1365. https://doi.org/ 10.3390/cells10061365 Keywords: osteoarthritis; degenerative changes; knee; adipose tissue; mesenchymal stem cells; stromal vascular factor y Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis https://doi.org/ 10.3390/cells10061365 Academic Editors: Kunlin Jin, Huanxing Su and Guo-Yuan Yang Received: 13 April 2021 Accepted: 28 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4 0/) Citation: Agarwal, N.; Mak, C.; Bojanic, C.; To, K.; Khan, W. Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis. Cells 2021, 10, 1365. https://doi.org/ 10.3390/cells10061365 Academic Editors: Kunlin Jin, Huanxing Su and Guo-Yuan Yang Received: 13 April 2021 Accepted: 28 May 2021 Published: 1 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Citation: Agarwal, N.; Mak, C.; Bojanic, C.; To, K.; Khan, W. Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis. Cells 2021, 10, 1365. https://doi.org/ 10.3390/cells10061365 y Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis wal 1 , Christopher Mak 2 , Christine Bojanic 2, Kendrick To 2 and Wasim Khan 2,* Nikhil Agarwal 1 , Christopher Mak 2 , Christine Bojanic 2, Kendrick To 2 and Wasim Kh 1 MBChB Ofﬁce, University of Aberdeen College of Life Sciences and Medicine, Foresterhill Rd, Aberdeen AB25 2ZD UK; nikagarwal@live co uk Aberdeen AB25 2ZD, UK; nikagarwal@live.co.uk 2 Division of Trauma & Orthopaedic Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK; chcm2@cam.ac.uk (C.M.); cbojanic@doctors.org.uk (C.B.); kendrick.to@doctors.org.uk (K.T.) , ; g 2 Division of Trauma & Orthopaedic Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK; chcm2@cam.ac.uk (C.M.); cbojanic@doctors.org.uk (C.B.); kendrick.to@doctors.org.uk (K.T.) g 2 Division of Trauma & Orthopaedic Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK; chcm2@cam.ac.uk (C.M.); cbojanic@doctors.org.uk (C.B.); kendrick.to@doctors.org.uk (K.T.) g * Correspondence: wasimkhan@doctors.org.uk Abstract: Osteoarthritis (OA) is a degenerative disorder associated with cartilage loss and is a leading cause of disability around the world. In old age, the capacity of cartilage to regenerate is diminished. With an aging population, the burden of OA is set to rise. Currently, there is no deﬁnitive treatment for OA. However, cell-based therapies derived from adipose tissue are promising. A PRISMA systematic review was conducted employing four databases (MEDLINE, EMBASE, Cochrane, Web of Science) to identify all clinical studies that utilized adipose tissue derived mesenchymal stem cells (AMSCs) or stromal vascular fraction (SVF) for the treatment of knee OA. Eighteen studies were included, which met the inclusion criteria. Meta-analyses were conducted on fourteen of these studies, which all documented WOMAC scores after the administration of AMSCs. Pooled analysis revealed that cell-based treatments deﬁnitively improve WOMAC scores, post treatment. These improvements increased with time. The studies in this meta-analysis have established the safety and efﬁcacy of both AMSC therapy and SVF therapy for knee OA in old adults and show that they reduce pain and improve knee function in symptomatic knee OA suggesting that they may be effective therapies to improve mobility in an aging population. Citation: Agarwal, N.; Mak, C.; Bojanic, C.; To, K.; Khan, W. Meta-Analysis of Adipose Tissue Derived Cell-Based Therapy for the Treatment of Knee Osteoarthritis. Cells 2021, 10, 1365. 1.1. The Burden of Osteoarthritis Osteoarthritis (OA) is a progressive degenerative joint disorder associated with aging. It is a leading cause of disability around the world. In 2019, the Global Burden of Disease Study reported that musculoskeletal disorders account for over 5% of worldwide disability adjusted life years (DALY) [1]. The World Health Organization (WHO) estimate that approximately 10% of all men and 18% of all women aged over 60 have OA [2]. Out of these individuals, they estimate that 80% have limitations in movement and 25% cannot perform major daily activities of life [2]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. p j y In addition to physical symptoms, there is evidence to suggest that OA is associated with mental health problems as well. A longitudinal cohort study, conducted by the Osteoarthritis Initiative, found that there was a greater risk of developing depressive symptoms in patients with hip or knee OA than those without [3]. Another observational study found that OA was associated with 1.27 times increase odds of suicidal ideation [4]. There is also evidence that OA increases the risk for myocardial infarction, with one meta-analysis reporting a 1.31 times increased risk for myocardial infarction [5]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). These, among many other studies have highlighted the burden OA has on the indi- vidual. In addition to this, OA carries signiﬁcant economic burden on societies across the world. When adjusted for age, sufferers are shown to be at high risk of sick leave and https://www.mdpi.com/journal/cells Cells 2021, 10, 1365. https://doi.org/10.3390/cells10061365 Cells 2021, 10, 1365 2 of 31 disability pension due to knee OA. This was especially the case for those working in health care, childcare and in cleaning [6]. In the United States, annual healthcare costs from OA exceed $45 billion [7]. In France, annual costs from OA can be as high as €2 billion [8]. In Spain costs may be as high as €4.738 billion annually [9]. A signiﬁcant proportion of these costs are associated with joint replacement surgeries. j p g Economic, societal, and individual burdens caused by OA are set to rise. 1.1. The Burden of Osteoarthritis The preva- lence of OA is increasing because of an aging population and an increased incidence of obesity. The United Nations estimates that by 2050, 1 in 6 people in the world will be over 65 years of age [10]. As such incidence rates of OA will naturally also increase. Several projection studies have been performed in different countries. In Australia, the number of people with OA is estimated to increase from 2.2 million in 2015, to 3.1 million by 2030 [11]. In Sweden it is estimated that from between 2012 and 2032, the percentage of people aged over 45 with OA will rise from 26.6% to 29.5% [12]. In the United States, this number is set to grow from 47.8 million in 2005 to 67 million by 2030 [13]. These ﬁgures show there is a global rising prevalence of this disease. Coupled with the debilitating nature of OA, this must be addressed before the disease overburdens healthcare systems worldwide. There is currently no cure to prevent or slow the progression of OA. Presently, it is ﬁrst managed via conservative means through exercises, weight loss and occupational therapy. When this is inadequate, paracetamol and non-steroidal anti-inﬂammatory drugs (NSAIDs) are used for symptom control [14]. Intra articular corticosteroid injections are then used if the aforementioned therapies do not provide relief. For end-stage OA, joint replacement surgery (total knee arthroplasty) is the gold standard of treatment. Despite being a highly successful operation, joint replacement surgery carries signiﬁcant risk, and nearly one in ﬁve knee replacements will not last beyond 25 years [15]. OA pathogenesis is predominantly driven by inﬂammatory mediators such as in- terleukin 1 (IL-1) and tumour necrosis factor alpha (TNF-α) [16]. Both are present in the synovial ﬂuid of patients with OA [17,18]. IL-1 has been shown to encourage the produc- tion of molecules such as nitric oxide, cytokines and prostaglandin E2 [19]. Furthermore, these inﬂammatory molecules promote the release of matrix metalloproteinases. These encourage the catabolism of articular cartilage [20]. This process of cartilage catabolism and loss is associated with aging, [21] alongside the diminishing ability of cartilage to repair itself [22]. While these mechanisms are central to cartilage depletion and eventually OA and pain, blockade of these mediators have failed to demonstrate efﬁcacy in clinical trials [23,24]. 1.2. Mesenchymal Stem Cells MSCs are becoming increasingly popular in tissue engineering due to their multipotent potential to differentiate into different lineages of mesenchymal tissue types [25]. These include bone, fat, cartilage, tendon, and muscle. MSCs are of interest as an OA therapy due to their immunoregulatory function and potential to repair cartilage. This is particularly useful as OA predominantly affects individuals in old age, who have limited ability to repair cartilage. MSC-based therapies can result in promotion of macrophage polarization from an M1 to M2 phenotype [20,26,27]. This enables macrophages in the cartilage to display anti-inﬂammatory properties which leads to down-regulation of the inﬂammatory milieu mentioned above in their role in triggering and sustaining osteoarthritic changes [28]. MSCs have also been shown to be capable of suppressing T-cell proliferation. MSCs do not express HLA class II on their surface and only express low levels of HLA class I and have demonstrated safety and low immunogenicity through various routes of administra- tion [29–31]. Although laboratory studies have suggested that MSCs, both intrinsic and transplanted may promote cancer cell activity, in-human clinical trials of transplantation have yet to show evidence of carcinogenic effect [32,33]. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy recommends a minimal criterion to deﬁne human MSCs. There are three Cells 2021, 10, 1365 3 of 31 elements to these standards. Firstly, MSCs must be adherent to plastic when they are maintained in standard culture conditions. Secondly, MSCs must express the following markers: CD105, CD73 and CD90. In addition, they must not express the following: CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Thirdly MSCs must be able to differentiate into the following cell types in vitro: osteoblasts, adipocytes and chondroblasts [34]. MSCs can be derived from a variety of locations including skeletal muscle, syn- ovium [35,36] and periosteum [37,38]. However, the most popular cell sources for MSC harvest, are bone marrow [39–42] and adipose tissue [43,44]. MSCs were ﬁrst isolated from bone marrow before any other source [45,46] and bone marrow derived MSCs remain one of the top choices for MSCs due to their high cell-yield and proliferative capacity in vitro [47]. However, despite its advantages, extraction of bone marrow to acquire MSCs for autologous use is a highly invasive and painful procedure that can cause long term pain at the donor site. Thus, such a procedure is not always ideal. 1.3. Adipose Tissue Derived MSCs The most common harvest location for Adipose tissue derived MSCs (AMSCs) is the abdomen due to the high tissue fat content. The harvesting process by which AMSCs are collected have been described extensively in the past [44–50]. AMSCs have a greater regenerative proﬁle than bone marrow derived MSCs [51]. AMSCs were also found to promote greater neovascularisation, display greater resistance to hypoxia induced apoptosis and higher telomerase activity [52]. Unlike bone marrow derived MSCs, which lose differentiation capacity with age, AMSCs do not [53]. AM- SCs maintain their chondrogenic potential and their expansion properties [54]. This is very important to consider in MSC therapies targeted for OA, since these are directed at older patients. p AMSCs have been found to have greater anti-inﬂammatory properties compared to bone marrow derived MSCs and produce much higher levels of IL-1 receptor antagonist and tissue protective protein tumour-necrosis factor stimulated gene-6 (TSG-6) [55]. When assessed in their role for OA, AMSCs were able to adapt the environment and exerted anti-inﬂammatory effects on chondrocytes and synoviocytes via prostaglandin E2 [56]. The AMSCs caused polarization of Mo non-polarized macrophages and mature dendritic cells, towards anti-inﬂammatory and phagocytic phenotypes [57]. Adipose tissue derived stem cells can also be derived from the stromal vascular fraction (SVF) which has the advantage of greater ease of harvest. However, these cells are not plated to select for cells which are plastic adherent [58,59]. As such, cells from the SVF cannot strictly be considered MSCs according to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy criteria [34]. The aim of this systematic review and meta-analysis was to determine the effectiveness of AMSCs and SVF for the use of treatment in osteoarthritis. 1.2. Mesenchymal Stem Cells Hence, other sources of MSCs have been sought out, the most popular being adipose tissue derived MSCs. These are accessible as a surgical waste tissue and are associated with lower donor site morbidity than bone marrow [48]. The low rejection rates coupled with the anti-inﬂammatory properties of MSCs makes them an appealing therapeutic solution for OA. 2. Materials and Methods 2.1. Database and Inclusion Criteria 2.2. Quality Assessment Each study was critically appraised to ensure relevance. Studies were appraised by two independent reviewers using either the Risk of Bias in Nonrandomised Studies 1 (ROBINS-1) or the Risk of Bias 2 (RoB2) for randomised studies tools. Upon completion of the appraisal, data was stratiﬁed according to the tools used and was collated into tables (Supplementary Tables S1 and S2). Any uncertainty was solved through discussion between the reviewers. 2.1. Database and Inclusion Criteria A systematic review was conducted, based on the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [60]. Using the PICOS model, (patient, intervention, control, outcome, study), inclusion and exclusion criteria were created. A comprehensive literature search was conducted using four databases: Medline (1946 to 4 June 2020), EMBASE (1974 to 29 June 2020), Cochrane library (1946 to June 2020), and Web of Science (1900 to 2020). Cells 2021, 10, 1365 4 of 31 The following was used as inclusion criteria for all studies screened: The following was used as inclusion criteria for all studies screened: 1. Any studies which investigated use of AMSCs on humans for the treatment of knee joint osteoarthritis 2. Any study that included the use of stromal vascular fraction (SVF) or microfragmented adipose tissue 3. Any study which was clinical in nature Consequently, the following was used as exclusion criteria for all studies screened. Consequently, the following was used as exclusion criteria for all studies screened. 1. Any study not conducted on human 2. Studies which investigated use of MSCs which were not of adipose origin y 4. Studies in which the data sets were either incomplete or inaccessible such as confer- ence abstracts and ongoing randomised controlled trials (RCTs) 4. Studies in which the data sets were either incomplete or inaccessible such as confer- ence abstracts and ongoing randomised controlled trials (RCTs) A search strategy was created, on the basis of the Cochrane Highly Sensitive Search Strategy. This included but was not limited to the following terms: ‘mesenchymal’, or ‘stem cell’ and ‘osteoarthritis, knee’ and ‘adipose’. Full search strings can be found in the Appendix A. Restrictions were applied to the search to only include studies conducted on humans and in the English language. Study selection was carried out by two reviewers independently. The titles of the articles were reviewed for relevance. Abstracts were then screened to check if they met inclusion criteria. Full-text manuscripts were then retrieved and analysed. A manual search was also performed on associated review articles to identify any articles that could have been missed by the search. The combined results of the comprehensive search strategy are shown in Figure 1. 2.3. Statistical Analysis Pre and post treatment Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were extracted from each study for each of the follow-up times. Percentage changes in these scores were then calculated for each follow-up time. Forest plots were created for each of the follow-up time periods. A summary forest plot was created to determine the overall statistical signiﬁcance of treatment on WOMAC scores. We sought to elucidate whether the pooled effect of treatment resulted in minimum clinically important differences in WOMAC scores, which has been deﬁned as 12, [61] to this end, we do not compare this against the various control groups used across the studies. All statistical analysis was conducted on R software through the ‘metafor’ package. The I2 test was used to test for heterogeneity. To account for heterogeneity (all-cause) between studies, assuming effects assuming that effects between studies are either similar or not, Fixed effect models were used for analyses with I2 < 25%. Random effect models were used for analyses with I2 > 25%. All WOMAC percentage changes were calculated with a 95% conﬁdence interval. 5 of 31 Cells 2021, 10, 1365 Cells 2021 10 x Figure 1. Overview of the screening and selection process of studies for the systematic review. Figure 1. Overview of the screening and selection process of studies for the systematic review. 3 R lt Figure 1. Overview of the screening and selection process of studies for the systematic review. gure 1. Overview of the screening and selection process of studies for the systematic review. Figure 1. Overview of the screening and selection process of studies for the systematic review. 3. Results gure 1. Overv 3. Results 2. Quality Assessment Each study was critically appraised to ensure relevance. Studies were appraised by wo independent reviewers using either the Risk of Bias in Nonrandomised Studies 1 ROBINS-1) or the Risk of Bias 2 (RoB2) for randomised studies tools. Upon completion f the appraisal, data was stratified according to the tools used and was collated into tables Supplementary Tables S1 and S2). Any uncertainty was solved through discussion be- ween the reviewers. 3. Statistical Analysis Pre and post treatment Western Ontario and McMaster Universities Osteoarthritis ndex (WOMAC) scores were extracted from each study for each of the follow-up times. ercentage changes in these scores were then calculated for each follow-up time. Forest lots were created for each of the follow-up time periods. A summary forest plot was reated to determine the overall statistical significance of treatment on WOMAC scores. The search conducted on Medline (1946 to 4 June 2020), EMBASE (1974 to 29 June 2020), Cochrane library (1946 to June 2020), and Web of Science (1900 to 2020) included 117 po- tentially relevant articles after title screening, and 94 studies were excluded after abstract screening as these were non-human, preclinical studies or did not assess the treatment of interest. After full textual analysis ﬁve studies were removed as these did not in- clude quantitative treatment outcomes relevant to our study, only eighteen studies were then included in this review. One of the 18 studies was a two-year follow-up of another included study [62]. The original study was a prospective cohort study which investi- gated the effects of three different AMSC dose injections [63]. Out of the studies, 16 were prospective [43,49,62–73] and two were retrospective [74,75]. Only ﬁve were randomised controlled trials [48,49,64,69–72]. The general characteristics of each AMSC study can be seen in Table 1. All studies investigated autologous treatments. Characteristics for SVF studies can be found in Table 2. Cellular characteristics for AMSC studies and SVF studies can be found in Tables 3 and 4, respectively. Outcomes measured, and complications documented have been collated for each study in Table 5. The overall bias determined in all studies included in this systematic review were found to be low, indicating high quality studies. Cells 2021, 10, 1365 6 of 31 Table 1. Study characteristics for AMSC studies, n = 11. Authors Type of Study Treatment vs. Control No. of Patients in Control Group (Gender) No. gure 1. Overv 3. Results of Patients in Treatment Group (Gender) Age, Mean Location of Defect Grade of OA (Grading Classiﬁcation) Lee et al. (2019) [64] Prospective double blinded RCT AMSC at 1 dose, control 12 (3M, 9F) 12 (3M, 9F) 62.7 Knee II–IV (K-L) Freitag et al. (2019) [76] Prospective non blinded RCT AMSC at 2 doses, control 10 (5M, 5F) 10 (7M, 3F) for 1 injection. 10 (4M, 6F) for 2 injections 53.6 Knee II–III (K-L) Song et al. (2018) [48] Prospective double blinded RCT AMSC at 3 doses, no control N/A 18 (4M, 14F) 54.8 Knee II–III (K-L) Roato et al. (2019) [73] Prospective single arm study AMSC at 1 dose, no control N/A 20 (9M, 11F) 59.6 Knee I–III (K-L) Hudetz et al. (2019) [63] Prospective non-randomised trial AMSC injection, no control N/A 20 (15M, 5F) Not speciﬁed Knee III–IV (K-L) Spasovski et al. (2018) [65] Prospective single arm study AMSC at 1 dose, no control N/A 9 (3M, 6F) 63 Knee B-D (IKDC) Jo et al. (2017) [62] Prospective cohort study AMSC at 3 doses, no control N/A 18 (3M, 15F) 61.8 Knee III–IV (K-L) Bansal et al. (2017) [77] Prospective interventional AMSC injection, no control N/A 10 (6M, 4F) 58.4 Knee I–II (BS) Pers et al. (2016) [72] Prospective single arm study AMSC at 3 doses, no control N/A 18 (8M, 10F) 64.6 Knee III–IV (K-L) Jo et al. (2014) [63] Prospective cohort study AMSC at 3 doses, no control N/A 18 (3M, 15F) 61.8 Knee III–IV (K-L) Yokota et al. (2019) [75] Retrospective cohort study AMSC vs SVF, no control N/A 80 (16M, 64F) 71.4 Knee II–IV (K-L) AMSC = Adipose Tissue Derived Mesenchymal Stem Cell, F = Female, IKDC = International Knee Documentation Committee, K-L = Kellgren-Lawrence, M= Male, N/A = Not Applicable, OA = Osteoarthritis, Table 1. Study characteristics for AMSC studies, n = 11. p y , , , g , , / pp , , RCT = Randomised Controlled Trial. Table 2. Study characteristics for SVF studies, n = 7. Authors Type of Study Treatment vs. Control No. of Patients in Control Group (Gender) No. of Patients in Treatment Group (Gender) Age, Mean Location of Defect Grade of OA (Grading Classiﬁcation) Garza et al. (2020) [69] Prospective double blinded RCT High dose SVF vs low dose SVF vs placebo 13 (6M, 7F) 13 (4M, 9F) for low dose SVF. gure 1. Overv 3. Results 13 (7M, 6F) for high dose SVF. 59.0 Knee II–III (K-L) RCT = Randomised Controlled Trial. Table 2. Study characteristics for SVF studies, n = 7. Authors Type of Study Treatment vs. Control No. of Patients in Control Group (Gender) No. of Patients in Treatment Group (Gender) Age, Mean Location of Defect Grade of OA (Grading Classiﬁcation) Garza et al. (2020) [69] Prospective double blinded RCT High dose SVF vs low dose SVF vs placebo 13 (6M, 7F) 13 (4M, 9F) for low dose SVF. 13 (7M, 6F) for high dose SVF. 59.0 Knee II–III (K-L) 7 of 31 Cells 2021, 10, 1365 Table 2. Cont. Authors Type of Study Treatment vs. Control No. of Patients in Control Group (Gender) No. of Patients in Treatment Group (Gender) Age, Mean Location of Defect Grade of OA (Grading Classiﬁcation) Hong et al. (2019) [70] Prospective RCT One knee with SVF and the other with hyaluronic acid placebo 16 (3M, 13F) 16 (3M, 13F) Not speciﬁed Knee II-III (K-L) Tran et al. (2019) [78] Prospective non-randomised trial Arthroscopic microfracture vs arthroscopic microfracture and injection of SVF 15 (3M, 12F) 18 (5M, 13F) 58.64 Knee II–III (K-L) Yokota et al. (2017) [68] Prospective uncontrolled Injection of SVF, no control N/A 13 (2M, 11F) 74.5 Knee III–IV (K-L) Nguyen et al. (2016) [67] Prospective unblinded, non-randomised trial Arthroscopic fracture vs arthroscopic fracture and injection of SVF and PRP. 15 (3M, 12F) 15 (3M, 12F) 58.4 Knee II–III (K-L) Koh et al. (2013) [66] Prospective cohort study SVF at 1 dose with PRP, no control N/A 18 (6M, 12F) 54.6 Knee III–IV (K-L) Panni et al. (2019) [74] Retrospective single arm study SVF at 1 dose following arthroscopy (for chondral shaving/abrasion and/or meniscal regularization), no control N/A 52 (22M, 30F) 57.3 Knee 0–II (K-L) BS = Brandt Radiographic Grading Scale for Osteoarthritis, F = Female, K-L = Kellgren-Lawrence, M= Male, N/A = Not Applicable, OA = Osteoarthritis, RCT = Randomised Controlled Trial, SVF = Stromal Vascular Fraction. Table 2. Cont. Cells 2021, 10, 1365 8 of 31 Table 3. Cellular characteristics for AMSC studies, n = 11. Authors Number of Cells Used/Multiple Injections Method of Delivery of Cells MSC Pre-Treatment Follow-Up Period (Weeks) Harvest Site Method of Harvest MSC Surface Marker Validation Via Flow Cytometry t al. gure 1. Overv 3. Results (2019) [64] 1 × 108 cells Intra articular injection under USS guidance into unspeciﬁed joint space Adipose tissues were treated with collagenase I and were centrifuged to obtain a pellet which was resuspended in culture media. The cells were cultured for up to 5 days in media until conﬂuent and were then harvested at passage 3 26 Abdomen Liposuction AMSCs were positive for CD73, CD90. AMSCs were negative for CD31, CD34, CD45 et al. (2019) [49] 100 × 106 cells, single and double injection Intra articular injection under USS guidance into unspeciﬁed location in joint space Lipoaspirate was digestion followed by centrifugation. MSCs were cultured under hypoxic conditions with standard growth media until 80% conﬂuency and was expanded to passage 2. 52 Abdomen Liposuction AMSCs were positive for CD73, CD90, CD105, AMSCs were negative for CD14, CD19, CD34, CD45 et al. (2018) [48] 1 × 107, 2 × 107 and 5 × 107 cells, three injections Intra articular injection under USS guidance into unspeciﬁed location in joint space Lipoaspirated suspensions were digested and centrifuged, then cells were culture-expanded to passage 4. 96 Not speciﬁed Liposuction AMSCs were positive for CD29, CD49d, CD70, CD90 and were negative for actin, CD13, CD34, CD45, HLA-DR et al. (2019) [73] Not speciﬁed Intra articular injection under arthroscopic guidance into chondral defect site Lipoaspirate was treated with Collagenase. The resulting cell pellet was then resuspended into culture media and counted. 78 Abdomen Liposuction AMSCs were positive for CD73, CD90, CD105, IgG1, IgG2a AMSCs were negative for CD44, CD45 z et al. (2019) [71] Unspeciﬁed Intra articular injection into unspeciﬁed location in joint space Samples were digested with collagenase and samples were ﬁltered through a 100 µm cell strainer and centrifuged. The cell pellet was resuspended in DMEM. 48 Abdomen Liposuction AMSCs were positive for CD70, CD90, CD105, CD146. AMSCs were negative for CD31, CD34, CD45. ki et al. (2018) [65] 0.5–1 × 107 cells Intra articular injection into unspeciﬁed location in joint space MSCs were digested using collagenase, expanded in standard culture media and harvested between passage 2 and 4. 78 Abdomen Liposuction AMSCs were positive for CD73, CD90, CD105. AMSCs were negative for CD34, CD45 Table 3. Cellular characteristics for AMSC studies, n = 11. Lee et al. (2019) [64] Cells 2021, 10, 1365 9 of 31 Table 3. Cont. gure 1. Overv 3. Results Authors Number of Cells Used/Multiple Injections Method of Delivery of Cells MSC Pre-Treatment Follow-Up Period (Weeks) Harvest Site Method of Harvest MSC Surface Marker Validation Via Flow Cytometry al. (2017) [62] 1 × 107, 5 × 107 and 1 × 108 cells Intra articular injection under arthroscopic guidance into unspeciﬁed location in joint space Aspirated tissues were digested with collagenase I. Cells were cultured for 4-5 days until conﬂuent. All AMSCs used in this study were collected at passage 3. 104 Abdomen Liposuction AMSCs were positive for CD73, CD90. AMSCs were negative for CD14, CD34, CD45 et al. (2017) [77] 1 × 106 cells Intra articular injection into unspeciﬁed location in joint space The adipose tissues was ﬁltered and centrifuged. The cell pellet was re-suspended in culture medium and the media was changed every 3-4 days until the cells achieved 90% conﬂuency. 96 Abdomen Liposuction AMSCs were positive for CD70, CD90, CD105. AMSCs were negative for CD34, CD45, HLA-DR et al. (2016) [72] 2 × 106, 10 × 106 and 50 × 106 cells Intra articular injection under USS guidance into unspeciﬁed location in joint space Adipose tissue was digested with collagenase solution and plated in culture medium. Cells were passaged and then cultured in CCM for 14 days with media changes every 3–4 days until conﬂuence. 26 Abdomen Liposuction AMSCs were positive for CD73, CD90, CD105, IgG1 AMSCs were negative for CD31, CD34, CD45 al. (2014) [63] 1 × 107, 5 × 107 and 1 × 108 cells Intra articular injection under arthroscopic guidance into unspeciﬁed location in joint space Aspirated tissues were digested with collagenase and cells were resuspended in media until conﬂuent. AMSCs used were collected at passage 3. 26 Abdomen Liposuction AMSCs were positive for CD73, CD90. AMSCs were negative for CD31, CD34, CD45 ra et al. (2019) [75] 12.75 × 106 cells, unknown for SVF Intra articular injection into unspeciﬁed location in joint space The collected aspirate was digested with collagenase. Cells were cultured in medium that was replaced every 3 days thereafter. When cells reached 80% conﬂuency they were passaged up to four times. SVF cells were produced without culture in a sterile single-use functionally-closed system, requiring approximately 2–2.5 h from lipoaspirate. gure 1. Overv 3. Results 26 Abdomen Liposuction Not speciﬁed SC = Adipose Tissue Derived Mesenchymal Stem Cell, BSA = Bovine Serum Albumin, CDU = Collagen Digestion Units, DPBS = Dulbecco’s Phosphate-Buffered Saline, DMEM = Dulbecco’s Modiﬁed Eagle ium, EDTA = Ethylenediaminetetraacetic acid, FBS = Foetal Bovine Serum, MSC = Mesenchymal Stem Cell, PBS = Phosphate-Buffered Saline, SVF = Stromal Vascular fraction, USS = Ultrasound. Table 3. Cont. Cells 2021, 10, 1365 10 of 31 Table 4. Cellular characteristics for SVF studies, n = 7. Authors Number of Cells Used/Multiple Injections Method of Delivery of Cells SVF Pre-Treatment Follow-Up Period (Weeks) Harvest Site Method of Harvest MSC Surface Marker Validation Via Flow Cytometry Garza et al. (2020) [69] 3.0 × 107, 1.5 × 107, 0 cells Intra articular injection under USS guidance into unspeciﬁed joint space SVF from dissociated tissue was centrifuged and the SVF cell pellet was extracted, resuspended for injection. 48 Abdomen Liposuction Not speciﬁed Hong et al. (2019) [70] 7.45 × 106 cells Intra articular injection under arthroscopic guidance into unspeciﬁed location in joint space The SVF from the lipoaspirate was isolated by means of collagenase digestion. The SVF was then washed twice with PBS to remove collagenase. 48 Abdomen Liposuction Not speciﬁed Tran et al. (2019) [78] 9–12 × 107 cells Intra articular injection under arthroscopic guidance into chondral defect site The SVF from the lipoaspirate was isolated through collagenase treatment. The SVF was then diluted with normal saline 0.9% to obtain 6 mL of solution containing 90–120 million cells to administer via injection. 96 Abdomen Liposuction Not speciﬁed Yokota et al. (2017) [68] Unknown, however estimated to be 3 × 107 cells Intra articular injection into unspeciﬁed location in joint space Autologous SVF cells were collected in a sterile single-use functionally-closed system, requiring approximately 2–2.5 h. 4 Abdomen Liposuction Not speciﬁed Nguyen et al. (2016) [67] 1 × 107 cells Intra articular injection under arthroscopic guidance into chondral defect site The adipose tissue was digested using collagenase and centrifuged, the pellet was suspended in PBS for cell counting before injection. 72 Abdomen Liposuction Not speciﬁed Table 4. Cellular characteristics for SVF studies, n = 7. Intra articular injection into unspeciﬁed location in joint space Intra articular injection under arthroscopic guidance into chondral defect site Intra articular injection under arthroscopic guidance into chondral defect site Cells 2021, 10, 1365 11 of 31 Table 4. Cont. gure 1. Overv 3. Results Authors Number of Cells Used/Multiple Injections Method of Delivery of Cells SVF Pre-Treatment Follow-Up Period (Weeks) Harvest Site Method of Harvest MSC Surface Marker Validation Via Flow Cytometry Koh et al. (2013) [66] 1.18 × 106 cells Intra articular injection into unspeciﬁed location in joint space SVF was derived from fat pad tissue and mixed with 3.0 mL of platelet-rich plasma for injection. 97.2 Infrapatellar fat pad Surgical excision of infrapatellar fat pad Not speciﬁed Panni et al. (2019) [74] Not speciﬁed Intra articular injection under arthroscopic guidance into unspeciﬁed location in joint space The harvested fat was processed with the Lipogems® ortho kit. The ﬁnal product was transferred directly to syringes for injection. 61.2 Abdomen Liposuction Not speciﬁed AMSC = Adipose Tissue Derived Mesenchymal Stem Cell, CDU = Collagen Digestion Units, DPBS = Dulbecco’s Phosphate-Buffered Saline, DMEM = Dulbecco’s Modiﬁed Eagle Medium, FBS = Foetal Bovine Serum, MSC = Mesenchymal Stem Cell, PBS = Phosphate-Buffered Saline, SVF = Stromal Vascular fraction, USS = Ultrasound. Table 5. Outcomes and complications, n = 18. Authors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events Nature of Complications Lee et al. (2019) [64] WOMAC, VAS, KOOS, ROM, K-L, Joint space width of medial and lateral compartment and HKA angle Baseline WOMAC score was 60.0 (±17.0 SD) At 6 months post procedure WOMAC scores were 26.7 (±13.3 SD) Single injection of AD-MSCs led to a 55% reduction in the WOMAC total score, 59% in the pain score, 54% in the stiffness score, and 54% in the physical function score at 6 months. Signiﬁcant improvements in the VAS, KOOS, ROM scores were also seen. K-L grade, joint space width of medial and lateral compartment, and HKA angle did not change signiﬁcantly over 6 months in either groups. No evidence of signiﬁcant cartilage regeneration in MRI at 6 months after the injection. 8AEs 6 cases of arthralgia and 2 cases of joint swelling after the procedure. Table 4. Cont. Table 5. Outcomes and complications, n = 18. Authors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events Nature of Complications Lee et al. 7 AEs and 1 SAE in the one injection group. 8AEs and 1 SAE in the ﬁrst injection of the two-injection group. 10 AEs in the second injection in the two-injection group. gure 1. Overv 3. Results (2019) [64] WOMAC, VAS, KOOS, ROM, K-L, Joint space width of medial and lateral compartment and HKA angle Baseline WOMAC score was 60.0 (±17.0 SD) At 6 months post procedure WOMAC scores were 26.7 (±13.3 SD) Single injection of AD-MSCs led to a 55% reduction in the WOMAC total score, 59% in the pain score, 54% in the stiffness score, and 54% in the physical function score at 6 months. Signiﬁcant improvements in the VAS, KOOS, ROM scores were also seen. K-L grade, joint space width of medial and lateral compartment, and HKA angle did not change signiﬁcantly over 6 months in either groups. No evidence of signiﬁcant cartilage regeneration in MRI at 6 months after the injection. 8AEs 6 cases of arthralgia and 2 cases of joint swelling after the procedure. Table 5. Outcomes and complications, n = 18. 12 of 31 Cells 2021, 10, 1365 Table 5. Cont. uthors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events Nature of Complications al. (2019) [49] NPRS-11, WOMAC, KOOS, MOAKS WOMAC scores were 59.6 (±17.9 SD) for the one injection group and 54.4 (±18.2 SD) for the two-injection group WOMAC scores were 84 (±9.4 SD) for the one injection group and 87.3 (±8 SD) for the two-injection group at 12 months. NPRS-11 scores were greater when compared with baseline (< 0.05) throughout all time points in all treatment groups. There was no difference however between treatment groups. KOOS and WOMAC improved in all subscales during follow-up to 12 months. Two-thirds of the control group showed cartilage loss. 30% of the one-injection group had further cartilage loss, 50% had progression of osteophyte formation at 12 months. 89% in the two-injection group had either no progression or improvement in cartilage loss. 7 AEs and 1 SAE in the one injection group. 8AEs and 1 SAE in the ﬁrst injection of the two-injection group. 10 AEs in the second injection in the two-injection group. Mild AEs: minor discomfort, bruising and/or swelling after the injection. SAEs were classiﬁed as pain and sweeling for 4 weeks after injection which impacted the daily activities of life for the patient. al. WOMAC scores were 84 (±9.4 SD) for the one injection group and 87.3 (±8 SD) for the two-injection group at 12 months. gure 1. Overv 3. Results (2018) [48] WOMAC, NPRS-11, SF-36, WOMAC scores were 34.75 (±17.05 SD) at baseline WOMAC scores were 25.94 (±16.09 SD), 20.38 (±19.89 SD), 22.77 (±22.72 SD), 15.00 (±11.36 SD) and 12.44 (±8.99 SD) in the 12th, 24th, 48th, 72nd and 96th week. WOMAC scores improved with time leading up to follow-up in all groups. Signiﬁcant improvements in the NPRS-11 scores in the low- and high-dose groups were ﬁrst observed at three months following treatment. A statistically signiﬁcant reduction in SF-36 scores were only found in the 12th and 96th week of follow-up The volume of knee cartilage increased over the course of follow-up. This was more apparent in the high-dose group 8 AEs in the low dose group (66.67%). 7 AEs in the middle dose group (58.33%). 6 AEs in the high dose group (50%). No SAEs or deaths. All complications were AEs. These were most commonly transient pain and swelling of joints, which were mild to moderate and were spontaneously relieved within 7 days without special treatment. One patient experienced mild oedema and cramps of bilateral lower extremities, which were relieved in 21 days without treatment and not related to the MSC treatment. al. (2019) [73] WOMAC, VAS, K-L WOMAC score was 45.91 (±2.8) pre procedure. (NO SE OR SD GIVEN) WOMAC scores were 27.47 (±3.02), 15.84 (±2.5) and 12.97 (±2.3) at 3 months, 6 months and 18 months post procedure. (NO SE OR SD GIVEN) Signiﬁcant improvement of VAS and WOMAC scores, with a signiﬁcant pain reduction and increased mobility at 3, 6, and 12 months follow-up. No increase in the thickness of cartilage at 18 months. 1 SAE Swelling persisted two months after surgery t al. (2019) [71] KOOS, WOMAC, VAS WOMAC baseline score was 55.38 (±18.8 SD) WOMAC scores after 12 months was 32.25 (±14.6 SD) All scores signiﬁcantly improved after treatment. 0 AEs or SAEs N/A et al. (2018) [65] KSS, HSS, Lysholm score, VAS, MOCART N/A N/A All outcomes signiﬁcantly improved at 3 and 6 months. However, there was no further improvement beyond 12 or 18 months after treatment. N/A N/A Table 5. Cont. WOMAC scores were 25.94 (±16.09 SD), 20.38 (±19.89 SD), 22.77 (±22.72 SD), 15.00 (±11.36 SD) and 12.44 (±8.99 SD) in the 12th, 24th, 48th, 72nd and 96th week. WOMAC scores were 24.6 (±8.6 SD), 22.0 (±8.5 SD) and 30.1 (±8.9 SD) at 1 week, 3 months and 6 months respectively for the low dose group. WOMAC scores were 45.8 (±9.1 SD), 52.8 (±9.6 SD) and 42.6 (±9.1 SD) at 1 week, 3 months and 6 months respectively for the middle dose group. WOMAC scores were 61.1 (±15.3 SD), 38.4 (±16.0 SD) and 42.6 (±16.0 SD) at 1 week, 3 months and 6 months respectively for the high dose group. Statistically signiﬁcant improvements in WOMAC, VAS, KOOS and SAS scores were only found in the low dose group at 1 week, 3 months and 6 months. No improvements in the SF-36 in any groups. Post Treatment WOMAC Scores WOMAC scores were 43.3 12.7 SE) for the low dose oup, 69.0 (±5.9 SE) for the dose group and 54.2 (±5.2 ) for the high dose group. WOMAC scores were 25.3 (±19.5 SE), 14.7 ± (12.7 SE) and 17.0 (±9.8 SE) at 6 months, 1 year and 2 years respectively for the low dose group. WOMAC scores were 48.5 (±9.5 SE), 13.1 (±10.0 SE) and 25.1 (±11.0 SE) at 6 months, 1 year and 2 years respectively for the middle dose group. WOMAC scores were 32.8 (±6.3 SE), 16.0 (±4.4 SE) and 19.0 (±5.5 SE) at 6 months, 1 year and 2 years respectively for the high dose group. 2 WOMAC scores were 25.3 (±19.5 SE), 14.7 ± (12.7 SE) and 17.0 (±9.8 SE) at 6 months, 1 year and 2 years respectively for the low dose group. WOMAC scores were 48.5 (±9.5 SE), 13.1 (±10.0 SE) and 25.1 (±11.0 SE) at 6 months, 1 year and 2 years respectively for the middle dose group. WOMAC scores were 32.8 (±6.3 SE), 16.0 (±4.4 SE) and 19.0 (±5.5 SE) at 6 months, 1 year and 2 years respectively for the high dose group. The WOMAC, VAS and KSS scores improved in the high-dose group at 6 months and 1 year. Non-signiﬁcant trends in the low and middle dose groups. Signiﬁcant improvement in KSS scores in the low dose groups up to one year. The sports subscore of the KOOS improved until 2 years for the high-dose group. No statistically signiﬁcant improvements were found in the quality-of-life subscore of the KOOS for any of the dose groups. WOMAC scores were 43.3 (±12.7 SE) for the low dose group, 69.0 (±5.9 SE) for the mid dose group and 54.2 (±5.2 SE) for the high dose group. WOMAC scores were 43.3 (±12.7 SE) for the low dose group, 69.0 (±5.9 SE) for the mid dose group and 54.2 (±5.2 SE) for the high dose group. Signiﬁcant changes in the WOMAC and 6MWD scores were noted in both the subsets and the total after 2 years as compared to the baseline. MRI evaluation demonstrated that cartilage thickness improved. Conclusions Based on Outcomes Adverse Events Nature of Complications NPRS-11 scores were greater when compared with baseline (< 0.05) throughout all time points in all treatment groups. There was no difference however between treatment groups. KOOS and WOMAC improved in all subscales during follow-up to 12 months. Two-thirds of the control group showed cartilage loss. 30% of the one-injection group had further cartilage loss, 50% had progression of osteophyte formation at 12 months. 89% in the two-injection group had either no progression or improvement in cartilage loss. Mild AEs: minor discomfort, bruising and/or swelling after the injection. SAEs were classiﬁed as pain and sweeling for 4 weeks after injection which impacted the daily activities of life for the patient. WOMAC scores were 25.94 (±16.09 SD), 20.38 (±19.89 SD), 22.77 (±22.72 SD), 15.00 (±11.36 SD) and 12.44 (±8.99 SD) in the 12th, 24th, 48th, 72nd and 96th week. Cells 2021, 10, 1365 13 of 31 Table 5. Cont. Table 5. Cont. Authors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Score Jo et al. (2017) [62] WOMAC, VAS, KSS, KOOS, K-L, Joint space width of the medial compartment, mechanical axis with weight bearing line, and anatomical axis WOMAC scores were 43.3 (±12.7 SE) for the low dose group, 69.0 (±5.9 SE) for the mid dose group and 54.2 (±5.2 SE) for the high dose group. WOMAC scores were 25.3 (±19.5 SE), 14.7 ± (12.7 SE) and 17.0 (±9.8 SE) at 6 months, 1 yea and 2 years respectively for the low dose group. WOMAC scores were 48.5 (±9.5 SE), 13.1 (±10.0 SE) and 25 (±11.0 SE) at 6 months, 1 year an 2 years respectively for the middle dose group. WOMAC scores were 32.8 (±6.3 SE), 16.0 (±4.4 SE) and 19. (±5.5 SE) at 6 months, 1 year an 2 years respectively for the high dose group. Bansal et al. (2017) [77] WOMAC, 6MWD, cartilage thickness WOMAC score was 64 at baseline (NO SE OR SD GIVEN) WOMAC scores were 52, 46, 42 38 and 41 at 3 months, 6 months 12 months, 18 months and 24 months respectively. (NO SE OR SD GIVEN) Pers et al. (2016) [72] WOMAC, VAS, PGA, SAS, KOOS, OARSI, SF-36 WOMAC scores were 63.2 (±4.1 SD) for the low dose group, 65.5 (±8.1 SD) for the mid dose group and 65.2 (±2.3 SD) for the high dose group. WOMAC scores were 24.6 (±8.6 SD), 22.0 (±8.5 SD) and 30 (±8.9 SD) at 1 week, 3 months and 6 months respectively for th low dose group. WOMAC scores were 45.8 (±9.1 SD), 52.8 (±9.6 SD) and 42 (±9.1 SD) at 1 week, 3 months and 6 months respectively for th middle dose group. WOMAC scores were 61.1 (±15.3 SD), 38.4 (±16.0 SD) and 42.6 (±16.0 SD) at 1 week, 3 months and 6 months respectively for the high dose group. MAC Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events WOMAC scores were 43.3 (±12.7 SE) for the low dose group, 69.0 (±5.9 SE) for the mid dose group and 54.2 (±5.2 SE) for the high dose group. Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Nature of Complications In the low dose group, the AE was an individual case of nasopharyngitis, and the SAE was a urinary calculus. In the mid dose group AEs were individual cases of nasopharyngitis, arthralgia and chest pain. In the high dose group AEs were individual cases of nasopharyngitis, arthralgia, back pain, cough and hypertriglyceridemia. 1 AE and 1 SAE in the low dose group (66.6%). 2 AEs in the mid dose group (66.6%). 5 AEs in the high dose group (41.66%). 3AEs in the ASC group and 26 AEs in the SVF group. WOMAC scores were 44.0 (±4.4 SE), 30.0 (±12.0 SE), 38.7 (±24.7 SE) and 25.3 (±19.5 SE) at 1, 2, 3 and 6 months respectively for the low dose group. WOMAC scores were 72.3 (±4.3 SE), 51.3 (±6.5 SE), 51.3 (±6.7 SE) and 48.5 (±11.0 SE) at 1, 2, 3 and 6 months respectively for the mid dose group. WOMAC scores were 45.5 (±4.5 SE), 40.1 (±6.0 SE), 37.0 (±6.8 SE) and 32.8 (±6.3 SE) at 1, 2, 3 and 6 months respectively for the high dose group. Signiﬁcant improvement of the WOMAC and VAS at 6 months compared with baseline in the high-dose groups. This was not seen in the other treatment groups. Knee subsection of KSS signiﬁcantly increased in the low-dose and the high-dose groups, but improvements in the function subsection of seen in the low-dose group only. Other parameters did not change signiﬁcantly at 6 months in any groups. The ICRS grade of the cartilage defect signiﬁcantly improved in the medial femoral and tibial condyle in the high-dose group at second-look arthroscopy. No signiﬁcant change was found in the lateral parts of the joint. Table 5. Cont. Table 5. Cont. thors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores WOMAC scores were 43.3 (±12.7 SE) for the low dose group, 69.0 (±5.9 SE) for the mid dose group and 54.2 (±5.2 SE) for the high dose group. Post Treatment WOMAC Scores Signiﬁcant changes in the WOMAC and 6MWD scores were noted in both the subsets and the total after 2 years WOMAC scores were 52, 46, 42, 38 and 41 at 3 months, 6 months, WOMAC score was 64 at baseline (NO SE OR SD GIVEN) 12 months, 18 months and 24 months respectively. (NO SE OR SD GIVEN) y as compared to the baseline. MRI evaluation demonstrated that cartilage thickness improved. 14 of 31 Cells 2021, 10, 1365 In the ASC group, there was 1 case of joint swelling after the injection and 2 cases of abdominal induration after harvest. These were all self-limiting. In the SVF group, there were 3 cases of joint swelling after the injection. There were 6 cases of abdominal pain, 5 cases of abdominal swelling and 12 cases of abdominal induration after harvest. All groups displayed a reduction in total WOMAC score from baseline at 6 month follow-up. All treated groups continued to demonstrate lower total WOMAC scores 1 year after injection as compared with baseline scores and sixth month scores. There was no change in cartilage thickness detected at six month follow-up. In the treated group, all scores including VAS, WOMAC pain, WOMAC stiffness, and knee ROM was founded to be signiﬁcantly improved at one, three, six, and 12-months follow-up as compared with baseline scores within the treated groups and against control groups. Both WORMS and MOCART MRI scores showed a statistically signiﬁcant improvement in the treatment group, while a deterioration in the control group. Baseline WOMAC pain score was 9.50 (±3.92 SD) for the control group and was 9.44 (±3.90 SD) for the treatment group. Baseline WOMAC stiffness score was 3.00 (±1.55 SD) for the control group and was 3.31 (±1.82 SD) for the treatment group. WOMAC pain scores were 8.94 (±4.98 SD), 11.56 (±6.84 SD), 12.88 (±5.73 SD) and 15.19 (±4.29 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC stiffness scores were 4.38 (±2.22 SD), 4.94 (±2.49 SD), 5.44 (±2.56 SD) and 5.69 (±2.57 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC pain scores were 6.25 (±3.02 SD), 2.13 (±3.52 SD), 1.5 (±3.84 SD) and 1.44 (±4.77 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. WOMAC stiffness scores were 1.75 (±1.59 SD), 1.12 (±1.80 SD), 0.81 (±1.59 SD) and 1.06 (±2.11 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. WOMAC scores for the placebo was 26.0, 22.9, 37.2 and 41.9 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 23.0(14.2–37.4), 20.0 (16.0–32.0), 30.2 (21.4–55.2), 41.0 (19.5–55.2). WOMAC scores for the low dose group was 24.8, 19.7, 23.7 and 21.8 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 20.0 (10.7–37.4), 14.0 (5.3–35.6), 26.7 (8.9–32.0), 12.5 (7.1–35.6) WOMAC scores for the high dose group was 25.7, 26.5, 20.0 and 13.2 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 27.0 (14.2–36.0), 27.0 (10.7–34.7), 8.9 (3.6–32.0), 3.6 (0.0–26.7) Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Nature of Complications Adverse Events Signiﬁcant improvement of the WOMAC and VAS at 6 months compared with baseline in the high-dose groups. This was not seen in the other treatment groups. Knee subsection of KSS signiﬁcantly increased in the low-dose and the high-dose groups, but improvements in the function subsection of seen in the low-dose group only. Other parameters did not change signiﬁcantly at 6 months in any groups. The ICRS grade of the cartilage defect signiﬁcantly improved in the medial femoral and tibial condyle in the high-dose group at second-look arthroscopy. No signiﬁcant change was found in the lateral parts of the joint. Change in KOOS symptoms occurred earlier in the AMSC group than the SVF group, with signiﬁcant improvement detected at 3 months follow-up. The extent of VAS improvement after injection was greatest in patients with mildest. Patients in the AMSC group had a greater improvement in VAS than patients in the SVF group, regardless of the extent of OA at baseline. The proportion of patients who responded to treatment as determined by the OMERACT-OARSI responder criteria was greater in the AMSC group than the SVF. In the ASC group, there was 1 case of joint swelling after the injection and 2 cases of abdominal induration after harvest. These were all self-limiting. In the SVF group, there were 3 cases of joint swelling after the injection. There were 6 cases of abdominal pain, 5 cases of abdominal swelling and 12 cases of abdominal induration after harvest. Th ll lf li i i Yokota et al. (2019) [75] KOOS, VAS, OARSI, K-L N/A N/A These were all self-limiting. Cells 2021, 10, 1365 15 of 31 15 of 31 Table 5. Cont. 10 AEs 4 AEs relating to abdominal pain after harvest which resolved after 1 week. 6 cases of pain and swelling in both knees after surgery. These all resolved after 2 weeks with analgesia. Table 5. Cont. Authors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Garza et al. (2020) [69] WOMAC, OS Baseline WOMAC scores were 49.3 for the placebo group, 56.2 for the low dose group and 47.1 for the high dose group (THIS WAS THE MEAN. NO SE OR SD WAS GIVEN) Median was 49.8 (37.4–57.0), 51.6 (46.3–62.3) and 49.8 (35.6–55.2) for the placebo, low dose and high dose groups. WOMAC scores for the placebo was 26.0, 22.9, 37.2 and 41.9 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 23.0(14.2–37.4), 20.0 (16.0–32.0), 30.2 (21.4–55.2), 41.0 (19.5–55.2). WOMAC scores for the low dose group was 24.8, 19.7, 23.7 and 21.8 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 20.0 (10.7–37.4), 14.0 (5.3–35.6), 26.7 (8.9–32.0), 12.5 (7.1–35.6) WOMAC scores for the high dose group was 25.7, 26.5, 20.0 and 13.2 at 6 weeks, 3 months, 6 months and 1 year respectively. Median values were 27.0 (14.2–36.0), 27.0 (10.7–34.7), 8.9 (3.6–32.0), 3.6 (0.0–26.7) Hong et al. (2019) [70] VAS, WOMAC, ROM, WORMS, MOCART Baseline WOMAC pain score was 9.50 (±3.92 SD) for the control group and was 9.44 (±3.90 SD) for the treatment group. Baseline WOMAC stiffness score was 3.00 (±1.55 SD) for the control group and was 3.31 (±1.82 SD) for the treatment group. WOMAC pain scores were 8.94 (±4.98 SD), 11.56 (±6.84 SD), 12.88 (±5.73 SD) and 15.19 (±4.29 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC stiffness scores were 4.38 (±2.22 SD), 4.94 (±2.49 SD), 5.44 (±2.56 SD) and 5.69 (±2.57 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC pain scores were 6.25 (±3.02 SD), 2.13 (±3.52 SD), 1.5 (±3.84 SD) and 1.44 (±4.77 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. WOMAC stiffness scores were 1.75 (±1.59 SD), 1.12 (±1.80 SD), 0.81 (±1.59 SD) and 1.06 (±2.11 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events For KL OA grade 2 patients, WOMAC scores were 24.25 (±19.77 SD) and 18.25 (±20.07 SD) at 12 and 24 months respectively. For KL OA grade 3 patients, WOMAC scores were 18.21 (±8.20 SD) and 9.00 (±8.46 SD) at 12 and 24 months respectively. Table 5. Cont. Table 5. Cont. Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events Nature of Complications 78] VAS, WOMAC, OS, BME, K-L WOMAC scores were 52.0 (±18.26 SD) and 42.64 (±12.51 SD) at baseline for patients with KL OA grade 2 and 3 respectively. For KL OA grade 2 patients, WOMAC scores were 24.25 (±19.77 SD) and 18.25 (±20.07 SD) at 12 and 24 months respectively. For KL OA grade 3 patients, WOMAC scores were 18.21 (±8.20 SD) and 9.00 (±8.46 SD) at 12 and 24 months respectively. No signiﬁcant difference was found between the VAS scores of the treatment and placebo groups at 12 months.A decreasing trend in the VAS and WOMAC scores of the treatment group was observed up to 24 months compared to controls. Between 12 and 24 months, the VAS scores increased in the placebo group. MRI results showed that after 24 months of treatment, bone marrow oedema was decreased in both the placebo and the SVF treatment groups, the latter demonstrated a greater effect. The Outbridge score also decreased in the SVF-treated group. N/A N/A [68] JKOM, WOMAC, VAS Baseline WOMAC scores were 49.6 (±20.4 SD) WOMAC scores were 43.0 (±17.4 SD) and 36.5 (±21.9 SD) at 1 month and 6 months after treatment respectively JKOM, WOMAC, and VAS scores were signiﬁcantly improved compared to baseline one month following treatment. This effect was also observed at the six-month visit. JKOM scores improved by an average of 35% over baseline compared to a 32% improvement in WOMAC, and 40% for VAS. 26 AEs All patients experienced pain and swelling at the fat harvest and injection sites. These however resolved after a few days with analgesia. [67] WOMAC, Lysholm score, VAS, OS, BME, JMA, WOMAC scores for the placebo group was 47.27 (±17.13 SD). WOMAC scores for the treatment group was 42.87 (±16.29 SD) WOMAC scores for the placebo group was 23.27 (±15.61 SD) and 25.60 (±19.69 SD) at 6 and 12 months respectively. WOMAC scores for the treatment group were 19.27 (±14.87 SD) and 17.33 (±14.91 SD) at 6 and 12 months respectively. WOMAC scores signiﬁcantly decreased compared with baseline scores at 6 and 12 months.WOMAC scores between the treatment and placebo groups were not signiﬁcantly different at 12 months, but a signiﬁcant difference was seen at 18 months. Post Treatment WOMAC Scores All groups displayed a reduction in total WOMAC score from baseline at 6 month follow-up. All treated groups continued to demonstrate lower total WOMAC scores 1 year after injection as compared with baseline scores and sixth month scores. There was no change in cartilage thickness detected at six month follow-up. 0 AEs or SAEs N/A (3.6–32.0), 3.6 (0.0–26.7) WOMAC pain scores were 8.94 (±4.98 SD), 11.56 (±6.84 SD), 12.88 (±5.73 SD) and 15.19 (±4.29 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC stiffness scores were 4.38 (±2.22 SD), 4.94 (±2.49 SD), 5.44 (±2.56 SD) and 5.69 (±2.57 SD) at 1 month, 3 months, 6 months and 12 months respectively for the control group. WOMAC pain scores were 6.25 (±3.02 SD), 2.13 (±3.52 SD), 1.5 (±3.84 SD) and 1.44 (±4.77 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. WOMAC stiffness scores were 1.75 (±1.59 SD), 1.12 (±1.80 SD), 0.81 (±1.59 SD) and 1.06 (±2.11 SD) at 1 month, 3 months, 6 months and 12 months respectively for the treatment group. In the treated group, all scores including VAS, WOMAC pain, WOMAC stiffness, and knee ROM was founded to be signiﬁcantly improved at one, three, six, and 12-months follow-up as compared with baseline scores within the treated groups and against control groups. Both WORMS and MOCART MRI scores showed a statistically signiﬁcant improvement in the treatment group, while a deterioration in the control group. 10 AEs 4 AEs relating to abdominal pain after harvest which resolved after 1 week. 6 cases of pain and swelling in both knees after surgery. These all resolved after 2 weeks with analgesia. 10 AEs 4 AEs relating to abdominal pain after harvest which resolved after 1 week. 6 cases of pain and swelling in both knees after surgery. These all resolved after 2 weeks with analgesia. Cells 2021, 10, 1365 16 of 31 WOMAC scores for the placebo group was 23.27 (±15.61 SD) and 25.60 (±19.69 SD) at 6 and 12 months respectively. WOMAC scores for the treatment group were 19.27 (±14.87 SD) and 17.33 (±14.91 SD) at 6 and 12 months respectively. WOMAC scores were 43.0 (±17.4 SD) and 36.5 (±21.9 SD) at 1 month and 6 months after treatment respectively Table 5. Cont. VAS and Lysholm scores improved in the treatment group compared to pre treatment scores at all follow-up timepoints. 0 AEs or SAEs N/A 66] WOMAC, lysholm score, VAS, WORMS Baseline WOMAC score was 49.9 (±12.6 SD) After ﬁnal follow-up post procedure WOMAC scores were 30.3 (±9.2 SD) WOMAC scores decreased in the treatment group over the follow-up period. Greater changes in WOMAC score were seen in subjects injected with greater cell numbers. Lysholm and VAS scores also signiﬁcantly improved over the follow-up period. Signiﬁcant reduction was observed in the WORMS cartilage subscore. 1 AE Notable pain and swelling after injection for 2 weeks. This was self-limiting. Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores WOMAC scores were 52.0 (±18.26 SD) and 42.64 (±12.51 SD) at baseline for patients with KL OA grade 2 and 3 respectively. WOMAC scores were 52.0 (±18.26 SD) and 42.64 (±12.51 SD) at baseline for patients with KL OA grade 2 and 3 respectively. JKOM, WOMAC, and VAS scores were signiﬁcantly improved compared to baseline one month following treatment. This effect was also observed at the six-month visit. JKOM scores improved by an average of 35% over baseline compared to a 32% improvement in WOMAC, and 40% for VAS. WOMAC scores were 43.0 (±17.4 SD) and 36.5 (±21.9 SD) at 1 month and 6 months after treatment respectively WOMAC scores for the placebo group was 47.27 (±17.13 SD). WOMAC scores for the treatment group was 42.87 (±16.29 SD) After ﬁnal follow-up post procedure WOMAC scores were 30.3 (±9.2 SD) Koh et al. (2013) [66] WOMAC, lysholm score, VAS, WORMS Baseline WOMAC score was 49.9 (±12.6 SD) 17 of 31 Cells 2021, 10, 1365 17 of 31 Table 5. Cont. Authors Outcome Measures Pre-Treatment WOMAC Scores Post Treatment WOMAC Scores Conclusions Based on Outcomes Adverse Events Nature of Complications Panni et al. (2019) [74] IKS, VAS, N/A N/A 96.2% of treated subjects reported improvements in knee function and/or pain. A subset (62%) achieved complete or near-complete function recovery and/or pain relief. Two (3.9%) patients reported slight reduction of the pain. Table 5. Cont. 3AEs Transient haematoma after harvest 6MWD = 6 Minute Walk Distance, BMA = Bone Marrow Oedema, HKA = hip-knee-ankle, HSS = Hospital for Special Surgery Knee score, ICRS = International Cartilage Repair Society, IKS = International Knee Society, JKOM = Japanese Knee Osteoarthritis Measure, JMA = Joint Motion Amplitude, KOOS = Knee Injury and Osteoarthritis Outcome Score, K-L = Kellgren-Lawrence, KSS = Knee Society Score, MOAKs = MRI Osteoarthritis Knee Score, MOCART = 2D Magnetic Resonance Observation of Cartilage Repair Tissue, NPRS = Numeric Pain Rating Scale, OARSI = Osteoarthritis Research Society International, OS = Outerbridge Classiﬁcation System, PGA = Patient Global Assessment score, ROM = range of motion, SAS = Short Arthritis Assessment scale, SF-36 = Short Form-36, WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index, WORMS = Whole-Organ MRI Score, VAS = visual analogue scale. 6MWD = 6 Minute Walk Distance, BMA = Bone Marrow Oedema, HKA = hip-knee-ankle, HSS = Hospital for Special Surgery Knee score, ICRS = International Cartilage Repair Society, IKS = International Knee Society, JKOM = Japanese Knee Osteoarthritis Measure, JMA = Joint Motion Amplitude, KOOS = Knee Injury and Osteoarthritis Outcome Score, K-L = Kellgren-Lawrence, KSS = Knee Society Score, MOAKs = MRI Osteoarthritis Knee Score, MOCART = 2D Magnetic Resonance Observation of Cartilage Repair Tissue, NPRS = Numeric Pain Rating Scale, OARSI = Osteoarthritis Research Society International, OS = Outerbridge Classiﬁcation System, PGA = Patient Global Assessment score, ROM = range of motion, SAS = Short Arthritis Assessment scale, SF-36 = Short Form-36, WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index, WORMS = Whole-Organ MRI Score, VAS = visual analogue scale. Cells 2021, 10, 1365 18 of 31 18 of 31 Forest plots were created, according to the follow-up time periods utilised, to an yse changes in WOMAC scores post AMSC and SVF treatment (Figure 2) in the tre ment group. Cells 2021, 10, x 17 of 30 Figure 2. Table 5. Cont. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2- inj = 2 injections, 3-inj = 3 injections, CI = confidence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Figure 2. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2-inj = 2 injections, 3-inj = 3 injections, CI = conﬁdence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Forest plots were created, according to the follow-up time periods utilised, to anal- yse changes in WOMAC scores post AMSC and SVF treatment (Figure 2) in the treat- ment group. 17 of 30 Figure 2. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2- inj = 2 injections, 3-inj = 3 injections, CI = confidence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Figure 2. Table 5. Cont. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2-inj = 2 injections, 3-inj = 3 injections, CI = conﬁdence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Figure 2. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2- inj = 2 injections, 3-inj = 3 injections, CI = confidence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Figure 2. Forest plots showing percentage changes in WOMAC score less than one month after treatment (a), approxi- mately two months after treatment (b), three months after treatment (c), six months after treatment (d), twelve months after treatment (e), eighteen months after treatment (f) and twenty-four months after treatment (g) (1-inj = 1 injection, 2-inj = 2 injections, 3-inj = 3 injections, CI = conﬁdence intervals, df = degrees of freedom, HD = high dose, LD = low dose, MD = middle dose, N = number, RE = random effects, Tx = treatment group). Cells 2021, 10, 1365 mont in W 19 of 31 ment other 19 of 31 ment other Figure 2a shows all four studies which documented WOMAC scores less than 1 month after treatment [63,68,70,72,78]. There was a statistically signiﬁcant improvement in WOMAC scores in the individual arm studies, and in the high dose groups of the other studies. The pooled analysis showed a −20.24% change [95% CI −35.70, −4.78] suggesting that use of AMSCs and SVF resulted in a statistically signiﬁcant improvement in WOMAC scores less than a month after treatment [Q = 232.46, p < 0.0001]. Table 5. Cont. Figure 2b–g show all studies which evaluated WOMAC scores at approximately two months, three months, six months, twelve months, eighteen months and twenty-four months after treatment, respectively [48,49,62–64,66–73,78] The plots show that there are statistically signiﬁcant improvements in WOMAC scores in all arms of all treatment groups across the follow-up periods between two and twenty-four months. The pooled analysis was −37.69% change [95% CI −50.30, −25.08, Q = 25.85, p < 0.0001] post two month treatment, −41.84% [95% CI −53.52, −30.17, Q = 200.89, p < 0.0001] post three month treatment, −40.07% [95% CI −54.44, −39.65, Q = 273.47, p < 0.0001] post six month treatment, −58.44% [95% CI −66.41, −50.47, Q = 85.71, p < 0.0001] post twelve month treatment, −65.69% [−80.05, −51.32, Q = 5.26, p = 0.022] post eighteen month treatment and −62.11% [95% CI −72.68, −51.54, Q = 93.51, p < 0.0001] post twenty-four month treatment. SCs and SVF resulted in a statistically significant improvement in WOMAC n a month after treatment [Q = 232.46, p < 0.0001]. Figure 2b–g show all valuated WOMAC scores at approximately two months, three months, six months, eighteen months and twenty-four months after treatment, respec- –64,66–73,78] The plots show that there are statistically significant im- WOMAC scores in all arms of all treatment groups across the follow-up n two and twenty-four months. The pooled analysis was −37.69% change −25.08, Q = 25.85, p < 0.0001] post two month treatment, −41.84% [95% CI Q = 200.89, p < 0.0001] post three month treatment, −40.07% [95% CI −54.44, 47, p < 0.0001] post six month treatment, −58.44% [95% CI −66.41, −50.47, Q 001] post twelve month treatment, −65.69% [−80.05, −51.32, Q = 5.26, p = hteen month treatment and −62.11% [95% CI −72.68, −51.54, Q = 93.51, p < enty-four month treatment. ot was created to show a summary of the pooled analyses for the respective s (Figure 3) A forest plot was created to show a summary of the pooled analyses for the respective follow-up times (Figure 3). s (Figure 3). showing pooled analyses of percentage changes in WOMAC scores across the different follow-up ce intervals df = degrees of freedom N = number RE = random effects Tx = treatment group) Figure 3. Table 5. Cont. Forest plot showing pooled analyses of percentage changes in WOMAC scores across the different follow-up times (CI = conﬁdence intervals, df = degrees of freedom, N = number, RE = random effects, Tx = treatment group). showing pooled analyses of percentage changes in WOMAC scores across the different follow-up e intervals df = degrees of freedom N = number RE = random effects Tx = treatment group) Figure 3. Forest plot showing pooled analyses of percentage changes in WOMAC scores across the different follow-up times (CI = conﬁdence intervals, df = degrees of freedom, N = number, RE = random effects, Tx = treatment group). showing pooled analyses of percentage changes in WOMAC scores across the different follow-up e intervals, df = degrees of freedom, N = number, RE = random effects, Tx = treatment group) Figure 3. Forest plot showing pooled analyses of percentage changes in WOMAC scores across the different follow-up times (CI = conﬁdence intervals, df = degrees of freedom, N = number, RE = random effects, Tx = treatment group). showing pooled analyses of percentage changes in WOMAC scores across the different follow-up e intervals df = degrees of freedom N = number RE = random effects Tx = treatment group) Figure 3. Forest plot showing pooled analyses of percentage changes in WOMAC scores across the different follow-up times (CI = conﬁdence intervals, df = degrees of freedom, N = number, RE = random effects, Tx = treatment group). , , , g p) hows the pooled analyses for all the follow-up time periods. In every fol- reatment, there was an improvement in WOMAC scores. This improvement me went on, between less than month and up to 18 months post-treatment. ght reduction in improvement at 24 months, compared to 18 months. These Figure 3 shows the pooled analyses for all the follow-up time periods. In every follow- up, post-treatment, there was an improvement in WOMAC scores. This improvement increased as time went on, between less than month and up to 18 months post-treatment. There was a slight reduction in improvement at 24 months, compared to 18 months. These analyses were further pooled showing −48.02% [−59.16, −36.88, Q = 34.33, p < 0.0001]. Cells 2021, 10, 1365 20 of 31 20 of 31 This suggests that overall, there was a statistically signiﬁcant improvement in WOMAC scores post treatment. 3.2. Follow-Up The most common follow-up period was 6.5 months [63,64,72,74]. Three studies had a follow-up of 12 months [69–71]. Three studies had a follow-up of 24 months [38,60,61]. One study had a follow-up of one month [68]. Five studies had a follow-up between 12 and 24 months [49,65,67,73,74]. Two studies had a follow-up greater than 24 months [62,66]. 3.3. Adverse Events Only two studies have been found which did not report whether adverse events (AEs) or severe adverse events (SAEs) occurred during the clinical study [65,78] The rest of the studies all reported AEs. Two studies observed no AEs or SAEs during the study [67,71]. One of these studies however had four cases of complications which were deemed unrelated to the treatment regimen. These complications were high blood pressure, chest pain, dyspnoea, and urinary retention [67]. Eleven studies reported that subjects commonly experienced either transient pain or swelling of the joint after injection of the AMSCs or SVF [48,49,64–70,72–74,77]. In most patients this resolved spontaneously. In some patients, paracetamol was administered after which this resolved. Some studies also reported that subjects experienced discomfort at the site of lipoharvest [49,68,74]. However, this was resolved on further follow-up. Three studies reported patients experienced internal haematomas at the site of lipoharvest [49,74]. Four studies reported SAEs [49,63–66,72]. One subject in one study had a urinary stone. The subject had a past medical history of stones, and this was subsequently treated [63]. In another study, a subject experienced angina pectoris. However, they had risk factors of hypertension and hyperlipidaemia which predisposed them to the condition. In the remaining two studies, two patients and one patient respectively experienced severe pain and swelling following the procedure [49,66]. The two patients recovered after four weeks, while the single patient recovered after two weeks. 3.1. Classiﬁcation of Osteoarthritis Most studies in the literature used the Kellgren-Lawrence (KL) radiological classiﬁca- tion of osteoarthritis to grade the severity of OA in patients [48,49,62–64,66–71,73–75,78]. Due to the subjective nature of clinical diagnoses of OA, this was not used in any stud- ies. Several of the included studies used this classiﬁcation in their inclusion and ex- clusion criteria where for example, patients with a KL grade of one would not be in- cluded [48,49,63,64,66–69,71,72,75,78] in the study. Some studies also only included pa- tients who had an average pain intensity of four or more on the 10-point visual analogue scale for at least four months [48,63,64,70]. One study used the IKDC classiﬁcation to grade OA [65] and another used the Brandt Radiographic Grading Scale for Osteoarthritis [77]. 4. Discussion End-stage knee OA is currently managed with joint replacement surgery. This, how- ever, does not target the underlying disease process of OA, but rather the end stage symp- toms. Treatment options such as cartilage repair and osteotomy can delay the progression of OA, but do not modify the disease [79,80]. Recently, use of AMSCs has sprung into the clinical purview. AMSCs have the potential to regenerate new healthy articular cartilage and thus alleviate the symptoms of knee OA. The results of this systematic review and meta-analysis demonstrate that use of both AMSC and SVF treatments signiﬁcantly reduce WOMAC pain scores. This suggests that these treatments provide improved function and a reduction in pain. p Numerous advantages of AMSCs have been described in the literature [76,81]. How- ever, despite this, there is limited information on this topic in the literature, especially with regards to human studies. Animal studies are more widespread since the safety of use of AMSCs had to ﬁrst be established, this was ﬁrst conducted in mice. ter Huurne et al. (2012) conducted such a study (C57BL/6 mice), with early-stage collagenase induced OA. They found that injection of AMSCs into the knee joints of these mice, led to a reduction and inhibition of cartilage destruction and formation of enthesophytes. In addition, there was reduced synovial thickening and the treatments were safe. Use of mice studies, have allowed the evaluation of safety of AMSC transplantation to treat knee OA. Song et al., (2018) conducted a human clinical trial using adipose tissue derived stem cells. However, they ﬁrst conducted preclinical safety tests in vitro and on BALB/c-nu nude mice. After conﬁrming the safety of administering AMSCs isolated through their methodologies, they enrolled 18 patients in their clinical study. These patients were separated into three groups: low-dose (1 × 107), mid-dose (2 × 107), and high-dose (3 × 107). Each patient was injected three times and followed up over a course of 96 weeks. They found that use of AMSCs is safe for human use. y This study and several others documented AEs and SAEs. However, despite these occurrences, they did not cause long term detriments to the patients’ quality of life and in most cases spontaneously resolved. As such several studies deemed AMSC therapy to be safe [48,49,63,65,72–74]. 3.4. Outcome Measures Several studies recorded two primary outcomes: clinical and radiological outcomes. Studies utilised patient reported outcome measures (PROMs) to document clinical out- comes and the PROMs used varied greatly between studies (Tables 4 and 5). The most widely employed scoring systems were the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and the visual analogue scale (VAS). The former was used by ﬁfteen studies, while the latter was used by fourteen studies. The Knee Injury and Osteoarthritis Outcome Score (KOOS) was used by six studies. The Knee Society Score (KSS) was used by three studies. The Lysholm score was also employed by three studies. The Numeric Pain Rating Scale (NPRS), range of motion (ROM) and bone marrow oedema (BME) scoring were utilized by two studies. The Short Form-36 (SF-36), Hospital for Special Surgery Knee score (HSS), Patient Global Assessment score (PGA), Short Arthritis Assess- ment scale (SAS), International Knee Society (IKS) score, Japanese Knee Osteoarthritis Cells 2021, 10, 1365 21 of 31 21 of 31 Measure (JKOM), Joint Motion Amplitude (JMA) and the six minute walking distance score (6MWD) were each utilized by one study. Fewer papers assessed radiological outcomes. Six studies used radiographs to assess KL grades of patients after the said treatment was given. Several studies utilised Magnetic Resonance Imaging (MRI) to assess cartilage defects in the knee of patients. However, only seven of these used standardised radiological scoring systems. Freitag et al., (2019) used the MRI Osteoarthritis Knee Score (MOAKs). Koh et al., (2013) utilised the Whole-Organ MRI Score (WORMS) [66]. Spasovski et al. (2018) used the 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score [65]. Hong et al., (2019) used both WORMS and MOCART [70]. Nguyen et al., (2016), Garza et al., (2020) and Tran et al., (2019), all used the Outerbridge Classiﬁcation System (OS) [67,69,78]. Only two studies conducted a second look arthroscopy and subsequent histological analysis of the cartilage. Jo et al., (2014) used the International Cartilage Repair Society (ICRS) score for histological grading [63], while Pers et al. (2016) used Osteoarthritis Research Society International (OARSI) for histological grading [72]. 4. Discussion AMSC or SVF post treatment outcomes were determined by changes in WOMAC scores in fourteen of the eighteen studies included in this systematic review. One study conducted by Bansal et al. (2017) also used WOMAC scores, however since standard deviation or error ﬁgures were not provided, this was excluded from quantitative meta- analysis [77]. In the main pooled analysis, it was demonstrated that use of AMSCs and SVF in knee osteoarthritic joints improved WOMAC scores. All studies that documented WOMAC scores between two months and 24 months after treatment found that there was a sta- Cells 2021, 10, 1365 22 of 31 22 of 31 tistically signiﬁcant improvement in WOMAC scores after treatment. This was the case, regardless of the dose of AMSC or SVF used, or number of injections administered, sug- gesting that the less laborious preparation of SVF compared to MSCs may be an advantage as both therapies achieve good clinical outcomes. Furthermore, the pooled analyses in Figure 3 illustrates that there is a statistically signiﬁcant improvement in WOMAC scores across all follow-up times, suggesting disease modiﬁcation that persists and long-term efﬁcacy without the need for repeat administration of treatment. These improvements in- creased as time from initial treatment increased between less than one month and eighteen months post treatment. After this, at twenty-four months there was a slight decrease in improvements in WOMAC scores. Overall, this suggests that the therapies act beyond short-term analgesia, and lead to changes in the disease process. Improvements in these scores suggest that these AMSC and SVF treatments reduce pain and improve knee func- tion in patients with knee OA. Due to the low number of studies that compared AMSC and SVF therapies, we were unable to conduct a meta-analysis to directly compare these therapies. As there were also signiﬁcant heterogeneity in the studies, subgroup analyses of AMSC and SVF separately could not be conducted to yield meaningful results. The pooled analysis in this review offers ﬁndings that are generalizable to multiple adipose derived cell-based therapies. p Out of the eighteen studies included in this review, ﬁve investigated the effects dose of AMSCs, had on outcomes [48,62,63,72]. In four of the studies, patients were divided into three groups: a low dose group, medium dose group and high dose group. 4. Discussion Three of these studies found that patients in the high dose group gained greater clinical improvements than those in the low and medium dose groups. This suggests that there is a relationship between the number of AMSCs administered and the therapeutic effect gained. This seems intuitive, as joint-native MSCs in OA patients may have diminished capacity to proliferate and repair cartilage [82]. Thus, provision of healthy, functional MSCs could prevent further cartilage loss and repair existing defects. This is in line with conclusions made by two studies which investigated radiological outcomes. They discovered greatest reduction in cartilage defects were found in the high dose groups [62,63]. One can posit that this suggests that increasing dose is correlated with greater regenerative potential. Conclusions drawn by Pers et al., (2016) differed with the aforementioned studies, ﬁnding that the lowest dose group had the best improvements in clinical outcomes. This may have arisen, since the authors documented that the patients with the highest levels of inﬂammation were those in the low dose group [72]. AMSCs could have been primed to exert their immunoregulatory functions more efﬁciently in this more prominent inﬂammatory background. However other studies in the literature show similar ﬁndings, implying that lower dose administration of AMSCs may be more effective than high dose [83,84]. If low doses of AMSCs, are just as or more effective than high doses, then clinical and radiological improvements do not correlate with the number of cells used. Using less cells may reduce the laborious preparation required. Moreover, this may cut down on costs, making AMSCs more appealing for clinical use. Since very few studies investigated doses on outcomes, subgroup analyses were not conducted. Freitag et al., (2019) compared the effect one injection of 100 × 106 cells, to two injections of 100 × 106 cells. Both were compared to a control group. They concluded that two injections of AMSCs achieved more consistent OA stabilisation than one injection [49]. They were the only study to do this, which represents a gap in the literature. This indicates that multiple low dose injections of AMSCs may provide superior clinical and radiological improvements than a singular dose. These results may be reﬂective of the overall increased number of cells used in the double injection group. On the other hand, this increased efﬁcacy of the double injection treatment could be due to spaced out exposure to AMSCs. 4. Discussion Therefore, this suggests that the beneﬁts expressed by SVF is not dose dependent. Additionally, there was no visible quantiﬁable changes detected in cartilage thickness on the MRI scans between all three groups. The result from the radiological outcomes suggests that unlike AMSCs, which improve cartilage defects, SVF do not. Therefore, SVF treatment has no impact on the disease process of OA and only plays a role in symptomatic relief. This could be potentially explained by the fact that AMSCs have a higher number of colony forming ﬁbroblast units (CFU-F) and greater differentiation potential [58]. This study also further illustrates the limitations of hyaluronic acid for the treatment of knee OA. This review has found a gap in the literature, which must be addressed with studies investigated SVF dose and outcomes, to further determine the role of SVF therapy in the treatment of knee OA. py Koh et al., (2013), Bansal et al. (2017) and Nyugen et al. (2016) were the only studies which used a growth factor alongside SVF [66,67,77]. They all used a platelet rich plasma (PRP) scaffold. All studies concluded that there was signiﬁcant improvement in clinical scores long term post treatment. Use of PRP may have improved the efﬁcacy of treatments. PRP is known to enhance MSC proliferation and chondrocyte differentiation, and as such could bolster cartilage degeneration [85,86]. However, none of these studies compared use of PRP alone against SVF and PRP. As such the efﬁcacy of PRP cannot be quantiﬁed. Only with further studies, comparing AMSCs and SVF with and without PRP can we deﬁnitively determine the role of PRP in therapy for knee OA. If use of growth factor proves to increase efﬁcacy, these could be applied to AMSC therapy as well. Many of the studies included in this systematic review used K-L grading of OA as an inclusion and exclusion criterion in the recruitment of their patients. Out of all studies, only one included patients with a K-L grade of 1 [73]. As such most of the studies could not determine the efﬁcacy of treatments on low grade knee OA. Since, there is reduced levels of inﬂammation in the earlier stages of OA than end stages, AMSC and SVF therapy may be more effective. Alternatively, these therapies may be more effective in end stage OA, since the high inﬂammatory environment could modulate cells to exercise their immunoregulatory function more effectively. 4. Discussion Repetitive low dose spaced out injections of AMSCs may prove to be more successful than a single large dose injection. Nevertheless, there is also a possibility that there is a ceiling on the correlation between numbers of injections and improvements seen. Moreover, Freitag et al., (2019) reported an increase in the number of moderate AEs in the second Cells 2021, 10, 1365 23 of 31 injection group. This implies that increased numbers of injections are linked to increased AEs, which may affect the tolerate of the therapy in question. injection group. This implies that increased numbers of injections are linked to increased AEs, which may affect the tolerate of the therapy in question. y py q Yakota et al. (2019) was the only study that compared the efﬁcacy of AMSCs with SVF [74]. They found that both therapies improved osteoarthritic symptoms and pain. However, when they analysed the clinical scores, they found that the improvements were more signiﬁcant and occurred earlier in the AMSC group. Earlier clinical improvements suggest that AMSCs have a faster mechanism of action. More signiﬁcant clinical improve- ments suggest that AMSCs are superior to SVF for symptomatic control of knee OA. No radiological outcomes were investigated in this study. Therefore, no conclusions can be drawn regarding the cartilage regenerative potential of either treatment. However, other studies have shown that AMSCs have great cartilage regenerative potentials, and reduce cartilage defects [62,63]. Yakota et al., (2019) also discovered that there was a higher fre- quency of knee effusion and minor complications related to the harvesting of adipose tissue in the SVF patient group [74]. As such iatrogenic complications may be higher in SVF. This may affect tolerate and favourability of such treatment in the future. More clinical studies need to be conducted to make robust conclusions regarding which treatment is superior. g g p Garza et al., (2020) was the only study to compare the effects of different doses of SVF on clinical and radiological outcomes. They led a study in which they compared a placebo group using hyaluronic acid, with a low dose SVF group (1.5 × 107 cells) and a high dose SVF group (3.0 × 107 cells) [69]. They discovered both doses of SVF resulted in improved WOMAC scores compared to the placebo. Nevertheless, there was no statistical difference between the high and low dose groups. 4. Discussion To conﬁdently determine the role of AMSC treatment in knee OA, we must understand the qualities and mechanism of cartilage repair involved. p Out of all the studies, six used arthroscopies prior to injection of the treatment. Roata et al. (2019) and Jo et al., (2017) only used arthroscopy as guidance for injection of the AMSCs into the osteoarthritic site [62,73]. However, the rest of the studies performed arthroscopic debridement prior to injection of the treatment [67,70,74]. As such it is possible that this led to bias of outcome. Due to the heterogeneity in treatment modality between the studies, and the low number of studies that examined arthroscopic delivery, sub-group analysis was not conducted. It should be noted that arthroscopy for the treatment of knee OA has been shown to be largely ineffective [90,91]. However, arthroscopic debridement removes inﬂammatory synovial ﬂuid which can interfere with AMSC adhesion in vitro, and therefore increasing the effectiveness of said treatment [73]. As such, arthroscopic debridement may prime the joint to become more responsive to injections. Consequently, one cannot deﬁnitively rule out the effect arthroscopy has on the clinical and radiological outcomes of AMSC treatment. Five of the studies included, utilised ultrasound to aid guidance of the AMSC or SVF injections. When the outcomes of these studies are compared to those which used arthroscopy or utilised neither, there is no difference in clinical outcomes. As mentioned previously, only two studies conducted second look arthroscopies. Hence, we cannot determine whether use of image guidance leads to greater cartilage regeneration. Future studies should directly compare use of image guidance against blind injection. In addition, studies should perform radiological and histological analysis on all patients to determine if imaging guidance has any bearing on cartilage regeneration. There was a lack of long-term studies carried out, as shown in the results. The average follow-up across all studies was 60.1 weeks. The longest follow-up period was 104 weeks [62]. This would be considered a short-term follow-up. Thus, there is a gap in the literature for such long-term studies. As a result, the effect AMSC injections and SVF injections have on long term knee OA is unknown. The importance of this was made evident by Park et al., (2016). They conducted a study in which they investigated cartilage regeneration in OA patients, through use of umbilical cord derived MSCs. They discovered that three years after treatment, cartilage repairs persisted. 4. Discussion In addition, only eight studies included patients with K-L grade 4 [48,62–64,68,71,72,74]. As such there is less data on the effects of AMSC treatment on end stage severe knee OA, and more on middle stage knee OA. Furthermore, most studies did not stratify patients according to the severity of the OA. As such they could not determine whether the severity of knee OA has any bearing on the Cells 2021, 10, 1365 24 of 31 24 of 31 efﬁcacy of the MSC treatment. Nonetheless, three of the studies did make observations based on this. Tran et al., (2019) inferred that treatment was more effective in patients with KL grade 3 than with grade 2 [78]. In the higher severities of OA, there is greater inﬂammation. On the other hand, Nyugen et al., (2016) and Yokota et al., (2019) found that lower K-L grades had greater clinical improvements, indicating that efﬁcacy was greater in patients with less severe OA [67,74]. If this is the case, more studies need to be conducted to include K-L grade 1 patients. g p In addition to K-L grading, studies did not stratify patient cohorts according to age or BMI. Thus, the impact these could have on the efﬁcacy of outcomes is unknown. It is possible that in younger patients, who have superior regenerative potential, the quality and therefore efﬁcacy of AMSC and SVF is superior to elderly patients [87]. Maredziak et al. (2016) illustrated that there is reduced CFU-F, proliferation rates, and quantiﬁed chondro- genic and osteogenic differentiation in aged AMSC cells. Furthermore, aged cells seem to shift more in favour of adipogenic differentiation [88]. In addition, it has been shown that there is a biological role of adipose inﬂammation in obese patients and OA [89]. As such, it is possible, this mechanism of OA may respond differently to AMSC and SVF treatments, compared to age related articular cartilage degeneration. Therefore, stratifying patients into different BMI groups, may be of beneﬁt. However, this must be investigated further before deﬁnitive conclusions can be made. The gold standard of evaluating new-born cartilage in the face of cartilage repair, is second look arthroscopy and histological biopsy. It is important to determine quantify the size of cartilage regeneration as well as the constitution of the cartilage. Nevertheless, only two studies performed such procedures [63,72]. 4. Discussion This implied that use of such Cells 2021, 10, 1365 25 of 31 25 of 31 MSCs could provide a long-term solution to OA. Conversely, Jo et al., (2017) found that two years post treatment, cartilage deterioration was apparent. As such, it is possible that the effect of AMSC and SVF treatment may be limiting, and further injections may be required for persistence of cartilage regeneration. Alternatively, it could be possible after several years, the knee joint becomes unresponsive to AMSC and SVF treatment injections. Only with long term studies, can the lasting implications of this treatment be determined. Understandably, the studies included in this review are very recent, as such long-term studies with 5–10-year follow-ups are not possible at this stage. There are signiﬁcant variations in the outcome measures utilised by studies. In terms of clinical outcomes, most studies using WOMAC and VAS. However, several did not. As a result, the clinical outcomes recorded are harder to compare between studies. Scoring systems such as WOMAC and VAS are non-speciﬁc. Perhaps creation of a PROM speciﬁcally for post intra-articular injection would be more beneﬁcial. Use of a handful of scoring systems rather than a wide array may allow for better comparison between studies and therefore allow scientists to determine what the most effective treatment for knee OA. SVF can be prepared through various methods. It has been shown that different methods have different compositions and properties [92]. This has not been addressed by any of the studies included in this review which investigated SVF. As a result, it may be unwise and inaccurate to compare the results of studies which used differing methods to produce their SVF. The gold standard in medical research when testing the efﬁcacy of a new treatment is a double blinded randomised controlled trial (RCT). However, use of AMSCs is very novel. Many of the studies included in this systematic review were carried out as pilot studies. Out of these studies, only six used a control group [49,64,67,69,70,78] As such the other studies are unable to truly deﬁne the efﬁcacy of the treatments tested. Many of these studies recognised this, however their principal goal was to determine the safety of AMSCs rather than its effectiveness. They stated that future studies should include control arms in clinical trials. 4. Discussion However, Freitag et al., (2019) and Pers et al., (2016) believed there may be ethical concerns with conducting an RCT [48–78]. All patients would have to undergo a lipoharvest procedure before randomisation. This procedure is not without complications. A study conducted by Comella et al. (2017) found that such complications are low in lipoharvest procedures [93]. Since these studies were pilot in nature, their sample sizes were also very small, with the largest being 80 patients, and the average being 30.5 across all studies. 5. Conclusions The studies in this systematic review have established the safety and efﬁcacy of both AMSC therapy and SVF therapy for knee OA in humans. In addition, the meta-analyses show that use of AMSC and SVF therapy for knee OA deﬁnitively improves WOMAC scores up to two-years, with improvements increasing with time. This suggest that AMSC and SVF treatments reduce pain and improve knee function in patients with severe knee OA. Future clinical studies must now incorporate control arms and have larger sample sizes to successfully determine the effectiveness of these treatments. Speciﬁcally, there is little to no literature on studies comparing use of single vs multiple AMSC injections, comparing AMSC treatments with SVF treatments and the effect of different doses of SVF on outcomes. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/cells10061365/s1, Table S1 Critical appraisal of the non-randomised studies included in this systematic review, using the ROBINS-1 tool n = 13, Table S2. Critical appraisal of the randomised studies included in this systematic review, using the RoB-2 tool n = 5 Author Contributions: N.A. wrote the manuscript under the supervision of K.T. and W.K. K.T. and W.K. conceptualized the study. C.B. and C.M. analysed the data and conducted risk of bias analysis. Cells 2021, 10, 1365 26 of 31 26 of 31 All authors reviewed the data, evaluated, drafted and approved the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not Applicable. Institutional Review Board Statement: Not Applicable. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Acknowledgments: In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A Databases searched: OVID MEDLINE®: 1946 TO JUNE WEEK 4 2020 Date of search: 29/06/20 Date range searched: January 1946 to June 2020 SEARCH STRATEGY 1. exp cartilage/ or exp “bone and bones”/ 2. exp Injections, Intra-Articular/ 3. exp Osteoarthritis, Hip/ or exp Osteoarthritis/ or exp Osteoarthritis, Spine/ or exp Osteoarthritis, Knee/ 4. 5. Conclusions exp Mesenchymal Stem Cells/ or exp Mesenchymal Stem Cell Transplantation/ 5. exp Transplantation, Homologous/ or exp Stem Cell Transplantation/ or exp Trans- plantation, Autologous/ or exp Mesenchymal Stem Cell Transplantation/ 6. exp Adipose Tissue/ or adipose.mp. 7. adipo.tw. 8. exp Autografts/ 9. exp Heterografts/ 10. 4 or 5 or 8 or 9 11. 1 or 2 or 10 12. 6 or 7 13. 11 and 12 14. 3 and 13 15. Limit 14 to (English language and humans) EMBASE: 1974 TO 2019 JUNE 26 Date of search: 29/06/20 Date range searched: January 1974 to June 2019 SEARCH STRATEGY 1. exp cartilage/ 2. exp intraarticular drug administration/ 3. exp knee osteoarthritis/ 4. exp mesenchymal stem cell transplantation/ or exp mesenchymal stem cell/ 5. exp autograft/ 6. exp adipose tissue/ 7. adipo.tw. 8. 4 or 5 9. 1 or 2 or 8 10. 6 or 7 11. 9 and 10 12. 3 and 11 13. Limit 12 to (human and English language) Appendix A Databases searched: OVID MEDLINE®: 1946 TO JUNE WEEK 4 2020 Date of search: 29/06/20 Date range searched: January 1946 to June 2020 SEARCH STRATEGY 1. exp cartilage/ or exp “bone and bones”/ 2. exp Injections, Intra-Articular/ 3. exp Osteoarthritis, Hip/ or exp Osteoarthritis/ or exp Osteoarthritis, Spine/ or exp Osteoarthritis, Knee/ 4. exp Mesenchymal Stem Cells/ or exp Mesenchymal Stem Cell Transplantation/ 5. exp Transplantation, Homologous/ or exp Stem Cell Transplantation/ or exp Trans- plantation, Autologous/ or exp Mesenchymal Stem Cell Transplantation/ 6. exp Adipose Tissue/ or adipose.mp. 7. adipo.tw. 8. exp Autografts/ 9. exp Heterografts/ 10. 4 or 5 or 8 or 9 11. 1 or 2 or 10 12. 6 or 7 13. 11 and 12 14. 3 and 13 15. Limit 14 to (English language and humans) EMBASE: 1974 TO 2019 JUNE 26 Date of search: 29/06/20 Date range searched: January 1974 to June 2019 SEARCH STRATEGY 1. exp cartilage/ 2. exp intraarticular drug administration/ 3. exp knee osteoarthritis/ 4. exp mesenchymal stem cell transplantation/ or exp mesenchymal stem cell/ 5. exp autograft/ 6. exp adipose tissue/ 7. adipo.tw. 8. 4 or 5 9. 1 or 2 or 8 10. 6 or 7 11. 9 and 10 12. 3 and 11 13. Limit 12 to (human and English language) #1: (TS = (cartilage or inject)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #2: (TS=(osteoarthritis)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #3: (TS=(mesenchymal stem cell or stem cell or homologous or autologous or autograft or heterograft)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #4: (TS=(adipose)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #5: #3 or #1 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #6: #5 and #2 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #7: #6 and #4 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #1: (TS = (cartilage or inject)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #2: (TS=(osteoarthritis)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #3: (TS=(mesenchymal stem cell or stem cell or homologous or autologous or autograft or heterograft)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #4: (TS=(adipose)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #5: #3 or #1 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #6: #5 and #2 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #7: #6 and #4 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 CLINICAL TRIALS.GOV: 1900 TO 2020 Date of search: 29/06/20 References 1. Vos, T.; Lim, S.S.; Abbafati, C.; Abbas, K.M.; Abbasi, M.; Abbasifard, M.; Abbasi-Kangevari, M.; Abbastabar, H.; Abd-Allah, F.; Abdelalim, A.; et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1204–1222. [CrossRef] y 2. WHO. Chronic Rheumatic Conditions; WHO: Geneva, Switzerla 2. WHO. Chronic Rheumatic Conditions; WHO: Geneva, Switzerland, 2016. 2. WHO. Chronic Rheumatic Conditions; WHO: Geneva, Switzerland, 2016. O. Chronic Rheumatic Conditions; WHO: Geneva, Switze 3. Veronese, N.; Stubbs, B.; Solmi, M.; Smith, T.O.; Noale, M.; Cooper, C.; Maggi, S. Association between lower limb osteoarthritis and incidence of depressive symptoms: Data from the osteoarthritis initiative. Age Ageing 2017, 46, 470–476. [CrossRef] p y p g g g , , 4. Kye, S.-Y.; Park, K. Suicidal ideation and suicidal attempts among adults with chronic diseases: A cross-section 2017, 73, 160–167. [CrossRef] [PubMed] 5. Schieir, O.; Tosevski, C.; Glazier, R.H.; Hogg-Johnson, S.; Badley, E.M. Incident myocardial infarction associated with major types of arthritis in the general population: A systematic review and meta-analysis. Ann. Rheum. Dis. 2017, 76, 1396–1404. [CrossRef] 6. Hubertsson, J.; Turkiewicz, A.; Petersson, I.F.; Englund, M. Understanding Occupation, Sick Leave, and Disability Pension Due to Knee and Hip Osteoarthritis from a Sex Perspective. Arthritis Rheum. 2017, 69, 226–233. [CrossRef] p p 7. Zhao, X.; Shah, D.; Gandhi, K.; Wei, W.; Dwibedi, N.; Webster, L.; Sambamoorthi, U. Clinical, humanistic, and economic burden of osteoarthritis among noninstitutionalized adults in the United States. Osteoarthr. Cartil. 2019, 27, 1618–1626. [CrossRef] 8. Salmon, J.; Rat, A.; Achit, H.; Ngueyon-Sime, W.; Gard, C.; Guillemin, F.; Jolly, D.; Fautrel, B. Health resource use and costs of symptomatic knee and/or hip osteoarthritis. Osteoarthr. Cartil. 2019, 27, 1011–1017. [CrossRef] 7. Zhao, X.; Shah, D.; Gandhi, K.; Wei, W.; Dwibedi, N.; Webster, L.; Sambamoorthi, U. Clinical, humanistic, and economic burden of osteoarthritis among noninstitutionalized adults in the United States. Osteoarthr. Cartil. 2019, 27, 1618–1626. [CrossRef] of osteoarthritis among noninstitutionalized adults in the United States. Osteoarthr. Cartil. 2019, 27, 1618–1626. [CrossRef] 8. Salmon, J.; Rat, A.; Achit, H.; Ngueyon-Sime, W.; Gard, C.; Guillemin, F.; Jolly, D.; Fautrel, B. Health resource use and costs of symptomatic knee and/or hip osteoarthritis. Osteoarthr. Cartil. 2019, 27, 1011–1017. [CrossRef] 8. Salmon, J.; Rat, A.; Achit, H.; Ngueyon-Sime, W.; Gard, C.; Guillemin, F.; Jolly, D.; Fautrel, B. g g p // g/ /g / g g ( y ) 11. Ackerman, I.N.; Pratt, C.; Gorelik, A.; Liew, D. Projected Burden of Osteoarthritis and Rheumatoid Arthritis in Australia: A Population-Level Analysis. Arthritis Rheum. 2018, 70, 877–883. [CrossRef] Appendix A Appendix A Databases searched: OVID MEDLINE®: 1946 TO JUNE WEEK 4 2020 Date of search: 29/06/20 Date range searched: January 1946 to June 2020 SEARCH STRATEGY 1. exp cartilage/ or exp “bone and bones”/ 2. exp Injections, Intra-Articular/ 3. exp Osteoarthritis, Hip/ or exp Osteoarthritis/ or exp Osteoarthritis, Spine/ or exp Osteoarthritis, Knee/ 4. exp Mesenchymal Stem Cells/ or exp Mesenchymal Stem Cell Transplantation/ 5. exp Transplantation, Homologous/ or exp Stem Cell Transplantation/ or exp Trans plantation, Autologous/ or exp Mesenchymal Stem Cell Transplantation/ 6. exp Adipose Tissue/ or adipose.mp. 7. adipo.tw. 8. exp Autografts/ 9. exp Heterografts/ 10. 4 or 5 or 8 or 9 11. 1 or 2 or 10 12. 6 or 7 13. 11 and 12 14. 3 and 13 15. Limit 14 to (English language and humans) EMBASE: 1974 TO 2019 JUNE 26 Date of search: 29/06/20 Date range searched: January 1974 to June 2019 SEARCH STRATEGY 1. exp cartilage/ 2. exp intraarticular drug administration/ 3. exp knee osteoarthritis/ 4. exp mesenchymal stem cell transplantation/ or exp mesenchymal stem cell/ 5. exp autograft/ 6. exp adipose tissue/ 7. adipo.tw. 8. 4 or 5 9. 1 or 2 or 8 10. 6 or 7 11. 9 and 10 12. 3 and 11 13. Appendix A Limit 12 to (human and English language) 27 of 31 Cells 2021, 10, 1365 COCHRANE LIBRARY: 1946 TO JUNE 2020 Date of search: 29/06/20 Date range searched: January 1946 to June 2020 SEARCH STRATEGY #1 MeSH descriptor: [Cartilage] explode all trees #2 MeSH descriptor: [Injections] explode all trees #3 MeSH descriptor: [Osteoarthritis] explode all trees #4 MeSH descriptor: [Mesenchymal Stem Cells] explode all trees #5 MeSH descriptor: [Stem Cells] explode all trees #6 MeSH descriptor: [Adipose Tissue] explode all trees #7 MeSH descriptor: [Autografts] in all MeSH products #8 MeSH descriptor: [Heterografts] in all MeSH products #9 #1 or #2 or #3 #10 #4 or #5 or #6 or #7 or #8 #11 #10 and #9 #12 #11 and #6 WEB OF SCIENCE: 1900 TO 2020 Date of search: 29/06/20 Date range searched: 1900 to June 2020 SEARCH STRATEGY #1: (TS = (cartilage or inject*)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #2: (TS=(osteoarthritis)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #3: (TS=(mesenchymal stem cell or stem cell or homologous or autologous or autograft or heterograft)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #4: (TS=(adipose)) Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #5: #3 or #1 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #6: #5 and #2 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 #7: #6 and #4 Indexes=SCI-EXPANDED, CPCI-S Timespan=1900–2020 CLINICAL TRIALS.GOV: 1900 TO 2020 Date of search: 29/06/20 Date range searched: 1900to July 2019 #1 Osteoarthritis, knee #2 Adipose References Martel-Pelletier, J.; Lajeunesse, D.; Pelletier, J.-P. Arthritis and Allied Conditions: A Textbook of Rheumatology, 15th ed.; Williams & Wilkins: Philadelphia, PA, USA, 2005. set, M.; Berenbaum, F.; Gabay, C. The Role of IL-1 and IL-1Ra in Joint Inﬂammation and Cartilage Degradat 06, 74, 371–403. [CrossRef] 19. Jacques, C.; Gosset, M.; Berenbaum, F.; Gabay, C. The Role of IL-1 and IL-1Ra in Joint Inﬂammation Vitam. Horm. 2006, 74, 371–403. [CrossRef] 20. Li, H.; Shen, S.; Fu, H.; Wang, Z.; Li, X.; Sui, X.; Yuan, M.; Liu, S.; Wang, G.; Guo, Q. Immunomodulatory Functions of Mesenchymal Stem Cells in Tissue Engineering. Stem Cells Int. 2019, 2019, 1–18. [CrossRef] [PubMed] y g g 21. Hui, W.; Young, A.D.; Rowan, A.; Xu, X.; Cawston, E.T.; Proctor, C.J. Oxidative changes and signalling pathways are pivotal in initiating age-related changes in articular cartilage. Ann. Rheum. Dis. 2014, 75, 449–458. [CrossRef] 22. Loeser, R.F. Aging and osteoarthritis: The role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthr. Cartil. 2009, 17, 971–979. [CrossRef] [PubMed] 23. Vincent, T.L. IL-1 in osteoarthritis: Time for a critical review of the literature. F1000Research 2019, 8, 934. [CrossRef] [PubMed] 24. Philp, A.; Davis, E.T.; Jones, S. Developing anti-inﬂammatory therapeutics for patients with osteoarthritis. Rheumatology 2016, 56, 869–881. [CrossRef] 24. Philp, A.; Davis, E.T.; Jones, S. Developing anti-inﬂammatory therapeutics for patients with osteoarthritis. Rheumatology 2016, 56, 869–881. [CrossRef] 25. Pittenger, M.F.; Mackay, A.M.; Beck, S.C.; Jaiswal, R.K.; Douglas, R.; Mosca, J.D.; Moorman, M.A.; Simonetti, D.W.; Craig, S.; Marshak, D.R. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999, 284, 143–147. [CrossRef] [PubMed] 26. Cho, D.-I.; Kim, M.R.; Jeong, H.-Y.; Jeong, H.C.; Jeong, M.H.; Yoon, S.H.; Kim, Y.S.; Ahn, Y. Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages. Exp. Mol. Med. 2014, 46, e70. [CrossRef] [PubMed] 27. Abumaree, M.H.; Al Jumah, M.A.; Kalionis, B.; Jawdat, D.; Al Khaldi, A.; Abomaray, F.M.; Fatani, A.S.; Chamley, L.W.; Knawy, B.A. Human Placental Mesenchymal Stem Cells (pMSCs) Play a Role as Immune Suppressive Cells by Shifting Macrophage Dif- ferentiation from Inﬂammatory M1 to Anti-inﬂammatory M2 Macrophages. Stem Cell Rev. Rep. 2013, 9, 620–641. [CrossRef] [PubMed] 28. Saldaña, L.; Bensiamar, F.; Vallés, G.; Mancebo, F.J.; García-Rey, E.; Vilaboa, N. Immunoregulatory potential of mesenchymal stem cells following activation by macrophage-derived soluble factors. Stem Cell Res. Ther. 2019, 10, 58. [CrossRef] [PubMed] 29. Tse, W.T.; Pendleton, J.D.; Beyer, W.M.; Egalka, M.C.; Guinan, E.C. References Health resource use and costs of symptomatic knee and/or hip osteoarthritis. Osteoarthr. Cartil. 2019, 27, 1011–1017. [CrossRef] p p 10. United Nations. Ageing\|United Nations. Available online: https://www.un.org/en/global-issues/ag p p 10. United Nations. Ageing\|United Nations. Available online: https://www.un.org/en/global-issues/ageing (accessed on 5 May 2021). 10. United Nations. Ageing\|United Nations. Available online: https://www.un.org/en/global-issues/ageing (accessed on 5 May 2021). 11. Ackerman, I.N.; Pratt, C.; Gorelik, A.; Liew, D. Projected Burden of Osteoarthritis and Rheumatoid Arthritis in Australia: A Population-Level Analysis. Arthritis Rheum. 2018, 70, 877–883. [CrossRef] 11. Ackerman, I.N.; Pratt, C.; Gorelik, A.; Liew, D. Projected Burden of Osteoarthritis and Rheumatoid Arthritis in Australia: A Population-Level Analysis. Arthritis Rheum. 2018, 70, 877–883. [CrossRef] Cells 2021, 10, 1365 28 of 31 28 of 31 12. Turkiewicz, A.; Petersson, I.; Björk, J.; Hawker, G.; Dahlberg, L.; Lohmander, L.; Englund, M. Current and future impact of osteoarthritis on health care: A population-based study with projections to year 2032. Osteoarthr. Cartil. 2014, 22, 1826–1832. [CrossRef] 13. Hootman, J.M.; Helmick, C.G. Projections of US prevalence of arthritis and associated activity limit 54, 226–229. [CrossRef] [PubMed] 14. Sen, R.; Hurley, J.A. Osteoarthritis; StatPearls Publishing: Treasure Island, FL, USA, 2020. y g 15. Evans, J.T.; Walker, R.W.; Blom, A.W.; Sayers, A.; Whitehouse, M.R. How long does a knee replacement last? A systematic review and meta-analysis of case series and national registry reports with more than 15 years of follow-up. Lancet 2019, 393, 655–663. [CrossRef] [ ] 16. Kapoor, M.; Martel-Pelletier, J.; Lajeunesse, D.; Pelletier, J.-P.; Fahmi, H. Role of proinﬂammatory cytokines in the pathophysiology of osteoarthritis. Nat. Rev. Rheumatol. 2010, 7, 33–42. [CrossRef] 17. Kobayashi, M.; Squires, G.R.; Mousa, A.; Tanzer, M.; Zukor, D.J.; Antoniou, J.; Feige, U.; Poole, A.R. Role of interleukin-1 and tumor necrosis factor α in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum. 2005, 52, 128–135. [CrossRef] 18 M l P ll i J L j D P ll i J P A th iti d Alli d C diti A T tb k f Rh t l 15 h d Willi & 17. Kobayashi, M.; Squires, G.R.; Mousa, A.; Tanzer, M.; Zukor, D.J.; Antoniou, J.; Feige, U.; Poole, A.R. Role of interleukin-1 and tumor necrosis factor α in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum. 2005, 52, 128–135. [CrossRef] 18. Martel-Pelletier, J.; Lajeunesse, D.; Pelletier, J.-P. Arthritis and Allied Conditions: A Textbook of Rheumatology, 15th ed.; Williams & Wilkins: Philadelphia, PA, USA, 2005. 18. References Suppression of allogeneic T-cell proliferation by human marrow stromal cells: Implications in transplantation. Transplantation 2003, 75, 389–397. [CrossRef] [PubMed] 30. Le Blanc, K.; Tammik, L.; Sundberg, B.; Haynesworth, S.E.; Ringden, O. Mesenchymal Stem Cells Inhibit and Stimulate Mixed Lymphocyte Cultures and Mitogenic Responses Independently of the Major Histocompatibility Complex. Scand. J. Immunol. 2003, 57, 11–20. [CrossRef] 31. Najar, M.; Rouas, R.; Raicevic, G.; Boufker, H.I.; Lewalle, P.; Meuleman, N.; Bron, D.; Toungouz, M.; Martiat, P.; Lagneaux, L. Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: The importance of low cell ratio and role of interleukin-6. Cytotherapy 2009, 11, 570–583. [CrossRef] [PubMed] p y py 32. Castro-Oropeza, R.; Vazquez-Santillan, K.; Díaz-Gastelum, C.; Melendez-Zajgla, J.; Zampedri, C.; Ferat-Osorio, E.; Rodríguez- González, A.; Arriaga-Pizano, L.; Maldonado, V. Adipose-derived mesenchymal stem cells promote the malignant phenotype of cervical cancer. Sci. Rep. 2020, 10, 14205. [CrossRef] p 33. Zhao, M.; Sachs, P.C.; Wang, X.; Dumur, C.I.; Idowu, M.O.; Robila, V.; Francis, M.P.; Ware, J.; Beckman, M.; Rizki, A.; et al. Mesenchymal stem cells in mammary adipose tissue stimulate progression of breast cancer resembling the basal-type. Cancer Biol. Ther. 2012, 13, 782–792. [CrossRef] [PubMed] 34. Dominici, M.; Le Blanc, K.; Mueller, I.; Slaper-Cortenbach, I.; Marini, F.C.; Krause, D.S.; Deans, R.J.; Keating, A.; Prockop, D.J.; Horwitz, E.M. Minimal criteria for deﬁning multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006, 8, 315–317. [CrossRef] 35. Fan, J.; Varshney, R.R.; Ren, L.; Cai, D.; Wang, D.-A. Synovium-Derived Mesenchymal Stem Cells: A New Cell Source for Musculoskeletal Regeneration. Tissue Eng. Part B Rev. 2009, 15, 75–86. [CrossRef] [PubMed] 36. Vandenabeele, F.; De Bari, C.; Moreels, M.; Lambrichts, I.; Dell’Accio, F.; Lippens, P.L.; Luyten, F.P. Morphological and immunocy- tochemical characterization of cultured ﬁbroblast-like cells derived from adult human synovial membrane. Arch. Histol. Cytol. 2003, 66, 145–153. [CrossRef] 29 of 31 Cells 2021, 10, 1365 29 of 31 37. Choi, Y.-S.; Noh, S.-E.; Lim, S.-M.; Lee, C.-W.; Kim, C.-S.; Im, M.-W.; Lee, M.-H.; Kim, D.-I. Multipotency and growth characteristic of periosteum-derived progenitor cells for chondrogenic, osteogenic, and adipogenic differentiation. Biotechnol. Lett. 2007, 30, 593–601. [CrossRef] [ ] 39. Vega, A.; Martín-Ferrero, M.A.; Del Canto, F.; Alberca, M.; García, V.; Munar, A.; Orozco, L.; Soler, R.; Fuertes, J.J.; Huguet, M.; et al. Treatment of Knee Osteoarthritis with Allogeneic Bone Marrow Mesenchymal Stem Cells. Transplantation 2015, 99, 1681–1690. [CrossRef] 40. References [CrossRef] [PubMed] 52. El-Badawy, A.; Amer, M.; Abdelbaset, R.; Sherif, S.N.; Abo-Elela, M.; Ghallab, Y.H.; Elhamid, H.A.; Ismail, Y.; El-Badri, N. Adipose Stem Cells Display Higher Regenerative Capacities and More Adaptable Electro-Kinetic Properties Compared to Bone Marrow-Derived Mesenchymal Stromal Cells. Sci. Rep. 2016, 6, 37801. [CrossRef] y p i, R.P.; Salim, A.; Longaker, M.T. The Osteogenic Potential of Adipose-Derived Mesenchymal Cells Is ging. Plast. Reconstr. Surg. 2005, 116, 1686–1696. [CrossRef] [PubMed] 53. Shi, Y.-Y.; Nacamuli, R.P.; Salim, A.; Longaker, M.T. The Osteogenic Potential of Adipose-Der Maintained with Aging. Plast. Reconstr. Surg. 2005, 116, 1686–1696. [CrossRef] [PubMed] ained with Aging. Plast. Reconstr. Surg. 2005, 116, 1686–1696. [CrossRef] [PubMed] 54. Beane, O.S.; Fonseca, V.C.; Cooper, L.L.; Koren, G.; Darling, E.M. Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells. PLoS ONE 2014, 9, e115963. [CrossRef] 55. Song, W.-J.; Li, Q.; Ryu, M.-O.; Ahn, J.-O.; Bhang, D.H.; Jung, Y.C.; Youn, H.-Y. TSG-6 Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells Ameliorates DSS-induced colitis by Inducing M2 Macrophage Polarization in Mice. Sci. Rep. 2017, 7, 5187. [CrossRef] 56. Manferdini, C.; Maumus, M.; Gabusi, E.; Piacentini, A.; Filardo, G.; Peyraﬁtte, J.-A.; Jorgensen, C.; Bourin, P.; Fleury- Cappellesso, S.; Facchini, A.; et al. Adipose-Derived Mesenchymal Stem Cells Exert Antiinﬂammatory Effects on Chondrocytes and Synoviocytes From Osteoarthritis Patients Through Prostaglandin E2. Arthritis Rheum. 2013, 65, 1271–1281. [CrossRef] y y g g 57. Ortiz-Virumbrales, M.; Menta, R.; Pérez, L.M.; Lucchesi, O.; Mancheño-Corvo, P.; Avivar-Valderas, Á.; Palacios, I.; Herrero- Mendez, A.; Dalemans, W.; De La Rosa, O.; et al. Human adipose mesenchymal stem cells modulate myeloid cells toward an anti-inﬂammatory and reparative phenotype: Role of IL-6 and PGE2. Stem Cell Res. Ther. 2020, 11, 462. [CrossRef] 58. Bourin, P.; Bunnell, B.A.; Casteilla, L.; Dominici, M.; Katz, A.J.; March, K.L.; Redl, H.; Rubin, J.P.; Yoshimura, K.; Gimble, J.M. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 2013, 15, 641–648. [CrossRef] py ( ) y py 59. Lin, K.; Matsubara, Y.; Masuda, Y.; Togashi, K.; Ohno, T.; Tamura, T.; Toyoshima, Y.; Sugimachi, K.; To Characterization of adipose tissue-derived cells isolated with the Celution™system. Cytotherapy 2008, 60. Page, M.J.; McKenzie, J.E.; Bossuyt, P.M.; Boutron, I.; Hoffmann, T.C.; Mulrow, C.D.; Shamseer, L.; Tetzlaff, J.M.; Moher, D. References Charbord, P. Bone Marrow Mesenchymal Stem Cells: Historical Overview and Concepts. Hum. Gene Ther. 2010, 21, 1045–1056. [CrossRef] [PubMed] 41. Pontikoglou, C.; Deschaseaux, F.; Sensebé, L.; Papadaki, H.A. Bone Marrow Mesenchymal Stem Cells: Their Role in Hematopoiesis and Hematopoietic Stem Cell Transplantation. Stem Cell Rev. Rep. 2011, 7 p p p p 42. Lin, H.; Sohn, J.; Shen, H.; Langhans, M.T.; Tuan, R.S. Bone marrow mesenchymal stem cells: Aging and tissue engineering applications to enhance bone healing. Biomaterials 2019, 203, 96–110. [CrossRef] [PubMed] 43. Song, Y.; Du, H.; Dai, C.; Zhang, L.; Li, S.; Hunter, D.J.; Lu, L.; Bao, C. Human adipose-derived mesenchymal stem cells for osteoarthritis: A pilot study with long-term follow-up and repeated injections. Regen. Med. 2018, 13, 295–307. [CrossRef] 44. Freitag, J.; Wickham, J.; Shah, K.; Tenen, A. Effect of autologous adipose-derived mesenchymal stem cell therapy in the treatment of an osteochondral lesion of the ankle. BMJ Case Rep. 2020, 13, e234595. [CrossRef] p 45. Laffey, J.; Hayes, M.; Friedenstein, A.J.; Gorskaja, J.F.; Kulagina, N.N. Faculty of 1000 evaluation for Fibroblast precursors in normal and irradiated mouse hematopoietic organs. F1000—Post. Publ. Peer Rev. Biomed. Lit. 2011, 4, 267–274. [CrossRef] 46. Prockop, D.J. Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues an gene therapy. J. Cell. Biochem. Suppl. 1998, 30–31, 284–285. [CrossRef] g py 47. Harrell, C.R.; Markovic, B.S.; Fellabaum, C.; Arsenijevic, A.; Volarevic, V. Mesenchymal stem cell-based therapy of osteoarthritis: Current knowledge and future perspectives. Biomed. Pharmacother. 2019, 109, 2318–2326. [CrossRef] g p p 48. Si, Z.; Wang, X.; Sun, C.; Kang, Y.; Xu, J.; Wang, X.; Hui, Y. Adipose-derived stem cells: Sources, potency, and implications for regenerative therapies. Biomed. Pharmacother. 2019, 114, 108765. [CrossRef] [PubMed] 49. Freitag, J.; Bates, D.; Wickham, J.; Shah, K.; Huguenin, L.; Tenen, A.; Paterson, K.; Boyd, R. Adipose-derived mesenchymal stem cell therapy in the treatment of knee osteoarthritis: A randomized controlled trial. Regen. Med. 2019, 14, 213–230. [CrossRef] [PubMed] 50. Freitag, J.; Shah, K.; Wickham, J.; Boyd, R.; Tenen, A. The effect of autologous adipose derived mesenchymal stem cell therapy in the treatment of a large osteochondral defect of the knee following unsuccessful surgical intervention of osteochondritis dissecans—A case study. BMC Musculoskelet. Disord. 2017, 18, 298. [CrossRef] y 51. Mazini, L.; Rochette, L.; Amine, M.; Malka, G. Regenerative Capacity of Adipose Derived Stem Cells (A Mesenchymal Stem Cells (MSCs). Int. J. Mol. Sci. 2019, 20, 2523. References Nguyen, P.D.; Tran, T.D.-X.; Nguyen, H.T.-N.; Vu, H.T.; Le, P.T.-B.; Phan, N.L.-C.; Vu, N.B.; Phan, N.K.; Van Pham, P. Comparative Clinical Observation of Arthroscopic Microfracture in the Presence and Absence of a Stromal Vascular Fraction Injection for Osteoarthritis. Stem Cells Transl. Med. 2016, 6, 187–195. [CrossRef] [PubMed] Clinical Observation of Arthroscopic Microfracture in the Presence and Absence of a Stromal Vascular Fraction Injection for Osteoarthritis. Stem Cells Transl. Med. 2016, 6, 187–195. [CrossRef] [PubMed] 68. Yokota, N.; Yamakawa, M.; Shirata, T.; Kimura, T.; Kaneshima, H. Clinical results following intra-articular injection of adipose- derived stromal vascular fraction cells in patients with osteoarthritis of the knee. Regen. Ther. 2017, 6, 108–112. [CrossRef] [PubMed] [ ] 69. Garza, J.R.; Campbell, R.E.; Tjoumakaris, F.P.; Freedman, K.B.; Miller, L.S.; Maria, D.S.; Tucker, B.S. Clinical Efﬁcacy of Intra- articular Mesenchymal Stromal Cells for the Treatment of Knee Osteoarthritis: A Double-Blinded Prospective Randomized Controlled Clinical Trial. Am. J. Sports Med. 2020, 48, 588–598. [CrossRef] [PubMed] p 70. Hong, Z.; Chen, J.; Zhang, S.; Zhao, C.; Bi, M.; Chen, X.; Bi, Q. Intra-articular injection of autologous adipose-derived stromal vascular fractions for knee osteoarthritis: A double-blind randomized self-controlled trial. Int. Orthop. 2018, 43, 1123–1134. [CrossRef] [PubMed] [ ] [ ] 71. Hudetz, D.; Bori´c, I.; Rod, E.; Jeleˇc, Ž.; Kunovac, B.; Polašek, O.; Vrdoljak, T.; Pleˇcko, M.; Skelin, A.; Polanˇcec, D.; et al. Early results of intra-articular micro-fragmented lipoaspirate treatment in patients with late stages knee osteoarthritis: A prospective study. Croat. Med. J. 2019, 60, 227–236. [CrossRef] y 72. Pers, Y.-M.; Rackwitz, L.; Ferreira, R.; Pullig, O.; Delfour, C.; Barry, F.; Sensebe, L.; Casteilla, L.; Fleury, S.; Bourin, P.; et al. Adipose Mesenchymal Stromal Cell-Based Therapy for Severe Osteoarthritis of the Knee: A Phase I Dose-Escalation Trial. Stem Cells Transl. Med. 2016, 5, 847–856. [CrossRef] [PubMed] 73. Roato, I.; Belisario, D.C.; Compagno, M.; Lena, A.; Bistolﬁ, A.; Maccari, L.; Mussano, F.; Genova, T.; Godio, L.; Perale, G.; et al. Concentrated adipose tissue infusion for the treatment of knee osteoarthritis: Clinical and histological observations. Int. Orthop. 2019, 43, 15–23. [CrossRef] 74. Panni, A.S.; Vasso, M.; Braile, A.; Toro, G.; De Cicco, A.; Viggiano, D.; Lepore, F. Preliminary results of autologous adipose-derived stem cells in early knee osteoarthritis: Identiﬁcation of a subpopulation with greater response. Int. Orthop. 2019, 43, 7–13. [CrossRef] [ ] 75. Nakamura, N.; Yokota, N.; Hattori, M.; Ohtsuru, T.; Otsuji, M.; Lyman, S.; Shimomura, K. References The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. BMJ 2021, 372, n71. [CrossRef] Cells 2021, 10, 1365 30 of 31 61. Clement, N.D.; Bardgett, M.; Weir, D.; Holland, J.; Gerrand, C.; Deehan, D.J. What is the Minimum Clinically Important Difference for the WOMAC Index After TKA? Clin. Orthop. Relat. Res. 2018, 476, 2005–2014. [CrossRef] [PubMed] p 62. Jo, C.H.; Chai, J.W.; Jeong, E.C.; Oh, S.; Shin, J.S.; Shim, H.; Yoon, K.S. Intra-articular Injection of Mesenchymal Stem Cells for the Treatment of Osteoarthritis of the Knee: A 2-Year Follow-up Study. Am. J. Sports Med. 2017, 45, 2774–2783. [CrossRef] 62. Jo, C.H.; Chai, J.W.; Jeong, E.C.; Oh, S.; Shin, J.S.; Shim, H.; Yoon, K.S. Intra-articular Injection of Mesenchymal Stem Cells for the 62. Jo, C.H.; Chai, J.W.; Jeong, E.C.; Oh, S.; Shin, J.S.; Shim, H.; Yoon, K.S. Intra articular Injection of Mesenc Treatment of Osteoarthritis of the Knee: A 2-Year Follow-up Study. Am. J. Sports Med. 2017, 45, 2774–2 p y p Shin, W.H.; Kim, H.; Chai, J.W.; Jeong, E.C.; Kim, J.E.; Shim, H.; Shin, J.S.; Shin, I.S.; et al. Intra-Articular 63. Jo, C.H.; Gil Lee, Y.; Shin, W.H.; Kim, H.; Chai, J.W.; Jeong, E.C.; Kim, J.E.; Shim, H.; Shin, J.S.; Shin, I.S.; et al. Intra-Articular Injection of Mesenchymal Stem Cells for the Treatment of Osteoarthritis of the Knee: A Proof-of-Concept Clinical Trial. Stem Cells 2014, 32, 1254–1266. [CrossRef] [PubMed] 64. Lee, W.; Kim, H.J.; Kim, K.; Kim, G.B.; Jin, W. Intra-Articular Injection of Autologous Adipose Tissue-Derived Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis: A Phase IIb, Randomized, Placebo-Controlled Clinical Trial. Stem Cells Transl. Med. 2019, 8, 504–511. [CrossRef] [PubMed] 65. Spasovski, D.; Spasovski, V.; Bašˇcarevi´c, Z.; Stojiljkovi´c, M.; Vre´ca, M.; Andjelkovi´c, M.; Pavlovi´c, S. Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis. J. Gene Med. 2018, 20, e3002. [CrossRef] 66. Koh, Y.-G.; Jo, S.-B.; Kwon, O.-R.; Suh, D.-S.; Lee, S.-W.; Park, S.-H.; Choi, Y.-J. Mesenchymal Stem Cell Injections Improve Symptoms of Knee Osteoarthritis. Arthrosc. J. Arthrosc. Relat. Surg. 2013, 29, 748–755. [CrossRef] 66. Koh, Y.-G.; Jo, S.-B.; Kwon, O.-R.; Suh, D.-S.; Lee, S.-W.; Park, S.-H.; Choi, Y.-J. Mesenchymal Stem Symptoms of Knee Osteoarthritis. Arthrosc. J. Arthrosc. Relat. Surg. 2013, 29, 748–755. [CrossRef] g T.D.-X.; Nguyen, H.T.-N.; Vu, H.T.; Le, P.T.-B.; Phan, N.L.-C.; Vu, N.B.; Phan, N.K.; Van Pham, P. Comparative 67. References Comparative Clinical Outcomes After Intra-articular Injection with Adipose-Derived Cultured Stem Cells or Noncultured Stromal Vascular Fraction for the Treatment of Knee Osteoarthritis: Response. Am. J. Sports Med. 2020, 48, NP19–NP20. [CrossRef] 76. Freitag, J.; Bates, D.; Wickham, J.; Shah, K.; Huguenin, L.; Tenen, A.; Paterson, K.; Boyd, R. Evaulation of intra-articular adipose derived mesenchymal stem cell therapy in the treatment of symptomatic knee osteoarthritis—A randomised controlled trial. Cytotherapy 2019, 21, S69. [CrossRef] 77. Bansal, H.; Comella, K.; Leon, J.; Verma, P.; Agrawal, D.; Koka, P.; Ichim, T. Retracted Article: Intra-articular injection in the knee of adipose derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. J. Transl. Med. 2017, 15, 141. [CrossRef] [PubMed] 78. Tran, T.D.X.; Wu, C.-M.; Dubey, N.K.; Deng, Y.-H.; Su, C.-W.; Pham, T.T.; Le, P.B.T.; Sestili, P.; Deng, W.-P. Time- and Kellgren– Lawrence Grade-Dependent Changes in Intra-Articularly Transplanted Stromal Vascular Fraction in Osteoarthritic Patients. Cells 2019, 8, 308. [CrossRef] [PubMed] 79. Zamborsky, R.; Danisovic, L. Surgical Techniques for Knee Cartilage Repair: An Updated Large-Scale Systematic Review and Network Meta-analysis of Randomized Controlled Trials. Arthrosc. J. Arthrosc. Relat. Surg. 2020, 36, 845–858. [CrossRef] [PubMed] 80. Cao, Z.; Mai, X.; Wang, J.; Feng, E.; Huang, Y. Unicompartmental Knee Arthroplasty vs High Tibial Osteotomy for Knee Osteoarthritis: A Systematic Review and Meta-Analysis. J. Arthroplast. 2018, 33, 952–959. [CrossRef] 81. Damia, E.; Chicharro, D.; Lopez, S.; Cuervo, B.; Rubio, M.; Sopena, J.J.; Vilar, J.M.; Carrillo, J.M. Adipose-Derived Mesenchymal Stem Cells: Are They a Good Therapeutic Strategy for Osteoarthritis? Int. J. Mol. Sci. 2018, 19, 1926. [CrossRef] 82. Murphy, J.M.; Dixon, K.; Beck, S.; Fabian, D.; Feldman, A.; Barry, F. Reduced chondrogenic and adipogenic activity of mesenchy- mal stem cells from patients with advanced osteoarthritis. Arthritis Rheum. 2002, 46, 704–713. [CrossRef] 31 of 31 Cells 2021, 10, 1365 31 of 31 83. Qu, H.; Sun, S. Efﬁcacy of mesenchymal stromal cells for the treatment of knee osteoarthritis: A meta-analysis of randomized controlled trials. J. Orthop. Surg. Res. 2021, 16, 11. [CrossRef] p g 84. Wang, J.; Zhou, L.; Zhang, Y.; Huang, L.; Shi, Q. Mesenchymal stem cells—A promising strategy for treating knee osteoarthritis. Bone Jt. Res. 2020, 9, 719–728. [CrossRef] mala, P.; King, A.; Lee, B.; Kraus, M.; Tse, V.; Jacobs, C.R. Buffered Platelet-Rich Plasma Enhances Mesenchy 85. Mishra, A.; Tummala, P.; King, A.; Lee, B.; Kraus, M.; Tse, V.; Jacobs, C.R. References Buffered Platelet-Rich Plasma Enhances Mesenchymal Stem Cell Proliferation and Chondrogenic Differentiation. Tissue Eng. Part C Methods 2009, 15, 431–435. [CrossRef] Mishra, A.; Tummala, P.; King, A.; Lee, B.; Kraus, M.; Tse, V.; Jacobs, C.R. Buffered Platelet-Rich Plasma En Stem Cell Proliferation and Chondrogenic Differentiation Tissue Eng Part C Methods 2009 15 431 435 [C Mishra, A.; Tummala, P.; King, A.; Lee, B.; Kraus, M.; Tse, V.; Jacobs, C.R. Buffered Platelet-Rich Plasma En Stem Cell Proliferation and Chondrogenic Differentiation. Tissue Eng. Part C Methods 2009, 15, 431–435. [C 85. Mishra, A.; Tummala, P.; King, A.; Lee, B.; Kraus, M.; Tse, V.; Jacobs, C.R. Buffered Platelet-Rich Plasma Enhances Mesenchymal Stem Cell Proliferation and Chondrogenic Differentiation. Tissue Eng. Part C Methods 2009, 15, 431–435. [CrossRef] Cell Proliferation and Chondrogenic Differentiation. Tissue Eng. Part C Methods 2009, 15, 431–435. [CrossRe 86. Krüger, J.P.; Hondke, S.; Endres, M.; Pruss, A.; Siclari, A.; Kaps, C. Human platelet-rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells. J. Orthop. Res. 2011, 30, 845–852. [CrossRef] [PubMed] 86. Krüger, J.P.; Hondke, S.; Endres, M.; Pruss, A.; Siclari, A.; Kaps, C. Human platelet rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells. J. Orthop. Res. 2011, 30, 845–852. [CrossRef] [PubMed] 87. Fossett, E.; Khan, W.S.; Pastides, P.; Adesida, A.B. The Effects of Ageing on Proliferation Potential, Differentiation Potential and Cell Surface Characterisation of Human Mesenchymal Stem Cells. Curr. Stem Cell Res. Ther. 2012, 7, 282–286. [CrossRef] [PubMed] 88. Mar˛edziak, M.; Marycz, K.; Tomaszewski, K.A.; Kornicka, K.; Henry, B.M. The Inﬂuence of Aging on the Regenerative Potential of Human Adipose Derived Mesenchymal Stem Cells. Stem Cells Int. 2016, 2016, 2152435. [CrossRef] [PubMed] p y 89. Urban, H.; Little, C.B. The role of fat and inﬂammation in the pathogenesis and management of osteoarthritis. Rheumatology 2018, 57, iv10–iv21. [CrossRef] [PubMed] 90. Katz, J.N.; Brownlee, S.A.; Jones, M.H. The role of arthroscopy in the management of knee osteoarthritis. Best Pract. Res. Clin. Rheumatol. 89. Urban, H.; Little, C.B. The role of fat and inﬂammation in the pathogenesis and management of osteoarthritis. Rheumatology 2018, 57, iv10–iv21. [CrossRef] [PubMed] 89. Urban, H.; Little, C.B. The role of fat and inﬂammation in the pathogenesis and management of osteoarthritis. Rheumatology 2018, 57, iv10–iv21. [CrossRef] [PubMed] 90. Katz, J.N.; Brownlee, S.A.; Jones, M.H. The role of arthroscopy in the management of knee osteoarthritis. Best Pract. Res. Clin. Rheumatol. 2014, 28, 143–156. References [CrossRef] [PubMed] S.A.; Jones, M.H. The role of arthroscopy in the management of knee osteoarthritis. Best Pract. Res. Clin. Rheumatol CrossRef] [PubMed] 91. Grieshober, J.A.; Stanton, M.; Gambardella, R. Debridement of Articular Cartilage: The Natural Course. Sports Med. Arthrosc. Rev. 2016, 24, 56–62. [CrossRef] [PubMed] 92. Aronowitz, J.A.; Ellenhorn, J.D.I. Adipose Stromal Vascular Fraction Isolation. Plast. Reconstr. Surg. 2013, 132, 932e–939e. [CrossRef] 93. Comella, K.; Parlo, M.; Daly, R.; Depasquale, V.; Edgerton, E.; Mallory, P.; Schmidt, R.; Drake, W.P. Safety Analysis of Autologous Stem Cell Therapy in a Variety of Degenerative Diseases and Injuries Using the Stromal Vascular Fraction. J. Clin. Med. Res. 2017, 9, 935–942. [CrossRef] [PubMed]
W4309432016.txt	https://zenodo.org/records/7339563/files/302066.pdf	en		10 Things You Learned In Kindergarden That'll Help You With Refleksoloji Masaj Aleti	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,015	Refleksoloji masaj aleti Anksiyete ve stresi azaltmaya yardımcı olan refleksoloji masajı seans ücretleri hastalığa için bileğmeselemektedir. Refleksoloji masajı yaptıranların yorumlarına bakmış olduğumızda tedaviye payanda için, sağlamlığı bakmak midein evet da rahatlama yağdırmak kucakin uygulanabilir. Ayrıca hastalıkları önlemeye yardımcı olur. Refleksoloji masaj aleti, bir hastanın ruhsal gerilim mi yoksa kaygı mı yaşadığını anlayabilir ve hastalarının vücutlarını dengelemeye yardımcı olur. Ayağın belirli bölgelerine matbuatç uygulanır ve ortaya çıkabilecek dertların ilişkilendirilen organlarla ait problemlerı gösterdiği varsayılır. 5 Killer Quora Answers On Refleksoloji Masaj Aleti Fiyatları Refleksoloji masaj aleti Yurtiçi turları veya yurtdışı turları ile yaşamınızdaki en aldatıcı andaçları evetşayacağınız tenzilatlı tur fırsatları Fırsat Bu Fırsat ile karşınızda. Titreşimli Masaj Aleti kullanıcı kişilerin esrar kabız yalnızçlı matraklarına revan bir cihaz olması taçı aralık kullanıcıların bazı bölgelerinde olan balkıları, krampları bastırmak derunin yeğleme edilen bir maşa de kabil. Kullanıcılar titreşimli masaj aletini vücutlarında istedikleri yerlerde kullanabilirler. örneğin kullanıcının boynunda kâin kulunç adı maruz adele ortamında bek düğümcüklerden oluşan vecayı ve ateşyı titreşim masaj aleti kullanılarak giderilebilir yahut azaltabilir iken üste kullanıcı eşhas keyfi olarak boyunlarına veya sırtlarına titreşimli masaj aletini kullanabilir. Kullanıcı kişiler titreşimli masaj aleti satın almadan önce ürünü kendilerine için nerede ne amaç ile kullanacağını değerlendirebilir ve ona bakılırsa bir tercihte bulunabilir. örneğin kullanıcılar yüzlerinde kâin oda altı torbalarının giderilmesi kucakin darbeli titreşim masaj aletini tercih edebilir iken boyunları veya sırtları midein farklı bir modele ehil olan titreşimli masaj aletini yeğleme edebilir. Kullanıcı eşhas titreşimli masaj aleti satın almadan önce nerede kullanılmasının münasip bulunduğunu okuması ve değerlendirmesi önemli olur. çünkü titreşimli masaj aleti vücudun farklı tasarruf bölgelerine farklı miktarlarda titreşim verebilir ve bu yer da kullanıcılar muhtevain zararlı mümkün. How To Explain Refleksoloji Masaj Aleti To Your Mom Refleksoloji masaj aleti özel bir masaj tekniği olan refleksoloji, bazı farmakolojik tedavilere dayak olmaktadır. Fizik terapi ve rehabilitasyona yardımcı olan bu masaj tekniği, vecaların azalmasını sağlamlarken aynı zamanda egzersizlerin henüz basit kuruluşlmasını sağlamlamaktadır. Meslek Enerjisi Teorisi: Hayatımızın her simultane var olan stresin hayat enerjisindeki akışı engellediği bu teori ile açıklanmıştır. Refleksoloji ile varlık enerjisi temizışı düzeltilebilir. Doğrusu varlık dâhilin vacip dengenin sağlamlanması esastır. Masaj aletleri, ağrı hissedilen noktalar bağırsakin kullanıldığı kabilinden düzenli cilt bakımı yürekin bile tercih edilir. Cildi sıkıcalaştırmak, evetğ yakımını kolaylaştırmak ve göz astı noktalarını iyileştirmek maksadıyla nazire konusu cihazların kullanıldığı görülür. Bu sayede cihazların mizaç ve rahatlama tesiri bile yakından hissedilebilir. 5 Bad Habits That People In The Refleksoloji Masaj Aleti Fiyatları Industry Need To Quit Refleksoloji masaj aleti Bunun yanı sıra mizaç problemlerı ya da organize masaj muhtevain alet satın alacaksanız da ürünler arasından ihtiyacınız olan masaj aletini burayı kontrol et satın alabilirsiniz. Refleksoloji masaj kompetanı, bölgesel ya da genel el getirmek yürekin yabancı ayak ve kulaklardaki noktalara odaklanır. Eğer ki saat kısıtlıysa ve kişinin rahatlaması gerekiyorsa spesiyalist can kulak üzerinden çallıkışabilir. Titreşimli masaj aleti ne nöbete yarar? Masaj aletlerinin çhileışma şekilleri bile birbirinden farklıdır. özellikle baskınülit masajı yaparken evet da balkılarınız olan bir bölgeye masaj açmak istediğinizde titreşimli masaj aletlerini kullanabilirsiniz. Titreşimli masaj aletlerinde farklı seviyeler evet. Jademaster ayak masaj aleti Hummalı ve tempolu bir iş sundurmaı, stres, şehir yaşamının koşunşturması derken hijyenikşam eve erişince kimsenin kolunu dahi kıpırdatacak hali kalmıyor. Tutulmuş dal kasları, eklem vecaları ve tüm vücudu kaplayan halsizlik âdem evladıı gülümserından bezdiriyor. Masaj tabancası olarak tercih edebileceğiniz ürünler ortada MEDISANA 48310 infrared terapili ülke tipi masaj aletini bile sayabiliriz. Sporcu masaj aleti olarak da kullanılabilen bu ürün kısaca süre zarfında hun deveranını hızlandırır. Refleksoloji ‘balans’ sağlayan bir terapidir. Refleksoloji Terapisi kişinin kendisini, fiziksel, hissî ve ruhsal falmdan elleme hissetmesini esenlar ve kişiye natürel dengesini kazanmıştırrır. Jademaster Ayak Masaj Aleti üzerine Bütün Zamanların En İyi 15 Pinterest Panosu Jademaster ayak masaj aleti Hun sirkülasyonını hızlandırır ve darbeli masaj sayesinde en derindeki kasları da gevşetir, Açılıp kapatılabilir kızılötesi ışık sayesinde infrared terapi, Isıtma fonksiyonu, Ergonomik şekil Jakuzi: Burada söz konusu olan, ayaklar bağırsakin konstrüksiyonlmış ve ab ile doldurulan küçümencik boyuttaki küvetlerdir. Burada dip yüzeyi modele bandajlı olarak farklı şekillerde bir strüktürya sahiptir. örneğin zeminde ruloları olan yahut tırtıklı mimarisı yardımıyla ayak masajı yapan modeller var veya titreşim evet da ısıtıcı kırmızı ışık kabilinden özelliklere iye olan cihazlar da var. From Around The Web: 20 Fabulous Infographics About Refleksoloji Masajı Aleti Refleksoloji masaj aleti Okazyon Bu Punt ayrıcalığıyla; dizge edinmenize yardımcı olacak onlarca ağırşak ve sertifikalı eğitimleri meraklı kurumların güvencesiyle, düzlükında spesiyalist eğitmenlerden iskontolu fiyata iktibas şansına sahip olacaksınız. hanımlar bendeübü alışveriş siteleri cinsilatif alışdoneş siteleri kadınlar yürekin tuzakışveriş kadınlar verişte ne hileır bayanların kırmızıışmutaş allıkışhunrizkları nisa aldatmaışdataş giyim by TemplatesYard Global ayak oğlan masaj dip doku ayak masaj ağrı stoper Plantar fasiit kabartma ayak masajı rulosu The Refleksoloji Ayak Masaj Aleti Case Study You'll Never Forget Refleksoloji masaj aleti Alan Teorisi: Bu teori esasen Refleksolojinin sorunleyişini belirtir. Refleksoloji haritasında halk ve ayakların farklı alanlarda makam almaktadır ve bu noktalarda her bir düzlük farklı organları rapor etmektedir. İsterseniz ısıtmalı, isterseniz de ısıtmasız olarak kullanabileceğiniz ürün esnek gestaltsı ile kişiye muazzam bir deneyim yaşatır. Neva konusuna encam olursak ultra suskun çkızılışan bir masaj aletidir. Dermatolog Dr. Whitney Bowe imzası taşıyan bu yeni cilt bakım trendi, peeling ve retinol uygulamalarının arkası sıra cilde iki temizşam süresince dinlenme fırsatı tanılamayor. Gate Kontrol Teorisi: 1965 senesinde Melzack ve Wall’un ortaya çıkardığı bu teori, vücudumuzun herhangi bir noktasındaki balkıların beyin aracılığıyla yaratıldığını söylemektedir. 9 Signs You Sell Refleksoloji Masaj Aleti Fiyatları For A Living Refleksoloji masaj aleti Ayak refleksoloji masajı, bu noktalar üzerinde yoğunlaşarak yavaş matbuatçlı bir şekilde sinir noktalarını uyararak uzmanlar aracılığıyla uygulanmaktadır. Hamam satış yoğurma titreşim Shiatsu ayak masajı elektrikli silindir oğlan ve ayak masaj makinesi tavsiyesi ilgisi ve hersey icin cok tesekkurler.ilk uygulamami yaptim ve sanki ayaklarim yeniden dunyaya geldi.O temel agrilarinin bu kadar hafifledigine inanamiyorum.İyi ki varsiniz .ilk firsatta masaj koltuklarina da gozumu diktim bilginize. Refleksoloji ‘balans’ sağlayan bir terapidir. Refleksoloji Terapisi kişinin kendisini, fiziksel, birey ve ruhsal bakımdan esen hissetmesini katkısızlar ve kişiye katıksız dengesini kazandırır. Refleksoloji masaj aleti Kez parmaklarının yönı aralık özel tasarlanmış çubuklarla da yapılan iş hastanın durumuna bakılırsa 45 zaman ile bir vakit beyninde sürmektedir. Umumi olarak ayak bölgenizden gıdıklanan biri iseniz endişelenmenize ister yoktur. Uygulamada baskı alelumum gıdıklanmaya vesile olmamaktadır. Refleksoloji masaj aleti özenle tasarlanmış diz masaj aparatı U şeklinde hava yastıklarıyla donatılmıştır. Isıtmalı diz masajı ile dizlerinizi gevşetmek yalnızçlanmıştır.
https://openalex.org/W1780158697	https://library.oapen.org/bitstream/20.500.12657/35309/1/340017.pdf	English	null	Citizenship Policies in the New Europe	null	2,007	cc-by	210,047	cetăţenie ждaнcтво  ,     ( .) Citizenship Policies in the New Europe Expanded and Updated Edition research imiscoe Citizenship Policies in the New Europe IMISCOE International Migration, Integration and Social Cohesion in Europe The IMISCOE Network of Excellence unites over 500 researchers from European institutes specialising in studies of international migration, integration and social cohesion. The Network is funded by the Sixth Framework Programme of the European Commission on Research, Citizens and Governance in a Knowledge-Based Society. Since its foundation in 2004, IMISCOE has developed an integrated, multidisciplinary and globally comparative research project led by scholars from all branches of the economic and social sciences, the humanities and law. The Network both furthers existing studies and pioneers new research in migration as a discipline. Priority is also given to promoting innovative lines of inquiry key to European policymaking and governance. The IMISCOE-Amsterdam University Press Series was created to make the Network’s findings and results available to researchers, policymakers and practitioners, the media and other interested stakeholders. High-quality manuscripts authored by IMISCOE members and cooperating partners are published in one of four distinct series. IMISCOE Research advances sound empirical and theoretical scholarship addressing themes within IMISCOE’s mandated fields of study. IMISCOE Reports disseminates Network papers and presentations of a time-sensitive nature in book form. IMISCOE Dissertations presents select PhD monographs written by IMISCOE doctoral candidates. IMISCOE Textbooks produces manuals, handbooks and other didactic tools for instructors and students of migration studies. IMISCOE Textbooks produces manuals, handbooks and other didactic tools for instructors and students of migration studies. IMISCOE Policy Briefs and more information on the Network can be found at www.imiscoe.org. Andre Liebich Introduction: Altneuländer or the vicissitudes of citizenship in the new EU states 21 1 New states and old concerns, or why there is not much plural citizenship in the Altneula¨nder 21 2 Old categories and new principles, or how ethnicity has trumped other grounds of citizenship 24 g p 4 3 Old wrongs and new rights, or how to use citizenship to correct history 31 4 Conclusion: The historical past, the ethnic present and the immigrant future 37 4 Conclusion: The historical past, the ethnic present and the immigrant future 37 Annex: Constitutional preambles (extracts) 38 Annex: Constitutional preambles (extracts) 38 List of figures and tables 11 List of figures and tables 11 Citizenship Policies in the New Europe Expanded and Updated Edition edited by Rainer Baubo¨ck Bernhard Perchinig Wiebke Sievers IMISCOE Research IMISCOE Research Cover design: Studio Jan de Boer BNO, Amsterdam Layout: The DocWorkers, Almere ISBN 978 90 8964 108 3 e-ISBN 978 90 4850 225 7 NUR 741 / 763 © IMISCOE / Amsterdam University Press, Amsterdam 2009 Cover design: Studio Jan de Boer BNO, Amsterdam Cover design: Studio Jan de Boer BNO, Amsterdam Layout: The DocWorkers, Almere ISBN 978 90 8964 108 3 e-ISBN 978 90 4850 225 7 NUR 741 / 763 © IMISCOE / Amsterdam University Press, Amsterdam 2009 All rights reserved. Without limiting the rights under copyright reserved above, no part of this book may be reproduced, stored in or introduced into a retrieval system, or transmitted, in any form or by any means (electronic, mechanical, photocopying, recording or otherwise) without the written permission of both the copyright owners and the authors of the book. Preface 15 Andre Liebich Introduction: Altneuländer or the vicissitudes of citizenship in the new EU states 21 PART I RESTORED STATES Priit Ja¨rve 1 Estonian citizenship: Between ethnic preferences and democratic obligations 45 1.1 History of Estonian nationality 45 1.2 Basic principles of the most important current modes of acquisition and loss of nationality 50 1.3 Current debates on nationality 53 1.4 Statistics on acquisition of nationality since 1992 58 1.5 Conclusions 60 Kristı¯ne Kru¯ma 2 Checks and balances in Latvian nationality policies: National agendas and international frameworks 67 2.1 History of nationality policy 67 2.2 Basic principles for the acquisition and loss of nationality 74 2.3 Current political debates 80 2.4 Statistics 83 2.5 Conclusions 88 CITIZENSHIP POLICIES IN THE NEW EUROPE 6 Kristı¯ne Kru¯ma 3 Lithuanian nationality: Trump card to independence and its current challenges 97 3.1 History of nationality policy 97 3.2 Basic principles of the most important current modes of acquisition and loss of nationality 106 3.3 Current political debates 112 3.4 Statistics 114 3.5 Conclusions 116 PART II STATES WITH HISTORIES OF SHIFTING BORDERS Agata Go´rny and Dorota Pudzianowska 4 Same letter, new spirit: Nationality regulations and their implementation in Poland 123 4.1 Polish nationality in historical perspective 124 4.2 Basic principles of current regulations on Polish nationality 128 4.3 The unresolved debate (1999-2001) 134 4.4 Acquisitions of Polish nationality in figures 136 4.5 Conclusions 141 Ma´ria M. Kova´cs and Judit To´th 5 Kin-state responsibility and ethnic citizenship: The Hungarian case 151 5.1 History of Hungarian policies on nationality since 1945 152 5.2 Current nationality legislation 154 5.3 Current political debates on (dual) citizenship 158 5.4 Trends in statistics 165 5.5 Conclusions 169 Constantin Iordachi 6 Politics of citizenship in post-communist Romania: Legal traditions, restitution of nationality and multiple memberships 177 6.1 History of Romanian nationality 178 6.2 Democratic transformation: Current regulations on ascription, acquisition and loss of nationality 184 6.3 Current political debates 191 6.4 Statistics on the restitution of nationality 198 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives 200 6.2 Democratic transformation: Current regulations on ascription, i i i d l f i li 8 acquisition and loss of nationality 184 6.4 Statistics on the restitution of nationality 198 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives 200 7 CONTENTS Daniel Smilov and Elena Jileva 7 The politics of Bulgarian citizenship: National identity, democracy and other uses 211 7.1 History 212 7.2 Current citizenship rules and practices 220 7.3 Current political debates 226 7.4 Current statistical trends 235 7.5 Conclusions 238 PART III POST-PARTITION STATES Andrea Barsˇova´ 8 Czech citizenship legislation between past and future 249 8.1 History of Czechoslovak citizenship policies 249 8.2 Basic principles of acquisition and loss of Czech citizenship 254 8.3 Current political debates 258 8.4 Statistics 261 8.5 Conclusions 263 Dagmar Kusa´ 9 The Slovak question and the Slovak answer: Citizenship during the quest for national self-determination and after 275 9.1 History of Slovak citizenship 276 9.2 Current regulations of acquisition and loss of Slovak citizenship 284 9.3 Current political debates and reform plans 289 9.4 Statistical trends (acquisition of Slovak citizenship since 1993) 294 9.5 Conclusions 296 Felicita Medved 10 From civic to ethnic community? The evolution of Slovenian citizenship 305 10.1 History of citizenship policies 305 10.2 Basic principles of acquisition and loss of Slovenian citizenship 313 10.3 Current political debates 320 10.4 Statistics 323 10.5 Conclusions 325 10.6 Epilogue 327 10 From civic to ethnic community? The evolution of Slovenian citizenship 305 10.1 History of citizenship policies 305 10.2 Basic principles of acquisition and loss of Slovenian citizenship 313 l l d b 10.3 Current political debates 320 10.3 Current political debates 320 10.4 Statistics 323 10.4 Statistics 323 10.5 Conclusions 325 10.5 Conclusions 325 10.6 Epilogue 327 10.6 Epilogue 327 CITIZENSHIP POLICIES IN THE NEW EUROPE 8 Francesco Ragazzi and Igor Sˇtiks 11 Croatian citizenship: From ethnic engineering to inclusiveness 339 11.1 The history of citizenship policy in Croatia since 1945 340 11.2 Acquisition and loss of Croatian citizenship: Current legal provisions 347 11.3 Current political debates and planned changes 352 11.4 Statistical developments 353 11.5 Conclusions 355 PART IV MEDITERRANEAN POST-IMPERIAL STATES Eugene Buttigieg 12 Malta’s citizenship law: Evolution and current regime 367 12.1 Historical background 367 12.2 Current modes of acquisition and loss of citizenship 373 12.3 Statistical developments 379 12.4 Recent reforms 381 12.5 Conclusions 383 Nicos Trimikliniotis 13 Nationality and citizenship in Cyprus since 1945: Communal citizenship, gendered nationality and the adventures of a post-colonial subject in a divided country 389 13.1 History of nationality policy since 1945 390 13.2 Modes of acquisition and loss of citizenship 397 13.3 Current debates: The challenges of gender equality, migration, Europeanisation and reunification 405 13.4 Statistical developments since 1985: The ‘politics of numbers’ and the ‘numbers game’ 408 13.5 Conclusions 409 Zeynep Kadirbeyoglu 14 Changing conceptions of citizenship in Turkey 419 14.1 History of Turkish citizenship law 419 14.2 Modes of acquisition and loss of Turkish citizenship 426 14.3 Statistics 428 14.4 Conclusions 431 14.5 Epilogue 432 Eugene Buttigieg 12 Malta’s citizenship law: Evolution and current regime 367 12.1 Historical background 367 12.2 Current modes of acquisition and loss of citizenship 373 12.3 Statistical developments 379 12.4 Recent reforms 381 12.5 Conclusions 383 Zeynep Kadirbeyoglu 14 Changing conceptions of citizenship in Turkey 419 14.1 History of Turkish citizenship law 419 14.2 Modes of acquisition and loss of Turkish citizenship 426 14.2 Modes of acquisition and loss of Turkish citizenship 426 14.3 Statistics 428 14.4 Conclusions 431 CONTENTS 9 9 Wiebke Sievers ‘A call to kinship’? Citizenship and migration in the new Member States and the accession countries of the EU 439 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia 440 2 Reaching out to emigrants and expatriates: Restitution laws, dual nationality and status laws 446 3 Excluding recent immigrants: Naturalisation requirements 452 4 Conclusions 455 List of contributors 459 List of figures and tables Figure 1.1 Estonian citizens and stateless persons in Estonia, 1992-1 January 2008, per cent of total population 48 Figure 1.2 Naturalisation in Estonia, 1992-2007, persons naturalised per year in thousands 59 Table 2.1 Changes in ethnic composition of Latvia’s population 83 Table 2.2 Citizens and non-citizens of Latvia 83 Table 2.3 Residents of Latvia on 1 July 2008 (by ethnic origin) 83 Table 2.4 Children of non-citizens and stateless persons born after 21 August 1991 who were granted Latvian nationality (31 May 2008) 84 Table 2.5 Numbers of naturalisations in Latvia per year 85 Table 2.6 Numbers of persons passing history and language exams for naturalisation 86 Table 2.7 Ethnic origin of applicants for naturalisation in Latvia (31 May 2008) 87 Table 2.8 Age of applicants for naturalisation in Latvia (31 May 2008) 87 Table 3.1 Ethnic composition in Lithuania 114 Table 3.2 Number of naturalised persons, 2003-2007 115 Table 3.3 Number of persons who have lost Lithuanian citizenship, by reasons 115 Table 3.4 Retention of the right to Lithuanian citizenship 115 Table 4.1 Foreigners granted Polish nationality by conferment in 1992-2001 and by conferment, acknowledgement and spousal acquisition in 2002-2006, by (former) nationality 137 Figure 4.1 Acquisitions of Polish nationality by conferment (1992-2006) 139 Table 4.2 Repatriation visas to Poland in 1997-2006, by repatriates’ previous country of residence 141 Table 5.1 Number of naturalisations and re-naturalisations as well as terminations of nationality in Hungary, 1985-1994 166 Table 5.2 Distribution of nationality law cases in Hungary, 1998-2008 167 CITIZENSHIP POLICIES IN THE NEW EUROPE 12 Table 5.3 Ratio of naturalisation decisions in Hungary in 2002, 2007 and 2008 168 Table 6.1 Naturalisations in Romania, 16 June 2000-29 October 2008 198 Figure 6.1 Duration in months of the process of naturalisation in Romania, by original nationality of applicants accepted in 2005, 2006 and 2007 199 Table 7.1 Number of decrees issued by the Vice-President of the Republic of Bulgaria for change of citizenship for the period 1 January 2001-31 December 2006 236 Table 7.2 Number of applications for a change of citizenship for the period 1 January 2000-31 December 2006 237 Table 8.1 Conceptual scheme of Act No. 40/1993 Coll. List of figures and tables on the Acquisition and Loss of Citizenship of the Czech Republic 252 Table 8.2 Naturalisations (excluding Slovak nationals) in the Czech Republic, 1993-2007 262 Table 8.3 Number of Slovak and other nationals acquiring Czech citizenship from 2001-2007 262 Table 8.4 Slovak nationals who acquired Czech citizenship by declaration (Section 18a of Act No. 40/1993 Coll.) 262 Table 8.5 Former Czechoslovak nationals who (re-)acquired Czech citizenship by declaration under Act No. 193/1999 Coll. 263 Table 9.1 Number of persons who acquired citizenship of the Slovak Republic, 1993-2008 294 Figure 9.1 Czechs and other foreign nationals who acquired citizenship of the Slovak Republic, 1995-2008 296 Table 9.2 Refugees and asylum seekers in the Slovak Republic, 1995-2008 296 Table 10.1 Admission to Slovenian citizenship based on supplementary and corrective initial determination rules (art. 40, 1991, art. 19, 2002) and on standard provisions for naturalisation by country of origin, 25 June 1991-31 December 2008 324 Table 10.2 Regular and facilitated naturalisations by groups of persons in Slovenia, 1991-2008 324 Table 10.3 Regular and facilitated naturalisations in Slovenia per year, 1991-2008 324 Table 10.4 Reasons for granting exceptional naturalisations in Slovenia 324 Table 10.5 Exceptional naturalisations in Slovenia per year, 1991-2008 325 LIST OF FIGURES AND TABLES 13 Table 10.6 Release from Slovenian citizenship per year, 1991-2008 325 Table 12.1 Acquisition of Maltese nationality by registration for 1998-2008 according to the grounds for registration 380 Figure 12.1 Number of naturalisations and registrations in Malta, 1990-2008 380 Table 13.1 Acquisitions of Cypriot citizenship by modes, 1985-3 March 2009 408 Table 14.1 Automatic acquisition of Turkish citizenship, 1990-2008 429 Table 14.2 Acquisition of Turkish citizenship through a decision of the authorities, 1990-2008 429 Table 14.3 Loss of Turkish citizenship by a decision of the authorities, 2000-2008 430 Table 14.4 Previous loss of citizenship by those who have reacquired Turkish citizenship according to three main categories, 2000-2004 431 Preface Nevertheless, one of the basic rationales of this publication has re- mained the same. While there has been a growing interest in compara- tive research on citizenship policies in major countries of immigration, both overseas and in Europe, most comparative studies are limited in geographic scope to a small number of already well-researched coun- tries. The present volume looks at countries that are rarely included in these studies.2 Why concentrate only on the twelve new Member States plus Croatia and Turkey. There are various reasons for this choice. The initial idea for analysing the nationality regulations in this particular group of countries originated in another EU-funded project called The Acquisi- tion of Nationality in EU Member States: Rules, Practises and Quanti- tive Developments, or ‘NATAC’ for short. NATAC provided the first strictly comparative analysis of the rules and practices regulating the acquisition and loss of nationality in the fifteen ‘old’ EU Member States. Unlike earlier, similar studies, it was not limited to country re- ports but used a new methodology that facilitates the comparison of the regulations across countries. The results of this project are pub- lished in two volumes (Baubo¨ck, Ersbøll, Groenendijk & Waldrauch 2006). Volume 1 contains comparative reports with chapters on the modes of acquiring and losing nationality, the statistics on nationality, the trends in nationality laws and the statuses of denizenship and qua- si-citizenship in the fifteen states. Volume 2 supplies specific back- ground information on the historical and political evolution of the na- tionality legislation in each individual country, structured according to a common grid in order to facilitate comparative analyses. g y This book represents a first attempt at adapting the methodology de- veloped in the NATAC project to the new Member States and the acces- sion countries of the EU. The country reports included in this volume are structured according to a common grid that is similar to the one used for Volume 2 of the above mentioned publication. Each chapter contains a historical outline of nationality policy since 1945 that pro- vides a broad overview of developments with special emphasis on im- portant reforms, breaks from basic principles of nationality acquisition and loss and regulations for special groups of people (e.g. an ethnic diaspora). Subsequently, the authors summarise the basic principles of the most important current modes of acquisition and loss of nationality in their respective countries. Preface It is not often that academic books are published in a second edition within less than two years from the date of first publication. Some- times the reason is that a book turns out to be a bestseller and reaches beyond a specific audience. Unfortunately, we cannot pretend that this applies to the present volume. Why, then, did we still think that it was worth putting out an expanded and thoroughly revised second edition? Firstly, the European Union was enlarged again in 2007. We now in- clude the newest Member States, Bulgaria and Romania, in our com- parison. The analyses of these two countries further confirm the com- plexity of citizenship arrangements in the post-communist countries of Central and Eastern Europe, which use their citizenship legislations to build and maintain external relations with ethnic kin populations and diaspora groups in neighbouring states. Secondly, we had already included Turkey in the first edition; the new edition also contains a chapter on Croatia and thus fully covers the official candidates for membership, which are currently negotiating their accession to the European Union.1 This allows us to study the on- going impact of enlargement on concepts and policies of citizenship. Moreover, both Turkey and Croatia are major source countries of immi- gration for the fifteen pre-2004 EU countries. These traditions as send- ing countries are reflected in the Turkish and Croatian citizenship leg- islations, which interact with the citizenship policies of countries where the Turkish and Croatian emigrants have settled. Finally, the two accession states both share several important features or historical con- nections with the newer Member States. The conceptions of citizenship in Turkey and Cyprus still show traces of the Ottoman legacy and Tur- key also shares some Mediterranean commonalities with Malta, while Croatia has had to cope with challenges of state formation in the post- Yugoslavian context that are similar to those of Slovenia. Thirdly, one of the main results of our comparative studies on the acquisition and loss of nationality in the European Union is that citi- zenship policies have become thoroughly politicised and are therefore also subject to frequent changes. This increased volatility of reforms means that published results can be quickly superseded by new events. We have therefore offered all of the authors the opportunity to update CITIZENSHIP POLICIES IN THE NEW EUROPE 16 and revise their chapters so that they cover significant new develop- ments. Preface A third section looks at current political debates and any changes planned for the future. Finally, the reports present the statistical developments since 1985, describe which modes of acquisition and loss of nationality are dealt with by the available sta- tistics and explain important breaks in the numbers of acquisitions PREFACE 17 and loss of nationality in their country. Like the country reports gath- ered for the NATAC project, the reports included in this volume do not primarily aim at a legal comparison but concentrate on the historical and political background of current regulations for the acquisition and loss of nationality. A further question guiding our research was the im- pact of the EU and other international bodies on the evolution of these regulations. g Nevertheless, this book on citizenship in the new Europe is a publi- cation in its own right with a very specific focus. The concepts of na- tionality and citizenship in the fourteen countries under discussion in this volume generally differ quite strongly from those prevalent in Wes- tern Europe. By and large, citizenship in these countries is still closely linked to an ethnic interpretation of nationality, transmission to subse- quent generations is exclusively based on descent, there is greater hos- tility towards multiple nationality, and greater emphasis is laid on citi- zenship links with ethnic kin-minorities in neighbouring countries and expatriates. Indeed, emigration has played a more important role for recent citizenship reforms in these countries than immigration. Yet, a few among them are already experiencing another transition, from a sending country to a transit country and finally to a receiving country of new immigration. Moreover, eleven of these countries have also un- dergone a transition from communist to democratic rule. A final fun- damental contrast with the old fifteen EU Member States is that none of these countries has enjoyed continuous independence within the present state borders for more than 60 years. In addition to dealing with individual acquisition and loss of citizenship, these countries therefore had to resolve the puzzling problems of initial collective citi- zenship determination for large populations in the context of state re- storation, of new establishment after partitioning or secession or of geographic relocations of borders. Preface Again, this has sometimes implied a return to ethnic roots and the exclusion of long term residents and their children for political reasons, such as the restrictive access to Es- tonian and Latvian citizenship for Russian immigrants who settled there after 1940. 9 This book does not include a detailed comparison of specific provi- sions in nationality law, as provided by Harald Waldrauch in volume 1 of the NATAC project on the fifteen ‘old’ Member States. However, we have added two comparative chapters to the fourteen case studies: a re- vised and updated version of Andre Liebich’s introduction to the first edition, which traces the historical trajectories of citizenship in the post-communist states, and a new comparative overview of the main features of current citizenship legislation by Wiebke Sievers that con- cludes the present volume. Both chapters emphasise the specific chal- lenge of new state formation and initial determination of citizenship. 18 CITIZENSHIP POLICIES IN THE NEW EUROPE What we provide in this book is not a definitive account of compara- tive citizenship studies in this European region, but a starting point for further inquiries. There are four main areas where we see a need for future research. Firstly, in order to fully understand how citizenship policies affect specific groups of migrants or minorities, we need to break down the laws into their components and study then how they are implemented and how the legislation of different countries interacts in generating le- gal statuses or bundles of rights and obligations. The research line in- itiated by the NATAC project should therefore eventually also be fully applied to the new Member States and accession countries.3 pp Secondly, we need to go beyond comparing legislative output to pro- mote studies of the policy formation process that leads to citizenship reform. Political parties, associations lobbying for specific interests, in- ternational organisations and public discourses in the mass media are all important factors that need to be researched in order to better un- derstand how and why citizenship issues enter the political agenda. y g Thirdly, building on these solid empirical foundations, one can try to condense the comparison into quantitative indices that measure how open or closed a citizenship regime is for newcomers or for external af- filiations of emigrants and kin minorities. Preface In the past, such standar- dised comparisons were often based on a few selected indicators, such as provisions on ius soli, residence requirements for naturalisation and toleration of dual citizenship. By studying the citizenship laws of the new EU Member States we can see why this approach is often mislead- ing. Formal residence requirements do not always tell us how easy ac- cess to naturalisation is. Some countries only start counting residence periods from the day the applicant has acquired a residence permit, which may take several years depending on the status of the applicant and the country of residence. In other countries, discretion regarding naturalisation is fairly unlimited. Moreover, toleration of dual citizen- ship often applies only to emigrants, not immigrants. We are happy to report that recent attempts to standardise the comparison between citi- zenship policies have improved, partly as a result of the findings of the NATAC project. We want to mention here specifically the revised ver- sion of Marc M. Howard’s Citizenship Policy Index (Howard 2010), Sara Wallace Goodman’s CIVIX index that measures language, country knowledge and value commitment requirements (Wallace Goodman 2010), as well as the new version of the Migrant Integration Policy In- dex MIPEX (British Council 2008). Fourthly, the point of standardised comparison is often to encourage policy changes oriented towards best practices in other countries. From a social science perspective, the main goal, however, is to explain why countries adopt or change the policies they have. The NATAC volumes 19 PREFACE and the present book provide much material for contextual policy ex- planation, but have not attempted a systematic comparative explana- tion. Much of the older literature in this field is based on extrapolating trends from a few case studies. Using NATAC results, recent studies have revised some earlier hypotheses about a general trend towards lib- eral convergence and have suggested some new ideas on how to ex- plain variations between countries (see e.g. Joppke 2007, 2008; de Groot & Vink 2010). Comparative analyses of larger numbers of cases using quantitative indicators in order to identify causal variables for policy change are still rare. The aforementioned recent studies by Ho- ward and Wallace Goodman illustrate that citizenship policies are now examined by the whole range of disciplinary and methodological ap- proaches available in the social sciences. Preface We hope that the present volume will encourage many more re- searchers to overcome the Western bias that has shaped the field of ci- tizenship studies for such a long time by including the new Member States and accession countries in their comparative studies. We also hope that this book will alert policymakers to the fact that the common citizenship of the European Union connects the citizenship regimes of Member States. In Europe, distinct historic legacies and sovereign powers of self-determination in matters of citizenship are increasingly challenged through growing interdependence between states connected through migration and supranational integration. Rainer Baubo¨ck, Bernhard Perchinig and Wiebke Sievers Vienna and Florence, August 2008 CITIZENSHIP POLICIES IN THE NEW EUROPE 20 citizenship in Europe within the European Union Democracy Observatory (EUDO), which is currently being built by the Robert Schuman Centre of the European University Institute. Notes 1 The Former Yugoslav Republic of Macedonia has also been a candidate since 9 November 2005, but accession negotiations have not commenced due to its conflict with Greece over the name ‘Macedonia’. 1 The Former Yugoslav Republic of Macedonia has also been a candidate since 9 November 2005, but accession negotiations have not commenced due to its conflict with Greece over the name ‘Macedonia’. 2 The first edition of this book was based on papers presented at a conference held in Vienna from 30 June to 2 July 2005. This conference was organised within the framework of the EU-funded Network of Excellence IMISCOE (International Migration, Integration and Social Cohesion in Europe) by a thematic research cluster that focuses on migration and citizenship. We would like to thank the European Committee for funding this meeting, the IMISCOE Editorial Committee and Amsterdam University Press for providing a platform for the dissemination of our work, all reviewers for their helpful comments and, last but not least, Karina Hof for her meticulous work on the proofs. 2 The first edition of this book was based on papers presented at a conference held in Vienna from 30 June to 2 July 2005. This conference was organised within the framework of the EU-funded Network of Excellence IMISCOE (International Migration, Integration and Social Cohesion in Europe) by a thematic research cluster that focuses on migration and citizenship. We would like to thank the European Committee for funding this meeting, the IMISCOE Editorial Committee and Amsterdam University Press for providing a platform for the dissemination of our work, all reviewers for their helpful comments and, last but not least, Karina Hof for her meticulous work on the proofs. 3 This is one of the major aims of the project Access to Citizenship in Europe (EUCITAC), which is financed by The European Fund for the Integration of Third Country Nationals and coordinated by Rainer Baubo¨ck together with Jo Shaw from the University of Edinburgh. EUCITAC aims to install a web-based observatory on Bibliography Baubo¨ck, R., E. Ersbøll, K. Groenendijk & H. Waldrauch (eds.) (2006), Acquisition and Loss of Nationality. Policies and Trends in 15 European Countries. Vol. 1: Comparative Analyses, Vol. 2: Country Analyses. Amsterdam: Amsterdam University Press. British Council (2008), Migrant Integration Policy Index. www.integrationindex.eu. Groot, R. de & M. Vink (2010), ‘Citizenship Attribution in Western Europe: International Framework and Domestic Trends’, Journal of Ethnic and Migration Studies 36 (4) (forthcoming). Howard, M.M. (2010), ‘Liberalizing Change or Continued Restrictiveness? Explaining Variation in Citizenship Laws in 11 EU Countries’, Journal of Ethnic and Migration Studies 36 (4) (forthcoming). Joppke, C. (2007), ‘Transformation of Citizenship: Status, Rights, Identity’, Citizenship Studies 11 (1): 37-48. Joppke, C. (2008), ‘Comparative Citizenship: A Restrictive Turn in Europe?’, Law & Ethics of Human Rights 2 (1): 1-41. Wallace Goodman, S. (2010), ‘Integration Requirements for Integration’s Sake? Identify- ing, Categorizing, and Comparing Civic Integration Policies’, Journal of Ethnic and Migration Studies 36 (4) (forthcoming). Introduction: Altneuländer or the vicissitudes of citizenship in the new EU states Andre Liebich Altneuland is the title of a novel written over a century ago by the Zio- nist leader Theodor Herzl. The old-new land Herzl had in mind was Palestine but the term is apposite for the lands with which this volume is concerned, in particular, the former communist states that have re- cently joined the European Union. From the point of view of the issue of citizenship, these countries display a peculiar blend of antiquity and novelty that deserves careful consideration. In this introduction, I therefore propose to make a survey of the preconditions and conditions of citizenship in the new EU Member States through the prism of ‘old’ and ‘new.’ Applying these terms, I shall first consider the specificities of statehood in this group of countries; I shall then look at the evolu- tion of principles of political membership, and, finally, I shall consider the efforts to incorporate the past into the present citizenship provi- sions. 1 New states and old concerns, or why there is not much plural citizenship in the Altneuländer When the First World War broke out, less than a century ago, only two of the twelve new members of the EU enjoyed statehood. Bulgaria had acquired sovereignty barely six years earlier and Romania, though a fully sovereign state for a generation, was to change boundaries so sub- stantially as an outcome of the First World War that it was practically recreated. Of the remaining countries, five (Poland, Hungary, Lithua- nia, Latvia, Estonia) arose as a result of the Paris Peace Treaties (1919). Only one (Hungary) has enjoyed uninterrupted statehood since 1918 and, in the case of the Baltic states, their statelessness in this period has lasted longer than their statehood.1 Two European states (Cyprus and Malta) acquired independence in the course of Cold War decolo- nialisation.2 Three other countries (Slovenia, the Czech Republic, Slo- vakia as well as potential EU Member State Croatia) have only become states in the last fifteen years (though Slovakia and Croatia had a brief and not very happy experience as states during the Second World War). 22 ANDRE LIEBICH ANDRE LIEBICH To be sure, at least two countries, Poland and Hungary, have long been acknowledged, even when absent from the map and even by such sceptics as Marx and Engels, as one-time historic states (Rosdolsky 1986; Connor 1984). Other countries, notably Bulgaria, Lithuania and the Czech Republic, might make weaker claims to a distant statehood in a more or less misty past. The contrast with the situation of the ‘old’ fifteen EU Member States could not be more striking. Although the ‘old’ EU also numbers three countries that only arose after the First World War (Finland, Austria, Ireland) and one whose existence was in- terrupted (Austria), twelve of the fifteen old EU countries have known uninterrupted statehood for periods running from well over a century (Germany, Luxemburg, Italy, Belgium, Greece) to many hundreds of years (United Kingdom, the Netherlands, Spain, Portugal, Sweden, Denmark, France). , ) The recent and discontinuous statehood that characterises the new EU states has broad implications for political attitudes and identity. The Hungarian public intellectual Istvan Bibo´ has spoken of the ‘dis- tress of the small states of Eastern Europe’, by which he means ‘an- guish at the perspective of the disappearance of one’s own people and country’ (Bibo´ 1991 [1946]: 13-69). This anxiety is based on the histori- cal realities noted above but it is reinforced by demography. 1 New states and old concerns, or why there is not much plural citizenship in the Altneuländer Though there are smaller states in the old EU, some of the new Member States are very small indeed (Slovenia, Estonia, Latvia and Lithuania together are smaller in population than Belgium or Portugal) and all of them combined, excepting Poland, have the population of one of the larger EU Member States, such as France or Italy. Even Poland, whose popu- lation is larger than all the other new EU adherents put together, is it- self only about the size of Spain. And, as though to underline that in East Central Europe even a population of 38 million nationals does not spare one from brooding on the survival of the state, Poland’s hymn still begins, somewhat ominously, ‘Poland has not yet perished while we are alive’ (further on these arguments, Liebich 2008). The fragility of statehood in East Central Europe drives all these countries in the direction of a state-reinforcing overcompensation. The preambles to most constitutions or other foundational documents (see annex) evoke ancient genealogies and historical continuities. Moreover, the Baltic states’ insistence on the legal fiction of uninterrupted state- hood, despite a half century of statelessness, leads them to adopt con- stitutional and legislative dispositions that transform a fixed date into a marker of timelessness. Legitimacy apparently reposes, at least in part, upon antiquity and continuity and the search for these is a serious task (Liebich 1995). One would imagine that this kind of tenacious attachment to a re- cent and therefore tenuous type of statehood would be reflected in the 23 INTRODUCTION philosophy and provisions regarding plural citizenship (Liebich 2000). This is indeed the case, though the variations are many and are them- selves significant. Generally speaking, one sees a spectrum running from qualified or court-imposed interdiction (Czech Republic and Lithuania) to unqualified (Hungary) or almost unqualified (Slovakia) acceptance of plural citizenship.3 In between, we find the countries that tend strongly towards acceptance (Poland and Slovenia) and a growing number of countries that do not allow plural citizenship for naturalised citizens but authorise it for members of their own ethnic group who repatriate in their country of origin, for citizens by birth who naturalise abroad or even for co-ethnic kin groups residing abroad. The situation in many of these countries appears to be in flux. 1 New states and old concerns, or why there is not much plural citizenship in the Altneuländer Three countries (Latvia, Poland and Slovenia) stipulate either that nationals will only be ‘considered’ citizens of the respective country or that na- tionals will ‘not be recognised’ as citizens of another country. In the cases of Poland and Slovenia, administrative authorities may require a candidate for naturalisation to relinquish previous citizenships but they apparently rarely do so. In Latvia, the courts have ruled that plural citi- zenship can be prohibited, but this applies only for naturalised citi- zens. Lithuania, where the Constitution states that no one may be a ci- tizen of both Lithuania and another state (art. 12, para. 2), moved to- wards the acceptance of plural citizenship for its pre-1940 nationals and their descendants, even those who did not repatriate. These inno- vations have since been struck down by the national courts as unconsti- tutional and discriminatory, leaving Lithuania with no provision for plural citizenship. Estonia, somewhat casuistically, does not accept plural citizenship on the basis of the 1995 citizenship law but does ac- cept it on the basis of its 1938 law. This effectively means that only nat- uralised citizens are required to relinquish their previous nationalities. q q p Whether a country edges towards the position of accepting or limit- ing plural citizenship, it generally looks out for its own ethnic kin, waiving most requirements, sometimes including language or resi- dency requirements, on behalf of expatriates and even their descen- dants or on behalf of minorities abroad. In addition to the cases men- tioned above, this policy of ethnic preference is particularly blatant in the cases of Bulgaria and Croatia, where the purpose is to establish the symbolic borders of the nation, beyond those of the state. This is also the thrust of the Romanian policy of extending citizenship to those per- sons and their descendants who were Romanian citizens before Roma- nia’s territorial losses in 1940. In contrast to other cases, however, this extension also includes non-Romanian ethnics living in these areas. 1 New states and old concerns, or why there is not much plural citizenship in the Altneuländer All three countries, but particularly Romania, which has thus offered its ci- tizenship to many if not most of the inhabitants of Moldova and some 24 ANDRE LIEBICH inhabitants of Ukraine, have pulled back from this policy – a policy that could be considered generous or imperialistic, as one would have it – since these countries have found themselves swamped with ‘oppor- tunistic’ applications that defeat the original ethnic in-gathering inten- tion of the policy. p y All in all, one may identify a tendency, however weak, towards the relaxation of the injunctions against plural citizenship.4 States have ab- rogated the communist-era bilateral conventions on elimination of cases of dual nationality. This relaxation does not, however, bring these states closer to the spirit of the most recent European Convention on Nationality (1997), which accepts plural citizenship as much as earlier conventions discouraged it. Most of these states reserve plural citizen- ship for their own ethnic kin. They are therefore not reacting to pres- sures for euro-compatibility but rather to pressures from their own e´migre´ communities and from nationalist politicians. As a conse- quence of the fall of communism, these countries have reconciled themselves with their historical e´migre´ communities, just as these communities abroad have reconciled themselves with their countries of origin. A recent development is that these countries are producing a significant new wave of emigration. Part brain drain, part cheap labour, stimulated by globalisation as well as by EU enlargement, these new emigrants are even more interested in maintaining ties with their home countries than their predecessors. The meanderings of policies regarding plural citizenship, which have provoked intense public debate in many of these countries, bring to the fore two characteristics of citizenship in the post-communist states. First, the triumph of ethnic conceptions of citizenship over others; second, the use of citizenship policy as a means to right histori- cal wrongs. These are the themes of the following two sections of this introduction. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship 2 The classic distinction between civic and ethnic conceptions of citizen- ship, as well as that between citizenship founded upon ius soli and ius sanguinis, apply to the countries under discussion here too.5 Signifi- cantly, what might be considered the more enlightened variant of citi- zenship, civic citizenship (or at least a prototype of civic citizenship), as well as the more progressive principle of membership, ius soli, belong to these countries’ past rather than to their present. In the two countries of the area under discussion that have the strongest state tradition, Poland and Hungary, a medieval conception 25 INTRODUCTION of political citizenship prevailed well after it had disappeared else- where. In both countries, as in some other parts of Europe, the gentry or equestrian estate was seen as constituting the nation, that is, the po- litically enabled and active part of the population. If, from the perspec- tive of global justice, ‘citizenship in Western liberal democracies is the modern equivalent of feudal privilege’ (Carens 1987: 252), then feudal privilege may well be the medieval equivalent of citizenship. The ori- ginality of the Polish and Hungarian cases was that this estate, largely made up of the landowning squirearchy or even the landless petty gen- try, though still only a small minority, comprised a far broader section of the overall population than it did, for example, in Western Europe.6 Here, as in pre-modern Western Europe, estate identity overrode lin- guistic or ethnic criteria. In the vast multiethnic entities that were the Polish and Hungarian kingdoms, referred to as the Polish-Lithuanian Commonwealth and the Lands of the Crown of Saint Stephen, social status thus trumped the multitude of potentially competing blood con- nections. The Polish szlachcic (noble or gentleman) was proud to de- clare himself natione polonus, gente ruthenus (or lituanus), thus affirm- ing that Polish political identity was compatible with Ruthenian (that is, Ukrainian, in modern terms) or Lithuanian primordial ties. As these terms may recall, a neutral dead language, Latin, was the political lin- gua franca of this class well into the eighteenth century in the case of Poland and into the middle of the nineteenth century in Hungary (Wa- licki 1994; Ba´ra´ny 1990: 201). Political citizenship was thus a function of estate membership. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship In Hungary, the crown was theoretically elective; in Poland, it was effec- tively elective until the disappearance of the Polish state in 1795, with the electorate consisting of the gentry estate. In spite of huge dispari- ties of wealth and power, members of this estate cultivated a formal equality to such an extent that in Poland all titles of nobility were out- lawed (Davies 1982: 239ff). In both countries as well as in Bohemia, Diets made up of members of the nation met regularly, deliberated vo- ciferously and exercised power to various degrees (Schramm 1996). In Bohemia, the estate system proved weaker and decayed earlier than in Hungary and Poland. As in these two countries, the estate sys- tem here did not originally differentiate among ethnic or linguistic identities, in this case, between Germans and Slavs. It cultivated a Bo- hemian ‘land patriotism [...] the patriotism of our aristocracy’ (Sayer 1998: 57ff). Only in the wake of the seismic events of 1848, did Bohe- mian and local identities change into ethnic ones. Bohemians and Bud- weisers became Czechs or Germans, to quote the title of a recent study which emphasises that ‘ethnicity was only one form of nationhood among several in Habsburg Central Europe, yet one that came to domi- nate the others’ (King 2002: 10). However, in Bohemia, territorial- 26 ANDRE LIEBICH based identity remained strong. During the First World War, Thomas Masaryk originally founded his case for Bohemian independence on the state rights of the historic Kingdom of Bohemia. He put aside this argument only when he saw that it did not impress British decision makers, who were indifferent to antiquarian constitutional niceties in countries other than their own. He also downplayed it as he realised that it did not further the project of uniting Slovakia with the Czech lands in a future Czechoslovak state (Agnew 2000; Galandauer 1993). ( g 993) The territorial demarcation of political membership, intimately con- nected to citizenship based on ius soli, was firmly anchored elsewhere in East Central Europe as well.7 From the early Middle Ages, the Hun- garian comitat gave local territorial content to the principle of gentry self-government (Bak 1990: 66; Holub 1958). After 1848 the comitat remained a fundamental and prestigious administrative unit. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship p y g p In the post-First World War peace treaties the victors were thus ob- liged to make concessions to the principle of ethnicity, at least as an al- ternative criterion for the determination of citizenship. The treaties al- lowed for a right of citizenship option. In the case of the Treaty of Ver- sailles with Germany, individuals could determine their citizenship not only on the basis of habitual residence but also by virtue of their place of birth, on condition that their parents were domiciled in that place at the time of their birth. As one commentator stated, ‘it [was] impossible that there be any question of race or language [italics in original]’ in set- ting criteria for optants ‘since Poland counts masses of Jews among its nationals [ressortissants] speaking a special jargon, and more than one third of the citizens of Czechoslovakia are of the German language’ (Brustlein 1922: 35). In fact, race and language were precisely the cri- teria applied for the successor states of the Habsburg empire. Indivi- duals having Heimatrecht anywhere in the former Austria-Hungary could choose, instead of the citizenship of the state in which their com- mune now lay, the citizenship of the state where the majority of the po- pulation was made up of people speaking their language and was of their ‘race’. In a sort of counterpart to the Treaty of Versailles provision that citizenship could also be based on one’s place of birth in addition to one’s current place of residence, the Treaty of Saint Germain and the Treaty of Trianon allowed citizenship to be claimed on the basis of an earlier Heimatrecht just previous to one’s current Heimatrecht (Sub- botitch 1926; Brustlein 1922). 9 9 ) One is tempted to see in the differential dispositions with regard to Germany and Austria-Hungary an expression of the different percep- tions of these two countries, Germany being seen as governed by more civic and Austria-Hungary by more ethnic considerations. Confirma- tion of such an approach might be sought in the fact that the Treaty of Versailles speaks of Czechoslovaks (art. 85) and Poles (art. 91) who are German nationals, yet does not define a Czechoslovak or a Pole, unlike Trianon and Saint Germain, which specifically evoke ‘race and lan- guage’. It may be simply the logic of Heimatrecht that leads in this di- rection. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship The Pol- ish Dietines, assemblies of local gentry, were the effective units of gov- ernment in pre-partition Poland from the fifteenth century to the late eighteenth century (Davies 1982: 323). Polish exiles after 1830, having abandoned the now obsolete idea of a Polish gentry nation, defined the Polish nation in territorial terms, as consisting of all those who lived in the territory of Poland before the first partition of 1772 (Kukiel 1955; Liebich 2004). Restoration of the Polish state within these borders was still the demand of Polish activists at the time of the First World War.8 Finally, until 1918, throughout the whole territory of the Austro-Hun- garian empire (with the partial exception of Bosnia-Herzegovina), Hei- matrecht (indige´nat, pertinenza), an original form of communal citizen- ship, was the basic building block of state citizenship (Redlich 1907).9 This institution, which deserves the attention of historians of citizen- ship, appears to have survived unto the present day only in Switzer- land.10 The Allied and Associated Powers, victors in the First World War, had fought, purportedly, for the rights of small nations and for the principle of national self-determination. Their objective was the crea- tion of national states, that is, states that were, if not ethnically homo- geneous, at least responsive to the aspirations of ethnic nations in East Central Europe. In setting down the rules for acquisition of citizenship in the successor states, however, the victorious powers resorted to terri- torial criteria. Anyone habitually resident (in the case of former Ger- man or Russian territory) or possessing communal Heimatrecht (in the case of former Austria-Hungary) within the new frontiers of a state was entitled to that citizenship. The solution did not preclude citizen- ship conundra for individuals who were not of the majority ‘race and language’ and who did not possess – or could not prove – present or past Heimatrecht in their state of residence – for example, some Hun- garians in Slovakia (Napier 1932). This was perhaps the last time that a INTRODUCTION 27 territorial principle predominated over ethnic criteria in determining citizenship in the countries with which we are concerned. Henceforth, territoriality, like social status in an earlier period, became a criterion of the past and ethnicity took the lead in regards to citizenship. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship As Heimatrecht replaces birth place and encourages the cult of a petite patrie or a spirit of subjective belonging, it may be expected that Heimatrecht-based citizenship on a state scale would edge away from a strictly impersonal basis of citizenship, such as birthplace, and seek out other criteria for belonging. Since 1918, the prevailing conceptions of identity in all of the coun- tries in question have led them to look towards ethnic criteria in defin- 28 ANDRE LIEBICH ing those entitled to citizenship, as we have already seen in the discus- sion about the selective authorisation of plural citizenship. On a formal level, the governing principle of the citizenship laws presented in this volume is descent without reference to ethnicity, albeit, in the case of the Baltic states, with a strict time reference. Bulgaria even proclaims an apparent ius soli principle that is, in fact, negated in the same breath because, according to the Constitution, any child born on Bul- garian territory is a citizen, unless it acquires another citizenship by origin (art. 25, para. 1; my italics). Ius soli is, in fact, so marginalised that in some countries (i.e. Latvia, Romania, Hungary), restrictions are made on the naturalisation of foundlings, who, one might assume, would be automatically granted citizenship.11 In addition to explicit ethnic criter- ia with regard to naturalisation, the underlying ethnic concept of citi- zenship can be found explicitly in schemes that attempt to establish a quasi-citizenship or a special connection with co-nationals abroad. The most famous recent case of such an attempt at quasi-citizenship – ‘fuzzy’ citizenship as one scholar has called it – is that of the Hungar- ian Status Law of 2001 (Fowler 2002). This measure provoked an enor- mous storm in the states concerned, the countries of the Hungarian diaspora. The question was examined by international bodies, notably the Venice Commission12 and the Council of Europe. The Hungarian Status Law was finally adopted in significantly modified form, having served as a reminder of the passions that issues of citizenship can arouse (see Ka´ntor, Majtenyi, Ieda, Vizi & Hala´sz 2004). The Hungarian Status Law provides a certain number of advantages to its beneficiaries. When in Hungary status holders enjoy the same cultural and educational benefits as Hungarian citizens, as well as sub- sidised travel, and some social security and health service benefits. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship They can work for up to three months a year in Hungary without re- striction. The Law provides additional advantages to Hungarian tea- chers living abroad (not just teachers of Hungarian but those teaching in Hungarian) and subsidies to families abroad who send their chil- dren to local Hungarian schools. State subsidies are guaranteed for Hungarian-language institutions and for Hungarian community orga- nisations abroad. The Hungarian Status Law is not unique. Overcoming lengthy bu- reaucratic and political obstacles, Poland finally introduced a Polish Card in 2007. Slovenia has adopted the Republic of Slovenia and Slove- nians Abroad Act (2006), which serves a similar purpose. Slovakia has adopted a full-fledged Law on Expatriate Slovaks (1997). The benefici- aries can reside in Slovakia ‘for a long period’ of time and can be em- ployed – apparently, for an unlimited period – without a work permit and without permanent residence status. They receive assistance ‘to maintain their Slovak identity,’ wherever they may be. There is some INTRODUCTION 29 alleviation of provisions governing social security contributions while elderly expatriates receive travel subsidies within Slovakia. Lithuania’s citizenship law (2002) provides for a certificate of indefinite ‘retention of the right to citizenship’ for pre-June 1940 Lithuanian citizens as well as ‘persons of Lithuanian descent’ – the term is unspecified – residing abroad. Why did the Hungarian status law provoke a storm abroad whereas there does not appear to have been any such adverse reaction to, say, the corresponding Slovak law? The answer seems to lie in the respec- tive definitions of prospective beneficiaries. Significantly, and perhaps paradoxically, the Slovak law has not caused international concern be- cause it defines its beneficiaries in ethnic terms whereas the Hungar- ian law is vague on ethnic requirements and precise on territorial con- ditions. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship Slovak expatriate status may be granted to an individual without Slo- vak citizenship who has ‘Slovak nationality or Slovak ethnic origin and Slovak cultural and language awareness.’ Slovak ethnic origin is ob- tained if at least one ancestor ‘up to the third generation had Slovak nationality.’ ‘Cultural and language awareness’ depends on ‘at least pas- sive knowledge of the Slovak language and basic knowledge of Slovak culture or declaring himself/herself actively for the Slovak ethnic [sic].’ I do not propose to ponder the ambiguities of the expression ‘declaring [oneself] actively for the Slovak ethnic.’ Rather, let me cite, for pur- poses of comparison, the definition contained in the Hungarian Status law: This Act shall apply to persons declaring themselves to be of Hungarian nationality, who are not Hungarian citizens and who have their residence in the Republic of Croatia, the Federal Re- public of Yugoslavia, Romania, the Republic of Slovenia, the Slo- vak Republic or the Ukraine and who have lost their Hungarian citizenship for reasons other than voluntary renunciation. (art. 1, para. 1) Simply declaring oneself to be ‘of Hungarian nationality,’ as certified by a recognised Hungarian community organisation abroad, is suffi- cient to obtain the ‘Certificate of Hungarian Nationality’ provided for in the Status Law. Note also that by referring only to persons of Hun- garian nationality rather than Hungarian ancestry or descent, the Sta- tus Law might be seen as thinking in terms of a state of affairs that disappeared in 1920. Underlying the difference in reactions provoked by the Hungarian Status Law and the (non) reaction to the Slovak Expatriate Law, is his- torical experience. For almost a millennium, Hungary, even when its 30 ANDRE LIEBICH own sovereignty was impaired, dominated the Danubian basin and out- lying areas. All or parts of the countries mentioned in the Status Law belonged to the Crown of Saint Stephen. For centuries, the Hungarian nobility – the Hungarian nation in the feudal sense, as we have seen above – owned and governed these territories. After having long ma- gyarised local elites, in the nineteenth century the Hungarian state also launched a sweeping campaign of general magyarisation. The Slovaks have been, in contrast, a dominated nation par excellence (dominated, in fact, by Hungarians). The subjects of the Slovak Expatriate Law are, above all, Slovak emigrants in America and elsewhere. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship The law also concerns Slovaks in the Czech Republic – the largest minority in that state since the dissolution of Czechoslovakia – as well as the small, and much assimilated, Slovak minority in Hungary, sometimes invoked by Bratislava to counter Budapest’s complaints about the treatment of the Hungarian minority in Slovakia. The overwhelming importance of history in determining reactions to the respective status and expatriate laws is confirmed by the Polish ex- ample. While the project of a Polish Card was stalling, Poland promul- gated a law on Repatriation (2000) that formalised a procedure pre- viously adumbrated in the Nationality Act. I shall deal with other as- pects of this law in the following section, but here let me point out that although this Polish law does dwell on ethnicity, like the Slovak law, it also has a determining territorial component, like the Hungar- ian statute. Crucially, however, the territorial scope of the Polish law is defined in such a way to exclude any former Polish territories. It con- cerns Poles ‘in the Asian part of the former Union of Soviet Socialist Republics’ – that is the three Caucasian Republics, the Asian part of the Russian Federation and the Central Asian Republics (art. 9, para. 1). The Polish Sejm (Lower House of Parliament) specifically rejected the senate’s proposed amendment that repatriation provisions be ex- tended to Poles in all states of the former socialist bloc.13 The Repatria- tion Law thus excludes Ukraine, Byelorussia and Lithuania, all of which were integrated in the pre-1795 Polish Commonwealth (Rzecz- pospolita) and parts of which were still included in the Polish ‘Second Republic,’ i.e. the interwar Polish state. Although there are consider- able numbers of Poles in these countries and, at least in the case of Byelorussia, they may have not only economic but serious political rea- sons for seeking repatriation, Poland seems to be bending over back- wards to avoid the suspicion that it is thinking in terms of its imperial past or historical boundaries. In the case of Hungary, there is a strong suspicion that this is precisely the thinking behind the Status Law.14 In summary, the new EU states go to considerable lengths to include those whom they consider their own. Old categories and new principles, or how ethnicity has trumped other grounds of citizenship This is usually understood as fa- cilitating naturalisation – sometimes to the extent of dropping virtually 31 INTRODUCTION all standard requirements in exchange for a declaration of national con- sciousness (as in the case of Bulgaria or EU candidate Croatia). Whether ‘one’s own’ are co-ethnics, as is usually the case, or inhabi- tants of lost territories, as in Romania, the twofold purpose is the same. First, as we have argued, the purpose is to define the boundaries of one’s imaginary community; and, second, the purpose is to rectify the wrongs of the past. It is to the latter objective that we shall now turn. 3 Old wrongs and new rights, or how to use citizenship to correct history The law also covers, perhaps inad- vertently, those individuals (and their descendants) who emigrated will- ingly to some of the peripheries of the Russian Empire or of the USSR; for example, as employees in Siberian development projects or in the military or civil service of the Russian or Soviet state.15 The primary tar- get of the law, however, are those families who were deported in 1939- 1940 from Soviet-occupied Eastern Poland (even though these areas are no longer part of Poland) as well as earlier exiles and deportees; members of the Polish minority in the USSR transferred in the Stali- nist era to areas far from the Polish border; and Polish nationalists and revolutionaries exiled under the tsar, especially after the 1905 Revolu- tion in the Russian Empire and the 1863 Insurrection in the Polish and Lithuanian lands, but perhaps even earlier and under other cir- cumstances. The number of persons affected by the Repatriation Act is insignificant. Between 1997 and 2006, the total number of the repa- triated, including non-Polish spouses, was some five thousand, the lar- gest number of whom came from Kazakhstan (see Go´rny & Pudzia- nowska in this volume). However, the symbolic significance of the act as an affirmation that the act of conferring citizenship may be used to right the wrongs of history, far back into the past, is enormous. Although attempts to replace the communist-era Polish citizenship law (1962) bogged down in legislative paralysis, the bill proposed by the Sejm to the senate in 2000 gives further insight into the hypoth- esis formulated above regarding the objective of righting historical wrongs through citizenship law.16 According to the proposed bill, the President may confer Polish citi- zenship, upon his or her own decision, on foreigners who did military service during the 1939-1945 war in the Polish army or in Polish mili- tary formations attached to Allied forces on all fronts, or who served in Polish underground formations and organisations, including those in partisan units attached to such organisations. These individuals do not need to have possessed Polish citizenship in the past (art. 17, paras. 1.1, 1.2, 2). 3 Old wrongs and new rights, or how to use citizenship to correct history 3 A peculiarity of the new EU states is that citizenship laws and related provisions are formulated with the intention of redressing past wrongs. The compensatory or restitutional function – Wiedergutmachung, in the literal sense of the term – is particularly strong with respect to the re- cent communist past, though it extends to earlier periods as well. The Polish Repatriation Act mentioned above is a prime example of an attempt at such historical redress. The preamble to the Act begins by ‘recognising that the duty of the Polish state is to allow the repatria- tion of Poles who had remained in the East […] due to deportations, ex- ile and other ethnically motivated forms of persecution.’ Repatriates enjoy significant benefits. They acquire Polish citizenship on the day they cross the Polish border (art. 4). Their costs of resettlement are un- derwritten by the Polish state. Repatriates are of ‘Polish extraction [and] declaring Polish national- ity.’ Polish extraction is defined as having at least one parent or grand- parent or two great grandparents who held Polish citizenship or who cultivated ‘Polish traditions and customs’ (art. 5). Polish nationality is ascertained by demonstrating ‘links with Polish provenance, in particu- lar by cultivating Polish language, traditions, and customs.’ Knowledge of Polish is, obviously, an advantage but it is not a requirement to the same degree as ‘traditions and customs’, since the latter suffice to con- firm the Polish nationality of one’s forbearers. These traditions and customs remain undefined, allowing wide latitude for consular officials who, according to the law, decide whether an individual meets criteria for repatriation. One supposes that some of the most evocative tradi- tions for the vast majority of today’s Polish population would be reli- gious; for example, celebration of Christmas in the Polish style. This might encompass non-Catholic Christian Poles and even non-believers but it would exclude members of other faiths, such as observant Jews. In spite of what one might expect from the preamble and spirit of the law, proof of deportation, forced exile or persecution are not condi- ANDRE LIEBICH 32 tions for obtaining repatriate status. 3 Old wrongs and new rights, or how to use citizenship to correct history The bill also provides for restitution of citizenship, without pre- sidential intervention and simply on the basis of a declaration before a consular official within a specified time period, for some individuals who left Poland between 1 September 1939 and 4 June 1989 – the lat- ter date being identified as the beginning of the end of communist rule. Those reinstated include individuals who, in order to leave Po- land, were forced to renounce their citizenship under threat of ‘repres- sions and chicaneries’, including arrest, loss of work or dwelling, or ex- pulsion from schools, including universities (art. 28, para. 1.1.b). The specific victims of communist persecution covered in this provision would seem to be, above all, those students, intellectuals and others purged as ‘Zionists’ in 1968. Reinstatement is not granted to those INTRODUCTION 33 who left Poland voluntarily on the basis of a declaration that they be- longed to a non-Polish ethnic group and who ‘obtained the citizenship of the native country of their nationality’ (art. 28, para. 2.4.). This pro- vision is aimed at preventing ethnic Germans or others from benefit- ing from the reinstatement granted to the victims of the 1968 purges. Finally, the bill offers restitution of citizenship to those who had lost it by enlisting in the armed forces of Great Britain, the US or France after (!) 9 May 1945. In a sense, the bill appears to be saying that Po- land – the real Poland which is now able to express itself – had never taken a stand against its wartime allies during the Cold War. g g The Czech Republic, also keen to underline and correct the injus- tices of the communist era, adopted restitution laws. The Law on the Citizenship of Some Former Czechoslovak Citizens (1999) opens with the following, somewhat grandiose declaration: Parliament, in order to assuage the legacy of certain wrongs that occurred in the period 1948 to 1989, and realising that Czechs and compatriots living abroad contribute to maintaining and cul- tivating the national cultural heritage as well as to deepening ties of common belonging with the Czech Republic and realis- ing that Czech emigrants developed, in exile, notable spiritual, political and cultural activity in favour of renewal of freedom and democracy in its homeland and that this activity deserves ex- traordinary recognition. 3 Old wrongs and new rights, or how to use citizenship to correct history In fact, however, the law benefits all individuals and their descendants who lost their Czechoslovak citizenship during this period for whatever reason, including by virtue of the prohibition on plural citizenship in Czechoslovakia or by virtue of naturalisation in a state that prohibited dual citizenship but, presumably, no longer does so now. Restitution of citizenship here may thus be seen as a favour or as a sort of citizenship amnesty offered to all Czechs, whatever the circumstances of their loss of citizenship. The law did have a cut-off period that expired in 2004; however, the deadline was abolished (Seitlova´ 2005: 11; Barsˇova´ in this volume). With regard to the numerically significant and politically vo- cal Czech-American lobby, the bilateral convention dating back to 1928 between the US and Czechoslovakia prohibiting dual citizenship had already been invalidated by a government decree in 1997, without re- ference to the 1993 Czech Citizenship Law article (art. 17) prohibiting dual citizenship in general. In practical terms, the 1999 law on former Czechslovak citizens would therefore be superfluous for this important group. This law, as well as other legal dispositions, takes care to in- clude some categories of Slovaks among its beneficiaries and dual Czech and Slovak citizenship is authorised, again as an exception to a In fact, however, the law benefits all individuals and their descendants who lost their Czechoslovak citizenship during this period for whatever reason, including by virtue of the prohibition on plural citizenship in Czechoslovakia or by virtue of naturalisation in a state that prohibited dual citizenship but, presumably, no longer does so now. Restitution of citizenship here may thus be seen as a favour or as a sort of citizenship amnesty offered to all Czechs, whatever the circumstances of their loss of citizenship. The law did have a cut-off period that expired in 2004; however, the deadline was abolished (Seitlova´ 2005: 11; Barsˇova´ in this volume). With regard to the numerically significant and politically vo- cal Czech-American lobby, the bilateral convention dating back to 1928 between the US and Czechoslovakia prohibiting dual citizenship had already been invalidated by a government decree in 1997, without re- ference to the 1993 Czech Citizenship Law article (art. 17) prohibiting dual citizenship in general. In practical terms, the 1999 law on former Czechslovak citizens would therefore be superfluous for this important group. 3 Old wrongs and new rights, or how to use citizenship to correct history This law, as well as other legal dispositions, takes care to in- clude some categories of Slovaks among its beneficiaries and dual Czech and Slovak citizenship is authorised, again as an exception to a ANDRE LIEBICH 34 general prohibition. One could argue, however, that the latter provi- sions are no longer prompted by considerations of historical justice but, rather, amount to housecleaning operations dealing with some of the messy aspects of the Czech-Slovak divorce. y p In the case of the Baltic states, the very reemergence of these coun- tries is itself seen as a redress for historical injustice. Naturally, citizen- ship laws also serve the purpose here of correcting past iniquities and they do so largely by legally abolishing the time period during which injustice was perpetrated. The Estonian Nationality Law (1995) does not mention specific dates, yet this does not mean that it is neutral with regard to them. At the time of registration for Estonian citizen- ship in 1989, those who had an a priori legal claim to citizenship were only those who were themselves, or one of those whose forbearers was, an Estonian citizen on 16 June 1940, the date of the Soviet ultimatum leading to occupation. In accordance with the thesis on state continuity, in 1992 the Estonian Parliament voted to reapply the Citizenship Act of 1938, as amended up to 16 June 1940 (Thiele 2002). The latter qua- lification deprived a number of resident non-Estonian nationals of elig- ibility for facilitated naturalisation. Like certain other countries, Estonia specifically states that it will not grant or restore citizenship to those who have acted against the interests of the state (art. 21, para. 3). Inde- pendent of this provision, the Estonian law, until 2005, also denied citi- zenship (by denying permanent residence status, which is a precondi- tion for citizenship) to individuals and spouses of individuals who en- tered Estonia ‘in conjunction with the assignment of military personnel into active service, the reserve forces or retirement’ (art. 21, para. 6). An exception was made in the law for individuals who had re- tired from the armed forces of a foreign state and had been married for at least five years (and were still married) to a natural-born Estonian citizen. (One wonders how many such cases there may be.) Apparently, historical injustices may be righted not only by conferring citizenship but also by denying it. 3 Old wrongs and new rights, or how to use citizenship to correct history Latvia’s Citizenship Law (1994) also identifies citizens, in the first instance, as those who were citizens on 17 June 1940 and their descen- dants, unless they had become citizens of another state after 4 May 1990 (art. 2, para. 1). Naturalisation by Latvians abroad during the peri- od of occupation is thus distinguished from naturalisation since the re- acquisition of independence. This is in accordance with the idea that, since the occupation was illegal, no change of a citizen’s legal status could occur in that period and thus those who were citizens in 1940 continued to be citizens in 1990, whatever they had done in the mean- time. The law also considers as citizens women and their descendants who lost Latvian citizenship by virtue of a law of 1919 concerning wo- men who married foreigners. This provision too – variants of which INTRODUCTION 35 may be found in other citizenship laws – represents the correction of a historical injustice, though one related to gender rather than commu- nist rule. Restrictions on who can obtain Latvian citizenship are more severe in some respects than those in Estonia. Citizenship will not be granted to those whom courts have identified as propagating, after 4 May 1990, racist or totalitarian ideas (the latter comprising communist ideas) as well as those who, after 13 January 1991, acted against the Re- public of Latvia through participation in the Communist Party (CPSU [LCP]) or front organisations, including the Organisation of War and Labour Veterans. In Latvia, neither retired Soviet military and police personnel, albeit only those who came to Latvia directly after demobili- sation, nor former employees or even informants for the Soviet security services are eligible for citizenship. This also holds true for military personnel who came to Latvia on active service. Brief mention is made of persons who entered Latvia in accordance with the Mutual Assis- tance Pact between Latvia and the USSR of 5 October 1939, the follow- up to the Molotov-Ribbentrop Pact, but this only seems to exclude the possibility of exceptional rather than regular naturalisation (art. 13, para. 1.3). Lithuania adopted the earliest and most liberal policies of naturalisa- tion among the Baltic states. 3 Old wrongs and new rights, or how to use citizenship to correct history It did, however, distinguish between citi- zens by right and ‘potential citizens’, the latter being Soviet-era immi- grants with no criminal record and with a legal income, who were en- titled to opt for Lithuanian citizenship within two years upon adoption of the law. Citizens by right, even according to the most recent Lithua- nian Citizenship Law (2002), were considered to be those who held ci- tizenship prior to 15 June 1940, and their descendants. The aim of his- torical redress is revealed in an article (art. 14, para. 2) that makes it ea- sier for deportees or political prisoners who married Lithuanian citizens, as well as for their children born in exile, to become natura- lised. In these cases, naturalisation is facilitated even further (com- pared to other spouses of Lithuanian citizens) by shortening the resi- dency period from seven to five years. Here legislators could also not resist introducing historical memory into citizenship law. g y In most countries, redressing historical wrongs and favouring co-eth- nics are intimately connected matters and the issue of plural citizen- ship is grafted onto this connection. In some cases, however, redres- sing wrongs may inflect the ethnic connection while creating a breach with regards to the issue of plural citizenship. The Bulgarian and the Romanian cases illustrate this inter-relationship. Bulgaria opened the door to plural citizenship when it naturalised or renaturalised Bulgarian ethnic Turks who had been expelled in 1989, in the last gasp of an increasingly chauvinistic but dying communist regime. A newly de-communised Bulgaria thus sought to make 36 ANDRE LIEBICH amends for this brutal treatment of its citizens. In the meantime, how- ever, these Turks had been granted Turkish citizenship, making them ineligible for Bulgarian citizenship according to the provisions of the communist-era Citizenship Law (1968) that was still in force and even according to the new democratic Constitution (1991). Bulgaria’s new Citizenship Law (1998), however, made provisions for allowing Turks to reclaim their Bulgarian citizenship. Considerations of restitutive jus- tice thus overrode the reluctance to tolerate plural citizenship for any- one other than ethnic Bulgarians and, indeed, nudged Bulgaria towards a more liberal naturalisation policy for ethnic Bulgarians. Even after historical justice had been done, however, the question of the ethnic Turks who had been driven out remained a burning political issue. 3 Old wrongs and new rights, or how to use citizenship to correct history The dispute within the Bulgarian political spectrum became one that had to do with the voting rights of ethnic Turks who had reacquired Bulgarian citizenship but were not residing permanently within Bulgaria. There- fore, it cannot be said, even after redress was achieved, that the matter has been successfully closed with no further chance of sequels (see Smilov & Jileva in this volume). Romania, as we have seen, chose to redress the wrongs committed by its dismemberment in 1940 by extending an offer of citizenship to all individuals who had been or were descended from Romanian citi- zens, as defined by the extended border that emerged in the wake of the First World War. This was an offer that could be taken up via a sim- plified procedure, without any obligation of repatriation or renuncia- tion of other citizenships. There was, however, something paradoxical about this initiative. Romania had been quick to recognise the sover- eignty of the Republic of Moldova, whose borders correspond to a large part of Romania’s lost Bessarabia. Romania did not, however, make ter- ritorial claims against Moldova, nor against Ukraine, which, during the time it was a Soviet republic, acquired part of Bessarabia and part of the province of Bukovina that had been Austrian before it became Ro- manian in 1919. This curiously subdued form of irredentism mirrors, to some extent, the Bulgarian position regarding the naturalisation of Macedonians. Bulgaria instantly recognised the sovereignty of the new post-Yugoslav Macedonian state but refused to recognise the Macedo- nian language (creating difficulties for the authentication of bilateral agreements) and thus, given the importance of language in defining nationality in this part of Europe, took an ambiguous stance with re- gard to the existence of a Macedonian nation. Romania explained its extension of citizenship not as a negation of Moldovan or Ukrainian sovereignty but as a protest against – and redress of – the iniquity of the Molotov-Ribbentrop Pact of 1939, which made the dismemberment of Romania possible. INTRODUCTION 37 The Romanian effort to redress historical wrongs also provoked un- desired consequences, as did the corresponding Bulgarian policy de- scribed here. Perhaps the Romanian government believed that its offer of citizenship would not be taken up, inasmuch as both Moldova (at the time) and Ukraine did not authorise dual citizenship? 3 Old wrongs and new rights, or how to use citizenship to correct history Somewhat cynically, the expected result would be that the inhabitants of these two ‘lost lands’ would regret the change of borders and would yearn for their former homeland! In fact, the Romanian offer was taken up so enthusiastically that somewhere between 100,000 and 300,000 indivi- duals, mostly Moldovans, became Romanian citizens. Apparently, Ro- manian naturalisation became something of a business, involving du- bious third-party brokers. In the face of an avalanche of applications, Romania at first suspended the process (2001) and then tightened its requirements (2003). A further cause of this change must have been the realisation that the policy of naturalisation was not doing anything to promote Romanian consciousness or any other Romanian interests in Moldova. The modifications transformed the restitution of citizen- ship to former citizens (irrespective of their ethnicity) into a policy that involves the repatriation of ethnic Romanians into Romania. Thus, even when there is the initial desire to right wrongs in territorial terms, without regard to ethnicity, the issue is eventually still defined in eth- nic terms. Conclusion: The historical past, the ethnic present and the immigrant future 4 All of the countries surveyed in this introduction, with the exception of Poland, have adopted new citizenship laws in the post-communist era. All citizenship laws and related instruments, including Poland’s, have been subject to substantial tinkering or, indeed, fundamental revisions. From the studies collected in this volume we may tentatively suggest that the issue of redressing historical wrongs, especially those dating from the communist period, is now losing its saliency. This concern with the historical past has now blended with a powerfully asserted emphasis on ethnic identity. It is this emphasis that has cleared away the earlier barriers to plural citizenship, thus confirming the tenet that one’s kin will always remain one’s own, wherever they may be and whatever other allegiances they may have. The new Europe has not, however, turned its attention to any of the future issues regarding citizenship, notably those raised by what will undoubtedly be a rising wave of immigration in the years to come. A number of countries, perhaps most acutely among the Baltic states, are already experiencing a labour shortage caused by widespread emigra- 38 ANDRE LIEBICH tion, whether it is temporary or permanent. All of the countries, with- out exception, are undergoing a demographic decline of more or less drastic proportions. This vacuum is most likely to be filled by immi- grants from further East or even from the former colonial world. This will be a completely unprecedented situation for countries that have al- ways been exporters of labour and have never possessed colonial em- pires. p The countries of the new Europe are barely completing the process of updating their understandings of citizenship from those that existed in the pre-communist and communist periods to ones that correspond to their present self-image. One wonders how they will undertake the transition to yet another conception of citizenship encompassing a new reality that is only now emerging. Annex: Constitutional preambles (extracts) Croatia (1990) The millennial national identity of the Croatian nation and the continuity of its statehood, confirmed by the course of its en- tire historical experience in various political forms and by the perpetua- tion and growth of state-building ideas based on the historical right to full sovereignty of the Croatian nation, manifested itself: – in the formation of Croatian principalities in the 7th century; – [twelve other historical dates follow to confirm Croatian statehood]. Czech Republic (1993) We, the citizens of the Czech Republic in Bohe- mia, Moravia, and Silesia, at the time of the renewal of an independent Czech state, being loyal to all good traditions of the ancient statehood of Czech Crown’s Lands and the Czechoslovak State. Estonia (1992) Unwavering in their faith and with an unswerving will to safeguard and develop a state which is established on the inextin- guishable right of the Estonian persons to national self-determination and which was proclaimed on February 24, 1918 […] which shall guar- antee the preservation of the Estonian nation and its culture through- out the ages, the Estonian people adopted, on the basis of art. 1 of the Constitution which entered into force in 1938, by Referendum held on June 28, 1992 the following Constitution. Hungary (1989) In order to facilitate a peaceful political transition to a constitutional state, establish a multi-party system, parliamentary de- mocracy and a social market economy, the Parliament of the Republic of Hungary hereby establishes the following text as the Constitution of 39 INTRODUCTION the Republic of Hungary, until the country’s new Constitution is adopted. the Republic of Hungary, until the country’s new Constitution is adopted. Latvia (1990)17 The independent state of Latvia, founded on 18 Novem- ber 1918, was granted international recognition in 1920 and became a member of the League of Nations in 1921. The Latvian Nation’s right to self-determination was implemented in April 1920, when the people of Latvia gave their mandate to the Constituent Assembly chosen by uni- versal, equal, direct and proportional elections. In February 1922, the Assembly adopted the Constitution of the Republic of Latvia, which is still in effect de iure. Lithuania (1992) The Lithuanian Nation having established the State of Lithuania many centuries ago […] having for centuries defended its freedom and independence […] having preserved its spirit, native lan- guage, writing, and customs. Annex: Constitutional preambles (extracts) Poland (1997) Recalling the best traditions of the First and the Second Republic […] Obliged to bequeath to future generations all that is valu- able from our over one thousand years’ heritage, bound in community with our compatriots dispersed throughout the world. Slovakia (1992) mindful of the political and cultural heritage of our forebears, and of the centuries of experience from the struggle for na- tional existence and our own statehood, in the sense of the spiritual heritage of Cyril and Methodius and the historical legacy of the Great Moravian Empire. Slovenia (1991) [Proceeding …] from the historical fact that in a centu- ries-long struggle for national liberation we Slovenes have established our national identity and asserted our statehood. Notes 1 One might group Poland along with Hungary as being a state with uninterrupted continuity since 1918. Poland was partitioned in 1939 and its demise was decreed by the partitioning (and occupying) powers, Germany and the USSR. Its disappearance was not acknowledged by the other Great Powers who recognised its government in exile until the restoration of the country in 1945. 2 This chapter focuses on comparing the histories of post-communist countries of East Central Europe. The post-colonial states Cyprus and Malta as well as Turkey require separate treatment. They are, however, included in the comparative overview of migration and citizenship (see Sievers in this volume). 3 In the Czech Republic, the fundamental principle of exclusive citizenship has been breached by a number of exceptions, notably the acceptance of dual citizenship for ex-Czechoslovaks who have become Slovaks as well as for repatriates (see below). In 40 ANDRE LIEBICH ANDRE LIEBICH Slovakia, the provision that renouncement of former citizenship is ‘in favour’ of naturalisation is so weak as to be self-negating. Slovakia, the provision that renouncement of former citizenship is ‘in favour’ of naturalisation is so weak as to be self-negating. 4 As we see from the chapter by Eugene Buttiegieg, this tendency, conceived in ethnic terms and directed at a diaspora, encompasses Malta as well. 4 As we see from the chapter by Eugene Buttiegieg, this tendency, conceived in ethnic terms and directed at a diaspora, encompasses Malta as well. 5 I take the point that the civic-West/ethnic-East stereotype ‘when true is only weakly true, and according to several measures is false’ (Shulman 2002: 554). With respect to conceptions of citizenship, however, the civic/ethnic distinction seems to me a useful heuristic device in tracing a historical evolution. g 6 In Poland, 11-13 per cent of the population belonged to the equestrian estate in the sixteenth century, 9-10 per cent in the eighteenth century. In France, under the July Monarchy (1830-1848), 1.5 per cent of the population was enfranchised. In Britain, the corresponding figure at that time (1828) was 3.2 per cent (figures cited by Walicki 1982: 16). In Hungary, the gentry numbered 3-5 per cent of the population by the fifteenth century (Engel 1990: 43). 6 In Poland, 11-13 per cent of the population belonged to the equestrian estate in the sixteenth century, 9-10 per cent in the eighteenth century. Notes Austrian and Hungarian citizenship laws (the legal regimes were entirely separate) did not require candidates for naturalisation to prove that they had lost their previous nationality but, as of 1870, they did require such proof of Hungarians seeking to become Austrians and vice versa (Soubbotitch 1926: 15). 10 A recent reference source refers to indige´nat as ‘a second-degree nationality preserved by nationals of a federal entity’ – which neither Hungary nor Austria ever was – and describes it as ‘vieilli’ or archaic (Salmon 2001). Even today, however, to ‘be a Swiss citizen implies a cantonal indige´nat and a communal citizenship. The three levels are inseparable. No one can be a national (ressortissant) of a canton without communal ci- tizenship or Swiss without a cantonal indige´nat’ (http://etat.geneve.ch). The same document refers to ‘nationalite´ (indige´nat) genevoise’. In addition to a droit de cite´ communal there is also a concept in some localities of bourgeoisie communale that im- plies a co-proprietorship of communal assets. 10 A recent reference source refers to indige´nat as ‘a second-degree nationality preserved by nationals of a federal entity’ – which neither Hungary nor Austria ever was – and describes it as ‘vieilli’ or archaic (Salmon 2001). Even today, however, to ‘be a Swiss citizen implies a cantonal indige´nat and a communal citizenship. The three levels are inseparable. No one can be a national (ressortissant) of a canton without communal ci- tizenship or Swiss without a cantonal indige´nat’ (http://etat.geneve.ch). The same document refers to ‘nationalite´ (indige´nat) genevoise’. In addition to a droit de cite´ communal there is also a concept in some localities of bourgeoisie communale that im- plies a co-proprietorship of communal assets. 11 Latvia restricts citizenship to foundlings born on Latvian territory after 21 August 1991, the date of the Moscow coup that might be seen as the last gasp of the USSR (see Kru¯ma in this volume). Romania grants foundlings citizenship not on the basis of birth on Romanian territory but under the assumption that their parents (or at least one of them) held Romanian citizenship (see Iordachi in this volume). Hungary grants foundlings nationality on a conditional basis; the presumption of statelessness or Hungarian parentage may be rebutted without any time limit (Kova´cs & Toth in this volume). Notes In France, under the July Monarchy (1830-1848), 1.5 per cent of the population was enfranchised. In Britain, the corresponding figure at that time (1828) was 3.2 per cent (figures cited by Walicki 1982: 16). In Hungary, the gentry numbered 3-5 per cent of the population by the fifteenth century (Engel 1990: 43). 7 The two new Balkan states of the EU present contrasting situations. As soon as Bulgaria acquired autonomy in 1878 it conferred citizenship on all of its inhabitants. At approximately the same time, Romania instituted a citizenship policy based on ethnicity that also applied for naturalisation (see Smilov & Jileva as well as Iordachi in this volume). 8 According to Soubbotitch (1926: 55), many (unnamed) Polish jurists argued that Poland had never ceased to exist because the partitions were in fact occupations. The analogy with present-day Baltic positions is striking (see section 3 of this chapter). I have not found examples of such Polish arguments but their existence is confirmed, a contrario, through a vehement attack on such arguments by Scha¨tzel (1921). 8 According to Soubbotitch (1926: 55), many (unnamed) Polish jurists argued that Poland had never ceased to exist because the partitions were in fact occupations. The analogy with present-day Baltic positions is striking (see section 3 of this chapter). I have not found examples of such Polish arguments but their existence is confirmed, a contrario, through a vehement attack on such arguments by Scha¨tzel (1921). 9 It might be noted, too, that there was no Austro-Hungarian citizenship. Austrian and Hungarian citizenship laws (the legal regimes were entirely separate) did not require candidates for naturalisation to prove that they had lost their previous nationality but, as of 1870, they did require such proof of Hungarians seeking to become Austrians and vice versa (Soubbotitch 1926: 15). 9 It might be noted, too, that there was no Austro-Hungarian citizenship. Austrian and Hungarian citizenship laws (the legal regimes were entirely separate) did not require candidates for naturalisation to prove that they had lost their previous nationality but, as of 1870, they did require such proof of Hungarians seeking to become Austrians and vice versa (Soubbotitch 1926: 15). 9 It might be noted, too, that there was no Austro-Hungarian citizenship. Notes 11 Latvia restricts citizenship to foundlings born on Latvian territory after 21 August 1991, the date of the Moscow coup that might be seen as the last gasp of the USSR (see Kru¯ma in this volume). Romania grants foundlings citizenship not on the basis of birth on Romanian territory but under the assumption that their parents (or at least one of them) held Romanian citizenship (see Iordachi in this volume). Hungary grants foundlings nationality on a conditional basis; the presumption of statelessness or Hungarian parentage may be rebutted without any time limit (Kova´cs & Toth in this volume). 11 g y y ( ) 12 The European Commission for Democracy through Law, an advisory commission of the Council of Europe. 2 The European Commission for Democracy through Law, an advisory commission of the Council of Europe. 3 Radio Free Europe/Radio Liberty, ‘Polish Repatriation to Focus ‘‘Mainly’’ on Compatriots from Kazakhstan’, Daily Report, 9 November 2000. 13 Radio Free Europe/Radio Liberty, ‘Polish Repatriation to Focus ‘‘Mainly’’ on Compatriots from Kazakhstan’, Daily Report, 9 November 2000. 14 Countering the claim that Hungary defines the scope of its Status Law in terms of its historic boundaries, one might note that the Status Law does not extend to Austria, though part of historic Hungary today lies within Austria. This exception is INTRODUCTION 41 not sufficient to alleviate suspicions. In fact, it nourishes other grounds for resentment: the Status Law does not cover Austria because one of the tacit intentions of the Law is to minimise the effects for expatriate Hungarians of Hungary’s entry into the EU. The resulting inequality of status for citizens of Romania (until 2007), Serbia, Ukraine, etc. was one of the principal grounds for international reservations vis-a`-vis the law. not sufficient to alleviate suspicions. In fact, it nourishes other grounds for resentment: the Status Law does not cover Austria because one of the tacit intentions of the Law is to minimise the effects for expatriate Hungarians of Hungary’s entry into the EU. The resulting inequality of status for citizens of Romania (until 2007), Serbia, Ukraine, etc. was one of the principal grounds for international reservations vis-a`-vis the law. 15 Some such individuals would be covered by the provision that repatriation cannot be offered to anyone who ‘during their stay outside the Republic of Poland acted against the vital interests of the Republic of Poland or participated in human rights violations’ (arts. Notes 8.3 a and b). The law also excludes those who repatriated from Poland between 1944 and 1957 to some Soviet Republics. Presumably, however, descendants of all these individuals are still eligible for repatriation if they meet other requirements. 16 Ustawa z dnia 29 czerwca 2000 r. o obywatelstwie polskim, Tekst ustawy przekazany do Senatu zgodnie z art. 48 regulaminu Sejmu (nie zakon´czony proces legislacyjny) [Statute of 29 June 2000 regarding Polish citizenship, text of statute transmitteed to Senate according to article 48 of the Senate Regulation (uncompleted legislative process)]. http://orka.sejm.gov.pl. 17 Declaration on the Renewal of Independence. 17 Declaration on the Renewal of Independence. Bibliography Agnew, H. L. (2000), ‘New States, Old Identities? The Czech Republic, Slovakia and His- torical Understandings of Statehood’, Nationalities Papers 28 (4): 619-650. Bak, J. (1990), ‘The Late Medieval Period, 1382-1526’, in P. F. Sugar, P. Hana´k & T. Frank (eds.), A History of Hungary, 54-82. London: I. B. Tauris. Ba´ra´ny, G. (1990), ‘The Age of Royal Absolutism, 1790-1848’, in P. F. Sugar, P. Hana´k & T. Frank (eds.), History of Hungary, 174-208. London: I. B. Tauris. ), History of Hungary, 174-208. London: I. B. Taur Bibo´, I. (ed.) (1991), ‘The Distress of the East European Small States [1946]’, in his De- mocracy, Revolution, Self-Determination, ed. by K. Nagy, 13-69. Boulder: Atlantic Re- search and Publications. Brustlein, A. (1922), ‘La Notion de l’indige´nat dans les traite´s de paix de Versailles, de Saint Germain et de Trianon’, Journal du droit international 49: 34-53. Carens, J. (1987), ‘Aliens and Citizens: The Case for Open Borders’, Review of Politics 49 (2): 251-273. Connor, W. (1984), The National Question in Marxist-Leninist Theory and Strategy. Prince- ton: Princeton University Press. Davies, N. (1982), God’s Playground: A History of Poland, vol. 1: The Origins to 1795. New York: Columbia University Press. Engel, P. (1990), ‘The Age of the Angevines, 1301-1382’, in P.F. Sugar, P. Hana´k & T. Frank (eds.), A History of Hungary, 34-53. London: I. B. Tauris. Fowler, B. (2002), ‘Fuzzing Citizenship, Nationalising Political Space. A Framework for Interpreting the Hungarian ‘‘Status Law’’ as a New Form of Kin-State Policy in Central and Eastern Europe’, Working Paper 40. ESRC (Economic and Social Research Council), One Europe or Several? ESRC European Research Programme. www.one-europe.ac.uk. Galandauer, J. (1993), ‘30 kve˘tna 1917: Slovensko poprve´ v programu legitimnı´ ceske´ poli- ticke´ representace (jak cˇesi prˇekrocˇili rˇeku litavu)’ [Slovakia for the the first time in a programme of legitimate Czech political representation (how the Czechs crossed the Leitha river)], Sbornı´k Vojenske´ Akademie V Brne˘ S1 (348): 135-142. ANDRE LIEBICH 42 ANDRE LIEBICH Holub, J. (1958), ‘La Repre´sentation politique en Hongrie au Moyen Age,’ in Studies pre- sented to the International Commission for the History of Representatives and Parliamen- tary Institutions, Tenth International Congress of Historical Sciences (Rome, 1955), 79-121. Louvain: Edition Nauwelaerts. Ka´ntor, Z., B. Majtenyi, O. Ieda, B. Vizi & I. Hala´sz (2004), The Hungarian Status Law: Nation building and/or Minority Protection. Hokkaido: Slavic Research Centre. http://src-h.slav.hokudai.ac.jp. King, J. Bibliography (2002), Budweisers into Czechs and Germans: A Local History of Bohemian Politics 1848-1948. Princeton: Princeton University Press. Kukiel, M. (1955), Czartoryski and European Unity 1770-1861. Princeton: Princeton Univer- sity Press. Liebich, A. (1995), ‘Nations, States, Minorities: Why is Eastern Europe Different?’, Dissent 42 (3): 313-318. Liebich, A. (2000), ‘Plural Citizenship in Post-Communist States’, International Journal of Refugee Law 12 (1): 97-107. Liebich, A. (2004), ‘Czartoryski and the Quest for Polish Statehood’, ASN and Studjum Europy Wschodniej Conference Paper, Warsaw, July 2004. Liebich, A. (2008), ‘How Different is the New Europe? Perspectives on the New Europe’, CEU Political Science Journal 3 (3): 269-292 Napier, W. (1932), ‘Nationality in the Succession States of Austria-Hungary’, Transactions of the Grotius Society 18: 1-16. Redlich, J. (1907), ‘The Municipality. 1. Austria. The Commune System’, Journal of the So- ciety of Comparative Legislation 8 (1): 12-40. Rosdolsky, R. (1986) [1934], Engels and the ‘Non-Historic’ Peoples: The National Question in the Revolution of 1848. Translated, edited and with an introduction by J.-P. Himka. Glasgow: Critique Books. g q Salmon, J. (ed.) (2001), Dictionnaire de droit international public. Bruxelles: Bruylant. Sayer, D. (1998), The Coasts of Bohemia: A Czech History. Translated from the Czech by A. Sayer. Princeton: Princeton University Press. Scha¨tzel, W. (1921), ‘Polens Untergang und Wiedererrichtung vom vo¨lkerrechtlichen Standpunkt’, Archiv des o¨ffentlichen Rechts 40 (1): 73-86. Schramm, G. (1996), ‘Polen-Bo¨hmen-Ungarn: U¨ bernationale Gemeinsamkeiten in der politischen Kultur des spa¨ten Mittelalters und der fru¨hen Neuzeit’, in J. Bahlcke, H.- J. Bo¨melburg & N. Kersken (eds.), Sta¨ndefreiheit und Staatsgestaltung in Ostmitteleuro- pa: U¨ bernationale Gemeinsamkeiten in der politischen Kultur vom 16.-18. Jahrhundert, 13- 38. Leipzig: Universita¨tsverlag. Seitlova´, J. (2005), ‘Sena`t navrhuje lhu˚ tu pro navra´cˇenı´ obcˇanstvi’ [Senate proposes to open a timeline for restoring citizenship], Verˇejna spra´va 16 (6): 11. Shulman, S. (2002), ‘Challenging the Civic/Ethnic and West/East Dichotomies in the Study of Nationalism’, Comparative Political Studies 35 (5): 554-585. Soubbotitch, I. V. (1926), Effets de la dissolution de l’Autriche-Hongrie sur la nationalite´ de ses ressortissants. Paris: Rousseau & Cie. Thiele, C. (2002), ‘Staatsangeho¨rigkeit in den baltischen Staaten: Das Beispiel Estland’, Osteuropa 52 (6): 729-742. Walicki, A. (1982), Philosophy and Romantic Nationalism: The Case of Poland. Oxford: Clar- endon Press. Walicki, A. (1994), Poland between East and West: The Controversies over Self-Definition and Modernization in Partitioned Poland. Harvard University: Ukrainian Research Insti- tute. 1 Estonian citizenship: Between ethnic preferences and democratic obligations Priit Ja¨rve The most important reform in the nationality policy of Estonia after 1945 was the restoration of the pre-1940 nationality in 1992 by reintro- ducing the 1938 Citizenship Act with slight changes. In 1995, Estonia adopted a new Citizenship Act which did not change the basic princi- ples of the acquisition and loss of Estonian nationality but established more demanding requirements for the acquisition of nationality by nat- uralisation. 1.1.1 Nationality policy since 1945 The Republic of Estonia was established in 1918. In 1940, it was an- nexed to the Soviet Union as the Estonian Soviet Socialist Republic un- der threat of military force. As a result, the citizens of the Republic of Estonia were incorporated into the Soviet citizenry. Estonian nationality was replaced by Soviet nationality. Between 1941 and 1944, Estonia was occupied by Nazi Germany. In 1944, Estonia was re-conquered by the Red Army, and Soviet nationality was once again imposed upon the people on its territory. Estonian nationality ceased to exist de facto.1 Instead, the Soviet passports, which were issued in Estonia after the Second World War, included a mandatory line with ethnic identifica- tion of the carrier. ‘Estonian’ became one of such identifications to be used in Soviet internal passports (Soviet passports for travel abroad did not mention ethnicity). In Estonia, as opposed to many internal re- gions of the USSR, everyone was issued Soviet internal passports upon reaching the age of sixteen. These passports, not valid for travel abroad, gave the holders relative freedom to travel within the Soviet Union. The authorities stamped the carrier’s domicile registration (propiska) and marital status into the passport. The Soviet Union sought to merge the different ethnic nations and groups living in the country into a new civic identity – the Soviet peo- ple. While the Soviet authorities claimed that such an identity was emerging, and some citizens reported that they already regarded them- selves as ‘Soviets’, the official registration of different ethnic identities 46 PRIIT JA¨RVE PRIIT JA¨RVE was not discarded. Thus, Estonians had the inscription ‘Estonian’ in their passports until the dissolution of the Soviet Union,2 though, this inscription could not be automatically converted into Estonian national- ity after independence. Since 1992, only pre-1940 nationals and their descendants, regardless of their ethnic identification, were entitled to acquire Estonian nationality by registration. Those Estonians who settled in Estonia after 1940 and their descendants (with ‘Estonian’ in their Soviet passports) could not acquire Estonian nationality by simple registration but had to take the path of naturalisation. At the same time, pre-1940 nationals and their descendants of non-Estonian ethnic origin (with ‘Jew’, ‘Russian’, ‘Latvian’, ‘Pole’, etc. in their Soviet pass- ports) could acquire Estonian nationality by registration. In new Esto- nian passports the registration of ethnic identity was dropped. 1.1.1 Nationality policy since 1945 g y The debate on nationality between liberal and conservative camps started in Estonia at the end of the 1980s when the national indepen- dence movement was gathering momentum. In 1989, the campaign of registering the citizens of the pre-war Republic of Estonia and their descendants was carried out by the Estonian Citizens’ Committees, vo- luntary associations established during the perestroika era to sustain the idea of the legal continuity of the pre-war Estonian state. On the posi- tive side, this campaign helped restore the awareness of the link between the individual and the state. At the same time, being led by national conservatives, it firmly introduced the exclusive approach towards Estonian nationality. The conservatives pointed at drastic changes in the ethnic composition of the population of Estonia due to a considerable influx of Russian-speaking immigrants under the Soviet regime. These settlers had pushed the share of non-Estonians in the population up from around 10 per cent in 1940 to unprecedented 38.5 per cent in 1989. In 1992, the conservatives emerged as winners in the debate on na- tionality. As a result, the Citizenship Act of 1992 was based on the principle of the restitution of the pre-1940 nationality. Only those who were citizens in 1940 and their descendants (regardless of ethnicity) were granted Estonian nationality by registration, those who settled in Estonia after 1940 were offered the possibility of becoming Estonian nationals through naturalisation. As an immediate consequence of this Act the majority of non-Estonians as well as a small number of Esto- nians were not granted the right to participate in the national referen- dum on the country’s new Constitution in 1992 and in the first parlia- mentary elections after independence later the same year. Estonia’s new political leadership considered the great number of non-Estonian settlers as a threat to the nation. Under these conditions, nationality became an instrument for the attainment of national homogeneity and for the political containment of Soviet era settlers. The interests of the ESTONIAN CITIZENSHIP 47 Estonian ethnic nation, as then understood, were given priority over full democratic participation. In Estonia these exclusionary policies enjoyed relatively wide support as a reaction to the changes in the ethnic composition of the popula- tion. 1.1.1 Nationality policy since 1945 A survey of public opinion, carried out in the Baltic states in 1993, showed that the principle of limiting nationality to descendants of the pre-1940 citizens was supported by 44 per cent of Estonian, 52 per cent of Latvian and 12 per cent of Lithuanian respondents (Rose & Maley 1994: 31-34). These differences among the Baltic respondents correlated very clearly with the demographics of the respective coun- tries: the bigger the share of non-titular groups in a given state, the stronger the reluctance to let them participate in political life. g p p p The restoration of pre-1940 nationality had profound political conse- quences. The exclusion of the majority of non-Estonians from the for- mation of state institutions and from the process of adoption of crucial legal documents, including the Constitution, enabled Estonians to en- trench themselves firmly in all the major posts of the state avoiding power-sharing with minorities. During the referendum on indepen- dence in Estonia in March 1991 there were 1,144,309 persons with the right to vote. During the referendum on the Estonian Constitution in the summer 1992, after the adoption of the first Citizenship Act, the reported number of eligible voters was 689,319, or only about 60 per cent of the 1991 figure. Consequently, 454,990 adults had been disen- franchised (Semjonov 2000: 15). It was therefore not surprising that the Parliament elected in 1992 was 100 per cent Estonian. The restoration of pre-1940 nationality caused mass statelessness of non-Estonians, which harmed the relations between different ethnic communities inside Estonia, caused tension in the relations with Rus- sia (the absolute majority of non-citizens were Russians), and evoked criticism, usually disguised as ‘recommendations’, from prominent in- ternational and regional organisations such as the United Nations, the OSCE, the Council of Europe and the European Union. Between 1992 and 1 January 2008, the share of stateless residents in Estonia declined from 32 to 9 per cent (see Figure 1.1). However, the inability and/or the lack of motivation of older cohorts of non-citizens to master the Estonian language at the necessary level raises doubts of whether the problem of statelessness will be easily overcome in the near future if the conditions of naturalisation remain the same. 1.1.2 Restoration of Estonian nationality3 Thus, a large part of the population, especially Rus- sians, did not have the right to vote or the right to run for office in the parliamentary election of 20 September 1992 and were therefore ex- cluded from political participation, giving rise to further tensions in a ESTONIAN CITIZENSHIP 49 situation that was already strained. These tensions were somewhat eased by the right of non-citizens to vote at the local elections after 1996. After some changes in the 1992 Citizenship Act, a new Citizenship Act was passed on 19 January 1995 and entered into force on 1 April 1995.4 The new Act integrated all regulations on nationality and intro- duced some new conditions for naturalisation (residence in Estonia on the basis of a permanent residence permit issued at least five years prior to the date of written application for Estonian nationality and at least one year after the registration of the written application; and a test on the knowledge of the Estonian Constitution and the Citizenship Act). According to the initial version of the 1995 Citizenship Act, children of stateless parents born in Estonia could not acquire Estonian nation- ality after birth. This was in violation of the International Covenant of Civil and Political Rights (art. 24(3)) and the Convention on the Rights of the Child (art. 7(1)), both of which Estonia had ratified. These provi- sions proclaim the right of the child to acquire a nationality. This con- troversy triggered a heated discussion. Some politicians and lawmakers saw the danger of compromising the governing principle of nationality acquisition (ius sanguinis) by adding the ius soli principle to it. After political and academic debates, in which the role of recommen- dations issued by international actors should not be underestimated, an amendment to the Citizenship Act was finally adopted in December 1998, which entered into force on 12 July 1999. Pursuant to this amendment, children under the age of fifteen born on Estonian terri- tory after 26 February 1992 can acquire the Estonian nationality on the basis of a declaration if their parents are stateless and have been legal residents of Estonia during the previous five years. This new regulation did not include children between the ages of fifteen and eighteen who are under the protection of art. 1 of the Convention on the Rights of the Child and children born before 26 February 1992. 1.1.2 Restoration of Estonian nationality3 On 26 February 1992, the Supreme Council of the Republic of Estonia put the version of 16 June 1940 of the Citizenship Act of 1938 into force. The main features of this nationality regulation were the ius san- 48 PRIIT JA¨RVE Figure 1.1 Estonian citizens and stateless persons in Estonia, 1992-1 January 2008, per total population 68 80 81 82.3 83 32 12.4 12 10.4 9 0 10 20 30 40 50 60 70 80 90 1992 2000 2003 2005 2008 Estonian cizens Stateless Source: Estonian Ministry of the Interior and Statistical Office of Estonia Figure 1.1 Estonian citizens and stateless persons in Estonia, 1992-1 January 2008, per cent of total population Source: Estonian Ministry of the Interior and Statistical Office of Estonia guinis principle and the avoidance of dual nationality. Pursuant to art. 3 of this Law, every person who possessed or whose parents possessed Estonian nationality before 16 June 1940 – the day of the Soviet ulti- matum followed by the annexation of Estonia – had a legal claim to Es- tonian nationality. About 80,000 non-Estonians thereby acquired Esto- nian nationality. Russians and others who came to Estonia after 16 June 1940, all in all almost one third of the entire population in 1992, were automati- cally excluded from Estonian nationality. In essence, they were mostly immigrant workers but perceived by many as colonial settlers with no right to automatic acquisition of Estonian nationality. The only way for them to acquire Estonian nationality was through naturalisation. As a precondition for naturalisation, the applicant had to have his or her permanent place of residence in the Estonian territory (as proved by propiska) for at least two years before and one year after the application date (residence census ‘two plus one’) and had to prove their knowl- edge of the Estonian language. The earliest date for establishing the permanent place of residence was set at 30 March 1990. The required time period was counted only from that day onwards, so that 30 March 1993 was the earliest date when one could acquire Estonian nationality by naturalisation. 1.1.2 Restoration of Estonian nationality3 Thiele (1999) ar- gues that this domestic regulation was not fully in line with Estonia’s international obligations. g Some changes in the legislation on nationality have made the natur- alisation process easier for certain groups of applicants. For example, in June 2002, the Estonian Parliament adopted amendments to the Citizenship Act, which created special conditions for acquisition of Estonian nationality through naturalisation by persons with severe or moderate disabilities (such as a visual, hearing or speech impairment). Disabled persons who have appropriate medical certificates are not ob- liged to pass exams on knowledge of the language or of the Estonian Constitution and the Citizenship Act. There are also other measures being taken to facilitate naturalisation such as free-of-charge language 50 PRIIT JA¨RVE courses since the beginning of 2009 (financed by the European Com- mission) and recognition of Estonian language and civics exams taken by students of Russian-language schools as valid for naturalisation. 1.2 Basic principles of the most important current modes of acquisition and loss of nationality The basic principles of Estonian nationality are stipulated in art. 8 of the Constitution as follows: every child with at least one parent who is an Estonian national shall have the right, by birth, to Estonian nation- ality; any person who as a minor lost his or her Estonian nationality shall have the right to have his or her nationality restored; no person may be deprived of Estonian nationality acquired by birth; no person may be deprived of Estonian nationality because of his or her beliefs. As further specified by art. 8, the conditions and procedures for the ac- quisition, loss and restoration of Estonian nationality shall be estab- lished by the Citizenship Act. The basic constitutional principles of na- tionality are reiterated in arts. 5(1), 16(1), 28(3) and 28(2) of the 1995 Citizenship Act respectively. 1.2.1 Acquisition of nationality Acquisition of Estonian nationality is stipulated by Chapters 2 and 3 (arts. 5 through 15) of the 1995 Citizenship Act. This includes acquisi- tion of nationality by birth, by naturalisation and for achievements of special merit. Nationality by naturalisation and for achievements of special merit shall be granted by a decision of the Estonian govern- ment. According to art. 5, nationality is acquired by birth if at least one of the child’s parents holds Estonian nationality at the time of the child’s birth. Nationality is also acquired by birth if the child is born after the death of his or her father and if the father held Estonian nationality at the time of his death. If a child of unknown parents is found in Esto- nia, a court can declare that the child has acquired Estonian nationality by birth upon application by the guardian of the child or a guardian- ship authority, unless the child is proven to be a citizen of another state. According to art. 5, nobody shall be deprived of Estonian nation- ality acquired by birth. Arts. 6 through 15 establish conditions for acquisition of Estonian nationality by naturalisation and for achievements of special merit. The conditions for acquisition of nationality by naturalisation differ depend- ing on whether a person is at least fifteen years of age or under that age. ESTONIAN CITIZENSHIP 51 An alien5 who is at least fifteen years of age and wishes to acquire Estonian nationality by naturalisation shall have stayed in Estonia on the basis of a permanent residence permit for at least five years prior to the date on which he or she submits an application for Estonian na- tionality and for one year from the day following the date of registra- tion of the application. Additionally, he or she must have knowledge of the Estonian language and of the Constitution of the Republic of Esto- nia and the Citizenship Act. 1.2.1 Acquisition of nationality has served as a professional member of the armed forces of a for- eign state or who has been assigned to the reserve forces thereof or has retired there from, nor shall Estonian nationality be granted to or resumed by the spouse of such a person. 6. has served as a professional member of the armed forces of a for- eign state or who has been assigned to the reserve forces thereof or has retired there from, nor shall Estonian nationality be granted to or resumed by the spouse of such a person. 6. has served as a professional member of the armed forces of a for- eign state or who has been assigned to the reserve forces thereof or has retired there from, nor shall Estonian nationality be granted to or resumed by the spouse of such a person. Thus, art. 21(6) clearly targets those non-Estonians (together with their spouses) who are not Estonian nationals by birth and who remained in Estonia after they retired from the Soviet Army.6 However, the same art. 21 also offers them one possibility of acquiring Estonian national- ity. It stipulates that Estonian nationality may be resumed by, or granted to, a person who has retired from the armed forces of a foreign state if the person has been married for at least five years to a person who acquired Estonian nationality by birth and if the marriage has not been terminated by divorce. 1.2.1 Acquisition of nationality In accordance with the requirements pro- vided for in this Act, he or she must also have a permanent legal in- come which ensures his or her own subsistence and that of his or her dependants, be loyal to the Estonian state, and take the following oath: ‘In applying for Estonian citizenship, I swear to be loyal to the consti- tutional order of Estonia.’ For a minor to acquire Estonian nationality by naturalisation, an ap- plication by his or her parents, or by a single or adoptive parent of Es- tonian nationality, accompanied by specific documents, is required. After the amendments to the Citizenship Act, which entered into force on 12 July 1999, a minor’s stateless parents and stateless single or adoptive parent(s) also have the right to apply for nationality by natura- lisation for a minor. Estonian nationality can be acquired for achievements of special merit to the Estonian state, which are defined as ‘achievements which contribute to the international reputation of Estonia in the areas of cul- ture or sports or in other areas’ (art. 10). Proposals for the granting of nationality for achievements of special merit may be submitted by members of the Estonian government. The government is required to approve the granting of citizenship for achievements of special merit. According to the amendment which entered into force in November 1995 (seven months after the Citizenship Act entered into force), Esto- nian nationality for achievements of special merit may be granted to not more than ten persons per year. However, in some cases, naturalisation is ruled out. According to art. 21 of the 1995 Citizenship Act, Estonian nationality shall not be granted to or resumed by a person who: 1. submits false information upon application for Estonian nationality; 2. does not observe the constitutional order and laws of Estonia; 3. has acted against the Estonian state and its security; 4. has committed a criminal offence for which a punishment of impri- sonment of more than one year was imposed and whose criminal record has not expired or who has been repeatedly punished under criminal procedure for intentionally committed criminal offences; y 5. has been employed or is currently employed by foreign intelligence or security services; 52 PRIIT JA¨RVE PRIIT JA¨RVE 6. 1.2.2 Loss of nationality Conditions and procedures for loss of Estonian nationality are stipu- lated in Chapter 6 of the 1995 Citizenship Act (arts. 22 through 30). According to these stipulations, a person shall cease to be an Estonian national 1) through release from Estonian nationality; 2) through depri- vation of Estonian nationality, and 3) upon acceptance of the citizen- ship of another state. A person who wishes to be released from Estonian nationality shall submit an application, identification documents, a certificate which proves that he or she has acquired the citizenship of another state or will acquire the citizenship of another state in connection with his or her release from Estonian nationality, and pay the state fee.7 According to art. 26, release from Estonian nationality may be refused to a person if: 1) the person would become stateless as a result; 2) he or she has unfulfilled obligations towards the Estonian state; 3) he or she is in ac- tive service in the Estonian Defence Forces. Decisions on release from Estonian nationality shall be taken by the government. y y g According to art. 28, a person shall be deprived of Estonian nation- ality by an order of the Estonian Government if he or she 1) as an Es- tonian national, enters state public service or military service of a for- eign state without permission from the Estonian Government; 2) joins the intelligence or security service of a foreign state or foreign organisation which is armed or militarily organised or which engages in military exercises; 3) forcibly attempts to change the constitutional order of Estonia; 4) upon the acquisition of Estonian nationality by naturalisation or upon resumption of Estonian nationality submits false information and thereby conceals facts which would have pre- cluded the granting of Estonian nationality to him or her or which ESTONIAN CITIZENSHIP 53 would have precluded him or her from resuming Estonian nationality; 5) is a citizen of another state but has not been released from Esto- nian nationality. This latter provision makes it possible to deprive nat- uralised dual citizens of their Estonian nationality if they have ac- quired another nationality. Since Estonian law is in principle opposed to dual nationality, such persons are obliged to apply for release from their Estonian nationality. y Art. 28(3) establishes an important difference between nationals by birth and by naturalisation. 1.2.2 Loss of nationality It stipulates that the reasons for depriva- tion of nationality listed in art. 28 do not apply to persons who acquire Estonian nationality by birth. It means that those who have acquired nationality by naturalisation are vulnerable – they can be deprived of their newly obtained nationality. y y Art. 29 addresses the loss of Estonian nationality upon acceptance of citizenship of another state or renunciation of Estonian nationality. It stipulates that a person is deemed by the government agency authorised by the Estonian Government to have ceased being an Esto- nian citizen upon acceptance of the citizenship of another state or upon renunciation of Estonian nationality in favour of the citizenship of another state. Nevertheless, in light of these stipulations, it remains unclear what happens if an Estonian national by birth does not de- clare his or her wish to be released from Estonian nationality after he or she has acquired, or is going to acquire another nationality. While the 1995 Citizenship Act rules out multiple nationality (arts. 2 and 3) the state has been quite tolerant in cases of the resumption of Esto- nian nationality by emigrants under art. 16(1) which grants everyone who loses Estonian nationality as a minor the right to resume Esto- nian nationality. Several such Estonians holding multiple nationalities have been members of the Estonian Government and elected to Par- liament. 1.3.1 The focus of the debate From the very outset of Estonian nationality policy in 1992, the ap- proaches of Estonians and Russian-speakers to the issue of nationality have been almost diametrically opposed to each other. The approach characteristic of the Estonians draws heavily on history and underlines that the changes in the ethnic composition during the Soviet years, when the share of Estonians fell from 90 per cent to almost 60 per cent between 1940 and 1989, were dangerous for the survival of the Estonian nation. Therefore, refusal to grant nationality to Soviet-era set- tlers by registration was regarded by many Estonians as an adequate re- PRIIT JA¨RVE PRIIT JA¨RVE 54 action to these changes in the population. The Estonian side also ar- gues that in comparison with the citizenship laws of other countries the Estonian requirements for nationality are quite liberal by current international standards. The opposite approach, taken by the Russian-speaking minorities and by several international actors, maintains that history and nation do not matter as much as the Estonians think they do. Rather, one should start with the present multi-ethnic situation and think about in- dividuals. As a characteristic example of this view, Helsinki Watch pointed out that it ‘rejects the argument that all those who came to Es- tonia after 1940 did so illegally and therefore were never citizens. Their residency was legally established under the applicable law at the time they entered the territory of Estonia. Those who settled in Estonia after 1940 must be treated as individuals, not as instruments of state policy, however reprehensive that policy may have been’ (Helsinki Watch 1993: 14). According to the proponents of this view, stateless people are a se- curity risk, since the interests of these individuals are not properly re- presented at the state level, and their behaviour can be unpredictable. The underlying implication of this argument is usually that Estonia should grant nationality more generously by further simplifying its conditions for naturalisation, especially the language requirements. Most of the ensuing debate has been about the political acceptability of such simplifications, and in most cases the Estonian legislators have rejected the proposals to that end. After more than fifteen years of de- bates, the opposition between the two approaches has somewhat sof- tened but is still far from having disappeared. As long as there remain many tens of thousands of stateless persons, the debate will probably continue. 1.3.2 International debate Estonia was regularly encouraged by international actors to speed up naturalisation to reduce the proportion of non-citizens in the popula- tion, especially during the country’s accession to the European Union. Estonia had to discuss its nationality issues with international partners and to even make changes in its Citizenship Act to bring it into align- ment with the country’s international obligations and to promote nat- uralisation. Several international and regional organisations, foreign embassies in Estonia, and international NGOs not only participated in the debate but also provided necessary know-how and financial assis- tance to their Estonian interlocutors. However, under the conditions set by the 1995 Citizenship Act naturalisation slowed down for several years. In 1997, international partners persuaded the Estonian authori- ESTONIAN CITIZENSHIP 55 ties to launch a policy of integration for non-Estonians. A special gov- ernment agency (Bureau of the Minister of Population Affairs8) and a special foundation for the integration of non-Estonians9 were estab- lished, which started to work out and to implement integration pro- grammes and action plans to resolve the problem of statelessness.10 g p p After several years of modest yields, the numbers of naturalised citi- zens briefly surged after Estonia joined the EU in 2004. Estonia inter- preted the admission to the EU as the ultimate international approval of its nationality policies. The EU and other international actors vir- tually stopped issuing recommendations on how Estonia should devel- op its nationality policy. Only Russia has not dropped the problem of statelessness in Estonia from its political agenda. It remains to be seen to what degree Russia can internationalise this issue in its contacts with the EU, in the framework of the OSCE and in the Council of Eur- ope. In the wake of Estonia’s admission to the EU, inputs from interna- tional actors have ceased to inform the domestic debate on nationality issues. Since then, this debate has been shaped more than ever before by internal incentives. 1.3.3 Domestic debate Estonian policy on nationality has remained conservative ever since in- dependence, without major ‘home-made’ debates after the Citizenship Act of 1992 was adopted. Instead, the mainstream political parties have regularly declared prior to national elections that, regardless of the elec- tion results, the Citizenship Act and the corresponding policies will not be changed. g The Estonian political elite deemed that the initial non-inclusion of Soviet-era settlers into the citizenry served the interests of the survival of the Estonian ethnic nation and its culture. According to a statement by a former Estonian minister, the ultimate hope for the future of the non-Estonians was ‘that a third or so will become Estonian citizens, a third may remain here with Russian citizenship, and at least a third will leave’ (Lieven 1993: 377). By 2000, these hopes had only partially materialised, mainly because the formation of a persistent contingent of stateless residents had not been anticipated. The results of the popu- lation censuses of 1989 and 2000 showed that 29 per cent of non-Es- tonians from 1989 had become Estonian citizens by 2000 and 14 per cent had obtained Russian citizenship, while the total number of non- Estonians had decreased from 602,381 to 439,833, or by 27 per cent be- tween the two censuses.11 In 2000, 173,539 non-Estonians, or 39 per cent of their number in 2000, were Estonian citizens, 86,067 non-Es- tonians, or 20 per cent, were Russian citizens and 170,349 non-Esto- 56 PRIIT JA¨RVE nians, or 39 per cent, were stateless residents (Statistical Office of Esto- nia 2001: 13-14). By the end of 2007, the number of stateless residents had fallen to 116,248, which was 19 per cent of the number of non-Es- tonians in 1989 and 26 per cent of their number in 2000.12 9 9 In 1995, minority members won six seats of the 101 in the Estonian Parliament, for the first time since independence, as representatives of the so-called Russian parties (minority parties). They organised a sepa- rate faction which tried to initiate changes in the Citizenship Act in or- der to make the acquisition of nationality easier for stateless Russian- speakers. However, all those attempts were systematically aborted by firm resistance from the Estonian majority in the Parliament. 1.3.3 Domestic debate As a re- sult, the minority parties were compromised in the eyes of Russian- speaking voters and during the 2002 national elections these parties were unable to surpass the 5 per cent threshold to get into the Parlia- ment. In 2002, nine and in 2007 eight candidates of minority origin were elected to the Estonian Parliament on the lists of the so-called Es- tonian parties, which have started to compete among themselves for the votes of naturalised non-Estonians. As members of mainstream parties, minority MPs hope to be more successful than before in de- fending the interests of non-Estonians, by promoting naturalisation, minority education and the public use of minority languages. 1.3.4 Changes in public opinion Many Estonians, influenced by history, came to perceive Russia and Russians as threats. Surveys of public opinion and sociological research of the early 1990s showed that Estonians tended to support the official nationality policies which sought to control the participation of Rus- sians in Estonian politics with the help of the Citizenship Act. Approxi- mately one fifth of Estonians thought that the official policies, includ- ing the language requirements for obtaining nationality were not harsh enough. In 2000, 46 per cent of Estonians believed that Estonia would benefit if non-Estonians left the country (Kruusvall 2001). y ( ) The majority of Russian-speakers in Estonia have considered the of- ficial policies on nationality, let alone the more radical views reflected in various media outlets, internet chat-rooms and elsewhere, as unfair and discriminatory. Nevertheless, the data from integration monitoring in 2000 showed that non-Estonians were predominantly oriented to- wards acquiring Estonian nationality: it was desired by 80 per cent of the family members of Estonian citizens who were without nationality, by 62 per cent of the family members of non-citizens, and by 61 per cent of the family members of Russian citizens. Estonian nationality was desired in the first place for children, but also for spouses and par- ents. At the same time, 12 per cent of the family members of non-citi- ESTONIAN CITIZENSHIP 57 zens did not want citizenship, and 16 per cent had not made up their minds. It might well be that a certain number of non-citizens had re- signed themselves to their status and did not see any particular reason (or possibility) to change it (Hallik 2001). While the official Estonian view on nationality has remained basi- cally the same since 1992, the public opinion of Estonians has changed due to an increase in overall tolerance and the proliferation of related values. Most remarkably, the integration monitoring of 2005 showed that already as much as 54 per cent of Estonians have agreed to grant nationality to Russians born in Estonia on simplified terms. Only about one third of Estonians held this view in 2000 (37 per cent in 2002).13 Thus, by 2005, the majority of Estonians no longer perceived the Rus- sians as a grave threat. Moreover, ordinary Estonians were more toler- ant with regard to nationality issues than official policies. These changes in public opinion might have facilitated new policy initiatives to overcome the problem of statelessness. 1.3.4 Changes in public opinion However, in 2007, the situa- tion took a different turn. On 27 April 2007, just two weeks before the Russians traditionally celebrate the anniversary of the defeat of Nazi Germany, the Estonian government clumsily relocated a Soviet-era war memorial from the centre of Tallinn, provoking street riots by mostly Russian-speaking youths who felt insulted. Approximately 1,200 people were arrested, while many were injured and one Russian citizen was stabbed to death. Attitudes of the Estonians and the Russian speakers towards each other hardened and various integration efforts seemed to be se- verely compromised. The riots and their aftermath, such as the siege of the Estonian Embassy in Moscow by a youth organisation, drew broad international attention. In March 2008, the Russian Embassy in Tallinn reported a doubling in the number of applicants for Russian citizenship between August 2007 and March 2008 compared with the same periods in 2006 and 2007. According to embassy officials, many applicants noted that they had ‘lost confidence in Estonian democracy’. The applicants were also motivated by the opportunities of visa-free travel and of finding jobs in both the Schengen area and Russia.14 This change in non-Estonians’ attitudes had already been detected by a July 2007 Estonian survey, which showed that after the relocation of the war memorial, the trust that non-Estonians had in Estonian state institutions had fallen to 24 per cent (among non-Estonian youths to 11 per cent; while among Esto- nians it remained as high as 62 per cent). This is in sharp contrast with figures from 2005, when trust in Estonian institutions among non-Estonians was similar to that of Estonians (Heidmets 2008: 59). Given these changes, the numbers of stateless residents in Estonia may continue to decline, as it is predicted, but it is hard to see how in- 58 PRIIT JA¨RVE creasing numbers opting for Russian nationality can be perceived as in Estonia’s best interests, particularly in light of the August 2008 armed conflict in Georgia. 1.4 Statistics on acquisition of nationality since 1992 Estonian statistics on acquisition of nationality date from after the 1992 Citizenship Act was adopted. The introduction of this Act granted Esto- nian nationality by registration to 68 per cent of the population who, or whose predecessors, were Estonian nationals before 17 June 1940. The rest of the population (32 per cent) who, or whose predecessors, were not Estonian nationals before that date, were given the status of aliens. Over 95 per cent of those aliens were not of Estonian descent. In 1993, after several reorganisations at governmental level, the Esto- nian Citizenship and Migration Board (CMB) was established.15 The CMB is a government agency acting within the administrative area of the Ministry of Internal Affairs and its main tasks include: determin- ing the status of persons living in Estonia either as Estonian citizens or as aliens and issuing identity documents to the residents of Estonia, as well as receiving and processing applications for acquiring and restor- ing Estonian nationality, as well as for exemptions from Estonian na- tionality, and preparing the respective material for the government of the Republic to decide on these applications (CMB 2003: 4). Currently, the CMB provides the most reliable statistics on national- ity and naturalisation in Estonia. According to these data, between 1992 and 2005 as many as 138,246 persons acquired Estonian nation- ality via naturalisation (CMB 2006: 19).16 Two special categories of ap- plicants account for more than one-third of that number. Between 1992 and 1995, a simplified fast-track procedure for naturalisation without a language exam was available for those aliens (Soviet-era set- tlers) who had participated in the elections of the Estonian Citizens’ Congress in 1990 and had registered as applicants for nationality prior to March 1990 (of those, 24,102 were naturalised), as well as for Esto- nians living outside Estonia, of whom 25,293 used this simplified pro- cedure (CMB 2006: 20). Besides those two special categories, the CMB has provided statistics on the following categories of persons naturalised between 1992 and 2005: 1. those who acquired Estonian nationality based on general condi- tions, i.e. who passed all of the exams (58,016 persons); 2. minors under fifteen years of age (29,461); 3. those without active legal capacity and the disabled (648); 4. those granted nationality for achievements of special merit (726). 1.4 Statistics on acquisition of nationality since 1992 ESTONIAN CITIZENSHIP 59 Moreover, 2,679 persons lost their Estonian nationality, while the gov- ernment also refused to grant nationality to 605 applicants during this same period (CMB 2006: 20). The process of naturalisation has not been a homogeneous flow of applications and their approval. After the Citizenship Act of 1992, the tempo of naturalisation was much higher than in the wake of the 1995 Citizenship Act, which changed the conditions of naturalisation by making the language exam more rigorous and by adding an exam on the Constitution and the Citizenship Act which also had to be taken in the Estonian language. Thus, between 1992 and 1996, as many as 87,712 persons naturalised under the conditions set by the first Citizen- ship Act, or 60 per cent of all persons who have naturalised between 1992 and 2007 (see Figure 1.2). In 1996, 16,740 persons passed the ci- tizenship language exam, which followed the old rules and require- ments. In 1997, only 2,099 persons passed an upgraded language exam (UNDP 1999: 42). However, in spite of the complications related to naturalisation, such as language exams which are considered difficult by the applicants, and the growing share of non-Estonians among the citizens, who are eyed with suspicion by ethnic conservatives, no political force in Esto- nia has proposed stopping the process. As a result, in November 2005, the overall number of naturalised persons (137,199) finally surpassed the number of stateless persons (136,533).17 Figure 1.2 Naturalisation in Estonia, 1992-2007, persons naturalised per year in thousands 0 5 10 15 20 25 5.4 20.4 22.5 16.7 22.8 8.1 9.9 4.5 3.4 3.1 4.1 3.7 6.5 7.1 4.8 4.2 Source: Estonian Citizenship and Migration Board Figure 1.2 Naturalisation in Estonia, 1992-2007, persons naturalised per year in thousands Source: Estonian Citizenship and Migration Board PRIIT JA¨RVE 60 1.5 Conclusions The current naturalisation process in Estonia is a politically sensitive and cautious inclusion of non-citizens in which international ‘suppor- tive pressure’ has played an important role. Naturalisation has brought new members to Estonian citizenry, made it ethnically more diverse and moved the country closer to full democratic participation. It is esti- mated that about 20 per cent of all Estonian nationals are non-Esto- nians. More than half of them acquired nationality after 1992 through naturalisation. However, 116,000 permanent residents of Estonia still had no nationality by the end of 2007. This means that sustained prac- tical efforts to promote integration and naturalisation are still needed in Estonian society for years to come. Both non-Estonians and Esto- nians should be targeted in order to promote better mutual under- standing and cultural accommodation. Further attempts at prudent sta- tesmanship are required to reduce the negative effects of the events of April 2007 and to restore the image of Estonian nationality in the Rus- sian-speaking community. Steps should also be taken in developing legal instruments and stan- dards concerning nationality and statelessness. While Estonia has signed and ratified the majority of international instruments aimed at combating racial and ethnic discrimination,18 it has so far failed to sign and ratify a number of international treaties dealing with issues of na- tionality and statelessness such as the UN Convention of the Status of Stateless Persons (1954); the UN Convention on the Nationality of Mar- ried Women (1957); the UN Convention on the Reduction of Stateless- ness (1961); the Convention of the International Commission of Civil Status to Reduce the Number of Cases of Statelessness (1973); and the European Convention on Nationality (1997). One is inclined to hope that membership in the EU and the prolif- eration of democratic values will motivate Estonia to sign and ratify more international treaties in the near future to help overcome state- lessness and promote the political participation of minorities through citizenship. 1998 Citizenship Act Amendment Act of 8 December 1998 (entered into force 12 July 1999) www.legislationline.org; www.legaltext.ee (in Estonian) 1999 Identity Documents Act 2002 Citizenship Act Amendment Act of 19 June 2002 (entered into force 1 August 2002) 2001 Citizenship Act Amendment Act of 14 November 2001 (entered into force 1 February 2002) 2002 Citizenship Act Amendment Act of 5 June 2002 (entered into force 1 July 2002) www.legislationline.org; www.legaltext.ee (in Estonian) 2002 Citizenship Act Amendment Act of 15 October 2002 (entered into force 10 November 2002) 2002 Citizenship Act Amendment Act of 15 October 2002 (entered into force 10 November 2002) 2003 Citizenship Act Amendment Act of 29 January 2003 (entered into force 1 March 2003) 2003 Citizenship Act Amendment Act of 29 January 2003 (entered into force 1 March 2003) 1.5 Conclusions Chronological list of citizenship-related legislation in Estonia Date Document Content Source 1992 Constitution of Estonia www.legislationline.org; www.legaltext.ee (in Estonian) 1992 Citizenship Act www.legaltext.ee (in Estonian) Chronological list of citizenship-related legislation in Estonia www.legislationline.org; www.legaltext.ee (in Estonian) www.legaltext.ee (in Estonian) 1992 Citizenship Act ESTONIAN CITIZENSHIP 61 Date Document Content Source 1993 Aliens Act (consolidated in 2003) Regulates the entry of aliens into Estonia, their stay, residence and employment in Estonia and the bases for legal liability of aliens www.legislationline.org; www.legaltext.ee (in Estonian) 1995 Citizenship Act (adopted 19 January 1995, entered into force 1 April 1995) Replaces the Citizenship Act of 1992; does not change the basic principles of acquisition and loss of nationality but establishes more demanding requirements for the acquisition of nationality by naturalisation www.legislationline.org; www.legaltext.ee (in Estonian) 1995 Citizenship Act Amendment Act of 18 October 1995 (entered into force 20 November 1995) Establishes that citizenship for achievements of special merit may be granted to no more than ten persons per year www.legislationline.org; www.legaltext.ee (in Estonian) 1995 Language Act Establishes the Estonian language as the only official language of Estonia; regulates the requirements for proficiency in the Estonian language and the use of Estonian and foreign languages in Estonia www.coe.int; www.legaltext.ee (in Estonian) 1997 Aliens Act Amendment Act Establishes new conditions for issuing permanent residence permits (at least three years residence within the last five years on the basis of a temporary residence permit) www.legislationline.org; www.legaltext.ee (in Estonian) 1998 Citizenship Act Amendment Act of 8 December 1998 (entered into force 12 July 1999) Provides for acquisition of citizenship for children of stateless single or adoptive parents; introduces deprivation of citizenship in case of submission of false information in the process of application and loss of citizenship upon acceptance of the citizenship of another state ww.legislationline.org; www.legaltext.ee (in Estonian) 1999 Identity Documents Act Establishes an identity document requirement and regulates the issue of www.legislationline.org; www.legaltext.ee (in Estonian) www.legislationline.org; www.legaltext.ee (in Estonian) www.legislationline.org; www.legaltext.ee (in Estonian) PRIIT JA¨RVE 62 Date Document Content Source identity documents to Estonian citizens and aliens by the Republic of Estonia 2000 Citizenship Act Amendment Act of 14 June 2000 (entered into force 10 July 2000) Amends the requirements for naturalisation for a person with a severe, profound or moderate disability www.legislationline.org; www.legaltext.ee (in Estonian) 2001 Penal Code Article 174 Establishes penalties for the alteration of a child’s descent by substituting a child with a child of another person for personal gain, or if causing alteration of the child’s citizenship www.legislationline.org; www.legaltext.ee (in Estonian) 2001 Citizenship Act Amendment Act of 14 November 2001 (entered into force 1 February 2002) Revises the wording of some articles as a result of changes in other civil laws www.legislationline.org; www.legaltext.ee (in Estonian) 2002 Citizenship Act Amendment Act of 5 June 2002 (entered into force 1 July 2002) Specifies rules for the naturalisation of children whose parents are dead, missing or have restricted active legal capacity or whose parents are deprived of their parental rights www.legislationline.org; www.legaltext.ee (in Estonian) 2002 Citizenship Act Amendment Act of 19 June 2002 (entered into force 1 August 2002) Rules that the government of the Republic shall substantiate the granting of citizenship for achievements of special merit (but not the refusal to grant citizenship on these grounds); regulates the fees for the acquisition of citizenship by naturalisation, for resumption of and for release from citizenship www.legislationline.org; www.legaltext.ee (in Estonian) 2002 Citizenship Act Amendment Act of 15 October 2002 (entered into force 10 November 2002) Regulates the naturalisation of persons with a severe, profound or moderate disability www.legislationline.org; www.legaltext.ee (in Estonian) 2003 Citizenship Act Amendment Act of 29 January 2003 (entered into force 1 March 2003) Regulates the procedures for acquisition of citizenship for adopted children www.legislationline.org; www.legaltext.ee (in Estonian) Date Document www.legislationline.org; www.legaltext.ee (in Estonian) www.legislationline.org; www.legaltext.ee (in Estonian) www.legislationline.org; www.legaltext.ee (in Estonian) ESTONIAN CITIZENSHIP 63 Date Document Content Source 2003 Citizenship Act Amendment Act of 10 December 2003 (entered into force 1 January 2004) Establishes the procedures for reimbursement of language training expenses to persons who passed the citizenship exams www.legislationline.org; www.legaltext.ee (in Estonian) 2004 Citizenship Act Amendment Act of 11 February 2004 (entered into force 20 March 2004) Specifies residence periods required for naturalisation (five years on the basis of a permanent residence permit prior to application and six months from the day following the date of registration of the application); establishes time limits and obligations for the applicants and authorities in processing applications www.legislationline.org; www.legaltext.ee (in Estonian) 2004 Citizenship Act Amendment Act of 23 November 2004 (entered into force on 1 April 2005) Specifies conditions for disabled applicants at the naturalisation exams www.legislationline.org; www.legaltext.ee (in Estonian) 2005 Citizenship Act Amendment Act of 19 October 2005 (entered into force on 1 January 2006) Simplifies the definition of legal income www.legislationline.org; www.legaltext.ee (in Estonian) ate Document Content Source 003 Citizenship Act Amendment Act of 10 December 2003 (entered into force 1 January 2004) Establishes the procedures for reimbursement of language training expenses to persons who passed the citizenship exams www.legislationline.org; www.legaltext.ee (in Estonian) 004 Citizenship Act Amendment Act of 11 February 2004 (entered into force 20 March 2004) Specifies residence periods required for naturalisation (five years on the basis of a permanent residence permit prior to application and six months from the day following the date of registration of the application); establishes time limits and obligations for the applicants and authorities in processing applications www.legislationline.org; www.legaltext.ee (in Estonian) 004 Citizenship Act Amendment Act of 23 November 2004 (entered into force on 1 April 2005) Specifies conditions for disabled applicants at the naturalisation exams www.legislationline.org; www.legaltext.ee (in Estonian) 005 Citizenship Act Amendment Act of 19 October 2005 (entered into force on 1 January Simplifies the definition of legal income www.legislationline.org; www.legaltext.ee (in Estonian) Shortens the waiting time after the registration of citizenship application from one year to six months; allows, as an exception, acquisition and restoration of citizenship by a person who has been repeatedly punished under criminal procedure for intentionally committed criminal offences 1 Although the 1977 Constitution of the Estonian SSR used the term ‘citizens of the Estonian SSR’, it was merely a synonym for the mandatory Soviet registration of domicile (in Russian: propiska). www.legaltext.ee (in Estonian) 2004 Citizenship Act Amendment Act of 23 November 2004 (entered into force on 1 April 2005) 2005 Citizenship Act Amendment Act of 19 October 2005 (entered into force on 1 January 2006) 2006 Citizenship Act Amendment Act of 15 June 2006 (enacted 8 July 2006) ( ) 2 In this chapter the terms ‘Estonian’, ‘Russian’, etc. designate ethnicity. The term ‘non-Estonians’ refers to all individuals whose ethnic origin is different from that of 2006 Citizenship Act Amendment Act of 15 June 2006 (enacted 8 July 2006) Notes 64 PRIIT JA¨RVE PRIIT JA¨RVE Estonians. The term ‘Russian-speakers’ stands for those non-Estonians whose mother tongue, or predominantly used language, is Russian. Estonians. The term ‘Russian-speakers’ stands for those non-Estonians whose mother tongue, or predominantly used language, is Russian. g p y g g 3 This subsection draws on Thiele (1999: 14-16). 3 This subsection draws on Thiele (1999: 14-16). 3 This subsection draws on Thiele (1999: 14-16). 4 An English translation is available at www.legislationline.org. 4 An English translation is available at www.legislationline.org. 5 Estonian law uses the term ‘alien’ rather than ‘foreign national’ to categorise a person who is not an Estonian citizen (Aliens Act of 1993, art. 8). The category of ‘aliens’ also applies to stateless persons who form a large group among Estonia’s non- citizens. The Estonian identification document issued to a stateless person is called an ‘Alien’s passport’ which many stateless persons who were born in the country consider as inappropriate, if not insulting. In Estonian political discourse the stateless persons are characterised differently from the legal jargon as individuals ‘who have undetermined citizenship’ which gives the whole issue a slightly more positive twist. 5 Estonian law uses the term ‘alien’ rather than ‘foreign national’ to categorise a person who is not an Estonian citizen (Aliens Act of 1993, art. 8). The category of ‘aliens’ also applies to stateless persons who form a large group among Estonia’s non- citizens. The Estonian identification document issued to a stateless person is called an ‘Alien’s passport’ which many stateless persons who were born in the country consider as inappropriate, if not insulting. In Estonian political discourse the stateless persons are characterised differently from the legal jargon as individuals ‘who have undetermined citizenship’ which gives the whole issue a slightly more positive twist. 6 According to some estimates, this group, which the authorities consider to be a threat to state security, is comprised of approximately 30,000 persons (including family members). Their pensions and health insurances are paid by the Russian Federation. Many of them are also citizens of the Russian Federation. Until 2006, before the new EU regulations were enacted, Estonia provided Soviet Army retirees with temporary residence permits. Now, they enjoy the right to permanent residence permits as nationals of third states who have legally resided in an EU Member State for five years or more. Notes Paradoxically, after Estonia was fully integrated into the Schengen area in 2008, those permanent residents who hold a Russian passport can travel without a visa from the Pacific Ocean to the Atlantic Ocean, while Estonian citizens still need a visa to travel to Russia. 7 In May 2008, the state fee for naturalisation as well as for release from Estonian nationality was 200 Estonian kroons (13 euros), while the minimum monthly salary was 3,600 Estonian kroons (230 euros). Applicants do not usually consider this fee to be a significant obstacle. 7 In May 2008, the state fee for naturalisation as well as for release from Estonian nationality was 200 Estonian kroons (13 euros), while the minimum monthly salary was 3,600 Estonian kroons (230 euros). Applicants do not usually consider this fee to be a significant obstacle. 8 See www.rahvastikuminister.ee. 8 See www.rahvastikuminister.ee. 9 See www.meis.ee (Non-Estonians’ Integration Foundation). 10 In 2000, the government of Estonia adopted the state programme ‘Integration in Estonian Society 2000-2007’; in April 2008, the government adopted the second state programme of integration for 2008-2013 (see www.rahvastikuminister.ee). 11 After 1991, depopulation became a firm trend in Estonia. The censuses of 1989 and 2000 show that while all minority groups diminished in size, only the Ukrainians, Byelorussians, Tatars, Jews and Germans lost more than one third of their population. At the same time, the most numerous group – the Russians in Estonia – had decreased from 475 to 351 thousand, or only by one fourth. All in all, the absolute number of non-Estonians went down 27 per cent between the two censuses while the absolute number of Estonians decreased by only 12 per cent. As a result, the share of Estonians in the total population went up 6.4 percentage points from 61.5 to 67.9. According to the census of 2000, the total population of Estonia was 1,370,052 (in 1989: 1,565,622) (Statistical Office of Estonia 2001: 14). 11 After 1991, depopulation became a firm trend in Estonia. The censuses of 1989 and 2000 show that while all minority groups diminished in size, only the Ukrainians, Byelorussians, Tatars, Jews and Germans lost more than one third of their population. At the same time, the most numerous group – the Russians in Estonia – had decreased from 475 to 351 thousand, or only by one fourth. Notes All in all, the absolute number of non-Estonians went down 27 per cent between the two censuses while the absolute number of Estonians decreased by only 12 per cent. As a result, the share of Estonians in the total population went up 6.4 percentage points from 61.5 to 67.9. According to the census of 2000, the total population of Estonia was 1,370,052 (in 1989: 1,565,622) (Statistical Office of Estonia 2001: 14). 12 See www.rahvastikuminister.ee. 12 See www.rahvastikuminister.ee. 13 See the results of Integration Monitoring 2000, 2002, and 2005 at www.meis.ee. 3 See the results of Integration Monitoring 2000, 2002, and 2005 at www.meis.ee. 13 See the results of Integration Monitoring 2000, 2002, and 2005 at www.meis.ee. 14 See ‘Huvi vene kodakondsuse vastu on kasvanud [Interest in Russian citizenship has grown]’, www.delfi.ee, 23 March 2008; Madis Taras, ‘Vene kodakondsust taotletakse varasemast aktiivsemalt [People apply for Russian citizenship more actively than before]’, Eesti Pa¨evaleht [Estonian Daily], 24 March 2008. www.epl.ee. 14 See ‘Huvi vene kodakondsuse vastu on kasvanud [Interest in Russian citizenship has grown]’, www.delfi.ee, 23 March 2008; Madis Taras, ‘Vene kodakondsust taotletakse varasemast aktiivsemalt [People apply for Russian citizenship more actively than before]’, Eesti Pa¨evaleht [Estonian Daily], 24 March 2008. www.epl.ee. 15 Estonians, worried by growing immigration, had already started introducing measures during the pre-Gorbachev era to bring this process under control. Thus, in the early 1980s, the municipality of Tallinn, the capital of Estonia, started to limit the 15 Estonians, worried by growing immigration, had already started introducing measures during the pre-Gorbachev era to bring this process under control. Thus, in the early 1980s, the municipality of Tallinn, the capital of Estonia, started to limit the ESTONIAN CITIZENSHIP 65 number of workers that industries and other enterprises were allowed to bring into Estonia, charging them considerable fees for every worker who eventually settled in Tallinn. It is interesting that the legality of these improvised methods was not challenged by Moscow, possibly because the growing inter-ethnic tensions had already sparked public unrest among the youth in Tallinn in the autumn of 1980. However, a more systemic foundation for the immigration policy was laid in 1990, when the Supreme Council of the Estonian Soviet Socialist Republic (Estonian SSR) established the National Migration Board of the Estonian SSR, the predecessor of the CMB. This agency’s task was to carry out state control of migration and issue residence and work permits. Notes For that purpose the Supreme Council adopted the ‘Immigration Law of the Estonian SSR’, which entered into force on 1 July 1990. This law established the requirement that any alien who wanted to settle in Estonia must apply for a residence permit. The first permits were issued in January 1991. pp y p p J y 99 16 Between 1992 and 31.12.2007, 147,228 persons were naturalised in Estonia according to updates available at www.rahvastikuminister.ee. 16 Between 1992 and 31.12.2007, 147,228 persons were naturalised in Estonia according to updates available at www.rahvastikuminister.ee. S h ik i i 17 Source: www.rahvastikuminister.ee. 18 These documents include the Convention on the Prevention and Punishment of the Crime of Genocide (entry into force in Estonia 19 January 1992), the International Convention on the Elimination of All Forms of Racial Discrimination (20 November 1991), the International Covenant on Economic, Social and Cultural Rights (21 January 1992), the International Covenant on Civil and Political Rights (21 January 1992), the Optional Protocol to the Covenant on Civil and Political Rights (21 January 1992), the Convention on the Elimination of All Forms of Discrimination against Women (20 November 1991), the Convention on the Rights of the Child (20 November 1991), the Framework Convention for the Protection of National Minorities (1 February 1998). 18 These documents include the Convention on the Prevention and Punishment of the Crime of Genocide (entry into force in Estonia 19 January 1992), the International Convention on the Elimination of All Forms of Racial Discrimination (20 November 1991), the International Covenant on Economic, Social and Cultural Rights (21 January 1992), the International Covenant on Civil and Political Rights (21 January 1992), the Optional Protocol to the Covenant on Civil and Political Rights (21 January 1992), the Convention on the Elimination of All Forms of Discrimination against Women (20 November 1991), the Convention on the Rights of the Child (20 November 1991), the Framework Convention for the Protection of National Minorities (1 February 1998). Bibliography CMB (Estonian Citizenship and Migration Board) (2006), Yearbook 2006. Tallinn. www.mig.ee. Hallik K. (2001), ‘Citizenship – Precondition To or Result of Integration?’, in Integration Monitoring 2000, CD-ROM Integrating Estonia, Minister Katrin Saksa Bu¨roo. Heidmets M. (ed.) (2008), Eesti Inimarengu Aruanne 2007 [Estonian Human Develop- ment Report 2007]. Tallinn: Eesti Koosto¨o¨ Kogu. Helsinki Watch (1993), Integrating Estonia’s Non-Citizen Minority. www.hrw.org. Kruusvall, J. (2001), ‘Understanding Integration in Estonian Society’, in Integration Mon- itoring 2000, CD-ROM Integrating Estonia, Minister Katrin Saksa Bu¨roo. OM Integrating Estonia, Minister Katrin Saksa Bu¨r Lieven, A. (1993), The Baltic Revolution. New Haven and London: Yale University Press. Rose, R. & W. Maley (1994), ‘Conflict or Compromise in the Baltic States?’, RFE/RL Re- search Report 3 (28). Semjonov, A. (2000), ‘Estonia: Nation Building and Integration. Political and Legal As- pects’, COPRI Working Paper 8. Statistical Office of Estonia (2001), 2000 Population and Housing Census, 2. Tallinn: Statis- tical Office of Estonia. Thiele, C. (1999), ‘The Criterion of Citizenship for Minorities: The Example of Estonia’, ECMI Working Paper 5. UNDP (United Nations Development Programme) (1999), Estonian Human Development Report 1999. Tallinn. www.gi.ee. 2 Checks and balances in Latvian nationality policies: National agendas and international frameworks1 Kristı¯ne Kru¯ma Latvia, upon the restoration of its independence in 1991, strictly fol- lowed the principle of state continuity. This has also been reflected in nationality policies which followed the ex iniuria ius non oritur princi- ple. However, Latvia had to take the framework of international law that existed when independence was restored into account and had to deal with a large number of Soviet-era settlers. This led to the creation of a specific category of persons in international law, namely so-called non-citizens, which has become the main issue of international debates on Latvian nationality policies. 2.1.1 Nationality policy prior to regaining independence An important step in the process of consolidating the new statehood proclaimed on 18 November 1918 was the adoption of the Law on Citi- zenship in 1919. This Law was not repealed after the occupation of Lat- via by the Soviet Union in 1940. At the same time, Latvian nationals became nationals of the USSR by way of automatic imposition of the latter’s nationality. y There were different views regarding the status of Baltic nationals after the Second World War. In some of the lawsuits initiated by Baltic nationals concerning their nationality they were still considered Baltic nationals by the courts of other states. The varying treatment of Baltic nationals by other states prevailed until 1991 when the Baltic states re- gained their independence. Upon the restoration of independence in 1990 the decision-makers were faced with the dilemma of the two main options available regard- ing nationality. Under the first option it was argued that the original state had disintegrated or disappeared and that a new state had been founded. The newly-founded state could therefore determine its na- tionals on the basis of its territory – a ‘zero option’. As far as this op- tion is concerned, one may add, however, that the codification efforts of the International Law Commission at the United Nations concern- 68 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA ing the nationality of persons in situations of state succession showed that awarding nationality to all residents by successor states that emerged from the dissolution of a predecessor state is by no means an automatic or established rule of international law.2 It would have been a preferred solution, especially in view of the existing obligation not to create statelessness, but state practices continue to vary. p y The second option emanated from the concept of state continuity, which implies the continuity of the nationality of the state in question (Thiele 1999: 12).3 When adopting nationality legislation Latvia was guided by the principle of the continuity of the state and the humani- tarian principles prohibiting the imposition of the nationality of the oc- cupying country upon nationals of the occupied country. 2.1.1 Nationality policy prior to regaining independence It was argued that automatic conferral of USSR nationality on the population of the Baltic states as a consequence of their occupation in 1940 was unlaw- ful under international law as long as the Baltic states were presumed to exist (Kalvaitis 1998: 231; Ziemele 2001: 233).4 Therefore, Latvian nationals recovered de facto rights and obligations deriving from their Latvian nationality but those USSR nationals who arrived in Latvia as a result of its foreign occupation were made subject to the naturalisation procedure according to relevant legal provisions. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 69 gistered by 1 July 1992; (2) persons who were Latvian nationals on 17 June 1940 and their descendants if they did not reside in Latvia or were nationals of another state and had submitted an expatriation permit; (3) persons born and residing in Latvia if their parents were unknown. The process of naturalisation was also made easy for those living in Latvia on 17 June 1940 without Latvian nationality. This approach was based on the premise that if Latvia had not been occupied, these per- sons could have acquired nationality (Ziemele 1998: 208). It was considered that only the nationals proper, as defined by the 1919 Law, could legitimately restore the political system of Latvia and thus take part in the elections for the Fifth Parliament in 1993. Others who did not qualify for nationality could apply for naturalisation under the 1919 Law and the Resolution. Since the requirements for naturali- sation were high, including inter alia sixteen years of residence, natura- lisation based on the Resolution never occurred (Ziemele 1998: 208; Kalvaitis 1998: 255). 2.1.2 Restoration of nationality During this period, the political institutions of the Soviet era were still in place. However, their freedom to act was significantly restricted. Since Latvia was guided by the principle of state continuity it had to re- store not only nationality but also its pre-1940 institutions, including its parliament. The post-Soviet institutions acting during this period had a limited capacity. Their authority was only to preserve continuity until the fifth legitimately elected Parliament started functioning. g y g According to the state continuity thesis, the aggregate body of Lat- vian nationals was re-established in accordance with the 1919 Law on Citizenship, as amended in 1927. It was again considered applicable with the adoption of the 15 October 1991 Resolution on the Renewal of the Republic of Latvia’s Citizens’ Rights and Fundamental Principles of Naturalisation by the Supreme Council. The presumption was that Lat- vian nationality had continued to exist, irrespective of the loss of inde- pendence in 1940. The Decree on the Order in which the Citizens of the Soviet Socialist Republics Lithuania, Latvia and Estonia are Granted USSR Citizenship (1940) on the basis of which Soviet nationality was imposed on Latvian nationals was declared null and void ab initio. According to the Resolution, the following groups of individuals were recognised as nationals: (1) those who were Latvian nationals on 17 June 1940 and their descendants, if they had lived in the country and had re- 2.1.3 Basis for current nationality policy During the parliamentary election campaign in 1993, nationality was the most important issue. Proposals ranged from repatriation of all Soviet-era settlers to a zero option. The elected Parliament in a way re- presented the opinion of Latvian nationals as to how the state should proceed in this matter. Initial proposals were very strict. According to the first model adopted by Parliament, the first applications for naturalisation would have been accepted in 2000 and then only at a rate of 0.1 per cent of the previous year’s total number of nationals. This would have resulted in approximately a thousand new nationals annually. This draft was heavily criticised by Western democracies and by international organisations. As a result, the President of Latvia re- fused to sign the adopted law. Complex nationality issues became even the reason for postponing Latvian membership of the Council of Europe. The new Law on Citizenship was adopted only on 22 July 1994. Ac- cording to art. 2, as amended in 1995,5 nationals of Latvia are: (1) those who were nationals on the date of occupation and their descendants, unless they had acquired the nationality of another state after Latvia proclaimed its independence on 4 May 1990; (2) Latvians and Livs6 who reside permanently in Latvia, do not hold the nationality of an- other state or have received an expatriation permit; (3) women who re- side permanently in Latvia and had lost their nationality according to the Law on Citizenship of 1919 as well as their descendants unless they had acquired the nationality of another state after 4 May 1990; (4) nat- uralised persons; (5) children who are found in the territory of Latvia 70 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA whose parents are unknown; (6) orphans living in an orphanage or a boarding school in Latvia; (7) children born of parents both of whom were nationals of Latvia at the time of such birth, irrespective of the place of birth of the children; (8) those who permanently reside in Lat- via and are duly registered and who have completed a full educational course in general education schools in which Latvian was the language of instruction, or in mixed language schools, if they are not nationals of another state or have received an expatriation permit. 2.1.3 Basis for current nationality policy As argued by Ineta Ziemele, the latter category broadens the scope of Latvian na- tionals in that it includes those former USSR nationals who may have integrated into Latvian society, irrespective of their place of birth (Zie- mele 2001: 235). The right of a child to acquire Latvian nationality was ensured by providing that, if at least one parent is a Latvian citizen, the child will acquire Latvian nationality, subject to mutual agreement by the parents. Those who did not belong to the abovementioned groups had to nat- uralise according to the procedures established by law and the regula- tions of the Cabinet of Ministers.7 Although naturalisation require- ments were made easier, they were still exclusionary. The law provided for gradual naturalisation, the so-called ‘window-system’, thus limiting the rights of individuals to freely choose the timing for naturalisation. It provided that persons will be naturalised in stages starting in 1996 and ending in 2003 (Kalvaitis 1998: 231). After 2003, anyone would have the right to apply.8 This approach was adopted because it was ex- pected that considerable numbers of non-citizens would apply for Lat- vian nationality and civil servants would therefore be unable to ensure proper application of the law. However, the number of applications turned out to be much lower than expected. The reasons for the low in- terest were only analysed after the law was adopted. The main reasons identified were (1) lack of knowledge of the Latvian language; (2) un- willingness to enter into obligatory military service; (3) the easier re- quirements for obtaining a Russian visa for non-citizens; (4) the num- ber of rights already granted; (5) political mistrust and disappointment at not having been granted nationality automatically and (6) an identity crisis after the collapse of the USSR. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 71 ties and most notably the European Union,9 Latvia amended its Citi- zenship Law in 1998 (Tomasˇevski 2000: 340). The amendments were confirmed in a referendum and became effective in November 1998.10 These amendments abolished the ‘window-system’ and provided na- tionality for children born in Latvia after 21 August 1991 to stateless persons or non-citizens. In accordance with art. 3 of the Citizenship Law, the parents of the child were required to submit an application for the acquisition of nationality before the child reached the age of fif- teen.11 In addition to these amendments, the naturalisation procedure was simplified, i.e. several groups of individuals were identified for ex- emption from the naturalisation process or who did not have to pass the naturalisation exams. Thus, for instance, applicants over the age of 65 were exempted from the history test.12 5 p y Western countries and international organisations provided consider- able assistance to Latvia with the objective of overcoming the main bar- riers which kept the numbers of applications for nationality low. Spe- cial attention was paid to language training. About 50 different sets of learning and informational material were published and 45 projects to facilitate naturalisation were initiated, an information centre was estab- lished and a number of campaigns were organised. Notwithstanding the amendments and campaigning, the numbers of non-citizens are still quite high. By July 2008, there were about 365,164 non-citizens in Latvia (in 1995, the number was 735,000). However, in the period between the start of the naturalisation process in 1995 and 30 April 2008, only 128,825 persons were granted Latvian citizenship, including 13,639 minors (the rest were either repatriated or acquired Russian nationality while remaining residents of Latvia). Various attempts to speed up the naturalisation of non-citizens have had limited success. Within the last ten years the number of non-citi- zens has not decreased very much.13 The reasons for the lack of inter- est are changing however. For instance knowledge of the language and military service are no longer mentioned in public opinion polls as im- portant barriers to naturalisation. The Naturalisation Board expects that naturalisation could be completed in five years but there will still be about 130,000 persons who will choose to remain non-citizens for the rest of their lives. However, this projection seems to be overly optimis- tic, as the naturalisation process has almost come to a halt. 2.1.4 Recent developments of nationality policy There were many assessments on the compliance of Latvia’s laws with applicable international standards in the area of nationality. These were accompanied by numerous recommendations, in particular concerning facilitation of access to nationality for Soviet-era settlers. In view of the constant pressure of the UN Commission on Human Rights, the Council of Europe, the OSCE High Commissioner on National Minori- 2.1.5 The status of non-citizens When Latvia regained its independence in 1991 it inherited large Rus- sian-speaking communities who had arrived there from the ex-USSR. The Soviet central authorities had encouraged large-scale immigration of the labour force, to meet the local demands of Soviet industrialisa- 72 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA tion and ethnic politics. Consequently, the collapse of the Soviet Union affected mostly the Russian people and other Eastern Slav groups such as Byelorussians and Ukrainians (Berg & van Meurs 2001: 139). The historical minorities of Slav origin living in the Baltic states before the Soviet invasion were treated differently. The collapse of the Soviet Union and the ensuing independence of Latvia created problems for persons living in Latvia who suddenly rea- lised that they were nationals of a state that no longer existed. Various international organisations were criticising Latvia for having too many inhabitants without nationality. This was due to the fact that former USSR nationals were not automatically granted Latvian nationality, nor did they apply for Russian nationality or the nationality of another state. Western European countries and international organisations con- sidered that a large number of persons without any factual nationality could constitute a risk for internal stability and could provoke ethnic conflicts. They could not be extradited as settlers from an occupying state because this would be contrary to human rights law which prohi- bits the expulsion of aliens en masse. Nor could these persons be classi- fied as stateless because that would be against the principle on the re- duction of statelessness. Under the circumstances, a special status of non-citizen was intro- duced. Non-citizens are those who were USSR nationals but who, after 1991, did not qualify for Latvian nationality and did not acquire Rus- sian or any other nationality. The Former USSR Citizens Act in art. 1 states: The persons governed by this Act – ‘non-citizens’ – shall be those nationals of the former USSR, and their children, who are resident in Latvia […] and who satisfy all of the following criter- ia: 1. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 73 Special rights given to non-citizens of Latvia can be summarised as follows. Non-citizens are given a special passport. The passport not only grants them the special status of belonging to the state, thus giv- ing them the constitutional right to return, but it has also been recog- nised by the EU as valid for visa-free travel (Regulation 1932/2006/ EC). Moreover, Russia has decided to provide holders of the non-citizen passport with visa-free travel to Russia (see sect. 2.3 below). In accor- dance with art. 2 of the Former USSR Citizens Act, non-citizens of Lat- via cannot be deported, which is not the case with third-country na- tionals. When ratifying international conventions, Latvia as a rule sub- mits a declaration requesting the equal treatment of citizens and non- citizens. For instance, upon ratification of the European Convention on Extradition and its Protocols in 1997, Latvia stated that it shall apply to both citizens and non-citizens. Non-citizens enjoy human rights granted to nationals and this has been submitted by Latvia and ac- cepted by a number of international treaty monitoring bodies. More- over, in accordance with art. 2 of the Law on Diplomatic and Consular Service, they enjoy diplomatic protection of Latvia. Latvia does not allow non-citizens the right to be elected at national and municipal levels or to hold public office. Moreover, non-citizens in Latvia are restricted from practising certain professions like those of: judge, court bailiff, notary, prosecutor, policeman, state security officer, land surveyor, fireman, national guard, captain of a crew, private detec- tive, attorney, or employee in diplomatic and consular service. There are also restrictions on possessing land and repatriation. Although, un- like immigrants in the EU, non-citizens are not nationals of any other state, they are treated as long-term resident third-country nationals in the EU framework in accordance with the provisions of Directive 2003/109/EC.14 This approach has been criticised by experts15 and raises questions about the extent that Latvia can live up to its interna- tional human rights obligations, i.e. especially those that fall under the International Covenant on Civil and Political Rights. Up to now there have been several attempts to classify non-citizens under a heading recognised by international law.16 Since Latvia’s acces- sion to the European Union there has been little or no pressure from international organisations regarding Latvia’s citizenship policy and the issue of its non-citizens. 2.1.5 The status of non-citizens on 1 July 1992 they were registered as being resident within the territory of Latvia, regardless of the status of their resi- dence; or their last registered place of residence by 1 July 1992 was in the Republic of Latvia; or a court has established that before the abovementioned date they had been resident within the territory of Latvia for not less than ten years; 2. they do not hold Latvian nationality; 3. they are not and have not been nationals of any other state. This provision recognises non-citizens as a special category whose legal status in some areas provides them with more rights and guarantees than, for example, proper permanent residents, however non-citizens are not yet nationals of Latvia. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES Moreover, Latvian courts have recently given an authoritative interpretation of the status of non-citizens, the most important of which is the ruling of the Constitutional Court.17 The Constitutional Court had to review the amendments made to the Former USSR Citizens Act which provided for the revocation of the status of non-citizen for persons who acquired the status of perma- nent residence in another country after 1 June 2004. Before these amendments were instituted, the status could only be renounced on KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA 74 condition that a nationality had been acquired. The Court regarded the amendments as unconstitutional. It started analysing the adoption of the Former USSR Citizens Act in historical and political context and concluded that the opinion that Latvia had a duty to grant nationality automatically to those individuals and their descendants who have never been Latvian nationals and arrived during occupation is un- founded (para. 13). The Court acknowledged that the introduction of the status of non-citizen was a complicated political compromise as a result of which a category unknown in international law was created. The Court has noted that Latvia has consistently defended its position that non-citizens cannot be qualified as stateless persons and this view has been accepted by the international monitoring bodies (Ziemele & Kruma 2003).18 In its judgment (para. 17) the Court defined the status of non-citizen in the following way: The status of non-citizens is not and cannot be considered as a mode of Latvian nationality. However, the rights given to non-ci- tizens and the international obligations which Latvia has under- taken in relation to these persons, signify that the legal link of non-citizens to Latvia is recognised to a certain extent and based on it mutual obligations and rights have emerged. This is de- rived from art. 98 of the Constitution which inter alia states that anyone who possesses a Latvian passport has a right to protec- tion by the state and the right to freely return to Latvia. The court therefore confirmed that non-citizens have a special link to Latvia which entails mutual rights and obligations. Those are, however, different from the ones that nationals have. It can be argued that non- citizens possess ‘functional Latvian nationality’, i.e. they have many of the same rights as nationals except for political rights and the right to hold certain positions but they cannot be defined as nationals. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES p y Latvia has adopted a so called ‘carrot-stick’ policy towards non-citi- zens, i.e. if they want to enjoy the rights of EU nationals, then they have to become nationals of a Member State. The current problem lies in the fact that the number of non-citizens is considerable and it is not decreasing fast enough. 2.2.1 Acquisition of nationality General principles According to the Citizenship Law of 1994 Latvian nationality is ac- quired on the basis of the ius sanguinis principle. Moreover, Latvian p p According to the Citizenship Law of 1994 Latvian nationality is ac- quired on the basis of the ius sanguinis principle. Moreover, Latvian CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 75 nationality legislation maintains the continuity of Latvian nationality, as identified in 1919. This is evident in the 1991 Resolution which re- fers to the restoration of the rights of Latvian nationals and not to a re- storation of the institution of ‘nationality’, which is presumed to exist. In addition to the ius sanguinis principle, there are groups of indivi- duals who are granted nationality almost automatically.19 Firstly, cer- tain ethnic groups: Latvians and Livs are nationals if they live perma- nently in Latvia and hold no other nationality. However, if they immi- grate from other countries they will be subject to a simplified naturalisation procedure. Secondly, persons who completed education in schools with Latvian as a language of instruction. Thirdly, women who lost their nationality in accordance with the archaic rule on revoca- tion of nationality upon marriage with a person of another nationality. Fourthly, children, whose parents are unknown, and orphans. Lastly, children born after 21 August 1991 to persons who are state- less or non-citizens. In order to apply for nationality in the case of sta- telessness a child should be: (1) a permanent resident; (2) stateless or a non-citizen ‘for the entire time’ of its life prior to application; (3) fluent in Latvian which is verified by a document from an educational estab- lishment or by the Commission of the Naturalisation Board; (4) over the age of fifteen. The applicant also should not have a criminal record of more than five years of imprisonment. Until the child reaches the age of fifteen, the application can be submitted by both parents jointly or separately, or by the adoptive parents of a child, if they are stateless or non-citizens and have resided in Latvia for at least five years. It shall be noted that a certificate of language proficiency shall be submitted only by those minors who have not been registered by their parents un- til the age of fifteen. Moreover, after they have reached the age of eigh- teen general naturalisation requirements apply. Art. 2.2.1 Acquisition of nationality 13 provides for the admission to nationality for special meritor- ious service beneficial to Latvia. A decision must be made by parlia- ment on each individual case. A person cannot acquire dual nationality by the application of art. 13, and the restrictions of art. 11 are applicable (see below). Dual nationality Dual nationality is, in principle, not permitted in Latvia. The 1994 Citi- zenship Law does not, however, exclude this possibility if the person has registered his or her Latvian nationality. This means that Latvia will not create dual nationality, while acknowledging that other states may do so. The Citizenship Law is indeed ambiguous in relation to dual nation- ality. Art. 9 provides that a person who acquires Latvian nationality cannot be a dual national. Para. 2 of the same article states that in the 76 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA case where a person is considered to be a national of another state, in his or her relations with Latvia the person is only considered to be a ci- tizen of Latvia. Art. 24 provides the possibility to revoke nationality by court decision if a person has acquired the nationality of another state without renunciation of his or her Latvian nationality. The possibility to hold dual Latvian nationality and that of another state is set out in the Transition Regulations of the Citizenship Law. They provided that those Latvian nationals who, during the period from 17 June 1940 until 4 May 1990, left Latvia as refugees or were deported and their descen- dants could register as Latvian nationals until 1 July 1995. This provi- sion is gender neutral meaning that descendants of either parent could register. However, it does not mention that they have to renounce their current nationality. y The Latvian Constitutional Court has ruled that dual nationality can arise at birth and is prohibited only in cases of naturalisation. Moreover, a child cannot be refused dual citizenship on the basis of formal require- ments.20 However, the question as to whether children must renounce their other citizenships when they come of age remains unclear. Naturalisation Individuals who have registered with the Residents’ Register are con- sidered to reside lawfully in Latvia and are entitled to acquire national- ity through naturalisation if they have received a permanent residence permit. The naturalisation requirements are the following: (1) perma- nent residence in Latvia for five years counting from 4 May 1990;21 (2) knowledge of the Latvian language, the Constitution,22 the anthem and the history of Latvia; (3) a loyalty oath to the Republic of Latvia; and (4) legal source of income (art. 12). The Law provides for a special naturalisation procedure in cases where applicants have been nationals of Lithuania, Estonia or Poland before the USSR intervention and have lived in Latvia for at least five years. These rules also include their descendants (art. 14).23 The special procedure also applies to persons married to Latvian citizens for not less than ten years, who have been residing in Latvia for at least five years, even if the spouse has passed away (art. 14). A special procedure provides that these applications are considered expediently. Upon application, a person shall declare that he or she does not hold any other nationality and that none of the restrictions apply as speci- fied in art. 11 of the Citizenship Law. Article 11 establishes restrictions for naturalisation, if a person: Article 11 establishes restrictions for naturalisation, if a person: – has acted against the independence of Latvia and its powers, which has been established by the courts; – propagated totalitarian ideals or ethnic or racial hatred, which has been established by the courts; CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 77 – served in the institutions of another state, including the armed forces; – served in the institutions of another state, including the armed forces; – served in the institutions of another state, including the armed forces; – served in the USSR army and was called-up from outside Latvia; – has been employed by the KGB, the security or the intelligence or similar service of another state; – has been sentenced in Latvia or another state for a crime that is also a crime in Latvia; – has, after 13 January 1991, worked in any organisations against Latvia.24 – has, after 13 January 1991, worked in any organisations against Latvia.24 This Article seems to follow a rather exclusionary approach. Naturalisation For in- stance, if a person has been convicted for any crime (even if imprison- ment was only for a year) he or she can never apply for Latvian nation- ality. Also, the restrictions in relation to the affiliation with the KGB could be challenged as to their legitimacy and proportionality since there are nationals who have had the affiliation but who were nationals or acquired nationality by registration. Children up to the age of sixteen acquire nationality together with the naturalised parent without undergoing the naturalisation process as established in art. 12. This is also the case if the parents have not reached an agreement but the child permanently resides in Latvia or in cases of adoption. Nationality is granted to a minor from fourteen to eighteen years of age only with his or her written consent (art. 16). If a minor’s nationality has changed and his or her consent has not been obtained, he or she can, within a year of coming of age, renew Latvian nationality irrespective of the period of residence in Latvia (art. 16, para. 2). If the nationality of a child has changed as a result of the mar- riage of (one of) its parents, the naturalisation procedure will not be applicable if the child wishes to renew his or her Latvian nationality. y In accordance with art. 4 of the Citizenship Law, all Latvian nationals are equal irrespective of the way nationality has been acquired. This is a constitutional principle confirmed by the Constitution in art. 91 stat- ing that all are equal before the law and human rights shall be re- spected without any discrimination. The Naturalisation Board, working under the auspices of the Minis- try of Justice, is responsible for the examination of applications for nat- uralisation. During the naturalisation procedure the Board co-operates with other institutions with the aim of verifying the information sub- mitted by the applicants. Its decisions are subject to appeal in court.25 During court proceedings the naturalisation process is suspended until the decision of a final instance or until the case is dropped. The proce- dure of naturalisation is set out in detail in a number of regulations of the Cabinet of Ministers. Naturalisation The Regulations on the Procedure of Accep- tance and Review of Naturalisation Applications include application 78 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA forms and specify the procedure for submission of applications and the documents to be submitted.26 Naturalisation takes place in regional units of the Naturalisation Board. In 2004, the procedure for submit- ting documents was liberalised and the requirement that documents must be submitted in the regional unit of the registered place of resi- dence of the applicant was lifted. The naturalisation procedure is rela- tively easy and takes no more than six months from the date of applica- tion. The fee for naturalisation has also been lowered several times. Since 2003, it has been set at 20 Lats (approximately 30 euros) and at 3 Lats (4 euros) for certain groups of applicants.27 Persons may with- draw their applications at any stage of the naturalisation procedure. pp y g p The requirements for the examinations are set out in the Regula- tions on the Examination of Proficiency in the Latvian Language and the Examination of Knowledge of the Basic Principles of the Constitu- tion, the Text of the National Anthem and the History of Latvia for Per- sons Who Wish to Acquire the Citizenship of Latvia through Naturali- sation. The regulations provide that knowledge of the language, of the Constitution, the anthem and history shall be tested by an examination commission established by the Naturalisation Board.28 Persons exempt from the tests are those who: (1) have acquired primary, secondary or higher education in educational institutions with Latvian as the lan- guage of instruction, (2) have disabilities. Persons over the age of 65 shall be subject to the Latvian language test only.29 According to sect. 4, the employees of the Naturalisation Board, the members of the Standing Committee on the Implementation of the Ci- tizenship Law of the Parliament as well as representatives from other organisations and institutions shall be allowed to be present in the ex- aminations as observers if they have received permission from the head of the Naturalisation Board. The examination of language profi- ciency takes place within two months from the day when all the neces- sary documents have been submitted, and the examination of the other topics two months after passing of the language exam (sect. 6). Naturalisation If the applicant does not attend or fails the exam he or she can retake the exam after three months in the case of the language exam and after one month in the case of the so-called knowledge exam (sect. 9). The language proficiency exam has a written and an oral part (sect. 11). According to sect. 22, the examination commission shall assess the applicant’s ability to read, write, listen and understand talks on topics of everyday life. Applicants above the age of 65 only take the oral lan- guage test (sect. 21). Language proficiency has often been mentioned as the main obstacle to naturalisation. Therefore, in 1996, the State Programme for Latvian Language Learning was initiated. In the framework of the programme a number of language courses and information campaigns on naturali- CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 79 sation were conducted by the Naturalisation Board with financial assis- tance from various international organisations and Western countries. Overall figures for the success rate remain high; between 1996 and May 2008 only 12 per cent of the applicants failed the exam. However, over the past four years, the passing rate has shown a steady decline. In 2007, for instance, 21 per cent of the applicants failed the language test. The applicant can choose whether to take the knowledge exam orally or in writing (sect. 23). The success rate is similar to the language exam. The overall failure rate is about 4 per cent. However, in 2007, 10 per cent of those who took the exam failed. The decreasing success rates can no longer be explained in terms of numbers of applicants, which skyrocketed in 2004 (when Latvia ac- ceded to the EU), but fell back to 1998 levels thereafter. 2.3 Current political debates The nationality issue still appears in public debates and is referred to by both left-wing and right-wing parties, although in different contexts. Recent studies by Latvian scholars conclude that the so-called Compa- triots Policy, which was established by the Russian Federation, seeks to minimise non-citizen interest in naturalising and integrating into Lat- vian society. The Compatriots Policy supports Russian-speakers resid- ing abroad, which is a concept that has been successfully ‘sold’ to the West. Other common designations used by the state authorities of the Russian Federation include ‘Russian compatriots’ or ‘Russian dias- poras’. Since 1992, Russia has continuously tried to introduce ‘issues involving Russian Compatriots’ onto the agendas of all the major regio- nal and international organisations (Muizˇnieks 2006: 121-122). The Russian Compatriots Policy was first outlined by the so-called Karaga- nov Doctrine, conceived by Sergey Karaganov, an adviser to the Presi- dent of the Russian Federation, on 6 October 1992. The doctrine per- ceives the territory of the former USSR as a region of special interest and mandates for Russia.30 Recently it has been reconfirmed officially by the Russian Foreign Policy Concept (adopted by the Russian Presi- dent on 12 July 2008). The Concept distinguishes a separate foreign policy dimension called the ‘humanitarian trend’ of Russian foreign policy, which inter alia includes the protection of the interests of ethnic Russians living abroad. The approach was even further sharpened after the conflict in Georgia. Thus, the so-called Medvedev Doctrine refers to protection of lives and dignity of Russian citizens, wherever they may be.31 The Presidential Administration, the Russian Foreign Affairs Min- istry and the Russian State Duma (the parliament) are the key institu- tions that provide support for non-citizen organisations in Latvia (Ler- his & Kudors 2008: 72). 7 ) Another factor that increases divisions within Latvian society is the growing importance of the Russian media, which have become largely self-sufficient and in terms of their size have (for some time now) eclipsed the Latvian-language media. The most popular and influential media for non-citizens in Latvia is television, which broadcasts pro- grammes produced and transmitted in Russia. The main Russian TV channels, censored by the Kremlin, are available in every cable TV package. Print media and the internet, however, play a less significant role in the Russian socialisation of non-citizens (Zepa 2005: 5). 2.2.2 Loss of nationality Latvian nationality is lost in cases of renunciation or revocation. Ac- cording to art. 23, renunciation can take place if a person has been guaranteed the nationality of another state except if he or she has un- fulfilled obligations towards the state or has not fulfilled mandatory military service. The clause on the fulfilment of obligations towards the state is unclear, i.e. whether it involves fiscal or other obligations. Such a broad formulation may make it possible to arbitrarily deny the right to change nationality (Ziemele 1998: 248). Moreover, since 2004 Latvia has a professional army and mandatory military service has been abolished. Art. 24 provides for three cases when nationality can be revoked by a decision of a regional court, namely, if a person (1) has acquired the na- tionality of another state without renouncing Latvian nationality; (2) continues to serve in foreign armed forces or similar institutions with- out permission from the Cabinet of Ministers; or (3) has acquired na- tionality by fraud. The provision applies equally to all nationals, except for those who hold dual nationality and are thus exempted from the ap- plication of art. 24 (Ziemele 1998: 247). Family members are also not affected by such proceedings. These grounds comply with those identi- fied in the Convention on the Reduction of Statelessness. If a person continues to reside permanently in Latvia for five years then this revo- cation does not affect future naturalisation (art. 25, para. 2). 80 KRISTI¯NE KRU¯ MA 2.3 Current political debates Due to the popularity of Russian TV channels in Latvia, the local editions of Russian-language newspapers and magazines have to follow general information protocol from Russia. Announcements and proclamations made by the Ministry of Foreign Affairs of the Russian Federation and other Russian institutions are published via the official news agencies CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 81 ‘ITAR-TASS’ and ‘Interfax’, and routinely reprinted in Latvia by the Russian-language newspapers Chas and Vesti Segodnya. Four attempts to produce bilingual newspapers have thus far failed because they were unable to meet the needs of their various audiences. ‘ITAR-TASS’ and ‘Interfax’, and routinely reprinted in Latvia by the Russian-language newspapers Chas and Vesti Segodnya. Four attempts to produce bilingual newspapers have thus far failed because they were unable to meet the needs of their various audiences. Russia has also decided to simplify the entry procedure for former USSR citizens currently living in Latvia and Estonia who have not ob- tained the citizenship of any country. The decree, signed by President Dmitry Medvedev on 18 June 2008, grants these persons visa-free tra- vel to Russia provided they have a valid travel document – a non-citizen passport in the case of Latvia and an aliens passport in the case of Esto- nia. Minors must present either a valid travel document or a birth certi- ficate if they are listed in the passport of an accompanying guardian. The decree became effective upon signing and the government has adopted all the necessary measures for its implementation.32 The Lat- vian Ministry of Foreign Affairs has protested this decision as it may bring the naturalisation of non-citizens to a halt.33 Latvian right-wing parties are discussing the need to amend the Citi- zenship Law for two main reasons. Firstly, it has been acknowledged that certain technical amendments are necessary. This holds true for the so-called ‘forgotten children’, i.e. children whose parents have nat- uralised but have forgotten to naturalise their children. Other proposals concern state security considerations with regard to terrorists and those working in the security services of other states. However, these propo- sals have not been specific enough and the debate remains general. 2.3 Current political debates Secondly, the proponents of amendments identify a need to liberalise Latvia’s dual citizenship policy.34 This debate was provoked by numer- ous studies by Latvian researchers and claims made by Latvian citizens who have made use of their free-movement rights after Latvia joined the EU (Kru¯ma 2006; Strategic Analysis Commission 2006). The strict policy regarding dual citizenship has been criticised by Latvian citizens living abroad, especially those in other EU Member States. Ac- cording to recent estimates, about 86,000 Latvian citizens have left Latvia since 2004 and the numbers continue to steadily increase (Indans & Kru¯ma 2007: 165). The depopulation problem is further aggravated by the fact that Latvia has Europe’s lowest birth rate. The Latvian government has responded to these demographic changes by establishing a working group to draft proposals for a new regulation re- garding dual citizenship. The debate initially concerned only children.35 However, the Minister of Integration has suggested that those who were forced to leave or were deported during the occupation period and those who acquired another citizenship while residing in another state should also be granted the right to hold dual nationality.36 He thus proposed extending the initial deadline envisaged in the Transi- tional Regulations of the Citizenship Law, which required that the for- 82 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA mer group register by 1 July 1995. In the course of the debate, the pos- sibility of holding dual citizenship was also suggested, mentioning in particular those former Latvian citizens who hold the citizenship of an- other EU Member State. Yet another discussion concerns a method to establish whether a person has acquired another nationality without in- forming Latvian authorities. One of the main problems is that verifica- tion requires the cooperation of other states. The Office of Citizenship and Migration Affairs estimates that currently 30,793 Latvian citizens also hold another citizenship. This number includes persons who have been granted dual citizenship according to the Citizenship Law, i.e. those who registered until 1 July 1995 irrespective of place of resi- dence.37 Finally, further debates can be expected concerning two cases at the European Court of Human Rights (ECHR). The recent ruling of the Grand Chamber in the Andrejeva case established that Latvia has dis- criminated against non-citizens concerning the calculation of their pen- sions. 2.3 Current political debates The ECHR considered it disproportionate to disregard employ- ment periods of non-citizens in Russian or Ukrainian enterprises before 1991 in pension calculations. It dismissed the Latvian govern- ment’s argument that such pension claims shall be subject to interna- tional agreements on social security. The Latvian judge present at the hearing disagreed with this ruling, arguing that the ECHR disregarded the historical context of the case. So far, only the Russian media have extensively reported on this case but further debates are to be ex- pected.38 The second case concerns Jurijs Petropavlovskis, who is a non-citizen and a member of the radical group Headquarters for the Protection of Russian Schools, which organised various protests against an education reform requiring more subjects to be taught in Latvian. He has declared that he would run as a candidate in local gov- ernment elections after being naturalised. According to various media sources, he publicly advocated the use of violence, bloodshed and ter- rorism and threatened to act on these after his naturalisation.39 The Cabinet of Ministers refused his application for nationality based on the argument that he is not loyal to the state. This was a precedent con- firmed by the Administrative Court Senate, which stated that the Cabi- net of Ministers has wide discretion in granting citizenship and that the decision is predominantly political. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 83 2.4 Statistics 2.4.1 Status and ethnic composition of Latvian inhabitants 2.4.1 Status and ethnic composition of Latvian inhabitants The following tables on status and ethnic composition of Latvian inha- bitants illustrates both Latvian national sentiments from the 1930s when they were a considerable majority and the current situation where Latvia is still hosting large numbers of non-citizens.40 g g As is evident from Table 2.1, the ethnic composition of Latvia’s resi- dents changed considerably during the occupation. g y g p Table 2.2 shows that, notwithstanding various efforts to liberalise naturalisation requirements, the numbers of non-citizens have not de- creased significantly since the beginning of the 1990s. Table 2.3 illustrates that it was mainly residents of Russian or East- ern Slav origin who became stateless or non-citizens after the restora- tion of independence in 1990. The situation has not changed since then and the naturalisation process is generally slow. Table 2.1 Changes in ethnic composition of Latvia’s population 1935 1995 2008 Latvians 75.5% 55.1% 59.0% Non-Latvians 24.5% 44.8% 41.0% Sources: Ziemele 2001: 236; Naturalisation Board, www.np.gov.lv Table 2.1 Changes in ethnic composition of Latvia’s population Sources: Ziemele 2001: 236; Naturalisation Board, www.np.gov.lv Table 2.2 Citizens and non-citizens of Latvia 1993 2008 Nationals 1,715,930 (71.8%) 1,858,718 (81.8%) Non-citizens and foreign nationals ,673,398 (28.2%) 412,889 (18.2%) Total 2,389,328 2,271,617 Sources: Ziemele 2005: 156, 365; Naturalisation Board, www.np.gov.lv; Register of Residents of the Department of Citizenship and Migration, www.pmlp.gov.lv Sources: Ziemele 2005: 156, 365; Naturalisation Board, www.np.gov.lv; Register of Residents of the Department of Citizenship and Migration, www.pmlp.gov.lv Table 2.3 Residents of Latvia on 1 July 2008 (by ethnic origin)41 2.4.2 Acquisition of nationality by children As a result there was an increase in the number of applications (Brands-Kehre & Puce 2005: 24).41 Experts have sug- gested replacing the current system with the automatic registration of children born to parents who are stateless or non-citizens as nationals. 2.4.2 Acquisition of nationality by children 2.4.2 The discussions before the 1998 referendum on the possibility of granting nationality to children of non-citizens and stateless persons were heated and there were arguments that large numbers of children would acquire nationality without being sufficiently integrated. Cur- rently available statistics tell the opposite. Table 2.4 Children of non-citizens and stateless persons born after 21 August 1991 who were granted Latvian nationality (31 May 2008) Ethnic origin Number Latvians, Livs 2 Lithuanian 168 Estonian 33 Russian 5,386 Polish 293 Byelorussians 500 Ukrainian 549 Not indicated 235 Other 369 Recognised as citizens 7,394 Source: Naturalisation Board, www.np.gov.lv Table 2.4 Children of non-citizens and stateless persons born after 21 August 1991 who were granted Latvian nationality (31 May 2008) Ethnic origin Number Latvians, Livs 2 Lithuanian 168 Estonian 33 Russian 5,386 Polish 293 Byelorussians 500 Ukrainian 549 Not indicated 235 Other 369 Recognised as citizens 7,394 Source: Naturalisation Board, www.np.gov.lv e 2.4 Children of non-citizens and stateless persons born after 21 August 1991 who were granted Latvian nationality (31 May 2008) Source: Naturalisation Board, www.np.gov.lv In total 7,394 children, of whom 5,386 are of Russian origin, have benefited from the amendments to the Citizenship Law in 1998. This figure is unsatisfactory considering that there are altogether about 20,000 children who have the right to acquire nationality according to the provisions of the Citizenship Law. In 2004, the Minister for Inte- gration, together with the Minister for Children and Family Affairs, conducted an information campaign sending information to the par- ents of these children. As a result there was an increase in the number of applications (Brands-Kehre & Puce 2005: 24).41 Experts have sug- gested replacing the current system with the automatic registration of children born to parents who are stateless or non-citizens as nationals. In total 7,394 children, of whom 5,386 are of Russian origin, have benefited from the amendments to the Citizenship Law in 1998. This figure is unsatisfactory considering that there are altogether about 20,000 children who have the right to acquire nationality according to the provisions of the Citizenship Law. In 2004, the Minister for Inte- gration, together with the Minister for Children and Family Affairs, conducted an information campaign sending information to the par- ents of these children. Table 2.3 Residents of Latvia on 1 July 2008 (by ethnic origin)41 Table 2.3 Residents of Latvia on 1 July 2008 (by ethnic origin) Citizens Non-citizens Foreigners or stateless individuals Total % Latvians 1,340,824 1,667 1,162 1,343,653 59.1 Russians 367,181 240,784 26,194 634,159 27.9 Byelorussians 31,207 49,085 2,734 83,036 3.7 Ukrainians 17,670 34,635 4,687 56,992 2.5 Poles 40,428 12,432 859 53,719 2.4 Lithuanians 18,388 9,943 2,272 30,603 1.4 Estonians 1,492 558 448 2,498 0.1 Other (including undeclared or unkown) 41,528 16,060 9,369 66,957 2.9 Total 1,858,718 365,164 47,725 2,271,617 100.0 Source: Naturalisation Board, www.np.gov.lv. 84 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA .4.2 Acquisition of nationality by children Naturalised persons per year As argued above, naturalisation rates remain low but with positive ten- dencies. The respective statistics allow some general conclusions to be drawn regarding the motivation of the potential applicants for national- ity to start on the naturalisation process. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 85 Table 2.5 Numbers of naturalisations in Latvia per year Year Persons applying for naturalisation Naturalised persons 1995 4,543 984 1996 2,627 3,016 1997 3,075 2,992 1998 5,608 4,439 1999 15,183 12,427 2000 10,692 14,900 2001 8,672 10,637 2002 8,370 9,844 2003 11,268 10,049 2004 21,297 16,064 2005 19,807 19,736 2006 10,581 16,439 2007 3,308 6,826 2008 2,601 3,004 Total 127,632 131,357 Source: Naturalisation Board, www.np.gov.lv Table 2.5 Numbers of naturalisations in Latvia per year Source: Naturalisation Board, www.np.gov.lv The biggest wave of naturalisation started after the window system was abolished. In 1998, only 4,439 persons were naturalised; the number rose to 12,427 persons in 1999. This increase might also be due to a number of campaigns for naturalisation taking place at the time. The second wave of naturalisations started after it became clear that Latvia would become a member of the European Union. From 2003 to 2004, the number of naturalisations rose from 10,049 to 16,064. In 2005, 19,736 persons were naturalised. However, since 2005, the num- bers of applications have decreased. Moreover, a growing number of applicants have failed the naturalisation exams (see Table 2.6). The authors of the report ‘Democracy Audit’ (Brands-Kehre & Pu¯ce 2005) believe that the decrease in applications is due to a lack of motivation to naturalise on the part of non-citizens. Firstly, non-citizens consider that they automatically deserve nationality. Secondly, there are certain benefits in retaining the status of non-citizen, mainly less travel restric- tions to the CIS countries. Thirdly, there is the fear of the naturalisa- tion exams. The last reason is the fee which, although it has been low- ered, is still relatively high for many people in Latvia. Ethnic origin of applicants for naturalisation The ethnic origin of applicants for naturalisation is indicative of the fact that Latvia is still dealing with its post-occupation legacies. The mi- gration rates are insignificant and applicants for naturalisation are So- viet-era settlers. Naturalised persons per year 86 86 KRISTI¯NE KRU¯ MA Table 2.6 Numbers of persons passing history and language exams for naturalisation Exam Year 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Total History Pass 1,408 1,790 3,628 13,273 10,668 7,717 8,208 8,953 18,912 19,269 10,588 3,418 107,832 History Fail 121 40 16 37 72 162 293 317 743 987 887 400 4,075 Language Pass 1,488 1,740 3,598 12,938 10,503 7,392 7,880 8,533 17,735 17,198 8,531 2,527 100,063 Language Fail 80 29 65 115 1,046 910 1,387 1,055 2,004 3,292 2,166 686 12,445 Source: Naturalisation Board, www.np.gov.lv Table 2.6 Numbers of persons passing history and language exams for naturalisation Exam Year 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Total History Pass 1,408 1,790 3,628 13,273 10,668 7,717 8,208 8,953 18,912 19,269 10,588 3,418 107,832 History Fail 121 40 16 37 72 162 293 317 743 987 887 400 4,075 Language Pass 1,488 1,740 3,598 12,938 10,503 7,392 7,880 8,533 17,735 17,198 8,531 2,527 100,063 Language Fail 80 29 65 115 1,046 910 1,387 1,055 2,004 3,292 2,166 686 12,445 Source: Naturalisation Board, www.np.gov.lv CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 87 Table 2.7 Ethnic origin of applicants for naturalisation in Latvia (31 May 2008) Ethnic origin Total Latvians, Livs 71 Lithuanians, Estonians 4,536 Russian 82,820 Polish 5,056 Byelorussian 12,542 Ukrainian 11,247 Not indicated 61 Other 5,154 Source: Naturalisation Board, www.np.gov.lv Table 2.7 Ethnic origin of applicants for naturalisation in Latvia (31 May 2008) During the Soviet-era, large numbers of ‘blue-collar socio-economic’ profile immigrants were sent to Latvia. At that time the Soviet central government put emphasis on the promotion of economic industrialisa- tion. Latvia has suffered under this policy because (1) Latvia hosted the headquarters of the Soviet army for the Baltic region and (2) the Lat- vian political elite was most sympathetic compared to other Baltic states. Age of applicants for naturalisation Age of applicants for naturalisation g f pp f Most applicants are found in the age groups of eighteen to 30 and 31- 40. These statistics exemplify that if the ‘window system’ had been maintained the numbers would be different because the age groups starting at 41 represent a considerable proportion of those who applied for naturalisation. Table 2.8 Age of applicants for naturalisation in Latvia (31 May 2008) Age of applicants Number % 15-17 10,625 8.7 18-30 38,428 31.6 31-40 24,082 19.8 41-50 25,006 20.6 51-60 15,136 12.4 61 and older 8,310 6.9 Source: Naturalisation Board, www.np.gov.lv Source: Naturalisation Board, www.np.gov.lv Nationality granted for special meritorious service for the benefit of Latvia With regard to granting of nationality for special meritorious service two periods can be distinguished. From 1995 to 1998 there were 199 cases of naturalisation due to special services, whereas the number has dropped to only twelve since 1999. This decline is explained by changes in the Citizenship Law in 1998 when the so-called ‘window system’ was dropped. Therefore, those who 88 KRISTI¯NE KRU¯ MA want to become nationals can apply for naturalisation and they do not have to rely on the special procedure for the extension of nationality by parliament. This procedure most often is used for sportsmen. 2.5 Conclusions Latvian nationality policy is based on the concept of state continuity. The rights attached to nationality were therefore restored to those who were nationals at the time of the occupation of Latvia in 1940 and their descendants. This policy led to the situation that a large group of peo- ple who settled in Latvia during occupation remained stateless. Due to international pressure to comply with the international legal frame- work, especially regarding the reduction of statelessness, Latvia intro- duced the status of non-citizen. A so-called carrot-and-stick policy has been adopted with regards to this group. Non-citizens are denied politi- cal rights and the right to hold certain posts or to be employed in cer- tain professions. In order to enjoy these rights they have to naturalise. Taking into account that nationality is a politically sensitive topic in Latvia, it is doubtful that radical changes will occur in the near future. The difficult compromise made in 1998 is satisfactory for the ruling centre-right parties. However, the question of the fate of non-citizens in the framework of EU law remains unresolved. Chronological list of citizenship-related legislation in Latvia Date Document Content Source 1919 Law on Citizenship (amended in 1927) Defines the basic principles of acquisition and loss of nationality during the interwar period 1922 Constitution of the Republic of Latvia (adopted 15 February 1922 with latest amendments on 15 December 2005) Is restored after restoration of independence www.ttc.lv (in Latvian) 1940 Decree on the Order in which the Citizens of the Soviet Socialist Republics Lithuania, Latvia and Estonia are Granted USSR Citizenship Imposes Soviet nationality on nationals of the three Baltic states automatically 1990 Declaration on the Renewal of Independence of the Republic of Latvia (4 Restores the authority of the 1922 Constitution and suspends it immediately www.humanrights.lv (in Latvian) 2004 Regulation No. 378 www.legislationline.org CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 89 Date Document Content Source May 1990) except for a few provisions that could only be suspended by a referendum 1991 Resolution on the Renewal of the Republic of Latvia’s Citizens’ Rights and Fundamental Principles of Naturalisation Aims at reconstituting the body of nationals who could elect a legitimate parliament; based on the 1919 Law 1994 Law on Citizenship Provides a ‘window system’ limiting the right to naturalise on the basis of age www.coe.int; www.ttc.lv (in Latvian) 1995 Amendments of Citizenship Law Provides for the right to citizenship for Latvians and Livs who have registered domicile in Latvia, persons who have acquired education in Latvian as well as women who lost their citizenship by marriage in accordance with the 1919 Law 1995 Law on the Status of Former USSR Citizens Who Are Not Citizens of Latvia or Any Other State (amended in 1997, 1998, and 2000) Introduces the status of non-citizen www.humanrights.lv (in Latvian) 1997 Amendments of Citizenship Law Makes technical amendments 1998 Amendments of Citizenship Law Repeals ‘window system’; liberalises access to Latvian nationality for children of non-citizens and the stateless 1999 Regulation No. 32 on the Procedure for the Acceptance and Review of the Application on the Recognition of a Child as a Citizen of Latvia Specifies the procedure and documents to be submitted to the Naturalisation Board with an application for the recognition of a child as a citizen www.legislationline.org 1999 Regulation No. 33 on the Examination of Proficiency in the Latvian Language and the Examination of Knowledge of the Basic Principles of the Constitution, the Text of the National Anthem and Provides for the procedure to be followed during examinations; identifies the persons to be exempted from tests; specifies the competences and obligations of the examination commissions www.np.gov.lv (in Latvian) 90 KRISTI¯NE KRU¯ MA Date Document Content Source the History of Latvia for Persons Who Wish to Acquire the Citizenship of Latvia through Naturalisation (with amendments 2000, 2001, 2003, 2004, 2006) 1999 Regulation No. 34 on the Procedure for the Acceptance and Review of Naturalisation Applications (with amendments 2000, 2001, 2003, 2004, 2005, 2006) Establishes the procedure and documents to be submitted for naturalisation; amendments bring the Regulation in line with other laws adopted in the meantime, such as the Immigration Law, the Law on the Declaration of Residence, the Law on Personal Identity Documents, the Administrative Procedure Law, etc. www.legislationline.org CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES www.legislationline.org 1999 Law on the Status of Stateless Persons in the Republic of Latvia Does not apply to those who hold non-citizen status www.ttc.lv (in Latvian) 2000 Regulation No. 410 on the State Duty Payable for Documenting Renunciation of the Citizenship of Latvia and Restoration of the Citizenship of Latvia Introduces a fee of 15 Lats for renunciation or restoration of nationality www.legislationline.org 2001 Regulation No. 234 on the State Duty Payable for Submission of a Naturalisation Application (with amendments 2002, 2003) Provides for three different categories of applicants and the amount of state duty each of these groups has to pay (20 Lats, 3 Lats, exempt from paying) www.legislationline.org 2001 Regulation No. 13 on the Procedure for Documenting Loss and Restoration of the Citizenship of Latvia (with amendments 2004) Sets guidelines for the procedure on loss and restoration of citizenship; specifies the documents to be submitted by the applicant and the respective decisions to be taken by the Naturalisation Board; amendment brings the Regulation in line with www.legislationline.org Date Document Content Source the History of Latvia for Persons Who Wish to Acquire the Citizenship of Latvia through Naturalisation (with amendments 2000, 2001, 2003, 2004, 2006) 1999 Regulation No. 34 on the Procedure for the Acceptance and Review of Naturalisation Applications (with amendments 2000, 2001, 2003, 2004, 2005, 2006) Establishes the procedure and documents to be submitted for naturalisation; amendments bring the Regulation in line with other laws adopted in the meantime, such as the Immigration Law, the Law on the Declaration of Residence, the Law on Personal Identity Documents, the Administrative Procedure Law, etc. www.legislationline.org 1999 Law on the Status of Stateless Persons in the Republic of Latvia Does not apply to those who hold non-citizen status www.ttc.lv (in Latvian) 2000 Regulation No. 410 on the State Duty Payable for Documenting Renunciation of the Citizenship of Latvia and Restoration of the Introduces a fee of 15 Lats for renunciation or restoration of nationality www.legislationline.org Date Document Content 1999 Law on the Status of Stateless Persons in the Republic of Latvia 2000 Regulation No. 410 on the State Duty Payable for Documenting Renunciation of the Citizenship of Latvia and Restoration of the Citizenship of Latvia 2001 Regulation No. 234 on the State Duty Payable for Submission of a Naturalisation Application (with amendments 2002, 2003) 2001 Regulation No. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 91 Date Document Content Source Regarding Passports for Latvian Citizens and Aliens as well as Travel Documents for Stateless Persons procedures for passports and the contents of each document 2004 Regulation No. 1011 on the Procedure to Determine the Status of Latvian Non- citizens Provides for the procedure to be followed by applicants and the Office of Citizenship and Migration Affairs regarding decisions as to whether a person satisfies all the conditions to qualify for the status www.pmlp.gov.lv (in Latvian) 2007 Regulation No. 353 on the Examination of Proficiency in the Latvian Language and the Examination of Knowledge of the Basic Principles of the Constitution, the Text of the National Anthem and the History of Latvia provided in the Citizenship Law Replaces Regulation No. 33; consolidates the amendments CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 13 on the Procedure for Documenting Loss and Restoration of the Citizenship of Latvia (with amendments 2004) on the or cation 002, Provides for three different categories of applicants and the amount of state duty each of these groups has to pay (20 Lats, 3 Lats, exempt from paying) www.legislationline.org the nd (with Sets guidelines for the procedure on loss and restoration of citizenship; specifies the documents to be submitted by the applicant and the respective decisions to be taken by the Naturalisation Board; amendment brings the Regulation in line with the new Law on Administrative Procedure www.legislationline.org Sets out the application www.ttc.lv (in Latvian) Notes 12 Apart from that, the requirements of exams and the fee for naturalisation have been lowered a number of times. 12 Apart from that, the requirements of exams and the fee for naturalisation have been lowered a number of times. 13 See the section on statistics at the end of this chapter. 13 See the section on statistics at the end of this chapter. 14 The EU accession negotiations avoided the issues related to the status and rights of non-citizens. The Commission of the European Union, when interpreting the scope of the application of the so called Third-country Nationals’ Directive (Council Directive 2003/109/EC of 25 November 2003 Concerning the Status of Third- country Nationals who are Long-term Residents, Official Journal, L 016, 23 January 2004, pp. 0044-0053) stated that ‘the expression ‘‘third-country national’’’ covers all persons who are not citizens of the Union in the sense of Article 17 paragraph 1 of the EC Treaty, that is to say those who do not have the nationality of an EU Member State. This indicates that those with undetermined citizenship fall within the scope of the directive. Letter from the Directorate-General of Justice and Home Affairs, Eur- opean Commission to the Permanent Delegation of Latvia in the EU institutions, 23 June 2003. This places non-citizens at a disadvantage compared to the status they have enjoyed so far. 14 The EU accession negotiations avoided the issues related to the status and rights of non-citizens. The Commission of the European Union, when interpreting the scope of the application of the so called Third-country Nationals’ Directive (Council Directive 2003/109/EC of 25 November 2003 Concerning the Status of Third- country Nationals who are Long-term Residents, Official Journal, L 016, 23 January 2004, pp. 0044-0053) stated that ‘the expression ‘‘third-country national’’’ covers all persons who are not citizens of the Union in the sense of Article 17 paragraph 1 of the EC Treaty, that is to say those who do not have the nationality of an EU Member State. This indicates that those with undetermined citizenship fall within the scope of the directive. Letter from the Directorate-General of Justice and Home Affairs, Eur- opean Commission to the Permanent Delegation of Latvia in the EU institutions, 23 June 2003. This places non-citizens at a disadvantage compared to the status they have enjoyed so far. 18 See Consideration of Reports Submitted by States Parties under Article 9 of the Convention. Concluding observations of the Committee on the Elimination of Racial Discrimination, 55th Session 2-27 August 1999, CERD/C/304/Add.79 12 April 2001, paras 12-14, and 63rd Session, 4-22 August, CERD/C/63/CO/7 10 December 2003, paras 12-13. Consideration of Reports Submitted by States Parties under Article 40 of Notes 1 I would like to thank Prof. Ineta Ziemele for her comments on the draft of this article. The usual disclaimer applies. 1 I would like to thank Prof. Ineta Ziemele for her comments on the draft of this article. The usual disclaimer applies. 2 UN Doc. A/RES/55/153 (Nationality of Natural Persons in relation to the Succession of States), 30 January 2001. 3 For a detailed treatment of this principle, see Ziemele 2005. 4 Latvia, like the other Baltic states, was guided by the principle ex iniuria ius non ori- tur, which has been seen as a rather inflexible approach. 5 In 1995, grounds (2), (3) and (8) were included. 6 Livs are a historic indigenous group of Finno-Ugric descent living near the Baltic sea. 7 See the part on naturalisation in section 2 in this chapter. 8 For instance a person who was 45 years of age and born in Latvia could apply for naturalisation in 2000, while a person who was twenty could apply in 1996. 9 The European Union ‘expressed grave concern at certain aspects of the […] law on citizenship adopted in Latvia’ (European Commission, General Report on the Activities of the European Union 1994 Brussels/Luxembourg 1995, para. 759). See also the Opi- nion No. 183 (1995) on Latvia’s application for membership in the Council of Europe; stars.coe.fr. Latvia was also cited three times under the UN 1503 procedure concern- ing gross and persistent violations of Human Rights (in 1995, 1997 and 2000). 92 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA 10 The amendments were adopted on 22 June 1998. The referendum was held on 3 October 1998 and about 53 per cent of the electorate voted for the adoption of the amendments. 10 The amendments were adopted on 22 June 1998. The referendum was held on 3 October 1998 and about 53 per cent of the electorate voted for the adoption of the amendments. 11 Only in exceptional cases can such an application be submitted by a single parent, i.e. by a mother if there is no entry regarding the father in the birth record or by the remaining parent if one parent is deceased. 11 Only in exceptional cases can such an application be submitted by a single parent, i.e. by a mother if there is no entry regarding the father in the birth record or by the remaining parent if one parent is deceased. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES Concerning the legality of similar limitations for running for public office, see the case of Zˇ danoka v. Latvia, application No. 58278/00, Judgment of Grand Chamber of ECHR, 16 March, 2006, especially paras. 119 and 120. 25 The Naturalisation Board is considered as one of the best performing institutions in Latvia. In relation to court cases the statistics show that out of 338 court cases the Naturalisation Board has lost only five. 25 The Naturalisation Board is considered as one of the best performing institutions in Latvia. In relation to court cases the statistics show that out of 338 court cases the Naturalisation Board has lost only five. 26 A special procedure is provided by the Regulations on the Procedure for the Acceptance and the Review of the Application on the Recognition of a Child to be a Citizen of Latvia. The documents submitted are subject to verification by the Office of Citizenship and Migration Affairs and the Ministry of the Interior if a child has reached the age of fourteen (minimum age for criminal liability). Any other state and self government institution can be approached by the Board (sect. 19). 27 Regulations on the State Duty Payable for Submission of a Naturalisation Application, Regulations No. 234 (Record No. 26, para. 43), Riga, 5 June 2001. The rate is lowered to 3 Lats for: (1) members of poor families or poor persons; (2) unemployed; (3) members of families with more than three under age children; (4) persons receiving old-age pension; (4) disabled persons with a certain degree of disability; (5) pupils and students; (6) full-time students of tertiary education establishments. Persons exempted are: (1) politically repressed; (2) severely disabled persons; (3) orphans and children who are not under their parents’ charge; (4) persons sheltered by social care institutions of the state or self-government. The fees were changed in 1997, 2001 and 2002. 27 Regulations on the State Duty Payable for Submission of a Naturalisation Application, Regulations No. 234 (Record No. 26, para. 43), Riga, 5 June 2001. The rate is lowered to 3 Lats for: (1) members of poor families or poor persons; (2) unemployed; (3) members of families with more than three under age children; (4) persons receiving old-age pension; (4) disabled persons with a certain degree of disability; (5) pupils and students; (6) full-time students of tertiary education establishments. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 24 of the Immigration Law, permanent residence can be acquired after five years of residence in Latvia with a temporary residence permit. This means that a person shall reside five years in Latvia in order to obtain permanent residence and a further five years with permanent residence to acquire the right to apply for citizenship. Exceptional cases provide for a shorter residence requirement as permanent residence permits can be issued in certain cases immediately after arrival (for instance, family reunification, former citizens and non- citizens and alike). 22 The Law states that a person shall know the basic principles of the Constitution of the Republic of Latvia and the Constitutional Law Rights and Obligations of a Citizen and a Person. However, this law became obsolete on 6 November 1998 when the Constitution was supplemented with a chapter on human rights. 22 The Law states that a person shall know the basic principles of the Constitution of the Republic of Latvia and the Constitutional Law Rights and Obligations of a Citizen and a Person. However, this law became obsolete on 6 November 1998 when the Constitution was supplemented with a chapter on human rights. g 23 In the cases of Estonia and Lithuania, they had to be citizens of the respective countries on 17 June 1940, but in the Polish case on 1 September 1939. 23 In the cases of Estonia and Lithuania, they had to be citizens of the respective countries on 17 June 1940, but in the Polish case on 1 September 1939. 24 These include the Communist Party of the Soviet Union, the Latvian Communist Party, the Working People’s International Front of the Latvian SSR, the United Council of Labour Collectives, the Organisation of War and Labour Veterans, the All- Latvia Salvation of Society Committee or their regional Committees or the Union of Communists of Latvia. Concerning the legality of similar limitations for running for public office, see the case of Zˇ danoka v. Latvia, application No. 58278/00, Judgment of Grand Chamber of ECHR, 16 March, 2006, especially paras. 119 and 120. 24 These include the Communist Party of the Soviet Union, the Latvian Communist Party, the Working People’s International Front of the Latvian SSR, the United Council of Labour Collectives, the Organisation of War and Labour Veterans, the All- Latvia Salvation of Society Committee or their regional Committees or the Union of Communists of Latvia. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 93 the Covenant, Human Rights Committee, 79th Session, CCPR/CO/79/LVA, 1 Decem- ber 2003, paras 16-18. the Covenant, Human Rights Committee, 79th Session, CCPR/CO/79/LVA, 1 Decem ber 2003, paras 16-18. the Covenant, Human Rights Committee, 79th Session, CCPR/CO/79/LVA, 1 Decem- ber 2003, paras 16-18. 19 Almost automatic acquisition means that a person shall approach the regional office of the Naturalisation Board and submit documents testifying that the person permanently resides in Latvia as well as supporting documents confirming that the person belongs to one of the groups of persons qualifying for almost automatic citizenship (for instance, diploma of secondary education in Latvian). 19 Almost automatic acquisition means that a person shall approach the regional office of the Naturalisation Board and submit documents testifying that the person permanently resides in Latvia as well as supporting documents confirming that the person belongs to one of the groups of persons qualifying for almost automatic citizenship (for instance, diploma of secondary education in Latvian). 20 Decision of the Latvian Constitutional Court, Case No. 2007-07-01, 21 August 2007. 20 Decision of the Latvian Constitutional Court, Case No. 2007-07-01, 21 August 200 21 According to para. 4 of art. 24 of the Immigration Law, permanent residence can be acquired after five years of residence in Latvia with a temporary residence permit. This means that a person shall reside five years in Latvia in order to obtain permanent residence and a further five years with permanent residence to acquire the right to apply for citizenship. Exceptional cases provide for a shorter residence requirement as permanent residence permits can be issued in certain cases immediately after arrival (for instance, family reunification, former citizens and non- citizens and alike). 21 According to para. 4 of art. 24 of the Immigration Law, permanent residence can be acquired after five years of residence in Latvia with a temporary residence permit. This means that a person shall reside five years in Latvia in order to obtain permanent residence and a further five years with permanent residence to acquire the right to apply for citizenship. Exceptional cases provide for a shorter residence requirement as permanent residence permits can be issued in certain cases immediately after arrival (for instance, family reunification, former citizens and non- citizens and alike). 21 According to para. 4 of art. Notes 15 See the conclusions by an EU Network of Independent Experts on Fundamental Rights, Synthesis report for 2003, p. 88. The experts regret that the situation of non- citizens has not been resolved during the entry negotiations between Latvia and the EU. 16 For instance, Kees Groenendijk suggested calling them ‘denizens’, a term describing residents enjoying a status between alien and citizen (Groenendijk 1993: 15). 17 See Constitutional Court Case 2004-15-0106, Official Gazette No. 40, 9 March 2005. Most of the other rulings (approximately 200 during 2004-2008) were passed by the administrative courts. The numbers of cases concerning access to status are limited. The most illustrative case is that of Gal¸ina Bakriseva, who, in the Court’s opinion, may have the right to non-citizen status notwithstanding her service in the Russian armed forces. Other cases concern children whose parents – either one or both – are foreign citizens who have agreed to register their child as a non-citizen of Latvia (see, for example, the Zaharov case, No. A42348705 AA 934-06/10, 28 April 2006). More- over, even if parents have acquired non-citizen status as a result of fraudulent meth- ods, this is not a valid enough reason to deprive their children of their non-citizen status (Case No. A42051204 SKA-24/2008, 14 February 2008). Most cases deal with the revocation of the non-citizen status. The majority of these concern non-citizens who have acquired another citizenship but failed to inform Latvian authorities. How- ever, the courts have been cautious when confirming the authorities’ revocation deci- sions. Thus, the Supreme Court Senate declared the following in the Saakjan case: The link of a non-citizen to the Republic of Latvia is closer than is that of a stateless person or alien. Therefore, the revocation of the status of non-citizen means a signifi- cant limitation of the rights of the respective person (Decision of 2004, No. SKA-89, C27261801). 18 18 See Consideration of Reports Submitted by States Parties under Article 9 of the Convention. Concluding observations of the Committee on the Elimination of Racial Discrimination, 55th Session 2-27 August 1999, CERD/C/304/Add.79 12 April 2001, paras 12-14, and 63rd Session, 4-22 August, CERD/C/63/CO/7 10 December 2003, paras 12-13. Consideration of Reports Submitted by States Parties under Article 40 of CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES Other measures include: 1) promoting not the return of ‘Russian-speaking’ residents to Russia, but their stay in the former Soviet Republics, since they can then be used to realise Russia’s long-term interests in the region; 2) expanding Russian investments as a tool to increase political influence; 3) applying economic sanctions and other means of pressure against ‘disobedient’ former USSR Republics; 4) using human rights and ethnic minority rights with regard to Russian minorities as a weapon against the former USSR Republics; 5) advocating a ‘zero solution’ in the citizenship laws of the former Soviet Republics. i.e. an automatic granting of citizenship in the respected states (for more detail, see Lerhis & Kudors 2008: 36-62). 30 The doctrine seeks to reintegrate this region by increasing Russia’s influence in the former USSR Republics and does not rule out the use of force ‘within the limits of the law’ if necessary. Other measures include: 1) promoting not the return of ‘Russian-speaking’ residents to Russia, but their stay in the former Soviet Republics, since they can then be used to realise Russia’s long-term interests in the region; 2) expanding Russian investments as a tool to increase political influence; 3) applying economic sanctions and other means of pressure against ‘disobedient’ former USSR Republics; 4) using human rights and ethnic minority rights with regard to Russian minorities as a weapon against the former USSR Republics; 5) advocating a ‘zero solution’ in the citizenship laws of the former Soviet Republics. i.e. an automatic granting of citizenship in the respected states (for more detail, see Lerhis & Kudors 2008: 36-62). 31 The Foreign Policy Concept of the Russian Federation is available at www.kremlin.ru. See also ‘Russia’s new foreign policy strategy will continue to defend Russian nationals’ interests in the Baltics’, LETA [National News Agency], 17 July 2008; and the report on the Russian Federation Foreign Policy (ОБЗОР ВНЕШНЕЙ ПОЛИТИКИ РОССИЙСКОЙ ФЕДЕРАЦИИ) at www.un.int. 32 ‘Visa waiving for Latvia’s ‘‘non-citizens’’ jeopardizes Russia-EU talks’, Ria Novosti [Russian national news agency], 18 June 2008. en.rian.ru. 32 ‘Visa waiving for Latvia’s ‘‘non-citizens’’ jeopardizes Russia-EU talks’, Ria Novosti [Russian national news agency], 18 June 2008. en.rian.ru. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES Persons exempted are: (1) politically repressed; (2) severely disabled persons; (3) orphans and children who are not under their parents’ charge; (4) persons sheltered by social care institutions of the state or self-government. The fees were changed in 1997, 2001 and 2002. 28 It was common practice that language proficiency had to be verified even after a person had passed the exam in case he or she wanted to hold public office. This practice was changed after the decision of the Human Rights Committee in the Ignatane case (Communication No. 884/1999, 31 July 2001). Antonina Ignatane was 28 It was common practice that language proficiency had to be verified even after a person had passed the exam in case he or she wanted to hold public office. This practice was changed after the decision of the Human Rights Committee in the Ignatane case (Communication No. 884/1999, 31 July 2001). Antonina Ignatane was 28 It was common practice that language proficiency had to be verified even after a person had passed the exam in case he or she wanted to hold public office. This practice was changed after the decision of the Human Rights Committee in the Ignatane case (Communication No. 884/1999, 31 July 2001). Antonina Ignatane was 94 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA deleted from the list of candidates for local government elections after language inspectors conducted an unexpected language examination at her place of work concluding that her level of language proficiency did not correspond to the highest degree necessary to be elected to local government. See also Podkolzina v. Latvia, application No. 46726/99 at the ECHR, 9 July 2002. 29 These exceptions were introduced in 1998. 30 The doctrine seeks to reintegrate this region by increasing Russia’s influence in the former USSR Republics and does not rule out the use of force ‘within the limits of the law’ if necessary. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 95 ropavlovskis: Mu¯rniece I., ‘Intervija ar Juriju Petropavlovski: “Mums vajadzı¯gs starp- tautisks skanda¯ls!” [Interview with J. Petropavlovskis: ‘We need International Scan- dal’]’, Latvijas Avı¯ze [daily newspaper], 20 December 2004. Russian sources can be found at: www.2004.novayagazeta.ru. 40 I. Rubule, ‘A¯rzeme¯s atbalsta dubultpilsonı¯bas ieviesˇanu’ [Outside Latvia there is support for the introduction of dual citizenship], Portal of Latvians living abroad, 2 August 2008. www.latviesi.com. 41 The Table is based on data on ethnic origin as indicated by residents. At the beginning of the 1990s, all residents were required to declare their ethnic origin which was mentioned both in their passports and in the Register of Residents. Current practice is that those applying for naturalisation are required to declare their ethnic origin on an application form that they submit to the Naturalisation Board. This requirement is optional as is the reference to ethnic origin in the passport. 41 The Table is based on data on ethnic origin as indicated by residents. At the beginning of the 1990s, all residents were required to declare their ethnic origin which was mentioned both in their passports and in the Register of Residents. Current practice is that those applying for naturalisation are required to declare their ethnic origin on an application form that they submit to the Naturalisation Board. This requirement is optional as is the reference to ethnic origin in the passport. 42 The number of applications received during 2004 was equal to the numbers received between 1998-2004. 42 The number of applications received during 2004 was equal to the numbers received between 1998-2004. CHECKS AND BALANCES IN LATVIAN NATIONALITY POLICIES 33 ‘A¯rlietu ministrijas pazin¸ojums par Krievijas Federa¯cijas le¯mumu atcelt vı¯zu rezˇı¯mu dal¸ai Latvijas iedzı¯vota¯ju [Announcement of the Ministry of Foreign Affairs concerning the decision of the Russian Federation to lift visa regime for a group of Latvia’s inhabitants]’, press release, Latvian Ministry of Foreign Affairs, 18 June 2008. www.am.gov.lv. 34 Z. Stankevicˇa, ‘Latvija zaude¯ izcilus pilson¸us likuma burta de¯l¸ [Latvia loses outstanding citizens because of the strict law]’, Neatkarı¯ga¯ Rı¯ta Avı¯ze [daily newspa- per], 15 October 2007. 35 I. Matisane, ‘Dubultpilsonı¯bu be¯rniem vare¯tu ieviest jau sˇogad [Dual citizenship for children could be introduced already this year]’, Official Gazette, 25 July 2007. www.lv.lv; G. Laganovskis, ‘Tieslietu ministrs: dubultpilsonı¯bai nepieciesˇams jauns li- kums [Minister of Justice: dual citizenship requires new law]’, Official Gazette, 14 May 2008. www.lv.lv; G. Laganovskis, ‘Tieslietu ministrs piel¸auj dubultpilsonı¯bu [Minister of Justice allows dual nationality]’, Official Gazette, 11 July 2008. www.lv.lv. [ y] ff y 36 A. Erin¸a, ‘Turpina gatavot prieksˇlikumus par pilsonı¯bas piesˇk¸irsˇanu a¯rvalstı¯s dzimusˇo Latvijas pilson¸u be¯rniem [Drafting of proposals for granting citizenship to children of Latvian citizens born abroad continues]’, LETA, 24 October 2007; A. Eri- n¸a, ‘Pilsonı¯bas likuma¯ rosina paredze¯t dubultpilsonı¯bu a¯rzeme¯s dzı¯vojosˇo Latvijas pil- son¸u be¯rniem [Suggestion to make provisions in the Citizenship Law for dual citizen- ship of children of Latvian citizens living abroad], LETA, 24 April 2004. 37 This data is available at www.pmlp.gov.lv. 38 Application No. 55707/00, Grand Chamber Judgment 18 February 2009. 39 There are several publications concerning these allegations, such as, ‘Sˇta¯ba brı¯dina¯jumus par sadursme¯m uzskata par provoka¯ciju [Warnings from Stab about clashes with police considered as provocation]’, Diena [daily newspaper], 14 August 2004. This publication refers to an earlier article where Petropavlovskis listed 160 combatants under his command (21 February 2004). See also the interview with Pet- Bibliography Berg, E. & W. van Meurs (2001), ‘Legacies of the Past, Ethnic and Territorial Conflict Po- tentials’, in I. Kempe (ed.), Beyond EU Enlargement, vol. 1, The Agenda of Direct Neigh- bourhood for Eastern Europe. Gu¨tersloh: Bertelsmann Foundation Publishers. Brands-Kehre, I. & I. Pu¯ce (2005), ‘Politiska¯ na¯cija un pilsonı¯ba’ [Political nation and citizenship], in Demokra¯tijas audits, SPPI, SAK, LU Apga¯ds. Also available at www.politika.lv. Groenendijk K. (1993), ‘Nationality, Minority and Statelessness: The Case of the Baltic States’, Helsinki Monitor 4 (3): 13-25. Inda¯ns I. & K. Kru¯ma (2007), ‘Vienoti daudzveidı¯ba¯: imigra¯cija un integra¯cija [United in Diversity: Immigration and Integration]’, in Save¯jie un Svesˇie. Sabiedrı¯bas Radikaliza¯ci- jas Tendences Latvija¯, Eiropa¯ un Pasaule¯ [Belonging and Remaining Outside. Tenden- cies of Radicalization of Society: Latvia, Europe and the World], Zina¯tniski pe¯tnieciskie raksti 6 (16): 149-176. Kalvaitis, R. (1998), ‘Citizenship and national identity in the Baltic States’, Boston Univer- sity International Law Journal 16: 231-271. Kru¯ma K. (2006), Dubulta¯ pieeja dubultpilsonı¯bai [Dual approach to dual citizenship]. Pub- lic policy portal. www.politika.lv. Lerhis, A. & A. Kudors (2008), Outside influence on the ethnic integration process in Latvia. Riga: Centre for East European Political Studies. Muizˇnieks N. (2006), ‘Russian Foreign Policy Towards “Compatriots” in Latvia’, in N. Muizˇnieks (ed.), Latvian-Russian Relations: Domestic and International Dimensions. Rı¯ga: LU Apga¯ds and Baltijas Socia¯lo zina¯tn¸u institu¯ts, Integra¯cijas prakse un per- spektı¯vas, 119-130. www.politika.lv. Strategic Analysis Commission under auspices of the State President (2006), Latvija un brı¯va darbaspe¯ka kustı¯ba: I¯rijas pieme¯rs [Latvia and the free movement of workers: The Irish case]. www.president.lv. Thiele, C. (1999), ‘The Criterion of Citizenship for Minorities: The Example of Estonia’, ECMI Working Paper 5. Tomasˇevski, K. (2000), Responding to Human Rights Violations. The Hague: Martinus Nijhoff Publishers. Zepa, B. (2005), Ethnopolitical tension in Latvia: In search of a conflict solution. Riga: Baltic Institute of Social Sciences. 96 KRISTI¯NE KRU¯ MA Ziemele, I. (1998), State Continuity and Nationality in the Baltic States: International and Constitutional Law Issues. PhD Dissertation. Wolfson College Cambridge. Ziemele, I. (2001), ‘State Continuity, Human Rights and Nationality in the Baltic States’, in T. Jundzis (ed.), The Baltic States at Historical Crossroads, 224-248. Riga: Academy of Sciences of Latvia. Ziemele, I. (2005), State Continuity and Nationality: The Baltic States and Russia. Past, Pre- sent and Future as Defined by International Law. Leiden, Boston: Martinus Nijhoff Pub- lishers. Ziemele, I. & K. Bibliography Kru¯ma (2003), ‘Eiropas Savienı¯bas pilsonı¯ba un Latvijas nepilson¸i’ [Eur- opean Union citizenship and Latvian non-citizens], Latvijas Ve¯stnesis, pielikums Jurista Va¯rds 33. 3 Lithuanian nationality: Trump card to independence and its current challenges1 Kristı¯ne Kru¯ma There are slight differences between the Latvian and Lithuanian ap- proaches as far as the transition from the Soviet to democratic institu- tions is concerned. Lithuania could be said to have used the Soviet le- gal and institutional basis for the adoption of the decisions necessary at the time more than Latvia did. However, it will be argued that these differences do not challenge the underlying principle of ex iniuria ius non oritur followed also by Latvia and Estonia. In comparison to other Baltic states, Lithuania escaped close interna- tional scrutiny of its nationality policies (see Ja¨rve and Kru¯ma in this volume). Therefore, nationality has, until recently, not created any ma- jor international controversies. Only after Lithuania encountered hur- dles related to the presidential discretion for granting nationality has the issue attracted attention, especially on the national political agenda. Since then there have been heated debates on the need to change the Lithuanian approach to dual citizenship. 3.1.1 General overview of nationality policy The same scenario of imposing Soviet nationality upon their nationals was applied in all three Baltic states, including Lithuania. However, So- viet Citizenship Law did allow the Soviet republics much authority re- garding nationality matters (Kalvaitis 1998: 240). This was seized by Lithuania in 1989 when it enacted its first Citizenship Law. Guided by the principle of ex iniuria ius non oritur, Lithuania, having declared independence on 11 March 1990, first reinstated the 1938 Constitution and simultaneously suspended some of its articles as they were incompatible with democratic principles or the institutions pro- vided for no longer existed. Following the full suspension of the 1938 Constitution, the Provisional Basic Law was enacted, accounting for present-day realities (Kalvaitis 1998: 243). The 1992 Constitution was carefully drafted with reference to laws in force before 1940 and with an emphasis on constitutional continuity (Ziemele 2005: 40). How- ever, the enactment of the 1989 Citizenship Law before adoption of the 98 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA Constitution is the main difference to the approaches adopted in the two other Baltic states because Lithuania was guided by the conflicting principle ex factis ius oritur, at least to a certain extent. This means that the new Constitution was adopted by an extended body of nationals in comparison to the citizenship laws prior to 1940. Constitution is the main difference to the approaches adopted in the two other Baltic states because Lithuania was guided by the conflicting principle ex factis ius oritur, at least to a certain extent. This means that the new Constitution was adopted by an extended body of nationals in comparison to the citizenship laws prior to 1940. p p p 94 The development of Lithuanian nationality legislation can be divided into three main phases. The first phase started with the Law adopted in 1989 providing for liberal conditions upon which Lithuanian nation- ality could be acquired. This phase ended with the Law of 1991 when Lithuania had already restored its independence and stricter criteria for the acquisition of nationality were introduced. This second phase is problematic and confusing because there were various attempts to find a balance between compliance with the principle of continuity of na- tionality and the avoidance of double nationality. The third phase was initiated by the new 2002 Law on Citizenship. 3.1.1 General overview of nationality policy It attempts to stream- line provisions of the 1991 Law and its numerous amendments and to liberalise the regulation of dual nationality. 3.1.2 The 1989 Citizenship Law The first Lithuanian nationality law was adopted on 3 November 1989. The Law identified four categories of persons who were or could be- come nationals of Lithuania: – Those who held Lithuanian citizenship prior to 15 July 1940 includ- ing their children and grandchildren, as well as those who were per- manent residents in the territory of the Lithuanian SSR on 15 July 1940 and their children and grandchildren provided that they are or have been permanent residents of the Lithuanian SSR. p – Those who had a permanent place of residence in the Lithuanian SSR if they were born in the territory of the Lithuanian SSR or can prove that at least one of their parents or grandparents was born there and if they are not citizens of another state. – Other persons who at the time of the adoption of the Law were per- manent residents for at least two years and had employment or other legal source of support in Lithuania. Thus, the law allowed those who arrived in Lithuania during the Soviet period to acquire Lithuanian nationality (with some exceptions, such as Soviet army officers). They had to declare their intention to become nationals within two years following the entry into force of the law2, i.e. until November 1991. Upon registration they had to swear an oath of al- legiance to the Lithuanian Constitution and laws (Kalvaitis 1998: 244, 261). This principle applied irrespective of their nationality or language abilities. LITHUANIAN NATIONALITY 99 – Those who had acquired the Lithuanian nationality in accordance with the law. – Those who had acquired the Lithuanian nationality in accordance with the law. – Those who had acquired the Lithuanian nationality in accordance with the law. – Those who had acquired the Lithuanian nationality in accordance with the law. According to the Constitutional Court of Lithuania in Case 7/94, the Law differentiated between existing and potential holders of Lithuanian nationality.3 Persons who were nationals prior to occupation, their des- cendants and permanent residents on 15 June 1940 who continued to reside in the country when the Law entered into force, were considered nationals ipso facto. The same applied to persons born in the territory of Lithuania and still residing there, and those whose parents were born or resided in that territory. 3.1.2 The 1989 Citizenship Law These persons were considered as hav- ing a permanent legal relationship with Lithuania; a principle which was considered particularly important in Lithuania for its nationality policies (Ziemele 1998: 223). Finally, those who were deported from the territory of Lithuania or emigrated after the occupation, as well as their children and grandchildren, also retain the right to Lithuanian ci- tizenship. Soviet-era immigrants were only considered potential nationals as they were guaranteed the right to freely decide on their nationality. After they accepted nationality they all had to take a pledge of loyalty to Lithuania (Kalvaitis 1998: 261). In case 7/94, the Constitutional Court emphasised that there were differences between this category of per- sons and other nationals. The latter never had permanent legal rela- tions with Lithuania and they were immigrants holding Soviet nation- ality. After the restoration of an independent Lithuania, they became foreigners if they did not use the option provided for by the 1989 Law. It has to be recalled that this choice was not obvious or easy at the time. In 1989 or even 1990, it was still difficult to foresee the collapse of the Soviet Union. Taking an oath of allegiance to Lithuania required certain convictions. 90 per cent (87 per cent according to other sources) of non-Lithuanian permanent residents registered as nationals under these provisions. Only 1 per cent of the pre-independence electo- rate chose not to become nationals of the Republic of Lithuania (Kalvai- tis 1998: 261). The law did not provide for dual nationality. This was confirmed in the Provisional Basic Law in art. 13 which stated that as a rule, a citizen of Lithuania may not at the same time be a citizen of another state. The subsequent amendments on 16 April 1991 confirmed that Lithua- nian nationality is lost upon the acquisition of the nationality of an- other state. However, a number of exceptions existed at that time as well. These concerned those who were nationals of Lithuania prior to 15 June 1940 and their descendants. 100 KRISTI¯NE KRU¯ MA The 1991 Citizenship Law 3.1.3 The second Citizenship Law in Lithuania was adopted on 10 December 1991.4 It established who are to be considered Lithuanian nationals. The new law ended the liberal period when any resident could apply for nationality after two years of residence and introduced stricter re- quirements. 3.1.2 The 1989 Citizenship Law The Constitutional Court stated: LITHUANIAN NATIONALITY 101 Such a decision meant that the consequences of occupation and annexation with regard to citizens of Lithuania on whom citizen- ship of the Soviet Union had been forced against their will, were being undone. It goes without saying, that such a decision on the part of the state could only be adopted regarding its citizens, and the state could by no means resolve issues concerning the citizenship of another state. According to art. 12 of the 1991 Lithuanian Constitution, with the ex- ception of cases established by law, no person may be a citizen of the Republic of Lithuania and another state at the same time. The Citizen- ship Law deals with the issue of dual citizenship in a confusing way which is closely connected with a certain conflict between the princi- ples of continuity of nationality and the principle of effective link that the Law tries to accommodate. Relevant provisions of the Law have been amended several times to clarify who can and who cannot acquire dual citizenship. Concerns were expressed by the Lithuanian nationals who could not obtain Lithuanian passports because they had in the meantime acquired another nationality. They were therefore denied the possibility to restore their nationality because dual nationality was pro- hibited. The Council of Europe characterised this situation as unsatis- factory (Ziemele 1998: 220). Explanations were given by the Constitutional Court in Case 7/94 when it dealt with questions pertaining to the right of members of the Soviet armed forces to acquire Lithuanian nationality. The Constitu- tional Court was approached by a group of MPs who challenged the va- lidity of the Resolution of the Parliament which provided that members of the USSR army, who had terminated their service before 1 March 1992 and 4 November 1994, and had been issued a Citizen Certifica- tion Card, could acquire citizenship. The Court declared the provisions of the Resolution unconstitutional. It clarified that the 1989 Citizen- ship Law ‘did not provide an option for a citizen of Lithuania to be at the same time a citizen of another state’. This was supported by an- other general principle of the 1989 Law, providing that Lithuanian na- tionality is lost with the acquisition of another nationality (Ziemele 1998: 220). The Court noted that there is only one exception to this general rule, i.e. 3.1.2 The 1989 Citizenship Law It was subsequently amended several times: 19 November 1992, 16 July 1993, 3 October 1995 and 6 February 1996. The follow- ing comments on the 1991 Law take these amendments into account. g 99 The law identified groups of individuals eligible for Lithuanian na- tionality. Initially those included: y y – nationals of Lithuania prior to 15 June 1940 including their children and grandchildren if they had not acquired nationality of another state; – permanent residents of Lithuania between 9 January 1919 and 15 June 1940 within the territory of the present Lithuania, their chil- dren or grandchildren, if they continue to reside in Lithuania and are not nationals of another country; – persons of Lithuanian origin who left Lithuania prior to 16 Febru- ary 1918, if they have not acquired nationality of another state;5 – persons who acquired nationality in accordance with the Law on Ci- tizenship effective prior to 1991; – other persons who acquired nationality under the Law (naturalised). The Supreme Council in the Resolution on the Procedure for Imple- menting the Republic of Lithuania Law on Citizenship of 10 December 1991, clarified in sect. 5 that persons serving in the armed forces, inter- nal troops and state security structures, as well as other law enforce- ment and repressive structures of the Soviet Union must not be con- sidered as permanently residing or employed in Lithuania. This was in line with the Supreme Council Resolution on 1939 Treaties between Germany and the USSR and Elimination of their Consequences for Lithuania (7 February 1990) and the Supreme Council Declaration on the Status of Soviet Armed Forces in Lithuania (19 March 1990). They stated that servicemen of the occupation army were not entitled to the right to participate in elections organised in Lithuania, with the excep- tion of those who under the 1989 Law on Citizenship could be recog- nised as nationals of Lithuania. A descendant of a Lithuanian citizen, as identified prior to 15 June 1940, who had served in the Soviet army, was not excluded from Lithuanian nationality. The USSR nationality was declared null and void with respect to these individuals as it was for all other Lithuanian nationals. 3.1.2 The 1989 Citizenship Law those who were nationals of the Republic of Lithuania prior to 15 June 1940 and their descendants. The latter explanation re- lates to the application of the principle of continuity of nationality while the prohibition of dual nationality is linked in principle to the understanding of effective link by Lithuania as concerns its decisions on nationality issues. According to this ruling of the Constitutional Court, the Law was amended in 1995. Art. I provided that nationals of Lithuania prior to 15 102 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA June 1940 and their children are nationals of Lithuania if they have not repatriated to their ethnic homeland (this new requirement was not changed in response to the court decision. In fact, it was declared unconstitutional by the court in 2006). The requirement that they are not permitted to be nationals of another state was lifted. However, this condition was still applicable to their grandchildren until the amend- ments of 2 July 1997, which permitted these, too, to retain their other nationality. This amendment also affected arts. 17 and 18 of the 1991 Law. Art. 17 stated that the right to nationality of Lithuania shall be retained for an indefinite period for (1) those who were nationals prior to 15 June 1940 and their children provided that they have not repatriated i.e. de- parted to their ethnical homeland and (2) persons of Lithuanian origin residing in other states.6 According to the 1995 amendments a person with one Lithuanian parent or grandparent and who is Lithuanian him or herself shall be considered a person of Lithuanian origin. The same amendments provided for differentiation between the above mentioned categories (1) and (2). While the first group could retain another na- tionality, the second had to renounce the nationality of another state and return to Lithuania for permanent residence in order to be granted Lithuanian nationality. Thus, the Law grouped persons according to their ethnic origin and according to whether they had departed for their ethnic homeland or another country. In both cases, those who were entitled to citizenship but resided abroad were not considered Lithuanian citizens ex lege. They had to express the corresponding in- tention and meet the requirements established by law.7 Art. 18 stated that all persons mentioned in art. 17 should renounce the nationality of another state. 3.1.2 The 1989 Citizenship Law Moreover, persons of Lithuanian origin residing in other states shall become permanent residents as well as take the oath to Lithuania in order to acquire nationality.8 Such a com- plicated scheme reflects the problems caused by the prohibition of dual nationality when the independence of a state, which was suppressed for a considerable time, is restored. It was only when amendments were made in 1993 that those who were deported or left Lithuania dur- ing occupation and their children who had not acquired nationality of another state by birth and lived in other states could recover Lithuanian nationality by presenting a written notice to the authorities. Before these amendments neither provisions of art. 17 or art. 18 provided pos- sibilities to acquire dual nationality. Therefore, on the one hand, the Law identifies nationals with respect to whom the prohibition of dual nationality does not apply, i.e. groups of individuals whose right to nationality is retained for an indefinite period without renouncing their present nationality. On the other hand, there are groups of persons who have the right to nationality but LITHUANIAN NATIONALITY 103 the right can only be exercised when they renounce their present na- tionality (Ziemele 2001: 235-236). Distinctions apply also to different categories of children, i.e. those who are considered nationals by birth and those who have to acquire nationality although they are born in Lithuania. A child born to parents one of whom is a Lithuanian citizen shall be a citizen irrespective of his or her place of birth if at least one parent has permanent residence in Lithuania (art. 9). If, however, both parents reside outside Lithuania they shall reach an agreement on the child’s nationality until he or she is eighteen years of age. Foundlings shall be considered nationals while children born to stateless persons who are permanent residents in Lithuania shall acquire Lithuanian nationality (arts. 10 and 11). The arti- cles do not specify whether these children have to be born in Lithuania, which may imply that the Law means children who have arrived in the country with their parents. Both articles draw a distinction between children who shall be nationals by birth and those who have to acquire nationality (Ziemele 1998: 237). In relation to spouses of Lithuanian nationals art. 14 provided a sim- plified procedure for the acquisition of nationality, i.e. 3.1.2 The 1989 Citizenship Law three years of re- sidence in Lithuania while married, the passing of exams on language and the Constitution as well as the renunciation of their previous na- tionality. The amendments of 1992 added another category of persons subject to a simplified procedure. It stated that those who are married to Lithuanian nationals who were deportees or political prisoners and their children born in exile shall be granted Lithuanian nationality if they are married for at least three years and have moved for permanent residence to Lithuania together with their spouse. These persons would only have to renounce their previous nationality and to pass an exami- nation on the Constitution of Lithuania. Lithuania’s general approach to the regulation of nationality, espe- cially in the early 1990s, can be considered as more liberal compared to the other Baltic states. First, most of the Soviet-era settlers acquired nationality on the basis of the 1989 Law while Latvia and Estonia re-es- tablished the body of nationals on the basis of the legislation of the pre-occupation period. Second, Lithuania did not introduce any quota system while Latvia only abolished its quota system in 1998.9 Third, Lithuania included residence during the Soviet period as valid for na- tionality purposes. Latvia and Estonia took into account only the resi- dence after restoration of independence. Therefore, Lithuania managed to avoid criticism which continues to be addressed to Latvia and Esto- nia. This has often been explained by the different proportion of non- indigenous populations residing in Lithuania when independence was restored. 104 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA The Lithuanian approach cannot be qualified as a ‘pure zero option’ because there were distinctions made between different groups of per- sons. The principle of continuity of nationality remained the main point of departure for deciding how to identify nationals. That is the reason why some of the groups were not considered nationals ipso facto and were subject to naturalisation according to the 1989 Law. However, the procedure was very simple and a majority of the groups affected by this clause, mainly former USSR nationals, naturalised. As a consequence, the 1991 Law did not really have to address the issues concerning the former USSR nationals, except when these did not use the 1989 Law option (Ziemele 1998: 225). However, concerning nationals, a distinc- tion was made between the execution of the right to nationality and the restoration of nationality. 3.1.2 The 1989 Citizenship Law Restoration concerns situations where the original nationality was not retained throughout the occupation or when some actions are needed to re-instate it (Ziemele 1998: 224). 3.1.4 The 2002 Citizenship Law The third Law on Citizenship was adopted on 17 September 2002 and entered into force on 1 January 2003. It repealed the 1991 Law and in- corporated certain related laws (such as the Law on the Validity of Citi- zenship Documents). The Law was subsequently amended in 2003 and 2004.10 One of the main issues which was publicly debated was the question of Lithuanian e´migre´s holding dual nationality.11 Emi- grants voiced their discontent with the fact that they were stripped of their Lithuanian nationality when acquiring the nationality of another state. The new Law accommodated their requests and in addition pro- vided for possibilities to have their nationality status re-instated. How- ever, some national minorities protested against the Law. They argued that permitting dual nationality only to ethnic Lithuanians contravenes the Constitution and international norms. The new Law on the Implementation of the Republic of Lithuania Law on Citizenship was adopted in 2003 (amended in 2004). One of the main provisions stated that those who applied for nationality under the 1989 Law, but did not receive a document confirming their status and were residing abroad, lost their nationality on 31 December 2003. This decision was made by the Minister of the Interior. The new Citizenship Law slightly amended art. 1 defining the cate- gories of nationals. It now includes references not only to children and grandchildren of persons who were nationals prior to 15 June 1940 or permanent residents from 9 January 1919 to 15 June 1940 but also to their great-grandchildren. Moreover, those who were nationals prior to 15 June 1940 and their descendants (including great-grandchildren) do not have an obligation to renounce a nationality held from another LITHUANIAN NATIONALITY 105 state. Reference to 16 February 1918 has been lifted and, thus, any per- son of Lithuanian descent is a Lithuanian citizen if he or she does not have any other nationality. Hence, the Law expands the category of per- sons who have an inherent right to nationality of Lithuania up to the fourth generation, and introduces conditions for simplified restoration of nationality for those who lost their Lithuanian nationality but have an inherent right to it. The former art. 17 has been simplified and art. 18 has been deleted altogether. The conditions upon which a child is considered a citizen if only one parent is a Lithuanian citizen have also been slightly changed. 3.1.4 The 2002 Citizenship Law 40/03 of 30 December 2003 regarding the granting of nationality by way of exception.13 A Seimas resolution asked the Court’s ruling on the possible violation of the constitutional principle of equality by the Pre- 106 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA sident when he granted nationality by exception to one of his advisors, Jurij Borisov. These events were heatedly debated and subsequently led to an impeachment procedure against the President. In summary, the Constitutional Court ruled that in cases where the President grants na- tionality by way of exception he or she shall verify the service which was given to Lithuania as a state, establish whether the person has per- manent factual links with Lithuania, whether the applicant is not sub- ject to any exceptions mentioned in the Law as well as his or her possi- bilities to recover nationality on his or her own initiative in accordance with the Law. Moreover, the Court emphasised that the legislator can- not deny the nature and meaning of the institution of citizenship and the relevance of the Constitution, which allows dual nationality only in individual cases provided for by the law. This means that cases of dual citizenship must be extraordinarily rare and exceptional. Generally the Law streamlines the conditions for the acquisition and retention of nationality of the previous Law which due to its numerous amendments became too cumbersome. A number of provisions are ex- cluded because they do not relate to the acquisition or retention of na- tionality but rather dealt with the conditions for entry and residence in the territory of Lithuania. The Law is clearer regarding the continuity and restoration of nationality as well as dual nationality. Moreover, it brings the conditions in line with the requirements of human rights law (groups excluded from acquiring nationality, loss of nationality due to invalid passport, etc.) and provides for stricter requirements in cer- tain cases (spouses). Finally, the Law grants more authority to the Min- ister of the Interior and clarifies a number of provisions in relation to the naturalisation procedure. These latest provisions came into force on 1 April 2006.14 3.2 Basic principles of the most important current modes of acquisition and loss of nationality 3.2.1 General principles of the acquisition of nationality 3.1.4 The 2002 Citizenship Law Ac- cording to the new art. 9, a child shall be a citizen if born in the terri- tory of Lithuania and one of the parents is a national. In the case of a child born outside Lithuania, his or her nationality is to be determined by an agreement between the parents (of whom one must be a Lithua- nian national) until he or she reaches eighteen years of age. This shall be done irrespectively of their place of permanent residence. Art. 10 provides ius soli acquisition of nationality for children whose parents are stateless persons permanently residing in Lithuania. The conditions for acquiring Lithuanian nationality were made stric- ter for spouses (art. 14). Firstly, the 2002 Law provided that only those spouses who had been married for at least five years and had been resi- dent in Lithuania for that period were to be granted nationality. They had to pass exams on language and the Constitution and were not al- lowed to hold another nationality. Thus, stricter requirements were in- troduced as previously only three years of residence were required. With the amendments effective from January 2005, the residence re- quirement for spouses of Lithuanian nationals has been raised even further to seven years.12 Secondly, persons married to Lithuanian na- tionals who were deportees or political prisoners and their children born in exile are no longer exempt from the Lithuanian language exam. They also have to reside in Lithuania for five, not three years as before. Thirdly, the Law has introduced conditions upon which a per- son can acquire nationality in the case of his or her spouse being de- ceased, if they were married for more than a year with residence in Lithuania. In these cases a person could acquire Lithuanian nationality after three years of residence provided that he or she passes the exams on language and the Constitution and renounces his or her previous nationality. However, after the amendments effective from January 2005 the residence requirement was raised to five years. It shall be noted that at least some amendments were introduced be- cause of a ruling of the Constitutional Court of Lithuania in case No. LITHUANIAN NATIONALITY LITHUANIAN NATIONALITY 107 naturalisation; (4) international treaties; (5) reference to other grounds provided in legislation. Reference to international treaties is unclear. It can be argued that in cases where the Citizenship Law contravenes Lithuania’s international obligations the norms of the treaties would then be directly applicable. 3.2.1 General principles of the acquisition of nationality Art. 12 of the Constitution proclaims that ‘citizenship of the Republic of Lithuania shall be acquired by birth or on other grounds established by law’. However, the Citizenship Law does not mention the principle of reducing statelessness as a possible guideline for the nationality pol- icy of the state (Ziemele 1998: 248). There is no support for the argu- ment that Lithuania has adopted the ius soli principle in addition to the ius sanguinis principle as basis for the acquisition of nationality in the Law. Art. 7 enumerates the grounds on which the nationality of Lithuania can be acquired by: (1) birth; (2) exercising the right to nationality; (3) 3.2.2 Right to nationality The Lithuanian Citizenship Law identifies a number of groups who are considered Lithuanian nationals by birth and by exercising the right to nationality. The Law provides for the retaining of the right to citizenship. It was designed to prevent persons residing in foreign states who had held citizenship prior to the occupation from losing their link to Lithuania, irrespective of their ethnic origin. Those who re- tained the right to citizenship could become citizens in two ways – by claiming the right to citizenship or by restoring their citizenship.15 Na- tionals are, firstly, those individuals who were nationals by right, i.e. they were nationals before 15 June 1940 or are of Lithuanian descent. However, a distinction is made within this category of people between nationals ipso facto who do not have to renounce the nationality of an- other state and those who have to do so in order to become nationals of Lithuania. In both cases their right is preserved indefinitely. Sec- ondly, nationals are people who were born in the territory and have subsequently resided there (Ziemele 2001: 237). They are given the right to acquire nationality on the basis of application because their links with Lithuania are not considered as obvious (Ziemele 2001: 237). Otherwise, they are regarded as foreigners, albeit with the right to permanent residence. In comparison with the first group, their right to opt for nationality is not preserved indefinitely (Ziemele 1998: 222). Thirdly, children born to Lithuanian parents and foundlings shall be Lithuanian nationals while children born to stateless persons have the right to acquire nationality. The provision is neutral regarding the gen- der of the parents. Nationals residing outside Lithuania can submit their applications to diplomatic and consular missions. The Minister of the Interior has the authority to submit a recommendation to recognise a person as having lost nationality, and to receive applications for retention of nationality by persons who were nationals prior to 15 May 1940 and those of Lithuanian descent. According to art. 29 if a person fails to obtain the necessary documents attesting Lithuanian nationality held prior to 15 June 1940 or his or her Lithuanian descent, the Minister of the Inter- ior or Minister of Foreign Affairs and institutions authorised by them may apply to the Presidential Citizenship Commission for a verifica- tion of facts. 3.2.2 Right to nationality The Commission presents its recommendatory findings 108 KRISTI¯NE KRU¯ MA to the Minister of the Interior or the institution authorised by him. Ac- cording to art. 31, repeated applications shall be accepted no earlier than one year after the adoption of the previous decision. 3.2.3 Naturalisation Art. 12 lays down several requirements to be met in order to acquire Lithuanian nationality. y The requirements are the following: y The requirements are the following: – passing the Lithuanian language exam (speaking and reading);16 – ten years of permanent residence in Lithuania; – permanent employment contract or a constant legal source of sup- port; – knowledge of the Constitution; – lack of any other nationality; and – agreement to take the oath to Lithuania. Amendments in 1995 lifted the requirements of the language exam and the knowledge of the Constitution exam for persons over 65 years of age, disabled persons of group I and II17 and the sick with grave chronic mental diseases. These exceptions were upheld in the 2002 Law. In addition, this Law has lifted the requirement that refugees have to be stateless or renounce their nationality prior to applying for Lithuanian nationality. Art. 12 provides that interests of the state have to be taken into con- sideration when nationality is granted. The application of this provision is unclear and open to discretion. The terms ‘permanent residence in the territory of Lithuania’ and ‘constant legal source of support’ were clarified in sect. 3 of the Su- preme Council Resolution on the Procedure for Implementing the Re- public of Lithuania Law on Citizenship (adopted on 10 December 1991). A person shall be considered as permanently residing in Lithua- nia if he or she has been registered in the register of permanent resi- dents, has accommodation, and is employed in Lithuania under an em- ployment contract or has another paid occupation in Lithuania and pays taxes there. A person will also be considered as permanently resid- ing if he or she is somebody’s dependent or is paid a pension legally due to him or her in Lithuania. Residency is counted including the per- iod 1940-1991 (Kalvaitis 1998: 264). Lithuanian practice as confirmed by the Constitutional Court in Case 7/94 has established that neither an occupying army nor repressive structures of a foreign state which resided in Lithuania without con- sent of Lithuania’s authorities could be considered as lawfully residing for the purpose of the permanent residence requirement of the Citizen- LITHUANIAN NATIONALITY 109 ship Law (Ziemele 2001: 236). Likewise, service in such foreign forces cannot be considered as legal employment. Moreover, this interpreta- tion is valid also in the context of the 1989 Law. According to art. 3.2.3 Naturalisation 16, the President has the right to grant nationality by exception. The requirements for this option are, first, significant contribution to strengthening of Lithuanian statehood by the person in question. Second, the person has to contribute to Lithuania’s power and its authority in the international community. Third, the person should be integrated into the Lithuanian society, i.e. he or she must have permanent factual links with Lithuania. Fourth, according to the ruling of the Constitutional Court in Case 40/03, art. 13 is applicable in these cases. Art. 13 identifies the groups of persons precluded from acquiring na- tionality. Those include persons who (1) have committed crimes against humanity or acts of genocide; (2) have taken part in criminal activities against Lithuania; (3) before coming to Lithuania have been tried for a deliberate crime for which the criminal liability is imposed in Lithua- nia or have been sentenced in Lithuania; (4) are chronic alcoholics or drug addicts and (5) have especially dangerous infectious diseases. The exclusion of alcoholics, drug addicts and criminals applied until the adoption of the 2002 Law. Criminals who were convicted before the adoption of the Law were subject to an ex post facto penalty to their punishment. The exclusion from naturalisation of alcoholics and drug addicts was particularly pernicious because it discouraged them from seeking needed treatment.18 Since 1 January 2005, groups (1) and (2) have been broadened and now include not only those persons who committed aforementioned crimes but also those who were preparing or attempting to commit those crimes and acts. Moreover, the amendments added that those who do not have the right to reside in Lithuania cannot be granted na- tionality. Since then, art. 13 on conditions for withholding nationality has been changed to bring it in line with human rights requirements. Firstly, it no longer states that chronic alcoholics, drug addicts or those ill with especially dangerous infectious diseases cannot become nationals. Sec- ondly, the scope of persons who have had criminal charges against them has been minimised. The Law no longer refers to persons who, before coming to Lithuania, have been tried for a deliberate crime but only to persons who, before coming to Lithuania, have had a custodial sentence imposed on them for a premeditated crime. Also, in relation to those convicted in Lithuania, reference is made to premeditated crimes, not deliberate ones. 3.2.3 Naturalisation The procedures for resolving issues related to nationality are set out in chapter V of the Law. The chapter includes detailed information as 110 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA to what documents shall be submitted in each case when a person ap- plies for nationality. According to para. 10 of art. 28 all applications for the acquisition, renunciation and restoration of nationality shall be submitted to the President through the executive institution of the mu- nicipality. Applications for nationality are reviewed by the Citizenship Commission which is established by the President. It submits propo- sals for a decision to the President. Decrees by the President should be co-signed by the Minister of the Interior. According to the Constitu- tional Court, the responsibility for these decrees lies with the ministers. The reason for this is that the President can be removed from office only for grave violations of the Constitution. In the case of a denial, an applicant is provided with a reasoned decision in writing. According to art. 30, decrees by the President concerning the granting, retention, re- storation or loss of nationality as well as declaring an act on the grant- ing of nationality invalid are published in the Official Gazette. 3.2.4 Loss of nationality The grounds for loss of Lithuanian nationality are outlined in art. 18. It provides that nationality is lost (1) upon renunciation; (2) upon acquisi- tion of nationality of another state; (3) on grounds provided for by inter- national agreements to which Lithuania is a party.19 Paragraph 2 of the same article states that nationality should not be lost in cases when a person acquires the citizenship of another state with which Lithuania has concluded a contract on dual citizenship. A person may be recog- nised as having lost nationality if he or she is in the military service of another state or is employed in the public service of a non-EU Member State without permission of the Lithuanian authorities. According to the amendments in 2004 (effective of 1 April 2006), a Lithuanian citi- zen working in another state under the permission of the Lithuanian authorities would lose Lithuanian nationality if he or she were to injure the interests of Lithuania. The formulation is wide and it is unclear what is meant by damaging Lithuanian interests. Acquiring the nation- ality of another state does not result in loss of nationality for two groups of persons, i.e. those who were nationals prior to 15 June 1940 and their descendants as well as persons of Lithuanian descent.20 A person may be recognised as having lost nationality if he or she is in the military service of another state or is employed in the public service of another state without permission of the Lithuanian authorities. Moreover, there are a number of grounds on which naturalisation can be invalidated. Art. 21 provides that, firstly, naturalisation will be deemed invalid if nationality has been acquired by fraud or if a person has committed international crimes provided for by the international treaties or customary law (aggression, genocide, crimes against human- LITHUANIAN NATIONALITY 111 ity, war crimes or crimes against Lithuania). Secondly, the act of natur- alisation will be declared invalid if the court establishes that after 15 June 1940 a person has ‘organised or carried out deportation or exter- mination of the residents, suppressed the resistance movement in Lithuania’ (official translation). The same result would be reached if a court establishes that a person took part in the actions against indepen- dence and territorial integrity of Lithuania after 11 March 1990. 3.2.4 Loss of nationality g y 99 The act of invalidation applies to naturalised nationals and Lithua- nian descendants, if they have opted for Lithuanian nationality (Ziemele 1998: 221). A declaration of invalidity may be used as an additional pen- alty in relation to other criminal charges, e.g. if a person has committed crimes against humanity, acts of genocide or crimes against the Repub- lic of Lithuania, prior to or after acquisition of nationality, as determined by the court decree. Original nationals cannot be subjected to such an additional penalty in similar circumstances (Ziemele 1998: 250). The ar- ticle does not set any precise time limit within which charges brought against a person for crimes against Lithuania could affect naturalisation. This again opens the possibility of arbitrary decisions which could result in statelessness and would run contrary to the rule prohibiting arbitrary deprivation of nationality (Ziemele 1998: 250). Art. 30 provides that invalidation of nationality is enacted by the Pre- sident of Lithuania who issues a decree to that effect. The decree is not subject to judicial scrutiny. As the Lithuanian Citizenship Law is both complex and cumbersome on a number of issues the right to appeal decisions would be more than desirable (Ziemele 1998: 222). According to art. 19, everyone has a right to renounce their national- ity. However, the same article provides for exceptions. Thus application for renunciation may not be considered if the person has been charged with a criminal act or if a court judgement passed on the person has be- come effective and enforceable. Setting the absence of criminal charges as a condition for the renunciation of nationality may also raise human rights considerations. The person may still be entitled to renounce na- tionality, which would not affect procedures employed by the state as long as the person remains within its jurisdiction (Ziemele 1998: 249). The Law on Citizenship places additional safeguards in order not to allow cases of statelessness to arise. Art. 20 provides that a person who has lost nationality as a result of renunciation or on grounds provided in an international treaty may be reinstated with Lithuanian nationality if he or she submits an application while having perma- nently resided in Lithuania for at least ten years, has a legal source of support and is stateless. 3.3 Current political debates The most recent proposals in the field of nationality policies aim to re- solve issues concerning dual nationality. Over the years, the group en- titled to dual citizenship has been considerably expanded. p y p According to the Citizenship Law, the right to nationality is retained by, and dual nationality allowed for, those who were nationals prior to 15 June 1940, their children, grandchildren and great-grandchildren who have not repatriated, as well as persons of Lithuanian descent. Thus, several categories can be distinguished. Firstly, persons of Lithua- nian origin are considered to be Lithuanian citizens and retain the right to citizenship irrespective of where they reside abroad. Secondly, per- sons of other ethnic origin are divided into two groups depending on where they reside. If their place of residence is their ethnic homeland, they are not considered citizens of Lithuania. For instance, if Poles holding Lithuanian citizenship moved to Poland during the period of Soviet annexation they no longer qualify for Lithuanian citizenship. According to art. 12, para. 2 of the Lithuanian Constitution, no one may be a citizen of both the Republic of Lithuania and another state at the same time except in individual cases provided for by law. This arti- cle forms part of Chapter One of the Constitution, which, according to art. 147, can only be altered by referendum, meaning that more than half of the electorate must vote in favour of a change. However, recent amendments to the Citizenship Laws have provided large groups of persons with the right to dual nationality. In response to a petition pre- sented by an Israeli citizen who also claimed to be a Lithuanian citizen this reform was challenged in the Constitutional Court by a group of parliamentarians and by the Vilnius Regional Administrative Court. p y g The Court emphasised that art. 12 of the Constitution should be in- terpreted narrowly, i.e. that cases of dual citizenship must be extraordi- narily rare and exceptional, and that the Constitution does not permit the widespread granting of dual citizenship. In cases involving the granting of citizenship based on special merits, the Court has stressed that candidates must prove permanent factual links to the state and must be integrated into society before being granted citizenship. The Court considers the provisions for dual citizenship in the Citizenship Law to be highly controversial, inconsistent and confusing. 3.2.4 Loss of nationality Special conditions are provided for those who were nationals or permanent residents before 15 June 1940 and their descendants as well as persons of Lithuanian descent. If they have lost nationality as a result of renunciation or on the basis of an 112 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA international treaty, Lithuanian nationality is restored automatically on the basis of application. 3.3 Current political debates Some provi- sions are barely compatible with each other while others are phrased ambiguously. As a consequence, the Court declared those provisions of the Citizenship Law that could lead to dual nationality as unconstitu- LITHUANIAN NATIONALITY 113 tional. While the Court noted that it is not against the Constitution to grant a right to Lithuanian citizenship, the relevant articles providing for the right to retain another nationality when acquiring or regaining Lithuanian nationality were found to be in violation of art. 12 of the Constitution. This conclusion is confusing because in its earlier ruling in case 7/94, the Court accepted the practice of dual citizenship in rela- tion to pre-1940 nationals and their descendants. Moreover, the Court declared that categorising people according to their ethnic origins to es- tablish whether their right to citizenship has been retained or not (in the case of repatriation) violates art. 29 of the Constitution, which pro- vides for equality before the law.21 q y During the spring and summer of 2007, several proposals for amendments were tabled. For instance, it was suggested that dual citi- zenship should be allowed for children born abroad if at least one par- ent is a Lithuanian citizen. The draft amendments were criticised by ex- perts as being too general and ambiguous and became bogged down in various parliamentary committees. Meanwhile, the Lithuanian parlia- mentary Social Liberals successfully collected the 40 signatures of members of parliament necessary to initiate a referendum on the legali- sation of dual citizenship.22 This referendum was planned to coincide with parliamentary elections in the fall of 2008 but did not take place.23 However, the amendments were reconsidered in 2008 and adopted by the Human Rights Committee on 4 June and subsequently by par- liament on 30 June 2008. The amendments concerned seven groups. Most notably among these amendments was the one that prevented persons who had acquired citizenship of EU and NATO Member States or of a country with which Lithuania has signed a mutual agreement on dual citizenship from losing their Lithuanian citizenship.24 Seventy- eight MPs voted in favour of the amendments, four against them and nine abstained. Nevertheless, the Lithuanian legal scholar Dainius Zali- mas has argued that these amendments violate the Constitution and devalue the significance of Lithuanian citizenship. He has suggested solving the problems that emigrants encounter by regulating the right to citizenship rather than citizenship itself. 3.3 Current political debates He has also advocated for the adoption of regulations similar to the Polish card system (see Go´rny & Pudzianowska in this volume), which would allow for the pre- servation of certain benefits for former Lithuanian citizens who have acquired citizenship in another country.25 President Valdas Adamkus vetoed certain provisions in the amend- ments, including the provisions on dual citizenship for those who hold a passport of an EU or a NATO Member State. According to the Presi- dent, the amendments broaden too widely the possibilities for granting (retaining) dual citizenship and ‘create legal prerequisites for dual citi- 114 KRISTI¯NE KRU¯ MA zenship that allow a rare exemption to become a widespread phenom- enon’. The parliament agreed with the President and amended the law again on 15 July 2008. The vote was 66 parliamentarians approving the President’s amendments, while ten voted against them and seven abstained. The adopted amendments expanded the possibilities for dual citizenship, e.g. for children who acquire Irish or Lithuanian citi- zenship by birth. However, this is not the end of the story. With the presidential veto, the current law became the provisional law, valid only until 1 January 2010. Since neither the government nor interest groups have come up with a new law, the president entrusted a group of law- yers with the task of presenting solutions. The group concluded that it is highly unlikely that a referendum on the amendment of art. 12 of the Constitution would succeed (the law can only be changed if a ma- jority of at least 50 per cent of all eligible voters agree). The group of lawyers therefore drafted a new Law on Citizenship, presented to the president on 9 February 2009. 3.4 Statistics Taking into account that nationality and statelessness issues were not high on the political agenda due to the liberal approach adopted in the 1989 Law, no exact statistics were maintained. Lithuanian statistical in- formation is therefore rather poor. Table 3.1 shows that the share of the titular ethnic group has been rather high in Lithuania compared to Estonia and Latvia. Table 3.1 Ethnic composition in Lithuania 1979 1989 2001 2007 Lithuanians 80% 79.6% 83.5% 84.7% Non-Lithuanians 20% 20.4% 16.5% 15.4% Sources: Statistikos Departmentas, 1979, 1989, 2001: population censuses, 2007: estima- tion carried out based on the data of the Residents’ Register central database, www.stat. gov.lt Data for the period from 1993 onwards are not available because no ex- act statistics were maintained during that period. Data on naturalisa- tion has only become available since 2002. The statistics show that the number of persons acquiring Lithuanian citizenship was insignificant between 2003 and 2007. Most of the new Lithuanian citizens were for- mer Russian citizens or stateless persons. For instance, out of the 370 persons granted Lithuanian citizenship in 2007, 113 were former Rus- sian citizens and 184 were former stateless persons. LITHUANIAN NATIONALITY 115 Table 3.2 Number of naturalised persons, 2003-2007 Year 2003 2004 2005 2006 2007 Number 389 610 435 467 370 Source: Migration Department 2008: 56 Table 3.2 Number of naturalised persons, 2003-2007 A relatively large number of persons lost nationality between 2003 and 2007. Moreover, the number increased over the years, especially due to acquisition of nationality of another state (see Table 3.3). 3.4 Statistics Table 3.3 Number of persons who have lost Lithuanian citizenship, by reasons Grounds for loss of citizenship Number of persons 2003 2004 2005 2006 2007 Withdrawal of Lithuanian citizenship 235 315 207 216 280 Acquiring citizenship of another state 372 386 265 485 610 The person lawfully decided on Lithuanian citi- zenship by 4 November 1991 in accordance with the Law on Citizenship of 3 November 1989, resides in a foreign state and has not, before 31 December 2003, approached institu- tions authorised by the Minister of the Interior or Lithuanian consular offices in foreign coun- tries for issue of a document evidencing Lithuanian citizenship - 96 283 197 123 Severance of actual ties with Lithuania 1 1 - - - Children under fourteen, if both parents lose citizenship - - - - 2 Total 608 798 755 898 1,015 Source: Migration Department 2008: 58 Table 3.3 Number of persons who have lost Lithuanian citizenship, by reasons The retention of the right to citizenship did not play a significant role between 2003 and 2007. However, the numbers increased in 2007. Most applicants are Russian and Byelorussian citizens. Table 3.4 Retention of the right to Lithuanian citizenship 2003 2004 2005 2006 2007 Decisions on retention of the right of aliens to citizenship 145 90 20 47 163 Number of certificates issued for retention of the right to citizenship 99 67 35 49 201 Source: Migration Department 2008: 61 Table 3.4 Retention of the right to Lithuanian citizenship 116 KRISTI¯NE KRU¯ MA 1992 Constitution of the Republic of Lithuania www.legislationline.org (excerpts) 1991 Law on the Validity of Citizenship Documents issued by the Republic of Lithuania and on the Supplement to the Law on Citizenship (void since 1 January 2003) www.coe.int; www.legislationonline.org 3.5 Conclusions VIII-391) requirements and a complicated system in relation to dual nationality 1991 Supreme Council of the Republic of Lithuania Resolution on the Procedure for Implementing the Republic of Lithuania Law on Citizenship (11 December 1991, No. I-2080) Clarifies the application of the Citizenship Law in relation to inter alia per- sons who served in armed troops and other state se- curity structures of the So- viet Union www.litlex.lt (in Lithuanian) 1991 Law on the Validity of Citizenship Documents issued by the Republic of Lithuania and on the Supplement to the Law on Citizenship (void since 1 January 2003) Confirms that Citizen’s Certification Card and Certification Testifying Decision to Acquire Citizenship shall be valid until the person is issued with new citizens’ passport but no longer than 1 July 1993; supplements Citizenship Law of 1991; provides that new Citizenship Law shall replace 1989 Law on 11 December 1991 www.legislationline.org (excerpts) 1992 Constitution of the Republic of Lithuania Confirms the basic principles of acquisition of Lithuanian nationality (ius sanguinis) and a negative position in relation to dual nationality www.litlex.lt (in Lithuanian) 2002 Law on Citizenship (as last amended by the Law of 3 April 2003, No. IX-1456) Repeals the 1991 Law; extends the category of persons who have inherent right to nationality and simplifies the restoration of nationality; clarifies Lithuania’s position on dual nationality; introduces ius soli for children whose parents are stateless and reside in Lithuania; introduces higher requirements for spouses of Lithuanian nationals www.coe.int; www.legislationonline.org 2002 Law on Implementation of th L Citi hi ( Clarifies the procedural i t i f www.coe.int; l lt (i Lith i ) requirements and a complicated system in relation to dual nationality Clarifies the application of the Citizenship Law in relation to inter alia per- sons who served in armed troops and other state se- curity structures of the So- viet Union 1991, No. 2002 Law on Citizenship (as last amended by the Law of 3 April 2003, No. IX-1456) 3.5 Conclusions The Lithuanian approach to nationality issues has been considered as more liberal than the regulations in other Baltic states because it did not apply pre-1940 citizenship laws in order to reconstitute its body of nationals. The Citizenship Law of 1989 provided easy criteria for acqui- sition of nationality. As a result of these liberal laws, most of its resi- dents acquired Lithuanian nationality. However, the Citizenship Law of 1991, enacted immediately after restoration of independence, intro- duced much stricter requirements for the acquisition of Lithuanian na- tionality. These are still in existence and in certain cases are more strin- gent than the regulations in other Baltic states, such as the conditions on residence before applying for citizenship. pp y g p One of the major areas currently debated is the regulation of dual nationality in Lithuania. Dual nationality became restricted ever since the adoption of the 1991 Citizenship Law and Constitution. Although it was extended over the years, Lithuania was still criticised for the ex- clusionary nature and unclear application of dual citizenship. The si- tuation has become even more acute since the Constitutional Court of Lithuania delegitimised nearly 30 statutory and sub-statutory provi- sions and omissions in the nationality legislation on dual citizenship. The possible solutions remain unclear, but they will have to be found by January 2010 when the now provisional Citizenship Law will be- come invalid. Another problem of Lithuanian nationality policy is the wide discre- tion given to the president concerning the granting of nationality. Not- withstanding the amendments introduced after the decision of the Constitutional Court in the Paksas case, the judicial review of all deci- sions related to nationality would be a welcome development. This would also facilitate Lithuania’s ratification of the European Conven- tion on Nationality which has not yet been signed. Chronological list of citizenship-related legislation in Lithuania Date Document Content Source 1989 Supreme Soviet of the Lithuanian Soviet Socialist Republic Law on Citizenship (3 November 1989, No. XI-3329) Reconstitutes the body of nationals by restoring nationality to those who were nationals before the 1940 occupation and their descendants; provides for liberal naturalisation of residents www.uta.edu 1991 Republic of Lithuania Law on Citizenship Introduces stricter naturalisation www.uta.edu Chronological list of citizenship-related legislation in Lithuania LITHUANIAN NATIONALITY 117 Date Document Content Source (5 December 1991, No. I-2072 as amended on 2 July 1997, No. www.coe.int; www3.lrs.lt (in Lithuanian) 2004 Amendments to the Law on the Implementation of the Law on Citizenship (11 November 2004, No. IX- 1079) Provides for dual citizenship for certain groups as a result of the 2006 Constitutional Court Ruling, which declared the norms on dual citizenship to be unconstitutional 3.5 Conclusions I-2080) 1991 Law on the Validity of Citizenship Documents issued by the Republic of Lithuania and on the Supplement to the Law on Citizenship (void since 1 January 2003) Confirms that Citizen’s Certification Card and Certification Testifying Decision to Acquire Citizenship shall be valid until the person is issued with new citizens’ passport but no longer than 1 July 1993; supplements Citizenship Law of 1991; provides that new Citizenship Law shall replace 1989 Law on 11 December 1991 www.legislationline.org (excerpts) 1992 Constitution of the Republic of Lithuania Confirms the basic principles of acquisition of Lithuanian nationality (ius sanguinis) and a negative position in relation to dual nationality www.litlex.lt (in Lithuanian) 2002 Law on Citizenship (as last amended by the Law of 3 April 2003, No. IX-1456) Repeals the 1991 Law; extends the category of persons who have inherent right to nationality and simplifies the restoration of nationality; clarifies Lithuania’s position on dual nationality; introduces ius soli for children whose parents are stateless and reside in Lithuania; introduces higher requirements for spouses of Lithuanian nationals www.coe.int; www.legislationonline.org 2002 Law on Implementation of the Law on Citizenship (as last amended by the Law of 21 January 2003, No. IX- 1298) Clarifies the procedural requirements in cases of dual nationality www.coe.int; www3.lrs.lt (in Lithuanian) 118 KRISTI¯NE KRU¯ MA Date Document Content Source 2003 Ruling of the Constitutional Court of Lithuania on the Compliance of the President of the Republic of Lithuania with Decree No. 40 on Granting Citizenship of the Republic of Lithuania by way of Exception of 11 April 2003 States that citizenship of the Republic of Lithuania is granted to Jurij Borisov by way of exception in compliance with the Constitution of the Republic of Lithuania and para. 1 of art. 16 of the Republic of Lithuania’s Law on Citizenship (30 December 2003) www.lrkt.lt (in Lithuanian) 2004 Amendments to the Law on Citizenship (12 September 2004, No. IX- 1078) Brings the law in line with human rights standards (regarding refugees, alcoholics, drug addicts); takes into account the ruling of the Constitutional Court in the Borisov case in relation to procedural aspects of nationality policies www3.lrs.lt (in Lithuanian) 2004 Amendments to the Law on the Implementation of the Law on Citizenship (11 November 2004, No. 2004 Amendments to the Law on Citizenship (12 September 2004, No. IX- 1078) www3.lrs.lt (in Lithuanian) 3.5 Conclusions IX- 1079) Streamlines the procedures for the acquisition of nationality attempting to address problems as envisaged in the Borisov case www3.lrs.lt (in Lithuanian) 2006 Ruling of the Constitutional Court of Lithuania on the Compliance of the Repeals provisions in the Citizenship Law concerning dual nationality Content 2006 Ruling of the Constitutional Court of Lithuania on the Compliance of the Provisions of Legal Acts Regulating the Citizenship Relations with the Constitution of the Republic of Lithuania of 13 November 2006 2008 Amendments to the Law on Citizenship (15 July 2008) LITHUANIAN NATIONALITY 119 Notes 1 I would like to thank Prof. Ineta Ziemele and Prof. Egidijus Kuris for their comments on the draft of this article. The usual disclaimer applies. 1 I would like to thank Prof. Ineta Ziemele and Prof. Egidijus Kuris for their comments on the draft of this article. The usual disclaimer applies. 2 Permanent residence was determined by so-called propiska which is similar to a resi- dence permit nowadays. It had to be obtained before individuals could move to an- other place. This was applicable not only in between republics but also within repub- lics. Report on Lithuania, European Commission against Racism and Intolerance. Strasbourg, September 1997, ECRI (97) 56 para. 6. 3 Case No. 7/94 of 13 April 1994 (1994) 1 E.E.C.R.C.L.255. Available at www.lrkt.lt. 4 The texts used here are available at www.litlex.lt and www.minelres.lv. 5 This option was inserted with amendments of 3 October 1995. 6 This article was slightly amended on 6 February 1996. Prior to these amendments, the law provided that citizenship shall be retained by children who had Lithuanian citizenship until 15 June 1940 and who were born in Lithuania or in refugee camps but are at present residing in other states. 7 Constitutional Court case No. 45/03-36/04 7 Constitutional Court case No. 45/03-36/04 8 Before amendments of 7 December 1993, this condition was also applicable to children of those who held Lithuanian citizenship until 15 June 1940. 9 See chapter 2 on Latvia in this volume. 9 See chapter 2 on Latvia in this volume. 10 Certain provisions of amendments have been effective since 1 April 2006, but some since 1 January 2005. 10 Certain provisions of amendments have been effective since 1 April 2006, but some since 1 January 2005. 11 For details on these debates, see the articles ‘Lithuanian e´migre´s unhappy with citizenship loss’, ELTA [National News Agency], 6 June 2001, ‘New Law will entitle Lithuanian emigrants to keep citizenship’, ELTA, 17 September 2002, and ‘Adamkus signed controversial citizenship law’, ELTA 30 September 2002. 12 The proposed amendments were even stricter and debate was reopened after the President of Lithuania intervened with proposals to liberalise the procedure for naturalisation of spouses in Lithuania. The compromise reached was that the required term of residency would be increased from five to seven years, but not to ten years as foreseen in the draft law (86 in favour, five against, seven abstentions). Notes 18 Provisions at issue were closely monitored, at least, by Human Rights Watch, which on a number of occasions condemned their application and advocated their abolition. See the reports on human rights developments in Latvia, Lithuania and Estonia available at www.hrw.org. 19 Before the 2002 Law was adopted, it was possible to lose one’s citizenship on the basis of severance of actual links with Lithuania. A person who has lived abroad with an invalid passport for more than three years or who has entered foreign military or public service without the permission of the competent authorities was considered to have severed links with Lithuania. 19 Before the 2002 Law was adopted, it was possible to lose one’s citizenship on the basis of severance of actual links with Lithuania. A person who has lived abroad with an invalid passport for more than three years or who has entered foreign military or public service without the permission of the competent authorities was considered to have severed links with Lithuania. 20 However, it is not entirely clear because according to art. 1, para. 3, persons of Lithuanian descent can acquire Lithuanian citizenship if they are not citizens of any other state. The only plausible explanation can be that according to art. 18 they do not lose their right to acquire Lithuanian citizenship because they possess the citizenship of another state. p 21 Lithuanian Constitutional Court Ruling in Case No. 45/03-36/04, 13 November 2006. p 21 Lithuanian Constitutional Court Ruling in Case No. 45/03-36/04, 13 November 20 22 ‘Lithuanian parliament collects signatures to initiate referendum on dual citizenship’, BNS [National News Agency], 15 September 2007. 23 Radzevicˇu¯te A., ‘Lietuva¯ iespe¯jams referendums par dubultpilsonı¯bu [The referendum on dual nationality in Lithuania is possible]’, Diena [Latvian daily news- paper], 7 September 2007. Notes 24 The other six groups are: (i) political deportees and prisoners and three generations of their descendants; (ii) those who left Lithuania during the Soviet era as well as three generations of their descendants; (iii) persons of Lithuanian descent living in countries sharing a common border with Lithuania; (iv) persons already in possession of passports from two other countries, granted by special decree of the President; (v) offspring born to Lithuanian citizens anywhere in the world; (vi) Lithuanians living in any nation that has signed an international agreement with Lithuania on dual citizenship (‘Dual citizenship now allowed in Lithuania’, The Baltic Times, 1 July 2008, www.baltictimes.com). l l h l d ll l 25 ‘Seimas’ Decision on multiple citizenship is unconstitutional and illogical’, ELTA, 30 June 2008. 25 ‘Seimas’ Decision on multiple citizenship is unconstitutional and illogical’, ELTA, 30 June 2008. Notes The President also opposed the additional requirement that the couple should have minor children who are Lithuanian citizens. In his view, this would contradict the principle of equality contained in the Constitution. The proposals made by the President were harshly criticised by conservative members of parliament. They saw the proposals as a threat to the survival of the Lithuanian nation and national identity. Moreover, they were afraid that liberal citizenship procedures might stimulate marriages of convenience, often referring to Denmark to illustrate this point. See ‘Seimas approves more liberal procedures for admission to citizenship via marriage’, ELTA, 9 December 2004. 13 Concerning this case, see also ‘Lithuanian Practice in International Law 2004’, as reported in the Baltic Yearbook of International Law 5, 2005: 329-332. 14 In addition, on 1 April 2006 the authority on questions of citizenship, formerly attributed to municipal institutions, was transferred to the Department of Migration of the Ministry of the Interior. 14 In addition, on 1 April 2006 the authority on questions of citizenship, formerly attributed to municipal institutions, was transferred to the Department of Migration of the Ministry of the Interior. 15 Constitutional Court case No. 45/03-36/04. 16 On 11 February 2004, the Ministers of Education and Science and Justice confirmed the programme of exams on the basics of the Lithuanian Constitution and language. The procedure for the organisation and implementation of the exams was confirmed by both ministers on 1 March 2004. 120 KRISTI¯NE KRU¯ MA KRISTI¯NE KRU¯ MA 17 There are three disability groups of which group I is the most serious. The group is assigned by a special commission on the basis of a diagnosis. Assignments can be for a defined period or for life. Group I is as a rule assigned for life. 17 There are three disability groups of which group I is the most serious. The group is assigned by a special commission on the basis of a diagnosis. Assignments can be for a defined period or for life. Group I is as a rule assigned for life. 18 Provisions at issue were closely monitored, at least, by Human Rights Watch, which on a number of occasions condemned their application and advocated their abolition. See the reports on human rights developments in Latvia, Lithuania and Estonia available at www.hrw.org. Bibliography Kalvaitis, R. (1998), ‘Citizenship and national identity in the Baltic States’, Boston Univer- sity International Law Journal 16: 231-271. y J 3 7 Migration Department (2008), Migration Yearbook 2007. Vilnius: Migration Department. Ziemele, I. (1998), State Continuity and Nationality in the Baltic States: International Constitutional Law Issues. PhD Dissertation. Wolfson College Cambridge. Ziemele, I. (2001), ‘State Continuity, Human Rights and Nationality in the Baltic States’, in T. Jundzis (ed.), The Baltic States at Historical Crossroads, 224-248. Riga: Academy of Sciences of Latvia. Ziemele, I. (2005), State Continuity and Nationality: The Baltic States and Russia. Past, Pre- sent and Future as Defined by International Law. Leiden, Boston: Martinus Nijhoff Pub- lishers. 4 Same letter, new spirit: Nationality regulations and their implementation in Poland Agata Go´rny and Dorota Pudzianowska The development of the legal notion of Polish citizenship has gone through twists and turns, shaped by the history of the country. Belong- ing to the Polish nation has not always meant belonging to the Polish state. Radical reconfigurations of Poland’s borders in the last century explain this conceptual inconsistency as much as substantial political and economic emigration from Poland does. Moreover, the Polish Peo- ple’s Republic promoted the communist idea of a single socialist com- munity comprised of inhabitants of Soviet Bloc countries. Thus, geo- politics defined a concept of the nation that was far removed from how many Polish people construed their own identity. y y In the Polish case, it is, therefore, justified to differentiate between the distinct concepts of ethnicity and nationality/citizenship. The latter concept refers to the affiliation to the state and thus denotes the legal bond between a citizen and the state. Ethnicity constitutes more of a subjective feeling of belonging to an ethnic group or to a given nation, along with concurrent objective criteria relating to a person’s ancestry.1 Such a distinction is necessary in an examination of Polish nationality regulations and practice. In our opinion, ethnicity was very important in the formulation of the law on nationality in the Polish People’s Re- public and still plays a role in current Polish legislation. The goal of this chapter is to demonstrate evolutions in the field of Polish nationality, particularly focusing on its acquisition and loss. We present changes not only in written law, but also in administrative practice regarding Polish nationality. This is necessary because of the high level of discretion that the Polish public authorities have in this field. Analyses of regulations are further enriched with selected statis- tics on the acquisition of Polish nationality in order to better represent the nature of the phenomenon in Poland. Apart from analysing legal acts and statistics, we also devote some space to recently proposed bills on Polish nationality in order to indi- cate the direction in which the approach towards Polish nationality has been evolving since the 1990s. We argue that contemporary Polish na- tionality policy (if we can talk about one) still puts the emphasis on emigrants and the diaspora rather than on immigrants. 4.1.1 Introductory remarks There have been three acts on Polish nationality – enacted in 1920,4 19515 and 19626 – that share important elements. First of all, the acqui- sition of Polish nationality by birth has always been driven by the prin- ciple of descent (ius sanguinis), with the territorial principle (ius soli) merely playing an auxiliary role. Secondly, due to radical changes in Poland’s international borders and long periods of emigration from Po- land, establishing whether a given individual holds Polish nationality has always been crucial to Polish legislation on this matter. Finally, rules concerning foreigners’ naturalisation in Poland have been of sec- ondary importance in the debates and legislation on Polish nationality, despite considerable growth in immigration to Poland since the early 1990s. 4 Same letter, new spirit: Nationality regulations and their implementation in Poland Our argument is supported by the debate and the work on new legislation relating to AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 124 matters of Polish nationality in the Polish Parliament during 1999- 2001. The focus on the diaspora is also evident when we consider the two most recent acts adopted in the field related to nationality, namely the Repatriation Act (2000)2 and the Act on the Polish Ethnicity Card (2007)3 (Ustawa o Karcie Polaka). The chapter opens with a historical overview of developments in na- tionality legislation from the post-war era to the present. It then dis- cusses basic rules governing the acquisition and loss of Polish national- ity in contemporary Poland. The third section analyses debates regard- ing new regulations and their underlying orientations as well as trends in Polish nationality policy. Finally, selected statistics on the acquisition of Polish nationality are provided and discussed. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 125 zens, and automatically lost their foreign nationality upon repatriation in Poland. The repatriation agreements signed with the Soviet Union also concerned the resettlement of Polish citizens of non-Polish (Ukrai- nian, Belarusian, Russian, etc.) ethnicity to the USSR. Thus, in both re- patriation actions ethnicity constituted a decisive criterion. Nevertheless, the biggest national group expatriated from Poland in the post-war period, on the basis of the Potsdam agreements, were Ger- mans. They were officially excluded by the Act on the Exclusion of Per- sons of German Ethnicity from Polish Society (1946).7 This applied to Germans not verified as Polish nationals or those manifesting their German origins.8 Ethnic Poles, even those who had been German citi- zens before the Second World War, were entitled to stay in Poland. Spe- cial public bodies were established and appropriate legal rules intro- duced to verify the Polish ethnicity of those who wished to stay in Po- land.9 Two pivotal legal acts announced at that time were the Act on Polish Nationality of Persons of Polish Ethnicity Inhabiting the Re- gained Territories (1946)10 and a similar decree for inhabitants of the former Free City of Gdan´sk (Danzig) (1947).11 These acts directly linked a person’s nationality to his or her ethnicity. Verification of ethnicity and objective ethnicity criteria were also in- cluded in the 1951 Act on Polish Nationality. The Act obliged the inha- bitants of the Regained Territories and the former Free City of Gdan´sk to obtain adequate documents certifying their Polish ethnicity. It also gave the right to Polish nationality to all Polish repatriates. Again, Pol- ish citizenship was based primarily on ethnic criteria. This link was also reflected in two subsequent legal acts concerning the permission for the renunciation of Polish nationality for people of German (1956)12 and Jewish (1958)13 ethnicities who left for their ethnic home- lands (Albiniak & Czajkowska 1996: 324-325). Such acts were designed to simplify the renunciation of Polish nationality. Behind these acts, however, lay the idea of expelling those with non-Polish ethnicity from the country. The fact that this pressure was directed towards selected ethnic groups is symptomatic of this trend. 4.1.2 Post-war arrangements (1945-1962) The end of the Second World War and agreements signed between Sta- lin and other allied leaders radically altered Polish territory. This in- volved two major changes – loss of (formerly) eastern Polish lands in- habited by Polish citizens and the acquisition of eastern German lands populated largely by German citizens (the ‘Regained Territories’). The loss of the eastern Polish territories brought with it the problem of re- patriating Polish citizens from the new Soviet territory. This act was based on several Polish-Soviet mutual repatriation agreements signed in the 1940s and in 1957. On the basis of these agreements, people of Polish and Jewish ethnicity, who had been Polish citizens as of 17 Sep- tember 1939, were entitled to move and resettle within Poland’s new borders (Łodzin´ski 1998). All repatriates were treated as Polish citi- 4.1.3 Stabilisation (1962-1989) Another Act on Polish Nationality was passed in 1962. This Act re- mained in effect, without any major amendments, until the end of the communist era in Poland. It did not challenge the rules of acquisition and loss of Polish nationality included in the 1951 Act. The 1962 Act did not directly address the issue of Polish ethnicity, although it still ac- corded special rights to repatriates returning to Poland.14 The link between the ethnicity of a person and his or her right to Polish nationality was made an issue in the late 1960s. Polish authori- AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 126 ties officially challenged the loyalty of Polish citizens of Jewish origin. These people, who were perceived as having ‘dual loyalties’ and had of- ten been active in some way in political life, were forced to leave Po- land after signing a document expressing their intention to renounce their Polish nationality upon acquisition of Israeli nationality (Stola 2000). The legal basis for this ‘action of mass renunciation of Polish nationality’ was the aforementioned Decree of 1958. It is not within the scope of this analysis to present the comprehensive political back- ground behind this move asking Jews to repudiate their Polish nation- ality.15 This episode demonstrates, however, how the concept of Polish ethnicity and its tight connection with the right to Polish nationality was exploited in Poland under the communist regime. Furthermore, the communist authorities often required Polish emi- grants to relinquish their Polish nationality whenever they came to visit Poland. If they refused to do so, they risked being imprisoned in Po- land for illegally overstaying abroad. Here ‘a need to renounce’ Polish nationality was justified not in terms of the ethnicity criterion, but in terms of a lack of loyalty towards the Polish People’s Republic and its ideology. In general, the analysis of legal acts on Polish nationality, alone, does not allow for a thorough understanding of the issues of nationality in communist Poland. This is due to the authorities’ high level of discre- tionary powers regarding Polish nationality at that time, which was par- ticularly evident in how ethnicity was used in administrative decisions. Although absent from the 1962 Act on Polish Nationality, ethnicity was a factor in decisions regarding Polish nationality and played a par- ticular role in relation to German and Jewish minorities. 4.1.3 Stabilisation (1962-1989) Special de- crees designed for these two groups in 1956 and 1958 were in force un- til 1984 (Albiniak & Czajkowska 1996: 326). NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 127 The most significant amendments to the 1962 Act were introduced in 1997-1998. Rules regarding the loss of Polish nationality were chan- ged since one of the clauses of the Act – namely that ‘acquisition of a foreign nationality results in the loss of Polish nationality’ – violated the 1997 Polish Constitution17 (Jagielski 2000). An amendment was passed to make it impossible to deprive anybody of Polish nationality unless he or she expressed the desire to renounce it. A definition of the type of stay (permanent residence permit) was added to the five- year residence requirement for naturalisation. Although the exact peri- od of total legal residence in Poland varies for different groups of for- eigners, in practice, this change amounts to at least ten years of resi- dency before a foreigner can apply for naturalisation, because it takes at least five years to obtain a permanent residence permit. Changes were also introduced to the procedure of acquiring nationality by mar- riage. Whereas this path to Polish nationality was previously open only to foreign women married to Poles, foreign men can now also acquire Polish nationality by marrying a Polish woman. In 2001, provisions concerning repatriation were removed from the Act on Polish National- ity. A separate legal act known as the Repatriation Act that dealt with this issue was implemented in 2000. p The Ordinance of the President of the Republic of Poland put into force in 200018 was a step towards reducing discretion in decisions re- garding the acquisition and loss of Polish nationality, although it did not change the procedures significantly. However, while these proce- dures previously had no legally binding basis, the 2000 Ordinance lists all of the documents and forms required by the Presidential Chancel- lery to process appropriate applications. The President initiated another significant change to Polish nationality policy. In 1999, he expressed his will (in the form of a legal act) to terminate all of the remaining conventions with other former communist countries concerning pre- vention of dual nationality.19 These conventions had affected foreign- ers’ naturalisation processes by creating inequality among applicants for Polish nationality. Most former Soviet Bloc citizens were not al- lowed to retain their previous nationalities upon naturalisation in Po- land, whereas other foreigners were subject to the discretionary deci- sion by the Polish President. 4.1.4 Political and economic transition (the post-1989 era) The end of the Polish People’s Republic and the establishment of the Third Republic of Poland necessitated deep economic and political re- forms in the country. Likewise, policymakers claimed as early as 1989 that there was need for a new nationality law. However, the 1962 Act on Polish Nationality, with some amendments in the late 1990s and at the beginning of the 2000s, is still in force.16 Nevertheless, some pol- icy changes regarding Polish nationality have been introduced. These changes in policy take advantage of the way provisions of the 1962 Act on Polish Nationality were formulated. Therefore, an approach based on a high level of discretion of public officials in conferring Polish na- tionality has been continued in the Third Republic of Poland. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 4.2 Basic principles of current regulations on Polish nationality 4.2.1 Principles concerning Polish nationality The legal regulations concerning Polish nationality can be found in the amended 1962 Act on Polish Nationality as well as in the Repatriation Act of 2000. The 1997 Polish Constitution formulated two principles concerning nationality (art. 34). One states that the basic mode of ac- quisition of Polish nationality is by birth to parents who are Polish citi- zens (the ius sanguinis principle).21 The other principle stipulates an absolute guarantee that no one can be deprived arbitrarily of his or her Polish nationality. Other principles are stated in the Act on Polish Nationality itself. The principle of the continuity of Polish nationality (art. 1) translates the idea of the persistence of Polish nationality in time (from the mo- ment of its acquisition until the moment of its loss) into law. Under this principle, the nationality status acquired under a given statute sur- vives all subsequent changes in the statute, i.e. it is always considered under the law in force at the moment of acquisition. The principle of exclusivity of Polish nationality (art. 2) means that a person with dual or multiple nationality is treated (domestically but also abroad) as a Polish national by the Polish public authorities; it cannot be interpreted as forbidding dual nationality. The principle of equal citizenship rights for both husband and wife (art. 3) means that a conclusion of marriage by a Polish national with a person who is not a Polish national does not produce an automatic change in nationality for either husband or wife and a change of nationality of one spouse does not induce the change of nationality of the other.22 NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND Since 2002, as a consequence of the Pre- sident’s initiative, Poland has ceased to be a party to those conventions.20 AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 128 NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 129 land to foreign parents do not acquire Polish nationality unless they would otherwise be stateless. This means that ius soli is treated as an auxiliary principle to determine the nationality of a child found or born in the Polish territory if the child’s parents are unknown, stateless or their nationality cannot be established (art. 5). All changes in the deter- mination of a parent of a child or with regard to the nationality of one or both parents will be considered in determining the nationality of a child only if they occur within one year from the birth of the child (art. 7.1) Hence, acquisition of nationality by legitimisation, for instance, is possible until the child’s first birthday. Afterwards, legitimisation would have no effect on the child’s nationality. Automatic acquisition of nationality is also possible through repatria- tion. This is the only mode of acquiring Polish nationality not defined in the Act on Polish Nationality but in a separate statute, namely the Repatriation Act of 2000. According to this act, those holding a repa- triation visa automatically acquire Polish nationality on the day they cross the Polish border (art. 4). Repatriation visas are granted to those of Polish descent, which is further defined to include those who once had Polish nationality or who have at least one parent or grandparent or two great-grandparents who were ethnic Poles or held Polish nation- ality. Other conditions include a declaration by the person concerned that he or she is of Polish ethnicity and proof of attachment to Polish culture by nurturing Polish language, traditions and customs (art. 5). Thus, this law uses both an ethnic criterion (Polish descent) and a cul- tural criterion to determine a person’s belonging to the Polish nation.23 A foreigner may be granted Polish nationality by conferment (regu- lar naturalisation) if he or she has resided in Poland for at least five years on the basis of one of three types of permanent residence permit (art. 8).24 Since such a permit may only be acquired after at least five years of legal residence,25 it follows that the period after which a per- son is eligible for naturalisation is at least ten years. 4.2.2 Acquisition of Polish nationality Polish law provides for several modes of acquiring Polish nationality, which can be divided into three basic groups according to their legal form. The first group comprises modes of acquiring nationality ex lege (acquisition at birth and acquisition through the repatriation proce- dure). The second group comprises modes of acquisition through ap- plication (acquisition by conferment and acknowledgement). The third group unites modes of acquisition through declaration (by marriage, option and reacquisition). Acquisition of nationality at birth is mainly based on the ius sangui- nis principle. A child acquires Polish nationality irrespective of the place of birth when at least one parent is a Polish national (art. 4.2 and art. 6). If the child has only one parent who is a Polish national and ac- quires another nationality at birth, the parents can renounce the child’s Polish nationality within three months after birth. Children born in Po- NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND Applications are submitted via voivods (provincial governors) or consuls (for those living abroad) and these public authorities as well as the Minister of Internal Affairs (prior to 2007 the Head of the Foreigners’ Office) normally give their opinion on the application. Apart from information on the re- quired period of residence, applications have to include information on the parents’ nationality, sources of income, past employment, knowl- edge of the Polish language and services rendered to Poland (or Polish diaspora organisations, etc.).26 The power to grant nationality is a con- stitutional prerogative of the President of the Republic of Poland. The decision of the President is entirely discretionary since the criteria are unclear, especially with regard to the assessment of the additional in- formation.27 The President also does not have to grant nationality even if all of the conditions have been fulfilled. The President’s decision AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 130 may even be called arbitrary in the sense that the decisions do not have to be justified and there is no judicial review available,28 which may be considered contrary to the provisions of art. 11 and art. 12 of the Eur- opean Convention on Nationality (ECN) of 1997.29 In ‘particularly jus- tified cases’ the President can disregard the residence requirement (art. 8.2), but this only applies to achievement-based acquisition of national- ity. The conferment may be conditional upon providing proof of loss of the former nationality through withdrawal or renunciation (art. 8.3). From all of these provisions it is evident that the conferment procedure cannot be regarded as a legal entitlement and is instead based on the exercise of sovereign power by the head of state. A stateless person or a person whose nationality cannot be estab- lished can be acknowledged as a Polish citizen if he or she has resided in Poland for at least five years on the basis of one of three types of permanent residence permit (art. 9; also see note 24). As with the nat- uralisation of foreign nationals, this is a discretionary procedure. How- ever, in contrast to the regular naturalisation procedure, in this case the decisions are not made by the President but by voivods and consti- tute administrative case decisions that have to be justified. In these cases, administrative and judicial reviews are available. Under Polish law (art. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 131 Another mode of acquiring nationality by declaration is acquisition of nationality by option (art. 6.3). This mode addresses those who (hav- ing one parent who is a Polish national) lost their Polish nationality in childhood by parental declaration (loss of nationality by option). They can reacquire this nationality in the period between the time they turn sixteen and six months after coming of age upon making a declaration in front of the relevant public authorities, i.e. voivods or consuls. The third and last mode of acquiring nationality by declaration is re- acquisition of nationality (art. 11), also called reintegration. A Polish ci- tizen who lost Polish nationality by marrying a foreign citizen can re- gain his or her nationality if the aforementioned marriage ceases to ex- ist. In these cases a declaration made in front of the relevant public authorities, i.e. voivods or consuls, must also be accepted. As in the case of acquisition of nationality by marriage, the public authorities can make the acceptance of the declaration dependent upon the proof of loss of the former nationality. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 8.4-8.7) Polish nationality acquired by parents through conferment can be extended to their minor children. When both parents acquire Polish nationality by conferment, it is automati- cally extended to children under sixteen years of age, whereas the child’s consent is necessary if the child is sixteen or older. If only one parent acquires nationality, it may be extended to a child if other condi- tions are fulfilled – either the child is under this parent’s exclusive par- ental authority or the other parent is a Polish citizen or the other par- ent gives his or her consent for the child’s acquisition of nationality in front of a relevant public authority. The same rules apply to the filial extension of acquisition of Polish nationality through the acknowledge- ment procedure, provided that these children reside in Poland (art. 9.4). Finally, there are three modes of acquiring nationality by declaration. The first is spousal acquisition of nationality (art. 10). The spouse of a Polish national acquires Polish nationality upon making a declaration (which has to be accepted by the relevant public authority, i.e. voivods or consuls) when he or she has lived in Poland on the basis of a per- manent residence permit and has been married to the Polish national for at least three years. Compared to regular naturalisation, the mini- mum period of stay required is thus shortened by half (five instead of ten years). The voivod or consul can exercise full discretion whether to make the acceptance of the declaration conditional upon the proof of loss of the former nationality. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 4.2.3 Loss of Polish nationality As we have already mentioned, Polish nationality cannot be withdrawn against the will of the person concerned. A Polish citizen can lose his or her nationality if he or she renounces it; the loss is conditional upon the consent of the President of Poland (art. 13). The decision concern- ing the issuing of this consent is the President’s constitutional preroga- tive and is thus not subject to administrative or judicial review. A per- mit to renounce one’s Polish nationality issued by the President ex- tends to children who are under parental authority if both parents stand to lose their nationality. It is automatic unless the children are sixteen years of age or older (in which case their agreement is neces- sary). A permit given to only one parent extends to children under par- ental authority if the second parent has no parental authority, if he or she is not a Polish citizen or if he or she is a Polish citizen but con- sents to the child losing the Polish nationality in front of relevant pub- lic authorities. Polish nationality may also be lost shortly after birth due to parental decision (loss by option). For a child who has acquired Polish national- ity at birth, parents may choose the nationality of a foreign country of which one of the parents is a national. This must be done within three months of the child’s birth (art. 6.1), regardless of whether the child is born in Poland or abroad. However, as already explained, a child can reacquire his or her Polish nationality lost in this way (acquiring Polish nationality by option, art. 6.3). The optional renunciation of Polish na- tionality acquired by ius sanguinis, however, does not apply if, for ex- AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 132 ample, both parents are Polish nationals abroad and the child has ac- quired the nationality of his or her country of birth through ius soli. During the communist period, the rules governing the loss of Polish nationality were quite different. Many emigrants lost their nationality contrary to their wills and would now like to have their Polish national- ity restored. Unfortunately, there is no procedure to this end. Some emigrants use the procedure in front of the voivod, who is authorised to establish whether a given individual has acquired and currently holds Polish citizenship, has forfeited it or has never held it. 4.2.3 Loss of Polish nationality Through an administrative decision, this authority can certify that someone holds Polish nationality following proceedings that take into account the principle of continuity of Polish nationality. 4.2.4 Dual nationality Dual and multiple nationality is allowed under Polish law. There are, however, certain nuances concerning the possibility of acquiring this status. Dual nationals by birth are never required to choose one nation- ality over the other (neither upon reaching the age of majority nor at any other time). Naturalisation procedures, by contrast, distinguish be- tween, on the one hand, individuals seeking to become naturalised Pol- ish citizens and, on the other hand, Polish citizens seeking to natura- lise in another state. The law in force favours the latter and Polish citi- zens can freely obtain other nationalities while retaining their Polish citizenship. Foreigners seeking to access Polish citizenship, however, may be required – at the discretion of relevant public authorities – to relinquish other nationality ties. One is thus tempted to use Sanford Levinson’s formulation to say that Polish law ‘tolerates political bigamy so long as the second political marriage follows, rather than precedes, acquiring [Polish] citizenship’ (cited in Schuck 2002: 72). q g [ ] p ( 7 ) Although nowadays multiple nationality as a legal status is tolerated in Poland, this has not always been the case in the past. A ban on dual nationality was a guiding principle of the 1920 and 1951 Acts on Polish Nationality. This historical fact, as well as the wording of art. 2 of the 1962 Act on Polish Nationality, seems responsible for some confusion. The article states that ‘according to the law, a Polish national cannot be recognised as a national of another country’. The Highest Administra- tive Court interpreted this provision as a prohibition of dual or multi- ple nationality in one of its judgments.30 Such an interpretation seems, however, inconsistent with the wording of the very article as it uses the term ‘recognised’ (uznawany). This is obviously not the same as stating a prohibition of having multiple nationalities, and the 1962 Act re- frains from restating an explicit prohibition of multiple citizenship in earlier regulations. Therefore, the view that Polish law generally prohi- NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 133 bits dual nationality does not prevail and is – correctly – not shared by most legal scholars (Jagielski 1998; Ramus 1968; Mincer-Jas´kowska 1998). Art. 2 should be interpreted as a rule resolving a very particular problem, which arises in domestic law when a Polish national holds several nationalities. 4.2.4 Dual nationality Accordingly, a dual or multiple national will al- ways be treated as a Polish national by Polish public authorities inside the Polish territory as well as abroad. A multiple citizenship holder cannot claim rights that stem from an additional nationality status or avoid obligations stemming from Polish nationality or ask to be treated as a foreigner (Ramus 1968). In other words, foreign citizenship ties are considered irrelevant in front of Polish public authorities and he or she is treated as if he or she was Polish only. This reading of art. 2 also coincides with the specific rules governing acquisition of Polish nation- ality as thus far described. 4.2.5 Act on the Polish Ethnicity Card As with other countries in the Central and Eastern European region, Poland has recently passed a law giving certain benefits to members of its kin minorities living abroad. The Act on the Polish Ethnicity Card31 was passed in September 2007. The first bill of this law was discussed in parliament almost ten years earlier. The scope of the card is re- stricted to ethnic Poles who are not Polish nationals, but are nationals of one of the fifteen successor states of Soviet Union republics (art. 2.2). The Polish Ethnicity Card confirms a person’s belonging to the Polish nation and the right to benefits provided for by the Act (art. 3). It may be issued to a person who declares himself or herself to be a member of the Polish nation and who fulfils the conditions specified in the statute. These conditions are of mixed ethnic and cultural char- acter and it should be noted that the ethnic criterion (descent) is not obligatory. Instead of proving Polish descent a certificate issued by one of the non-governmental organisations operating in these countries (specified in the Prime Minister’s decree, obwieszczenie) may be pre- sented, confirming active engagement in activities concerning Polish language and culture or other involvement in Polish national minority communities for at least three years prior to the application. In any event, it is necessary to prove attachment to ‘Polishness’ through hav- ing at least basic knowledge of the Polish language, which must be considered by the applicant to be his or her native language, as well as knowledge and practice of Polish traditions and customs (art. 2.1). The law provides for different benefits, which include easier access to the labour market (holders of the card do not need a work permit and they can undertake economic activity in Poland on the same basis as Polish nationals), and some educational, cultural and health bene- AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 134 fits. In order to enter Polish territory, the holder of a Polish ethnicity card still has to apply for a visa, but may be exempted from the fee for a national visa (or this fee may be refunded) (arts. 5-8). The actual meaning of these benefits is different depending on whether a holder of the card is a citizen of a European Union country (in this case Lat- via, Lithuania, Estonia) or not. 4.2.5 Act on the Polish Ethnicity Card Third country nationals holding the Pol- ish ethnicity card enjoy free access to the labour market, free immedi- ate medical assistance and access to education at tertiary level, free en- try to state-run museums and 37 per cent travel reduction for train transport. For EU citizens, only the last two benefits are of any signifi- cance (Jagielski & Pudzianowska 2008). (J g ) The Act on the Polish Ethnicity Card was not easy to draft. It took over ten years of discussions as there was a range of controversies re- garding the shape of the successive bills (see Jagielski & Pudzianowska 2008). Many commentators claimed that it was discriminatory, and that it was dangerous to create a privileged group of people having spe- cial rights in Poland who are residents of foreign countries. This argu- ment was not specific to Poland and was present in all countries that adopted a kin minority legislation (see Kova´cs & To´th, Kusa´, Medved and Smilov & Jileva in this volume). Moreover, a number of practical arguments against the card were discussed during the parliamentary debates (see Go´rny, Grzymała-Kazłowska, Korys´ & Weinar 2003). These concerned high costs, the danger of abuse of the card by economic mi- grants, etc. Even though domestic controversies were extensive, it can be argued that the Polish Ethnicity Card did not produce many reac- tions on the international arena (unlike, for example, the Hungarian Status Law of 2001). The Belarusian government’s negative reaction is the exception to the rule that can be easily explained. On the one hand, apart from Ukrainians, Belarusian citizens are expected to be the main recipients of the Polish ethnicity cards. On the other hand, the negative reaction of Belarus is in line with the more general Belarusian policy towards the Polish minority in Belarus, marked as it is by a variety of more or less openly demonstrated conflicts and repressions from the Belarusian authorities. Interestingly enough, preparations for a Belaru- sian Ethnicity Card have recently been announced. 4.3 The unresolved debate (1999-2001) Changes in the Polish nationality law have been planned since 1989, but only selected goals have been achieved to date. The most important act – the Act on Polish Nationality – remains unchanged, although sev- eral bills on Polish nationality have already been proposed and dis- cussed in the Polish Parliament. Work on nationality legislation was NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 135 particularly intensive during the third parliamentary term (1997-2001), when post-Solidarity parties held a majority in the Polish Parliament. In the fourth parliamentary term (2001-2005), when post-communist parties held the majority, work on nationality legislation was postponed (see Go´rny, Grzymała-Kazłowska, Korys´ & Weinar 2003) only to reap- pear on the agenda during the fifth and sixth legislative terms under a majority of post-Solidarity parties (2005-2007 and 2007-present). Nevertheless, works on the Act on Polish Nationality did not enter the phase of parliamentary readings and discussions. Apart from the ideological rationale for introducing new legislation on nationality with democratic transition in all CEE countries, new so- cial currents require new solutions for the law on Polish nationality. For example, it is necessary to establish clear rules concerning rein- statement of Polish nationality for people who were deprived of it in various ‘historical contexts’. Moreover, issues relating to the repatria- tion of people of Polish descent from the territory of the ex-USSR again became prominent in the 1990s. Last but not least, increasing immi- gration to Poland requires that rules on naturalisation be reevaluated and made less discretionary. The repatriation problem was solved legislatively with the introduc- tion of the Repatriation Act in 2000. Seven years later, the Act on the Polish Ethnicity Card introduced some privileges for members of Pol- ish kin minorities. The enactment of this latter statute can also be re- garded as a partial solution to the problem of the reinstatement of Pol- ish nationality, as part of the diaspora considers it as a symbolic confir- mation of their belonging to the Polish nation. However, a new Act on Polish nationality is still missing. Its preparation started in the late 1990s and three bills on Polish nationality have been proposed. The most recent one, which was submitted by a working group comprising representatives of various parties in 2000, combined the two earlier proposals. 4.3 The unresolved debate (1999-2001) Certain issues included in the latest bill demonstrate the po- litical background and aims behind the formulation of a new Act on Polish Nationality and are worth noting. In the bill – as in all acts on Polish nationality – the basic principle for being recognised as a Polish citizen was the ius sanguinis principle. This newest bill foresaw special procedures for people who intended to reacquire their Polish nationality. In fact, as stated in the introduction to the bill, the problem of ‘reinstating Polish nationality to all those who have the right to it’ was considered very important by the bill’s authors. The proposed reinstatement procedure would have applied to those who had lost Polish nationality on the basis of previous Acts on Polish Nationality (1920, 1951, 1962) and whose relinquishing of Pol- ish nationality had not been ‘fully voluntary’.32 Applicants’ entitlement to this procedure would not have been conditional on living perma- AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 136 nently in Poland. The bill also proposed a procedure for granting na- tionality to a particular group of people of Polish origin: Polish veter- ans of the Second World War. As far as the naturalisation procedure is concerned, the Bill on Polish Nationality added to the requirement re- garding length of stay contained in the 1962 Act, criteria referring to the applicants’ level of social, economic and cultural integration into Polish society. They included adequate knowledge of the Polish lan- guage, evidence of the applicants’ ability to sustain themselves in Po- land and absence of a criminal record and of behaviour that does not violate loyalty towards the Polish state. These criteria were intended to set more precise and thereby less discretionary criteria for naturalisa- tion in Poland. The focus of discussions and political and legislative action regard- ing Polish nationality and related matters has been undoubtedly on the Polish diaspora and Polish emigrants, with immigration and naturali- sation being of secondary importance. The parliamentary debates in 1999-2001 on such proposals were fairly uncontroversial (Go´rny, Grzy- mała-Kazłowska, Korys´ & Weinar 2003) and can thus be considered in- dicative of a consensual approach to nationality matters observed in the contemporary Polish political scene. The reasons for the lack of con- sensus on the bill, which led to its withdrawal from the parliamentary agenda, are not clear. 4.3 The unresolved debate (1999-2001) It seems, however, that the issue dividing the Pol- ish Parliament was the problem of explicit acceptance of dual national- ity (Go´rny, Grzymała-Kazłowska, Korys´ & Weinar 2007), which was more a problem of a symbolic nature, rather than a legal one. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 137 three procedures: conferment, acknowledgement and spousal acquisi- tion. Finally, we also provide data on repatriation to Poland for the peri- od 1997-2006. 4.4.1 Comment on data Data on acquisitions of Polish nationality have just recently been inte- grated into the main statistical system and database on foreigners. For the years 1992-2001 we will therefore focus on acquisitions through only one procedure, namely the conferment of Polish nationality, for which nationwide data are available for the 1990s. To make our de- scription for that period more precise and informative, we have en- riched it with fragmented data regarding other procedures. These data include information on foreigners who were naturalised through ac- knowledgement and marriage procedures in 199733 and results of re- search carried out in the Warsaw voivodeship in 1999, when data on applicants for Polish nationality in 1989-1998 based on the three most important procedures – acquisition through conferment, acknowledge- ment and marriage – were collected.34 When describing later years (2002-2006) we refer to acquisitions of Polish nationality through all a We include the general category ex-USSR, since for as many as 804 persons the statistics do not indicate from which former Soviet Union republic they originate. 4.4.2 Naturalisations: Conferment and two other procedures In 1992-2001, two consecutive Polish presidents granted Polish nation- ality to 8,979 people under the conferment procedure. In this period, the biggest national group of newly admitted Polish citizens were Ger- mans (16 per cent). Other important groups were Israelis (8 per cent), Canadians (8 per cent), Bulgarians (5 per cent) and Americans (4 per cent) (see Table 4.1). However, as much as 20 percent of the applicants originated from the former Soviet Union. Table 4.1 Foreigners granted Polish nationality by conferment in 1992-2001 and by conferment, acknowledgement and spousal acquisition in 2002-2006, by (former) nationality Nationality Acquisitions by conferment procedure in 1992-2001 All b acquisitions in 2002-2006 Number of persons % Number of persons % German 1,416 16 328 4 Israeli 726 8 469 5 Canadian 676 8 184 2 Bulgarian 490 5 165 2 American 381 4 149 2 The former Soviet Union, including the Baltic statesa 1,778 20 4,392 51 Other 3,512 39 2,925 34 Total 8,979 100 8,612 100 a We include the general category ex-USSR, since for as many as 804 persons the statistics do not indicate from which former Soviet Union republic they originate. b Does not include acquisitions by repatriation, which are demonstrated in Table 4.2. Sources: Ke˛pin´ska 2007; data provided by the Polish President’s Chancellery Table 4.1 Foreigners granted Polish nationality by conferment in 1992-2001 and by conferment, acknowledgement and spousal acquisition in 2002-2006, by (former) nationality In the 1990s, most applicants for Polish nationality originated from countries constituting traditional areas of destination for Polish emi- grants: Germany, the US, Canada and various countries in Western Europe (e.g. France). The intensive Polish-Bulgarian student exchanges during the communist era resulted in many Polish-Bulgarian mar- riages and complicated nationality matters for their families. It is evi- dent that the conferment procedure has been used by successive Presi- dents to restore Polish nationality to Polish emigrants who had lost it. This also explains the high number of Israelis ‘naturalising’ in Poland. 138 AGATA GO´ RNY AND DOROTA PUDZIANOWSKA The number of ex-USSR citizens naturalising via the conferment procedure in Poland in the 1990s and 2000s is also quite high. It does not fully reflect, however, the predominance of ex-Soviet citizens in contemporary migration to Poland, since they were particularly likely to use the acknowledgement procedure in the 1990s. 4.4.2 Naturalisations: Conferment and two other procedures This was due to the requirement to relinquish their original nationality in accordance with bilateral conventions on the prevention of dual nationality, which were still in effect between Poland and countries of the former Soviet Bloc in the 1990s. As these persons were generally stateless, they qua- lified for the acknowledgement procedure. In 1989-1998, in the War- saw voivodeship, 76 per cent of ex-USSR citizens (stateless persons at the moment of applying) used the acknowledgement procedure and ci- tizens of this region constituted 94 per cent of all those applying for naturalisation under this procedure. The importance of applicants for Polish nationality originating from the ex-USSR is more visible in the data on acquisitions for 2002-2006, where all three procedures are included. Newly naturalised Polish citi- zens from this area constitute as much as 51 per cent of the total, with the Ukrainians being the leading group (27 per cent of the total num- ber of acquisitions and 54 per cent of the acquisitions by nationals of the former Soviet Union). Immigrants from the former Soviet Union have constituted the main segment of the ‘new wave’ of immigration to Poland, which began in the late 1980s, and these migrants began qualifying for naturalisation in the second half of the 1990s. The sub- sequent termination of bilateral conventions on the prevention of dual nationality with some Soviet Bloc countries allowed more and more ex- USSR citizens to use the conferment procedure. The chart showing the number of acquisitions of nationality by con- ferment in the whole period analysed here shows a roughly u-shaped curve (see Figure 4.1), so long as we exclude the latest figures for 2006. The highest annual numbers registered were 1,522 (in 1992), 1,791 (2004) and 2,625 (2005), whereas two among the three smallest annual figures (679 and 555) occurred in the mid-1990s (in 1996 and 1997), and the third (662) in 2006. The ‘boom’ of naturalisations re- gistered at the beginning of the 1990s was caused mainly by ‘early re- conferments’ of Polish nationality. For example, in the 1992-1995 peri- od, over one quarter of the people granted Polish nationality were Ger- man citizens, probably many or most of whom had lost their Polish na- tionality in the past. The dramatic increase (56 per cent) in the number of acquisitions in 1998 can be partly explained by factors described above. 4.4.2 Naturalisations: Conferment and two other procedures Among them, the increase in the number of applications by Israelis (and other Polish emigrants) seems to be important. The number of ‘naturalising’ Israe- lis rose in 1998 after President Kwas´niewski’s aforementioned promise NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 139 Figure 4.1 Acquisitions of Polish nationality by conferment (1992-2006) 1,522 751 679 555 871 975 756 988 1,471 1,791 2,625 834 1,036 662 1,000 0 500 1,000 1,500 2,000 2,500 3,000 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Sources: Kępińska 2007; data provided by the Polish President's Chancellery Sources: Kępińska 2007; data provided by the Polish President's Chancellery of a ‘broad and uncomplicated restoration’. In 1997, the President granted Polish nationality to only nineteen Israelis, whereas in 1998, the respective number was six times higher (114 persons). Then, as mentioned above, a gradual increase in the numbers of ex-USSR citi- zens using the conferment procedure also contributed to the increase in acquisitions. The sharp decrease in the number of acquisitions of Polish national- ity by conferment in 2006 deserves some explanation. Its main cause is the change of the Head of State: Lech Kaczyn´ski, who is close to the right-wing parties, replaced Aleksander Kwas´niewski, who came from the main post-communist party. In 2005 – the last year of the Kwas´- niewski presidency – many cases were processed faster than usual in order to be completed before his term came to an end. Thereafter, Pre- sident Kaczyn´ski appeared to be reluctant to grant Polish nationality, at least at the beginning of his term. Data on the conferment procedure certainly only describe part of the phenomenon of naturalisations in Poland though, in our opinion, they provide an accurate snapshot of national groups interested in Polish nationality, especially in the 2000s. In the 1990s, the number of appli- cants for the acknowledgement procedure was slightly higher than for the conferment procedure. In the 2000s, the first of these procedures almost totally lost its importance due to the abrogation of conventions on the prevention of dual nationality. In fact, between 2002 and 2004, AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 140 fewer than 200 people were naturalised through any procedure other than conferment. The remaining procedure, i.e. spousal acquisition, played a second- ary role in the 1990s and is still of only marginal importance. 4.4.2 Naturalisations: Conferment and two other procedures In 1997, for example, only 52 foreign women used this mode of acquisition. In the Warsaw voivodeship during the period 1992-1998, the number was 73 women. At the same time, the annual numbers of mixed marriages in Poland were much higher – between 3,000 and 3,500 were con- cluded in the 1990s and 2000s, respectively. Spousal acquisition gained more importance after 1999 when it started applying not only to women, but also to men, and when conditions regarding applica- tions became more ‘reasonable’. It is likely to increase in importance, since ex-USSR citizens no longer have to relinquish their foreign na- tionality upon naturalisation in Poland. In 2002, for example, from among 3,552 mixed marriages concluded in Poland, over 40 per cent involved citizens of post-Soviet countries. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 141 Table 4.2 Repatriation visas to Poland in 1997-2006, by repatriates’ previous country of residence Country of previous residence 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Total 316 281 278 662 804 613 301 269 252 239 Azerbaijan - - - - - - - - - 1 Belarus - 10 15 45 140 127 43 39 30 25 Czech Republic - - - - 2 4 1 1 1 1 Georgia - - - - - 1 3 - 3 3 Kazakhstan 316 245 172 361 216 194 156 122 155 125 Lithuania - - 11 16 20 3 - 1 1 1 Latvia - 1 1 10 - - - - - - Moldova - 1 2 10 9 5 2 - 2 1 Russian Federation - 7 8 10 36 31 11 35 32 40 Ukraine - 15 69 210 381 245 77 56 23 27 Uzbekistan - 2 - - - 2 8 15 5 14 Source: Central Statistical Office, from Ke˛pin´ska (2007: 95) According to the available fragmented data on acquisitions of Polish nationality, naturalisation remains a limited phenomenon in Poland. In the 1990s and 2000s, the annual numbers of persons granted Pol- ish nationality did not exceed 3,000, although the beginning of the 1990s produced a visible increase in naturalisations. For example, in the Warsaw voivodeship, 26 and 80 applicants had been granted Polish nationality in 199035 and 1991, respectively, whereas in 1992, the num- ber rose to 203, with no decrease evident in subsequent years. 4.4.3 Repatriation The repatriation procedure was introduced amidst much discussion. On the one hand, speculation about thousands of people of Polish des- cent (not always genuine) who would take advantage of the repatriation procedure was aired in the media and in parliament. On the other hand, virtually nobody dared to question Poland’s obligation to take care of its exiles in faraway Asiatic republics of the former Soviet Un- ion. The controversies surrounding repatriation influenced the final shape of the Repatriation Act of 2000 by limiting repatriation to a very small group of people. As a rule the repatriation procedure only applies to persons who have lived permanently in certain Asiatic republics prior to 2000. Thus, it is designed for those who did not manage to re- patriate themselves in the 1940s and 1950s. The requirement that a would-be repatriate has to be invited by an official institution or a pri- vate person further limits the accessibility of this procedure. y In all, during the 1997-2006 period, only 4,015 repatriation visas were issued and 5,293 repatriates arrived via the repatriation program (Ke˛pin´ska 2004, 2007). The actual number of people who acquired Polish nationality through the repatriation procedure is somewhere be- tween these two figures, since new arrivals include non-Polish mem- bers of repatriate families. From 2001 to 2006, 2,935 people acquired Polish nationality as repatriates. As demonstrated in Table 4.2, people from Kazakhstan represent the majority among repatriates (based on a count of visas issued). Residents of other former republics of the Soviet Union are in the minority and this relationship will persist due to the structure of the Repatriation Act. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 4.5 Conclusions There have been surprisingly few changes in Polish legislation on na- tionality since 1962. The post-communist Third Republic of Poland did not pass a new law on nationality despite expectations. All this does not mean, however, that nothing changed at the level of practice re- garding Polish nationality. Political attitudes also changed, a fact that is exemplified by President Kwas´niewski’s declaration in 1998 to return Polish nationality to those who had lost it during the communist era. In general, the characteristic feature of legislation on Polish national- ity is discretionary power given to public officials in making decisions. Consequently, changing policy in nationality matters does not necessa- rily require changes in the written law. At the same time, uncovering the mechanisms of this policy in practice requires looking beyond the written law. Even though the 1962 Act on Polish Nationality makes ac- quisition of Polish nationality conditional only on the duration and AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 142 type of an applicant’s stay in Poland, civil servants also take into ac- count other factors referring to a foreigner’s social and cultural integra- tion as well as his or her family and financial situation. Poles living abroad and/or returning to Poland and their right to Pol- ish nationality were the focus of the debate on reforms to Polish na- tionality law in the 2000s. It is important to remember that only some of the applicants for Polish nationality are immigrants. A visible pro- portion of new citizens – around half in the 1990s – are people who had lost Polish nationality under communism and repatriates. This is undoubtedly a temporary phenomenon. The proportion has already started to decline and will continue to do so as the pool of individuals interested in reacquiring their Polish nationality wanes and as the number of ‘typical immigrants’ who qualify for acquisition of Polish nationality, which is already relatively high today, gradually grows. Polish accession to the European Union boosted discussions on im- migration to Poland in the context of the shift of the EU border to Po- land’s eastern border. However, it did not affect the discourse on Polish nationality, which was absent from the political and public agenda in pre- and post-accession periods. 4.5 Conclusions This absence was probably due to the post-communist majority in the Polish Parliament between 2001 and late 2005, which was not eager to tackle nationality (and other) issues pertaining to how to ‘deal with the communist past’. Works on legisla- tion relating to nationality issues were resumed in the fifth (2005- 2007) and sixth (2007-2011) legislative periods when the post-Solidar- ity parties had a majority in Parliament. It is, however, too early to ana- lyse the proceedings relating to the most recent Bill on Polish National- ity since it has not been yet discussed in Parliament. It can be also argued that another consequence of Polish accession to the EU is the visible increase in the interest in Polish nationality among diaspora members. The number of applications for certifica- tion/recognition of Polish nationality submitted to Polish consulates abroad, especially outside Europe, grew in the 2000s. In 2000, only 765 such applications were registered, whereas in 2004 their number reached 3,807. In 2000-2002, the largest number of applications came from Germany. In 2003, Argentina was first with 505 applications (Centre of Migration Research 2005).36 We are certainly not talking about acquisitions of Polish nationality, but about situations whereby people, usually descendants of Polish emigrants, who are entitled to ci- tizenship but are not registered citizens (not having a national registry number and passport, possibly due to a lack of interest on their part), take advantage of this right.37 All in all, it seems that interest in Polish nationality, which was rather weak in the 1990s and at the beginning of the 2000s, has been growing recently. Moreover, it is likely to further increase in light of on- NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 143 going immigration to Poland and the fact that Polish nationality has entailed European Union citizenship since 2004. www.dziennik-ustaw.pl (in Polish) www.dziennik-ustaw.pl (in Polish) www.coe.int; www.uric.gov.pl 4.5 Conclusions Chronological list of citizenship-related legislation in Poland Date Legislative act Content Source 1920 Act on Nationality of Poland Regulates modes of acquisition and loss of Polish nationality www.dziennik-ustaw.pl (in Polish) 1938 Act on Deprivation of Polish Nationality Regulates modes of loss of Polish nationality www.dziennik-ustaw.pl (in Polish) 1946 Decree Concerning Exclusion of Persons of German Ethnicity from Polish Society Defines the framework for the exclusion and eventual deportation of persons of German ethnicity living on the Polish territory after the Second World War www.dziennik-ustaw.pl (in Polish) 1946 Act on Polish Nationality of Persons of Polish Ethnicity Inhabiting the Regained Territories Defines the conditions for entitlement to Polish nationality for persons living in North-Western Poland (territories belonging to Germany before the Second World War) www.dziennik-ustaw.pl (in Polish) 1947 Act on Polish Nationality of Persons of Polish Ethnicity Inhabiting the Former City of Gdan´sk Defines the conditions for entitlement to Polish nationality for persons living in the former city of Gdan´sk (Danzig) www.dziennik-ustaw.pl (in Polish) 1951 Act on Polish Nationality Regulates modes of acquisition and loss of Polish nationality www.dziennik-ustaw.pl (in Polish) 1956 Decree of the Council of the State No. 37/56 Concerning the Permission for German Repatriates to Renounce Polish Nationality (unpublished) Provides a fast track for the renunciation of Polish nationality for people leaving for Germany 1958 Decree of the Council of the State No. 5/58 Concerning the Permission for People Leaving for Israel to Renounce Polish Nationality (unpublished) Provides a fast track for the renunciation of Polish nationality for people leaving for Israel 1962 Act on Polish Nationality Regulates modes of acquisition and loss of www.coe.int; www.uric.gov.pl www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) www.legislationline.org www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) Defines the framework for the repatriation of people of Polish descent from the Asiatic republics of the ex-USSR 2000 Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving permission to renounce one’s Polish nationality, as well as specimen Defines the documents to be submitted and the exact procedures for the acquisition of nationality by conferment AGATA GO´ RNY AND DOROTA PUDZIANOWSKA AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 144 Date Legislative act Content Source Polish nationality (in Polish); www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 1997 Amendment of the Act on Polish Nationality Extends required time of residence in Poland (by five years) by introducing the clause that only stay on the basis of permanent residence permit be counted 1997 Constitution of the Republic of Poland (excerpts) Protects citizenship status: ‘A Polish citizen shall not lose Polish citizenship except by renouncing it’ (art. 34.2) www.legislationline.org 1998 Amendment of the Act on Polish Nationality Introduces equality in treatment of husbands and wives of Polish citizens with regard to acquisition of Polish nationality; removes all possibilities of losing Polish nationality against person’s will; makes resignation from Polish citizenship fully dependent on the will of its holder 1999 Act on Terminating the Convention, Being Effective in Polish- Belarusian Relations, between the Polish People’s Republic Government and the USSR Government Concerning the Prevention of Cases of Dual Nationality, signed in Warsaw on 31 March 1965 Expresses Poland’s will to terminate the Convention on Prevention of Dual Citizenship in force between Poland and Belarus www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 1999 Act on Terminating the Convention, Being Effective in Polish-Czech Relations between the Polish People’s Republic Expresses Poland’s will to terminate the Convention on Prevention of Dual Citizenship in force www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) www.legislation.org; www.uric.gov.pl (in Polish); www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) www.poland.gov.pl (in Polish, Russian and other languages) AGATA GO´ RNY AND DOROTA PUDZIANOWSKA AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 146 Date Legislative act Content Source certificates and applications 2001 Amendment of the Act on Polish Nationality Introduces changes relating to registration of acquisitions and losses of Polish nationality; removes rules applying to repatriation procedure 2003 Amendment of the Act on Polish Nationality Introduces procedural changes 2005-2007 Amendments of the Act on Polish Nationality Introduce minor changes resulting partly from the accession of Poland to the EU 2007 Act on Polish Ethnicity Card Defines the conditions for entitlement to the Polish Ethnicity Card; introduces certain benefits to members of Polish kin-minorities in fifteen post-Soviet states including easier access to the labour market and some educational, cultural and health benefits www.poland.gov.pl (in Polish, Russian and other languages) NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 145 Date Legislative act Content Source on Dual Nationality, signed in Warsaw on 17 May 1965 1999 Act on Terminating the Convention between the Polish People’s Republic and the Mongolian People’s Republic Concerning Regulations on Dual Nationality, signed in Ulan Bator on 23 May 1975 Expresses Poland’s will to terminate the Convention on Prevention of Dual Citizenship in force between Poland and Mongolia www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 1999 Act on Terminating the Convention, Being Effective in Polish-Slovak Relations between the Polish People’s Republic and the Czechoslovak Socialistic Republic Concerning Regulations on Dual Nationality, signed in Warsaw on 17 May 1965 Expresses Poland’s will to terminate the Convention on Prevention of Dual Citizenship in force between Poland and Slovakia www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 1999 Act on Terminating the Convention, Being Effective in Polish- Ukrainian Relations, between the Polish People’s Republic Government and the USSR Government Concerning Prevention of Cases of Dual Nationality, signed in Warsaw on 31 March 1965 Expresses Poland’s will to terminate the Convention on Prevention of Dual Citizenship in force between Poland and Ukraine www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 2000 Repatriation Act Defines the framework for the repatriation of people of Polish descent from the Asiatic republics of the ex-USSR www.legislation.org; www.uric.gov.pl (in Polish); www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) 2000 Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving permission to renounce one’s Polish nationality, as well as specimen Defines the documents to be submitted and the exact procedures for the acquisition of nationality by conferment www.dziennik-ustaw.pl (in Polish); www.abc.com.pl (in Polish) Defines the documents to be submitted and the exact procedures for the acquisition of nationality by conferment 4 Act on Nationality of Poland, Journal of Law 44, 1920, 44. Notes 1 It is beyond the scope of this chapter to discuss the complex relation between ethnicity and nationality/citizenship. Though following, for example, Thomas Hylland Eriksen (1999: 35), it is worth mentioning that the distinguishing mark of nationalism (when talking about nationality he refers to nationalism) is by definition its relation to the state. A nationalist holds that political boundaries should be coterminous with cultural boundaries, whereas many ethnic groups do not demand command over the state. 1 It is beyond the scope of this chapter to discuss the complex relation between ethnicity and nationality/citizenship. Though following, for example, Thomas Hylland Eriksen (1999: 35), it is worth mentioning that the distinguishing mark of nationalism (when talking about nationality he refers to nationalism) is by definition its relation to the state. A nationalist holds that political boundaries should be coterminous with cultural boundaries, whereas many ethnic groups do not demand command over the state. 2 Repatriation Act, Journal of Law 160, 2000, 1118. 2 Repatriation Act, Journal of Law 160, 2000, 1118. 3 Act on the Polish Ethnicity Card, Journal of Law 180, 2007, 1280. In the official Eng- lish translation, the Polish Ethnicity Card is sometimes called the ‘Polish Charter’ and there can be nothing more misleading. It is important to note that the Polish Ethnicity Card in the title of the act refers to the document that is issued under the act to certain subjects who are not Polish citizens, but are citizens of certain states entitled to certain privileges within Polish territory. The card thus does not ascertain the identity of the subject and can be compared to a student card inasmuch as it serves as proof of certain entitlements under the act. In its symbolic function, the card is deemed to ascertain that its holder ‘belongs to the Polish Nation’ defined in ethnic terms in this statute (art. 3). 4 Act on Nationality of Poland, Journal of Law 44, 1920, 44. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND Moreover, some of them remained not only in Poland but also in various Polish political structures. p 16 Act on Polish Nationality, Journal of Law 28, 2000, 353 (version including amend- ments). 16 Act on Polish Nationality, Journal of Law 28, 2000, 353 (version including amend- ments). 17 Constitution of the Republic of Poland, Journal of Law 78, 1997, 483. 17 Constitution of the Republic of Poland, Journal of Law 78, 1997, 483. 18 Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving permission to renounce one’s Polish nationality, as well as specimen certificates and applications, Journal of Law 18, 2000, 231. 18 Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving permission to renounce one’s Polish nationality, as well as specimen certificates and applications, Journal of Law 18, 2000, 231. 19 They include conventions signed with the Soviet Union (1965), Czechoslovakia (1965), Bulgaria (1972), Mongolia (1975) and the German Democratic Republic (1975) (Albiniak & Czajkowska 1996). 19 They include conventions signed with the Soviet Union (1965), Czechoslovakia (1965), Bulgaria (1972), Mongolia (1975) and the German Democratic Republic (1975) (Albiniak & Czajkowska 1996). ( 975) ( j 99 ) 20 At the time of writing, only the Ukrainian government has not ratified the termination of the convention. 20 At the time of writing, only the Ukrainian government has not ratified the termination of the convention. 21 This is a guiding principle for the legislature, meaning that the preference in the nationality regime should be given to the ius sanguinis principle and that other principles should only play an auxiliary role. 22 This refers only to automatic change of nationality as a consequence of marriage. Polish nationality law does provide for facilitated naturalisation of foreign spouses of Polish nationals. 23 It should be noted that these conditions are defined differently in the recently adopted Act on the Polish Ethnicity Card. 24 These are: 1) a permit to settle (zezwolenie na osiedlenie sie˛); 2) a long-term resident’s EC residence permit (zezwolenie na pobyt rezydenta długoterminowego Wspo´lnot Eur- opejskich) as defined in the Act on Aliens (arts. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 147 Act on Polish Nationality, Journal of Law 5, 1951, 5 Act on Polish Nationality, Journal of Law 5, 1951, 25. Act on Polish Nationality, Journal of Law 10, 196 7 Act on the Exclusion of Persons of German Ethnicity from the Polish Society, Journal of Law 66, 1946, 404. 7 Act on the Exclusion of Persons of German Ethnicity from the Polish Society, Journal of Law 66, 1946, 404. 8 The exclusion involved forced resettlement from the Polish territory and the loss of property in Poland. 8 The exclusion involved forced resettlement from the Polish territory and the loss of property in Poland. 9 To be positively verified as Polish, a person had to prove his or her coming from a Polish family and express his or her desire to belong to the Polish nation. 10 Act on Polish Nationality of Persons of Polish Ethnicity Inhabiting the Regained Territories, Journal of Law 15, 1946, 106. 11 Act on Polish Nationality of Persons of Polish Ethnicity Inhabiting the Former Free City of Gdan´sk, Journal of Law 65, 1947, 378. 12 Decree of the Council of the State No. 37/56 of 1956 Concerning t German Repatriates to Renounce Polish Nationality, (unpublished). 12 Decree of the Council of the State No. 37/56 of 1956 Concerning the Permission for German Repatriates to Renounce Polish Nationality, (unpublished). 13 Decree of the Council of the State No. 5/58 of 1958 Concerning the P People Leaving for Israel to Renounce Polish Nationality, (unpublished) 13 Decree of the Council of the State No. 5/58 of 1958 Concerning the Permission for People Leaving for Israel to Renounce Polish Nationality, (unpublished). 14 In fact, few people took advantage of this procedure, as there were no appropriate guidelines for its implementation. 14 In fact, few people took advantage of this procedure, as there were no appropriate guidelines for its implementation. 15 It would be an oversimplification to look for origins of that action only in the anti- Semitic attitudes of the Polish elites and society. Not all Jews were forced to leave Poland. Moreover, some of them remained not only in Poland but also in various Polish political structures. 15 It would be an oversimplification to look for origins of that action only in the anti- Semitic attitudes of the Polish elites and society. Not all Jews were forced to leave Poland. AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 148 26 Attachment to the Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving the permit to resign from Polish nationality and specimen certificates and applications, Journal of Law 18, 2000, 231. 26 Attachment to the Ordinance of the President of the Republic of Poland concerning the procedure in cases of conferment or giving the permit to resign from Polish nationality and specimen certificates and applications, Journal of Law 18, 2000, 231. 27 The study on positive and negative decisions on applications for Polish nationality in the Warsaw voivodship in 1989-1998 shows that Polish origins may influence a positive decision on nationality (Go´rny 2001). 28 Cf. the resolution of the Highest Administrative Court of 9 November 1998, OPD 4/ 98, ONSA 1999, book 1, item 6. 29 Art. 11 ECN requires that decisions relating to the acquisition, retention, loss, recovery or certification of nationality contain reasons in writing; while art. 12 demands that these decisions be left open to an administrative or judicial review. Poland has signed, but not yet ratified the ECN. 30 Judgment of 28 December 1994, V SA 1507/94; see Mincer-Jas´kowska 1996. 31 Act on the Polish Ethnicity Card, Journal of Law 180, 2007, 1280. 32 They had not expressed their will to decline Polish nationality (deprivation on the basis of the Act of 1920) or they were ‘forced’ to relinquish Polish nationality (deprivation on the basis of the Acts of 1951 and 1962). 33 Data from regional departments for 1997 were collected in one ad hoc action. For 2002-2006, we do not have exact data on the acknowledgement and marriage procedures but only on the total for all three procedures. Moreover, we have separate data only for the conferment procedure, but not for the years 2005-2006. 34 The database of 1,483 applicants, among whom 1,314 were granted Polish nationality, was compiled on the basis of personal data files. 35 The number for 1990 may be slightly underestimated, as files were checked according to the year of application. We started from 1989 and it is likely that somebody applying prior to 1989 and who received Polish nationality in 1990 was not registered in our database. 36 By 18 November 2004, 259 applications had been submitted in Argentina. AGATA GO´ RNY AND DOROTA PUDZIANOWSKA 37 Data collected by Agnieszka Weinar in the research project entitled ‘New Poles, new Europeans – dual nationality among descendants of Polish emigrants in Argentina’. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 64, 65); and 3) right of permanent re- sidence (prawo stałego pobytu) as defined in the Act on the Entry into, Residence in and Exit from the Republic of Poland of Nationals of the European Union Member States and their Family Members (Journal of Law 144, 2006, 1043). 24 These are: 1) a permit to settle (zezwolenie na osiedlenie sie˛); 2) a long-term resident’s EC residence permit (zezwolenie na pobyt rezydenta długoterminowego Wspo´lnot Eur- opejskich) as defined in the Act on Aliens (arts. 64, 65); and 3) right of permanent re- sidence (prawo stałego pobytu) as defined in the Act on the Entry into, Residence in and Exit from the Republic of Poland of Nationals of the European Union Member States and their Family Members (Journal of Law 144, 2006, 1043). 25 In some cases, the period after which a permanent residence permit may be acquired is longer. For example, for people who have been granted subsidiary protection it is seven years, which means that to be eligible for naturalisation such a person has to wait at least twelve years (art. 64 of the Act on Aliens, Journal of Law 128, 2003, 1175). 25 In some cases, the period after which a permanent residence permit may be acquired is longer. For example, for people who have been granted subsidiary protection it is seven years, which means that to be eligible for naturalisation such a person has to wait at least twelve years (art. 64 of the Act on Aliens, Journal of Law 128, 2003, 1175). AGATA GO´ RNY AND DOROTA PUDZIANOWSKA Stola, D. (2000), Kampania antysyjonistyczna w Polsce 1967-1968. Warsaw: ISP PAN. Bibliography Albiniak, M. & A. Czajkowska (1996), Przepisy o obywatelstwie polskim 1919-1995. Torun´: Wydawnictwo ‘Dom Organizatora’. Centre of Migration Research, Warsaw University, Centre for International Relations (2005), ‘Wzrost liczby wniosko´w o potwierdzenie polskiego obywatelstwa’, Biuletyn Migracyjny 1. www.migration-news.uw.edu.pl. Eriksen, T.H. (1999), ‘Ethnicity, race and nation’, in M. Guibernau & J. Rex (eds.), The Ethnicity Reader: Nationalism, Multiculturalism and Migration, 33-42. Cambridge: Po- lity Press. Go´rny, A. (2001), ‘Polish citizenship in relation to concepts of integration and transna- tionalism’, in J.W. Dacyl (ed.), Challenges of cultural diversity in Europe, 217-253. Stock- holm: Centre for Research in International Migration and Ethnic Relations. Go´rny, A., A. Grzymała-Kazłowska, P. Korys´ & A. Weinar (2003), ‘Multiple citizenship in Poland’, ISS UW Working Papers, Migration Series 53. www.migracje.uw.edu.pl. Go´rny, A., A. Grzymała-Kazłowska, P. Korys´ & A. Weinar (2007), ‘Selective tolerance? Regulation, practice and discussion regarding dual nationality in Poland’, in T. Faist (ed.), Dual Citizenship in Europe: From Nationhood to Societal Integration, 147-169. Avebury: Ashgate. NATIONALITY REGULATIONS AND THEIR IMPLEMENTATION IN POLAND 149 Jagielski, J. (1998), Obywatelstwo polskie. Zagadnienia podstawowe. Warsaw: Wydawnictwa Prawnicze. Jagielski, J. (1998), Obywatelstwo polskie. Zagadnienia podstawowe. Warsaw: Wydawnictwa Prawnicze. Jagielski, J. (2000), ‘Podstawowe instytucje oraz kierunki zmian polskiego prawa o oby- watelstwie’, Newsletter of the Information Office of the Council of Europe 4-5: 98-108. Jagielski, J. (2000), ‘Podstawowe instytucje oraz kierunki zmian polskiego prawa o oby- watelstwie’, Newsletter of the Information Office of the Council of Europe 4-5: 98-108. 5 9 Jagielski, J. & Pudzianowska, D. (2008), Komentarz do Ustawy o Karcie Polaka. Warsaw: Wolters Kluwer. Ke˛pin´ska, E. (2004), ‘Recent trends in international migration. The 2004 SOPEMI Report for Poland’, ISS UW Working Papers, Migration Series 56. www.migracje.uw.edu. pl. Ke˛pin´ska, E. (2007), ‘Recent trends in international migration. The 2007 SOPEMI Report for Poland’, ISS UW Working Papers, Migration Series 29/87. www.migracje.uw.edu.pl. Łodzin´ski, S. (1998), ‘Polish citizenship – ethnic boundaries and the issue of citizenship in Polish society’, in B. Balla & A. Sterbling (eds.), Ethnicity, nation and culture. Cen- tral and East European Perspectives, 149-164. Hamburg: Kramer. Mincer-Jas´kowska, M. (1996), ‘Glosa do wyroku NSA z 28 grudnia 1994, V SA 1507/94’, Orzecznictwo Sa˛do´w Polskich 7-8: item 131. 7 3 Ramus, W. (1968), Prawo o obywatelstwie polskim. Warsaw: Wydawnictwo Prawnicze Schuck, P. (2002), ‘Plural citizenships’, in R. Hansen & P. Weil (eds.), Dual Nationality, Social Rights and Federal Citizenship in the US and Europe, 61-100. New York: Berghan Books. Stola, D. Bibliography (2000), Kampania antysyjonistyczna w Polsce 1967-1968. Warsaw: ISP PAN. 5 Kin-state responsibility and ethnic citizenship: The Hungarian case Ma´ria M. Kova´cs and Judit To´th The preference for the naturalisation of ethnic Hungarians has been considered a counterbalance to the troubled history of a nation artifi- cially split among various states and as a tool for preserving cultural identity in the twentieth century. The principle of ethnicity has been observed directly in nationality legislation and migration law through regulations for visa, residence and employment permits, and asylum status (To´th 1995). Due to the ideology of a ‘threatened Hungarian eth- nic identity’ the relationship between the social and economic integra- tion of migrants, migration law, naturalisation and citizenship has never been publicly discussed (Fullerton, Sik & To´th 1997). Hungarian authorities need not give reasons for refusing an application for natura- lisation and there is no legal remedy against a negative decision. This is justified by referring to the sovereign power of the state and, in cases of rejection, by a presumption of the applicants’ missing ethnic and cultural ties to Hungary. An extension of preference in naturalisation to European Union citizens was smoothly passed in 2003, partly be- cause of the supposed ethnic proximity of applicants in adjacent states.1 Provisions supportive of family unity in nationality law are widely accepted and so are the discretionary powers in naturalisation proceedings that determine who is to be allowed to join this rather homogeneous society (To´th 2005). On the other hand, there are some contentious components of the nationality regulations in contemporary Hungary. – Naturalisation and its precondition, the authorisation of permanent residence, are criticised as being too time-consuming and expen- sive, and the requirements for documentation as too bureaucratic. In other words, ethnic Hungarians, being the largest group of appli- cants, do not see themselves as preferential beneficiaries when it comes to the attitude of the authorities or to procedural provisions. – Moreover, certain privileges of Hungarian citizenship were extended to EU nationals and migrants under the scope of Community law in the accession process (To´th 2004a). p ( 4 ) – The role of naturalisation in the process of migrant integration has been unclear. While the applicant is required to be highly integrated in a cultural, economic and social sense, integration programmes MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 152 do not exist at all, which means that integration can only be achieved by individual effort. 5 Kin-state responsibility and ethnic citizenship: The Hungarian case The applicant must also not endanger public order and is investigated in this regard in various ways. – Nationality as a basket of various rights and obligations is basically considered by the general public as a historical, cultural, ethnic and emotional issue without awareness of its existing legal and norma- tive status and its neutral significance in a democratic rule-of-law system. For this reason, public opinion is strongly divided into ‘nor- mativists’ and ‘nation builders’, representing different standpoints concerning voting rights, principles for the acquisition of national- ity, dual citizenship and never-ending citizenship for emigrants in the diaspora. p – As for ethnic Hungarians, the right to have the family and given name and the name of the applicants’ prior place of residence and birthplace in their original ethnic language was finally introduced in amendments related to the naturalisation and registry process.2 This causes certain confusion in the registration of foreigners and nationals since registration is, in theory, based on the authenticity and unaltered nature of existing identity documents. Moreover, this right is exclusively reserved for ethnic Hungarians; it does not apply to the non-Hungarian version of names of, for instance, naturalised refugees or stateless migrants belonging to a linguistic minority, which would be registered in the dominant language in their coun- tries of origin. – As for ethnic Hungarians, the right to have the family and given name and the name of the applicants’ prior place of residence and birthplace in their original ethnic language was finally introduced in amendments related to the naturalisation and registry process.2 This causes certain confusion in the registration of foreigners and nationals since registration is, in theory, based on the authenticity and unaltered nature of existing identity documents. Moreover, this right is exclusively reserved for ethnic Hungarians; it does not apply to the non-Hungarian version of names of, for instance, naturalised refugees or stateless migrants belonging to a linguistic minority, which would be registered in the dominant language in their coun- tries of origin. 5.1 History of Hungarian policies on nationality since 1945 Although the first Act on Hungarian Nationality (1879) became in- creasingly restrictive through amendments adopted during wars, its ius sanguinis principle has remained dominant up to the present day. This Act was in force until 1948. The history of Hungarian policies on na- tionality since 1945 can be divided into the following periods: 1945-1948: The Armistice Agreement concluded in Moscow (1945)3 annulled all the modifications of nationality that had come about as a result of the territorial changes of the Hungarian state between 1939 and 1945. Millions of former Hungarian citizens who ended up under the jurisdiction of neighbouring states lost their Hungarian nationality. The Peace Agreement fixed the borders of the Hungarian state along the frontiers as they had existed on the last day before the war began.4 Between 1945 and 1948 temporary regulations on nationality consid- ered all those residing in Hungary in 1945 as nationals except for those holding the nationality of another state. Bilateral agreements on popu- lation exchange initiated by Czechoslovakia and the expulsion of Ger- KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 153 mans resulted in the deprivation of nationality for those falling under these measures.5 Individuals who had not returned to Hungary follow- ing the conclusion of the war were deprived of their citizenship and, between 1946 and 1948, their property was confiscated.6 Finally, the ci- tizenship status of communists who had fled Hungary during the in- terwar years was settled.7 y 1948-1956: In 1946 a reform of the legal status and civil rights of children born out of wedlock established their full equality,8 but only the new Act on Hungarian Nationality of 19489 provided a coherent le- gal framework for the acquisition of nationality through changes in fa- mily and personal status. The Act provided for the equal treatment of children born out of wedlock and stipulated that all nationals residing abroad should be registered, without, however, creating techniques for registration in the absence of consular relations. The Act recognised the pending Hungarian nationality of undocumented persons who had been residing in Hungary for a given number of years. 5.1 History of Hungarian policies on nationality since 1945 1956-1989: This period witnessed the emancipation of spouses on the basis of the New York Convention of 1957 on married women,10 the principles of which were inserted into the third Act on Nationality adopted in 1957.11 The executive rules of the Act were not published and were implemented by confidential order, such as the one requiring emigrants to renounce their nationality and social insurance rights. Following the 1956 revolution and the mass emigration it triggered, a broad amnesty was proclaimed for returnees and a registry of nationals permanently abroad was established.12 1989-1993: After 1989, Hungary started reforms to establish the rule of law and constitutionalism. In 1989 the prohibition of deprivation of nationality was regulated in the modified Constitution.13 At the same time the nationality of expatriate nationals who had been deprived of their nationality arbitrarily was restored upon request.14 The Geneva Convention of 195115 inspired the preferential naturalisation of refugees that was inserted into the nationality law. The fourth Act on Nationality passed in 1993 made preconditions for naturalisation more restrictive but preferences based on ethnic and family ties were intended to com- pensate for this.16 Between 1989 and 1993 Hungary terminated bilat- eral agreements with former socialist states that excluded dual citizen- ship. 1994-2005: This period is marked by Hungary’s accession efforts to the EU and by political debates on the status of ethnic Hungarians liv- ing outside Hungary’s borders. During this time the Act on Nationality was amended three times,17 due to the ratification of the European Convention on Nationality (1997) and the UN Convention on Stateless Persons (1954).18 Eligibility for preferential naturalisation was extended to EU citizens and a super-preference was adopted in favour of ethnic 154 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH Hungarians in the shadow of the upcoming Schengen restrictions (To´th 2003). In the period under discussion there were three major breaks in ba- sic principles. Although from 1879 onwards Hungary tolerated multi- ple nationality, between 1946 and 1989 the main rule was the exclu- sion of dual citizenship through bilateral agreements with socialist states. Mixed couples had to choose one of their nationalities for their child. After 1989, the modified Constitution abolished the arbitrary de- privation of nationality. International principles of human rights rele- vant to nationality were inserted into the law, while a growing circle of preferences was defined as a core element of domestic legislation. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 155 unity (with respect to legal status) by various preferences for the natur- alisation of spouses and (adopted) minors. Refugees and stateless per- sons are also given priority for admission to citizenship. Hungarian regulations are special in granting preferential treatment to persons who are former Hungarian nationals and to ethnic Hungarians in the process of acquiring citizenship. p q g p Hungary tolerates multiple citizenship, and the state strives to create rules and enter into agreements to avoid conflicts between different le- gal systems. A person acquiring Hungarian nationality by naturalisa- tion need not renounce his or her prior citizenship. The circle of bilat- eral agreements and the European Convention regulate several legal relationships with respect to persons of multiple citizenship (e.g. with regard to military service or taxation). Furthermore, those having an- other citizenship are entitled to the same rights and obligations in the territory of Hungary as other nationals, with the exception of employ- ment in the police or security services (To´th 2004b). On the other hand, the principle of genuine link19 requires a factual, effective and close relationship between Hungary and the applicant for naturalisa- tion or other modes of acquiring citizenship, regardless of his or her existing other citizenship. However, for those in possession of Hungar- ian nationality and living abroad the genuine and effective link to Hun- gary is irrelevant. Since 1929, millions of (lawful) emigrants and their descendants have preserved their Hungarian nationality despite acquir- ing a second or third nationality, and despite the absence of close rela- tions, or cultural and ethnic affiliation to Hungary. Hungarian citizenship shall be certified with a valid document (iden- tity card, passport, citizen’s certificate). In case of doubt it will need to be either attested by the authorities or a certificate issued. Upon re- quest, the responsible minister issues a certificate on the existence of citizenship or its cessation, or verifies that the person concerned has never been a Hungarian national. The certificate is valid for one year from the date of issuance. The certificate’s contents may be contested before the Municipal Court by the person concerned, his or her lawful representative, the public prosecutor as well as the person’s guardian.20 p p p p g The regulatory principles and essence of the citizenship system in Hungary are in harmony with international legal norms. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE Hungary is a signatory to all important conventions that define the framework of the development of the law. However, some shortfalls in procedural guaran- tees are still apparent. 5.2.2 Current modes of acquisition and loss of nationality 5.2.1 Current principles in nationality legislation The Constitution contains a guarantee relating to citizenship, i.e. the prohibition of its arbitrary deprivation (art. 69). Other rules are to be settled in legislation to be adopted by a two-thirds voting majority. The two-thirds rule, however, does not apply to the ratification of interna- tional agreements on citizenship. The Nationality Act ensures the equality of rights of citizens. It guaran- tees that all citizens have identical legal standing irrespective of the le- gal title of acquisition of citizenship. The 1997 European Convention on Nationality obliges participating states to refrain from discrimina- tion between their citizens, whether they are nationals by birth or have acquired nationality subsequently. Discrimination is forbidden among Hungarian nationals, irrespective of the legal title under which their citizenship was granted. The Act contains only one exception with regard to withdrawal of citizenship which only applies to citizens by naturalisation. The right to change citizenship is also included in the Nationality Act. Withdrawal of citizenship is an exception. The more common proce- dure is renunciation by a person who lives abroad and thus would pre- sumably not become stateless. Measures aimed at the prevention of sta- telessness restrict the right of the individual to self-determination and the sovereignty of the state in accordance with the conventions of the UN and the European Convention. The only legitimate reason for the withdrawal of citizenship is if it was acquired in a manifestly fraudu- lent manner. Moreover, in the case of renunciation the person must prove that he or she has obtained another citizenship. Domestic law ensures the granting of citizenship at birth by descent (ius sanguinis) while ius soli is applied as an auxiliary principle for abandoned or stateless children. The Act on Nationality supports family KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 5.2.2 Current modes of acquisition and loss of nationality There are seven legal titles of acquisition of Hungarian nationality with different requirements: 156 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 1. The child of a Hungarian national obtains Hungarian citizenship by birth (ius sanguinis) regardless of the place of birth. 1. The child of a Hungarian national obtains Hungarian citizenship by birth (ius sanguinis) regardless of the place of birth. 1. The child of a Hungarian national obtains Hungarian citizenship by birth (ius sanguinis) regardless of the place of birth. 2. The child of a stateless immigrant in possession of a permanent re- sidence permit or an abandoned child of unknown parents shall be considered as a Hungarian national unless or until this presump- tion is rebutted (e.g. when he or she obtains a foreign citizenship due to the clarification of his or her parent’s identity and national- ity). There is no time limit for rebuttal; presumption of Hungarian nationality on the basis of ius soli is therefore conditional. 2. The child of a stateless immigrant in possession of a permanent re- sidence permit or an abandoned child of unknown parents shall be considered as a Hungarian national unless or until this presump- tion is rebutted (e.g. when he or she obtains a foreign citizenship due to the clarification of his or her parent’s identity and national- ity). There is no time limit for rebuttal; presumption of Hungarian nationality on the basis of ius soli is therefore conditional. y 3. Hungarian nationality of exiled nationals who were deprived of their nationality between 1945 and 1990 shall be restored upon re- quest. A declaration addressed to the President of the State rein- states the nationality of the exiled national immediately when it is made. Acquisition of nationality is also possible by declaration in case the applicant was born in Hungary and has not acquired an- other nationality through his or her parent by birth, provided that at the time of the person’s birth he or she resided in Hungary, he or she has lived without interruption in Hungary for a period of at least five years by the time of submission of the declaration and he or she is not older than nineteen years. Another ground for acquisi- tion applies if the applicant was born from a Hungarian national mother and a foreign father before 1 October 1957 and did not be- come a Hungarian national by birth. 5.2.2 Current modes of acquisition and loss of nationality L The permanent residence requirement is reduced to five years if the applicant was born on Hungarian territory or has established residence in Hungary before reaching legal age or is stateless. L The permanent residence requirement is reduced to three years, if the applicant has been married to a citizen for three years, or he or she has a minor child who is a Hungarian citizen, or if the applicant has been adopted by a Hungarian citizen or is an offi- cially recognised refugee. y g g L There is a permanent residence requirement if any of the appli- cant’s ascendants was a Hungarian national and he or she de- clares himself or herself to be an ethnic Hungarian. The permanent residence requirement can also be waived L in the case of the extension of naturalisation to a minor child, i.e. if the applicant is a minor and his or her application was sub- mitted along with that of a parent who qualifies for naturalisa- tion, L if the applicant is a minor and has been adopted by a Hungarian citizen, L if the President of the State or the Minister of Foreign Affairs de- termines that the applicant’s naturalisation is of ‘overriding inter- est’ to the Republic of Hungary (for instance, if he or she is a top-level artist, athlete, or scientist). The proven means of stable livelihood and residence of the applicant can also be waived by the President of the State. 7. Requirements for re-naturalisation include a permanent residence permit of the applicant whose nationality has ceased, a clean crim- inal record and no current criminal proceedings, proven means of stable livelihood and residence in Hungary, and the assurance that his or her naturalisation does not violate Hungarian national inter- ests. 5.2.2 Current modes of acquisition and loss of nationality y 3. Hungarian nationality of exiled nationals who were deprived of their nationality between 1945 and 1990 shall be restored upon re- quest. A declaration addressed to the President of the State rein- states the nationality of the exiled national immediately when it is made. Acquisition of nationality is also possible by declaration in case the applicant was born in Hungary and has not acquired an- other nationality through his or her parent by birth, provided that at the time of the person’s birth he or she resided in Hungary, he or she has lived without interruption in Hungary for a period of at least five years by the time of submission of the declaration and he or she is not older than nineteen years. Another ground for acquisi- tion applies if the applicant was born from a Hungarian national mother and a foreign father before 1 October 1957 and did not be- come a Hungarian national by birth. 4. Presumptive paternity ensures nationality by law for a child born out of wedlock if a parent who declares paternity or a judgement re- cognises paternity/maternity, or if the parents marry subsequently (family law facts). 5. Upon request the restitution of citizenship is ensured if the appli- cant could not obtain a new citizenship within one year of his or her renunciation of Hungarian citizenship. 6. Naturalisation implies a long procedure and is conditional on var- ious preconditions. Basic, non-preferential cases of naturalisation shall meet all of the following requirements: L permanent residence in Hungary for eight years in possession of a permanent residence permit or EEA citizens’ residence permit, L clean criminal record and no current criminal proceedings, L proven means of stable livelihood and residence in Hungary, L naturalisation must not violate national interest of the state, and L successful examination taken on basic constitutional issues in the Hungarian language. If the applicant attended a Hungarian language primary or secondary school or university either in Hungary or in another state, he or she is exempt from the exam. The requirements for preferential naturalisation differ from basic ones as follows: KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 157 L The permanent residence requirement is reduced to five years if the applicant was born on Hungarian territory or has established residence in Hungary before reaching legal age or is stateless. 5.3.1 The Hungarian Status Law and the referendum on dual citizenship Minority protection for ethnic Hungarians and nation building has in- spired debate in contemporary Hungary. There are numerous ramifica- tions of the political discussions on legal development but we will describe only two aspects briefly here and give a concrete example in order to highlight the interrelations between nationality law, migration law, external relations, European integration and nation building. p g g Although the list of states and criteria for visa obligations became part of Community control, bilateral agreements on visa-free travelling were maintained up to Hungary’s accession to the EU. Issuing visas, includ- ing a national visa (in the terminology of the Schengen regime), has just been reformed in favour of Hungarian minorities living in adja- cent third countries. In 2006 a visa allowing its holder to stay in Hun- gary and a multi-entry visa for ethnic Hungarian visitors has been in- troduced. This visa may be issued for five years to a foreign applicant who is capable of sustaining himself or herself, and wishes to use his or her stay in Hungary for practising the Hungarian language and cul- tural activities. Under this visa, employment or study in Hungary is not allowed. The text of the visa agreements is neutral but there are plans to reform them to reflect certain ethno-national priorities towards Romania, Ukraine and Serbia-Montenegro.21 In brief, the visa policy intends to secure the possibility for individuals belonging to the Hun- garian external kin-minorities to freely visit and enter Hungary in order to compensate for Community law and security requirements (To´th 2004b). The Act on Benefits for Ethnic Hungarians living in Neighbouring States of Hungary (usually called the Status Law) was adopted in 2001 after stormy political debates. It introduced a specific certificate for eth- nic Hungarians living in Slovakia, Romania, Ukraine, Slovenia, Serbia- Montenegro and Croatia. Because of constitutional inconsistency and international protests (Ka´ntor 2004), the law was modified in 2003 ending some of the individual benefits (employment, social insurance and public health) that were available in Hungary to holders of the Ethnic Hungarian Certificate (identity card).22 In December 2004, an- other support system (Homeland Fund) for community building was adopted.23 Naturally, this set of direct ethnically-based assistance by dia- spora law (To´th 2000) can legalise and inspire migratory movements toward Hungary. Loss of nationality shall be based on Loss of nationality shall be based on Loss of nationality shall be based on 1. Renunciation: A national residing abroad may renounce his or her nationality if he or she possesses another nationality or relies on the probability of its acquisition. 2. Withdrawal: Hungarian nationality may be withdrawn only if a per- son who has acquired nationality by naturalisation has violated the law on nationality, in particular by misleading the authorities by submitting false data or omitting data or facts. In practice, however, there have not been actual cases in which this provision would have been applied to persons that would have become stateless as a re- sult. Ten years after naturalisation, Hungarian nationality may no longer be withdrawn. 158 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 5.3.1 The Hungarian Status Law and the referendum on dual citizenship On 5 December 2004, Hungary held a referendum on whether it should offer Hungarian citizenship to Hungarians living outside the borders of the Hungarian state.24 The novel aspect of the proposal was not the introduction of dual citizenship itself, since the option of ob- KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 159 taining a Hungarian second citizenship had long been available for per- manent residents within the country. The innovation would have been to remove all residency requirements from the pre-conditions for ob- taining a Hungarian second citizenship. Ethnic Hungarians in neigh- bouring states, and possibly living elsewhere, were to be granted the opportunity of obtaining Hungarian citizenship merely by declaring themselves as of Hungarian linguistic affiliation, at a Hungarian con- sular office, or if they hold a Hungarian Certificate, confirming their Hungarian nationality. The proposal was thus directed at external co- ethnic minorities living in neighbouring states and at members of the Hungarian diaspora elsewhere in the world. g The text of the referendum question was as follows: ‘Do you think that Parliament should pass a law allowing Hungarian citizenship with preferential naturalization to be granted to those, at their request, who claim to have Hungarian nationality, do not live in Hungary and are not Hungarian citizens, and who prove their Hungarian nationality by means of a ‘‘Hungarian Identity Card’’ issued pursuant to Article 19 of Act LXII of 2001 or in another way to be determined by the law which is to be passed?’25 Although the referendum question left the criteria of eligibility open for future lawmaking, an approximation of potentially eligible clai- mants can be made on the basis of the size of the Hungarian popula- tion in the neighbouring states numbering around three million.26 As- suming that the majority of those made eligible by the reform would actually claim citizenship, the proportions of the resulting change would exceed the growth of Germany’s citizenry after unification, but of course, without the corresponding territorial enlargement. This then points to the second specificity of the Hungarian situation, namely that the dimensions of Hungary’s kin-minority problem are unusually large even for Europe. Nearly a quarter of all ethnic Hungarians live outside Hungary’s borders in neighbouring states. g y g g Political debates on the referendum within Hungary were tremen- dously polarised. 5.3.1 The Hungarian Status Law and the referendum on dual citizenship Indeed, in 2003, the initiative to call a referendum had not come from within the Hungarian political establishment, but from a radical and somewhat marginal organisation not well integrated into Hungarian politics, the World Federation of Hungarians (Debrec- zeni 2004).27 The Federation had contested the policies of the Hungar- ian Government on citizenship matters for years and had also set itself on a collision course with the more moderate Hungarian minority par- ties across the borders, especially when it mounted opposition against the Orba´n Government’s (1998-2002) efforts, supported by external Hungarian minorities, to provide an alternative solution to dual citizen- ship through the creation of the Status Law of 2001.28 The law estab- lished the certificate for ethnic Hungarians living in neighbouring 160 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH states,29 entitling its beneficiaries to a set of cultural and economic rights, including seasonal working permits in Hungary. However, the Federation insisted that the benefits provided by the law were no sub- stitute for what the Hungarians really needed, which was full Hungar- ian citizenship.30 p The Status Law provoked angry response in neighbouring states (see Kusa´ and Iordachi in this volume). Hungary was accused of irredentist nationalism, of creating a ‘veiled form of dual citizenship’, the ultimate effect of which was to call the sovereignty of the neighbouring states into question. Hungary was also criticised by the European Union for the unilateral adoption of the law, for not having consulted the states in question, and for the extraterritorial aspects of the law. But despite this negative response, the World Federation of Hungarians insisted that Hungary must proceed with the unilateral creation of non-resident trans-border citizenship for ethnic Hungarians.31 Responding to argu- ments that such a step would not be compatible with the terms of Hungary’s accession to the Union, in the spring of 2003, the federa- tion called on Hungarian voters to say ‘no’ to Hungary’s accession. Hungary should only join the EU if it could take trans-border Hungar- ians into the Union even if the state in which they live remains outside of it (Csergo˝ & Goldzeiger 2004). So, in October 2003, the Federation began collecting signatures for a referendum on establishing non-resi- dent citizenship for trans-border Hungarians. This points then to the third specificity of the Hungarian story, namely that the initiative for citizenship reform came from outside the Hungarian political establishment. 5.3.1 The Hungarian Status Law and the referendum on dual citizenship Only this feature can explain the puzzle of why any political actor would take the risk of launching an initiative that has only limited support within Hungary itself and there- fore carries the prospect of its own defeat. Initially, mainstream Hungarian parties on all sides reacted very cau- tiously to the initiative, along with the more moderate groups of trans- border minorities. Only after a few months did the mainstream right- wing parties (FIDESZ and MDF) along with the President of the Republic declare their support for the referendum, while the socialists and liberals turned against it.32 What followed was an agitated, occa- sionally hysterical, campaign leading up to the referendum that ful- filled the prophecy of its own failure ending up invalid on account of the low number of participants. Eventually, 63.33 per cent of the eligi- ble voters stayed away from the referendum. Among those who cast their ballots, 51.57 per cent voted in favour of the reform, 48.43 per cent against.33 No research is available on the question of what precisely motivated Hungarian voters in their choices. Welfare protectionism could well have played a role, given the fact that, apart from Slovakia, the living KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 161 standards of trans-border Hungarians are way below those of Hungar- ians, and that the arguments of the Socialist Party against dual citizen- ship relied primarily on the costs of the reform. An equally important motive may have been the fear of instability at the borders resulting from conflicts with Hungary’s neighbours. Voters may also have been influenced by the perception that dual citizenship would eventually lead to voting rights. What is sufficiently clear, however, is that, at least for now, trans-border dual citizenship could only be created in Hun- gary without the popular mandate of the Hungarian electorate, the mandate that the supporters of the initiative had hoped to obtain in the referendum. To quote one liberal opponent of the initiative (Kis 2004a: 4): ‘The offer was made to a nation of ten million to enlarge its home- land beyond the state-borders to the entire Carpathian basin. The na- tion refused to take the risk and accept the costs.’ But given the enormous disappointment of trans-border Hungarians with the result, the issues raised during the campaign will remain on the agenda of Hungarian politics for quite some time to come. 5.3.2 Implications of trans-border dual citizenship The arguments for the Hungarian trans-border dual citizenship initia- tive are fundamentally different from those advanced in favour of dual citizenship in the major immigration states of Western Europe. In the immigration states dual citizenship is an instrument used to integrate labour migrants into their country of immigration. Dual citizenship in this case works towards the decoupling of citizenship from ethnicity. In contrast, the Hungarian initiative is part of a counter-trend present in a number of European countries of re-linking citizenship with ethnicity. p g p y The Hungarian suggestion associates eligibility for extraterritorial dual citizenship with membership in an ethnically defined community. Dual citizenship would thus purposefully reaffirm the connection be- tween ethno-cultural nationality and citizenship, which is precisely the connection that most immigration states have been trying to weaken when tolerating dual citizenship (Fowler 2002). Advocates of the reform wish to overcome this difficulty by present- ing their plan as based on a traditional ius sanguinis concept rather than on ethnicity. In this view, trans-border citizenship is not some- thing that would be newly granted to ethnic Hungarians. Trans-border Hungarians would only ‘regain’ the citizenship of their ancestors who had been citizens of the Hungarian part of the Dual Monarchy before the First World War.34 However, there are several difficulties with this approach (Nagy 2004).35 The first difficulty is political. After the First World War, those Hun- garians who ended up as minorities in neighbouring states were ob- MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 162 liged by the Peace Treaties to opt for the citizenship of their new home state, or, if they declined to do so, to move to Hungary. Therefore, in the eyes of Hungary’s neighbours, any unilateral change in the citizen- ship status of minority Hungarians would amount to a unilateral breach of treaty obligations, to a revision of the terms of the peace treaty that still serves as the basis of international legitimacy for the current borders of these states. It was for a similar reason that the Ita- lian law of 2000 that offered Italian citizenship to the Italian diaspora did not extend this offer to the descendants of Italians in Dalmatia, Is- tria and Fiume, i.e. those regions that were ceded by Italy to Yugoslavia in the post-war treaties. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 163 people who had never even lived in Hungary so that their political choices would be made on a highly selective image of issues and candi- dates. For all these reasons, he concluded, ‘the victory of ‘‘yes votes’’ would pull us back to the murky nationalism of past ages, it would lock up Hungarian politics in the prison of revisionist nostalgia, it would poison public life within Hungary as well as our relationship with neighbouring states and with trans-border Hungarians, and it would damage the level of our acceptance within the European Union’.37 g p p In stark contrast to the liberals, advocates of the initiative argued that their proposal is modelled on concepts and processes that are part and parcel of an integrated Europe of the future, a de-territorialised world in which individuals with multiple identities are entitled to a legal ex- pression of the free choice of their nationality. Advocates argued that all European states accept ethnicity as part of the basis of citizenship, most even making provisions for the acquisition of benefits, including citizenship, for co-ethnics who are citizens of another state. The pro- blem with European norms and practices, they argued, is not that there is no connection between ethnicity and citizenship but that Europe is in a process of denial about this connection, treating ethnicity as though it was a disreputable relative on whom we rely secretly, but whom we hide from others (Scho¨pflin 2004). They pointed to plans or existing legislation on non-resident citizenship for co-ethnic kin within the European Union in Italy, Greece, Slovakia and the Czech Republic. A particularly relevant example is Silesian Germans who, from the early 1990s, were able to obtain German passports in addition to their Polish ones and, by implication, European citizenship, without having to take up residence in Germany. These precedents, they argued, point to the legitimacy, even within the core nations of the European Union, of using dual citizenship for the inclusion of trans-border co-ethnics in the citizenry of the homeland. y Liberal opponents challenged this interpretation of larger European processes and insisted that the EU would regard the ethnicist turn in Hungarian legislation as a breach of common principles laid down in European agreements (To´th 2004c).38 Secondly, they criticised the con- frontational attitude towards Hungary’s neighbours promoted by this policy. 5.3.2 Implications of trans-border dual citizenship p Second, trans-border populations whose ancestors bore the citizen- ship of a larger Hungarian state in the Dual Austro-Hungarian Monar- chy before the First World War include millions of non-Hungarians. So, even if the ius sanguinis view was applied, the only way to narrow down eligibility for Hungarian dual citizenship to those with a Hungar- ian ethno-cultural affiliation would be to apply an ethnic definition. A third feature of dual citizenship that emerged from the referen- dum initiative was the potentially weak distinction between active and inactive citizenship for dual citizens. In most immigration states, trans- national dual citizenship implies that only the citizenship of the cur- rent country of residence is active, so that the rights associated with the external citizenship are dormant (Faist 2005). However, in the case of Hungarian trans-border citizenship such clear-cut distinctions be- tween periods of active and inactive citizenship would be hard to make (Vizi 2003).36 Therefore, with regard to the potential content of non-re- sident trans-border citizenship, the general perception that has emerged in Hungary is that even if dual citizenship would initially be created without voting rights, it would only be a matter of time before large numbers of trans-border voters would begin casting their ballots. In view of these implications, it is hardly surprising that the proposal created passionate debates both within Hungary and among the Hun- garian minorities in the neighbouring states. For many participants the question at stake was whether Hungary should experiment with ideas that are pulling it away from, rather than bringing it closer to ‘main- stream’ Europe. As Ja´nos Kis summarised it, the victory of the ‘yes’ votes would mean nothing less than putting Hungarian parliamentar- ianism in danger and transforming the nature of Hungarian democ- racy. Since elections in Hungary are usually won by a narrow margin, the appearance of trans-border voters would most likely mean that ‘the outcome of Hungarian elections would regularly be decided by voters who do not pay taxes in Hungary and who are, in general, not subject to its laws’. A further element of ‘organised irresponsibility’ inherent in such a solution would be that those casting the swing votes may be KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE The problem with unilateral action is not so much that it vio- lates international law, but that it is self-defeating. To quote the above mentioned newspaper article by Ja´nos Kis again: The unilateral crea- tion of Hungarian citizens in the territory of other states is nothing but a ‘mirage’ that provokes ‘phony wars over phony questions and phony answers’. Thirdly, opponents argued, that the creation of dual citizenship can- not be justified by reference to the approval by trans-border minorities either, because these groups are themselves divided over the issue and 164 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH do not speak with a single voice.39 In the end, any unilateral move by Hungary to create dual citizenship would remain ‘a game of illusions played between Hungarian nationalists and a minority within the Hun- garian minority’ in a useless, but ‘ritual display of imagined political togetherness’ (Kis 2004b). g ( 4 ) Fourthly, critics objected that dual citizenship is incompatible with claims of autonomy raised by trans-border minorities.40 Concurring with Rainer Baubo¨ck they maintained that parallel ‘claims of multiple citizenship and territorial autonomy should be seen as mutually in- compatible. They would create fears in the host society about irreden- tist threats to its territorial integrity that cannot be easily dismissed as unreasonable’ (Baubo¨ck 2006: 159-160). ( 59 ) Therefore, according to the socialists and the liberals, Hungary must take a new look at its homeland policies regarding kin-minorities. The discourse advocated by the two mainstream right-wing parties aims at recreating a ‘unitary Hungarian nation’ over and above existing state- borders by means of creating legal bonds between parts of the Hungar- ian nation living in several countries (Stewart 2004). Hungary should step back from this confrontational approach because it relies on out- right ignorance about the sensitivities of other states. Instead, it should clearly articulate its policies in the conceptual framework of minority protection. Hungary must accept that trans-border Hungarians are the citizens of other states and should promote the protection of Hungar- ian minorities in their efforts to secure equal individual and collective rights in their home states. Finally, there are obvious ambiguities in the arguments of both sides in the debate. The idea of dual citizenship emerged in Hungary with reference to a larger international trend of increasing toleration of dual citizenship, partly within the European Union and partly within the East-Central European region. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE However, while in the immigration states of Europe the idea of dual citizenship is not associated with na- tionalist policies, in Hungary, as in many other states of the region, the demand for dual citizenship has mostly migrated to the nationalist right. In the Hungarian referendum debate, the battle over dual citi- zenship has been cast as a debate between the nationalist right as sup- porters, on the one hand, and the Europe-oriented liberals, as oppo- nents, on the other. However, this representation of the debate is, to some extent, self-made and arbitrary. In fact, in their support of dual ci- tizenship the nationalists have mainly been drawing on the arguments of European liberals. At the same time, liberals relied on counter-argu- ments they claimed to have extrapolated from relevant European norms and practices, but these practices are much too diverse to form the basis of a coherent interpretation. Unsurprisingly, by the end, both sides failed to present a fully convincing, coherent interpretation of KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 165 those international norms and practices that would support their re- spective positions. In the final analysis it is quite possible that the con- flicting stances of the two sides in the debate may stem from concerns that are only remotely connected to the problems of trans-border Hun- garians, namely from conflicting opinions, and concerns about the long-term stability of Hungary’s transitional democracy. After all, par- liamentary practices have not been firmly established in Hungary for much more than a decade. Yet, in the Hungarian context, the creation of trans-border non-resident dual citizenship would most likely amount to a mass enfranchisement of a new electorate that, similar to all epi- sodes of mass enfranchisement in the past, would introduce new un- certainties into the system and could lead to an internal destabilisation of Hungarian democracy itself. In this respect, both sides share the same intuition, namely that if instituted, trans-border citizenship would most likely have the effect of freezing the regular rotation of par- liamentary forces for some time to come in favour of the nationalist right: a prospect that is as welcome on one side as it is feared on the other. 5.4 Trends in statistics Data on trends of acquisition and termination of citizenship is infor- mation of public interest.41 Nevertheless relevant data is only partially available and in more detail only since 2001. Available data contain numbers on naturalisation, re-naturalisation and on the termination of nationality. Between 1958 and 1984 there were more cases of emigra- tion than immigration (To´th 1997), and the total number of naturalised and re-naturalised persons was 16,156 while at least 24,082 left the country. The yearly average of naturalisations and re-naturalisations was 622 while the average terminations of nationality was 926. During this time there was no change in citizenship law, so it is only by exam- ining legal and political practices that we can find an explanation for the growth in the rate of nationality loss after 1967. A substantial pro- portion of removal-upon-request came from female Hungarian spouses marrying husbands from European states that prohibited dual citizen- ship. Between 1985 and 1989, the number of terminations was even high- er than the number of naturalisations and re-naturalisations, but the difference between them decreased. The major groups of applicants for naturalisation were from the adjacent and socialist states (Romania, Czechoslovakia, Soviet Union and East Germany) while the direction of emigration/marriage migration was towards Austria and Yugoslavia. MA´ RIA M. 5.4 Trends in statistics KOVA´ CS AND JUDIT TO´ TH 166 Table 5.1 Number of naturalisations and re-naturalisations as well as terminations of nationality in Hungary, 1985-1994 Year Naturalisation/Re-naturalisation Removal/Renunciation 1990 3,170 1,184 Czech/Slovak 63 Czech/Slovak 2 Yugoslav 21 Yugoslav 18 Austrian 11 Austrian 169 Romanian 2,661 Romanian 1 Soviet 156 Soviet 1 East German 35 East German 70 Non-European 96 Non-European 1 1991 5,893 441 Czech/Slovak 25 Czech/Slovak 2 Yugoslav 22 Yugoslav 3 Austrian 18 Austrian 80 Romanian 5,114 Romanian – Soviet 306 Soviet – Stateless 13 Non-European 186 Non-European 1 1992 21,880 1,149 Czech/Slovak 249 Czech/Slovak 7 Yugoslav 1 Yugoslav 3 Austrian 7 Austrian 211 Romanian 20,624 Romanian – Ex-Soviet 569 Ex-Soviet – Stateless 7 Non-European 60 Non-European 3 1993 11,521 2,084 Czech/Slovak 55 Czech/Slovak 5 Yugoslav 309 Yugoslav – Austrian 20 Austrian 314 Romanian 9,956 Romanian – Ex-Soviet 843 Ex-Soviet – Stateless 7 Non-European 75 Non-European 3 1994 9,238 1,688 Czech/Slovak 40 Czech/Slovak 7 Yugoslav 888 Yugoslav – Austrian 1 Austrian 346 Romanian 6,254 Romanian – Ex-Soviet 1,730 Ex-Soviet – Stateless 1 Non-European 120 Non-European 2 Total 1985-1994 55,409 11,492 Yearly average 1985-1994 5,541 1,149 Source: www.bmbah.hu Since 1990, the number of naturalisations has increased. This is not only due to the larger number of ethnic Hungarian applicants but also to the changing interpretation of the legal rules in force. The constitu- Table 5.1 Number of naturalisations and re-naturalisations as well as terminations of nationality in Hungary, 1985-1994 Since 1990, the number of naturalisations has increased. This is not only due to the larger number of ethnic Hungarian applicants but also to the changing interpretation of the legal rules in force. The constitu- KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 167 tional reform, aimed at establishing rule of law, influenced the practice of the Ministry of the Interior. If an applicant met the legal require- ments the discretionary power of naturalisation had to be interpreted such that a positive decision on naturalisation was to be granted by the President. However, this practice of ‘self-limitation’ could not compen- sate for the more restrictive preconditions of naturalisation adopted by the Act on Hungarian Nationality in 1993. The number of non-Eur- opean applicants is growing, but has still remained marginal since the 1990s. 5.4 Trends in statistics Table 5.2 Distribution of nationality law cases in Hungary, 1998-2008 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Applications for naturalisation/ re-naturalisation 3,593 3,160 3,963 4,282 4,282 4,453 4,916 5,761 9,127 5,437 4,143 Applicants with citizenship (%): Romania 61 60 63 69.8 69.8 73.2 60 Yugoslavia/Serbia 17 15 13 9.5 5.8 8.9 12 Ukraine 11 15 13 8.5 8.8 9.2 15 Other European 6 14 14 9.2 11.8 5 3 Non-European 5 5 3 2.5 3.3 3.2 7 Stateless 1 1 1 0.5 0.5 0.5 0.5 Naturalised and re-naturalised persons 6,203 6,203 7,538 5,934 3,890 5,579 5,667 9,981 6,564 9,398 8,132 Applications for re-obtaining nationality upon declaration of expatriation, prior nationals (persons) 232 200 208 194 212 151 144 136 104 85 68 Applications for certificate of existing nationality (persons) 3,934 4,264 3,935 3,924 4,401 4,803 5,984 5,482 4,121 4,276 4,958 Reinstatement of nationality (persons) - - - 1 1 2 1 0 0 0 0 Applications for renunciation of nationality (cases) 893 728 748 684 609 463 189 135 126 82 69 Accepted renunciations of nationality (persons) 1,070 995 955 791 857 n/a 236 164 137 98 107 Note: n/a= not available Source: www.bmbah.hu Table 5.2 Distribution of nationality law cases in Hungary, 1998-2008 1998 1999 2000 2001 2002 2003 2004 2005 2006 20 Over the past number of years Hungary has become an immigration country for large numbers of ethnic Hungarians and, increasingly for others coming from more distant regions. There are three major chan- nels for immigrants to become nationals: (1) naturalisation, (2) prior nationals, mainly expatriates re-obtaining Hungarian nationality by de- 168 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH claration or re-naturalisation, (3) expatriates or their descendants living abroad who can prove Hungarian citizenship through a verification pro- cedure of existing citizenship (Certificate of Nationality). This restora- tion of legal ties with Hungary was made possible by political changes and new rules on rehabilitation and compensation for damages or harm committed against nationals by the socialist regime. Between 1998 and 2008, the number of naturalised and re-naturalised persons was below the number of applicants for a citizenship card, which serves to certify the holder’s Hungarian nationality. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 169 5.4 Trends in statistics The ratio of naturalisation according to legal titles for the years 2002, 2007 and 2008 proves that preferential cases vastly outweigh non-preferential ones, which repre- sented between 3 and 6 per cent of all cases (Table 5.3). Table 5.3 Ratio of naturalisation decisions in Hungary in 2002, 2007 and 2008 Type of legal titles Art. 2002 2007 2008 Total % Total % Total % No preference (basic decision) 4 § (1) 244 6.27 186 2.71 206 3.39 Weak preference (applicant was born in Hungary) 4 § (4) a. 3 0.0 90 1.31 70 1.15 Weak preference (applicant immigrated as minor to Hungary) 4 § (4) b. 2 0.0 136 1.98 136 2.24 Medium preference (applicant’s spouse is Hungarian national) 4 § (2) a. 325 8.35 402 5.87 380 6.26 Medium preference (applicant’s minor child is Hungarian national) 4 § (2) b. 49 1.25 123 1.79 118 1.94 Medium preference (applicant is a recognised refugee) 4 § (2) d. 17 0.4 43 0.62 22 0.36 Strong preference (applicant is a minor) 4 § (5) 9 0.2 27 0.39 11 0.18 Strong preference (applicant is a minor adopted by a national) 4 § (6) 30 0.7 4 0.05 7 0.11 Strong preference (applicant is an ethnic Hungarian) 4 § (3) 2,447 62.9 5,158 75.33 4,713 77.66 Re-naturalisation 5 § 764 19.6 676 9.87 403 6.64 Total 3,890 100.0 6,845 100.0 6,066 100.0 Source: www.bmbah.hu Table 5.3 Ratio of naturalisation decisions in Hungary in 2002, 2007 and 2008 Table 5.3 indicates that, beyond the ethnic immigration from the Car- pathian basin, family reunification and repatriation of prior nationals have added the largest numbers of new nationals. www.coe.int; www.bmbah.hu/ jogszabalyok.php (in Hungarian) Introduces an identity card for ethnic Hungarians; lists allowances and benefits granted to holders of such cards (mainly in Hungary) Allows ethnic Hungarians to have the family and given name in their original ethnic language; introduces facilitated 5.5 Conclusions In Hungary, the term ‘nation’ is interpreted and used in law as a con- cept referring to membership in the cultural, ethnic and linguistic community. But the substance of the term remains indefinable by law. This reveals contradictions between existing laws and the Constitution. On the one hand, art. 6 of the Constitution refers to the kin-state’s re- sponsibility for kin-minorities living across the borders. However, the definition of membership in the minority or ethnic community is va- gue, and various preferential provisions legally discriminate against certain categories of people despite the fact that the state is party to dozens of international treaties aimed at avoiding such discrimination. Furthermore, minorities living in Hungary are distinct participants in the state, in possession of subjective and collective constitutional rights, although, in their case as well, membership of a specific ethnic or national entity cannot be defined. Due to this problem neither statis- tics on membership of minorities living in Hungary, nor hard data on immigrants entering Hungary and enjoying legal preferences in the country are available. According to Rainer Baubo¨ck, ‘[h]istoric tradi- tions and the distinction between ethnic and civic nationhood are in- creasingly irrelevant for explaining legislative changes’.42 Despite a standard level of immigration, in the case of Hungary Baubo¨ck’s sug- gestion is less evident than among the old EU Member States (To´th & Sik 2003). The recently failed referendum of 5 December 2004 on ex lege citizenship being granted to ethnic Hungarian minorities living in adjacent states is a case in point as it would have used ethnic prefer- ences for granting non-resident citizenship to trans-border Hungar- ians. The role of nationality law in the integration process of migrants has not been discussed publicly and the need to harmonise Hungarian citizenship with that of other Member States of the European Union has not been put on the agenda. Chronological list of citizenship-related legislation in Hungary Date Document Content Source 1946 Act XV on Czech-Slovak- Hungarian Agreement Deprives those who fall under the bilateral agreements on population exchange of Hungarian nationality 1947 Government Decree 12.200 Deprives expelled Germans of Hungarian nationality 1947 Act X Deprives those who have not returned to Hungary Chronological list of citizenship-related legislation in Hungary MA´ RIA M. www.coe.int; www.huembwas.org; www.bmbah.hu/ jogszabalyok.php (in Hungarian) 1993 Government Decree 125/ 1993 on the Execution of Act No. LV of 1993 on Hungarian Nationality www.legislationline.org 2001 Act XXXII amending Act LV of 1993 on Hungarian Nationality 2001 Legislation on Kin- minorities (Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries) www.legislationline.org 5.5 Conclusions KOVA´ CS AND JUDIT TO´ TH 170 Date Document Content Source following the conclusion of the war of Hungarian nationality 1948 Act LX on Hungarian Nationality Like the previous Act of 1879, is based on ius sanguinis; provides for the equal treatment of children born out of wedlock; stipulates that all nationals residing abroad should be registered; recognises the pending Hungarian nationality of undocumented persons 1949 Constitution (excerpts) www.legislationline.org 1957 Act V on Hungarian Nationality Introduces the emancipation of spouses; includes executive rules, such as the one requiring emigrants to renounce their nationality and social insurance rights 1989 Act XXXI amending the Constitution of 1949 (excerpts) Prohibits arbitrary deprivation of nationality 1993 Citizenship Act (Act LV of 1993 on Hungarian Nationality) Provides that the nationality of expatriate nationals who have been arbitrarily deprived of their nationality is restored upon request; includes preferential naturalisation of refugees and stricter conditions for naturalisation, but also preferences based on ethnic and family ties www.coe.int; www.huembwas.org; www.bmbah.hu/ jogszabalyok.php (in Hungarian) 1993 Government Decree 125/ 1993 on the Execution of Act No. LV of 1993 on Hungarian Nationality Defines formats and procedural rules www.coe.int; www.bmbah.hu/ jogszabalyok.php (in Hungarian) 2001 Legislation on Kin- minorities (Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries) Introduces an identity card for ethnic Hungarians; lists allowances and benefits granted to holders of such cards (mainly in Hungary) 2001 Act XXXII amending Act LV of 1993 on Hungarian Nationality Allows ethnic Hungarians to have the family and given name in their original ethnic language; introduces facilitated Date Document KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 2005 Act XLVI amending Act LV of 1993 on Hungarian Nationality 2005 Act LXXXIII amending Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries 2008 Act LXXXII amending the Act LV of 1993 on Hungarian Nationality KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 171 Date Document Content Source acquisition of nationality for exiled nationals by declaration to the President of State 2001 Government Decree 103/ 2001 amending Government Decree 125/ 1993 on the Execution of Act LV of 1993 on Hungarian Nationality Provides fast-track naturalisation process for ethnic Hungarians and minors 2003 Act LVI amending Act LV of 1993 on Hungarian Nationality Extends preference in naturalisation to European Union citizens 2003 Act LVII amending Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries Cuts and restructures benefits and allowances for ethnic Hungarians 2003 Government Decree 128/ 2003 amending Government Decree 125/ 1993 on the Execution of Act LV of 1993 on Hungarian Nationality Introduces new formats in nationality procedures 2005 Act XLVI amending Act LV of 1993 on Hungarian Nationality Reduces the waiting period in naturalisation procedures for ethnic Hungarians; specifies exceptions from taking the examination on basic constitutional issues; allows the use of the ethnic version of a person’s place of birth in official documents (in combination with the Hungarian version) 2005 Act LXXXIII amending Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries Harmonises proceedings for the identity card for ethnic Hungarians with the new Code on Public Administration Processes 2005 Government Decree 119/ 2005 amending Government Decree 125/ 1993 on the Execution of Act LV of 1993 on Hungarian Nationality Determines the C 20 fee for the examination on basic constitutional issues in the naturalisation procedure; clarifies family unification rules in related immigration rules 2006 Act XXI amending Act LV of 1993 on Hungarian Nationality Introduces official notice on ceased Hungarian nationality of individuals to u ga a Nat o a ty 2005 Act XLVI amending Act LV of 1993 on Hungarian Nationality Reduces the waiting period in naturalisation procedures for ethnic Hungarians; specifies exceptions from taking the examination on basic constitutional issues; allows the use of the ethnic version of a person’s place of birth in official documents (in combination with the Hungarian version) 2005 Act LXXXIII amending Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries Harmonises proceedings for the identity card for ethnic Hungarians with the new Code on Public Administration Processes 2005 Government Decree 119/ 2005 amending Government Decree 125/ 1993 on the Execution of Act LV of 1993 on Hungarian Nationality Determines the C 20 fee for the examination on basic constitutional issues in the naturalisation procedure; clarifies family unification rules in related immigration rules 2006 Act XXI amending Act LV of 1993 on Hungarian Nationality Introduces official notice on ceased Hungarian nationality of individuals to y Act LV an Reduces the waiting period in naturalisation procedures for ethnic Hungarians; specifies exceptions from taking the examination on basic constitutional issues; allows the use of the ethnic version of a person’s place of birth in official documents (in combination with the Hungarian version) ng Act nic n tries Harmonises proceedings for the identity card for ethnic Hungarians with the new Code on Public Administration Processes e 119/ e 125/ on of ity Determines the C 20 fee for the examination on basic constitutional issues in the naturalisation procedure; clarifies family unification rules in related immigration rules Act LV an Introduces official notice on ceased Hungarian nationality of individuals to 2005 Act XLVI amending Act LV of 1993 on Hungarian Nationality Reduces the waiting period in naturalisation procedures for ethnic Hungarians; specifies exceptions from taking the examination on basic constitutional issues; allows the use of the ethnic version of a person’s place of birth in official documents (in combination with the Hungarian version) 2005 Act LXXXIII amending Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries Harmonises proceedings for the identity card for ethnic Hungarians with the new Code on Public Administration Processes 2005 Government Decree 119/ 2005 amending Government Decree 125/ 1993 on the Execution of Act LV of 1993 on Hungarian Nationality Determines the C 20 fee for the examination on basic constitutional issues in the naturalisation procedure; clarifies family unification rules in related immigration rules 2006 Act XXI amending Act LV of 1993 on Hungarian Nationality Introduces official notice on ceased Hungarian nationality of individuals to MA´ RIA M. 2007 Act I on entry and staying of persons in possession of rights on free movement and residence KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE KOVA´ CS AND JUDIT TO´ TH 172 Date Document Content Source the population registry of the Central Statistical Office and to the military service registry of the Ministry of Defence 2006 Act CIX amending the Act LV of 1993 on Hungarian Nationality Entitles the President of State to exempt applicants from some naturalisation requirements (e.g. subsistence, examination on constitutional basics); introduces several procedural changes regarding issuing of documents, the length of procedures, etc. 2007 Act I on entry and staying of persons in possession of rights on free movement and residence Defines period of residence in Hungary as starting with the address registration for permanent residence holders, recognised refugees and registered persons with right to free movement (i. e. EU citizens and their family members) 2008 Act LXXXII amending the Act LV of 1993 on Hungarian Nationality Defines the body responsible for the exam on the constitution Date Document Notes 1 Act LVI of 2003 amending Act LV of 1993 on Hungarian Nationality. It entered into force with the accession of Hungary to the European Union on 1 May 2004. 2 Act XXXII of 2001 amending Act LV of 1993 on Hungarian Nationality, Government Decree No. 125 of 22 September 1993, Decree of the Minister of the Interior No. 6 of 7 March 2003. 3 Concluded in Moscow on 20 January 1945 and published in Act V of 1945. 4 The Peace Agreement was concluded in Paris and published in Act XVIII of 1947. It entered into force by Government Decree 11.800 of 1947. 5 See details in Czech-Slovak-Hungarian Agreement published in Act XV of 1946 and Government Decree 12.200 of 1947. 6 In particular, Act X of 1947 and Act XXVI of 1948. 7 For instance, Prime Minister Decree 9.590 of 1945. 8 Act XXIX of 1946. 9 Act LX of 1948. 1 Act LVI of 2003 amending Act LV of 1993 on Hungarian Nationality. It entered into force with the accession of Hungary to the European Union on 1 May 2004. 2 Act XXXII of 2001 amending Act LV of 1993 on Hungarian Nationality, Government Decree No. 125 of 22 September 1993, Decree of the Minister of the Interior No. 6 of 7 March 2003. 7 3 3 Concluded in Moscow on 20 January 1945 and published in Act V of 1945. 4 The Peace Agreement was concluded in Paris and published in Act XVIII of 1947. It entered into force by Government Decree 11.800 of 1947. 4 The Peace Agreement was concluded in Paris and published entered into force by Government Decree 11.800 of 1947. 5 See details in Czech-Slovak-Hungarian Agreement published in Act XV of 1946 and Government Decree 12.200 of 1947. 6 In particular, Act X of 1947 and Act XXVI of 1948. For instance, Prime Minister Decree 9.590 of 19 8 Act XXIX of 1946. 9 Act LX of 1948. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE Visa requirements were introduced for Ukrainian and Serbian citizens, while the agreement with Romania introduced a maximum length of stay. 22 Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries. It was amended by Act LVII of 2003. Its administrative rules on financial, technical and procedural issues are laid down in ten government and ministerial Decrees. 22 Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries. It was amended by Act LVII of 2003. Its administrative rules on financial, technical and procedural issues are laid down in ten government and ministerial Decrees. 22 Act LXII of 2001 on Ethnic Hungarians Living in Neighbouring Countries. It was amended by Act LVII of 2003. Its administrative rules on financial, technical and procedural issues are laid down in ten government and ministerial Decrees. 23 Act II of 2005 on the Homeland Fund covers various community-building projects for kin-minorities living in adjacent states. 24 In Hungary, a referendum is valid if at least 25 per cent of the electorate returns identical votes, or if participation is higher than 50 per cent of the total number of eligible voters. In this case neither criterion was fulfilled. 24 In Hungary, a referendum is valid if at least 25 per cent of the electorate returns identical votes, or if participation is higher than 50 per cent of the total number of eligible voters. In this case neither criterion was fulfilled. g 25 Official translation provided by the Government’s Election Office (Orsza´gos Va´laszta´si Bizottsa´g), www.election.hu, last accessed 5 May 2005. 25 Official translation provided by the Government’s Election Office (Orsza´gos Va´laszta´si Bizottsa´g), www.election.hu, last accessed 5 May 2005. 26 According to the statistics published in 2004 by the Hungarian Government Office for Trans-Border Hungarians (Hata´ron Tu´li Magyarok Hivatala), the number of Hun- garians living in Romania, Ukraine, Serbia and Montenegro, Slovakia, Croatia and Slovenia as provided by the official censuses in these countries between 2000 and 2002 amounted to 2,429,000, among these in Romania 1,435,000; Ukraine 156,000; Serbia and Montenegro 293,000; Slovakia 516,000; Croatia 16,000 and Slovenia 8,500 (see www.htmh.hu, last accessed 5 May 2005). KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 173 10 Published in Law-Decree No. 2 of 1960. 11 Act V of 1957. 957 12 Law-Decree No. 11 of 1955, No. 7 of 1956, No. 11 of 1956; Ministerial Decree of the Interior 2 of 11 January 1956. 12 Law-Decree No. 11 of 1955, No. 7 of 1956, No. 11 of 1956; Ministerial Decree of the Interior 2 of 11 January 1956. 13 Act XXXI of 1989 introduced a substantially new Constitution but formally it was only an amendment. y 14 Provisions of Act XXVII of 1990 and Act XXXII of 1990 were inserted into the third Act on Nationality in 1993. Act on Nationality in 1993. 15 Published in Law-Decree No. 15 of 1989. 15 Published in Law-Decree No. 15 of 1989. 16 Act VL of 1993. 17 Acts XXXII of 2001, LVI of 2003 and XLVI of 2005. 17 Acts XXXII of 2001, LVI of 2003 and XLVI of 2005. 18 Published in Acts II and III of 2003. 19 This principle is a legal expression of the fact that the individual who obtains this citizenship – directly through the law or as a result of the action of the authorities – is in actual fact more closely related to the state whose citizen he or she is than to any other state (Lichtenstein v. Guatemala, 1995 WL 1 (International Court of Justice) generally known as the Nottebohm case). 19 This principle is a legal expression of the fact that the individual who obtains this citizenship – directly through the law or as a result of the action of the authorities – is in actual fact more closely related to the state whose citizen he or she is than to any other state (Lichtenstein v. Guatemala, 1995 WL 1 (International Court of Justice) generally known as the Nottebohm case). 20 Act on Hungarian Nationality, arts. 10-12. 21 Before accession, Hungary had agreements on visa-free travel with six neighbours, and a voucher system was defined with the Ukraine. For the sake of legal harmonisation these agreements were modified. Visa requirements were introduced for Ukrainian and Serbian citizens, while the agreement with Romania introduced a maximum length of stay. 21 Before accession, Hungary had agreements on visa-free travel with six neighbours, and a voucher system was defined with the Ukraine. For the sake of legal harmonisation these agreements were modified. 28 The Hungarian name of the federation is Magyarok Vila´gszo¨vetse´ge. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE The possibility of inheriting Hungarian citizenship applies only to people whose right to Hungarian citizenship is derived from their connection to the territory of the state of Hungary as delineated in the Paris Peace Treaty of 1947. 35 The dimension of the population potentially affected by the ius sanguinis transmission of citizenship is difficult to assess. Given the fact that in 1920, Hungary’s population had been reduced to half of what it had been before the war (with a corresponding reduction of two-thirds of its territory), the idea that ius sanguinis transmission could automatically create dual citizens after any number of generations would amount to the obligation to re-activate the ‘dormant’ citizenship of people whose numbers may surpass half of Hungary’s current population. The peace treaty of 1920 reduced Hungary’s population from 18.2 million to 7.9 million and its territory from 282,000 km2 to 93,000 km2. Trans-border Hungarians are estimated to number about 3.5 million, while people (with their offspring) who retain a ius san- guinis right to Hungarian citizenship (e.g. those who emigrated after 1939) are esti- mated to be about 1.5 million. 36 Hungarian trans-border citizenship, if ever instituted, is more likely to be in line with that of Croatia where trans-border dual citizens retain some of their rights associated with Croatian citizenship, including voting rights in Croatian elections, even at times when their alternate citizenship is active (see Ragazzi & Sˇtiks in this volume). But while trans-border Croats vote for a quota of expatriate seats, trans- border Hungarians would find it easy to vote for regular seats without putting their alternate citizenship to rest. This is because Hungarian regulations on the declaration of residence are extremely lax, requiring only three months of residence for a citizen to activate his or her right to vote. Moreover, in order to avoid the disenfranchisement of the homeless, voters can be admitted to the voters’ registry without actually possessing an address or residence permit by simply making a declaration of residence at a given locality at the municipal office. According to recent changes in Italian law, Italian non-resident citizens may also vote in referenda and national elections for a fixed number of seats. However, the numerical dimensions of the Italian case are radically different from that of Hungary. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE The estimate for the number of trans-border Hungarians potentially eligible for Hungarian citizenship based on ethnic identification is higher than these numbers, which is explained by the assumption that more people would be able to fulfil the criteria of Hungarian af- filiation than those who actually declare themselves Hungarian in government cen- suses. The number of potential claimants on such grounds globally was estimated at around five million by the Under Secretary for Foreign Affairs, Andra´s Ba´rsony (‘Ha- ta´rok ne´lku¨l’, Kossuth Ra´dio´, 16 January 2003. www.hhrf.org, last accessed 5 May 2005). Also see note 34. 26 According to the statistics published in 2004 by the Hungarian Government Office for Trans-Border Hungarians (Hata´ron Tu´li Magyarok Hivatala), the number of Hun- garians living in Romania, Ukraine, Serbia and Montenegro, Slovakia, Croatia and Slovenia as provided by the official censuses in these countries between 2000 and 2002 amounted to 2,429,000, among these in Romania 1,435,000; Ukraine 156,000; Serbia and Montenegro 293,000; Slovakia 516,000; Croatia 16,000 and Slovenia 8,500 (see www.htmh.hu, last accessed 5 May 2005). The estimate for the number of trans-border Hungarians potentially eligible for Hungarian citizenship based on ethnic identification is higher than these numbers, which is explained by the assumption that more people would be able to fulfil the criteria of Hungarian af- filiation than those who actually declare themselves Hungarian in government cen- suses. The number of potential claimants on such grounds globally was estimated at around five million by the Under Secretary for Foreign Affairs, Andra´s Ba´rsony (‘Ha- ta´rok ne´lku¨l’, Kossuth Ra´dio´, 16 January 2003. www.hhrf.org, last accessed 5 May 2005). Also see note 34. 27 J. Debreczeni, ‘Haza´rdja´te´k’ [Gambling], Ne´pszabadsa´g [daily newspaper], 27 Novem- ber 2004. 28 The Hungarian name of the federation is Magyarok Vila´gszo¨vetse´ge. MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 174 29 Since its adoption, approximately a quarter of all trans-border Hungarians applied for the Hungarian ID. There are about 850,000 card-holders today. 29 Since its adoption, approximately a quarter of all trans-border Hungarians applied for the Hungarian ID. There are about 850,000 card-holders today. g 5 y 30 As a result of the conflict surrounding the Status Law, the Orba´n Government withdrew public funding from the Federation. 30 As a result of the conflict surrounding the Status Law, the Orba´n Government withdrew public funding from the Federation. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 31 Soon after the announcement of the plan for the referendum it became clear that any legislation on dual citizenship would have to happen unilaterally, as the Romanian president promptly announced his country’s opposition. 31 Soon after the announcement of the plan for the referendum it became clear that any legislation on dual citizenship would have to happen unilaterally, as the Romanian president promptly announced his country’s opposition. 32 On 12 November 2004, President Ferenc Ma´dl, in a speech addressed to the Hungarian Permanent Assembly (MA´ E´RT), spoke of the perception of the referen- dum initiative by external minorities as an act of ‘historical justice’ and added: ‘I call upon Hungarians to use their votes to assume a sense of community with Hungar- ians outside of our borders’ (www.martonaron.hu, last accessed 17 February 2005). 32 On 12 November 2004, President Ferenc Ma´dl, in a speech addressed to the Hungarian Permanent Assembly (MA´ E´RT), spoke of the perception of the referen- dum initiative by external minorities as an act of ‘historical justice’ and added: ‘I call upon Hungarians to use their votes to assume a sense of community with Hungar- ians outside of our borders’ (www.martonaron.hu, last accessed 17 February 2005). 33 ‘A ketto˝s a´llampolga´rsa´gro´l, Adatok, a´lla´sfoglala´sok, elemze´sek’ [On dual citizenship, data, opinions and analyses]. www.martonaron.hu, last accessed 17 February 2005. 33 ‘A ketto˝s a´llampolga´rsa´gro´l, Adatok, a´lla´sfoglala´sok, elemze´sek’ [On dual citizenship, data, opinions and analyses]. www.martonaron.hu, last accessed 17 February 2005. p y ] 7 y 5 34 Hungarian citizens who had emigrated from Hungary retained their Hungarian citizenship. This, however, did not apply to former citizens of Hungary in the neighbouring states who had lost their Hungarian citizenship as a result of the peace treaties that redrew the borders of the Hungarian state. The possibility of inheriting Hungarian citizenship applies only to people whose right to Hungarian citizenship is derived from their connection to the territory of the state of Hungary as delineated in the Paris Peace Treaty of 1947. 34 Hungarian citizens who had emigrated from Hungary retained their Hungarian citizenship. This, however, did not apply to former citizens of Hungary in the neighbouring states who had lost their Hungarian citizenship as a result of the peace treaties that redrew the borders of the Hungarian state. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE Minorities them- selves do not speak with a single voice because the attitudes of the different groups of which they are composed are derivative of the long-term view each of these groups takes on the possibilities of negotiating a better status for themselves in their host states. Even if the idea of dual citizenship enjoyed the support of the majority of trans-border Hungarians, this support would be based on a demagogic-populist misrepresentation of what is actually possible. p y p 40 T. Bauer, ‘Ketto˝s Kapitula´cio´’ [Dual Capitulation], Ne´pszabadsa´g, 8 January 2004. y 40 T. Bauer, ‘Ketto˝s Kapitula´cio´’ [Dual Capitulation], Ne´pszabadsa´g, 8 January 2004. 40 T. Bauer, Kettos Kapitulacio [Dual Capitulation], Nepszabadsag, 8 January 2004. 41 Art. 61 of the Constitution provides a fundamental right to free expression and obtaining as well as freely disseminating information of interest to the general public. A separate law regulates its implementation (Act LXIII of 1992). 41 Art. 61 of the Constitution provides a fundamental right to free expression and obtaining as well as freely disseminating information of interest to the general public. A separate law regulates its implementation (Act LXIII of 1992). 42 See ‘Western European Countries Tend to Follow a Liberalizing Trend towards Citizenship Policies. Interview with Rainer Baubo¨ck’. www.migrationonline.cz. 42 See ‘Western European Countries Tend to Follow a Liberalizing Trend towards Citizenship Policies. Interview with Rainer Baubo¨ck’. www.migrationonline.cz. 42 See ‘Western European Countries Tend to Follow a Liberalizing Trend towards Citizenship Policies. Interview with Rainer Baubo¨ck’. www.migrationonline.cz. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE 175 37 J. Kis, ‘Mie´rt megyek el szavazni?’ [Why am I taking part in the voting?], Ne´pszabad- sa´g, 20 November 2004. 37 J. Kis, ‘Mie´rt megyek el szavazni?’ [Why am I taking part in the voting?], Ne´pszabad sa´g, 20 November 2004. 37 J. Kis, ‘Mie´rt megyek el szavazni?’ [Why am I taking part in the voting?], Ne´pszabad- sa´g, 20 November 2004. 38 Especially in the European Convention on Nationality (1997) ratified by Hungary in 2002 which stipulates in art. 2/a that ‘‘‘nationality’’ means the legal bond between a person and a State and does not indicate the person’s ethnic origin’, and restricts the ‘recovery of former nationality’ of a given state to those residing on its territory. 38 Especially in the European Convention on Nationality (1997) ratified by Hungary in 2002 which stipulates in art. 2/a that ‘‘‘nationality’’ means the legal bond between a person and a State and does not indicate the person’s ethnic origin’, and restricts the ‘recovery of former nationality’ of a given state to those residing on its territory. 39 For example, the biggest Hungarian party of the large Hungarian minority of Romania, which has substantial representation in the Romanian parliament and government, has traditionally been, at best, lukewarm about dual citizenship. However, the most vocal advocates of trans-border Hungarian citizenship also come from Romania and they also rely on a substantial constituency. Minorities them- selves do not speak with a single voice because the attitudes of the different groups of which they are composed are derivative of the long-term view each of these groups takes on the possibilities of negotiating a better status for themselves in their host states. Even if the idea of dual citizenship enjoyed the support of the majority of trans-border Hungarians, this support would be based on a demagogic-populist misrepresentation of what is actually possible. 39 For example, the biggest Hungarian party of the large Hungarian minority of Romania, which has substantial representation in the Romanian parliament and government, has traditionally been, at best, lukewarm about dual citizenship. However, the most vocal advocates of trans-border Hungarian citizenship also come from Romania and they also rely on a substantial constituency. KIN-STATE RESPONSIBILITY AND ETHNIC CITIZENSHIP: THE HUNGARIAN CASE There are altogether 2.7 million non-resident Italian citizens, which is equivalent to about 3 per cent of the resident citizenry of Italy, as opposed to the size of the trans-border Hungarian population that represents 30-35 per cent of Hungary’s current citizenry. MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH Minority Protection, 152-176. Hokkaido: Slavic Research Centre. http://src-h.slav.hokudai.ac.jp/coe21/publish/no4_ses/contents.html. Minority Protection, 152-176. Hokkaido: Slavic Research Centre. http://src h slav hokudai ac jp/coe21/publish/no4 ses/contents htm Nagy, B. (2004), Ketto˝s A´ llampolga´rsa´g: Nemzet, a´llam, polga´r: kise´rlet a fogalmi rendterem- te´sre [Dual Citizenship: Nation, State and Citizen: An Attempt at Conceptual Clarifi- cation]. www.martonaron.hu. Scho¨pflin, G. (2004), ‘Citizenship and Ethnicity: The Hungarian Status Law’, in Z. Ka´n- tor, B. Majte´nyi, O. Ieda, B. Vizi & I. Hala´sz (eds.), The Hungarian Status Law: Nation Building and/or Minority Protection, 87-104. Hokkaido: Slavic Research Centre. http://src-h slav hokudai ac jp/coe21/publish/no4 ses/contents html Stewart, M. (2004), ‘The Hungarian Status Law: A New European Form of Transnational Politics?’, in Z. Ka´ntor, B. Majte´nyi, O. Ieda, B. Vizi & I. Hala´sz (eds.), The Hungarian Status Law: Nation Building and/or Minority Protection, 120-151. Hokkaido: Slavic Re- search Centre. http://src-h.slav.hokudai.ac.jp/coe21/publish/no4_ses/contents.html. To´th, J. (1995), ‘Who are the Desirable Immigrants in Hungary under the Newly Adopted Laws?’, in M. Fullerton, E. Sik & J. To´th (eds.), Refugees and Migrants: Hungary at a Crossroads. Yearbook of the Research Group on International Migration, 57-68. Buda- pest: Institute for Political Science, Hungarian Academy of Sciences. To´th, J. (2000), ‘Diaspora in Legal Regulations: 1989-1999’, in I. Kiss & C. McGovern (eds.), New Diasporas in Hungary, Russia and Ukraine: Legal Regulations and Current Politics. Budapest: Open Society Institute/COLPI. To´th, J. (2003), ‘Connections of Kin-minorities to the Kin-state in the Extended Schengen Zone’, European Journal of Migration and Law 5: 201-227. p J f g 5 7 To´th, J. (2004a), Sta´tusjogok. Kisebbse´gkutata´s Ko¨nyvek. Budapest: Lucidus Kiado´. 04a), Sta´tusjogok. Kisebbse´gkutata´s Ko¨nyvek. Bud To´th, J. (2004b), ‘Aspects of Analysing the Minority Problem’, in L. Szarka (ed.), Hungary and the Hungarian Minorities: Trends in the Past and Our Time. Atlantic Studies on So- ciety in Change 122, 223-244. New York: Columbia University Press. To´th, J. (2004c), ‘Ketto˝s a´llampolga´rsa´got Ne´pszavaza´ssal?’ [Dual Citizenship by Referen- dum?], Fundamentum 2: 80-87. To´th, J. (2005), ‘Principles and Practice of Nationality Law in Hungary’, Regio 8: 21-39. To´th, J. & E. Sik (2003), ‘Joining an EU identity. Integration of Hungary or the Hungar- ians?’, in W. Spohn & A. Triandafyllidou (eds.), Europeanisation, National Identities and Migration. Changes in Boundary Constructions between Western and Eastern Europe. London: Routledge. To´th, P. P. (1997), Haza csak egy van? Meneku¨lo˝k, beva´ndorlo´k, u´j a´llampolga´rok 1988-1994 [Is there only one homeland? Refugees, immigrants, new citizens]. Budapest: Pu¨ski. Vizi, B. Bibliography Baubo¨ck, R. (2006), ‘Citizenship: International, State, Migrant and Democratic Perspec- tives’, in J. Palme & K. Tamas (eds.), Globalizing Migration Regimes, 144-166. Avebury: Ashgate. Csergo˝, Z. & J. Goldgeier (2004), ‘Nationalist Strategies and European Integration’, in Z. Ka´ntor, B. Majte´nyi, O. Ieda, B. Vizi & I. Hala´sz (eds.), The Hungarian Status Law: Nation Building and/or Minority Protection, 270-302. Hokkaido: Slavic Research Cen- tre. http://src-h.slav.hokudai.ac.jp/coe21/publish/no4_ses/contents.html. Faist, T. (2005), ‘Transnationalization in International Migration: Implications for the Study of Citizenship and Culture’, Transnational Communities Programme: Working Paper Series 8. www.transcomm.ox.ac.uk. Fowler, B. (2002), ‘Fuzzing citizenship, nationalising political space: A framework for in- terpreting the Hungarian ‘‘status law’’ as a new form of kin-state policy in East-Cen- tral Europe’, Centre for Russian and East European Studies, European Research Institute, University of Birmingham, Working Paper 40. www.one-europe.ac.uk. Fullerton, M., E. Sik & J. To´th (eds.) (1997), From Improvisation toward Awareness? Con- temporary Migration Politics in Hungary. Yearbook of the Research Group on Interna- tional Migration. Budapest: Institute for Political Science, Hungarian Academy of Sciences. Ka´ntor, Z. (2004), ‘Re-institutionalizing the Nation – Status Law and Dual Citizenship’, Regio 1: 40-49. Kis, J. (2004a), ‘Nemzetegyesı´te´s vagy kisebbse´gve´delem’ [Unification of the Nation or Minority Protection], E´let e´s Irodalom 51: 3-4. Kis, J. (2004b), ‘The Status Law: Hungary at the Crossroads’, in Z. Ka´ntor, B. Majte´nyi, O. Ieda, B. Vizi & I. Hala´sz (eds.), The Hungarian Status Law: Nation Building and/or MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH 176 MA´ RIA M. KOVA´ CS AND JUDIT TO´ TH (2003), ‘A hata´ron tu´li olaszokto´l a ku¨lfo¨ldo¨n e´lo˝ olasz a´llampolga´rokig – az olasz a´llampolga´rsa´g kiterjeszte´se az ezredfordulo´n’ [From trans-border Italians to Italian citizens living abroad], Kisebbse´gkutata´s 4: 685-694. 6 Politics of citizenship in post-communist Romania: Legal traditions, restitution of nationality and multiple memberships Constantin Iordachi A member of the European Union since January 2007, Romania has brought a rich historical experience into the union that goes all the way back to long-lasting Byzantine and Ottoman imperial legacies and to the more recent successive waves of Western- and Soviet-style mod- ernisation. Given Romania’s multiple historical legacies, which com- bine pan-European trends with Central and Southeast European regio- nal features, the history of Romanian citizenship legislation challenges the clear-cut and neatly defined analytical dichotomies, such as ‘old’ versus ‘new’ states and ‘civic’ versus ‘ethnic’ or ‘inclusive’ vs. ‘exclusive’ citizenship doctrines, which are erroneously regarded as corresponding to ‘Western’ vs. ‘Eastern’ historical experiences (for a critique of such views, see Iordachi 2006). This chapter focuses on the interplay of historical legacies in the evo- lution of Romanian citizenship, underlining continuities and ruptures in the transition from communist to post-communist policies of na- tional membership. Methodologically, I distinguish between two insti- tutional dimensions of citizenship: the legal category of nationality that defines membership in a state understood as a territorial and national organisation; and citizenship as rights and duties stemming from membership and participation in a political community. The first di- mension encompasses the construction of legal and political borders between state citizens and aliens. The second dimension refers to the civil, political and social entitlements of citizenship (Marshall 1950).1 p p ( 95 ) Like the other contributions in this book, this chapter focuses on the first legal dimension of nationality, which is regulated mainly by con- stitutions and citizenship laws. Particular attention is devoted to the most contested component of post-communist Romania’s nationality policy: the right to reacquisition of nationality by former citizens and their descendants living outside the state’s (post-1945) borders. This policy resulted in the massive (re-)naturalisation of Moldovan and Uk- rainian citizens stripped of their Romanian nationality following the 1940-1941/1944 Soviet occupation of Bessarabia and Northern Bukovi- na. I argue that Romania’s policy regarding the restoration of national- 178 CONSTANTIN IORDACHI ity should be placed in the political and analytical context of post-com- munist restitution. Restitution in its various forms has been an impor- tant component of legal systems since ancient times, referring to the return of a person to his or her original status and of restoration of his or her rights or property, prior to a loss, injury or abuse. 6 Politics of citizenship in post-communist Romania: Legal traditions, restitution of nationality and multiple memberships In post-com- munist Central and Eastern Europe, the concept took on a peculiar le- gal meaning because it denoted the process of undoing communist le- gal and political abuses and dispossessions. Restitution was central to post-communist legal and political transformation, which was aimed at the restoration of the status-quo ante (before the communist takeover). From this perspective, the legal ‘revolutions’ initiated in 1989, which led to the dismantling of the communist regimes, should be under- stood more in the original meaning of the term ‘revolution’, that is, as a movement of rotation, which returns to an original position. g p In post-communist East Central Europe, practices of restitution have been applied to a wide range of societal domains. Yet, to date, research has focused almost exclusively on the reconstruction of individual and communal property rights. An important but largely understudied as- pect is the restitution of nationality to former de-naturalised citizens. In the context of post-communist nationalist upsurge, this practice was not simply a necessary legal reparation for past injustices; it was also seen as a means of recreating the pre-communist citizenry and na- tional community and as a means for the restoration of national iden- tity, allegedly lost under communist rule, which was defined as a re- gime of Soviet occupation. The gap between political visions of recreat- ing the inter-war national ‘imagined community’ and the far-reaching practical complications this project generated led to a multitude of poli- tical, legal-procedural and diplomatic crises, with wide domestic and in- ternational implications. These complications and the debates sur- rounding them account for the numerous shifts and turns of Roma- nia’s policy regarding the restitution of nationality, which culminated in the temporary suspensions of the process of restitution during the period from 2001 to 2007. 6.1.1 The making of Romanian nationality: Pre-communist legacies 6.1.1 The making of Romanian nationality: Pre-communist legacies Modern Romania was established in 1859 through the state union of the principalities of Moldova and Wallachia. After their establishment in the fourteenth century, the two principalities were part of the Byzan- tine political tradition and Eastern Orthodox religious commonwealth. They fell under Ottoman domination in the fifteenth century and were subject to Ottoman suzerainty until 1878; and thereafter experienced POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 179 major stages of nation- and state-building during the ‘long nineteenth century’, with such landmarks as the Congress of Paris (1856), the Congress of Berlin (1879), and the Versailles Peace Treaties (1919- 1920), all part of successive geo-political reorganisations of Southeast Europe by the great European powers. One can identify several histori- cal periods in the development of Romanian citizenship, corresponding to major stages in the process of nation-building and state-building: 1859-1918, 1918-1937, 1937-1944, 1944-1989 and 1989 to the present. 59 9 9 937 937 944 944 9 9 9 9 p The legal bases of the modern Romanian nationality were set by the 1865 Civil Code, which emulated the French legal system put forward in the 1804 Code Civil, based on the ius sanguinis principle in ascrib- ing nationality at birth, and a selective policy of naturalisation of aliens, favouring those born and raised in the country. The French model was nevertheless amended in several respects: the Romanian Civil Code, soon supplemented but also partially (and restrictively) modified by a modern Constitution adopted in 1866, introduced Christian religion and Romanian ethnicity as criteria for naturalisation, both absent in the Code Civil. Firstly, until 1879, Jews were excluded from Romanian nationality, on the basis of their religion, even if born and raised in the country for generations; on this basis, they were deprived of substantial civil, social and political rights. In 1879, under pressure from the inter- national community, Jews were granted access to naturalisation; how- ever, instead of a fast and collective citizenship emancipation, Jews were only allowed to apply for individual naturalisation that could be granted by parliament by means of a special law adopted for each indi- vidual case. This lengthy and highly bureaucratic practice explains the small number of naturalisations before the First World War, the great majority of Jews remaining non-citizen permanent residents. 6.1.1 The making of Romanian nationality: Pre-communist legacies Secondly, the Romanian state pursued an active national policy: ethnic Roma- nians from neighbouring countries immigrating to the ‘mother coun- try’ were granted privileged access to nationality, without a naturalisa- tion stage, i.e. without the necessity of having lived in the country for a period of ten years. (They could prove their ethnic origin by means of witness accounts or certificates of ethnicity issued by Romanian com- munities abroad and further corroborated by their knowledge of the Romanian language.) This practice, euphemistically called ‘recognition’ of citizenship, was justified by the incomplete ethnic boundaries of the Romanian nation-state and legitimised an irredentist policy of incor- porating Romanians from Austria-Hungary, Russia and the Balkans. This legal model functioned until the First World War, with only minor modifications necessitated by the annexation of Northern and Southern Dobrogea/Dobrudja from the Ottoman Empire and from Bulgaria in 1878 and in 1913, respectively (Iordachi 2002). 180 CONSTANTIN IORDACHI The socio-political upheaval of the Great War brought significant changes to the Romanian citizenry. Firstly, interwar ‘Greater Romania’ almost doubled in size and population compared to the pre-war ‘Old Kingdom’ by incorporating the province of Bessarabia (situated be- tween the rivers Prut and Dniestr and annexed by Russia in 1812) and territories that had been previously part of Austria-Hungary – namely Bukovina, Transylvania, the Banat, Maramures¸ and the Partium. For the first time in their modern history, ethnic Romanians thus lived in a single ‘national and unitary state’, as Greater Romania was defined by the 1923 Constitution. Although dominated by Romanians, the new state also included a high ratio of ethnic and religious minorities: 28.1 per cent of the total population in 1930, including Hungarians (7.9 per cent), Germans (4.1 per cent), Jews (4.0 per cent), Ruthenians (3.2 per cent), Russians (2.3 per cent), Bulgarians (2.0 per cent), Gypsies (1.5 per cent), Turks (0.9 per cent) and Tartars (0.1 per cent) (Institutul Central de Statistica˘ 1940: 44-45). Secondly, the events of the war gen- erated an unprecedented liberalisation of access to citizenship. Under international pressure, Romania took final steps towards the full civil and political emancipation of Jews. Adopted in February 1924, the new law on nationality granted citizenship to all legal inhabitants of the Old Kingdom and the annexed territories. 6.1.1 The making of Romanian nationality: Pre-communist legacies It also preserved the main fea- tures of Romanian citizenship doctrine by stipulating three main ways of acquiring nationality: (1) by descent, according to the principle of ius sanguinis; (2) by marrying a Romanian man; and (3) by naturalisation, after having fulfilled a residence requirement of ten years following the declaration of intent to naturalise. Foreigners born and raised in Roma- nia were exempt from the mandatory residential stage, provided they requested naturalisation upon reaching maturity. Thirdly, the liberalisa- tion of access to citizenship was accompanied by major socio-political reorganisations of the country. Comprehensive reforms such as univer- sal male suffrage (1918), massive land redistribution (1921) and the new liberal Constitution (1923) remodelled the country into a multi- party parliamentary monarchy While the new liberal regime remained largely unconsolidated and marred by major regional and socio-political cleavages, it is important to note that political pluralism was preserved almost throughout the entire interwar period, free parliamentary elections being held as late as 1937, at a time when the European continent was mostly dominated by authoritarian political regimes. Ironically, the 1937 parliamentary elections turned out to be Romania’s last free elections before 1990. The Romanian citizenship doctrine suffered significant changes just prior to and during the Second World War, with long-term legal conse- quences. Firstly, under the joint pressure of right-wing organisations from below and the authoritarian tendencies of King Carol II (1930- POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 181 1940) from above, in 1938, the multiparty parliamentary regime col- lapsed, being replaced by a (short-lived) regime of royal dictatorship (1938-1940). The new political changes were also reflected in a new law on citizenship, adopted in 1939 at King Carol’s initiative. The law did not alter the main principles of ascribing citizenship, but intro- duced numerous changes in the procedure of naturalisation, placed under the control of the Ministry of Justice. The most important change was that naturalised citizens were granted full political rights only six years after the act of naturalisation. The law served as a basis of Romania’s nationality policy until 1947/1952, when it was amended and then fully abolished by the new communist regime; however, in the post-communist period, many of its provisions have been rein- stated. 6.1.1 The making of Romanian nationality: Pre-communist legacies Secondly, the political accession of the extreme right led to the massive de-naturalisation of Romanian Jews, their deprivation of sub- stantive political and civil rights during the royal dictatorship of King Carol II and their partial deportation and extermination during the dic- tatorial regime of Ion Antonescu (1940-1944). Thirdly, during the Sec- ond World War, Greater Romania suffered major territorial losses. In 1940 – under the new political conditions created by Nazi Germany’s military domination in Europe – Romania was forced to cede North- Western Transylvania to Hungary and Southern Dobrogea to Bulgaria. Following the 1939 ‘Ribbentrop-Molotov’ Non-Aggression Pact, which divided the spheres of influence between Nazi Germany and the USSR, on 28 June 1940 the Soviet army occupied the provinces of Bes- sarabia and Northern Bukovina. Romania ceded these provinces with- out resistance, but in June 1941, it joined Nazi Germany’s anti-Soviet war and managed to temporarily liberate the occupied territories (1941- 1944). In 1944, a coalition of communists and democratic parties ousted Antonescu from power, reinstated the 1923 Constitution, abolished all anti-Semitic discriminatory laws and restored citizenship to all denatur- alised inhabitants. The new democratic Romania also joined the anti- fascist military coalition and restored its control over Northwestern Transylvania. The return to the legal and territorial order of interwar Greater Romania was nevertheless hampered by several factors: firstly, in 1944, the Soviets reoccupied Bessarabia, which became the Molda- vian Soviet Socialist Republic (MSSR) and included also the Transnis- tria region (a long conundrum situated across the river Dniestr) and Northern Bukovina, which was granted to the Ukraine (together with the southern part of Bessarabia, detached from the MSSR). Secondly, the Soviets intervened in the political process by installing the tiny Communist Party in power and initiating the forceful Sovietisation of Romania. The understanding of the communist legacy is essential to 182 CONSTANTIN IORDACHI our effort to grasp the main features of citizenship policies in the post- communist period. 6.1.2 A new legal beginning: Nationality under the communist regime 6.1.2 A new legal beginning: Nationality under the communist regime The communist regime implemented radical changes to Romania’s le- gal and political system (1945-1989). 6.1.1 The making of Romanian nationality: Pre-communist legacies Through three consecutive consti- tutions (1948, 1952, 1965), major changes to the civil code and an all- encompassing set of laws regulating every sphere of activity, the new political regime broke with the past and redefined the nature of the state by emulating the Soviet model of development. As a legal bound- ary defining membership in the national and social-political commu- nity, citizenship legislation was an essential dimension of the commu- nist political transformation and was therefore subject to many revi- sions in 1947, 1948, 1952, 1954, 1956 and 1971, reflecting the shifts and turns of the political regime. The communist regime redefined the conditions of acquisition and loss of nationality. With the stroke of a pen, Decree No. 33/1952 abol- ished all existing laws on citizenship (art. 10); instead, in two pages and ten articles, it set new rules for the acquisition of Romanian na- tionality, defining the legal boundaries of the socialist nation. Roma- nian nationality was ascribed at birth iure sanguinis to children of at least one Romanian parent. In a major departure from the legal tradi- tion of the country, the decree thus allowed the transmission of nation- ality on the paternal as well as on the maternal line in mixed families, provided that at least one parent lived in Romania. This transmission could not result in dual citizenship: upon adulthood, children born into mixed marriages had to choose between the nationality of the mother or the father, by parental accord. Combined with Decree No. 130/1949 (which allowed official investigations of the paternity of children, thus eliminating ‘illegitimacy’ as an accepted legal category), these stipula- tions contributed to the formal legal equality of women, since they were legally enabled to transmit their own nationality to their children. The decree discontinued the traditional ius soli policy of naturalisa- tion of aliens born in the country and the privileged naturalisation of ethnic Romanians living abroad. Decisions on the naturalisations of aliens, as well as on the renunciations or withdrawals of nationality were taken by the Presidium of the Grand National Assembly, estab- lished in 1947 after the abolition of the monarchy and the proclama- tion of the republic. After 1958, political divergences with Moscow and the move of the Romanian leaders towards political autonomy and a ‘national’ path to building socialism led to significant changes in the official socialist ideology. 6.1.1 The making of Romanian nationality: Pre-communist legacies With the retreat of the Red Army (1958), Romanian leaders POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 183 renounced external sources of legitimisation and recuperated tradi- tional themes of nationalist ideology in an attempt to gain broader do- mestic support (Shafir 1985). Initiated under the last years of Gheorghe Gheorghiu-Dej’s rule, the nationalist turn of the regime, which was in- tensified during the rule of Nicolae Ceaus¸escu (1965-1989), resulted in a syncretism between nationalism and a ‘decayed Marxism,’ best de- scribed by the concept of ‘national-communism’ (Verdery 1991). y p ( y 99 ) The new nationalist orientation of the regime was also reflected in the definition given to the legal principle governing the ascription of nationality at birth. Adopted in 1971, the new Law on Romanian citi- zenship reconfirmed the principle of ius sanguinis as the very founda- tion of a homogeneous national community and imbued it with nation- alist connotations. Art. 5 of the law read: As an expression of the relationship between parents and chil- dren, of the uninterrupted continuity of the fatherland of previous generations that fought for social and national freedom, children born of Romanian parents on the territory of the Socialist Repub- lic of Romania are Romanian citizens. (emphasis added)2 This definition linked the application of the ius sanguinis principle to birth on the territory and uninterrupted continuity of the nation in its ‘fatherland’. It made reference not only to parents and children in the transmission of nationality, but also to generations. Other articles of the law made evident that this link operated only at symbolic-ideologi- cal level, the principle of ius sanguinis being in fact also applied to children of citizens born outside the country. The argument was never- theless meant to emphasise the ‘autochthonous’ roots of the Romanian people and the historical ‘symbiosis’ between the nation, its territory and the new socialist citizenry, thus alluding to the idea of organic na- tionalism elaborated by Romantic nationalist thinkers in the first half of the nineteenth century and brought to political prominence by right- wing organisations in the interwar period. In addition to ascription through ius sanguinis at birth, Romanian nationality could also be acquired by naturalisation, by adoption and by repatriation. 6.1.1 The making of Romanian nationality: Pre-communist legacies Naturalisation was granted at adulthood by the Council of State (a leading organ of the republic created in 1961) to persons who: a) were born in Romania and lived there at the time of their request; b) were born abroad but had lived uninterruptedly in Romania for at least five years; c) were married to a Romanian citizen and had lived in the country for at least three years. In addition to the residence condition, aliens were required to: 1. prove, through their behaviour and attitude, attachment to the Ro- manian state and the Romanian people; 1. prove, through their behaviour and attitude, attachment to the Ro- manian state and the Romanian people; 184 CONSTANTIN IORDACHI 2. be eighteen years of age or above; 2. be eighteen years of age or above; 2. be eighteen years of age or above; 2. be eighteen years of age or above; 3. undertake socially useful work or prove sufficient material means of subsistence; 4. renounce their foreign nationality or any commitment of loyalty to a foreign power and swear allegiance to Socialist Romania. The Romanian state reserved its right to unilaterally withdraw the na- tionality of those individuals who ‘broke with the fatherland by cross- ing the border clandestinely or, after relocating their domicile abroad, assumed a foreign nationality, worked against the interests of the coun- try or enrolled in a foreign army’ (art. 19). Access to nationality was firmly controlled by the executive power; Ceaus¸escu alone, as the presi- dent of the Council of State (from December 1967 to December 1989), could grant or withdraw Romanian nationality. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 185 6.2.1 Acquisition at birth The ascription of Romanian nationality at birth is governed solely by the principle of ius sanguinis, being granted to children who are: 1) born within the territory of the country to two Romanian citizen par- ents; 2) born within the territory of the country in mixed marriages with only one Romanian citizen; and 3) born abroad to at least one Ro- manian parent. That the principle of ius soli is of no relevance in the ascription of nationality at birth is made evident by the provisions con- cerning the nationality of newly-born children of unknown parents; they are granted citizenship not on the basis of their birth on Roma- nia’s territory, but under the assumption that their parents held Roma- nian nationality (art. 5). Evidence to the contrary results in loss of na- tionality, followed by the obligation of naturalisation (art. 30). In order to make it clear that this procedure does not constitute an ius soli as- cription of nationality, a 2003 amendment to the citizenship law re- phrased art. 5 to read that the child found on Romanian territory ‘is considered to be [instead of ‘is…’] a Romanian citizen’ (art. 5, 3/1; em- phasis added). 6.2 Democratic transformation: Current regulations on ascription, acquisition and loss of nationality The 1989 collapse of the communist regime and the gradual democra- tisation of the political system had a powerful impact on Romanian ci- tizenship legislation, resulting in the redefinition of the legal criteria of membership in the national community. Without significant dissident or reformist movements during the communist period on which to build the process of democratisation, post-communist Romania mod- elled its legal and political systems on the interwar political regime: the restitution of urban and land property, the recreation of political parties and parliament’s structure and organisation were all shaped by its pre- communist tradition. Yet, in many ways, the communist legacy deeply affected the society and could not be written off as a simple ‘parenth- esis’ in the country’s development. Citizenship legislation is a relevant example in this respect. Adopted in March 1991, the new Law on Romanian Citizenship was modelled on the 1939 Law, abrogated by the communist regime in 1952; yet it also preserved many provisions of the 1971 Law, resulting in a novel synthesis. The 1991 Law specified four main ways of acquiring nation- ality by different categories of inhabitants: 1. ascription at birth, through transmission iure sanguinis to descen- dants of citizens, provided at least one of the child’s parents holds Romanian nationality at the time of the child’s birth; 2. adoption of an alien child by a Romanian citizen; 3. by the act of repatriation of former citizens; and 4. upon request, by naturalisation of aliens born in Romania or who have lived there for a certain period of time. 6.2.2 Naturalisation The 1991 Law granted naturalisation, upon request, to adult aliens and their minor children, who were: a) born in Romania and lived there at the time of their request; b) born abroad but had lived uninterruptedly in Romania for at least five years; c) married to a Romanian citizen and had lived in the country for at least three years. In addition to the resi- dence requirement, applicants also had to: 1. prove, through their behaviour and attitude, their attachment to the Romanian state and people; 1. prove, through their behaviour and attitude, their attachment to the Romanian state and people; 2. be eighteen years of age or above; 3. prove they possess sufficient material means of existence; 4. have a clean criminal record; and 5. have ‘sufficient knowledge of the Romanian language’ in order to 5. have ‘sufficient knowledge of the Romanian language’ in order to be able to integrate into society. 5. have ‘sufficient knowledge of th be able to integrate into society. Although the naturalisation requirements have been amended several times since 1991 (see next section), the procedure of naturalisation, which is patterned on the 1939 Law, has remained the same. Applica- tions have to be filed personally or through authorised attorneys to a Commission of Citizenship set up by the Ministry of Justice and made up of five judges of the Bucharest Court, appointed for four years by the president of the court. Upon their registration, requests for natura- lisation are published in the Official Monitory of Romania, Part III, and 186 CONSTANTIN IORDACHI are subsequently examined by the Commission. Decisions on naturali- sation are taken by the Romanian government upon the recommenda- tion of the commission and are published in the Official Monitory of Romania, Part I. Naturalisation becomes effective upon the would-be citizens taking the oath of loyalty in front of the Ministry of Justice, a sub-secretary of state, or the chief of diplomatic missions abroad. y p Romanian citizenship legislation underwent substantial amend- ments, additions and modifications in 1999, 2002, 2003 and 2008 which were necessitated by the process of European integration and the intensification of immigration and emigration. As a reaction to growing migration tides, coupled with EU pressure to strictly control external acquisitions of Romanian citizenship, requirements for natur- alisation have been tightened. 6.2.2 Naturalisation The mandatory residence period for the naturalisation of foreigners was increased from five to seven years in 1999, and to eight years in 2003 (albeit reduced to five years for for- eigners married to Romanian citizens). In order to eliminate cases of fraud, a 1999 amendment demands a ‘continuous, stable and legal’ re- sidence, while a 2003 amendment requires foreigners applying for nat- uralisation to effectively relocate to Romania, spend at least six months per year in the country and pay taxes there.3 In 1999, the residency re- quirement was reduced to half for persons of international reputation, a privilege granted since 2003 also to those who have invested more than 500,000 euros in Romania. In 2008, this sum was increased to 5 million euros and the shorter residency requirement was extended also to refugees and to citizens of EU Member States. Moreover, besides the longer residency requirement, a 1999 amend- ment to art. 9 of the law introduced additional conditions for naturali- sation, such as sufficient knowledge of the Romanian language, of ‘ele- mentary notions of Romanian culture and civilisation,’ of the Constitu- tion and, since 2003, of the national anthem. Applicants for naturalisation also need to sign a declaration of loyalty to the Roma- nian state. Persons suspected of terrorism and those who present po- tential threats to national security are ineligible for naturalisation. 6.2.3 Loss of nationality According to the 1991 Law, Romanian nationality can be forfeited: 1) as a result of the unilateral withdrawal by the state; 2) through voluntary individual renunciation by citizens; 3) or in other special cases, such as the adoption of children by foreign citizens (art. 24). Firstly, the Roma- nian state could terminate the nationality of those individuals who had obtained their naturalisation by fraud, who worked abroad against the interests of the country or who enrolled in an enemy army (art. 25). Secondly, the Law allowed Romanian citizens to renounce their nation- POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 187 ality ‘for solid reasons’ according to a special procedure and pending of- ficial approval, provided they are not under trial and have no debts to private or public parties (art. 27). Since 2001, in particular, Romania has become a major source coun- try of labour migration, especially to Italy and Spain; taking advantage of the freedom of movement, an estimated two to two and a half mil- lion Romanian citizens currently live and work abroad either tempora- rily or permanently. The most recent stipulations on the loss of Roma- nian nationality express the Romanian state’s concern to preserve legal ties with its citizens living abroad and to reduce the number of external renunciations of nationality. To this end, in 2003, the Romanian state waived its right to unilaterally terminate the nationality of ‘natural citi- zens’ who had obtained it at birth (art. 24.d). In addition, in 2007, the procedure for the individual renunciation of citizenship became even more complex, costly and bureaucratic. These stipulations, combined with the fact that the principle of ius sanguinis operates externally without generational limits (so that Romanian nationality can be passed on indefinitely to subsequent generations born abroad even in cases of acquisition of a new nationality as long as parents do not re- nounce their nationality of origin), account for the fact that the number of individual renunciations or losses of Romanian nationality has been rather small – varying from 12,594 persons in 1999 to 10,938 persons in 2005 (National Institute of Statistics 2006: 81-83) – especially when compared to the massive number of Romanian citizens living abroad on a temporary or even permanent basis. 6.2.4 Restitution of nationality and multiple memberships The most debated provision of the post-communist citizenship legisla- tion was the right to restitution of nationality to former citizens. A tra- ditional feature of the Romanian modern legal system, the right to re- naturalisation survived in various forms under the communist regime as well. Although the Socialist Republic conceived of itself as a new state and granted citizenship to all inhabitants living in the country, it also (partially) employed the principle of restitution in order to recon- struct Romania’s interwar citizenry on new political bases. Thus, a law passed in 1947 restored Romanian nationality to all those denaturalised during the Second World War under discriminatory legislation or for- eign occupation. The 1952 decree granted Romanian nationality to all inhabitants who had settled in the country by 1920 (the ratification of the Peace Treaty with Austria) but who had failed to qualify for citizen- ship under previous laws. Another decree passed in 1954 reconfirmed Romanian nationality for all those who had held this legal status as of 28 June 1940 and had resided in Romania ever since. Under Soviet 188 CONSTANTIN IORDACHI pressure, the deadline for restitution was chosen specifically to exclude from this right the inhabitants of Soviet-occupied Bessarabia and Northern Bukovina, a clear indication of the limits of the communists’ policy on nationality restitution. p y y The communist regime also permitted the re-naturalisation of for- mer citizens, but granted this right according to strict political criteria. Art. 1.c of the 1952 Decree and art. 7 of the 1971 Law allowed former ci- tizens to reacquire their nationality upon request on the basis of a spe- cial authorisation issued by the Presidium of the National Assembly or, after 1969, by the Council of State. Re-naturalisation was conditional on the renunciation of the claimant’s foreign nationality, repatriation and ‘integration into the socialist society’ (i.e. integration into the work- force) as well as an attachment to the communist political regime, to be affirmed by an oath of loyalty. In exceptional cases, the Council of State authorised former citizens applying for re-naturalisation to main- tain their domicile abroad, but they were expressly required to re- nounce ‘in an authentic form’ their foreign nationality or – in case they did not hold a foreign nationality – ‘any commitment, obligation of fi- delity or oath of loyalty to a foreign state’ (1971 Law, art. 10 and art. 11). 6.2.4 Restitution of nationality and multiple memberships Due to massive violations of human rights and the deterioration of the standard of living, few former citizens applied for repatriation; on the contrary, in the late 1980s, numerous Romanian citizens fled abroad in order to escape political persecution and material hardship. Upon the collapse of the communist regime, the repatriation of pre- viously persecuted persons and the restitution of nationality to former citizens were the major concerns of the new revolutionary power, which was eager to resume ties with the Romanian Diaspora and kin- minorities abroad. On 31 December 1989, the National Salvation Front guaranteed the right of repatriation to all Romanian citizens residing abroad (Decree No. 7). In addition to the repatriation of Romanian citi- zens in exile, the decree also facilitated the reacquisition of nationality by former Romanian citizens living abroad (art. 2), by request, through the act of repatriation. Unlike the 1971 Law, the new decree did not re- quire former citizens re-naturalised in Romania to renounce their for- eign nationality, thus implicitly opening a gate to dual nationality. In May 1990, a new decree passed by the provisional government enlarged the rights to reacquisition of nationality by former citizens. While the 1989 Decree made re-naturalisation conditional on repatria- tion, the 1990 Decree granted former citizens the right to retrieve their Romanian nationality, upon request, ‘even if they hold another nation- ality and they do not establish their domicile in Romania.’4 In doing so, the decree explicitly allowed certain categories of citizens to hold dual nationality for the first time in Romania’s legal history. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 189 The provisions on repatriation and reacquisition of nationality were reconfirmed and enlarged by the 1991 Law on Citizenship. The law sti- pulated three modalities for the reacquisition of Romanian nationality: (1) by repatriation (art. 8); (2) re-naturalisation by request without repa- triation (art. 11); and (3) ‘restoration’ of nationality to former Romanian citizens (art. 35) living in the lost territories of the interwar Greater Ro- mania. Firstly, the law guaranteed former citizens the right to re-natur- alisation through repatriation: ‘The person who has lost Romanian citi- zenship can re-acquire it through repatriation, if he or she expresses a manifest desire to do so’ (art. 8). 6.2.4 Restitution of nationality and multiple memberships (Equally eligible were the inhabitants of Southern Dobrudja ceded by Romania to Bul- garia in 1940, yet no claims to Romanian citizenship were reported from this province). p ) Secondly, in a departure from the established legal tradition of the country that had prohibited dual nationality, the law allowed re-natura- lised former Romanian citizens to retain their foreign nationality as well as their domicile abroad. In doing so, the law generated a novel ca- tegory of non-resident dual citizens living in neighbouring countries. g y g g g Thirdly, compared to regular naturalisations, the restitution of na- tionality was subject to a simpler procedure: re-naturalisation requests could be sent by post or by third-party intermediaries to the Romanian embassies or consulates abroad. Applicants were exempt from consular taxes and the major conditions of naturalisation required of ‘regular’ aliens. Moreover, the process of re-naturalisation did not necessitate an official interview and the personal presence of the claimant in Buchar- est, as the oath of loyalty could be taken at Romania’s diplomatic repre- sentations abroad. It was thus technically possible for a descendant of a former citizen living abroad to ‘reacquire’ Romanian citizenship with- out ever travelling to the country. g y Overall, the legislation on the reacquisition of Romanian nationality was highly expansive, albeit legally ambiguous. It combined the right to re-naturalisation of expatriates and their repatriation with the princi- ple of restoration of nationality to former citizens and their descen- dants living in former historical provinces of interwar Greater Roma- nia, including their right to hold dual nationality. How can one account for these multiple forms of nationality restitution? According to the legislators, the motivations behind these provisions were democratic, as they were meant to redress communist injustice by allowing anti- communist political dissidents or expatriates to reacquire, upon re- quest, their lost rights. Adopted in anticipation of the imminent dis- memberment of the USSR, the March 1991 Law was also animated by implicit nationalist motivations, which aimed to symbolically undo the effects of the Soviet occupation of Bessarabia and Northern Bukovina and to reconstruct the interwar national community. Seen in a histori- cal retrospective, the law thus completed the process of restoration of the citizenship body of interwar Greater Romania. 6.2.4 Restitution of nationality and multiple memberships Secondly, in line with the 1990 De- cree, the 1991 Law allowed reacquisition of nationality by former Ro- manian citizens even without repatriation: ‘[f]ormer Romanian citizens who, before 22 December 1989, have lost their Romanian citizenship for different reasons,’ can reacquire Romanian citizenship by request even if they retain their foreign citizenship and their domicile abroad (art. 37; emphasis added). Thirdly, and most importantly, the 1991 Law introduced a new form of access to Romanian nationality that can be generically called ‘restoration’ or ‘restitution.’ An additional paragraph to art. 37 stipulated that the right to reacquisition of nationality is also granted to all those who ‘were stripped of Romanian citizenship against their will or for reasons beyond their control, and their descendants’ (em- phasis added). Due to the imprecise and ambiguous wording of the law, at first glance, the difference between the second and third forms of re-natura- lisation is not evident: the second referred to those who had lost Roma- nian citizenship ‘for various reasons’, while the third referred to those who had lost citizenship ‘against their will or for reasons beyond their control’. The official interpretation of the law, however, made it evident that the first paragraph referred to those who had lost nationality as a result of individual actions that unilaterally breached their citizenship contract with the Romanian state, while the second concerned those ci- tizens denaturalised en masse as a result of territorial changes. In so doing, the additional paragraph to art. 37 introduced several major in- novations into Romanian citizenship legislation: Firstly, the right to reacquisition of nationality was not restricted only to those persons who had emigrated due to political persecution or were stripped of citizenship by the communist regime; it was also granted to ‘all former citizens and their descendants’ regardless of when or under what conditions they had lost Romanian nationality. Although not specifically mentioned in the text of the law, the main beneficiaries of the policy of restoration of nationality have been the inhabitants of the former Soviet Socialist Republic of Moldova, and those of the pro- vinces of Northern Bukovina and Southern Bessarabia in the Ukraine. Following the Soviet wartime occupation, the inhabitants of these pro- 190 CONSTANTIN IORDACHI vinces were forcefully stripped of their Romanian nationality; the 1991 Law has enabled them to retrieve that legal status. 6.2.4 Restitution of nationality and multiple memberships Initiated in the post- 1944 period (see above the laws adopted in 1947, 1952 and 1954), this process had, under Soviet pressure, left out the inhabitants of Bessara- bia and Northern Bukovina. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 191 6.3 Current political debates The new policy on the restitution of nationality has triggered numer- ous domestic and international political debates. Far from concentrat- ing on legal technicalities, these debates were linked to major political issues such as the communist legacy, the ethnic-cultural boundaries of the nation, issues of state sovereignty and territoriality, diplomatic rela- tions with neighbouring countries and the compatibility of this policy with the European standard on nationality laws and minority protec- tion. 6.3.1 Dual nationality A first set of political debates concerned the right to dual nationality. Was dual nationality permitted only for former citizens reacquiring Ro- manian nationality or was it open to all Romanian citizens? Although the 1991 Law on Romanian Citizenship allowed re-naturalised Roma- nian citizens to hold dual nationality, this provision did not imply a generalised acceptance of dual nationality, but only a tolerance of dual membership of re-naturalised citizens. For other modes of naturalisation, the 1991 Law explicitly eliminated the tolerance of dual nationality. For example, children adopted by aliens lost their Romanian nationality upon acquiring the nationality of their adoptive parents (art. 29). Yet, as noted above, the law did not contain provisions referring to the legal status of Romanian citizens residing abroad who acquired another na- tionality, and did not expressly oblige them to renounce their Roma- nian nationality if the receiving state tolerated dual nationality in natu- ralisations, thus de facto allowing for dual membership. g p The most debated issue was the right of Romania’s ethnic Hungarians to hold dual nationality, mostly in connection with campaigns by politi- cal forces in Hungary for their access to Hungarian nationality (for de- tails on these campaigns, see Kova´cs & To´th in this volume). The Ro- manian-Hungarian post-communist debate over dual nationality was linked to a wider ideological controversy between the two countries over contrasting but also overlapping definitions of the nation (Iordachi 2004: 257-260). These debates originated in the separation of the citi- zenries of the two countries following the collapse of Austro-Hungary after the First World War, and led to numerous diplomatic and territor- ial conflicts. During the communist period, Hungary abandoned the idea of recovering lost territories but focused instead on the issue of kin-minority protection, legitimised by an ethno-cultural definition of the nation. To Hungary’s policy of treating its kin minorities abroad as an integral part of the Hungarian nation and its pretence of monitor- ing their treatment in neighbouring countries, Romania answered with 192 CONSTANTIN IORDACHI a statist definition of the nation according to which all inhabitants of the country – irrespective of their ethnicity – were Romanian citizens and full members of the socialist nation, ethnic Hungarians included. 6.3.1 Dual nationality The Romanian-Hungarian political-diplomatic conflict over the status of ethnic Hungarians in Romania reached a peak in the late 1980s, as became manifest, for example, during the meetings of the Conference for Security and Cooperation in Europe that took place in Vienna (1986-1989); it also continued in the post-communist period, even if at a lower intensity. Leading Romanian politicians criticised the stipula- tions of the 2001 Hungarian Status Law pertaining to Romanian citi- zens of Hungarian origin, agreeing to its implementation only after Hungary granted access to its labour market to all Romanian citizens, irrespective of their ethnicity; they also declared their opposition to the granting of dual nationality to Romania’s ethnic Hungarians. With the liberalisation of the status of dual citizens in Romania in 2003 (see next paragraph), on the one hand, and the failure of the 2004 national referendum in Hungary over granting dual nationality to ethnic Hun- garians living abroad, on the other hand, the debates over the issue faded away from the public agenda, with dual citizenship ceasing to be a matter of political contestation. In retrospect, it is important to note that, while rejecting the right to dual nationality for Romania’s ethnic Hungarians, Romanian policymakers defended this right in the case of Moldovan citizens opting for Romanian nationality. This contradiction can be explained by the fact that Romania acted simultaneously in a double role: as a ‘nationalising state’ in regard to the Hungarian minor- ity in Transylvania and as an ‘external homeland’ in relation to ethnic Romanians in Bessarabia and Bukovina (Iordachi 2004: 32; for a con- ceptualisation of these roles, see Brubaker 1996). p 99 ) A second set of debates concerned the legal status of dual citizens. The 1991 Constitution restricted the political rights of dual citizens, granting access to ‘public office or dignity, civil or military,’ only to per- sons ‘whose citizenship is only and exclusively Romanian, and whose domicile is in Romania’ (art. 16.3; emphasis added). Gradually, the great increase in the number of dual citizens led to a ‘normalisation’ of their status. In 2003, as part of numerous amendments to the Consti- tution, the restrictions on the political participation of dual citizens were lifted. 6.3.1 Dual nationality Currently, the only condition of eligibility to public office, including the parliament and the presidency, is ‘Romanian citizenship and domicile in the country.’ The restoration of Romanian nationality to Moldovan and Ukrainian citizens has generated a new category of non-resident dual citizens. What is the legal status of these absentee citizens? According to the Roma- nian legislation, non-resident dual citizens acquired automatic access to full social and political rights, except for the rights and obligations POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 193 that are temporarily discontinued for citizens residing abroad, such as the obligation to pay taxes and perform one’s military service (also dis- continued for resident citizens since 2006), and – until 2003 – eligibil- ity for public offices and awards (restricted for dual citizens; see above paragraph). It is intriguing to note, however, that the public debates over the restoration of Romanian nationality and the right to dual na- tionality focused on the national and geo-political effects of this policy, mostly in connection to Romania’s relation to the Republic of Moldova and, to a lesser extent, the Ukraine. The debates did not address the question of the new citizens’ potential socio-political integration into Romanian society or the devaluation of citizenship implied by the pol- icy of granting nationality to persons who have not proven their knowl- edge of the country’s legislation and might not have even visited Roma- nia. The massive numbers of restitutions of Romanian citizenship to Moldovan and Ukrainian citizens also generated international debates regarding issues of overlapping citizenries and the loyalty of dual citi- zens. Firstly, Romania’s policy on the restitution of nationality contra- dicted the internal legislation of two neighbouring states, since neither Moldova (until 2000) nor the Ukraine allowed their citizens to hold dual nationality. Secondly, Moldovan and Ukrainian policymakers ac- cused Romania of using dual nationality as a strategy of increasing its political influence in the region, with the final aim of reacquiring its lost territories. Romania’s citizenship policy was thus perceived as add- ing to regional instability rather than to retroactive justice and integra- tion. Diplomatic debates concentrated mainly on Romania’s relationship to the Republic of Moldova. With the establishment of the new state in 1991, Romania was trapped in ‘the dilemma of the Romanian-Roma- nian relations’ (Cojocaru 2001). 6.3.1 Dual nationality In the early 1990s, the diplomatic rela- tions between the two countries seemed to proceed in tune with the strategy of the ‘two Romanian states’, put forward by the Moldovan Popular Front and shared by numerous politicians in Romania, accord- ing to which Moldova’s independence represented the first step toward a gradual and negotiated process of political unification between the two countries. To this end, Romania inaugurated a policy of special partnership with Moldova, introduced visa-free and passport-free travel between the two countries, set up special educational programmes for Moldovan students and built a comprehensive network of inter-minis- terial consultations. However, at the political-diplomatic level, the two countries soon drifted further apart. That was mostly because the Re- public of Moldavia was tormented by internal inter-ethnic conflicts and secessionist movements, tacitly or openly supported by Moscow, which degenerated into a civil war in 1992 in the multiethnic province of 194 CONSTANTIN IORDACHI Transnistria (also known as the Trans-Dniestr region). Fearing that ethnic strife would lead to disintegration, Moldovan leaders decided to consolidate the statehood of the new republic by relying on the Soviet version of local identity, i.e. on Moldovenism rather than on the pan-Ro- manian national identity. This change in Moldova’s internal policy af- fected its relations to Romania. At the official level, the formula of the ‘two Romanian states’ was gradually abandoned, with Romania and Moldova defining themselves as ‘two brotherly states’ and then more neutrally as ‘two neighbouring states’. After the electoral victory of the Communist Party in 2001, the diplomatic relations between Romania and the Republic of Moldova worsened considerably. The new Moldo- van President Vladimir Voronin launched aggressive cultural polices meant to strengthen the Moldovan identity, to marginalise Greater Ro- manian unionist forces and to forcefully reduce Romania’s political in- fluence in the republic. Moreover, blaming Romania’s irredenta poli- cies, Voronin put forward his own plans for a Greater Moldova, raising territorial claims to Romania’s province of Moldova.5 This obstruction- ist policy led to an almost complete deadlock in the diplomatic rela- tions between the two countries. 6.3.2 The restoration of nationality in practice: Domestic and international constraints 6.3.2 The restoration of nationality in practice: Domestic and international constraints From 1991 to 2001, the policy of restoration of Romanian nationality was applied without major convulsions, resulting in massive (re-)natur- alisations of Moldovan citizens. Since 2001, however, Romania has considerably slowed down the process of restitution for two main rea- sons. Firstly, the number of applications from Moldova has increased dramatically since January 2001, when Romanian citizens were granted visa-free travel in the Schengen space, effectively clogging the bureaucratic process of restoration of nationality. Secondly, although the European Commission repeatedly stated that the policy of restitu- tion of citizenship is an internal matter for Romania, several EU agen- cies voiced concerns that, upon Romania’s accession in January 2007, the country’s policy on restitution of nationality could become an un- controllable gate of access to the Schengen space of non-EU citizens, bypassing restrictive immigration policies. These combined challenges generated a series of crises in the process of restitution, leading to its intermittent suspension from December 2001 to September 2007. According to official figures, between August and December 2001 alone the Commission for Citizenship received approximately 300 de- mands for nationality per day, or an aggregate of 19,000 in six months.6 Confronted with this massive influx of requests, the govern- ment temporarily suspended the provisions on the restoration of na- POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 195 tionality by emergency ordinances valid for two periods of six months each, from December 2001 to June 2002, and then again from Novem- ber 2002 to May 2003, subsequently approved by parliament. In justi- fying the approval of the second ordinance, a parliamentary report in April 2003 pointed out that the ‘explosive increase in the number of demands’ blocked the work of the commission, which was composed of only five magistrates. 6.3.2 The restoration of nationality in practice: Domestic and international constraints It also blamed the fact that, according to offi- cial statistics, most of the re-naturalisation demands were opportunis- tic, being made ‘in the new context created by the elimination of visa requirements for Romanian citizens who travel in the Schengen space, as well as in view of Romania’s prospective integration into the North Atlantic structures.’ Pointing out that ‘the reparatory character taken into account at the time of elaboration of the Law on Citizenship is present in fewer and fewer cases,’ the parliament asked the govern- ment to identify a legal solution ‘to eliminate re-acquisitions of nation- ality for a purpose alien to the original intention of the law.’7 At the end of each period of suspension of the restoration of nation- ality, the government implemented major alterations of the citizenship law by two emergency ordinances passed in June 2002 and April 2003. The first ordinance unified the provisions on the reacquisition of nationality with those on the restoration of nationality in a single arti- cle (art. 10) placed in the section dealing with naturalisation, thus im- plying that the restoration of nationality to former citizens was a privi- leged naturalisation granted by the Romanian state and not an auto- matic entitlement to nationality. The restoration of nationality continued to be exempted from consular taxes (art. 36, para. 2); in ad- dition, claimants were given the right to contest the decision of the Commission of Citizenship during an interval of fifteen days. p g y These new provisions did not offer an efficient solution to the flood of naturalisation demands, so that in November 2002 – just four months after its reinstatement – the government yet again suspended the restoration of nationality process for six months. In May 2003, upon the expiry of the new deadline, the law on citizenship was altered once more by a governmental ordinance approved by parliament with minor modifications in October 2003. The new ordinance reinstated the suspended provisions on the restoration of nationality as two addi- tions to art.10 dealing with the reacquisition of nationality (classified as art. 101 and 102), but introduced ample amendments to their imple- mentation: Firstly, applicants to the restoration of nationality became subject to almost all of the conditions for naturalisation demanded of aliens; the only condition waived for former citizens was the obligation to relocate to Romania and reside there for a mandatory period. 6.3.2 The restoration of nationality in practice: Domestic and international constraints Sec- ondly, the requests for nationality had to be handed in personally to the Commission of Citizenship in Bucharest and only ‘in thoroughly justi- 196 CONSTANTIN IORDACHI fied cases’ by attorneys or third parties. Until 2003, the requests for re- acquisition of Romanian nationality could be filed not only personally but also ‘by third parties’, either at the Ministry of Justice in Bucharest or at Romania’s consulates abroad. According to media reports, this procedure led to the creation of clientelist networks in Moldova for the collection of dossiers and their transport in huge packages to Buchar- est. In order to eliminate these practices, the new ordinance obliged ap- plicants to travel personally to Bucharest, sometimes for undetermined periods of time necessitated by the new bureaucratic procedures, thus increasing the costs of naturalisation. Thirdly, the ordinance introduced a new form of re-naturalisation of former citizens and their descen- dants: a new article 102 allowed persons eligible for the restoration of nationality to apply for naturalisation directly at the Ministry of Justice after four years of continuous residence in Romania. This provision was another indication that Romanian authorities intended to trans- form the restoration of nationality into a privileged form of naturalisa- tion of former citizens relocating to Romania. Fourthly, in order to eliminate opportunistic reacquisitions of nationality, the new amend- ments stripped new citizens of some of the most immediate advan- tages of Romanian nationality: art. 37 stated that former citizens who reacquire nationality and effectively live in Romania ‘cannot exercise their right to free movement of persons’, i.e. they are forbidden to tra- vel abroad with a Romanian passport during the first four years after their naturalisation. Exceptions to this rule were allowed only in emer- gency situations, such as periods of study abroad, family unification, medical treatment abroad, etc. This overt form of discrimination against a certain category of Romanian citizens because of the manner of their naturalisation was abolished in September 2007. p 7 These substantial amendments to the citizenship law revealed the government’s intention to discontinue the restitution of nationality to former citizens living in Moldova and the Ukraine, transforming it in- stead into a selective and privileged naturalisation of alien ethnic Roma- nians relocating to Romania, after a residence of four years. 6.3.3 The restoration of nationality reloaded 6.3.3 Predictably, the May 2003 amendments to the citizenship law led to an almost complete deadlock in the process of re-naturalisation, at a time when the number of applications was soaring. The restrictive policy of the government triggered incendiary reactions from the pro-Moldovan interest groups in Romania, who conducted media campaigns against the governmental policy and – with the help of naturalised Moldovans elected in the Romanian parliament – initiated bills for amending the ci- tizenship law. The most contested provision was the applicants’ obliga- tion to travel to Bucharest and file their dossiers in person. Following street protests in Bucharest of Moldovan citizens applying for naturalisa- tion, this condition was finally abrogated by parliament in June 2003. y g y p 3 In March and April 2006, two open letters signed by 25 non-govern- mental organisations from Romania and Moldova urged the govern- ment to unblock the process of nationality restitution. The petitioners argued that the bureaucratic blockage was ‘premeditated,’ with the Ministry of Justice deliberately creating obstacles to the restoration of nationality. In their view, the restoration of nationality was a ‘legitimate right’ of the Moldovans and its denial by the Romanian government violated basic human rights and established principles of international law. The petitioners also denounced the restrictions on free movement imposed on new citizens, arguing that this provision discriminated among Romanian citizens according to their place of residence and the manner of their naturalisation, in direct violation of the Constitution which stated that all citizens are equal before the law. In two consecutive responses, the Ministry of Justice pointed out that the restoration of nationality is not an automatic entitlement of former citizens but a right granted by the Romanian state under certain condi- tions. Acknowledging that the Commission for Citizenship was over- whelmed by the large number of applications, the Ministry pledged to consider potential solutions to the problem.9 In 2006, two parliamentary bills attempted to provide a legal solu- tion to the issue. 6.3.2 The restoration of nationality in practice: Domestic and international constraints Thus, while former citizens living abroad were required to fulfil additional conditions that made their naturalisation very lengthy and difficult, for- mer citizens working, studying or living in Romania were granted ac- cess to direct naturalisation after a residence period of four years, by means of a special procedure. In addition, by requiring applicants to possess ‘knowledge of the Romanian language and elementary notions of Romanian culture and civilisation’,8 the government made it more difficult for non-ethnic Romanian applicants to recover their lost na- tionality, fuelling suspicion that the new conditions were specifically meant as an obstacle to their re-naturalisation. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 197 6.3.3 The restoration of nationality reloaded In order to speed up the restoration of nationality pro- cess, they proposed simplifying the procedure, shortening the proces- sing time, allocating more magistrates to the task and making the Commission of Citizenship responsible for the resolution of demands within ‘reasonable administrative deadlines.’10 The senate nevertheless rejected both bills on the grounds that they contradicted Romania’s ob- ligations under the EU treaty of accession, and that they would lead to potential conflicts with the Commission of the European Union.11 More recently, however, mounting public and political pressure has convinced the government to again modify its citizenship laws. In Sep- tember 2007, a new governmental ordinance facilitated the restoration 198 CONSTANTIN IORDACHI of nationality by means of major procedural amendments. Firstly, the Commission of Citizenship now consists of five specially appointed jur- idical councillors on a full-time basis, who replace the regular part time judges. Applications for nationality can be sent by post as well; incom- plete files are not automatically rejected, with later additions also being permitted. Secondly, in order to speed up the process of naturalisation, the decisions are now also taken by the Minister of Justice and not so- lely by the entire Council of Ministers. Rejections can be appealed by applicants in local courts, not only in Bucharest; new requests for na- tionality can also be filed one year after a rejection. The ordinance also obliges naturalised citizens to take the oath of allegiance no later than three months after the decision to naturalise instead of within one year as previously requested. It is expected that these amendments will speed up the process of naturalisation and will increase the number of Moldovan citizens acquiring Romanian nationality. Source: Monitorul Oficial al României, Part I; calculated from official sources made available by Constantin Dolghier, available at http://cetatenie.info.tm 6.4 Statistics on the restitution of nationality Since there are no general official statistics available, the number of former citizens and their descendants re-naturalised to Romanian na- tionality is highly contested. Official sources acknowledge a number of 102,000 Moldovan and Ukrainian citizens naturalised until 2001; no general figures have been released for the period since then. According to alternative unofficial estimates, the Romanian government granted nationality to at least 300,000 Moldovan citizens belonging to various ethnic groups between 1991 and 2000 alone.12 The number of ‘restitu- tions’ of nationality to Moldovan and Ukrainian citizens dropped to in- significant levels in the period 2000 to 2003. Although the process of Table 6.1 Naturalisations in Romania, 16 June 2000-29 October 2008 Year Total number naturalisations Moldovan citizens Ukrainian citizens 2008 4,961 3,605 165 2007 1,150 615 51 2006 740 487 3 2005 1,789 1,592 13 2004 751 258 6 2003 281 8 0 2002 318 1 3 2001 348 1 1 2000 357 1 0 Total 2000-2008 10,695 6,568 242 Source: Monitorul Oficial al României, Part I; calculated from official sources made available by Constantin Dolghier, available at http://cetatenie.info.tm Table 6.1 Naturalisations in Romania, 16 June 2000-29 October 2008 POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 199 restitution of nationality was resumed in 2004, the annual number of (re-)naturalisations remains relatively low. Since there was neither a drop in the number of demands nor an in- crease in the number of rejections during this period, the low number of restitutions of nationality was clearly due to the considerable slow- down in the bureaucratic processing of applications. According to var- ious unofficial estimates, the number of accumulated applications for restoration of nationality filed by Moldovan citizens is currently be- tween 500,000 and 900,000.13 Among these, in the period 2001 to 2007, Romanian authorities officially registered only 31,764 requests (by publishing them in the Official Monitory), among which 29,276 ori- ginated in the Republic of Moldova and 838 in Ukraine. Overall, be- tween 16 June 2000 and 28 October 2008, the Romanian government awarded 10,695 naturalisations, of which 6,568 were awarded to Mol- dovan citizens and 242 to Ukrainian citizens.14 Although Moldovan citizens continue to dominate the number of persons acquiring Romanian nationality, their proportion within the to- tal number of naturalisations is much lower than the share of Moldo- vans among all applicants. 6.4 Statistics on the restitution of nationality As shown in Figure 6.1, this gap is due to the fact that since 2000 the procedure of restitution of Romanian nationality to Moldovan and Ukrainian applicants has been slower than that of the naturalisation of aliens. Figure 6.1 Duration in months of the process of naturalisation in Romania, by original nationality of applicants accepted in 2005, 2006 and 2007 14 15 15 20 39 14 17 20 18 18 44 16 25 40 40 32 40 36 0 5 10 15 20 25 30 35 40 45 50 Stateless Americans Germans Israelis Moldovan/ Ukrainian Swedes Duraon in months 2005 2006 2007 Source: Data compiled from official sources by Constantin Dolghier, available at http://cetatenie.info.tm Figure 6.1 Duration in months of the process of naturalisation in Romania, by original nationality of applicants accepted in 2005, 2006 and 2007 Figure 6.1 Duration in months of the process of naturalisation in Romania, by original nationality of applicants accepted in 2005, 2006 and 2007 Source: Data compiled from official sources by Constantin Dolghier, available at http://cetatenie.info.tm 200 CONSTANTIN IORDACHI On average, applicants from the Republic of Moldova and the Ukraine naturalised in 2005 (included in the graph under the label ‘Moldovan/ Ukrainian’) had to wait 39 months between the official registration of their requests and the decision regarding naturalisation; those natura- lised in 2006 had to wait 44 months between the two stages. At the same time, other categories of aliens only waited, on average, between fourteen and eighteen months. Since the procedure for the naturalisa- tion of aliens, according to the law, is in fact more complicated than that of the restitution of nationality, it becomes apparent that the length of the re-naturalisation process was not simply due to the record number of demands but also to a conscious political decision to halt the process by administrative means. p y The amendments to the nationality law enacted in September 2007 made possible a massive increase in the number of naturalisations. As a consequence, the duration of the naturalisation process for all cate- gories of applicants began to converge: while aliens had to wait longer (40 months at maximum), the waiting period for nationality restitution slightly decreased from 44 to 40 months (see Figure 6.1). 6.4 Statistics on the restitution of nationality Overall, be- tween 16 June 2000 and 14 September 2007, the Romanian govern- ment awarded 5,062 naturalisations, of which 2,664 were restitutions of nationality to Moldovan citizens and 58 to Ukrainian citizens.15 Since the adoption of the new amendments to the nationality law on 14 September 2007, the Romanian government awarded a record num- ber of 5,633 naturalisations (higher than the sum of 5,062 naturalisa- tions granted in the previous seven years); of this number, 672 were granted in the last quarter of 2007 and 4,961 in 2008 (until October 2008). Out of this total number of new citizens, there were 1,826 nat- uralisations of aliens and 3,807 ‘restitutions’ of nationality. Among the latter, 318 restitutions were registered between 14 September and 31 December 2007, 299 to Moldovan citizens and nineteen to Ukrainian citizens. A record number of 3,770 restitutions of nationality occurred during the period 1 January to 28 October 2008, among which were 3,605 to Moldovan citizens and 165 to Ukrainian citizens. These fig- ures prove that, after the de facto suspension during the period 2000- 2007, the process of restitution of Romanian citizenship to former citi- zens from Moldova and the Ukraine has been resumed under the new 2007 provisions of the nationality law. 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives The collapse of the communist regimes led to the reorganisation of na- tionality policies in Central and Eastern Europe. One can distinguish POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 201 two main clusters of nationality policies, in direct relation to patterns of nation-building and state-building in these regions: nationality poli- cies in successor states in former multi-ethnic federal states such as Czechoslovakia, Yugoslavia and the USSR; and in post-communist na- tion-states such as Albania, Poland, Romania, Bulgaria and Hungary. g g y The dismantling of the former federal systems generated numerous legal, political and territorial conflicts, most notably in Yugoslavia and the USSR (the ‘velvet divorce’ between Slovakia and the Czech Repub- lic was smoother, although it was not free of legal conflicts, see Liebich, Warner and Dragovic 1995). In his analysis of ‘citizenship struggle’ in the successor states of the former USSR, Rogers Brubaker differen- tiated between a ‘new state’ model of legislation on nationality and a ‘restored-state’ model (1992: 275-276). The former was enacted in So- viet republics that lacked a statehood tradition. Without a history of dis- tinctive citizenry, these republics, such as the Republic of Moldova, had to create their citizenship body by conferring rights on all of their resi- dents, on an inclusive basis. The latter, ‘restored-state’ type, was applied in republics that relied on a pre-Soviet statehood tradition, such as the Baltic states. Motivated by the fear that their nation would ‘die out’, these states revived their pre-Second World War nationality laws in or- der to restore the citizenry that had existed prior to the Soviet conquest and to initially exclude from citizenship all residents who immigrated to these countries in the post-1945 period. To these, I would add a third, hybrid category, represented by ‘new states’ that have also as- sumed a ‘restored’ state-dimension, such as the Ukraine. The Ukrai- nian legislation granted citizenship to all inhabitants residing within the republic’s territory, on a very inclusive basis. 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives At the same time, it also granted access to Ukrainian citizenship to former citizens and their descendants born or permanently residing within any territories which formed a part of the historical states of the Ukrainian People’s Repub- lic, the Western Ukrainian People’s Republic, the Ukrainian State, the Ukrainian Socialistic Soviet Republic, Trans-Carpathian Ukraine and Ukrainian Soviet Socialistic Republic (URSR), on the express condition that they renounce their foreign citizenship.16 A different category of nationality policies can be found in post-com- munist nation-states. Although these states did not suffer territorial changes or a massive influx of population after 1989, they have all re- vised their nationality laws in order to reflect the new political transfor- mations.17 New nationality laws in these states encompassed an impor- tant national dimension, repressed under the regime of Soviet domina- tion; after decades of political isolation from their kin-populations abroad, most of these states have resumed policies of ‘positive discrimi- nation’ towards their co-ethnics. 202 CONSTANTIN IORDACHI Romania’s post-communist nationality policy belongs to the second cluster mentioned above; yet, due to the country’s geo-political position and its territorial disputes with the Soviet Union during the Second World War, the Romanian policy combines elements characteristic of policies in East-Central European and former Soviet countries. The most important concept that dominated Romania’s post-communist ci- tizenship policy was that of the restoration of nationality in order to undo the effects of the territorial changes that took place during and after the Second World War, an issue declared taboo during the period of Soviet domination. Romania granted the right to re-naturalisation to all former citizens and their descendants, irrespective of their ethnic ori- gin, their form of de-naturalisation and the period of their attachment to the Romanian state. In doing so, Romanian legislation went beyond regular laws on repatriation, of the kind post-communist Poland passed in relation to former citizens of Polish ethnic origin deported to the Soviet Union at the end of the Second World War. It also went be- yond forms of privileged naturalisation of kin population abroad, as is the case with Hungary’s policy towards former citizens of Hungarian eth- nic origin relocating to the kin-state. 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives To a certain extent, Romanian legis- lation resembles the policy of the restoration of citizenship to former citizens and their descendants implemented by the Ukraine, with the notable difference that, unlike the Ukraine, Romania allowed new citi- zens to hold dual citizenship and retain their domicile abroad. Most clo- sely, Romanian citizenship legislation resembles the ‘restored-state’ po- licies of the Baltic states. This similarity is not surprising: Greater Ro- mania’s provinces of Bessarabia and Bukovina as well as the Baltic states were occupied by the Soviet Union in June 1940 as a direct con- sequence of the 1939 Ribbentrop-Molotov Pact. In the post-Soviet peri- od, Romania and the Baltic states officially denounced the ‘infamous pact’ and tried to undo its legal consequences by applying the principle of restitution of pre-Soviet nationality. The major difference in the ap- plication of this principle was that in Romania its provisions were not meant to discriminate against ‘internal foreigners’ as in the Baltic states, but to include former citizens living abroad. h l f l fl d f g Due to the legacy of territorial conflicts and competing projects of nation-building and state-building in post-communist East-Central Eur- ope, Romania’s policy on the restoration of nationality generated inter- state tensions, most evident in its relation to the Republic of Moldova. Given the complex and multifarious nature of this relationship, Roma- nian policymakers have been unable to put forward a coherent policy toward Moldova, oscillating between ‘sentimentalism’ and ‘pragma- tism’. On the one hand, Romanian politicians regard Moldova as Ro- mania’s former province, occupied as a result of the 1939 Ribbentrop- Molotov Pact, and reserve the country’s right to unilaterally restore Ro- POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 203 manian nationality to Moldovan citizens. On the other hand, Romania was the first country to recognise the independence of the new Repub- lic of Moldova upon its proclamation in August 1991. Unlike the Feder- al Republic of Germany, in relation to the former GDR, or Greece, in relation to the Former Yugoslav Republic of Macedonia, Romania con- tested neither the statehood nor the name of the new republic. It has treated Moldova as an independent and sovereign state, thus implicitly recognising its legitimate right to establish its own version of national identity and citizenship legislation. 1954 Decree No. 80 for the Reacquisition of Citizenship by Certain Categories of Persons 1954 Decree No. 296 for the Modification of art. 1 of the Decree No. 33 of 24 January 1952, Concerning Citizenship in the Popular Republic of Romania 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives The failure of the policy of special partnership inaugurated between the two countries in the early 1990s and the forceful suspension of the process of restitution of nationality following pressure from the EU (2001-2007), widened the gap between these two policy lines. p y What are the prospects of Romanian-Moldovan relations? Currently, both countries try to adapt their bilateral relations to the new realities created by Romania’s EU membership. On the one hand, Romania has reiterated its historical rights to Bessarabia. In June 1991, the Roma- nian parliament officially condemned the 1939 Ribbentrop-Molotov Pact as illegal, ab initio null and void of consequences for Romania; in September 2007, President Traian Ba˘sescu announced his intention to officially condemn (yet again) the pact in order ‘to give political force’ to this declaration.18 Moreover, in September 2007 Romania re- launched its policy of restitution of nationality. Facing criticism from various EU agencies,19 Romanian authorities presented this policy as part of the EU’s program of integration with neighbouring countries, arguing that it would enable the EU to boost its influence in the for- mer Soviet space, as well as to import a qualified Moldovan workforce in order to reduce its labour shortages. On the other hand, Moldova’s communist leadership, backed by Russia, took advantage of the securi- tisation of the EU border between Romania and Moldova and the im- position of travel visas to Moldovan citizens travelling to Romania in 2007 in order to discontinue socio-economic and cultural relations be- tween the two countries. Moreover, a new Law on the Status of the Public Functionary, adopted in 2007, excludes all Moldovan citizens, who hold dual citizenship or have their domicile abroad, from public office; this effectively stripped a large part of the population of impor- tant political rights. Officially justified by the need to reduce the num- ber of naturalisations abroad, to eliminate conflicts of interest with other states and to consolidate Moldova’s statehood, this controversial Law primarily targets Romanian-Moldovan dual citizens with a view to- ward weakening political opposition to the ruling Communist party and to countering Romania’s political influence in the republic. 1950 Decree No. 145 Concerning the Abrogation of Law No. 162 of 30 May 1947 for the Settlement of the Citizenship of Certain Categories of Inhabitants 1952 Decree No. 33 Concerning Citizenship in the Popular Republic of Romania www.cdep.ro (in Romanian) 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives At a diplomatic level, Moldova has also tried to bypass Romania as a media- tor in its relations with the EU (see the decision to prevent the opening 204 CONSTANTIN IORDACHI of new Romanian consulates in Moldova and to instead use Hungary’s embassy for granting of EU Schengen visas to Moldovan citizens). Although the effects of Romania’s policy on the restitution of national- ity and the response to it by the neighbouring states (mostly Moldova) should not be unduly exaggerated, it is likely that the interaction of citi- zenship policies in East Central Europe will continue to challenge in- ter-state relations in the region. Chronological list of citizenship related laws in Romania Date Document Content Source 1947 Law No. 162 for the Modification of arts. 9, 18, 25, 26, 28, 29, 30, 31, and 34 of the Law Concerning the Acquisition and Loss of Romanian Nationality of 19 January 1939 Restores citizenship to all those who were domiciled in Romania on 26 September 1920 (the ratification of the Treaty with Austria), Jews de- naturalised in the period 1937-1944 and inhabitants of Bessarabia who immigrated to Romania proper after the Soviet occupation (28 June 1940) www.cdep.ro (in Romanian) 1950 Decree No. 145 Concerning the Abrogation of Law No. 162 of 30 May 1947 for the Settlement of the Citizenship of Certain Categories of Inhabitants Abrogates Law No. 162; punishes false declarations under that law with long- term imprisonment www.cdep.ro (in Romanian) 1952 Decree No. 33 Concerning Citizenship in the Popular Republic of Romania Abolishes previous laws on state citizenship; redefines conditions of access and loss of Romanian citizenship based exclusively on the ius sanguinis principle; serves as a legal basis for communist citizenship doctrine www.cdep.ro (in Romanian) 1954 Decree No. 80 for the Reacquisition of Citizenship by Certain Categories of Persons Restores citizenship to all those denaturalised under previous special laws www.cdep.ro (in Romanian) 1954 Decree No. 296 for the Modification of art. 1 of the Decree No. 33 of 24 January 1952, Concerning Citizenship in the Popular Republic of Romania Modifies art. g 1952 Decree No. 33 Concerning Citizenship in the Popular Republic of Romania www.cdep.ro (in Romanian); www.legislationline.org (in French) 1990 Decree-Law No. 137 of 11 May 1990 Concerning certain Provisions Pertaining to Romanian Citizenship 6.5 (Re)constructing nationality in post-communist Romania: Comparative perspectives 1 of the 1952 Law; reconfirms Romanian citizenship to all those who held this status on 28 June 1940 and have resided in Romania since that date; excludes from Romanian www.cdep.ro (in Romanian) Chronological list of citizenship related laws in Romania Chronological list of citizenship related laws in Romania Date Document Content Abolishes previous laws on state citizenship; redefines conditions of access and loss of Romanian citizenship based exclusively on the ius sanguinis principle; serves as a legal basis for communist citizenship doctrine www.cdep.ro (in Romanian) Restores citizenship to all those denaturalised under previous special laws www.cdep.ro (in Romanian) Abolishes previous laws on state citizenship; redefines conditions of access and loss of Romanian citizenship based exclusively on the ius sanguinis principle; serves as a legal basis for communist citizenship doctrine www.cdep.ro (in Romanian) Restores citizenship to all those denaturalised under previous special laws www.cdep.ro (in Romanian) Modifies art. 1 of the 1952 Law; reconfirms Romanian citizenship to all those who held this status on 28 June 1940 and have resided in Romania since that date; excludes from Romanian www.cdep.ro (in Romanian) Modifies art. 1 of the 1952 Law; reconfirms Romanian citizenship to all those who held this status on 28 June 1940 and have resided in Romania since that date; excludes from Romanian ww (in 1998 Law No. 146 for the Abrogation of Decree-Law No. 7/1989 1999 Law No. 192 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 www.cdep.ro (in Romanian) www.cdep.ro (in Romanian) 1998 Law No. 146 for the Abrogation of Decree-Law No. 7/1989 1999 Law No. 192 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 2003 GEO No. 43 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 2003 Law No. 405 Concerning the Approval of the GEO No. 43/2003 2007 GEO No. 87 for the POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 160 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) 2003 GEO No. 43 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania www.cdep.ro (in Romanian) Date Document Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) Unifies the provisions on the reacquisition of citizenship with those on the restoration of citizenship www.cdep.ro (in Romanian) Approves the modification with minor amendments www.cdep.ro (in Romanian) Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) Introduces new conditions for naturalisation of former citizens; demands that www.cdep.ro (in Romanian) Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) Unifies the provisions on the reacquisition of citizenship with those on the restoration of citizenship www.cdep.ro (in Romanian) Approves the modification with minor amendments www.cdep.ro (in Romanian) w Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) 2002 GEO No. 68 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 2002 Law No. 542 Concerning the Approval of the GEO No. 68 of 13 June 2002 2002 GEO No. 160 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizenship No. 21/1991 2002 GEO No. 160 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizenship No. 21/1991 / 99 o. 165 Concerning pproval of the GEO 60 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) No. 43 for the ication and ons to the Law on nian Citizenship No. 91 Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania after four years of continuous residence; denies former citizens re- naturalised in Romania the right to travel abroad for four years www.cdep.ro (in Romanian) o. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 542 Concerning the Approval of the GEO No. 68 of 13 June 2002 Approves the modification with minor amendments www.cdep.ro (in Romanian) 2002 GEO No. 160 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizenship No. 21/1991 Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) 2003 Law No. 165 Concerning the Approval of the GEO No. 160 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) 2003 GEO No. 43 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania after four years of continuous residence; denies former citizens re- naturalised in Romania the right to travel abroad for four years www.cdep.ro (in Romanian) 2003 Law No. 405 Concerning the Approval of the GEO No. 43/2003 Approves the modifications with minor amendments www.cdep.ro (in Romanian) 2007 GEO No. 87 for the Streamlines the procedure www.cdep.ro Date Document Content Source residential stage of seven years, knowledge of the country’s language and legislation) 2001 GEO* No. 167 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizen- ship No. 21/1991 Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) 2002 Law No. 225 Concerning the Approval of the GEO no 167/2001 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) 2002 GEO No. 68 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Unifies the provisions on the reacquisition of citizenship with those on the restoration of citizenship www.cdep.ro (in Romanian) 2002 Law No. 542 Concerning the Approval of the GEO No. 68 of 13 June 2002 Approves the modification with minor amendments www.cdep.ro (in Romanian) 2002 GEO No. 160 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizenship No. 21/1991 Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) 2003 Law No. 165 Concerning the Approval of the GEO No. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 205 Date Document Content Source citizenship the inhabitants of Soviet-occupied Bessarabia 1956 Decree No. 63 on the Citizenship of Certain Categories of Persons Regulates the legal status of certain categories of persons www.cdep.ro (in Romanian) 1971 Law No. 24 Concerning Romanian Citizenship Redefines conditions of naturalisation, ascription and reacquisition of Romanian citizenship www.cdep.ro (in Romanian) 1989 Decree-Law No. 7 Concerning the Repatriation of Romanian Citizens and of Former Romanian Citizens Guarantees the right to repatriation of Romanian citizens living abroad; allows them duty-free transfer of goods and priority acquisition of state-owned real estate www.cdep.ro (in Romanian) 1990 Decree-Law No. 137 of 11 May 1990 Concerning certain Provisions Pertaining to Romanian Citizenship Allows reacquisition of Romanian citizenship by former citizens without renunciation of foreign citizenship and relocation to Romania www.cdep.ro (in Romanian) 1991 Law on Romanian Citizenship Redefines conditions of naturalisation, ascription and reacquisition of Romanian citizenship; introduces the right of restoration of citizenship to former citizens of interwar Greater Romania according to a simple procedure without renunciation of foreign citizenship and relocation to Romania www.cdep.ro (in Romanian); www.legislationline.org (in French) 1998 Law No. 146 for the Abrogation of Decree-Law No. 7/1989 Abrogates facilities granted upon the repatriation of citizens living abroad www.cdep.ro (in Romanian) 1999 Law No. 192 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Denies the state the right to withdraw state citizenship from those who acquired it at birth; abolishes repatriation as a mode of reacquisition of citizenship; transforms the reacquisition of citizenship into a simplified procedure of naturalisation; increases requirements for t li ti ( ti www.cdep.ro (in Romanian) 1991 Law on Romanian Citizenship CONSTANTIN IORDACHI 206 Date Document Content Source residential stage of seven years, knowledge of the country’s language and legislation) 2001 GEO* No. 167 Concerning the Suspension of Certain Provisions Concerning the Implementation of the Law on Romanian Citizen- ship No. 21/1991 Suspends art. 35 on the restoration of citizenship for a period of six months www.cdep.ro (in Romanian) 2002 Law No. 225 Concerning the Approval of the GEO no 167/2001 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) 2002 GEO No. 68 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Unifies the provisions on the reacquisition of citizenship with those on the restoration of citizenship www.cdep.ro (in Romanian) 2002 Law No. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 207 Date Document Content Source Modification of the Law on Romanian Citizenship No. 21/1991 of naturalisation now granted by the Ministry of Justice and not the Council of Ministers; reorganises the Commission of Citizenship under the leadership of the Ministry of Justice; enlarges the Commission to four members, but allows it to work with a quorum of at least three members (in Romanian) * GEO: Governmental Emergency Ordinance POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 405 Concerning pproval of the GEO 3/2003 Approves the modifications with minor amendments www.cdep.ro (in Romanian) No. 87 for the Streamlines the procedure www.cdep.ro Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania after four years of continuous residence; denies former citizens re- naturalised in Romania the right to travel abroad for four years www.cdep.ro (in Romanian) Approves the modifications with minor amendments www.cdep.ro (in Romanian) Streamlines the procedure www.cdep.ro g O Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) n No. Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania after four years of continuous residence; denies former citizens re- naturalised in Romania the right to travel abroad for four years www.cdep.ro (in Romanian) g O Approves the modifications with minor amendments www.cdep.ro (in Romanian) Streamlines the procedure www.cdep.ro 2003 Law No. 165 Concerning the Approval of the GEO No. 160 2003 Law No. 165 Concerning the Approval of the GEO No. 160 Approves the suspension of the right to restoration of citizenship for six months www.cdep.ro (in Romanian) 2003 GEO No. 43 for the Modification and Additions to the Law on Romanian Citizenship No. 21/1991 Introduces new conditions for naturalisation of former citizens; demands that applicants submit their requests personally in Bucharest; introduces a new form of re- naturalisation of former citizens living in Romania after four years of continuous residence; denies former citizens re- naturalised in Romania the right to travel abroad for four years www.cdep.ro (in Romanian) 2003 Law No. 405 Concerning the Approval of the GEO No. 43/2003 Approves the modifications with minor amendments www.cdep.ro (in Romanian) 2007 GEO No. 87 for the Streamlines the procedure www.cdep.ro Notes 1 This distinction also necessitates a terminological clarification: in Romania, the modern legal vocabulary of citizenship emulated the French legal terminology, employing nat¸ionalitate (nationality) in the sense of state citizenship. The semantic distinction between the terms ‘citizenship’ (meaning state membership) and ‘nation- ality’ (meaning ‘ethnic origin’) was only introduced in the official legal vocabulary after the Second World War, as part of the Soviet-style institutionalisation of ethnicity. On the one hand, due to its discriminatory use in the Second World War, the expres- sion ‘ethnic origin’ (originea etnica˘) was purged from the communist political vocabu- lary. On the other hand, in order to continue to acknowledge the legal existence and legitimate collective rights of various ethnic groups living in the country side by side with the dominant ethnic group, the communist legislation employed the term ‘na- tionality’ to denote the ethnic origin of a person and not his or her state citizenship, as had previously been the case. To differentiate the new denotation of the term from its previous legal meaning (state citizenship), ‘nationality’ was employed in the com- munist political language mostly in plural, as ‘co-inhabiting nationalities.’ In the post-communist period, Romanian legislation employs the term nat¸ionalitate along- side the expression ‘origine etnica˘’ (ethnic origin), and refers to ethnic groups mostly as minorita˘t¸i nat¸ionale (national minorities). The term ‘citizenship’ (ceta˘t¸enie) is em- ployed to refer to both the legal and participatory dimensions of this institution. This chapter uses the term ‘nationality’ to refer to state membership in line with the legal vocabulary employed in the current volume, irrespective of the previous or current understandings of the term ‘nat¸ionalitate’ in Romanian. g ¸ All translations of legal texts are the author’s, if not otherwise indicated. 2 All translations of legal texts are the author’s, if not otherwise indicated 3 See the parliamentary debates at www.cdep.ro. 4 Monitorul Oficial 75, 21 May 1990. 5 ‘Voronin: ‘Iˆn Romaˆnia sunt 10 milioane de moldoveni’, 24 February 2007, www.bbc.co.uk. 6 ‘Expunere de motive pentru aprobarea Ordonant¸ei de urgent¸a˘ a Guvernului nr. 160/ 2002’, 1, www.cdep.ro. 7 ‘Expunere de motive la Legea pentru aprobarea Ordonant¸ei de urgent¸a˘ a Guvernului nr. 160/2002,’ www.cdep.ro. 8 Monitorul Oficial, Partea I, 399, 9 June 2003. 9 The petitions and the official answers are available on the site www.curaj.net. 208 CONSTANTIN IORDACHI 10 Ilie Ilas¸cu, Session of the Senate, 19 March 2007, www.parlament.ro. Notes A former Moldovan citizen imprisoned in Transnistria for his opposition to the secessionist leadership of the region during the civil war, Ilas¸cu was later elected in the Moldovan and Romanian parliaments in absentia. Following international pressure, Ilas¸cu was released from prison in 2001 and migrated to Romania, where he agitates for dual ci- tizenship for all Moldovans, in his capacity as a member of the Romanian parlia- ment. On the case of Ilas¸cu, see Iordachi (2004: 249-252) 11 Petre Stra˘chinariu, Session of the Chamber of Deputies, 28 June 2007, www.parlament.ro. 12 Estimation by former Moldovan Prime Minister Mircea Druc, Evenimentul Zilei, 20 May 2000. See Iordachi (2004: 248). 13 For this estimation, see http://en.wikipedia.org/wiki/Traian_Basesc For this estimation, see http://en.wikipedia.org/w 14 Data calculated after the Official Monitory of Romania, processed and made available online by Constantin Dolghier at www.cetatenie.info.tm. 14 Data calculated after the Official Monitory of Romania, pro online by Constantin Dolghier at www.cetatenie.info.tm. 15 Data calculated after the Official Monitory of Romania, processed and made available online by Constantin Dolghier at www.cetatenie.info.tm. 16 See art. 8 of the 2001 Law on Citizenship of Ukraine, available at www.mfa.gov.ua. 16 See art. 8 of the 2001 Law on Citizenship of Ukraine, ava 17 This claim is valid for Poland, as well. Although the 1962 Polish Nationality Act has not been replaced by a new post-communist law, it was nevertheless amended in important points, while numerous procedural changes have also been implemented. See Go´rny & Pudzianowska in this volume. 18 ‘Traian Ba˘sescu va denunt¸a Pactul Ribbentrop-Molotov’, 21 December 2007, www.bbc.co.uk. 19 On 25 September 2007, President Ba˘sescu asked the government ‘to simplify to the maximum’ the naturalisation conditions for Moldovan citizens. His statement was criticised by Marianne Mikko, the President of the European Parliament Committee for the Cooperation between the EU and the Republic of Moldova, as lacking ‘political wisdom’. See ‘EP official: Basescu’s statements about citizenship for Moldovans “not wise”’, Nine O’Clock 4029, 28 September 2007, www.nineoclock.ro. In July 2007, Kalman Mizsei, the director of the EU representative office to Moldova, called on Romania to cancel its policy of nationality restitution for Moldovans as it contradicts the EU charter. See: ‘Romania asked to cancel easy citizenship for Moldo- vans’, New Europe: The European Weekly, 14 July 2007, www.neurope.eu. Bibliography Brubaker, R.W. (1992), ‘Citizenship Struggles in Soviet Successor States’, International Migration Review 26 (2): 269-291. Brubaker, R.W. (1996), Nationalism Reframed: Nationhood and the National Question in the New Europe. Cambridge: Cambridge University Press. Brubaker, R.W. (1996), Nationalism Reframed: Nationhood a New Europe. Cambridge: Cambridge University Press. Cojocaru, G. (2001), Colapsul URSS s¸i dilema relat¸iilor romaˆno-romaˆne. Bucharest: Omega. Cojocaru, G. (2001), Colapsul URSS s¸i dilema relat¸iilor romano-romane. Bucharest: Omega. Institutul Central de Statistica˘ (1940), Anuarul Stat in 1878 istic al Romaˆniei, 1939 s¸i 1940. Bucharest: Imprimeria Nat¸ionala˘. Iordachi, C. (2002), Citizenship, Nation and State-Building: The Integration of Northern Do- brogea in Romania, 1878-1913. Pittsburgh: University of Pittsburgh Press. Iordachi, C. (2004), ‘Dual Citizenship and Policies toward Kin-Minorities in East-Central Europe: A Comparison between Hungary, Romania, and the Republic of Moldova’, in Z. Ka´ntor, B. Majte´nyi, O. Ieda, B. Vizi & I. Hala´sz, (eds.), The Hungarian Status Law Syndrome: A Nation Building and/or Minority Protection, 239-269. Sapporo: Slavic Re- search Centre, Hokkaido University. POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA POLITICS OF CITIZENSHIP IN POST-COMMUNIST ROMANIA 209 Iordachi, C. (2006), ‘The Ottoman Empire: Syncretic Nationalism and Citizenship in the Balkans’, in T. Baycroft & M. Hewitson (eds.), What is a Nation? Europe, 1789-1914, 120-151. Oxford: Oxford University Press. Liebich, A., D. Warner & J. Dragovic (eds.) (1995), Citizenship, East and West. London: Ke- gan Paul International. Marshall, T.H. (1950), Citizenship and Social Class. Cambridge: Cambridge University Press. National Institute of Statistics (2006), Romanian Statistical Yearbook. Bucharest: National Institute of Statistics. Shafir, M. (1985), Romania: Politics, Economics, and Society: Political Stagnation and Simu- lated Change. London: Pinter Publishers. Verdery, K. (1991), National Ideology under Socialism: Identity and Cultural Politics in Ceau- sescu’s Romania. Berkeley: University of California Press. 7 The politics of Bulgarian citizenship: National identity, democracy and other uses Daniel Smilov and Elena Jileva1 It is a well-known paradox that a polity cannot define its membership in a democratic way; there must be an already defined membership in order for a democratic procedure to take place. Therefore, even in genu- inely democratic polities, the original membership rules are a complex mixture of normative egalitarian principles and historical contingency, which privileges certain groups. With the passage of time, the contin- gent privileges tend to acquire a self-perpetuating, normative status. Modern Bulgarian citizenship laws are no exception to this general pattern. Various groups controlling the government and the parliamen- tary majority in the country have at one point or another attempted to entrench their privileged status in Bulgarian legislation. Such groups were most successful in times when democracy gave way to authoritar- ian regimes with fascist leanings and communist rule. What is surpris- ing in the Bulgarian case, however, is the resilience of social pluralism, which has ultimately prevailed over such attempts. The overall result has been a certain normative incoherence of citizenship regulation in the country, which makes it capable of accommodating different histor- ical narratives and normative visions. This inclusive incoherence, we ar- gue, was and still is of crucial importance for the Bulgarian polity. g p g p y Bulgarian mediaeval states existed between the late seventh century and the Ottoman invasion in the fourteenth century – an event that left a substantial historical mark. Modern Bulgaria was established in 1878- 1879 as a fairly independent principality in the former territories of the Ottoman Empire. Its subsequent turbulent history, which fits within its mere 130 years of existence with some difficulty, has not been con- ducive to the formation of a natural and self-explanatory sense of be- longing to, and membership of, the Bulgarian polity. Ethnic Bulgarians (comprising today more than 80 per cent of the population), Turks, Roma, Greeks, Macedonians, Christians, Muslims and Jews – the main constituents of modern Bulgarian society – may differ in their interpre- tations of the past and their visions for the future of the country. Never- theless, despite this lack of homogeneity, the Bulgarian polity has pro- ven remarkably stable and, particularly over the last two decades, has made important strides towards the establishment of genuine liberal democracy. 7 The politics of Bulgarian citizenship: National identity, democracy and other uses 212 DANIEL SMILOV AND ELENA JILEVA The main goal of this chapter is to trace the citizenship policies, which have played an important role in these developments. The first section outlines the history of Bulgarian citizenship laws in the period between the liberation from Ottoman rule and the end of communist rule. The second section discusses the developments after the fall of the Iron Curtain by focusing specifically on the 1991 Constitution and the new citizenship laws. The third section provides an overview of the current political debates on citizenship in Bulgaria. The fourth section offers some statistical information about the current trends in the ac- quisition of Bulgarian citizenship. The chapter ends with some obser- vations about the overall adequacy of the current citizenship regulation. As this introduction shows, Bulgarian regulation in this field is rather backward-looking, as it mainly addresses problems characteristic of the twentieth century. The danger is that by preserving this focus, it re- mains oblivious to the ever more pressing demands of a globalising world. 7.1 History The history of the modern Bulgarian state begins with the liberation of the country in the Russo-Turkish war of 1877-1878. In the spring of 1879, the provisional Russian authorities in the mediaeval capital of Veliko Turnovo convoked a Constituent Assembly. It was given a man- date to adopt a constitution for the new Principality, which was practi- cally independent but formally remained in a vassal (tributary) relation- ship with the Turkish Sultan.2 The decisions of this Constituent As- sembly are very important from the point of view of citizenship policy in Bulgaria, and are thus worth examining in some detail (for the fol- lowing, see Vladikin 1994 and Balamezov 1993). g 994 993) Firstly, the delegates encountered the paradox outlined in the open- ing paragraph of this chapter: they faced the question of who had the right to participate in the Constituent Assembly – and whose interest it should represent. Of course, the Russian authorities had invited some of the Bulgarian nobility (notables), and had carried out impromptu elections in parts of the Bulgarian lands under their jurisdiction. How- ever, in addition to these rather haphazardly gathered (though formally legitimate) representatives, there were numerous delegations from other lands inhabited by Bulgarians. These lands were to remain out- side the territory of the Bulgarian Principality, according to the treaty concluded at the Berlin Congress of the Great Powers (1879).3 Before the formal opening of the Constituent Assembly, all of the de- legates – both legitimate ones and others – met to discuss the question of the ‘unity of the nation’. All of them agreed that the great powers THE POLITICS OF BULGARIAN CITIZENSHIP 213 had unjustly excluded certain Bulgarian territories from the Principal- ity.4 The disagreement was about the proper course of action; some ar- gued that the Constituent Assembly should be boycotted in protest against the Berlin Congress treaty. The adoption of a constitution, their argument went, would legitimise the partition of the Bulgarian lands. Others, strongly encouraged by the Russian authorities, insisted that a constitution should be adopted anyway in order to stabilise the new po- lity. Finally, common sense prevailed and, after numerous passionate patriotic speeches, the formally legitimised delegates began their work on the text of the Constitution. 7.1 History The discussions of the ‘national ques- tion’, however, left an indelible mark on the ensuing proceedings; there was a common understanding that the Constitution should defend the interests of all Bulgarians as much as possible – both those living in the Principality and those left outside of it. p y Secondly, another main issue, which became the focus of vigorous debate in the Constituent Assembly, was the scope of the (political) rights that were to be granted to citizens of the Principality. There were conservative voices in the Assembly who argued in favour of limited suffrage (based on education, wealth and property), or for special powers of the monarch to appoint members of parliament. These con- servative voices were drowned out by a sea of egalitarian sentiments. The end result was full male suffrage in the elections for parliament, no powers for the monarch to appoint MPs, and a single-chamber leg- islature (without a senate of the type of the House of Lords, for in- stance). These institutional arrangements – which were decidedly unty- pical of the period – determined the egalitarian, Rousseauian bias of the Turnovo Constitution. Male citizens5 were entitled to an impressive set of political rights of participation. g Thus, the concrete citizenship arrangements, which the 1879 Consti- tution embodied, should be read through the double lens of nationalist and egalitarian-Rousseauian concerns and ideas. On the face of it, ega- litarianism prevailed. Art. 54 stated that: All persons born in Bulgaria who have not obtained any other ci- tizenship as well as those born elsewhere of Bulgarian subjects are subjects of the Bulgarian Principality. This emphasis on the principle of ius soli revealed the reluctance of the delegates to draw distinctions among the people living in Bulgaria: all were entitled to citizenship status, regardless of their ethnic origin and religion. Moreover, the Constitution prohibited drawing further distinctions among citizens: all males received the same scope of politi- cal rights (art. 60), there were express prohibitions of different estates 214 DANIEL SMILOV AND ELENA JILEVA (casts) of citizens (art. 57) and of any titles of nobility, orders or other signs of distinction (art. 58).6 The constitutional defence of national ideals was by no means ne- glected, however. The main concern of the drafters was to ensure that Bulgaria preserved legitimate claims over lands inhabited by Bulgar- ians. 7.1 History This was done, somewhat ingeniously, in the provisions on reli- gion (arts. 37-42). It was here regulated that the dominant religion in the Principality would be Eastern Orthodox Christianity, and (more im- portantly) that the Principality ‘constitute[d] an inseparable part of the Bulgarian Church District’ and was ‘subject [in religious matters] to the power of the Bulgarian Church, regardless of its seat’. The purpose of these articles was to constitutionalise the Bulgarian Exarchate – the autonomous Bulgarian Church recognised by the Ottoman Empire in 1870 – whose seat was in Istanbul and whose jurisdiction extended well beyond the territory of the Principality; it covered both Eastern Ru- melia and Macedonia, as well as some lands ceded to Serbia in 1878. In this way, the articles regarding religion drew the informal bound- aries of the Bulgarian political community, and in a highly symbolic way articulated the territorial claims of the new Bulgarian state. It is important to stress this ‘territorial’ meaning of the regulations on religion; the entrenchment of Orthodox Christianity in this specific way was not designed to create religious discrimination against other faiths. The new Bulgarian state was by no means militantly religious. Art. 40 of the Constitution explicitly stated that all persons (citizens or not) residing (permanently or temporarily) in Bulgaria were entitled to religious freedom (as long as they did not violate the law). The real pur- pose of establishing Orthodox Christian faith was that it provided an opportunity for the Constituent Assembly to settle the ‘national ques- tion’ in a way acceptable to all delegates. On the one hand, citizens of the new Principality were granted a broad spectrum of civil and politi- cal rights in a rather egalitarian fashion (for its time). On the other hand, ethnic Bulgarians living abroad were given the consolation that their lands were symbolically ‘constitutionalised’ through references to the Bulgarian Exarchate. These Bulgarians were granted the equivalent of a symbolic citizenship and a promise that the new Principality would take care of their interests as well. This promise actually deter- mined the course of Bulgarian politics for the better part of the follow- ing century. Its influence is still evident in current citizenship law. Along with the symbolic gains for ethnic Bulgarians, there were some more tangible privileges for them as well. THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP 215 nia, Eastern Thrace and other regions. Instead, the founding fathers of modern Bulgaria would maintain the compact Bulgarian population in adjacent lands as a way of legitimising future territorial expansion. Yet, these Bulgarians were granted some privileges – mostly in the form of less strict requirements for acquiring Bulgarian citizenship through naturalisation. Based on the explanations given thus far, we will now provide a gen- eralised description of Bulgarian citizenship regulations, since all sub- sequent laws adopted in the country took into account the principles elaborated in the Turnovo Constitution of 1879. The first such law was adopted as early as 1880 (First Law on Bulgarian Nationality (podanstvo)).7 The second law was adopted by the State Council in 1883. The Third Law on Bulgarian Nationality came into force in 1904. They were all heavily reliant on the principle of ius soli: all persons born in the territory of the Principality became Bulgarian citizens by right. Such was the case with the existing minorities of Turks, Greeks and others: ‘Bulgarian subjects are all those individuals who at the time of the establishment of the Bulgarian Principality had residence in or were born on its territory’ (1880 Law).8 By comparison, the principle of ius sanguinis had more limited ap- plications. Persons born in foreign countries of parents who were Bul- garian subjects were granted citizenship by origin. Ethnic Bulgarians born or living abroad, however, did not automatically acquire citizen- ship, and this covered probably more than half of all Bulgarians living in the Ottoman Empire before the liberation of 1878. The principle of ius domicilii was also applied in two ways: firstly, as already mentioned, all former Ottoman subjects residing in the terri- tory of the Principality at the time of its creation were granted citizen- ship; secondly, residence became a ground for acquiring citizenship through naturalisation. The regular residence requirement was three years based on a permit for permanent domicile. Those who did not have a permit for permanent domicile had to prove ten years of contin- uous residence to become eligible for naturalisation. The regulations foresaw the facilitated naturalisation of foreigners of Bulgarian extrac- tion. They became entitled to citizenship one year after they obtained a grant for permanent domicile in the Principality. The same applied to men married to Bulgarian women and those who had performed a meritorious service to the country. 7.1 History The Constituent As- sembly did not accept a strong ius sanguinis principle; no one really wanted all ethnic Bulgarians (or Bulgarians by blood) to automatically acquire citizenship rights in the Principality. This would create incen- tives for a mass exodus of Bulgarians from Eastern Rumelia, Macedo- THE POLITICS OF BULGARIAN CITIZENSHIP (Needless to say, every alien woman who married a Bulgarian became ipso facto a Bulgarian.) Naturalisation was granted by the King’s decree (ukaz) upon a proposal from the Min- ister of Justice. Naturalised citizens enjoyed the civil and political rights of Bulgarian subjects with a few important exceptions: they were not eligible to membership in the National Assembly or any other elective public of- 216 DANIEL SMILOV AND ELENA JILEVA fice for fifteen years after naturalisation. This text of the law was actu- ally in conflict with the Turnovo Constitution, which did not introduce such restrictions,9 but since there was no mechanism for constitutional review, such conflicts were resolved in favour of the law. This was the model that lasted until 1940 without any important changes of principle. In 1885, the Bulgarian Principality was united with Eastern Rumelia, which almost doubled its territory and popula- tion. In 1908, the Principality gained full formal independence and be- came a kingdom – the monarch received the original mediaeval Bulgar- ian title of ‘tsar’. The defeats in the Balkan wars and the First World War paradoxically led to a certain enlargement of the territory of the kingdom; parts of Macedonia and Eastern Thrace were among the most important gains. The net result – territorially – was positive, although some lands of the Principality in Dobrudja were lost to Roma- nia. But the real problem was the cost of the territorial gains. The numbers of dead and wounded were staggering, and the economy was in ruins and burdened by war reparations for decades to come. Most importantly, from our perspective, the problem of refugees arose. Sig- nificant numbers of people from Macedonia, Eastern and Western Thrace and other neighbouring territories moved to the motherland as a result of the war and of post-war policies of ‘population exchange’, which was really just an internationally regulated version of ethnic cleansing. Immediately after the war, leading politicians attempted to abandon the territorial expansion paradigm (‘unification of all Bulgarian lands’, in the parlance of the time), which was at the root of so many political and social disasters. This proved virtually impossible, however. The po- pulist-agrarian Prime Minister Alexander Stambolijski, who tried to mend fences with neighbouring countries and Serbia in particular, was considered a national traitor by many. THE POLITICS OF BULGARIAN CITIZENSHIP Eventually he was brutally killed by Bulgarian-Macedonian nationalists, who insisted that the inclusion of Macedonian lands in Bulgaria should always be a top political prior- ity.10 y Although the desire for territorial expansion remained a key factor of Bulgarian politics after the Balkan wars and the First World War, the symbolic geography of the Bulgarian political community dramatically changed. Firstly, the religious jurisdiction of the Bulgarian Exarchate over Macedonia and other lands was lost. Serbia (or rather the King- dom of Serbs, Croats and Slovenes), Greece and other countries did not recognise this jurisdiction, and extended the jurisdiction of their own national churches over these lands. Secondly, the mass exodus of Bulgarians from Macedonia and Eastern Thrace led to important de- mographic changes in these regions. The end result was that the THE POLITICS OF BULGARIAN CITIZENSHIP 217 boundaries of the ‘Bulgarian lands’ were no longer so neatly defined as in the pre-war period. The changes in the symbolic geography of Bulgarian lands led to the increased importance of the concept of ‘Bulgarian origin’ or ‘Bulgarian extraction’. After the First World War, Bulgarian governments could no longer rely on the Exarchate and the Bulgarian schools to help preserve the Bulgarian national identity of those living in neighbouring coun- tries. In fact, the Greek and Serbian governments both pursued a course of aggressive – and often physically repressive – replacement of the markers of Bulgarian identity. The Bulgarian government could not do much in this situation; in fact, all it could do was increase the privi- leges for ethnic Bulgarians within its domestic jurisdiction. g g j The Fourth Law on Bulgarian Nationality adopted in 1940 was the primary example of this tendency. Formally, the law repeated some of the main provisions of previous legislation.11 However, there were many provisions in which the concept of ethnically defined ‘Bulgarian origin’ played a crucial role. Firstly, the law introduced a strongly ethnic definition of ‘Bulgarian origin’: ‘all persons born of Bulgarian parents’ (art. 4). Secondly, persons of Bulgarian origin were granted significant privileges vis-a`-vis other groups. For instance, previous legislation al- lowed foreigners of non-Bulgarian extraction to be naturalised three years after obtaining a residence permit,12 while the 1940 law raised the requirement to ten years (art. 9). At the same time, people of Bul- garian origin were entitled to naturalisation within a year (as in pre- vious laws). THE POLITICS OF BULGARIAN CITIZENSHIP Furthermore, and very tellingly, art. 15 of the law provided that Bulgarian subjects of non-Bulgarian origin who left the country would thereby lose their citizenship. Moreover, these individuals had to sell their property within three months of departure. These discrimina- tory provisions bear the mark of the time, and this mark was increas- ingly fascistic and paranoid. In his commentary on the law (prefaced by the then Minister of Justice Vasil Mitakov), Malinkov (1941: 42) ar- gues that these measures were necessary in view of the agreements with Turkey, Greece and Romania on population exchange.13 Another ‘reason’, cited by the author, was ‘the strong Zionist propaganda, which resulted in a great number of Jews, who were Bulgarian subjects, reset- tling in Palestine.’ g Thus, the 1940 law shifted the emphasis of citizenship regulation from ius soli to ius sanguinis. Ethnic Bulgarians were given some sig- nificant privileges. Furthermore, the state became increasingly para- noid about its citizens of non-Bulgarian extraction. During the Second World War, Bulgaria was governed by a pro-German authoritarian re- gime headed by Tsar Boris III. Although this regime never openly en- dorsed the Nazi totalitarian ideology as a whole, in some of its policies it clearly came close to it. For instance, under German pressure, laws 218 DANIEL SMILOV AND ELENA JILEVA restricting the rights of Jews were implemented; the infamous Law on the Protection of the Nation (1940) was the primary piece of legislation modelled on the Nuremberg laws. Much of the Bulgarian legislation re- mained loosely enforced, however, and the population at large sym- pathised with the Jews. These public sentiments, along with the deci- sive action of the politician Dimiter Peshev and a few other members of the establishment and the Orthodox Church, ultimately led to the saving of 50,000 Bulgarian Jews from deportation to the death camps (see Todorov 2001). This triumph of citizen decency in repressive times was marred, however, by the deportation of 11,000 Jews from the terri- tories occupied by the Bulgarian army in Greece, Macedonia and south- east Serbia to Treblinka and possibly other camps.14 y Thus, the regime did succumb to the fascist political fashion of its time, and the 1940 Law was a good example of this. Art. 21, for in- stance, envisaged various grounds for a loss of Bulgarian citizenship, including ‘acting against the security of the Bulgarian state’. THE POLITICS OF BULGARIAN CITIZENSHIP It is im- portant to note, that only Bulgarian subjects living abroad and Bulgar- ian subjects of non-Bulgarian origin could lose their citizenship in this way. The language in the provision for subjects of non-Bulgarian origin was particularly telling: they were to lose their citizenship ‘if they ha[d] proven unworthy of it or were considered dangerous for state security and public order.’ Needless to say, the vagueness of the provision was a guarantee for administrative abuses. In September 1944, the monarchical regime came to a crushing end through a communist takeover, which began the establishment of Soviet-sponsored communist rule in Bulgaria. This led to significant changes in Bulgarian nationality laws. The 1947 Constitution of the People’s Republic of Bulgaria did not have provisions pertaining to the acquisition, loss or restoration of nationality, but for the first time changed the legal status of Bulgarians by turning them from subjects to citizens – this was in line with the antimonarchical sentiments of the time. In 1948, the Grand National Assembly adopted the Law on Bul- garian Citizenship, which replaced the 1940 Law on Bulgarian Nation- ality. The reasons set out in the bill sent to the Assembly pointed out that there was a need to overcome an inadmissible differentiation of Bulgarian citizens into those of Bulgarian origin and those of non-Bul- garian origin who were treated less favourably. Therefore, the law re- verted back to the pre-1940 main principles in this area. Art. 1.1 stated that a ‘Bulgarian citizen by origin is any person whose parents are Bul- garian citizens’. The reference to ‘Bulgarian parents’ was eliminated, which turned the definition from an ethnic into a rather more civic one. Art. 2 introduced the principle of ius soli by stipulating that a: THE POLITICS OF BULGARIAN CITIZENSHIP 219 Bulgarian citizen by place of birth is every individual born or found inside the territory of the country, whose parents are un- known, or are of unknown citizenship or are without citizen- ship. However, this was a more limited application of ius soli compared to the Law of 1880, according to which practically every individual born in Bulgaria was considered a Bulgarian subject.15 The restriction of the scope of the place-of-birth principle can be explained by contextual con- siderations: in 1948, Bulgaria was a country with a growing population that had absorbed significant waves of refugees over the previous two decades. THE POLITICS OF BULGARIAN CITIZENSHIP There were practically no reasons for encouraging aliens to settle in Bulgaria. Paradoxically, however, this principle of limited ius soli became a permanent feature of subsequent legislation, even after the demographic context had completely changed. Moreover, although the communist rulers attempted to abandon the fascist-inspired conceptions of ‘Bulgarians by origin’, they still pre- served some of the most discriminatory practices embedded in the 1940 Law. Thus, immediately after the communist takeover on 9 Sep- tember 1944, many Bulgarians were deprived of Bulgarian citizenship for political reasons and their properties were confiscated (Aleksandrov 1995: 44-45). In 1968, a second Law on Bulgarian Citizenship was adopted. Under the 1968 Law all Bulgarians who refused to return behind the Iron Curtain were deprived of their citizenship. Similarly, anyone who in any way dared to express a negative opinion of the communist regime was also deprived of his or her citizenship.16 The 1968 Law, like the 1940 Law, did not tolerate dual citizenship: a number of special bilat- eral international agreements were signed with a view toward eliminat- ing or preventing dual citizenship (Valkanov 1978: 33-48; Tzankov 2004: 48-51).17 The 1968 Law did not explicitly require the renuncia- tion of a previous citizenship by aliens who sought naturalisation, but it did deprive Bulgarians naturalised elsewhere of their Bulgarian citi- zenship. This last clause was introduced at the time of the expulsion of the Bulgarian Turks (Liebich 2000: 105) – a shameful episode, which coin- cided with the end of communist rule in the country. Although crude analogies between fascist and communist rule are always misleading, it seems that in 1984 the Communist party leadership got infected with an ethnic paranoia that was very similar to that of their fascist pre- decessors. They decided to change the names of all ethnic Turks and Muslims into Bulgarian names (for more details, see Kalyonski & Gruev 2008). The massive administrative operation was accompanied by intense propaganda, which promoted the idea that these minorities DANIEL SMILOV AND ELENA JILEVA 220 were actually ethnic Bulgarians forcefully assimilated by the Ottomans. Few ethnic Turks were convinced, which led to a protracted period of tension and repression, culminating in 1989 in the successful attempt of the Communist regime to expel more than 300,000 Turks to neigh- bouring Turkey. The regime argued that the process was a ‘voluntary’ resettlement; the public called it, with some irony, ‘the Great Excur- sion’. THE POLITICS OF BULGARIAN CITIZENSHIP Thus, the regime which started with a fierce rejection of the fas- cist laws and policies, ended up endorsing similar ones, albeit consider- ably less sinister (without death camps and ‘a final solution’, for in- stance). In the same year, communist rule finally collapsed, as a result of which between one-third and one-half of the ‘excursion-goers’ even- tually returned home safely.18 On 20 December 1990, the Grand Na- tional Assembly passed a law to have citizenship restored to the victims of the repressions against the Turks and Muslims, after which they could also reclaim any property confiscated during their absence (for more details, see the discussion in section 7.4).19 THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP 221 should be Bulgarian ‘citizens by birth’,21 which, as a non-ethnic cate- gory, does not exclude representatives of minorities from running for this office. The inclusion of this provision was not a reflection of some grand ideology of Bulgarian nationhood, but was the result of a contex- tual, tactical political game among the major players in the Grand Na- tional Assembly, who wanted to block the candidacy for president of the then popular ex-communist leader Andrei Lukanov, who was born in the Soviet Union and originally had a Soviet passport. g y p p As often happens with such contingent political calculations, this one also had serious unintended consequences for its drafters from the ex-communist Bulgarian Socialist Party (BSP). In 1996, Georgi Pirins- ki, the official BSP candidate for president was practically disqualified from the race by the Constitutional Court, which ruled that for the pur- poses of the presidential election citizenship by birth was to be estab- lished according to the law in force at the time of birth of the candi- date.22 Thus, Pirinski was denied ‘citizenship by birth’ due to the vag- aries of the communist citizenship laws in force at the time of his birth (Pirinski was born in New York, and according to the then valid law was not a citizen by birth). The dubious constitutional reasoning of the justices – which gave priority to the communist legislation over the Constitution – can be explained by the politicisation of the Court dur- ing that period; most of the judges just happened to be opponents of the BSP. This example vividly shows that citizenship policy is never so- lely a reflection of coherent ideologies and grand principles because key decisions can often be explained by contextual factors of temporary importance. Another example was the amendment to the Constitution that stipulated five years of residence in the country for presidential candidates. In the mind of the drafters, this requirement was designed to prevent Tsar Simeon II from running for the presidency in 1991. In fact, it did prevent him from doing so, but only ten years later (in 2001), when many of the drafters of this amendment would have been perhaps more supportive of his candidacy. 7.2 Current citizenship rules and practices The current Bulgarian legal regulations regarding citizenship are based on the provisions of the 1991 Constitution. As we made clear in the previous section, the most repressive parts of the communist legisla- tion were amended even before the adoption of the new basic law. However, the Constitution was meant to embody a complete vision of all questions relating to membership in the Bulgarian polity. Not sur- prisingly, much of this new philosophy resembles the approach of the Constituent Assembly in Veliko Turnovo in 1879. Firstly, the Constitu- tion grants citizenship to all persons born in the territory of Bulgaria, unless they acquire another citizenship by origin, or born to at least one parent who is a Bulgarian citizen (art. 25 (1)). Compared to the 1880 Law, for instance, this is a more limited application of ius soli si- milar to the communist laws. Concessions to ethnic Bulgarians are also constitutionalised: ‘A person of Bulgarian origin shall acquire Bul- garian citizenship through a facilitated procedure’ (art. 25 (2)). In order to avoid some of the most repressive practices of previous regimes, the Constitution expressly prohibits depriving Bulgarian citizens by birth of their citizenship (art. 25 (3)) and the extradition or expatriation of ci- tizens (art. 25 (4)).20 Furthermore, the Constitution practically guarantees the full scope of rights to all Bulgarian citizens without differentiating between citi- zens by birth or by naturalisation. There are only some minor, but symbolically important, exceptions, like the requirement of art. 93 (2), which states that candidates for the office of President of the Republic THE POLITICS OF BULGARIAN CITIZENSHIP Leaving these unintended consequences aside, the Constitution had to solve one very serious problem indeed: the ongoing tension between the Bulgarian majority and the Turkish minority after the events of 1989. As mentioned above, the citizenship rights and property of Bul- garian Turks were restored, but many of them had already acquired Turkish citizenship and some had actually decided to resettle in Turkey permanently.23 The question about the status of these persons with double citizenship would require a major revision of citizenship poli- cies, as both the 1940 and the communist regulations had expressly prohibited dual citizenship. The 1991 Constitution altered this practice, mostly by remaining silent on the possibility of double citizenship.24 Thus, in order to remedy the former injustices against the Bulgarian DANIEL SMILOV AND ELENA JILEVA 222 Turks, Bulgaria became one of the few countries in Eastern Europe to recognise dual citizenship (Liebich 2000: 105). Despite these concessions to the Turkish minority, the 1991 Consti- tution remains fundamentally sceptical about minority rights. It espouses an attitude that closely resembles the German constitutional doctrine of ‘militant democracy’, especially in its application to minori- ties in contemporary Turkish constitutionalism.25 The Constitution is specifically concerned with ethnic and religious politics and in a num- ber of provisions it expressly prohibits the establishing political parties on an ‘ethnic, racial or religious basis’, (11(4)) or parties whose activities are directed against the integrity of the country, the unity of the nation, or the igniting of racial, national, ethnic, and religious hatred (44(2)). g g g (44( )) These provisions threatened to bring the Bulgarian polity to a crisis immediately after the first parliamentary elections under the new Con- stitution in 1991. The reason was the controversy over the Movement for Rights and Freedoms (MRF), a political party organised mostly by members of the Turkish ethnic minority, which successfully ran in the in parliamentary election and formed a coalition government with the Union of Democratic Forces (UDF), the anti-communist opposition. BSP deputies attacked the constitutionality of the party before the Con- stitutional Court.26 In a well-reasoned decision, the Court effectively re- laxed the prohibition of ethnic parties in art. 11 of the Constitution by arguing that only parties that are ethnically exclusive and threaten the constitutional order are to be banned. This decision legitimised the MRF, which gradually emerged as one of the major political parties in Bulgarian politics. THE POLITICS OF BULGARIAN CITIZENSHIP The MRF is currently probably one of the few ethnic parties in Europe that officially belong to the European family of liberal parties. The MRF has been part of the ruling coalitions in Bulgaria since 2001. The rationale of the 1991 Constitution was reflected in Bulgaria’s ci- tizenship legislation. The 1968 Law went through a series of amend- ments. The most recent Law on Citizenship was enacted in 1998. It took into consideration the new social developments of the transition period, as well as the prospect of EU membership. All of the remain- ing discriminatory positions included in the earlier Bulgarian citizen- ship laws, which affected those citizens who had left the country, were ultimately revoked by the 1998 Law.27 The 1998 Law restored the citi- zenship of Bulgarians whose citizenship had been withdrawn by de- crees during the period 1944 to 1947.28 7.2.1 Acquisition of Bulgarian citizenship The present law, as already stated, accommodates multiple citizenship. Dual citizens are treated as Bulgarian citizens only when they enter THE POLITICS OF BULGARIAN CITIZENSHIP 223 Bulgarian territory and acquire the rights and duties of Bulgarian citi- zens. According to art. 3 in the current law: Bulgarian territory and acquire the rights and duties of Bulgarian citi- zens. According to art. 3 in the current law: Any Bulgarian citizen who is also a citizen of another state shall only be considered a Bulgarian citizen in the application of the Bulgarian legislation unless otherwise provided for by law. There are very few disadvantages for people with dual citizenship, ac- cording to the Constitution: among the conditions for the election of deputies to the national parliament is the requirement that the ‘person must be a Bulgarian citizen who does not have another citizenship’; the same is true for presidential candidates. As Todorov points out, the 1991 Constitution defines two notions: ‘a person of Bulgarian origin’ (a Bulgarian) and ‘a Bulgarian citizen’. A Bulgarian is a person who by his or her origin is of Bulgarian ‘blood’. Para. 2 (1) of the 1998 Law specifies that ‘a person of Bulgarian origin is one whose ascendants (or at least one of these) are Bulgarian’. A Bul- garian is not necessarily a Bulgarian citizen. He or she could, for in- stance, be a Moldavian, a Macedonian or a Hungarian citizen. The no- tion ‘Bulgarian’ refers to an ethnic identity, not a legal status. The only privilege that the Constitution provides for ethnic Bulgarians is the ability to obtain Bulgarian citizenship through a facilitated naturalisa- tion procedure. By contrast, the notion of the ‘Bulgarian citizen’ is legal and civic in its content. Bulgarian citizens, including those by birth, can be ethnic Bulgarians but also individuals from other ethnic groups such as Turks, Armenians, Chinese, etc. (Todorov 1996: 7). Therefore, the ethnic definition of ‘Bulgarian’ plays a role only in the rules for naturalisation. There are two methods for the acquisition of Bulgarian citizenship by naturalisation: a general regime and a prefer- ential regime for certain categories of persons, including ethnic Bulgar- ians. The great majority of those who have acquired Bulgarian citizen- ship through naturalisation over the past few years have done so using the preferential regime (Tzankov 2005). Art. 12 of the 1998 Law establishes the general regime for naturalisa- tion. 7.2.1 Acquisition of Bulgarian citizenship The requirements are that the applicant: 1. is of lawful age; 2. was granted permission for permanent residence29 in the Republic of Bulgaria not less than five years before application; 3. has not been sentenced by a Bulgarian court for an intentional crime of a general nature and has not been the subject of criminal proceedings for such a crime unless the person concerned has been rehabilitated; 4. has an income and occupation enabling him or her to support him- self or herself in the Republic of Bulgaria; DANIEL SMILOV AND ELENA JILEVA 224 5. has a command of the Bulgarian language subject to verification in accordance with a procedure established by an order of the Minister of Education and Culture; and 5. has a command of the Bulgarian language subject to verification in accordance with a procedure established by an order of the Minister of Education and Culture; and 6. was released from his or her previous citizenship or will be released from his or her citizenship at the moment of acquiring Bulgarian citizenship.30 6. was released from his or her previous citizenship or will be released from his or her citizenship at the moment of acquiring Bulgarian citizenship.30 It is clear from these provisions that the general regime for obtaining citizenship by naturalisation is quite restrictive. Applicants (if they do not fall into some special category) are normally required to have legally resided in the country for ten years (five years to obtain a permit, and another five to be eligible for citizenship). The waiting period for the permanent residence permit can be waived for those who have invested more than US$ 500,000. Furthermore, the 1998 Law introduces for the first time the requirement of a clean criminal record, and, more im- portantly, knowledge of the Bulgarian language,31 employment and in- come or income guarantees for an applicant of foreign origin. It is not surprising, then, that between 2000 and 2006, there were only a total of 2,395 applications for Bulgarian citizenship through general natura- lisation, of which only 865 were granted – all in all, statistically insig- nificant numbers (see Table 1 and Table 2 in section 7.4). It is clear from these provisions that the general regime for obtaining citizenship by naturalisation is quite restrictive. 7.2.1 Acquisition of Bulgarian citizenship Applicants (if they do not fall into some special category) are normally required to have legally resided in the country for ten years (five years to obtain a permit, and another five to be eligible for citizenship). The waiting period for the permanent residence permit can be waived for those who have invested more than US$ 500,000. Furthermore, the 1998 Law introduces for the first time the requirement of a clean criminal record, and, more im- portantly, knowledge of the Bulgarian language,31 employment and in- come or income guarantees for an applicant of foreign origin. It is not surprising, then, that between 2000 and 2006, there were only a total of 2,395 applications for Bulgarian citizenship through general natura- lisation, of which only 865 were granted – all in all, statistically insig- nificant numbers (see Table 1 and Table 2 in section 7.4). In contrast to the general regime, the 1998 Law is much less restric- tive with regard to privileged groups. These can be divided into three ca- tegories. The first one is entitled to privileged naturalisation, which re- quires a minimum of three years residency after the acquisition of a permit for permanent residence in the territory of the Republic of Bul- garia. It includes persons married to Bulgarian citizens, persons having acquired permanent residence before the age of majority (art. 13) and re- fugees (art. 13a). Similar access is granted to stateless persons (art. 14). The second category of persons who can benefit from the regime of privileged naturalisation includes those who have made special contri- butions and performed meritorious service to the Republic of Bulgaria. They are granted achievement-based nationality. Most often, this criter- ion is used to facilitate the acquisition of Bulgarian citizenship for ath- letes.32 While refugees and foreign athletes must still satisfy a three-year re- sidence requirement for facilitated naturalisation, this condition is dropped for the third category. It is, in fact, the most important group in terms of numbers and consists of persons of Bulgarian origin. Ac- cording to art. 15 of the law, as amended in 2001, applicants of Bulgar- ian origin are exempted from all but two requirements: the minimum age and a clean criminal record. 7.2.1 Acquisition of Bulgarian citizenship It is important to note that the exemp- tion from language tests and residence requirements was adopted in 2001, which led to a significant increase in naturalisation proceedings, THE POLITICS OF BULGARIAN CITIZENSHIP 225 as discussed in section 7.4. The privileges for ethnic Bulgarian appli- cants also have financial implications; for instance, they pay a fee of only 5 BGN, whereas other foreigners pay 1,000 BGN.33 Any person who is not a Bulgarian citizen may acquire Bulgarian ci- tizenship through naturalisation provided that he or she is of Bulgarian origin.34 Establishment of Bulgarian origin is ethnic; the applicant has to show that at least one of his or her ancestors (ascendants – parents and grandparents) was an ethnic Bulgarian. The birth certificates of the parents and grandparents, their mother tongue, membership in Bulgarian institutions such as the Bulgarian Church, schools, former Bulgarian citizenship of the parents, etc., are relevant criteria for the establishment of the ethnic origin of the applicant through the ethni- city of his or her parents. It is important to note, however, that the law remains virtually silent about these more specific criteria of Bulgarian ethnicity, which leaves a significant degree of discretion to the adminis- trative authorities in the resolution of individual cases.35 An attempt to clarify the concrete conditions of proving ‘Bulgarian origin’ is made in the 2000 Law on Bulgarians Living outside the Re- public of Bulgaria, which states that Bulgarian origin can be proven by documents issued by a Bulgarian or foreign state institution, an organi- sation of Bulgarians living outside the Republic of Bulgaria approved by the authorised state institution or by the Bulgarian Orthodox Church (art. 3). However, even with these clarifications, the problem of definitional indeterminacy and administrative discretion remains.36 7.2.2 Loss of citizenship Following the Constitution’s rationale to avoid the excesses of the com- munist regime, the 1998 Law declares that Bulgarian citizens by birth can lose their citizenship only if they express an explicit wish for this and acquire another citizenship. Generally, there are three ways to lose one’s Bulgarian citizenship: release from Bulgarian citizenship, revoca- tion of naturalisation, and deprivation of citizenship. According to art. 20: any Bulgarian citizen who is permanently residing abroad may request to be released from Bulgarian citizenship, if he or she has acquired foreign citizenship or if there is information show- ing that a procedure for acquisition of foreign citizenship has been initiated. any Bulgarian citizen who is permanently residing abroad may request to be released from Bulgarian citizenship, if he or she has acquired foreign citizenship or if there is information show- ing that a procedure for acquisition of foreign citizenship has been initiated. Naturalisation can be withheld in a limited number of cases, for in- stance if the person has obtained it through the provision of false infor- mation or through the withholding of relevant information from the 226 DANIEL SMILOV AND ELENA JILEVA authorities (art. 22). Deprivation of citizenship of a naturalised person can take place if the person has committed grave crimes against Bul- garia (art. 24). Revoked Bulgarian citizenship can be restored under certain condi- tions. These are dealt with both in the provisions in the 1998 Law and the amendments of 2001. The most important of these conditions is a residence requirement: three years of lawful permanent residence after the submission of the application for the restoration of one’s citizen- ship (art. 26 (3)).37 7.3 Current political debates The discussion thus far has demonstrated that, at the level of the Con- stitution, Bulgarian legislation provides for a fairly egalitarian frame- work, which could be interpreted as an acceptable basis for a civic com- munity. However, this framework remains incomplete, and it allows for alternative interpretations through legislation; the main culprit in this regard is the provision for privileged naturalisation of Bulgarians by origin. The same is true of the provisions for multiple citizenship, which could also be interpreted as bestowing specific privileges on par- ticular groups of citizens. Thus, ultimately, the Constitution leaves it to the political process to determine the precise make up of the Bulgarian political community, and the exact scope of the privileges for specific groups. In this section we therefore examine the dynamics of the politi- cal processes, which have a bearing on the questions discussed. p g q The most important recent development in this regard is the rise of populist politics in the country. Bulgarian populism is marked by a cri- sis in the representative system, and the ‘mainstream parties’ in parti- cular, and the constant emergence of new players – such as the ex-Tsar Simeon’s NDSV movement (National Movement Simeon the Second)38 in 2001, Boyko Borissov’s GERB (Citizens for European Development of Bulgaria) in 2006, and even something like a radical nationalist party – Ataka – in 2005. These populist players are largely indifferent to traditional left-wing or right-wing political ideologies. They use other means for the mobilisation of their people, which often include anti- corruption campaigns and various milder or stronger versions of na- tionalism. The rise of populism in Bulgaria coincided with the transfor- mation of the MRF – the Turkish minority party – into a mainstream player consistently taking part in the government of the country.39 Thus, the anti-elite, anti-establishment rhetoric of the newcomers also acquired some ethnic overtones; they started to ethnicise corruption and to portray the MRF as the most problematic element in the party system. However, evidence shows that the MRF, although prone to pa- THE POLITICS OF BULGARIAN CITIZENSHIP 227 tronage and clientelistic practices, is no more corrupt than the other parties. In the face of populist attacks from these newcomers, the representa- tives of the ‘mainstream parties’, such as the BSP, had to revise their campaign strategies and react to the challenge. 7.3 Current political debates President of the Repub- lic Georgi Parvanov – who was the former chairman of the BSP and a historian by training – played a primary role in the design of the new political course, which could be described as the ‘mainstreaming’ of some mild forms of nationalism in order to reduce the appeal of the populists. As the head of state, Parvanov started taking public positions on issues of ‘historical importance’, such as the significance of the struggles for the liberation of Macedonia in Bulgarian history, the ‘mis- interpretations’ by domestic and foreign experts of specific events, such as the Batak massacre in 1876, and so on.40 Formal official rituals also underwent significant redevelopment in order to stress the historical continuity of the Bulgarian nation and the grandeur of the sacrifice made by its ancestors. Of course, President Parvanov played an important role in the for- mation of citizenship policy as well, though in this area, the ‘main- streaming’ of nationalism began even before the start of his mandate. Gradually, however, citizenship policy was included in the symbolic manifestations of mild nationalism, especially with regard to interna- tional relations with Macedonia. Below we demonstrate how the rise of populism and the reactive ‘mainstreaming’ of mild nationalism affected three specific policy areas: the voting rights of Bulgarian Turks residing in Turkey, the nat- uralisation policies and policies that address demographic problems and the need for foreign labour. 7.3.1 The voting rights of Bulgarian Turks living in Turkey As already discussed, in the 1980s, the then communist regime of Bul- garia adopted a policy towards the Turkish minority in Bulgaria, ac- cording to which Bulgarians with Turkish origin were forced to adopt Slavic names. This led to a great wave of emigration back to Turkey in 1989. After the one-party system collapsed in 1989, many reclaimed their Bulgarian citizenship, but only some of them resettled in Bulgar- ia, while others who regained Bulgarian passports preferred to remain in Turkey (see also Kadirbeyoglu in this volume). Among this group, there are many children and retirees who nowadays spend their sum- mers in Bulgaria. Some come back to pursue higher education or per- form military service in Bulgaria, given the general fear among Turks of having to do one’s military service in the Kurdish regions of Turkey. The two states have an agreement concerning the mutual recognition 228 DANIEL SMILOV AND ELENA JILEVA of one another’s military service (which became a moot point after the professionalisation of the Bulgarian army). In general, the relation- ships between Bulgaria and Turkey have begun to flourish and have reached an unprecedented level of civility, especially when, in 1997, the then newly elected Bulgarian President Peter Stoyanov delivered a speech to the Turkish National Assembly, asking for forgiveness for what had been done to the Turkish minority in his country (Petkova 2002: 52-54). According to official migration statistics, Turkish migrants with dual Bulgarian and Turkish citizenship form a community of around 380,000 people. Under the 1998 citizenship law, these migrants have the right to regain their Bulgarian citizenship while keeping their Turk- ish citizenship. As dual citizens, they develop and share dual loyalty, rights and obligations (O¨ zgu¨r-Baklacioglu 2006: 322). The rise of populism in Bulgaria meant that the voting rights of Bul- garian citizens of Turkish origin who are resident in Turkey came in- creasingly under attack. These rights are the same as those of other ci- tizens; that is, they can participate in parliamentary, presidential, local and EP elections in Bulgaria. Debates about these voting rights arose first in the 1990s. Thus, on the eve of parliamentary elections in De- cember 1994, the possibility of significant support for the MRF by Bul- garian Turkish voters in Turkey was strongly debated in the Bulgarian media. MRF supporters were estimated at 150,000 eligible voters. 7.3.1 The voting rights of Bulgarian Turks living in Turkey The main question was whether citizens with dual citizenship who were not resident in Bulgaria had the right to influence Bulgarian internal politics. However, this debate was not revived in the 1997 parliamen- tary elections when Bulgarians had much more serious crises to deal with, such as trying to survive after the economic crisis of 1996 (Iva- nov 1997). During all general elections held in the past fifteen years, the inter- ested parties (specifically the MRF) have organised large numbers of Bulgarian Turks with voting rights who reside permanently in Turkey to vote either in polling stations in Turkey (in over 70 locations in 2005) or to be transported by bus to Bulgaria to cast their votes there.41 Bus transportation has been widely available during municipal elec- tions in Bulgaria. In the June 2001 parliamentary election, the MRF re- ceived 38,840 of the 50,000 votes cast in Turkey. In this way, the votes of the dual citizens living in Turkey helped elect three MRF members to the Bulgarian Parliament (it needs to be noted, however, that dual ci- tizens have neither a special representation quota nor specially desig- nated MPs as their representatives). The local elections in 2003 showed similar results, when dual citizens contributed to MRF electoral vic- tories in twelve municipalities as well as the election of 695 local mu- THE POLITICS OF BULGARIAN CITIZENSHIP 229 nicipality council members and advisors (O¨ zgu¨r-Baklacioglu 2006: 328). nicipality council members and advisors (O¨ zgu¨r-Baklacioglu 2006: 328). Protests against the voting rights for dual citizens have been ex- pressed by political parties, public opinion as well as some scholars (e.g. Boyadjiev 1996).42 In 2007, however, these protests escalated into a campaign by all of the opposition parties, in which individual mem- bers of the ruling majority also took part (including the BSP chairman of parliament who in a crucial vote on some of the debated residence requirements, sided with the opposition). q pp ) The conflict over the voting rights for dual citizens emerged in rela- tion to the first European Parliament elections in May 2007. The ques- tion was whether the National Assembly should introduce specific resi- dence requirements for voters in European Parliamentary elections, which would disqualify the Bulgarian Turks living in Turkey. The oppo- sition parties proposed these requirements, which soon gained wide- spread public support. 7.3.1 The voting rights of Bulgarian Turks living in Turkey According to a study by the Alpha Research Agency, public opinion in Bulgaria was clear with some 78.7 per cent of all Bulgarians supporting residency requirements in these elections. Such a high rate of popular approval is comparatively rare in Bulgarian politics. The sole opponents of the new regulation were supporters of the MRF.43 In the ensuing heated parliamentary debates some members of the opposition even argued that MEPs elected with the help of Bulgarian voters in Turkey would in fact represent Turkey rather than Bulgaria. After four hours of debate over the controversial text, 80 opposition MPs walked out of the plenary hall. Ultimately, the ruling coalition al- lowed all citizens to be enfranchised regardless of where they resided. In the subsequent election, the MRF did exceptionally well and came in third with 20.26 per cent, just behind GERB (a centre-right populist party) with 21.68 per cent and the BSP with 21.41 per cent. The MRF received a total of 392,650 votes. 39 5 In the following local elections of 2007, attempts to introduce the re- sidency requirements succeeded initially, but were then dramatically watered down through various legal technicalities, which made it possi- ble to claim residency only on the basis of a permanent address regis- tered on one’s identity card, which virtually all people have, even if they live abroad. Thus, the practice of ‘electoral tourism’ continued una- bated. It is important to note, however, that the MRF is gradually losing the public debate on residency requirements, and if they were to join the opposition, a reversal of policy is very probable. What is striking in the emerging dominant public opinion is the lack of a principled vision of citizenship. Some who support the denial of voting rights argue that this measure will eliminate possibilities for vote-buying and electoral DANIEL SMILOV AND ELENA JILEVA 230 fraud; others see it as a punishment for the ‘corrupt’ MRF; while still others try to argue that only taxpayers should have political rights; and last, but not least, are the Ataka supporters, who believe that ethnic Bulgarians should be privileged in terms of political rights. This ca- cophony of angry voices is the mark of populist mobilisation. THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP 231 cedonians and a slightly greater number of Moldovans were granted Bulgarian citizenship. The total combined number of Russians, Israe- lis, Ukrainians and Serbs who received Bulgarian passports in 2005 was less than 700.45 Less than five months before Bulgaria joined the EU, the number of applicants awaiting approval exceeded 50,000. In 2007, that number rose to 60,000 persons waiting for Bulgarian citi- zenship.46 p All this is due to the privileged and facilitated naturalisation proce- dure for members of Bulgarian minorities applying for Bulgarian citi- zenship in neighbouring countries. Once it is granted to them, they en- joy all the rights to which Bulgarians are entitled, without being ob- liged to give up their other nationality or live in Bulgaria. But here lies the problem: it remains unclear what the actual contri- bution of these people to contemporary Bulgarian society is going to be. They are applying with the explicit expectation of the travel oppor- tunities that a Bulgarian passport affords, without having to live in the country. The fear is that the ultimate result of this policy might be the further extension of the ‘symbolic Bulgarian citizenship’ without ad- dressing the actual pressing social problems. There is also a European dimension to this issue: this type of external Union citizenship gener- ates immigration rights and rights to political participation at the EU level, which may conflict with the developing EU immigration regime. This problem, however, has not even been registered in Bulgarian pub- lic debates. As to the symbolic ‘gains’ that the policy offers, they come mostly in the form of settling historical scores with neighbouring countries. Ma- cedonia is the primary target here,47 and thus it is no surprise that cur- rent Bulgarian naturalisation policy is most negatively received there, especially among the nostalgic pro-Yugoslav local political establish- ment. The concept of ‘Bulgarian by origin’ contradicts the ‘official’ in- terpretation of Macedonian identity because the concept assumes that there are ethnic Bulgarians living within Macedonian borders. The offi- cial reaction of the Macedonian government to the Bulgarian policy has been ambivalent, however, if we leave aside the ongoing anti-Bul- garian bias in the local media. Unlike the Ukraine, Macedonia has not chosen to penalise its citizens who hold dual citizenship. 7.3.2 Dual citizenship for persons with Bulgarian origin At first sight, the contemporary naturalisation policy of Bulgaria has been a success – numbers of applicants and naturalised citizens con- tinue to rise. In a situation of negative demographic trends,44 this is undoubtedly good news. However, the problem is that this policy is not designed to help solve the demographic problem, or provide the neces- sary workforce, but mostly to win some symbolic battles over the past with neighbouring countries, as well as to mobilise domestic voters. The backward-looking nature of this policy means that its successes will hardly be sustainable. The majority of applications for the acquisition of Bulgarian citizen- ship are based on Bulgarian origin. Between 2001 and 2005 this amounted to 90 per cent of the total number of applications (Tzankov 2005). For the period 2000 to 2006, the largest number of applica- tions for citizenship were those based on origin and were filed by Bul- garians abroad, mainly from Macedonia, Moldova, Russia, Ukraine and Serbia. In total, there were 87,722 applications for citizenship based on origin. Of these, 32,702 came from Macedonia and 38,641 were filed by Moldovan citizens. During the same period, the number of citizens from Macedonia who were granted Bulgarian citizenship on the grounds of their Bulgarian origin by Vice-Presidential Decree was 10,850. The number for Moldova was 9,187 (see Table 1 and Table 2 in section 7.4). According to Stefan Nikolov from the State Agency for Bulgarians Abroad (SABA), ‘since 2001, Bulgaria has been attractive because of its Euro-Atlantic prospects, its stability and the travel opportunities that Bulgarian passports offer’. Nikolov also noted that the number of appli- cations has increased exponentially. In 2001, only 940 people acquired Bulgarian citizenship, but the number rose to 3,000 in 2002; 4,000 in 2003; 5,559 in 2004; 5,722 in 2005; and 6,511 in 2006 (see Table 2). One of the most prominent cases of Macedonians being granted Bul- garian citizenship was that of the former Prime Minister of Macedonia, Ljubcho Georgievski. More than 20,000 people have received Bulgarian passports since 2001, when the number of applicants for Bulgarian citizenship was al- most 5,500. That number rose to nearly 29,500 in 2004. In 2005 alone, when 23,200 applications were submitted, more than 2,400 Ma- THE POLITICS OF BULGARIAN CITIZENSHIP Nevertheless, Macedonia has intensified its claims that Bulgaria is violating the rights of Macedonians in Bulgaria and, in particular, their rights to association and assembly. Symbolic politics here has indeed led Bulgaria to violate the European Convention on Human Rights (ECHR). In 2000, the Bulgarian Constitutional Court (BCC) banned the tiny nationalist Macedonian party OMO-Ilinden because it was seen as a threat to the integrity of the state and the unity of the nation. The BCC’s decision48 strayed from its previous reasoned judgements DANIEL SMILOV AND ELENA JILEVA 232 concerning the MRF (see the discussion in section 7.2), and essentially ruled that certain speeches, letters and maps produced by the party were a substantial threat to the Bulgarian state. This paranoid reason- ing was judged to be in violation of ECHR art. 11 (in conjunction with art. 10) by the European Court of Human Rights in 2006.49 Since then, Bulgarian authorities have denied registration to OMO on various formal and procedural grounds. These decisions have recently been cri- ticised by the Committee of Ministers of the Council of Europe.50 y p It is difficult to say who has the upper hand in these friendly skirmishes in the symbolic warfare between Bulgaria and Macedonia. Naturalisation numbers and some famous ‘casualties’, such as the nat- uralised former PM of Macedonia, tilt the scales towards Bulgaria. But the OMO-Ilinden story and the general sympathy for the underdog seem to level the score. It is quite clear, however, that the target audi- ence of the symbolic warfare is domestic; it is used for mobilisation purposes by particular actors, and at least in the Bulgarian case, it cor- relates to the rise of populism and the reactions to it. Not surprisingly, the major actors in this symbolic warfare in Sofia are, on the one hand, the leaders of the nationalistic-populist parties (Ataka, Internal Macedo- nian Revolutionary Organisation (VMRO), among others); on the other hand, it is the President of the Republic himself, who apart from being a key figure in the administration of naturalisation policies, regularly expresses the official Bulgarian position on the symbolic front. In terms of more tangible international relations, Macedonia and Bulgaria, surprisingly, do not have any serious unresolved questions. THE POLITICS OF BULGARIAN CITIZENSHIP Despite the apparent mutual animosities, Bulgaria has always staunchly supported Macedonia both in difficult times, such as during the bombardment of Kosovo by NATO, and now when Macedonia is on its way to becoming a NATO member. Patronising as this behaviour may appear to politicians in Skopje, it is still markedly different from the behaviour of Macedonia’s other EU neighbour, Greece, which still refuses to recognise the official name of the country, and vetoed Mace- donian NATO membership in April 2008. THE POLITICS OF BULGARIAN CITIZENSHIP 233 A primary recent example of these efforts is the aforementioned Law on Bulgarians Living outside the Republic of Bulgaria (2000). This law is based on a specific ethnic definition of Bulgarians abroad, which is less inclusive than the definition of ‘Bulgarian by origin’ (O¨ zgu¨r-Bakla- cioglu 2006: 335). ‘Bulgarians by origin’, to remind the reader, are those persons who have a Bulgarian parent (mother or father). ‘A Bul- garian living outside the Republic of Bulgaria’ is a person permanently residing abroad who has at least one parent of Bulgarian origin and has Bulgarian national consciousness. The law in question is designed to: encourage the creation of favourable conditions for the free de- velopment of Bulgarians living outside […] Bulgaria, according to the principles of international law and the legislation of the respective state with the aim of protecting their rights and lawful interests. The Bulgarian state commits itself to support the organisation of Bul- garians outside Bulgaria whose activities are directed at the conserva- tion and development of the Bulgarian language, as well as cultural and religious traditions. Furthermore, the law provides certain entitle- ments for Bulgarians living abroad; for instance, free elementary and secondary education in the state and municipal schools in the Republic of Bulgaria, according to current conditions and regulations for Bulgar- ian citizens. Significantly, with regard to the pursuit of higher educa- tion, the law grants Bulgarians living abroad the right to apply for pub- lic financial assistance (art. 10). Apart from dealing with students, the law also regulates the state support for ‘the preservation and demonstration of the Eastern Ortho- dox faith as the traditional religious affiliation of Bulgarians and as a factor in preserving the Bulgarian national identity’. Moreover, it makes special provisions for Bulgarians living abroad who wish to settle in Bulgaria. It creates favourable conditions for their return by offering them state-owned lands or municipal agricultural lands for use during the early years after settlement. y y Although the law envisages the creation of a National Council for the Bulgarians Living Abroad, the state body responsible for relations with Bulgarians abroad is the State Agency for Bulgarians Abroad (SABA). This agency primarily establishes and maintains contact with and supports the activities of societies, associations, church communes, media and schools of the Bulgarian communities in dozens of coun- tries. 7.3.3 National dreams and the imperatives of a globalised world In this section, we further pursue the theme of an obsession with the past in the development of Bulgarian naturalisation policy, and raise the issue of its adequacy vis-a`-vis more contemporary challenges. Many of the Bulgarian government’s organisational efforts in this area can be understood as an attempt to symbolically restore the Bulgarian Exarch- ate through some modern surrogate, which would institutionalise links with the ethnic Bulgarians abroad. THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP In addition, SABA is an important element in the processes of ac- quiring Bulgarian citizenship, in obtaining permission for long-term residence in Bulgaria and for certifying the Bulgarian origin of persons DANIEL SMILOV AND ELENA JILEVA 234 who have applied for Bulgarian citizenship. The SABA, in essence, serves as a functional substitute for the now defunct Exarchate. The problem here is that because of the excessive focus on historical Bulgarian minorities in adjacent lands,51 the state has virtually ne- glected the close to a million people who have left the country since 1989, emigrating for economic reasons to Western Europe, North America and elsewhere. There is hardly any coherent policy concerning these people, who remain Bulgarian citizens in most cases. Many open questions concerning their situation – such as their health insurance contributions, for instance – are resolved ad hoc, if at all. The Council of Ministers discussed a special report on The Bulgarians Abroad and the State Policy towards Them on 20 December 2007 in an effort to re- spond to some of these problems.52 p p However, as the report shows, the state is making efforts mainly to attract Bulgarians from historical minorities, while relatively few mea- sures have been designed to motivate the Bulgarian emigrants living in Western Europe and North America to return. The report estimates that at present more than three million Bulgarians live abroad, of which one million are Bulgarian citizens. About 800,000 of these have emigrated to Western European or North American countries. Unfortu- nately, the report does not provide more detailed figures for the various regions. Nevertheless, it contains several interesting findings, such as the lack of a new migration of people to EU countries since accession. The policy measures envisaged by the report mainly involve preserving Bulgarian national identity abroad, and introducing educational and vo- cational advantages in the country for ethnic Bulgarians residing abroad with a view to curbing the worrying demographic trends; ac- cording to projections, the Bulgarian population will decrease from 7.7 million in 2004 to only 5.5 million by 2050, if current trends continue. y 5 5 y 5 While the interests of the historical Bulgarian communities are more or less addressed by the report and its recommendations, the problems of economic emigrants are largely neglected. Academic degrees and professional qualifications gained abroad still need to go through a complex procedure of domestic legalisation. THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP 235 At the end of his mandate, Prime Minister Ivan Kostov of the UDF proved to be an exception to the general trend of neglecting economic emigration by inviting ‘prominent Bulgarians’ living abroad (mainly in the EU and North America) to a grand seminar in Sofia. This initiative – episodic as it was – encouraged a number of young, well-educated Bulgarians to return to Bulgaria (ironically, most of them joined the Tsar’s movement in 2001 and became political opponents of Kostov). However, what is currently needed in this regard are not these kinds of episodic gatherings, but some major revisions in policies and institu- tional structures, which remain highly conservative and do not allow for ‘external’ competition. Restructuring institutions, ranging from the higher education system (which is highly inflexible to courses in for- eign languages, guest professorships, etc.) to the administration and the business communities, would not only motivate Bulgarian emi- grants who have left the country for economic reasons to return, but would also attract new highly skilled immigrants. Arguably, the latter are as important as the historical diaspora, since they could introduce newer skills and training, novel practices, etc. g p Finally, an important flaw in the current state policy in terms of the imminent problems of labour shortages (recognised both by the afore- mentioned report and increasing insistent calls from the Bulgarian business community), is its implicit assumption that labour shortages can be addressed primarily by attracting ethnic Bulgarians, be they his- torical or contemporary emigrants. However, it is questionable whether the three million ethnic Bulgarians are willing to return to the mother- land under the conditions Bulgaria can currently offer. Bulgaria may therefore have to look for alternative solutions to labour shortages such as non-ethnic immigration, which, in turn, would require dramatic im- migration and citizenship policy revisions. This is a debate that has yet to take place, however. THE POLITICS OF BULGARIAN CITIZENSHIP EU accession partially re- solved this problem for degrees and qualifications acquired in other EU countries, although, at the practical level, numerous problems per- sist.53 For North American emigrants, EU accession has not changed anything substantially. Moreover, Bulgarian employment and hiring practices in many areas (and especially in the public sector) remain highly clientelistic. The practice of widespread patronage in appoint- ments is definitely a serious hurdle for any person motivated to return from a period of economic emigration. Significantly, the above-men- tioned report fails to address any of these problems. 7.4 Current statistical trends As we have already mentioned, we can observe a marked increase both in the number of applications for Bulgarian citizenship and the num- ber of those granted citizenship since 2001. Between 2001 and 2005, the number of applications for citizenship increased by a factor of four, from 5,495 applicants in 2001 to 29,493 in 2004. There was a slight decrease in 2005 and 2006, but the sizeable difference between appli- cations filed and citizenships granted does not necessarily mean that those applications were denied. There are at present approximately 58,600 files still awaiting a decision, which might simply mean that there is an administrative backlog (Tchorbadjiyska 2007: 100-101). 7.4 Current statistical trends DANIEL SMILOV AND ELENA JILEVA 236 Table 7.1 Number of decrees issued by the Vice-President of the Republic of Bulgaria for change of citizenship for the period 1 January 2001-31 December 2006 Number of persons who obtained Bulgarian citizenship through naturalisation by Vice-Presidential decree Number of persons who obtained Bulgarian citizenship by origin by Vice-Presidential decree Year Total number of filed applications Macedonia Moldova Russia Israel Ukraine Poland Albania Serbia and Montenegro Total Russia Iraq Persons without citizenship Armenia Ukraine Afghanistan Jordan Lebanon Syria Total Number of applications for restoration of BG citizenship Number of applications for renunciation of BG citizenship 001 5,495 169 157 300 13 65 14 18 61 940 165 1 7 6 13 3 2 3 8 274 117 1,093 002 7,438 1,360 583 447 42 243 19 50 219 3,210 71 1 11 3 10 11 3 3 7 161 174 1,091 003 14,306 1,685 1,172 346 91 222 9 59 285 4,179 7 0 16 13 4 10 1 3 4 87 120 695 004 29,493 2,281 2,210 281 122 209 10 86 161 5,559 20 2 25 9 9 9 0 6 6 101 123 945 005 23,200 2,425 2,455 160 152 245 4 34 128 5,722 25 6 42 1 9 11 1 6 2 126 35 710 006 14,468 2,930 2,619 217 107 249 11 52 235 6,511 24 1 27 13 13 7 0 3 7 117 110 828 Total 94,400 10,850 9,187 1,751 527 1,233 67 299 1,089 26,121 312 11 128 45 58 51 7 24 34 865 679 5,362 Source: Ministry of Justice 2007 THE POLITICS OF BULGARIAN CITIZENSHIP 237 Table 7.2 Number of applications for a change of citizenship for the period 1 January 2000-31 December 2006 Number of applications for acquisition of Bulgarian citizenship by origin, submitted by citizens of the respective countries Number of applications for acquisition of Bulgarian citizenship through general naturalisation, submitted by citizens of the respective countries HE POLITICS OF Table 7.2 Number of applications for a change of citizenship for the period 1 January 2000-31 December 2006 Number of applications for acquisition of Bulgarian citizenship by origin, submitted by citizens of the respective countries Number of applications for acquisition of Bulgarian citizenship through general naturalisation, submitted by citizens of the respective countries Year Total number of filed applications Macedonia Moldova Russia Israel Ukraine Poland Albania Serbia and Montenegro Total Russia Iraq Persons without citizenship Armenia Ukraine Afghanistan Jordan Lebanon Syria Total Number of applications for restoration of BG citizenship Number of applications for renunciation of BG citizenship 2000 3,334 113 172 380 12 56 25 22 106 1,145 237 7 23 11 17 29 12 20 46 580 198 1,402 2001 5,495 1,471 613 512 60 243 17 57 239 3,506 180 2 27 11 28 18 4 17 33 407 218 1,347 2002 7,438 2,405 1,866 445 90 269 15 76 297 5,960 85 5 37 9 19 15 4 22 29 299 135 1,034 2003 14,306 4,165 6,124 387 243 465 12 127 345 13,128 57 1 20 19 25 19 1 13 17 227 140 767 2004 29,493 8,731 15,821 485 347 706 19 138 581 28,378 60 10 45 7 33 28 5 27 16 317 186 615 2005 23,200 7,577 11,578 496 651 567 12 255 784 22,129 59 10 35 22 29 16 6 13 17 265 114 692 2006 14,468 8,240 2,467 439 508 506 7 421 716 13,476 76 9 20 20 43 10 5 11 27 300 125 567 Total 97,734 32,702 38,641 3,144 1,911 2,812 107 1,096 3,068 87,722 754 44 207 99 194 135 37 123 185 2,395 1,116 6,424 238 DANIEL SMILOV AND ELENA JILEVA Table 7.1 and Table 7.2 provide detailed data on the numbers of ap- plications and the numbers of those granted citizenship status in Bul- garia between 2000 and 2006. 7.4 Current statistical trends They differentiate between normal nat- uralisation procedures and the preferential treatment of ethnic Bulgar- ians. According to Tchorbadjiyska (2007), one of the factors influencing the increase was a change in 2001 in the laws regarding Bulgarian citi- zenship. Until 2001, applicants of Bulgarian ethnic origin needed to demonstrate proficiency in the Bulgarian language and, most impor- tantly, had to either renounce their present nationality or commit themselves to doing so. As discussed, these two conditions were dropped after 2001. Thus, those Bulgarian minorities living abroad who might not have applied earlier because they did not want to lose their present nationality now have a possibility to acquire Bulgarian na- tionality while retaining their former citizenship. Apart from the change in legal conditions, the increase in citizenship applications is explained by changes in the visa regime. As most of the Bulgarian minorities abroad are citizens of countries that were either on the Bul- garian visa blacklist or were included in it upon entry to the EU, their travel options were seriously hampered. In this situation, the acquisi- tion of Bulgarian citizenship, especially under simplified procedures, became an obvious solution. 7.5 Conclusions The normative frame of Bulgarian citizenship discussed in this chapter is characterised by a degree of substantial incoherence. At the most ba- sic level, the Bulgarian polity combines two different competing and sometimes conflicting principles. On the one hand, it commenced as, and remains, a predominantly Bulgarian project. On the other hand, there has been a genuine attempt to create an egalitarian political com- munity, which does not differentiate between its members, be they of Bulgarian origin or not. This constitutive incoherence has resulted in a complex web of general equality norms, privileges and exceptions. In- deed, there were low points in the development of the Bulgarian polity, when the desire to entrench privileges led to discriminatory and repres- sive practices – especially in the authoritarian-totalitarian period of 1940-1989. However, in times when democracy had a real chance, the incoherent normative framework proved sufficiently inclusive and thus stimulated a rich plurality of voices in Bulgarian public life. This inclu- sive incoherence characterises the contemporary regulation of citizenship as well. THE POLITICS OF BULGARIAN CITIZENSHIP 239 Normative incoherence has one significant drawback: it does not lend itself easily to constitutionalisation and judicial interpretation. Even Dworkin’s super judge Hercules (Dworkin 1986) would find it difficult to construct a theory that eliminates the tension between egali- tarian and identity-based considerations in the Bulgarian case. In the absence of a judicially administrable citizenship rulebook, much has been left to the political process. It is an encouraging fact that at pre- sent this political process has provided for a robust representation of the main minorities in the country. However, as John Hart Ely (1980) predicted in another context, when you rely exclusively on the demo- cratic process, there may be some tiny and insular minorities whose rights will be neglected. In Bulgaria, the associational rights of Macedo- nians have proven Ely right, and it is good that the country is already in an internationally regulated environment; under pressure from the Council of Europe and other organisations this problem is currently being resolved. More worrying, however, are certain aspects of the internal dynamic of the political process related to the rise of populism. This phenomen- on focuses domestic politics on issues such as anti-corruption, personal charisma and, most importantly, national identity and historical symbo- lism. This seems a common Central European development. It does not affect only the marginal radical nationalist parties. 7.5 Conclusions In the emerging political trend, Bulgarian actors stand together with the Kaczynski brothers in Poland, Victor Orban in Hungary, Fico and Mecˇiar in Slova- kia, and so on. Populism threatens to turn the inclusive incoherence of the Bulgarian normative model into a cacophony of angry voices, in which some nasty overtones from authoritarian and totalitarian times are clearly discernible. Overall, although the record of the Bulgarian polity in its modern history has been, at certain points, mixed, it still provides reasons for confidence. In the absence of thoroughly principled solutions to ten- sions and conflicts, the achievement of modus vivendi compromises has been the norm in this history. For students of coherent normative the- ories, this political modus vivendi response perhaps seems unsatisfac- tory. This is not such a great problem, however, because the very idea that civilised relationships among members of a pluralistic polity de- pend on the existence of a coherent theory embodied in a citizenship rulebook strikes us as utterly bizarre. DANIEL SMILOV AND ELENA JILEVA 240 Chronological list of citizenship-related legislation in Bulgaria Date Document Content Source 1879 Turnovo Constitution Defines Bulgarian subjects as those being born in Bulgaria who have no other citizenship and those born of Bulgarian subjects elsewhere 1880 First Law on Bulgarian Nationality Is based on the ius soli principle; envisages four modes of acquisition (by origin, adoption, marriage, naturalisation) and five modes of losing Bulgarian nationality (by renunciation, decision of the authorities, absence, adoption and marriage) 1883 Second Law on Bulgarian Nationality Adds the ius domicilii to the ius soli principle (i.e. 1991 Constitution of the Republic of Bulgaria 7.5 Conclusions Bulgarian subjects are all Turkish subjects who were born on, or resided in, the territory of the Bulgarian Kingdom at the time of the liberation wars) 1903 Third Law on Bulgarian Nationality Maintains the main principles of the first two laws while the principle of ius sanguinis becomes more important 1940 Fourth Law on Bulgarian Nationality Stresses the ius sanguinis principle in addition to the ius soli principle 1947 Constitution of the People’s Republic of Bulgaria Changes the legal status of Bulgarians from subjects into citizens 1948 First Law on Bulgarian Citizenship Based on the ius sanguinis principle; declares that ‘enemies’ of the state and those who defected to the West can be deprived of Bulgarian citizenship 1968 Second Law on Bulgarian Citizenship Deprives Bulgarian citizenship of all those who refuse to return to Bulgaria; prohibits dual citizenship www.legislationline.org (as amended in 1989) 1991 Constitution of the Republic of Bulgaria Defines as Bulgarian citizens all those who have a parent with Bulgarian www.legislationonline.org Chronological list of citizenship-related legislation in Bulgaria Chronological list of citizenship-related legislation in Bulgaria Date Document Content THE POLITICS OF BULGARIAN CITIZENSHIP 241 Date Document Content Source citizenship and those born in the Bulgarian territory who are not entitled to any other citizenship; foresees a facilitated procedure of acquiring citizenship for those of Bulgarian descent 1998 Law on Bulgarian Citizenship Based on both ius soli and ius sanguinis; establishes four principles for the acquisition of Bulgarian citizenship (by origin, place of birth, naturalisation and restoration); accommodates multiple citizenship www.bulgarianembassy. org.uk 2001 Amendments Facilitate the procedures for acquisition of Bulgarian citizenship for ethnic Bulgarians; require persons of non-Bulgarian origin acquiring Bulgarian citizenship by naturalisation to be released of their previous citizenship Date Document 2001 Amendments DANIEL SMILOV AND ELENA JILEVA DANIEL SMILOV AND ELENA JILEVA 242 5 The Turnovo Constitution used subject (podanik) and citizen (grazhdanin) interchange- ably. With the fall of the monarchy and the establishment of communist rule, the term subject was eliminated from legal documents and acquired negative connota- tions in official public discourse. However, it was not true that the subjects to the Bul- garian monarch had fewer rights than the citizens of the communist republic. 5 The Turnovo Constitution used subject (podanik) and citizen (grazhdanin) interchange- ably. With the fall of the monarchy and the establishment of communist rule, the term subject was eliminated from legal documents and acquired negative connota- tions in official public discourse. However, it was not true that the subjects to the Bul- garian monarch had fewer rights than the citizens of the communist republic. 6 It is not only political rights that reflect the egalitarian bias of the Constitution. Art. 78, for instance, granted the right to free public primary schooling to all subjects (both male and female) of the principality. 6 It is not only political rights that reflect the egalitarian bias of the Constitution. Art. 78, for instance, granted the right to free public primary schooling to all subjects (both male and female) of the principality. 7 It envisaged four methods of obtaining Bulgarian nationality (by origin, adoption, marriage and naturalisation) and five methods for the loss of Bulgarian nationality (by renunciation, decision of the authorities, absence, adoption and marriage). The main principle embedded in the law again was the principle of ius soli. Art. 1 para. 1 stipulates: ‘All individuals born on the territory of the Bulgarian Kingdom who have not obtained foreign nationality count as Bulgarian nationals.’ 8 Art. 2 of the 1883 Law introduced a slightly more restrictive version – not all individuals, but only former Turkish subjects could make use of the principle stating that: ‘Bulgarian subjects are all those Turkish subjects who at the time of war for the liberation of Bulgaria had residence or were born on the territory of the Bulgarian Kingdom’. This restriction, which was contrary to the general language of the Constitution (art. 54), was eventually eliminated from later versions. 9 Art. 65 just stated: ‘Only Bulgarian subjects may occupy positions in the state, public and military service.’ 9 Art. Notes 1 The authors thank Rossitza Guentcheva and Ruzha Smilova for their very helpful comments. 1 The authors thank Rossitza Guentcheva and Ruzha Smilova for their very helpful comments. 2 This had implications from the point of view of international law; formally, until 1908, Bulgarians were still considered subjects of the Turkish Sultan (Geshkoff 1927). 3 Such delegations mostly came from Eastern Rumelia – an artificially created, semi- autonomous region in the Ottoman Empire – and Macedonia and Eastern Thrace (or the Vilayet of Adrianople), two other Ottoman regions which were to remain within the Empire without any special privileges for the compact localised masses of Bulgarians living there. Similar delegations came from other lands, such as Bessarabia in present-day Ukraine and Moldavia and Dobrudja in present-day Romania (see Vladikin 1994: 97). The delegates considered the Berlin treaty an illegitimate revision of the San Stefano Peace Treaty (March 1878) between Russia and Turkey, which created Grand Bulgaria, including territories in what is today Macedonia, Turkey, Serbia and Greece. The date of this treaty – 3 March – is currently the Bulgarian national holiday. San Stefano Bulgaria roughly coincided with the jurisdiction of the Bulgarian Exarchate, the Bulgarian Orthodox Church authorities in the Ottoman Empire. 4 The delegates considered the Berlin treaty an illegitimate revision of the San Stefano Peace Treaty (March 1878) between Russia and Turkey, which created Grand Bulgaria, including territories in what is today Macedonia, Turkey, Serbia and Greece. The date of this treaty – 3 March – is currently the Bulgarian national holiday. San Stefano Bulgaria roughly coincided with the jurisdiction of the Bulgarian Exarchate, the Bulgarian Orthodox Church authorities in the Ottoman Empire. DANIEL SMILOV AND ELENA JILEVA 65 just stated: ‘Only Bulgarian subjects may occupy positions in the state, public and military service.’ 10 This was the second assassination of a prime minister by Macedonian nationalists. In 1895, Stefan Stambolov was slain in the centre of Sofia, partly for the same reasons. 10 This was the second assassination of a prime minister by Macedonian nationalists. In 1895, Stefan Stambolov was slain in the centre of Sofia, partly for the same reasons. 11 According to art. 7, a ‘Bulgarian national is every person 1) whose father or (if the father does not have a nationality or is of unknown nationality) mother is a Bulgarian subject, regardless of their places of birth; 2) legitimated by a Bulgarian subject, 3) born out of wedlock whose Bulgarian subject’s origin is proved while he or she is a minor.’ Ius soli was still present in art. 8: a ‘Bulgarian subject by place of birth is every individual 1) born in the Kingdom of unknown parents or parents without nationality and 2) born of foreigners, if he or she had permanent residence in Bulgaria and has not declared foreign nationality one year after coming of lawful age’ (Valkanov 1978). 11 According to art. 7, a ‘Bulgarian national is every person 1) whose father or (if the father does not have a nationality or is of unknown nationality) mother is a Bulgarian subject, regardless of their places of birth; 2) legitimated by a Bulgarian subject, 3) born out of wedlock whose Bulgarian subject’s origin is proved while he or she is a minor.’ Ius soli was still present in art. 8: a ‘Bulgarian subject by place of birth is every individual 1) born in the Kingdom of unknown parents or parents without nationality and 2) born of foreigners, if he or she had permanent residence in Bulgaria and has not declared foreign nationality one year after coming of lawful age’ (Valkanov 1978). 12 In order to obtain a residence permit, applicants had first to prove that they had lived in the kingdom for a year, and then to provide information about their origin (parents), religion, material standing and other details (art. 27). 12 In order to obtain a residence permit, applicants had first to prove that they had lived in the kingdom for a year, and then to provide information about their origin (parents), religion, material standing and other details (art. 27). THE POLITICS OF BULGARIAN CITIZENSHIP 243 Republic in 1971, the People’s Republic of Poland in 1972 and the Czechoslovak Socialist Republic in 1975 (Zakon za bulgarskoto grazhdanstvo 1968). Republic in 1971, the People’s Republic of Poland in 1972 and the Czechoslovak Socialist Republic in 1975 (Zakon za bulgarskoto grazhdanstvo 1968). Republic in 1971, the People’s Republic of Poland in 1972 and the Czechoslovak Socialist Republic in 1975 (Zakon za bulgarskoto grazhdanstvo 1968). 18 From 3 June through 21 August 1989 (when Turkey closed its borders), 311,862 ethnic Turks managed to leave the country. After the fall of Todor Zhivkov and the communist regime in November 1989, some 125,000 returned to Bulgaria. By the end of 1989, the refugees’ back-and forth movements ceased and 245,000 refugees who had fled Bulgaria were granted Turkish citizenship (O¨ zgu¨r-Baklacioglu 2006: 321). 18 From 3 June through 21 August 1989 (when Turkey closed its borders), 311,862 ethnic Turks managed to leave the country. After the fall of Todor Zhivkov and the communist regime in November 1989, some 125,000 returned to Bulgaria. By the end of 1989, the refugees’ back-and forth movements ceased and 245,000 refugees who had fled Bulgaria were granted Turkish citizenship (O¨ zgu¨r-Baklacioglu 2006: 321). 19 The reversal of policy against the Turkish minority started immediately after the fall of the communist regime in November 1989: there were communist leadership decisions calling for the repeal of the repressive legislation as early as December of that same year. 19 The reversal of policy against the Turkish minority started immediately after the fall of the communist regime in November 1989: there were communist leadership decisions calling for the repeal of the repressive legislation as early as December of that same year. 20 The last provision was amended in 2005 in compliance with international treaties concluded by Bulgaria regarding international criminal tribunals: ‘No Bulgarian citizen may be surrendered to another State or to an international tribunal for the purposes of criminal prosecution, unless the opposite is provided for by international treaty that has been ratified, published and entered into force for the Republic of Bulgaria.’ g 21 ‘Bulgarian by birth’, according to the 1991 Constitution, is not an ethnic category; it refers to those born within the country’s borders or to Bulgarian citizen parents and includes thus ethnic Turks and other non-ethnic Bulgarians. 22 See Decision No. 12, 23 July 1996. DANIEL SMILOV AND ELENA JILEVA 12 In order to obtain a residence permit, applicants had first to prove that they had lived in the kingdom for a year, and then to provide information about their origin (parents), religion, material standing and other details (art. 27). 13 The treaty with Greece was ratified by Bulgaria on 4 October 1920. In September 1940, a similar treaty was signed with Romania. 13 The treaty with Greece was ratified by Bulgaria on 4 October 1920. In September 1940, a similar treaty was signed with Romania. 13 The treaty with Greece was ratified by Bulgaria on 4 October 1920. In September 1940, a similar treaty was signed with Romania. 14 It is still being debated in Bulgarian scholarly literature whether the Bulgarian authorities were fully responsible for these deportations: some argue that the occupied territories were virtually under German command. Be that as it may, the Bulgarian authorities did nothing, or at least not enough, to prevent these deportations. Most importantly, Jews from the occupied territories were apparently not granted Bulgarian citizenship, while Greeks and Macedonians were. 14 It is still being debated in Bulgarian scholarly literature whether the Bulgarian authorities were fully responsible for these deportations: some argue that the occupied territories were virtually under German command. Be that as it may, the Bulgarian authorities did nothing, or at least not enough, to prevent these deportations. Most importantly, Jews from the occupied territories were apparently not granted Bulgarian citizenship, while Greeks and Macedonians were. 15 Those born in Bulgaria to an alien were entitled to Bulgarian citizenship if at the time of their majority they were domiciled in Bulgaria. 16 Toni Nikolov, ‘The Right of Bulgarian Citizenship and the European Norms’, Demok- ratzija, 12 May 1998. 17 Such agreements were concluded between the People’s Republic of Bulgaria and the USSR in 1958, the People’s Republic of Hungary in 1959, the Socialist Republic of Romania in 1959, and again with the USSR in 1966, the German Democratic 17 Such agreements were concluded between the People’s Republic of Bulgaria and the USSR in 1958, the People’s Republic of Hungary in 1959, the Socialist Republic of Romania in 1959, and again with the USSR in 1966, the German Democratic THE POLITICS OF BULGARIAN CITIZENSHIP DANIEL SMILOV AND ELENA JILEVA DANIEL SMILOV AND ELENA JILEVA 244 30 This requirement was added in 2000. 30 This requirement was added in 2000. 31 Order No. 5 of 1999 of the Minister of Education and Science defines the procedure for the establishment of knowledge of Bulgarian language. The applicants either have to show documents proving that they have studied in Bulgarian schools or have valid certificates for the language proficiency, or they have to take a written exam before a special commission. They need to demonstrate that they have sufficient knowledge of the Bulgarian language to enable them to communicate at ‘an elementary level’ (art. 6(2)). 32 Until 2006, the proposal allowing foreign athletes to obtain Bulgarian citizenship was made by the Bulgarian vice prime minister responsible for sports. This provision was amended in 2006 when the government decided that the proposal should be made by the director of the State Agency for Youth and Sport (Telegraph, 8 June 2006). ) 33 Interview with Elitza Hristrova, State Agency for Bulgarians Abroad, 2 June 2007. ) 33 Interview with Elitza Hristrova, State Agency for Bulgarians Abroad, 2 June 2007 33 Interview with Elitza Hristrova, State Agency for Bulgarians Abroad, 2 June 2007. 34 Alternatively, (2) he or she has been adopted by a Bulgarian citizen on terms of full adoption; (3) one of his or her parents is a Bulgarian citizen or has passed away as a Bulgarian citizen (art. 15). 34 Alternatively, (2) he or she has been adopted by a Bulgarian citizen on terms of full adoption; (3) one of his or her parents is a Bulgarian citizen or has passed away as a Bulgarian citizen (art. 15). 35 While the 1998 Law envisions a preclusive deadline of three months for the facilitated naturalisation of foreigners with Bulgarian origin, the procedure usually takes two to three years due to the numerous inspections at various Bulgarian institutions. Such inspections are carried out by the Directorate for Migration at the Ministry of the Interior, the National Security Service, the State Agency for Bulgarians Abroad (SABA) and others (Interview, State Agency for Bulgarians Abroad, 2 June 2007). The problem of discretion in the administration of these checks remains quite acute, however. THE POLITICS OF BULGARIAN CITIZENSHIP 23 There are no reliable data for the number of permanent settlers, but estimates show that it was about 100,000. 24 The Constitution mentioned the possibility of dual citizenship in its prohibition on dual citizens running for parliament and the presidency. 25 The term ‘militant democracy’, which was first coined by Karl Loewenstein in the 1930s, refers to a constitutional legal doctrine according to which the democratic state should actively protect itself and its values against internal enemies and should prevent them from coming to power by using the democratic process. The doctrine requires instruments such as bans on extremist organisations and anti- system parties, loyalty requirements for civil servants, etc. The doctrine was first systematically implemented within the German Basic Law after the Second World War. For further information, see Sajo 2004. 26 Decision 4, 21 April 1992. 26 Decision 4, 21 April 1992. 26 Decision 4, 21 April 1992. 27 This process was ultimately finalised in 1998 because it was only then that the Union of Democratic Forces succeeded in the formation of a stable government. Until then, the country was run mainly by the ex-communist BSP, whose assessment of the communist period was rather ambivalent; in general, the party was unwilling to radically revise the policies of the communist regime. 28 Among those, for instance, were 43 former ambassadors of the Bulgarian Kingdom who had lost their Bulgarian citizenship by a decree issued by the erstwhile President Vassil Kolarov in 1947. They had refused to acknowledge the ‘people’s power’ of the communist regime (Rossitza Milanova, ‘The Citizenship of Those Bulgarians Whose Citizenship Was Denounced between 1944 and 1947 Is Restored’, Democrazija, 12 May 1998). 29 There are different procedures for obtaining this permission. In most cases, the applicants have to show that they have legally resided in the country for the previous five years or that they have invested more than US$ 500,000. Ethnic Bulgarians and foreigners born in Bulgaria are exempt from these conditions. Those who have performed meritorious service to the country are also exempted from the requirements. For more details, see the information at the site of the Ministry of Foreign Affairs: www.mfa.bg. DANIEL SMILOV AND ELENA JILEVA 40 In a curious development, in 2007, the Bulgarian President involved himself in a public debate with a junior art historian, who was supported in her research by a young German scholar. The art historian had written a paper interpreting the ‘myth of the Batak massacre’. Apparently, she had traced the reconstruction of specific events in the public memory from the Bulgarian uprising against the Ottomans in 1876, exploring the role that works of art played in this construction. The President interpreted the whole project as an attempt to deny that the massacre ever happened (an indisputable fact), and to misinterpret Bulgarian history. He even hosted an ‘open history class’ in the town of Batak, the main purpose of which was to discredit the art historian’s project. Leaving aside the historical substance of the debate, it is highly unusual for a head of state to take on the role of a guardian of national history, especially when his opponents are academics. 40 In a curious development, in 2007, the Bulgarian President involved himself in a public debate with a junior art historian, who was supported in her research by a young German scholar. The art historian had written a paper interpreting the ‘myth of the Batak massacre’. Apparently, she had traced the reconstruction of specific events in the public memory from the Bulgarian uprising against the Ottomans in 1876, exploring the role that works of art played in this construction. The President interpreted the whole project as an attempt to deny that the massacre ever happened (an indisputable fact), and to misinterpret Bulgarian history. He even hosted an ‘open history class’ in the town of Batak, the main purpose of which was to discredit the art historian’s project. Leaving aside the historical substance of the debate, it is highly unusual for a head of state to take on the role of a guardian of national history, especially when his opponents are academics. 40 In a curious development, in 2007, the Bulgarian President involved himself in a public debate with a junior art historian, who was supported in her research by a young German scholar. The art historian had written a paper interpreting the ‘myth of the Batak massacre’. DANIEL SMILOV AND ELENA JILEVA 35 While the 1998 Law envisions a preclusive deadline of three months for the facilitated naturalisation of foreigners with Bulgarian origin, the procedure usually takes two to three years due to the numerous inspections at various Bulgarian institutions. Such inspections are carried out by the Directorate for Migration at the Ministry of the Interior, the National Security Service, the State Agency for Bulgarians Abroad (SABA) and others (Interview, State Agency for Bulgarians Abroad, 2 June 2007). The problem of discretion in the administration of these checks remains quite acute, however. 36 The State Agency for Bulgarians Abroad (SABA) is also involved in the process of verifying Bulgarian origin: see the discussion in section 7.3.3 on this topic. Stefan Nikolov (SABA) said that officials are conducting ‘thorough inspections’ to prevent unlawful claims and abuses, since many of those waiting for their applications to be processed might have submitted ‘documents with dubious validity’ (‘Thousands Seeking Bulgarian Citizenship Ahead of EU Entry’, Southeast European Times, 14 Au- gust 2006, www.setimes.com). 36 The State Agency for Bulgarians Abroad (SABA) is also involved in the process of verifying Bulgarian origin: see the discussion in section 7.3.3 on this topic. Stefan Nikolov (SABA) said that officials are conducting ‘thorough inspections’ to prevent unlawful claims and abuses, since many of those waiting for their applications to be processed might have submitted ‘documents with dubious validity’ (‘Thousands Seeking Bulgarian Citizenship Ahead of EU Entry’, Southeast European Times, 14 Au- gust 2006, www.setimes.com). 37 Some usual provisos apply, such as the absence of a criminal record and the requirement of good, moral behaviour. 37 Some usual provisos apply, such as the absence of a criminal record and the requirement of good, moral behaviour. 38 The NDSV could only be interpreted as a populist party in is first two years of its existence; it gradually transformed into a ‘normal’ party, albeit at the cost of its popularity. 39 The reasons for this development are numerous, but one is that MRF can rely on the relatively stable ethnic support of Bulgarian Turks. In circumstances of declining turnout, the stability of the electorate of the MRF increases its relative influence. 39 The reasons for this development are numerous, but one is that MRF can rely on the relatively stable ethnic support of Bulgarian Turks. In circumstances of declining turnout, the stability of the electorate of the MRF increases its relative influence. DANIEL SMILOV AND ELENA JILEVA Apparently, she had traced the reconstruction of specific events in the public memory from the Bulgarian uprising against the Ottomans in 1876, exploring the role that works of art played in this construction. The President interpreted the whole project as an attempt to deny that the massacre ever happened (an indisputable fact), and to misinterpret Bulgarian history. He even hosted an ‘open history class’ in the town of Batak, the main purpose of which was to discredit the art historian’s project. Leaving aside the historical substance of the debate, it is highly unusual for a head of state to take on the role of a guardian of national history, especially when his opponents are academics. THE POLITICS OF BULGARIAN CITIZENSHIP THE POLITICS OF BULGARIAN CITIZENSHIP 245 41 Some Bulgarian elections have coincided with major Muslim holidays, in order to make the ‘electoral tourism’ possible. 41 Some Bulgarian elections have coincided with major Muslim holidays, in order to make the ‘electoral tourism’ possible. 42 In 2006, the radical nationalist party Ataka proposed amendments to the 1998 Law on Bulgarian Citizenship. These amendments were meant to eliminate dual citizenship. The party proposed that a Bulgarian citizen cannot be a citizen of another country, with the exception of citizens of Bulgarian ethnic origin and Bulgarian culture. These privileges would be confined to persons living permanently in neighbouring regions in Greece, Macedonia, Serbia and Romania, and also including Bessarabian Bulgarians. The Bulgarian Parliament rejected the proposal with nineteen votes ‘for’, 78 ‘against’ and 26 abstentions (Telegraph, 8 June 2007). h h f l b 43 Rumjana Bachvarova, ‘The Reticence of Power’, Capital, 23 February 2007, www.capital.bg. 44 The country is losing between 30,000-50,000 citizens per year, although the most recent statistics show that the decrease in population is slowing down. 45 Although there is a relatively large community of some 235,000 Bulgarians in the Ukraine, not many applications for Bulgarian citizenship are filed from this country, as according to Ukrainian law, they have to renounce their Ukrainian citizenship in the process. Serbians have been increasingly interested in obtaining Bulgarian citizenship since Bulgaria joined the EU. Since the beginning of 2007, nearly 1,200 Serbs (actually ethnic Bulgarians from Dimitrovgrad and Bosilegrad) have taken advantage of the right to obtain Bulgarian citizenship. According to the Bulgarian Ministry of Justice, another 3,400 had submitted applications and were awaiting decisions (‘Serbians Are Increasingly Interested in Obtaining Bulgarian Citizenship’, Sofia Echo, 6 August 2007, www.sofiaecho.com). 46 In contrast to Hungary, the privileges for ‘external citizens’ neither created heated public debates nor significant problems during EU accession negotiations. There are several reasons for this. Firstly, the Bulgarian diaspora in neighbouring countries is smaller than the Hungarian one, and much less organised. In most cases, the policy provides for individual exit strategies of Bulgarians abroad. Secondly, Bulgaria has complied with all of the formal requirements of the EU visa regulations, and has imposed visa restrictions on neighbouring countries in which Bulgarians live. Finally, the issue is not internally divisive, as is the case in Hungary; no major party, including the MRF, opposes the current policies. DANIEL SMILOV AND ELENA JILEVA 51 One of the main target groups of the SABA is the community of Bessarabian Bulgarians and Bulgarian minorities in Serbia, Moldova and Romania. g g 52 At the time of writing there is an ongoing debate organised by SABA on this document; see www.saba.government.bg. 52 At the time of writing there is an ongoing debate organised by SABA on this document; see www.saba.government.bg. 53 For instance, the so-called VAK (Supreme Commission of Attestation) still has the right to examine, in substantive terms, degrees earned in the EU. It is true that the Commission, as a rule, does not usually apply this right. But still, according to the law it can deny the domestication of an EU degree on substantive grounds by a secret vote! 53 For instance, the so-called VAK (Supreme Commission of Attestation) still has the right to examine, in substantive terms, degrees earned in the EU. It is true that the Commission, as a rule, does not usually apply this right. But still, according to the law it can deny the domestication of an EU degree on substantive grounds by a secret vote! THE POLITICS OF BULGARIAN CITIZENSHIP 46 In contrast to Hungary, the privileges for ‘external citizens’ neither created heated public debates nor significant problems during EU accession negotiations. There are several reasons for this. Firstly, the Bulgarian diaspora in neighbouring countries is smaller than the Hungarian one, and much less organised. In most cases, the policy provides for individual exit strategies of Bulgarians abroad. Secondly, Bulgaria has complied with all of the formal requirements of the EU visa regulations, and has imposed visa restrictions on neighbouring countries in which Bulgarians live. Finally, the issue is not internally divisive, as is the case in Hungary; no major party, including the MRF, opposes the current policies. 47 As O¨ zgu¨r-Baklacioglu argues, the Law on Bulgarians Abroad established kin minority protection and turned dual citizenship into an engine of intensive nation-building. Dual citizenship status for the migrant community in Turkey remains a subject of political debate, while it does not create problems in cases involving Macedonian applications. In other words, the accommodation of Macedonians as Bulgarian citizens has a special historical importance in building the Bulgarian nation. In nationalist circles, it is perceived as the return of the Macedonians to their ‘first’ origin, i.e. the Bulgarian one. As understood from the application procedure, Macedonian applications are handled under the provision for applicants with Bulgarian origin. Macedonians do not need a permanent resident status and do not have to show a certificate proving knowledge of the Bulgarian language. Among the initial set of application documents is a declaration that verifies Bulgarian cultural consciousness (O¨ zgu¨r-Baklacioglu 2006: 336). 48 Decision 1, 29 February 2000. 49 Case of the United Macedonian Organisation Ilinden and others v. Bulgaria, 19 January 2006. 50 See Svetoslav Terziev, ‘Strassbourg Harasses Bulgaria about OMO’, Sega, 27 March 2008, www.segabg.com. 246 DANIEL SMILOV AND ELENA JILEVA Bibliography Aleksandrov, E. (1995), Pravo na zadrganichnite bulgari. Sofia: Agency for Bulgarians Abroad. Balamezov, S. (1993), Konstitutsionno pravo: chast purva. Sofia: Sofi-R. Boyadjiev, M. (1996), ‘Dvojnoto grazhdanstvo i Bulgaria: zagubvane i pridobivane’, Mezh- dunarodni otnosheniya 2: 69-76. y 9 7 Dworkin, R. (1986), Law’s Empire. Cambridge, MA: Harvard University Press. Ely, J. H. (1980), Democracy and Distrust. Cambridge, MA: Harvard University Geshkoff, T. (1927), ‘An analysis of the Bulgarian Nationality Law’, American Journal of International Law 21 (3): 534 - 537 Ivanov, A. (2007), Minority Nationalism in the Balkans: the Bulgarian Case. www.ime-bg.org. Kalyonski, A. & M. Gruev (2008), Vuzroditelnijat process [The Rebirth Process]. Sofia: Insti- tute for the Study of Recent Past. Liebich, A. (2000), ‘Plural Citizenship in Post-Communist States’, International Journal of Refugee Law 12 (1): 97-107. f g ( ) 97 7 Malinkov, A. (1941) Komentar na Zakona za bulgarskoto podanstvo. Sofia. ¨ O¨ zgu¨r-Baklacioglu, N. (2006), ‘Dual Citizenship, Extraterritorial Elections and National Policies: Turkish Dual Citizens in the Bulgarian-Turkish Political Sphere’, in O. Ieda (ed.), Beyond Sovereignty: From Status Law to Transnational Citizenship?, 319-358. Sap- poro: Slavic Research Center. Petkova, L. (2002), ‘The Ethnic Turks in Bulgaria: Social Integration and Impact on Bul- garian-Turkish Relations, 1947-2000’, The Global Review of Ethnopolitics 1 (4): 42-59. Sajo, A. (ed.) (2004), Militant Democracy. Utrecht: Eleven International Publishing. State Agency for Bulgarians Abroad (2007), Introduction. www.aba.government.bg. Todorov, T. (1996), ‘Grazhdanstvo po rozhdenie po chl. 93, al. 2 ot Konstitutzijata’, Prav- na misul (4): 7-14. Todorov, T. (2001), The Fragility of Goodness: Why Bulgarian Jews Survived the Holocaust. Princeton, NJ: Princeton University Press. J y Tzankov, V. (2004), Bulgarsko grazhdanstvo. Sofia: Bulgarian Academy of Sciences. Tzankov, V. (2005), Az, grazhdaninut na Republika Bulgaria i na Evropejskija suyuz. Sofia: Ministry of Justice. Tchorbadjiyska, A. (2007), ‘Bulgarian experiences with visa policy in the accession pro- cess: A story of visa lists, citizenship and limitation of citizenship rights’, Regio, Mino- rities, Politics, Society 10: 87-107. Valkanov, V. (1978), Bulgarskoto grazhdanstvo. Sofia: Bulgarian Academy of Sciences. Vladikin, L. (1994), Istoriya na Turnovskata constitutsia. Sofia: Narodna kultu Zakon za bulgarskoto grazhdanstvo from 1968 (1982) In Dogovori za pravna pomosht, konventzii za urezhdane na dvojnoto grazhdanstvo i grugi aktove. Sofia: State Publishing House ‘Science and Art’. 8 Czech citizenship legislation between past and future1 Andrea Barsˇova´ The main contours of the Czech (formerly Czechoslovak) citizenship laws were shaped by momentous historic events. Administrators, judges and lawyers smoothed over rough outlines made by political forces, adding to them exceptions, interpretations and remedies. Con- sequently, Czech citizenship legislation has always been complex.2 This chapter follows the main trends of its development and gives a brief ac- count of Czech citizenship legislation and the politics and policies linked with it since 1918. It focuses on responses to unprecedented so- cial changes and challenges in the last two decades, which may be sti- mulating for the current debates on citizenship policies in the Eur- opean and transnational context. 8.1.1 Citizenship policies from 1918 to 1993 8.1.1 Citizenship policies from 1918 to 1993 Czechoslovak citizenship came into existence with the creation of Cze- choslovakia on 28 October 1918. It was linked to the municipal right of domicile that had been an important instrument regulating migration within the Habsburg monarchy.3 Former Austro-Hungarian nationals, who had a right of domicile in municipalities that became part of the Czechoslovak territory after the break-up of the Austro-Hungarian Em- pire, acquired Czechoslovak citizenship. The basic rule was modified by peace treaties4 and constitutional laws5 which regulated the issue of citizenship in order to protect ethno-national minorities and provided options to choose the citizenship of an ethnic kin state. The creation of Czechoslovakia led to the massive remigration of ethnic Czechs and Slovaks, in particular from Austria (Vaculı´k 2002: 38-39; Kristen 1989: 83). Apart from specific provisions linked to the creation of the new state, provisions of old Austrian laws on citizenship remained in force in the Czech lands of Bohemia and Moravia.6 This was the case, for in- stance, with the ius sanguinis principle laid down in the 1811 Austrian Civil Code. The end of the Second World War7 and the restoration of Czechoslo- vakia led to the adoption of ad hoc laws that introduced the criterion of 250 ANDREA BARSˇOVA´ ANDREA BARSˇOVA´ ethnicity into citizenship legislation. The new legislation was linked to post-war (both forced and voluntary) migration. Under the President’s Constitutional Decree No. 33/1945 Coll. Concerning Czechoslovak Citi- zenship of Persons of German and Hungarian Ethnicity (one of the so- called ‘Benesˇ decrees’), Czechoslovak nationals of German and Hun- garian ethnic origin were deprived of Czechoslovak citizenship.8 On the other hand, Constitutional Act No. 74/1946 Coll. Concerning the Naturalisation of Compatriots Returning to the Homeland and its im- plementing regulations provided for facilitated naturalisation of ethnic Czechs, Slovaks and members of other Slavonic nations who settled or re-settled in Czechoslovakia. Naturalisation was often linked to changes of names to Czech or Slovak ones (Vaculı´k 2002: 40-49). In the post- war years, more than 200,000 Czechs, Slovaks and members of other Slavonic nations immigrated to Czechoslovakia while more than 2,820,000 inhabitants of German ethnicity were expelled. After the communists seized power in Czechoslovakia in February 1948, deprivation of citizenship was introduced as a supplementary penalty for certain political offences. A complex new citizenship legis- lation was adopted in 1949. Act No. 194/1949 Coll. on Czechoslovak Citizenship modified by Act No. 8.1.1 Citizenship policies from 1918 to 1993 72/1958 Coll., replaced the old legis- lation, but preserved many of its features, such as the ius sanguinis principle and the principle of a single citizenship. Both in the commu- nist ideology and in legal theory, citizenship meant not only legal but also factual bonds between a citizen and the society. A legal textbook published in 1963 defines ‘socialist citizenship’ in the following way: ‘Socialist citizenship is not only a legal bond between a citizen and the state, but it means also belonging to a community of working people, who participate in the building of socialist (communist) society and in the building and defence of the socialist state; it means belonging to the community connected by shared dreams and ideals’ (Cˇerny´ & Cˇer- venka 1963: 19). 9 3 9) The law also provided for depriving people of Czechoslovak citizen- ship. It was applied as penalty to those citizens who lived abroad and had engaged in activities ‘which might endanger state interests’, those who had left the territory of Czechoslovakia ‘illegally’, those who had not returned to Czechoslovakia when requested to do so by the Minis- try of the Interior and those who lived abroad for more than five years without a ‘valid passport permitting its holder to live abroad’. These le- gal provisions existed until 1990. The Prague Spring of 1968, a movement towards the liberalisation of communist rule, was accompanied by the Slovak national move- ment.9 This movement demanded the introduction of a federal system within a multiethnic, but centralised, Czechoslovakia. As of 1 January 1969, the unitary Czechoslovak state was transformed into a federal CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 251 state, composed of the Czech and the Slovak Republics.10 At the level of citizenship legislation, this change was reflected by the adoption of Federal Act No. 165/1968 Coll. on the Principles of Acquisition and Loss of Citizenship, which was followed by the Act of the Czech Na- tional Council No. 39/1969 Coll. on the Acquisition and Loss of Citi- zenship of the Czech Socialist Republic, adopted in April 1969.11 The new legislation introduced, in addition to the (federal) Czechoslovak ci- tizenship, citizenship of the two (Czech and Slovak) Republics as the constituent entities of the Federation.12 Under this legislation, Czecho- slovak nationals automatically acquired the citizenship of either the Czech or Slovak Republic, based on their place of birth and some sup- plementary criteria. 8.1.1 Citizenship policies from 1918 to 1993 The new legislation gave people a right to change the republic-level citizenship at their discretion but this right was rarely exercised. The reason was trivial: the republic-level citizenship had no practical conse- quences whatever. In fact, most citizens were not even aware of their republic-level citizenship.13 In addition, the freezing period of ‘normali- sation’ in the 1970s and 1980s, which followed the suppression of the Prague Spring, pushed most people into private and family life as the only remaining space for meaningful activities, where the question of citizenship had no significance. g The fall of the communist regime in November 1989 prompted new developments in all spheres, including citizenship legislation. The first task for the new democratic government was to remedy injustices caused by deprivations of citizenship under the communist rule. In re- sponse to communist abuses of power, a constitutional provision was introduced, to stipulate that, ‘no one shall be deprived of his or her citi- zenship against his or her will’.14 In 1990, Act No. 88/1990 Coll. was adopted, which provided for the reacquisition of the Czechoslovak citi- zenship by emigrants who had lost it in the period of communist rule. The law, which was not free of certain restrictions and shortcomings,15 identified one strand of future development in the field of citizenship legislation that I will call restitution legislation. 8.1.2 Break-up of Czechoslovakia and creation of the Czech citizenship The demise of the communist regime opened a space for the resur- gence of nationalist feelings and politics.16 In Czechoslovakia, the re- birth of the Slovak nationalist movement led to a consensual break-up of the federal state. In the fall of 1992, as the break-up of Czechoslova- kia was increasingly becoming a realistic option (negotiated and carried through by the ruling political elite),17 many Czechoslovaks started to think about their future in terms of citizenship.18 The dormant provi- sions of the existing citizenship legislation, which allowed for a simple 252 ANDREA BARSˇOVA´ switch between the Czech and the Slovak republic-level citizenships, started to be widely invoked. Until the end of 1992, some 65,000 Slo- vak republic-level citizens applied for the Czech republic-level citizen- ship. On 1 January 1993, the Czech and the Slovak Republics were estab- lished as successor states to the former Czechoslovakia. In the Czech Republic, citizenship issues were regulated by the hastily drafted and adopted Act No. 40/1993 Coll. on the Acquisition and Loss of Citizen- ship of the Czech Republic.19 The primary aim of the law was to identi- fy nationals of the new state and to prevent dual (Czech and Slovak) ci- tizenship.20 p The provisions of the new legislation fell into two main categories. The first was a set of transitory provisions regulating initial determina- tion of nationals of the new state,21 complemented by provisions gov- erning the option for Czech citizenship. The other category involved rules of permanent nature, regulating e.g. acquisition of the Czech citi- zenship by birth, naturalisation or loss of Czech citizenship. Table 8.1 Conceptual scheme of Act No. 40/1993 Coll. on the Acquisition and Loss of Citizenship of the Czech Republic Norms regulating initial determination of citizenship Norms regulating standard procedures for acquisition and loss of citizenship (e.g. by birth, naturalisation) overall initial determi- nation of citizenship supplementary and cor- rective initial determi- nation time aspect automatic operation of laws taking effect on 1 January 1993 temporary application of norms permanent application personal scope core of nationals of the new state, the category was established by operation of law – all former Czech Republic- level citizens solves individual cases, takes into account the will of individuals concerned plurality of cases, not to be defined in advance Table 8.1 Conceptual scheme of Act No. 40/1993 Coll. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 253 law, the Czech republic-level citizenship acquired as of 1 January 1993 an international dimension and turned into full-fledged state citizen- ship.’ (Cˇerny´ & Vala´sˇek 1996: 99). The same approach as regards over- all (collective) initial determination was adopted by Slovak legislators. This prevented de iure statelessness in the wake of the break-up of Czechoslovakia.22 The primary rule was supplemented with a set of transitory provi- sions regulating the right of option and facilitating naturalisation for certain Slovak citizens. In the period from 1 January 1993 to 30 June 1994, 240,000 former Czechoslovak citizens acquired Czech citizen- ship under the option clauses. The criteria for exercising this right of option, however, included not only two years of permanent residence in the territory of the Czech Re- public, but also a clean criminal record.23 The application of the latter condition had a disproportionate impact on members of the Roma (Gypsy) minority.24 It was criticised by Czech human rights activists as well as by the international community. The criticism led to piecemeal adjustments and a softening of Act No. 40/1993 Coll. in relation to for- mer Czechoslovak (now Slovak) nationals.25 In the decade following the establishment of the independent Czech Republic, public and political discourse on citizenship matters was dominated by one issue: the intentional and accidental consequences of the break-up of Czechoslovakia. In the shadow of this central theme, some problems related to the restitution of Czechoslovak (now Czech) citizenship for emigrants were also discussed. In the autumn of 1998, with the change of government (from liberal-conservative to social-de- mocratic), a more profound reform of citizenship legislation was put on the government agenda. This led to (a) significant alterations of the tran- sitory provisions of the 1993 Citizenship Act, and (b) the adoption of Act No. 193/1999 Coll. on the Citizenship of Some of the Former Czecho- slovak Citizens, which was another piece of restitution legislation. p g (a) The former legislation mitigated the harsh consequences of the break-up of Czechoslovakia for some groups of former Czechoslovak nationals. A ruling of the Constitutional Court of the Czech Republic of 5 May 199726 also fostered this development. The Court held that one does not lose Czech citizenship by one’s declaration to opt for citi- zenship in the Slovak Republic. (These individuals became dual na- tionals.)27 Major amendments to the 1993 Citizenship Act were imple- mented by Act No. 194/1999 Coll. 8.1.2 Break-up of Czechoslovakia and creation of the Czech citizenship on the Acquisition and Loss of Citizenship of the Czech Republic Norms regulating initial determination of citizenship Norms regulating standard procedures for acquisition and loss of citizenship (e.g. by birth, naturalisation) overall initial determi- nation of citizenship supplementary and cor- rective initial determi- nation time aspect automatic operation of laws taking effect on 1 January 1993 temporary application of norms permanent application personal scope core of nationals of the new state, the category was established by operation of law – all former Czech Republic- level citizens solves individual cases, takes into account the will of individuals concerned plurality of cases, not to be defined in advance overall initial determi- nation of citizenship time aspect automatic operation of laws taking effect on 1 January 1993 personal scope core of nationals of the new state, the category was established by operation of law – all former Czech Republic- level citizens overall initial determi- nation of citizenship As regards the initial overall determination of citizenship, Act No. 40/ 1993 Coll. stipulated that, ‘natural persons, who were citizens of the Czech Republic as of 31 December 1992, are citizens of the Czech Re- public as of 1 January 1993.’ Leading Czech jurists explain the estab- lishment of Czech citizenship in the following way. ‘As a consequence of the disappearance of Czechoslovakia and the establishment of the Czech Republic as an independent entity under public international CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE which not only transformed this ruling into a statutory provision, but also allowed all Czech citizens who were former nationals of Czechoslovakia to acquire Slovak citizen- ship without losing their Czech citizenship. (This is an exception to one of the declared principles of the Czech citizenship legislation, i.e. the prevention of dual citizenship.) 254 ANDREA BARSˇOVA´ The law also introduced a simplified procedure for acquisition of Czech citizenship by declaration for former Czechoslovak nationals who had been living continuously in the territory of the Czech Repub- lic since the break-up of Czechoslovakia. This was a corrective provi- sion. It provided for acquiring Czech citizenship by those who for var- ious reasons (legal or personal) could not opt or apply for Czech citi- zenship before. The necessity of the remedy is demonstrated by this figure: 6,278 former Czechoslovak citizens acquired Czech citizenship in 1999 alone, by invoking the new provision.28 A subsequent amendment to Act No. 40/1993 Coll. adopted in 2003 (Act No. 357/2003 Coll.) introduced further remedial provisions. It gave former Czechoslovak nationals who were granted Slovak citizenship (i.e. were naturalised in Slovakia) in the period from 1 January 1994 to 1 September 1999 the right to (re-)acquire the lost Czech citizenship by declaration. The amendment also gave the right to acquire the Czech citizenship by declaration to certain groups of Slovak nationals who were minors at the time of the break-up of Czechoslovakia.29 (b) Act No. 193/1999 Coll. on the Citizenship of Some of the Former Czechoslovak Citizens, reintroduced and broadened the right of reac- quisition of Czech citizenship by declaration. It applied to emigrants who had lost Czechoslovak citizenship under communist rule, but for legal or practical reasons had not been able to make use of the first res- titution act of 1990. Originally, the applicability of the law was limited to five years after its entry into force. 8.2 Basic principles of acquisition and loss of Czech citizenship As stated above, the citizenship legislation has gone through a series of adjustments since 1993. While the greater part of the fine-tuning was related to the situation of former Czechoslovak nationals, there have been other changes, such as acquisition of citizenship by children or naturalisation. Some changes reflected reforms of the administrative structures. This section describes the main principles of Czech citizen- ship legislation in force as of 1 January 2008. It does so only selectively in so far as they reflect topical political discussions or indicate new trends. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 255 citizenship by being found in the territory of the Czech Republic and d) acquisition of Czech citizenship by naturalisation. Under the ius sanguinis principle, one acquires Czech citizenship at birth if at least one parent is a Czech citizen. The place of birth is not relevant. Ius soli applies if the parents are stateless and at least one of them is a permanent resident (i.e. a green-card holder).30 A natural person found in the territory of the Czech Republic is a Czech citizen unless it is proved that he or she acquired the citizenship of another state at birth. The conditions for naturalisation are strict:31 permanent residence for at least five years, clean criminal record,32 passing a Czech language test, renunciation of the previous citizenship,33 no infringement of im- migration law, and fulfilment of certain statutory duties, such as pay- ing taxes, health, social and retirement insurance. The permanent resi- dence status is an eligibility criterion that may not be waived. Under the immigration legislation in force until April 2006 an immigrant could apply for permanent resident status (i.e. a green card) only after ten years of continuous legal residence in the Czech Republic and after eight years in cases of family reunion. Exemptions were made only for those related to Czech citizens or permanent residents and on humani- tarian grounds. Thus, the waiting period for naturalisation for many immigrants was in fact fifteen years or more.34 There are statutory exemptions for certain categories (such as recog- nised refugees and stateless persons). For instance, refugees are eligi- ble for naturalisation without having to renounce their original citizen- ship. (They acquire permanent resident status by virtue of being granted asylum.) Most of the requirements can be waived at the discre- tion of the Ministry of the Interior if certain conditions are met. For in- stance, the Ministry may waive the five-year duration of permanent re- sidency (but not the permanent resident status as such) for applicants born in the Czech Republic, former Czech (or Czechoslovak) nationals, spouses of Czech nationals, children of Czech nationals, stateless per- sons or refugees. g There is a long list of discretionary exemptions from the require- ment to renounce one’s original citizenship. This list was extended by Act No. 357/2003 Coll. partly because of an initiative by the Human Rights Council (see below). 8.2.1 Acquisition of citizenship There are four ways of acquiring Czech citizenship: a) acquisition of ci- tizenship by former Czechoslovak citizens by option, declaration or fa- cilitated naturalisation (as described above), b) acquisition of citizen- ship by birth, adoption and establishment of paternity, c) acquisition of CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 8.2.2 Loss of citizenship Since the Czech Constitution prohibits deprivation of citizenship against one’s own will, it may not be imposed as a penalty. In confor- mity with the principle of ius sanguinis even later generations of Czech descent born and living abroad cannot lose Czech citizenship by mere operation of law. A Czech national can lose citizenship in two ways: by a declaration of renunciation and by acquisition of foreign citizenship at his or her re- quest. A Czech citizen may lose his or her citizenship by a declaration of renunciation if he or she stays abroad and at the same time pos- sesses the citizenship of a foreign state (cases of dual and multiple citi- zenship).36 A Czech citizen automatically loses Czech citizenship as a consequence of acquiring a foreign citizenship if the latter citizenship is acquired at his or her own request. (This does not apply if the acqui- sition of a foreign citizenship is automatic, for example by descent.) This mode of automatic loss of citizenship was introduced by the 1993 Citizenship Act and did not exist before 1 January 1993.37 This provi- sion became controversial in practice.38 Its constitutionality was also challenged with reference to the ban on deprivation of citizenship against one’s will but was eventually confirmed by the Constitutional Court.39 The 2003 amendment to the 1993 Citizenship Act (Act No. 357/ 2003 Coll.) introduced an exemption from the loss of citizenship in re- lation to marriage. If a Czech national acquires the citizenship of his or her spouse during the marriage, he or she will not lose Czech citi- zenship.40 CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE Applicants may keep their previous citizen- ship (and become dual or multiple nationals) if they are permanent re- sidents, have stayed legally in the territory for at least five years, have a genuine link to the Czech Republic and, in addition, satisfy one of the prescribed conditions. These are, for example, situations when the ap- plicant’s renunciation of the previous citizenship involves unreasonable fees or other demands not acceptable in a democratic state, when nat- uralisation is in the interest of the Czech Republic because of the ex- 256 ANDREA BARSˇOVA´ ANDREA BARSˇOVA´ pected significant contribution to the Czech society in science, societal life, culture or sports or when the applicant is a former Czech (or Cze- choslovak) national.35 There is also an exemption for applicants who have resided legally in the Czech Republic for at least twenty years and have held permanent resident status for five years or more. p y The relatively simple language test is waived for all Slovak nationals and for any one else at the discretion of the authorities. The Ministry also screens all applicants in order to assess the secur- ity risk that they pose to the state. Some authors consider passing the security test an additional condition for naturalisation (Vala´sˇek & Kucˇera 2006: 85). 8.2.3 Dual and multiple citizenship The Czech legislation is becoming generally more tolerant about dual (and multiple) citizenship. This trend is clearly visible in spite of the CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 257 fact that the official citizenship policy followed the principle of preven- tion of dual citizenship until recently. In reality, there are numerous dual and multiple nationals who acquired the status legally. These are e.g. former Czech and Czechoslovak citizens who reacquired Czech ci- tizenship by declaration under the restitution laws, people who became dual Czech and Slovak nationals due to the break-up of Czechoslova- kia, applicants for naturalisation for whom the Ministry of the Interior waived the requirement for them to renounce their former citizenship (including cases of achievement-based naturalisation), naturalised refu- gees, and Czech spouses of foreign citizens. There are those who ac- quired dual citizenship by descent and emigrants who acquired foreign citizenship but never lost their Czech (or Czechoslovak) citizenship. p ( ) p Moreover, there are many cases in the grey zone. For instance, if the Czech authorities are not informed of the acquisition of a foreign citi- zenship by a Czech citizen or by the foreign state concerned, which is often the case, they still treat the person as a Czech citizen (e.g. they will grant him or her a passport). 8.2.4 International treaties The Czech Republic is party to certain bilateral and multilateral treaties concerning citizenship. The current trend is toward accession to multi- lateral treaties41 as most bilateral treaties were terminated, primarily because they were not compatible with the provisions of the 1997 Eur- opean Convention on Nationality as regards the preservation of dual ci- tizenship for children whose parents have different citizenship. These terminated bilateral agreements were with the former Soviet Union and some of its successor states as well as the German Democratic Re- public, Hungary, Poland, Bulgaria, and Mongolia. p g y g g Politically, the most controversial of the bilateral agreements was the 1928 Naturalisation Treaty between the United States and Czechoslova- kia, which precluded dual citizenship of emigrants.42 The Treaty was valid until 20 August 1997. The application of the Treaty excluded many former Czechoslovak citizens from restitution of their property lost during the communist regime. 8.2.5 Procedure The Ministry of the Interior is responsible for citizenship issues. The Ministry decides on naturalisations. It maintains a central register of persons who have acquired or lost Czech citizenship. A decision by the Ministry may be appealed. In this case, the Minister of the Interior de- cides. The Minister receives the opinion of a special consultative com- mission, but is not bound by it.43 Decisions on citizenship are open to 258 ANDREA BARSˇOVA´ ANDREA BARSˇOVA´ judicial review.44 Fees are relatively high – 10,000 Czech crowns (ap- proximately 400 euros) for granted naturalisation. The acquisition of citizenship by declaration is free of charge. 8.3 Current political debates The proposed rule would at least CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 259 eliminate the need to apply for membership in the community where the applicant was born and grew up. On the other hand, the Ministry states that the strict conditions for nat- uralisation should be maintained or even be tightened. It proposes ex- cluding from naturalisation applicants who are likely to become a pub- lic burden. The Analysis also contemplates some changes in language testing: the testing should be more professional.48 g g p In July 2005, the government approved the Analysis.49 A year later, in June 2006, the Ministry of the Interior circulated the first frame- work draft of the new legislation. In addition to the changes mentioned above, the Ministry proposes to amend the Constitution so as to allow the withdrawal of citizenship in cases of false acquisition of citizenship (e.g. fraudulent use of documents). Another controversial proposal of the draft concerns an amendment to the Constitution introducing the clause that there is ‘no legal right to be granted citizenship’. This pro- posal can be regarded as the Ministry’s response to the criticism that it was making extensive use of discretion in naturalisation cases (see be- low). The parliamentary elections held in June 2006 and the subsequent establishment of a right- wing centrist government slowed down the legislative process, but they have so far not brought about a major reor- ientation.50 On 17 March 2008, the government discussed the new draft framework legislation submitted by the Minister of the Interior.51 The blueprint consisted of two pieces: the framework Constitutional Act on Citizenship of the Czech Republic and the framework Law on Citizenship of the Czech Republic.52 The primary aim of the proposed constitutional act is to implement the two controversial changes discussed above. It also proposes to ex- empt from judicial review a denial of naturalisation if it is justified on the grounds of possible risks to the sovereignty, security or democratic foundations of the state. Guided by the opinion of its Legislative Council, the government eventually rejected the proposed constitutional legislation. 8.3 Current political debates The gradual solution to the problem of the break-up of Czechoslovakia, which dominated the citizenship agenda in the first decade after 1993 created some space for fresh approaches to the fundamental issues of citizenship legislation. The shift of perspective was also due to a new phenomenon: increasing migration to the Czech Republic and evolving integration policies (Barsˇova´ 2005; Barsˇova´ & Barsˇa 2005: 231-238). g ( 5 5 3 3 ) An impetus for an overall revision of the citizenship legislation came from the government Council for Human Rights.45 Following an Ana- lysis of Dual Citizenship Issues submitted to the government and the adoption of major amendments to the 1993 Citizenship Act in 2003, the government decided that the Ministry of the Interior should pre- pare a comprehensive analysis of the citizenship legislation based on broad public consultation and submit it to the government by 30 June 2005. The Ministry circulated a consultation paper in the autumn of 2004 and subsequently, in May 2005, a draft Analysis of the Legisla- tion on the Acquisition and Loss of Citizenship.46 The Analysis discusses the fundamental principles of the new citi- zenship law. It suggests the following crucial moves towards liberalisa- tion: a) full toleration of dual (multiple) citizenship on both the entry and exit sides and b) facilitated acquisition of citizenship by second- and third-generation migrants. a. As regards the grounds for this profound reform, the Ministry re- fers to both the prevailing trends toward liberalisation abroad and practical aspects. There are countries which do not allow, in legisla- tion or in practice, renunciation of one’s citizenship. This is a source of undue administrative burden, according to the Ministry.47 b. The Ministry suggests that foreign nationals born in the territory of the Czech Republic should have the right to acquire Czech citizen- ship by declaration within two years after the age of majority if they are permanent residents and have a clean criminal record. The same should apply to those who have lived continuously in the Czech Republic since early childhood. Even if this proposal is not a very favourable solution in comparison with other options (such as the application of ius soli at birth), it is still a positive change in the national context. At present the Czech citizenship legislation does not have any provisions specifically addressing the issue of second and third generation immigrants. 8.3 Current political debates According to the opinion of the Council, it is neither necessary nor advisable to amend the constitution for the purposes of the intended reform.53 However, the government approved the framework citizenship law and decided that the Minister of the Interior should submit a full ver- sion of the bill for approval no later than 31 May 2009. The law should enter into force as of 1 January 2010. The approved blueprint builds upon the analysis and suggests mer- ging all legislation on citizenship into a single code. The transformation provisions of the existing citizenship law relating to the break-up of Czechoslovakia would be deleted. Broadly formulated provisions en- 260 ANDREA BARSˇOVA´ ANDREA BARSˇOVA´ abling former Czechoslovak and Czech citizens, as well as their descen- dants, to acquire Czech citizenship by declaration, would replace the existing specific restitution legislation. Excluded are, however, those who lost Czechoslovak citizenship under the Benesˇ decrees and their descendants. Acquisition of citizenship by declaration is also suggested for second-generation migrants. The acquisition of citizenship by de- claration is in both cases construed as a legal right with relatively sim- ple qualifying conditions. This is in marked contrast with the proposed naturalisation rules. Conditions for naturalisation are detailed and strict. They allow for a thorough screening of applicants and include, inter alia, a test of writ- ten Czech proficiency as well as knowledge and orientation tests, stric- ter requirements regarding one’s criminal record, and compliance with an extensive range of public law obligations, obligations in relation to the municipality where the applicant lives, and even some civil law ob- ligations. Because of the rigorous naturalisation requirements, activists from various human rights organisations have criticised the proposal. Martin Rozumek of OPU (Organisation for Aid to Refugees) argues: ‘The pro- posal requires that foreigners fulfil a number of conditions which most Czech citizens would find difficult to fulfil’.54 The blueprint includes an unrestricted acceptance of dual (multiple) citizenship on both the entry and exit sides. However, there are indica- tions that this liberal approach may change in the future, albeit only with regard to foreigners applying for naturalisation. In fact, the gov- ernment urged the Minister of the Interior to reconsider this particular issue in further stages of legislative drafting.55 The liberalisation on the exit side (that is in relation to Czech citizens who acquire foreign na- tionality) remains unchallenged. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 261 Some Former Czechoslovak Citizens merely abolished the deadline for making a declaration on the reacquisition of Czech citizenship. The senate, without support of the government, pushed the bill through parliament.58 p The link between the restitution or the retention of citizenship and the restitution of property also plays a key role in relation to the Benesˇ decrees, which deprived people of German and Hungarian ethnic ori- gin of their Czechoslovak citizenship. The interpretation of these de- crees still plays a role in some restitution cases and remains highly controversial.59 The prevailing public opinion on the restitution of Ger- man property is negative. Therefore, it is not surprising that the gov- ernment does not contemplate any change in the citizenship legislation in this regard. The decrees thus remain a sensitive political issue not only in Slovakia and Hungary, but also in the Czech Republic. Among the practical problems repeatedly brought to the attention of the government by non-governmental organisations and other actors, such as the Ombudsman, is the extensive discretion of the Ministry of the Interior in decisions on naturalisation and the way it is exercised. In practice, the Ministry’s negative decisions do not often constitute grounds for rejection of an application, but refer vaguely to administra- tive discretion as such and to the fact that there is ‘no legal right to be granted citizenship’. Until recently the courts conducting the judicial reviews have sustained the practice.60 It was only a few legal scholars who expressed doubts about whether ‘granting citizenship is, indeed, an act of mercy, exercised by the state at its own good will’ or is rather in the interest of society (Chlad 2004: 350; Molek & Sˇimı´cˇek 2005: 142-144). A cautious shift in the jurisprudence regarding the use of dis- cretion in naturalisation procedures was brought about by the Supreme Administrative Court (established in 2003).61 Most recently, the idea that discretionary naturalisation should be completed or replaced by granting a right to naturalisation once the specified conditions are met, is gaining some ground within the administration and the justice sys- tem. However, the Ministry of the Interior, a key player in the formula- tion of citizenship policies, still rejects the idea strongly.62 8.3 Current political debates y) g As one of the conditions for property restitution introduced in the early 1990s was the Czechoslovak (Czech) citizenship of the applicant, legislators and the general public have consistently viewed citizenship legislation and its changes in this context. Notwithstanding the criti- cism of various international bodies, including the UN Human Rights Committee,56 this restriction has been retained both by the legislature and by the Constitutional Court.57 The need to protect legal certainty, ownership rights, and the stability of the legal order were raised to de- fend the status quo. As a result, the link between restitution of citizen- ship and restitution of property often hampered the adoption of more inclusive citizenship legislation. Lukewarm attitudes towards Czech emigrants among part of the lo- cal population also influenced the discussion on the possible extension of the legislation on reacquisition of Czech citizenship. Finally, the 2006 amendment to Act No. 193/1999 Coll. on the Citizenship of 8.4 Statistics63 If we exclude Slovak nationals, the numbers of persons naturalised an- nually in the Czech Republic have been surprisingly stable in the last decade, with a maximum of 2,000 persons and a minimum of 837 per annum, as shown in Table 8.2. 262 Table 8.2 Naturalisations (excluding Slovak nationals) in the Czech Republic, 1993-2007 Year 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 1,469 1,412 2,000 1,380 837 1,128 1,031 1,059 1,121 1,150 1,267 1,495 1,177 1,355 1,027 Source: Ministry of the Interior The number of Slovak nationals granted Czech citizenship based on supplementary and corrective initial rules (see Table 8.1) is still signifi- cant (Act No. 40/1993 Coll., Sections 18a, 18b and 18c). Table 8.3 Number of Slovak and other nationals acquiring Czech citizenship from 2001-2007 Year 2001 2002 2003 2004 2005 2006 2007 Total 6,321 4,532 3,410 5,020 2,626 2,346 1,877 Declaration based on Act No. 139/1999 Coll. 1,607 1,273 1,154 1,784 190 205 225 Act No. 40/1993 Coll. – total 4,714 3,259 2,256 3,236 2,436 2,141 1,652 Slovakia – Act No. 40/1993 Coll. – total 3,593 2,109 989 1741 1,259 786 625 Slovakia – declaration section 18a* 3,378 1,862 850 627 565 375 268 Slovakia – declaration section 18b – – 55 364 123 63 49 Slovakia – declaration section 18c* – – 5 573 325 167 177 Slovakia – naturalisation section 7 215 247 79 177 246 181 131 Other naturalisations (section 7) – Act No. 40/1993 Coll. 1,121 1,150 1,267 1,495 1,177 1,355 1,027 Source: Czech Statistical Office * Former Czechoslovak nationals who had lived continuously in the territory of the Czech Republic since the break-up of Czechoslovakia Former Czechoslovak nationals who were naturalised in Slovakia in the period from 1 January 1994 to 1 September 1999 * Former Czechoslovak nationals born in Slovakia who were minors during the break-up of Czechoslovakia, but with at least one parent a Czech republic-level citizen Table 8.3 Number of Slovak and other nationals acquiring Czech citizenship from 2001-2007 Year 2001 2002 2003 2004 2005 2006 2007 In 2007, the total number of persons naturalised was 1,158. This num- ber included 424 Ukrainian nationals, 131 Slovak nationals, 102 Rus- sian nationals, 50 Polish nationals, 40 Vietnamese nationals, 39 Belar- usian nationals, 36 Romanian nationals, 33 Moldovan nationals, 31 Greek nationals and 28 Armenian nationals.64 In 2007, the total number of persons naturalised was 1,158. This num- ber included 424 Ukrainian nationals, 131 Slovak nationals, 102 Rus- sian nationals, 50 Polish nationals, 40 Vietnamese nationals, 39 Belar- usian nationals, 36 Romanian nationals, 33 Moldovan nationals, 31 Greek nationals and 28 Armenian nationals.64 Table 8.4 Slovak nationals who acquired Czech citizenship by declaration (Section 18a of Act No. ANDREA BARSˇOVA´ ANDREA BARSˇOVA´ CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 263 Table 8.4 shows that at the end of 1990s there were still a number of former Czechoslovak citizens living in the Czech Republic whose sta- tus was not adequately regularised. The decreasing numbers indicate that the problem is diminishing. Table 8.5 Former Czechoslovak nationals who (re-)acquired Czech citizenship by declaration under Act No. 193/1999 Coll. Year 1999 2000 2001 2002 2003 2004* 2005 2006 2007 798 1,899 1,607 1,273 1,154 1,784 190 205 225 Source: Ministry of the Interior * The deadline for making the declaration expired on 2 September 2004. The Act No. 46/2006 Coll. deleted the deadline and thus made the law operational again. It was pub- lished and entered into force on 27 February 2006. Table 8.5 Former Czechoslovak nationals who (re-)acquired Czech citizenship by declaration under Act No. 193/1999 Coll. Act No. 193/1999 Coll. concerns those who emigrated during the com- munist regime. As they live all over the world, the process requires time both for spreading the information on the right to reacquire the Czech citizenship and for the actual exercising of that right. The num- bers show that the deletion of the deadline for making the declaration is warranted. Table 8.2 Naturalisations (excluding Slovak nationals) in the Czech Republic, 1993-2007 Year 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 1,469 1,412 2,000 1,380 837 1,128 1,031 1,059 1,121 1,150 1,267 1,495 1,177 1,355 1,027 Source: Ministry of the Interior 40/1993 Coll.) Year 1999 2000 2001 2002 2003 2004 2005 2006 2007 6,278 5,377 3,378 1,862 850 627 565 375 268 Source: Ministry of the Interior Table 8.4 Slovak nationals who acquired Czech citizenship by declaration (Section 18a of Act No. 40/1993 Coll.) 8.5 Conclusions The consensual division of Czechoslovakia caused many problems re- garding citizenship. The new legislation did not generate de iure state- less persons, as was sometimes mistakenly contended by its critics. Rather, the consequence of the restrictive and inadequate citizenship legislation was that some former Czechoslovak citizens ended up with the citizenship of a successor state in which they did not live and to which they were only formally attached.65 This revealed the need to clarify international rules concerning cases of state succession.66 It also raised a more puzzling question: Does the right to a citizenship imply a right to choose one’s own citizenship? By the application of remedial provisions introduced in the period 1993-2003, most of the problems related to the break-up of Czechoslo- vakia have been solved. Nonetheless, the original intention of the legis- lators to avoid dual Czech and Slovak citizenship has not been fully achieved. On the contrary, the precarious position of some groups of ci- tizens shows that there are situations in which it is not justified to deny a person the right to dual citizenship. Cases of dual Czech and Slovak citizenship are numerous. Conditions are unfavourable as regards naturalisation except for for- eign nationals with strong family links to Czech citizens (spouses, chil- 264 ANDREA BARSˇOVA´ dren). They involve long waiting periods to fulfil the eligibility criteria as regards the residence requirement. Longer periods of absence are not tolerated and only formal continuous legal status counts. There is also a Czech language test (although kept at a reasonably easy level). As a rule, applicants have to relinquish their original citizenship and a number of additional criteria are tested. There are no specific provi- sions for automatic acquisition of the Czech citizenship by second- and third-generation immigrants (not even at the age of majority) or facili- tated naturalisation for this category. Decisions denying naturalisation are open to judicial review but both in theory and practice administra- tive discretion is applied very broadly in naturalisation cases. On the other hand, the legal status of naturalised citizens is secure as a ban on the deprivation of citizenship is guaranteed under the Constitution. At present, Czech citizenship legislation is at a crossroads. Issues re- lated to the break-up of Czechoslovakia, which have dominated the po- litical debates, are losing their topicality. New challenges are linked to increasing immigration. 8.5 Conclusions In particular, the restrictive nature of current as well as proposed naturalisation provisions, which reflects the surviv- ing parochial character of Czech society, is in conflict with the declared need for effective integration policies. The proposed comprehensive ci- tizenship reform should address the issue more adequately. It is also important that the considered liberal approach to former Czechoslovak and Czech citizens and their descendants is not in practice narrowed down to an ethnic preference. At a more general level, the proposed re- form can be seen as a key element in the transformation of the Czech self-image from an ethnic to a civic nation. Chronological list of citizenship-related legislation in Czechoslovakia/the Czech Republic Date Document Content Source 1920 Act of 29 February 1920, No. 121 Coll. of Acts of the Czechoslovak Republic, introducing the Constitutional Charter of the Czechoslovak Republic Provides for a single Czechoslovak citizenship and prohibits dual citizenship 1920 Constitutional Act of 9 April 1920, No. 236, Supplementing and Amending Existing Provisions on the Acquisition and Loss of Citizenship and on Domicile in the Czechoslovak Republic Implements provisions of the peace treaties concerning the state succession in relation to citizenship; determines who are Czechoslovak citizens; provides for the continuation of Austro- Hungarian citizenship legislation Chronological list of citizenship-related legislation in Czechoslovakia/the Czech Republic Date Document Content Source 1920 Act of 29 February 1920, No. 121 Coll. of Acts of the Czechoslovak Republic, introducing the Constitutional Charter of the Czechoslovak Republic Provides for a single Czechoslovak citizenship and prohibits dual citizenship 1920 Constitutional Act of 9 April 1920, No. 236, Supplementing and Amending Existing Provisions on the Acquisition and Loss of Citizenship and on Domicile in the Czechoslovak Republic Implements provisions of the peace treaties concerning the state succession in relation to citizenship; determines who are Czechoslovak citizens; provides for the continuation of Austro- Hungarian citizenship legislation Chronological list of citizenship-related legislation in Czechoslovakia/the Czech Republic Date Document Content Source CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 265 Date Document Content Source 1945 President’s Constitutional Decree No. 33/1945 Coll. Concerning Czechoslovak Citizenship of Persons of German and Hungarian Ethnicity Deprives most ethnic Germans and Hungarians of the Czechoslovak citizenship http://sudetengermans. freeyellow.com 1946 Constitutional Act No. 74/ 1946 Coll. Concerning the Naturalisation of Compatriots Returning to the Homeland Facilitates naturalisation of returnees who were ethnic Czechs, Slovaks or members of other Slavonic nations 1949 Act No. 194/1949 Coll. on the Acquisition and Loss of Czechoslovak Citizenship, amended by the Act No. 72/1958 Coll. Modifying the Regulations on the Acquisition and Loss of Czechoslovak Citizenship New citizenship code adopted after the communist coup d’e´tat in February 1948 1968 Constitutional Act No. 143/ 1968 Coll. on the Czechoslovak Federation Transforms centralised Czechoslovakia into a federation of two entities: the Czech Republic and the Slovak Republic 1968 Federal Act No. 165/1968 Coll. on the Principles of Acquisition and Loss of Citizenship Provides a framework for the introduction of republic-level (Czech and Slovak) citizenship 1969 Act No. 39/1969 Coll. of Czech National Council on Acquisition and Loss of Citizenship of the Czech Socialist Republic Introduces republic-level Czech citizenship 1990 Act No. 88/1990 Coll. Amending and Supplementing Legislation on Acquisition and Loss of Czechoslovak Citizenship Provides for the reacquisition of Czechoslovak Citizenship by emigrants who have lost it during the period of the communist rule; deletes the provisions on withdrawal of citizenship 1993 Constitution of the Czech Republic Contains the provision that ‘no one shall be deprived of his or her citizenship against his or her will’ www.psp.cz (in Czech and English) 1993 Act No. 40/1993 Coll. on Acquisition and Loss of Citizenship of the Czech New citizenship code which enters into force in the Czech Republic after www.legislationline.org Czechoslovak Citizenship 1968 Constitutional Act No. 143/ 1968 Coll. on the Czechoslovak Federation Transforms centralised Czechoslovakia into a federation of two entities: the Czech Republic and the Slovak Republic 1968 Federal Act No. 165/1968 Coll. on the Principles of Acquisition and Loss of Citizenship Provides a framework for the introduction of republic-level (Czech and Slovak) citizenship 1969 Act No. 39/1969 Coll. of Czech National Council on Acquisition and Loss of Citizenship of the Czech Socialist Republic Introduces republic-level Czech citizenship 1990 Act No. 88/1990 Coll. 2003 Act No. 357/2003 Coll., Amendment to the Act No. 40/1993 Coll. 1999 Act No. 194/1999 Coll., Amendment of the Act No. 40/1993 Coll. 1999 Act No. 193/1999 Coll. on the Citizenship of Some of the Former Czechoslovak Citizens 1996 Act No. 139/1996 Coll., Amendment of the Act No. 40/1993 Coll. 1995 Act No. 140/1995 Coll., Amendment of the Act No. 40/1993 Coll. www.legislationline.org www.legislationline.org www.legislationline.org Notes 1 The author and editors would like to thank Tibor Papp from the Institute of Informatics and Statistics in Bratislava for providing the most recent information on the draft bill in section 8.3 and the data for 2006 and 2007 in section 8.4. 1 The author and editors would like to thank Tibor Papp from the Institute of Informatics and Statistics in Bratislava for providing the most recent information on the draft bill in section 8.3 and the data for 2006 and 2007 in section 8.4. 2 See the leading handbooks on Czech citizenship law Cˇ erny´ & Vala´sˇek 1996 and Vala´sˇek & Kucˇera 2006. 3 Domicile (domovske´ pra´vo, Heimatrecht) refers to membership in a municipal commu- nity. In the Czech lands (Bohemia and Moravia) as parts of the Austro-Hungarian Empire, domicile was regulated by Act No. 105/1863 Coll. [Collection] of Acts of the Empire, as amended by Act No. 222/1896 Coll. 4 E.g. Treaty of Versailles (1919), Treaty of Saint-Germain-en-Laye (1919) and Treaty of Trianon (1920). 5 Constitutional Act of 9 April 1920, No. 236, Supplementing and Amending Existing Provisions on the Acquisition and Loss of Citizenship and on Domicile in the Czechoslovak Republic and Act of 29 February 1920, No. 121 Coll. of Acts of the Czechoslovak Republic, introducing the Constitutional Charter of the Czechoslovak Republic. The basic principles of the Czechoslovak citizenship were thus regulated by constitutional laws and treaty provisions. The system, however, failed to achieve the declared aim of protecting minorities and preventing statelessness. 5 Constitutional Act of 9 April 1920, No. 236, Supplementing and Amending Existing Provisions on the Acquisition and Loss of Citizenship and on Domicile in the Czechoslovak Republic and Act of 29 February 1920, No. 121 Coll. of Acts of the Czechoslovak Republic, introducing the Constitutional Charter of the Czechoslovak Republic. The basic principles of the Czechoslovak citizenship were thus regulated by constitutional laws and treaty provisions. The system, however, failed to achieve the declared aim of protecting minorities and preventing statelessness. 6 In Slovakia, provisions of former Hungarian laws remained in force. See also Kusa´ in this volume. 7 In October 1938, Czechoslovakia lost parts of its territory inhabited mainly by a German population. In March 1939, after the secession of Slovakia, the rest of the Czech lands were turned into the Protectorate Bohemia and Moravia. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE Amending and Supplementing Legislation on Acquisition and Loss of Czechoslovak Citizenship Provides for the reacquisition of Czechoslovak Citizenship by emigrants who have lost it during the period of the communist rule; deletes the provisions on withdrawal of citizenship 1993 Constitution of the Czech Republic Contains the provision that ‘no one shall be deprived of his or her citizenship against his or her will’ www.psp.cz (in Czech and English) 1993 Act No. 40/1993 Coll. on Acquisition and Loss of Citizenship of the Czech Republic New citizenship code which enters into force in the Czech Republic after the dissolution of Czechoslovakia www.legislationline.org 1993 Act No. 272/1993 Coll., Introduces changes to the www.legislationline.org 266 ANDREA BARSˇOVA´ Date Document Date Document Content Source Amendment of the Act No. 40/1993 Coll. right to opt for Czech citizenship with regard to certain Czechs born in the territory of Slovakia before 31 December 1939 1995 Act No. 140/1995 Coll., Amendment of the Act No. 40/1993 Coll. Facilitates the naturalisation of those who immigrated to the Czech Republic upon invitation by the government; concerns in particular the members of the Czech minority from the Chernobyl area www.legislationline.org 1996 Act No. 139/1996 Coll., Amendment of the Act No. 40/1993 Coll. Introduces discretionary waiver of the clean criminal record requirement in naturalisation procedures with regard to Slovak citizens who were former Czechoslovak citizens and have been living in the Czech Republic since the break-up of Czechoslovakia www.legislationline.org 1999 Act No. 193/1999 Coll. on the Citizenship of Some of the Former Czechoslovak Citizens Provides for the reacquisition of Czech citizenship by emigrants who have lost it in the period of communist rule and were not able to make use of Act No. 88/1990 Coll. www.legislationline.org 1999 Act No. 194/1999 Coll., Amendment of the Act No. 40/1993 Coll. Introduces significant remedial changes in relation to the situation of former Czechoslovak citizens living in the Czech Republic since the break- up of Czechoslovakia www.legislationline.org 2003 Act No. 357/2003 Coll., Amendment to the Act No. 40/1993 Coll. Introduces further remedial provisions with regard to former Czechoslovak nationals and certain liberal changes, in particular with regard to dual nationality CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 267 Date Document Content Source 2006 Act No. 46/2006 Coll., Amendment to Act No. 193/1999 Coll. on the Citizenship of Some of the Former Czechoslovak Citizens Removes the deadline for making declarations on reacquisition of Czech citizenship by some of the former Czechoslovak citizens (emigrants) Notes The provision offers stronger protection against deprivation of citizenship than art. 15(2) of the Universal Declaration of Human Rights, which only bans arbitrary deprivation of citizenship. 14 Constitutional Act No. 23/1991 Coll. introducing the Charter of Fundamental Rights and Freedoms. The Act amended art. 5 of Constitutional Act No. 143/1968 Coll. on the Czechoslovak Federation. The Act came into force on 8 February 1991. Later, it was transformed into art. 12(2) of the Czech Constitution. The provision offers stronger protection against deprivation of citizenship than art. 15(2) of the Universal Declaration of Human Rights, which only bans arbitrary deprivation of citizenship. 14 Constitutional Act No. 23/1991 Coll. introducing the Charter of Fundamental Rights and Freedoms. The Act amended art. 5 of Constitutional Act No. 143/1968 Coll. on the Czechoslovak Federation. The Act came into force on 8 February 1991. Later, it was transformed into art. 12(2) of the Czech Constitution. The provision offers stronger protection against deprivation of citizenship than art. 15(2) of the Universal Declaration of Human Rights, which only bans arbitrary deprivation of citizenship. 15 Act No. 88/1990 Coll. provided for the reacquisition of Czechoslovak citizenship by former Czechoslovak citizens who had lost Czechoslovak citizenship in the period between 1 October 1949 and 31 December 1989. The reacquisition took effect in certain cases through a simple declaration. However, two issues are important. First, the law did not go back to before 1948 to cover former Czechoslovak citizens who were deprived of Czechoslovak citizenship by the post-war Presidential Decrees (Germans and Hungarians). Second, the law provided a relatively short period to exercise the right to request the reacquisition of citizenship. It expired on 31 December 1993. 16 See Kusa´ in this volume. 16 See Kusa´ in this volume. 17 The Constitutional Act No. 542/1992 Coll. on the Dissolution of Czechoslovakia on 31 December 1992 was adopted by the Federal Assembly on 25 November 1992. 18 Since the establishment of Czechoslovakia in 1918, there has been much intra-state migration. For instance, in the period 1918-1938 many Czechs went to Slovakia as part of a new Czechoslovak administration. After 1945, there was continuous economic emigration from Slovakia to Bohemia and Moravia. One important element of the post-war internal movements of inhabitants was (both spontaneous and state-organised) resettlement of Slovak Roma in industrial towns and cities of Moravia and Bohemia. Notes The complex legal consequences in terms of citizenship are described by Verner (1947, Appendix II: 227-270). 7 In October 1938, Czechoslovakia lost parts of its territory inhabited mainly by a German population. In March 1939, after the secession of Slovakia, the rest of the Czech lands were turned into the Protectorate Bohemia and Moravia. The complex legal consequences in terms of citizenship are described by Verner (1947, Appendix II: 227-270). 8 The Presidential Decree exempted from withdrawal of citizenship those citizens of German and Hungarian ethnicity who had joined the fight for liberation or were persecuted by the Nazis. The legislation also established a possibility to apply for the restitution of Czechoslovak citizenship within six months after the entry of the Decree into force. Most of the Czechoslovak citizens concerned had actually acquired German or Hungarian citizenship in the period 1938-1945. 8 The Presidential Decree exempted from withdrawal of citizenship those citizens of German and Hungarian ethnicity who had joined the fight for liberation or were persecuted by the Nazis. The legislation also established a possibility to apply for the restitution of Czechoslovak citizenship within six months after the entry of the Decree into force. Most of the Czechoslovak citizens concerned had actually acquired German or Hungarian citizenship in the period 1938-1945. 9 See also Kusa´ in this volume. 11 The corresponding law regulating the same issue in the Slovak Republic was the Act of the Slovak National Council No. 206/1968 Coll. 12 In this paper, the term republic-level citizenship is used to denote membership in the constitutive entities of the federal state. The term (state) citizenship is used exclusively to indicate membership of a sovereign state. In Czech language and legal terminol- ogy, the term state citizenship (sta´tnı´ obcˇanstvı´) is used for both legal statuses. 268 ANDREA BARSˇOVA´ 13 The republic-level citizenship was not recorded in any official documents, such as birth certificates, ID cards or passports. On the other hand, the ID and other documents recorded the ethnic origin (na´rodnost), (e.g. Czech, Slovak, Hungarian), which was based, in principle, on one’s own declaration. 14 Constitutional Act No. 23/1991 Coll. introducing the Charter of Fundamental Rights and Freedoms. The Act amended art. 5 of Constitutional Act No. 143/1968 Coll. on the Czechoslovak Federation. The Act came into force on 8 February 1991. Later, it was transformed into art. 12(2) of the Czech Constitution. Notes 19 The drafting and the adoption of the law took place in exceptional circumstances. The whole process was finished within two months. 20 The possibility of dual (Czech and Slovak) citizenship was the most divisive issue between the ruling political elites – Slovak nationalists and Czech pragmatists. It was favoured by the former and denied by the latter. Since an agreement on state succession regarding citizenship had not been reached, two separate citizenship laws regulated the citizenship of the successor states. 21 For the concept of the initial determination (Erstabgrenzung), see the work by Krom- bach (1967). 21 For the concept of the initial determination (Erstabgrenzung), see the work by Krom- bach (1967). 22 The same criterion, i.e. republic-level citizenship, was used in some countries of former Yugoslavia (Slovenia, Croatia), while the countries of the post-Soviet Eurasia applied a permanent residency criterion instead. 22 The same criterion, i.e. republic-level citizenship, was used in some countries of former Yugoslavia (Slovenia, Croatia), while the countries of the post-Soviet Eurasia applied a permanent residency criterion instead. pp p y 23 The right to opt for Czech citizenship was restricted by the requirement that the person had not been convicted in the last five years for an intentional criminal offence. 23 The right to opt for Czech citizenship was restricted by the requirement that the person had not been convicted in the last five years for an intentional criminal offence. 24 Most Roma migrated to Czech lands from Slovakia after 1945. Consequently, many Czech Roma became Slovak citizens by the application of the general rules of initial determination. 24 Most Roma migrated to Czech lands from Slovakia after 1945. Consequently, many Czech Roma became Slovak citizens by the application of the general rules of initial determination. 25 The first significant change was introduced by Act No. 139/1996 Coll., which allowed for exceptions in naturalisation procedures from the clean criminal record requirement for former Czechoslovak citizens who had resided in the territory of the Czech Republic since the break-up of Czechoslovakia. See Linde 2006 for a 25 The first significant change was introduced by Act No. 139/1996 Coll., which allowed for exceptions in naturalisation procedures from the clean criminal record requirement for former Czechoslovak citizens who had resided in the territory of the Czech Republic since the break-up of Czechoslovakia. See Linde 2006 for a 25 The first significant change was introduced by Act No. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 326/1999 Coll., as amended (Aliens Act). The amendment of the Aliens Act, Act No. 161/2006, which entered into force on 27 April 2006, cut the waiting period for permanent resident status to five years (in order to implement EU Directive 2003/109/EC Concerning the Status of Third-country Nationals who are Long-term Residents). 34 Act No. 326/1999 Coll., as amended (Aliens Act). The amendment of the Aliens Act, Act No. 161/2006, which entered into force on 27 April 2006, cut the waiting period for permanent resident status to five years (in order to implement EU Directive 2003/109/EC Concerning the Status of Third-country Nationals who are Long-term Residents). 35 This does not apply to Slovak citizens. 35 This does not apply to Slovak citizens. 36 In order to avoid statelessness, there is no provision allowing for the renunciation of Czech citizenship if the person concerned is not a citizen of another state. 36 In order to avoid statelessness, there is no provision allowing for the renunciation of Czech citizenship if the person concerned is not a citizen of another state. 37 We assume that the intention of the drafters of the law was to reduce the cases of Czech citizens living abroad transferring Czech citizenship over several generations. 37 We assume that the intention of the drafters of the law was to reduce the cases of Czech citizens living abroad transferring Czech citizenship over several generations. 38 It had particularly precarious consequences for Czech women who married citizens of some Islamic countries. The status of non-citizens put them at a disadvantage with regard to inheritance rights, for example whereas the potential loss of Czech citizenship in the case of naturalisation would deprive them of diplomatic protection. Another category adversely affected are citizens who applied for a foreign citizenship before the law entered into force but were granted a foreign citizenship after 1 January 1993. 38 It had particularly precarious consequences for Czech women who married citizens of some Islamic countries. The status of non-citizens put them at a disadvantage with regard to inheritance rights, for example whereas the potential loss of Czech citizenship in the case of naturalisation would deprive them of diplomatic protection. Another category adversely affected are citizens who applied for a foreign citizenship before the law entered into force but were granted a foreign citizenship after 1 January 1993. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 269 comprehensive explanation of the causes that have led to the state’s changes in attitude. comprehensive explanation of the causes that have led to the state’s changes in attitude. 26 File No. IV US 34/97. 27 The ruling was confirmed by a subsequent ruling on 14 November 2000 (File No. I. US 337/99). The Court argued that exercising the right of option does not mean that a person acquired foreign, i.e. Slovak citizenship at his or her own request, which would lead to automatic loss of Czech citizenship. In practical terms, this ruling con- cerned mostly ethnic Czechs living in Slovakia. 27 The ruling was confirmed by a subsequent ruling on 14 November 2000 (File No. I. US 337/99). The Court argued that exercising the right of option does not mean that a person acquired foreign, i.e. Slovak citizenship at his or her own request, which would lead to automatic loss of Czech citizenship. In practical terms, this ruling con- cerned mostly ethnic Czechs living in Slovakia. 28 See also below Table 8.4. 29 See below Table 8.3, sections 18b, 18c. 29 See below Table 8.3, sections 18b, 18c. 30 The situation of children born to a stateless parent without permanent residence is not regulated adequately. 30 The situation of children born to a stateless parent without permanent residence is not regulated adequately. 31 I do not distinguish between eligibility and conditions for naturalisation. In Czech citi- zenship legislation, the conditions for naturalisation fall into two categories: a) condi- tions sine qua non, which cannot be waived, and b) conditions, which can be waived at the discretion of the authorities. 32 The applicant has not been convicted for an intentional crime in the last five years. 32 The applicant has not been convicted for an intentional crime in the last five years 33 The applicant has to submit a certificate of the loss of his or her previous citizenship or a certificate that by the acquisition of Czech citizenship he or she will lose his or her previous citizenship. 34 Act No. 326/1999 Coll., as amended (Aliens Act). The amendment of the Aliens Act, Act No. 161/2006, which entered into force on 27 April 2006, cut the waiting period for permanent resident status to five years (in order to implement EU Directive 2003/109/EC Concerning the Status of Third-country Nationals who are Long-term Residents). 34 Act No. Notes 139/1996 Coll., which allowed for exceptions in naturalisation procedures from the clean criminal record requirement for former Czechoslovak citizens who had resided in the territory of the Czech Republic since the break-up of Czechoslovakia. See Linde 2006 for a CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE T.), date of entry into 270 ANDREA BARSˇOVA´ force: 19 March 2002; 1954 UN Convention Relating to the Status of Stateless Persons (No. 108/2004 Coll. of I. T), date of entry into force: 17 October 2004. force: 19 March 2002; 1954 UN Convention Relating to the Status of Stateless Persons (No. 108/2004 Coll. of I. T), date of entry into force: 17 October 2004. force: 19 March 2002; 1954 UN Convention Relating to the Status of Stateless Persons (No. 108/2004 Coll. of I. T), date of entry into force: 17 October 2004. ( ) y 7 42 Published as Act No. 169/1929 Coll. The treaty established a rule that in the case of naturalisation, the citizenship of the state of origin is automatically lost. ( ) y 7 42 Published as Act No. 169/1929 Coll. The treaty established a rule that in the case of naturalisation, the citizenship of the state of origin is automatically lost. 43 In 2007, the Ministry issued 577 negative decisions regarding naturalisation. 319 unsuccessful applicants appealed. In 92 cases the Minister of the Interior overturned the negative decision (Ministry of the Interior 2008: 155). 43 In 2007, the Ministry issued 577 negative decisions regarding naturalisation. 319 unsuccessful applicants appealed. In 92 cases the Minister of the Interior overturned the negative decision (Ministry of the Interior 2008: 155). 44 Act No. 150/2002 Coll. on Judicial Reviews of Administrative Acts. For judicial review in naturalisation cases see section 8.3 of this chapter. 44 Act No. 150/2002 Coll. on Judicial Reviews of Administrative Acts. For judicial review in naturalisation cases see section 8.3 of this chapter. 45 The Council for Human Rights is an advisory body to the government. See Resolution of the Government of the Czech Republic No. 493/2002 Related to the Communication by the Council for Human Rights on the Citizenship of the Czech Republic. The communication concerned certain urgent issues, such as the incompatibility of the remaining bilateral agreements with the requirements of the European Convention on Nationality. It also brought to the attention of the government certain problems of interpretation and practice in the field of citizenship law. 46 Ministry of the Interior, Document No. VS – 473/50/2-2004. 46 Ministry of the Interior, Document No. VS – 473/50/2-2004. 47 The fundamental incentives for the switch towards toleration of multiple citizenship thus seem to be those described by Hagedorn (2003). CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE Obviously, citizens and immigrants campaign for dual citizenship for different reasons. Dual citizenship corresponds to the needs of both expatriates and immigrants and offers them a greater scope for individual choice. 47 The fundamental incentives for the switch towards toleration of multiple citizenship thus seem to be those described by Hagedorn (2003). Obviously, citizens and immigrants campaign for dual citizenship for different reasons. Dual citizenship corresponds to the needs of both expatriates and immigrants and offers them a greater scope for individual choice. 48 At present, the state authorities processing applications for naturalisation carry out the testing. This does not guarantee uniform standards of testing. 48 At present, the state authorities processing applications for naturalisation carry out the testing. This does not guarantee uniform standards of testing. 48 At present, the state authorities processing applications for naturalisation carry out the testing. This does not guarantee uniform standards of testing. 49 Ministry of the Interior, Analy´za u´pravy naby´va´nı´ a pozby´va´nı´ sta´tnı´ho obcˇanstvı´. [Ana- lysis of the regulation on acquisition and loss of state citizenship] Adopted on 13 July 2005 by Government Resolution No. 881. 50 The governing coalition consists of the liberal-conservative Civic Democratic Party, Christian Democrats and the Greens. In its programme declaration, the new government stated that it would ‘consider the possibility of simplifying the acquisition of dual nationality’. See the Programme Declaration of the government, available at www.vlada.cz. 51 Under the government’s legislative rules, a framework law (veˇcny´ za´meˇr za´kona) nor- mally precedes the full legislative draft if a law regulates new and complex issues. These blueprints represent a step in legislative drafting at government level. As such, they are not subject to consultation with, or decision by, the parliament. Consulta- tions on the draft legislation within the administration and with other stakeholders took place from July to November 2007. y 7 52 Ministry of the Interior, Na´vrh veˇcne´ho za´meˇru u´stavnı´ho za´kona o sta´tnı´m obcˇanstvı´ Cˇ eske´ republiky a na´vrh veˇcne´ho za´meˇru za´kona o sta´tnı´m obcˇanstvı´ Cˇ eske´ republiky, VS- 1283/50/2-2007 [The proposal of the framework for the Constitutional Act on Citi- zenship of the Czech Republic and the framework for the Act on Citizenship of the Czech Republic]. The government took its decision by Resolution of 17 March 2008, No. 254. 53 See Stanovisko Legislativnı´ rady vla´dy ze dne 7. u´nora 2008 [Opinion of the Legislative Council adopted on 7 February 2008]. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 39 In the Court’s opinion, there is a distinction between deprivation of citizenship, pro- hibited by the Constitution, and loss of citizenship. Those who apply for a foreign citi- zenship should be aware of the legal consequences attached to the act, which are pro- vided for by law. Thus, the loss of citizenship based on the acquisition of foreign citi- zenship does not constitute deprivation of citizenship against one’s will. See Ruling published under No. 6/1996 Coll. Concerning the Proposal to delete Section 17 of Act No. 40/1993 Coll. on the Acquisition and Loss of Citizenship of the Czech Re- public. 39 In the Court’s opinion, there is a distinction between deprivation of citizenship, pro- hibited by the Constitution, and loss of citizenship. Those who apply for a foreign citi- zenship should be aware of the legal consequences attached to the act, which are pro- vided for by law. Thus, the loss of citizenship based on the acquisition of foreign citi- zenship does not constitute deprivation of citizenship against one’s will. See Ruling published under No. 6/1996 Coll. Concerning the Proposal to delete Section 17 of Act No. 40/1993 Coll. on the Acquisition and Loss of Citizenship of the Czech Re- public. 39 In the Court’s opinion, there is a distinction between deprivation of citizenship, pro- hibited by the Constitution, and loss of citizenship. Those who apply for a foreign citi- zenship should be aware of the legal consequences attached to the act, which are pro- vided for by law. Thus, the loss of citizenship based on the acquisition of foreign citi- zenship does not constitute deprivation of citizenship against one’s will. See Ruling published under No. 6/1996 Coll. Concerning the Proposal to delete Section 17 of Act No. 40/1993 Coll. on the Acquisition and Loss of Citizenship of the Czech Re- public. 40 This provision existed previously, but the wording was not clear and, in practice, it was incorrectly applied only to rare cases of automatic acquisition of citizenship through marriage. 41 The Czech Republic is party to the following multilateral treaties: 1997 European Convention on Nationality (No. 76/2004 Collection of International Treaties, henceforth Coll. of I. T.), date of entry into force: 1 July 2004; 1961 UN Convention on the Reduction of Statelessness (No. 43/2002 Coll. of I. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 271 55 Government Resolution of 17 March 2008, No. 254, point II. 56 The Human Rights Committee expressed in its views in several cases, e.g. No. 516/ 1992 (Simunek et al.), 586/1994 (Joseph Adam), 857/1999 (Blazek et al.) and 747/ 1997 (Dr. Karel Des Fours Walderode), that ‘a requirement in the law for citizenship as a necessary condition for restitution of property previously confiscated by the authorities makes an arbitrary, and, consequently a discriminatory distinction between individuals who are equally victims of prior state confiscations, and constitutes a violation of article 26 of the Covenant [on Civil and Political Rights]’. 57 See Judgement of the Constitutional Court published under No. 185/1997 Coll. The European Court of Human Rights declared cases that concerned the restitution of property inadmissible. See European Court of Human Rights, Grand Chamber decisions of 10 July 2002 as to the admissibility of application No. 38645/97, Polacek v. the Czech Republic and application No. 39794/98, Gratzinger v. the Czech Republic. 58 The Chamber of Deputies passed the bill on 23 November 2005. One hundred and two deputies voted for the bill and 49 against. The Act No. 46/2006 Coll. was published and entered into force on 27 February 2006. 59 Some of the judicial cases concern the citizenship of deceased persons, as the right to restitution by heirs depends on this issue. In certain cases, the Ministry of the Interior completed legal proceedings that had started in the late 1940s, using the then valid citizenship legislation. (See e.g. Decision of the Supreme Administrative Court of 27 November 2003, ref. no. 6 A 90/2002-82 (www.nssoud.cz) and the Judgment of the Constitutional Court of 29 June 2005, 1U´ S 98/04 (www.concourt.cz). ( ) 60 See Molek & Sˇimı´cˇek 2005. 60 See Molek & Sˇimı´cˇek 2005. 61 Resolution by the Supreme Administrative Court of 23 March 2005, ref. no. 6A 25/ 2002-42 and Decision by the Supreme Administrative Court of 4 May 2006, ref. no. 2As 31/2005-78. (www.nssoud.cz). In the former case the Court decided that negative decisions on naturalisation can be reviewed by administrative courts. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 54 See Martin Rozumek, ‘Prˇipomı´nky Organizace pro pomoc uprchlı´ku˚ m k na´vrhu veˇcne´ho za´meˇru nove´ho za´kona o naby´va´nı´ a pozby´va´nı´ sta´tnı´ho obcˇanstvı´ Cˇ eske´ republiky [Comments of the Organisation for Aid to Refugees concerning the framework proposal of the new Czech Citizenship Law]’, 15 August 2007, www.migraceonline.cz. Bibliography Barsˇa, P. (2005), ‘National Identities and Migration Policies’, Tr@nsit online. www.iwm.at. Barsˇova´, A. (2003a), ‘Dvojı´ obcˇanstvı´: lidske´ pra´vo v post-naciona´lnı´m sveˇteˇ?’, Socia´lnı´ stu- dia 9: 51–64. Barsˇova´, A. (2003b), ‘Sta´tnı´ obcˇanstvı´’, in Pra´vnı´ komparativnı´ studie programu migrace, 37- Barsˇova´, A. (2003b), ‘Sta´tnı´ obcˇanstvı´’, in Pra´vnı´ komparativnı´ studie programu migrace, 37- 44. Prague: Poradna pro obcˇanstvı´ & OSF. 44. Prague: Poradna pro obcˇanstvı´ & OSF. Barsˇova´, A. (2004), Vı´cecˇetne´ sta´tnı´ obcˇanstvı´: teoreticke´ a prakticke´ pohledy. di k i i Barsˇova´, A. (2004), Vı´cecˇetne´ sta´tnı´ obcˇanstvı´: teoreticke´ a prakticke´ pohledy. www.diskriminace.cz. Barsˇova´, A. (2005), ‘Foreigners Integration Policies in the Czech Republic’, in Social an Cultural Diversity in Central and Eastern Europe, 30-31. Prague: MKC. www.mkc.cz. Barsˇova´, A. & P. Barsˇa (2005), Prˇisteˇhovalectvı´ a libera´lnı´ sta´t. Imigracˇnı´ a integracˇnı´ politiky v USA, za´padnı´ Evropeˇ a Cˇ esku. Brno: Masarykova univerzita, MPU. Baubo¨ck, R. (1994), Transnational Citizenship. Membership and Rights in International Mi- gration. Aldershot: Edward Elgar. Boucˇkova´, P. & M. Vala´sˇek (1999), ‘Slovensˇtı´ obcˇane´ v Cˇeske´ republice a trvaly´ pobyt’, Spra´vnı´ pra´vo 2: 93-111. ˇ ˇ Cˇ erny´, J. & M. Vala´sˇek (1996), Cˇ eske´ sta´tnı´ obcˇanstvı´. Prague: Linde. ˇ ˇ ˇ Cˇ erny´, J. & V. Cˇervenka (1963), Sta´tnı´ obcˇanstvı´ Cˇ SSR. Prague: Orbis. Chan, J. M. M. (1991), ‘The Right to a Nationality as a Human Right’, Human Rights Law Journal 12: 1-14. Chlad, L. (2004), ‘Sta´tnı´ obcˇanstvı´ v kontextu Evropske´ integrace’, Pra´vnı´k 143 (4): 333- 382. Faist, T. (2004), ‘Dual Citizenship as Overlapping Membership’, in D. Joly (ed.), Interna- tional Migration in the New Millennium, 210-231. Aldershot: Ashgate. g 3 g Grawert, R. (1973), Staat und Staatsangeho¨rigkeit. Verfassungsrechtliche Untersuchungen zur Entstehung des Staatsangeho¨rigkeitsrechts Berlin: Duncker & Humboldt Grawert, R. (1973), Staat und Staatsangeho¨rigkeit. Verfassungsrechtliche Untersu Entstehung des Staatsangeho¨rigkeitsrechts. Berlin: Duncker & Humboldt. Hagedorn, H. (2003), ‘Administrative Systems and Dual Nationality. The Information Gap’, in D. A. Martin & K. Hailbronner (eds.), Rights and Duties of Dual Nationals. Evolution and Prospects, 183-200. The Hague: Kluwer Law International. Hansen, R. & P. Weil (eds.) (2002), Dual Nationality, Social Rights and Federal Citizenship in the U.S. and Europe. The Reinvention of Citizenship. New York: Berghahn Books. Kaspersen, L. B. (1997), War, State, Sovereignty and Citizenship. PhD Thesis. Department of Political Science, Aarhus University. Kristen, V. (1989), ‘Vznik Cˇeskoslovenske´ republiky a rakousˇtı´ krajane´’, in Cˇ esˇi v cizineˇ 4: 77-87. Krombach, D. (1967), Erstabgrenzungen im Staatsangeho¨rigkeitsrecht im 19. Jahrhundert und am Anfang des 20. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE (The Ministry of the Interior as well as some courts were of the opinion that naturalisation cases, unlike other citizenship cases, can not be reviewed by courts because of the non existence of the legal entitlement to citizenship.) In the latter case the Court confirmed its opinion on the possibility of administrative review of naturalisation cases and rejected the whole theory of unlimited administrative discretion in these procedures. 62 In the discussion on the draft citizenship legislation in 2007-2008, the Ministry of Justice, the Supreme Administrative Court and the Minister of Human Rights and Minorities supported this idea. 63 As part of a new, more active approach to the issues of immigration and integration, the Czech Statistical Office started to gather and analyse data on naturalisation in a systematic manner. These statistics however only cover the period since 2001. They can be found at www.czso.cz. 64 See Czech Statistical Office, www.czso.cz. 64 See Czech Statistical Office, www.czso.cz. 64 See Czech Statistical Office, www.czso.cz. 65 In many cases, Slovak citizens living in the Czech Republic had even difficulties to acquire permanent resident status (Boucˇkova´ & Vala´sˇek 1999). In some of these cases, the situation developed into de facto statelessness. 66 The variety of solutions adopted in the numerous cases of state succession made it difficult to prove the presence of a concrete and detailed customary law on state succession and citizenship. It was only the 1997 European Convention on Nation- ality that introduced certain generally applicable rules on citizenship in cases of state succession. 272 ANDREA BARSˇOVA´ Bibliography Jahrhunderts. Inaugural Dissertation. Rechts- und Staatswis- senschafliche Fakulta¨t der Rheinischen Friedrich-Wilhelms-Universita¨t Bonn. Linde, R. (2006), ‘Statelessness and Roma Communities in the Czech Republic: Compet- ing Theories of State Compliance’, International Journal on Minority and Group Rights 13: 341-365. Martin, D. A. & K. Hailbronner (2003), Rights and Duties of Dual Nationals. Evolution and Prospects. The Hague: Kluwer Law International. g Ministry of the Interior (2008), Zpra´va o situaci v oblasti migrace na u´zemı´ Cˇ eske´ republiky v roce 2007. www.mvcr.cz. ˇ Molek, P. & V. Sˇimı´cˇek (2005), ‘Udeˇlova´nı´ sta´tnı´ho obcˇanstvı´ – na cesteˇ od milosti sta´tu k soudneˇ prˇezkoumatelne´mu spra´vnı´mu uva´zˇenı´’, Pra´vnı´k 144 (2): 137-156. Schmied, E. (1974), Das Staatsangeho¨rigkeitsrecht der Tschechoslowakei. 2nd edn. Frankfurt/ Main: Alfted Metzner Verlag. Vaculı´k, J. (2002), Cˇ esˇi v cizineˇ – emigrace a na´vrat do vlasti. Brno: Masarykova univerzita. CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE CZECH CITIZENSHIP LEGISLATION BETWEEN PAST AND FUTURE 273 Vala´sˇek, M. & V. Kucˇera (2006), Sta´tnı´ obcˇanstvı´. Komenta´rˇ. Prague: Linde. Verner, V. (1947), Sta´tnı´ obcˇanstvı´ a domovske´ pra´vo republiky Cˇ eskoslovenske´. Prague: Va´clav Tomsa. Verner, V. (1947), Sta´tnı´ obcˇanstvı´ a domovske´ pra´vo republiky Cˇ eskoslovenske´. Prague: Va´clav Tomsa. Weis, P. (1979), Nationality and Statelessness in International Law. Alphen aan den Rijn, Germantown: Sijthoff and Noordhoff. Wiessner, S. (1989), Die Funktion der Staatsangeho¨rigkeit: eine historisch-rechtsvergleichende Analyse unter besonderer Beru¨cksichtigung der Rechtsordnungen der USA, der UdSSR und der Bundesrepublik Deutschland. Tu¨bingen: Attempto Verlag. 9 The Slovak question and the Slovak answer: Citizenship during the quest for national self- determination and after1 Dagmar Kusa´ Citizenship is both a status and a praxis. As a status, it is defined by a collection of laws and regulations. In Slovakia, these have been shaped by both principles of ius soli and ius sanguinis, the latter gaining im- portance especially after the First and the Second World War. The praxis involves the civic and political participation by citizens as well as the policies of governments concerning the implementation of the law in relation to its citizens as well as to non-citizens. The latter depend strongly on the political situation of the times. The first two turbulent decades of the Czechoslovak Republic were marked by attempts to eth- nically homogenise the ‘Czechoslovak’ nation, targeting primarily the German and Hungarian minorities (but also Roma and others) as un- wanted elements, culminating in three years of ‘homelessness’ after the end of the Second World War. Only the communist government re- stored their civil and political rights. Yet it was unable to do away with the national sentiments of the Slovaks, striving to achieve national self- determination within or without Czechoslovakia. The Federation of 1968 (and the Warsaw Pact tanks that preceded it) quieted the national- ist voices until 1989, when they echoed through the public squares with all the more vigour. The dissolution of Czechoslovakia, which fol- lowed in 1993, made for a messy transition period in citizenship policy with the need to address both issues related to the end of the commu- nist regime and its victims, as well as to the status of Czech nationals in Slovakia. The last decade has also brought new challenges connected to the in- tegration of Slovakia into the European Union and marked by general globalisation processes. Slovakia is figuring out its relationship towards an influx of newcomers from parts of the world with which it had no cultural contact in the past. International institutions shape these poli- cies to a large degree, although the careful observation of Hungary’s – its closest neighbour and historic adversary – citizenship policies seems to have just as much impact on shaping the public debate and legal provisions taken in Slovakia. While we will be focusing in this chapter primarily on citizenship as a status, the political praxis of gov- 276 DAGMAR KUSA´ ernments does need some attention to complete our understanding of what shaped citizenship policies at different times. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 277 13. Is the language used on a daily basis identical with the mother ton- gue? Absolutely not. Czech employees […] use in their German employ- ment the German language instead of their mother tongue. German is their language of everyday use.’ (Zeman 1994: 37). In a similar manner Czech, Slovak and Ruthenian leaders appealed to their respective con- stituencies to enter their mother tongue. Data were collected by census officials, often with the aid of the army and police and accompanied by threats, blackmail or violence. The census remained important, especially in border disputes after 1918. The northern part of the Czech Teschen-Silesia region as well as the southern part of the Slovak borderlands with Hungary were heavily disputed after the war and nationality was used as a tool for demarca- tion policies. Polish representatives based their arguments on census data from before 1918, which showed a clear majority of ethnic Poles in those territories. As the populations here were ethnically mixed and their mother tongue was often Polish or Hungarian, the question in the 1921 census carried out by the Czechoslovak Government was promptly changed to ask directly about nationality. A Silesian national- ity was created (besides Polish and Czechoslovak). Respondents in this category were then automatically counted among Czechoslovak na- tionals. This resulted in a complete change of the population propor- tions. While the percentage of Poles fell to 25 per cent (from 139,000 to 69,000), the percentage of Czechoslovaks grew from 40 per cent to 65 per cent (from 123,000 to 177,000) (Paul 1998: 163). The fate of Teschen-Silesia was decided at the Paris Peace Confer- ence. Polish representatives succeeded in their demand for a plebiscite. If this had been carried out, Czechoslovakia might have lost some of these economically strong territories. However, the international com- mission overseeing the plebiscite could not agree on the conditions, the Red Army was already invading Poland, and legal norms in Cze- choslovakia were confusing due to the existing state of legal dualism where Czech lands inherited the legal system from Austria, and Slova- kia that of Hungary. A plebiscite was to be carried out not only in Sile- sia, but also in the northern Slovak areas of Spisˇ and Orava, which would result in implementing two plebiscites regulated by differing sets of laws. 9.1.1 Citizenship policies since the first Czechoslovak Republic Czechoslovak citizenship was created with the first Czechoslovak Re- public on 28 October 1918. The collective identity to which it referred was cumbersome, to say the least, and was a result of the historical path of the Czech and Slovak nation-building processes as well as of the peculiar nature of the new state that had resulted from the dissolu- tion of the Austro-Hungarian Empire and from the peace treaties fol- lowing the First World War. The Wilsonian principle of self-determina- tion influenced the understanding of the concept of citizenship and contributed to the growing role of ethnicity in its legal definition. Con- cepts of citizenship and ethnic nationality are often difficult to set apart neatly. They influence each other, and both depend heavily on political interpretations. The Czechoslovak Republic consisted of a multitude of ethnic groups and the leadership struggled with asserting the dominant position of the Czech and Slovak nations in their newly established Republic. National minorities, especially the three million Germans and close to a million Hungarians, formed 44 per cent of the total population. The Czechoslovak Government thus enforced an offi- cial Czechoslovak nationality2 (instead of separate Czech and Slovak nationalities). The sovereign nation needed to be propped up by some ‘objective’ quantifiable measures of dominance. Population censuses helped to provide these measures and also allowed citizens to be distinguished from foreigners.3 The power of numbers as represented in the census was becoming apparent to national leaders prior to the foundation of Czechoslovakia. With the growing turbulence over what was then called the ‘nationality question’ within the Habsburg Empire the cen- sus was becoming more and more powerful as an expression of ‘real’ power, as a ticket to future control over territory and as one of the de- terminants of state formation and boundaries. In 1900, for example, the German newspaper in Bohemia appealed to its readers: ‘Dear fel- low citizens! Please pay close attention to column 13 (Umgangssprache) in the census form. The future of our nation depends on this minor entry. 1. What is the language used on a daily basis? It is the language most commonly used by an individual. Daily use means the communi- cation in the family, among people that live together, in their employ- ment, with an employer. Wherever this communication happens in the German language, no other language should be entered into column THE SLOVAK QUESTION AND THE SLOVAK ANSWER The northern boundary was therefore finally decided upon the recommendation of the Allied Powers. Poland was compensated for much of Silesia with 25 settlements in Orava and Spisˇ (Klimko 1980; Peroutka 1991). Legal dualism was caused by differing practices in granting citizen- ship and domicile before 1918 following the Austro-Hungarian Com- promise of 1867. While in Austria domicile, i.e. a legal title of resi- dence in a municipality (Heimatrecht), was closely registered, it was not in the Hungarian part of the empire that included Slovakia.4 Even 278 DAGMAR KUSA´ DAGMAR KUSA´ though domicile was granted to all those born and residing in a muni- cipality, the gentry had a right to deny some people domicile even if they were born or had resided in the locality for a long time. Jurova´ (2002) maintains this was the fate of many Roma who moved from vil- lage to village. This was due to arts. 8-15 of the municipal law (XXVII/ 1886) that tied the acquiring of domicile of those who move and/or marry to fulfilling certain duties towards the municipality, thus giving the authorities opportunities for convenient interpretation. Further- more, Act No. 222/1896 amended some provisions of the 1863 munici- pal law that specified conditions under which a Roma could be granted domicile. The Roma and Hungarians were groups that succeeding Czechoslo- vak governments sought to minimise statistically after 1918. The cen- sus of 1921 shows a remarkable number of ‘foreigners’ without Cze- choslovak citizenship that still have domicile on Slovak territory. The extent to which these groups were affected by citizenship policies has unfortunately not been extensively researched and quantitative data in this area are missing (Jurova´ 2002). Czechoslovakia’s citizenship regulations were further disturbed by the events of the Second World War. Slovakia experienced its first (de- batably) independent statehood as a Nazi puppet state, while the Czech lands were occupied under the Third Reich’s Protectorate. The end of the Second World War and the restoration of Czechoslovakia led to the adoption of ad hoc laws that introduced the criterion of ethnicity into citizenship legislation. The new legislation was linked to the post-war massive emigration and population exchange. Under the President’s Constitutional Decree No. 33/1945 Coll. THE SLOVAK QUESTION AND THE SLOVAK ANSWER (Collection), Czechoslovak citi- zens of German and Hungarian ethnic origins were deprived of Cze- choslovak citizenship.5 This also meant their exclusion from official in- stitutions (Order 99/1945 of the Slovak National Council), as well as from reimbursement for war damages, and implied other practical con- sequences.6 Further decrees also disbanded German and Hungarian associations and organisations. g The transfers of ethnic Germans were agreed to by the Allied Powers at the Potsdam Conference in 1945. They did, however, not approve of applying the same policy based on a principle of collective guilt to Hungarians. The alternative solution found by the Benesˇ Government was a ‘voluntary exchange of populations’ between Czechoslovakia and Hungary. This plan resulted in the removal of 89,660 ethnic Hungar- ians, who were moved into Hungary, in return for receiving 73,273 eth- nic Slovaks (Vadkerty 2002: 32). Oral history projects document that the nature of the exchange was in many cases coercive. Another wave of transfers, labelled by the Czech historian Karel Kaplan as an ‘inter- nal colonisation’ (Kaplan, 1993: 9), was based on the Presidential De- THE SLOVAK QUESTION AND THE SLOVAK ANSWER 279 cree No. 88/1945 on universal labour service. Ethnic Hungarians were recruited for ‘voluntary agricultural work’7 into the then vacant Sude- tenland. Age limits imposed by the Decree were also frequently ig- nored and property left behind was confiscated (in direct violation of the Decree) (Kusa´ 2005). These policies were accompanied by a pro- gramme of re-Slovakisation, passed by the Slovak National Council in June 1946. This policy gave ethnic Hungarians an opportunity to ‘re- claim’ Slovak citizenship (based on the premise of previous coercive Magyarisation of Slovaks) within the time span of one year. Some 320,000 Hungarians were granted Slovak citizenship on this basis. However, as the census of 1960 shows, many returned to claiming Hungarian ethnicity in the census as soon as the political situation tol- erated it.8 This era has been dubbed as the ‘homeless years’ by Hungarian authors. Citizenship was eventually restored to the Germans and Hun- garians remaining in Czechoslovakia in 1948 by the newly established communist government; most Hungarians who had been transferred to Sudetenland have returned. Many, however, never recovered lost properties. THE SLOVAK QUESTION AND THE SLOVAK ANSWER The Benesˇ Decrees and their legal and practical conse- quences remain a painful open wound in Czech and Slovak political memory to this day and have been repeatedly debated, especially in connection with possible compensation for those affected and their descendants. Representatives of German and Hungarian communities sometimes called for an annulment of the Benesˇ Decrees, but due to the complexity of the political situation of interwar and post-Second World War years and a lack of political will in the Czech and Slovak Re- publics, it is unlikely that such a measure will be adopted. Some conci- liatory steps were taken by the Czech and Slovak governments in the past decade on the level of bilateral declarations (the Czech-German Declaration of 1997) or public speeches (e.g. Hrusˇovsky´ 2003). 9.1.2 Regulation of Czechoslovak citizenship in 1949-1968 and the ‘Slovak Question’ 9.1.2 Regulation of Czechoslovak citizenship in 1949-1968 and the ‘Slovak Question’ The rise of communist monopoly rule meant, ironically enough, the end of ‘homelessness’ for the Hungarians and Germans in Czechoslo- vakia. Citizenship laws were, however, misused for other political pur- poses, as one of the tools to keep the lid on the population, as a sort of preventive blackmail of those who might think of publicly voicing their disapproval of the communist regime. The legal process of acquisition and loss of Czechoslovak citizenship in the period following the February putsch of 1948 was governed by the Act on the Acquisition and Loss of Czechoslovak Citizenship No. 194/1949, as amended by the Act No. 72/1958 Modifying the Regula- 280 DAGMAR KUSA´ tions on the Acquisition and Loss of Czechoslovak Citizenship.9 Cze- choslovak citizenship could be acquired in four ways: 1) by birth: Cze- choslovak citizenship was transferred to the child by his or her parent citizens regardless of whether the child was born in the territory of the Czechoslovak Republic or abroad. If the child was born in the territory of the Czechoslovak Republic, it was sufficient if one of the parents was a Czechoslovak citizen;10 2) by marriage: A foreigner could acquire Czechoslovak citizenship on demand upon marrying a Czechoslovak citizen. This acquisition needed to be investigated and approved by a district National Committee within six months; 3) by grant: A foreigner could be granted Czechoslovak citizenship upon request after meeting two principal conditions: residing in the Czechoslovak territory for five consecutive years and abandoning his or her previous citizenship. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 281 Slovaks. Dissent in Slovakia was therefore more muted compared to the Czech region. The Soviets poured investment into the Slovak in- dustry in the post-1968 era further contributing thereby to different perceptions of the ‘normalisation’ period between the two nations. What was an era of darkness for most Czechs, was seen by many Slovaks as a repressed society, but with real industrialisation and fed- eration at least on paper. While this reality itself may not have had an immediate impact on citizenship laws and practice, it certainly reverb- erated on the political scene after 1989, when the cultural divide be- tween Czechs and Slovaks escalated into the ‘Velvet Divorce’. 9.1.2 Regulation of Czechoslovak citizenship in 1949-1968 and the ‘Slovak Question’ There was no legal entitlement to be granted citizenship; 4) by reacqui- sition: This applied to the acquisition of citizenship by the ‘homeless’ persons of German and Hungarian nationality ex lege after taking a citi- zenship oath without the need to apply or to fulfil other conditions.11 The loss of Czechoslovak citizenship was possible by 1) renunciation upon request,12 2) revocation by the state due to hostile acts against the Republic, illegal emigration, or not returning to the homeland for the period of five years or upon request of the Ministry of the Interior, 3) marrying and acquiring citizenship in another country (with a possibi- lity to request retention of Czechoslovak citizenship), 4) a court deci- sion as a penalty for ‘high treason, espionage, desertion of the Repub- lic, military subversive activities, war treason, assassination of a state official’,13 5) naturalisation in the United States of America, and 6) as a consequence of agreements on dual citizenship.14 During this period of time, and especially during the de´tente period of the 1960s, when literature and arts were flourishing after the de- nunciation of the Stalinist doctrine, Slovak leaders and intellectuals voiced their desire for self-determination of the Slovak nation in a fed- erative arrangement. They did not wish to be Czechoslovak citizens, but Slovak citizens of Czechoslovakia. While the Czech elite focused on market liberalisation and democratisation of the regime, Slovaks called for ‘first federalisation, then democratisation’ – a slogan that reap- peared repeatedly in public squares after 1989 in a much more malevo- lent form. This issue divided Czech and Slovak intellectuals during the entire duration of the communist regime, as the Czech cultural leaders failed to see the urgency of this issue for the Slovaks. The Soviet leader- ship, however, duly noted Slovak aspirations for federation. Thus when the tanks rolled into Prague and Bratislava on the 21 August 1968 it brought with it different realities for the two nations. While the oppres- sion following the Warsaw Pact invasion was equally suffocating in both parts of the country, it also brought the desired federation for the 9.1.3 Regulation of Czechoslovak citizenship in 1969-1992: Czechoslovak Socialist Federative Republic Until 1968, when the Czechoslovak Federation was established, Cze- choslovakia was a unitary state with a single Czechoslovak citizenship. The establishment of a federation also resulted in the creation of Czech and Slovak citizenships. Constitutional Law No. 143/1968 Coll. on the Czechoslovak Federation, which came into force on 1 January 1969, is based on the principle of individual preference when determining the citizenship of the two constituent republics.15 The original text contains a provision according to which every citi- zen of one of the republics is also a citizen of Czechoslovakia (art. 5). Citizenship was regulated by the Constitutional Act of the National Council of the Czechoslovak Socialist Republic No. 165/1968 Coll. on the Principles of Acquisition and Loss of Czech and Slovak Citizenship, followed by the Act No. 206/1968 Coll. of the Slovak National Council on Acquisition and Loss of Citizenship of the Slovak Socialist Republic. q p p Normally citizenship at the level of the two republics was deter- mined by the place of birth or by the citizenship of the parents, if that could be identified. Czech or Slovak citizens could, however, choose a different citizenship until 31 December 1969. The Act precluded dual citizenship, one had to choose one or the other. The Slovak National Council passed Act No. 206/1968 Coll. to apply these rules in domes- tic legislation. g Between 1969 and 1992 it was possible to acquire Slovak and Czech citizenship by birth,16 by choice (within one year after the establish- ment of the federation), by marriage, or by grant (after five consecutive years of residence for foreigners and two years for Czech citizens with permanent residence in Slovakia). Loss of citizenship in the ‘normalisation’ era was similar to previous regulations. It could be renounced, lost due to acquiring Czech citizen- ship, or one could still be deprived of it on the basis of art. 7 of Act No. 282 DAGMAR KUSA´ 194/1949 Coll., naturalisation in the US, or according to agreements on dual citizenship. After the fall of communism, both Czech and Slovak national elites struggled to assert the position of their nations within Europe. National identity had to be reconstructed and to a large extent even re-invented. Both elites turned to their past to seek linkages and justification for steps towards self-determination. Czechs and Slovaks, however, sought friendship with very different animals from their past. 9.1.3 Regulation of Czechoslovak citizenship in 1969-1992: Czechoslovak Socialist Federative Republic Even before the dissolution, the citizenship laws had been growing in THE SLOVAK QUESTION AND THE SLOVAK ANSWER 283 significance, and many Czechs and Slovaks were using their right to choose their republic-level citizenship. In Slovakia, the nationalist craze played out directly in many legal provisions that concerned anybody ‘other’ than ethnic Slovaks.19 Such was the case with the ‘Sign Law’ (a law regulating public inscriptions such as topographical names of towns, villages, streets and store signs), the Act on the Official State Language, which was passed without any provisions for the use of languages of the national minorities (which were adopted only in 1997), the ‘Territorial Arrangement’ that redrew district boundaries to lessen the percentage of ethnic Hungarians in areas where they were concentrated, and other legislation. This policy has also affected the practice of allowing access to those seeking asy- lum, with possible hopes for eventually acquiring citizenship in the Slovak Republic. While the legislation regulating the asylum proce- dures was not markedly different from other countries, the political en- vironment was palpably hostile. During the war in Bosnia and Herze- govina, Slovakia, just as many other countries, received an influx of re- fugees. The Migration Office of the Ministry of the Interior was at that time particularly untoward in granting anyone the status of a refugee. Many, if not most, displaced persons had to contend with a protective status of the United Nations High Commissioner for Refugees office in Slovakia, and most were turned back after a few months, not always into safe conditions. After the accession of the Slovak Republic to the European Union in 2004 and its becoming a part of the Schengen territory in 2007, a number of EU regulations were transposed into Slovak Law. With the amendment of the Asylum Law in 2007 the Slovak Republic imple- mented the EU provision on minimal standards for the granting and loss of the refugee status and also implemented the Dublin Treaty, which sets criteria for determining which EU Member State is respon- sible for processing a particular asylum application. The Slovak Repub- lic, unlike its neighbours, still lacks a coherent immigration and inte- gration strategy, although a plan for establishing Immigration and Nat- uralisation Services is underway. In 2007, the Slovak Government amended the Act on Citizenship, gearing up towards accession to the Schengen Treaty. 9.1.3 Regulation of Czechoslovak citizenship in 1969-1992: Czechoslovak Socialist Federative Republic Czechs built on Masaryk’s democratic ideals from the first interwar republic, while Slo- vaks viewed this era suspiciously with a memory of Czech ‘Pragocentr- ism’17 and of the refusal of the Czechoslovak Government to grant Slo- vakia the right to self-determination or autonomy in a federation. In- stead, Slovaks referred to the legacy of the Slovak puppet state created by the Nazis.18 The discrepancy in perceptions of the post-1968 era added to the rift between the two nations. This ‘failure to find a decent past’ together, as Igor Lukes (1995) coined it, contributed to the choice of separate paths for the future by the political elites, whose sentiments were, however, not reciprocated by the majorities of populations on either side of the new border. In the confused atmosphere of rampant nationalism that had anti- Czech, anti-Hungarian, anti-Semitic, and even anti-Western traits in the years prior to the Velvet Divorce, Slovak representatives raised many issues that seemed to be frivolously escalating the conflict into what became popularly known as the ‘hyphen war’, i.e. the war about the spelling of ‘Czechoslovakia’. Slovak delegates claimed that the term Czechoslovakia was discriminatory to the Slovaks, who are commonly mistaken for Czechs abroad. Claims were backed by invoking the myths of one thousand years of suffering by the Slovaks under the Hungarian yoke, only to be replaced by the Czech yoke in 1918. The Federative Assembly finally settled on ‘Czech and Slovak Federative Re- public’ as the name for the post-communist state. The Slovak Prime Minister Vladimı´r Mecˇiar conducted a policy of blackmail, threatening the Czech leadership with the possibility of se- cession over each major political issue. The Czech Prime Minister Klaus eventually called his bluff and startled Mecˇiar by accepting the proposal for separation. The divorce was decided at the top political le- vel without being ratified by popular participation, but also without strong protests by the Czech and Slovak public. Over half of the re- spondents in public opinion surveys voiced their desire to remain in the common state and/or to have an opportunity to decide its fate in a referendum (Nemcova´ 1992). It was instead decided by political elites. On 1 January 1993, the two nations started a new period in their his- tory and had to determine their identities and related policies anew. 9.1.3 Regulation of Czechoslovak citizenship in 1969-1992: Czechoslovak Socialist Federative Republic According to the Minister of Interior, Robert Kalinˇa´k, the new act is a response to ‘the growing danger of organised crime and international terrorism’.20 The new version of the citizenship act significantly tightens the naturalisa- tion requirements. According to this law, a foreigner may acquire Slo- vak citizenship after eight years of permanent residence in the Slovak Republic (as opposed to the previous five). The law also stipulates that foreigners applying for Slovak citizen- ship must pass a test where they must demonstrate a thorough knowl- 284 DAGMAR KUSA´ DAGMAR KUSA´ edge of the Slovak language, the basic historical and geographical facts regarding the Slovak Republic and Slovak culture. Applicants are inter- viewed about their person and family, and must further demonstrate ‘basic general knowledge’, and pass a written test where they are asked to summarise a random newspaper article. These new requirements, which demand that applicants ‘prove cultural acclimatisation,’ are per- haps more a reflection of the nature of the government that came to power in 2006 (a coalition consisting of the populist SMER, or the People’s Party – Movement for Democratic Slovakia, led by the notor- ious former prime minister Vladimı´r Mecˇiar, and the nationalist Slovak National Party) than of any purported requirements relating to acces- sion to the Schengen Treaty. 9.2 Current regulations of acquisition and loss of Slovak citizenship In the first years of the Slovak Republic, Slovak citizenship was either determined by law or could be individually chosen. Those who were ci- tizens of the Slovak Republic before 31 December 1992 automatically became citizens of independent Slovakia, as stipulated in Act No. 40/ 1993 Coll. on Citizenship of the Slovak Republic. Czech citizens could apply for Slovak citizenship until 31 December 1993 by way of a written request to the District Office in the territory of the Slovak Republic or to the Diplomatic Mission or Consular Office of the Slovak Republic abroad. This option was open to all citizens of the former Czech and Slovak Federative Republic. Those applying for Slovak citizenship had to provide proof that they were Czechoslovak citizens as of 31 Decem- ber 1992 and state their place of birth and permanent residence (art. 7). THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 285 nis.21 If citizenship cannot be established, a child is considered to be a citizen of the Slovak Republic if he or she was born or was found in the territory of the Slovak Republic and his or her parents are not known. If one of a child’s parents is a citizen of another country and the other is a citizen of the Slovak Republic, then the child is a citizen of the Slovak Republic even if it is later established that the child’s par- ent who is a citizen of the Slovak Republic is not the child’s natural parent. A child can also acquire citizenship when he or she is adopted by a Slovak citizen. In the case of a disagreement between the parents, Slovak citizenship can be determined by a court judgement on the ba- sis of one parent’s or a legal guardian’s request. Citizenship in the Slovak Republic can also be granted upon request to a foreigner. This requires consecutive permanent residence and phy- sical stay in the Slovak territory for at least eight years immediately prior to submitting an application for citizenship. Slovak law also re- quires sufficient basic proficiency in the Slovak language. Applicants must also have a clean criminal record, which means that they must not have been prosecuted for an intentional crime during those five years before the application, must not be under an administrative ex- pulsion order from the country of residence or subject to extradition proceedings.22 Facilitating factors in the application procedure are if an applicant is stateless or voluntarily renounces his or her previous citizenship. Furthermore, citizenship can be granted upon request to those who have entered into marriage with a Slovak citizen (after living in the Slo- vak Republic for a period of five consecutive years), or those who have made special contributions to the Slovak Republic through their achievements in the field of economy, science, culture or technology. y gy There are also special provisions for the restoration of citizenship to those who lost it according to previous legislation. THE SLOVAK QUESTION AND THE SLOVAK ANSWER A person whose for- mer Czechoslovak citizenship has expired or who has lost the Czecho- slovak citizenship due to a long absence or on the basis of citizenship law during the communist regime, may be granted citizenship of the Slovak Republic even if the abovementioned condition of five years consecutive permanent residence has not been met. Former Slovak citi- zens returning to live in Slovakia have to have permanent residence in the Slovak Republic for three years prior to filing an application for citi- zenship.23 9.2.1 Acquisition of citizenship Slovak citizenship can currently be acquired by birth, by adoption, or by grant. The laws regulating citizenship are comparatively generous towards individuals with Czech or Slovak roots, allowing for a plural ci- tizenship and extending considerable citizenship rights to the Slovak expatriates living abroad. Acquisition of citizenship by birth is firmly based on ius sanguinis except in those cases where a child would otherwise become stateless. In current legislation a child acquires Slovak citizenship only if at least one of the parents is a citizen of the Slovak Republic or if the child was born in the territory of the Slovak Republic to parents who are sta- teless or whose citizenship is not transmitted to the child iure sangui- 9.2.2 Loss of citizenship Slovak citizenship can be lost, only upon the holder’s own request, by releasing the person from the state bond. Only those can be released 286 DAGMAR KUSA´ DAGMAR KUSA´ who already possess another citizenship, or who will acquire another citizenship as soon as they are released from Slovak citizenship. A Slovak citizen cannot be released if he or she is being prosecuted, is currently serving a sentence or is due to serve a sentence or has out- standing taxes or other debts to pay to the state. The District Office, Diplomatic Mission or a Consular Office of the Slovak Republic makes the final decision on the loss of citizenship. Citizenship is lost on the day of receipt of the document stating his or her release from the state bond of the Slovak Republic. 9.2.4 International treaties Slovakia is party to many international multilateral and bilateral treaties that impact on domestic citizenship regulations. International treaties take precedence over domestic law – if they differ from the pro- visions in the Act No. 40/1993 Coll. on Citizenship of the Slovak Re- public, the legal regulations of international law outweigh domestic law (art. 17). ( 7) As in the case of the Czech Republic, the treaty with the United States that precluded naturalised American citizens of Czech and Slo- vak origin from holding dual citizenship (the 1928 Naturalisation Treaty) expired in 1997. This allowed many former citizens and their descendants to restore their Slovak citizenship and to file claims for restitution of property with the Slovak state. Among the other important bilateral treaties was the Agreement on Slovak-Hungarian Neighbourly Relations from 1995, which had impli- cations for the practical implementation of certain cultural and educa- tional rights of ethnic Hungarians in Slovakia. Many international pro- visions – including this one – were passed only because of extensive pressure from European institutions dangling the carrot of EU acces- sion in front of the Slovak leadership. The Slovak-Hungarian Treaty was passed at the peak of the Mecˇiar Government era, to the bewilder- ment of his followers and perhaps of himself, after Slovakia had re- ceived demarches from the OSCE High Commissioner on National Minorities and other international institutions regarding its practices concerning national minorities and foreigners. The international com- munity thus played a key role in shaping domestic policies in this tran- sition period keeping the ugly dragon of nationalism and xenophobia on a somewhat shorter leash. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 287 vak Constitution, laws and other legal rules and will duly fulfil all du- ties of a Slovak citizen.’24 If the applicant doesn’t pick up the Certificate of Acquisition within six months of receiving a written notification the Ministry will stop the procedure. If the Ministry rejects the application then the applicant can apply again after a minimum waiting period of one year. 9.2.3 Procedure Slovak citizenship acquired by naturalisation is awarded by the Minis- try of the Interior of the Slovak Republic based on a written applica- tion. This application has to be filed in person at a District Office, Diplomatic Mission or Consular Office of the Slovak Republic. It must include personal data about the applicant and must be accompanied by a dossier of documents including a brief curriculum vitae, an identifi- cation card, a birth certificate, a personal status certificate, and a certifi- cate of residence in the Slovak Republic. Former Czechoslovak citizens that qualify for restoration of citizenship have to provide a document stating the release from the state bond of the Czechoslovak Republic, the Czechoslovak Socialist Republic or the Slovak Socialist Republic (whichever applies). Former Slovak citizens applying for citizenship after two years of residence in Slovakia can submit a Slovak Status ID as a form of identification. The Ministry of the Interior can ask for other documents if required to render a decision. The application is accompanied by a questionnaire on the basis of which the authorities evaluate the applicant’s Slovak language skills. Verification has to be done in a way that takes the applicant’s circum- stances into account. The District Office has the right to request a statement from the police and will then forward the complete applica- tion with all documents and statements to the Ministry of the Interior for a final decision. When making its decision, the Ministry of the In- terior has to take into account the public interest as well as statements of state institutions and of the police. It has nine months from receipt of an application to issue a decision. If statements of state institutions and of the police are required, the processing period is prolonged to one year. Slovak citizenship is acquired by obtaining a Certificate of Acquisi- tion of Slovak Citizenship at the District Office, Diplomatic Mission or Consular Office of the Slovak Republic and after taking the obligatory oath. The citizenship oath reads: ‘I promise on my honour and con- science that I will be loyal to the Slovak Republic, I will respect the Slo- 9.2.5 Dual and multiple citizenship Slovak legislation tolerates dual citizenship. Regulations of dual and multiple citizenship on a European level are, however, developing slowly and with obstacles. The regime changes and successive creation of new states after 1989 created a need to come up with common regu- lations regarding citizenship policies that resulted in the European 288 DAGMAR KUSA´ DAGMAR KUSA´ Convention on Nationality (ETS No. 166), which entered into force on 1 March 2000. It was the first international document to establish core principles and rules applying to all aspects of citizenship to which the domestic law of the parties to the treaty should conform. The Conven- tion was opened for signature to Member States of the Council of Eur- ope as well as non-members on 6 November 1997. Slovakia signed and ratified the Convention, as did the Czech Republic. p Among other issues the Convention covers questions of multiple ci- tizenship. Art. 14 directly stipulates the right to dual citizenship in the case of acquiring citizenship of another country by marriage. The force of the Convention is, however, softened by arts. 15 and 16, which give the parties the right to determine whether their nationals who acquire or possess the nationality of another state retain or lose their citizen- ship; and the right of state parties to make the acquisition or retention of their citizenship conditional upon renunciation or loss of another ci- tizenship (unless it is not possible or cannot reasonably be required). These articles are often used in practice to preclude multiple citizen- ship. There have been speculations as to whether Slovakia could use them in this way if the Hungarian Parliament passes the law on dual citizenship for ethnic Hungarians living abroad (see Kova´cs & To´th in this volume). This would not be possible without amendments to the current law, which stipulates that the loss of Slovak citizenship results only from a person’s own request to be released from the state bond. The state cannot on its own initiative deprive any person of their Slo- vak citizenship. It is, however, possible that some ethnic Hungarians residing in Slovakia could be released from the state bond upon their own request after gaining Hungarian citizenship, thus becoming Hun- garian foreign nationals living in Slovakia. This status would, however, bring more inconveniences than benefits to the applicants. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 289 1993 did not lose their Czech citizenship, the Czech citizenship laws were amended to allow for reacquisition of the Czech citizenship for certain groups of people within a stipulated period. Further revisions of the Czech law were passed in September 2005 to allow for dual citi- zenship for Czechs living in Slovakia, who had lost their Czech citizen- ship by acquiring the Slovak nationality between 1 January 1994 and September 1999.26 Applications for dual citizenship can be submitted to the Consular Office of the Czech Embassy in Bratislava. The applica- tion process takes up to two months. Approximately five thousand peo- ple requested dual citizenship in 2005.27 9.3.1 The Hungarian Status Law and referendum on dual citizenship Slovak-Hungarian relations have been an inflammable issue on the Slovak political scene since the fall of communism. Much nationalist rage was directed against the former dominant nation, the Hungarian part of the dual monarchy. Policies of forceful Magyarisation in the late nineteenth and early twentieth century and the turbulent dissolution of the empire that left one third of the ethnic Hungarians outside the bor- ders of the Hungarian state, provide historical memories that shaped mutual relations in a controversial fashion. The myth of a thousand years of suffering under the Hungarian yoke has long been nurtured by Slovak nationalists and after 1989 it often served as a useful rallying point. The question of Hungary’s relationship with ethnic Hungarians liv- ing abroad, especially in the areas immediately bordering on Hungar- ian state territory, was therefore watched closely and suspiciously. The issue exploded in the Slovak media in 2001 when Hungary passed the Status Law (the law on Hungarians living abroad) and again in 2004 when a referendum was held on allowing ethnic Hungarians to acquire dual citizenship. The content and impact of these Hungarian initiatives are described in detail in Ma´ria M. Kova´cs’s and Judit To´th’s chapter on Hungarian citizenship in this book, so I will focus here only on the re- percussions in Slovakia. 9.2.5 Dual and multiple citizenship It is far more likely that, if Hungary passed the dual citizenship law, most eth- nic Hungarians in Slovakia would hold on to their Slovak citizenship. As was already mentioned, Czech and Slovak nationals could choose their citizenship for a period of one year after the dissolution of the Czechoslovak Federative Republic. This situation was not without com- plications. It rendered tens of thousands of Roma living in the Czech Republic stateless due to improper documentation, permanent resi- dence in Slovakia (many migrated from Slovakia to Czech lands before 1989), lack of information about the procedure (and the need to apply), a criminal record or other reasons.25 Furthermore, from 1994 it be- came harder for Czech or Slovak citizens to live and work in the other part of the former common republic. In 1999, after years of continu- ous pressure from European institutions and non-governmental orga- nisations, and following a Czech Supreme Court decision of 1997, which ruled that the Czech citizens who chose Slovak citizenship in The Hungarian Status Law The question of ethnic Hungarians living abroad was not used for a na- tionalist agenda in Slovakia alone. It also polarised the political scene in Hungary and deepened the left-right divide. Viktor Orba´n’s FIDESZ played on national sentiments of Hungarians about co-ethnic minori- ties in neighbouring countries and produced a bill on benefits for eth- 290 DAGMAR KUSA´ DAGMAR KUSA´ nic Hungarians living abroad, passed by the Hungarian Parliament in 2001. The first version of the law, which entered into force on 1 January 2002, provided for financial stipends for students of Hungarian eth- nic origin abroad. Members of Hungarian minorities could also ap- ply for Hungarian identity cards (Status ID), with which they can ac- cess further benefits such as discounts in Hungary for public trans- portation and entrance fees for museums and cultural and educational events. The Status ID was handed out on the basis of a recommendation from local cultural organisations representing Hun- garian minorities abroad by the newly established Office for Hungar- ians living abroad with its seat in Budapest. After the refusal of the Slovak and Romanian Governments to allow implementation of the Status Law in their states’ territories and after criticism by the Ve- nice Commission that was asked by the Council of Europe to exam- ine the matter,28 the law was amended in summer 2003. Since then the education stipend is no longer addressed to individuals, but to in- stitutions that offer education in the Hungarian language or on Hun- garian culture. The financial aid is thus accessible not only to ethnic Hungarians but to anybody who wishes to study Hungarian culture and history. The amended version was approved by a majority of the Hungarian Parliament, with the exception of the FIDESZ party, the originator of the law, and the FKGP, the Smallholders’ Party, which had lost seats due to a large corruption scandal involving its president. It was also accepted by the Venice Commission and Romanian Government. Slo- vak representatives, however, remained opposed to it, and the political parties of the ruling coalition (apart from the Party of Hungarian Coa- lition SMK) contemplated passing an ‘anti-law’, which would prevent the implementation of the Status Law in the territory of the Slovak Republic. The Hungarian Status Law The lengthy, emotionally charged squabble between Slovak and Hungarian leaders was finally resolved in December 2003 by the Slovak-Hungarian Agreement on Support for the National Minorities in the Areas of Culture and Education. An article on the Slovak-Hun- garian Agreement in the daily paper SME summarises its key points.29 The treaty identifies two specific cultural foundations that are permitted to distribute financial aid to cultural and educational institu- tions only (some university students qualify as an exception). It estab- lishes a principle of reciprocity, and the distribution of funds will be subject to annual control by a Slovak-Hungarian commission of ex- perts. The crux of the tensions, however, was apparently not in the law it- self. Old historical grievances were voiced in the circles of the law’s critics, accusing the political representation of Hungary of ‘soft irre- THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 291 dentism’, i.e. attempts to recreate the Hungary of the times of the Hun- garian kingdom on a psychological level, and of lurking historic revi- sionism among the Hungarian minorities themselves. Frantisˇek Miklosˇko, one of the most prominent Christian Democrats and the former Speaker of the National Council of the Slovak Republic, expressed views that can be attributed to Slovak representatives in gen- eral: ‘I voiced my opinion even on TV, and my Hungarian Colleagues hold it against me. I would say that the Status Law psychologically cre- ates the concept of a Great Hungary. The Slovak side made mistakes too, when the Law was debated we were sleeping and suddenly we were confronted with a done deed. There is one serious problem how- ever: Hungary is passing a law that is implemented in the territory of the Slovak Republic. We don’t mind if Hungarians have some advan- tages, but it seemed to be a precedent that would not be good, and the Venice Commission has also denounced it.’30 The representatives of the Party of the Hungarian Coalition in Slova- kia, which had seats in the Slovak coalition government, found them- selves between the grindstones as it were of the two national leader- ships. Both sides looked to them for resolution and they drew fire from Slovak nationalists for being ‘irredentist Hungarians’, as well as from Hungarian leaders in Hungary for being too passive. La´szlo´ Nagy, member of the SMK Presidium and chair of the Committee for Hu- man Rights, Nationalities, and Status of Women of the NCSR, laments: ‘One problem of the Law is that it became a part of the internal politi- cal game. We are not affected by it, but Dzurinda and others assume that the voter expects rejection of the Status Law by the Slovak political leaders, which may be an erroneous assumption. It has played a nega- tive role in Slovak-Hungarian relations that got decidedly chilly in 2002.’31 The subject of the Hungarian Status Law is divisive among the Slo- vak-Hungarian population of the Slovak south as well. Although ten- sions between Slovaks and Hungarians in this ethnically mixed region are usually less than in the rest of the country, they have been palpable concerning topics related to the quasi-citizenship of the Status Law and the question of dual citizenship, which emerged shortly afterwards. The question of dual citizenship for ethnic Hungarians The question of dual citizenship for ethnic Hungarians The question of dual citizenship for ethnic Hungarians living abroad emerged as a hot political issue in 2003. The first requests to the Hun- garian leadership came from the Hungarian minority in Vojvodina, la- ter accompanied by similar demands from Hungarians in Romania. The World Federation of Hungarians prepared a petition for a referen- dum about dual citizenship. Its goal was to achieve Hungarian citizen- 292 DAGMAR KUSA´ DAGMAR KUSA´ ship for all applicants who were already holders of a Status ID under the Hungarian Status Law. This initiative was supported by the opposition political parties in Hungary – the Young Democrats (FIDESZ) and the Hungarian Demo- cratic Forum (MDF), which managed to rally enough support to get the required number of signatures on the petition for a referendum that would decide whether to grant Hungarian citizenship to ethnic Hungarians from abroad. The referendum took place on 5 December 2004, but, since over 60 per cent of eligible voters decided to stay at home, the referendum results (in favour of dual citizenship by a small margin) were declared invalid.32 g ) Dual citizenship for ethnic Hungarians was justified mainly on the basis of empathy with ethnic kin. The press again debated attempts to repair the ‘Trianon Injustice’ that truncated the Hungarian nation after the First World War. On the other hand, the initiative was also designed to give practical advantages resulting from Hungarian nationality. This would be relevant especially for Hungarians living outside of the EU borders. The ruling parties MSZP and SZDSZ stood firmly against the referendum, appealing mostly against the costly consequences that im- plementation of the law would have. The situation was further compli- cated by the fact that Romanian and Ukrainian legislations preclude dual citizenship, thus ethnic Hungarians acquiring Hungarian citizen- ship would have to renounce their original citizenship, which could lead to an untenable situation for the Hungarian Government. The Slovak leadership watched the development leading to the refer- endum with a heightened sense of insecurity and antagonism. Accord- ing to diplomatic sources (report of Ministry of Foreign Affairs), Slova- kia was prepared to protest in the EU if the referendum was successful, based on its inconsistency with the Agreement on Slovak-Hungarian Neighbourly Relations from 1995, as well as with the principles of the EU of non-discrimination and democratic governance. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 293 kia), for treason because of his speech in favour of the dual citizenship initiative in the Hungarian Parliament.34 The ethnically charged de- bates about the Status Law and the referendum on dual citizenship have probably also contributed to support for Slovak nationalist and po- pulist platforms, which has grown over the past two years. 9.3.2 Comparison of the Slovak Act on Expatriate Slovaks with the Hungarian Status Law The question of dual citizenship for ethnic Hungarians g The SMK was once again caught in the middle. While the executive vice-president of the SMK, Miklo´s Duray, supported the idea of the re- ferendum, the official SMK position, as represented by its chairman Be´la Buga´r, was to support policies that will help ethnic Hungarians to stay in the country where they were born. He warned that the initiative might antagonise Hungarians living in Hungary and members of Hungarian minorities. ‘We find ourselves unwillingly amidst the Hun- garian internal political struggle and are receiving one slap after an- other. We have not received such slaps even in our native country. We want to remain in our native country, pay taxes there, etc.’33 The heated debate ended up in a Slovakian Court. The Slovak Na- tional Party (SNS) sued the Vice-Chairman of the SMK, Miklo´s Duray (one of the more radical leaders of the Hungarian minority in Slova- 9.3.2 Comparison of the Slovak Act on Expatriate Slovaks with the Hungarian Status Law The Hungarian Status Law is not a unique invention without parallel (as it sometimes appeared to be from the indignant reactions in the Slovak press). In 1997, the Slovak Republic passed Act No. 70/1997 on Expatriate Slovaks. Prior protection of Slovak nationals living abroad was guaranteed by a declaration of support in the Slovak Re- public’s constitution. The House of Expatriate Slovaks, founded by the Ministry of Culture of the Slovak Republic, has also been in existence since 1995, focusing on cultural cooperation and support of expatriate Slovak institutions. According to the Act No. 70/1997, it is sufficient to apply for the status of an expatriate Slovak or to be a direct descen- dant of a Slovak national. If the applicant cannot provide any documen- tation certifying his or her ethnic origin, a letter from an institution re- presenting Slovaks abroad or two witnesses that have the status of ex- patriate Slovaks will do. Application is submitted to the Ministry of Foreign Affairs (MFA) of the Slovak Republic and the application pro- cess takes two months. If it is successful the MFA issues an Expatriate Slovak Certificate. Among the benefits that this status brings is the per- mission to reside ‘for a long time’ in the territory of the Slovak Repub- lic and the opportunity of applying for permanent residence in Slova- kia. It is likewise possible to apply for studies at any Slovak university or to apply for a job without having permanent residence in Slovakia or the employment authorisation required by other foreign nationals.35 The Hungarian Status Law has inspired changes in the Slovak Status Law. In 2005 the National Council of the Slovak Republic passed an Amendment to the Act on Expatriate Slovaks36 (now properly labelled ‘Slovaks living abroad’) that established the Office for Slovaks Living Abroad, which is funded from the state budget and is responsible for carrying out the official state policy towards Slovakia’s external citizens. The Office also issues Certificates of Ethnic Slovaks Living Abroad (Slo- vak Status IDs) that make the process of claiming benefits related to the status easier. Financial support is tied to the areas of culture, edu- cation and research, information, and media. 9.3.2 Comparison of the Slovak Act on Expatriate Slovaks with the Hungarian Status Law Individuals and institu- tions can apply for funding in ‘activities that further the development of Slovak identity, culture, language, or cultural heritage in these coun- tries.’37 294 DAGMAR KUSA´ DAGMAR KUSA´ Hopefully, the amended law will help to provide assistance to Slo- vaks living abroad at the place of their residence. Some representatives of the Slovak institutions abroad complain that the direct result of the Slovak Status Law is a brain drain of young people who leave to study and work in Slovakia rather than financial support for Slovak publica- tions and cultural events in the areas where Slovaks living abroad are concentrated.38 The most remarkable difference between the Slovak and Hungarian Status Law in their current form is the territorial lim- itation of the latter, which restricts the implementation of the law to neighbouring countries with a large proportion of Hungarian minori- ties. The Slovak counterpart has no such stipulation. This is easily ex- plained by the fact that most of the Slovaks living abroad reside in the United States (over 1,200,000 Slovaks). THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 295 nationals among the successful applicants for citizenship was over- whelming. This proportion has gradually declined thereafter and was lowest in 1996 to 1998, which is probably due to the political situation in Slovakia. The numbers of Czech applicants rose again especially after the amendments to the citizenship law in 1999, and have also been growing in the years up to 2005. Since then, the number of Czech applicants has dropped rapidly. One explanation for this drop might be that both the Czech Republic and Slovakia joined the Euro- pean Union in 2004. p 4 For others the trends in the acquisition of citizenship are quite differ- ent. Notable again is the decline in numbers in the years 1995 and 1996, followed by an increase due to the influx of refugees fleeing from the countries of former Yugoslavia. There is a marked increase in the naturalisation of foreigners from outside former Czechoslovakia espe- cially since the year 2000, when more applicants from Asia and the Near East sought to settle in the Slovak Republic. The more detailed ap- plication procedure for citizenship specified in the Act on Citizenship No. 265/2005 Coll. and stricter conditions for naturalisation that were introduced in the Act No. 344/2007 may have contributed to the drops in the number of citizenship acquisitions in 2005 and 2008, although these drops were not more dramatic than in some of the previous years. Figure 9.1 illustrates the diverse trends in the two populations who have acquired Slovak citizenship over the past decade. (The years 1993 and 1994 have been excluded here due to the high number of Czech applications for citizenship resulting from the dissolution of Czecho- slovakia.) We can clearly see the impact of the Czech amendments to the citizenship law in 1999 in the resulting increase of Czech nationals applying for and receiving Slovak citizenship. The rapid increase in ci- tizenship granted to other foreign nationals cannot be readily explained on the basis of legislative changes, but rather on the basis of new mi- gration patterns. Compared to earlier times, many more foreigners looking both for asylum and for citizenship have settled in Slovakia. g y Among those who seek Slovak citizenship are people fleeing from per- secution, violence, civil war or other conditions threatening their lives and security in their home countries. 9.4 Statistical trends (acquisition of Slovak citizenship since 1993) After the fall of communism, Slovakia experienced tumultuous shifts in population, largely in connection with the dissolution of the Czech and Slovak Federative Republic, but undoubtedly also as a result of its strategic position as a bridge between Western and Eastern Europe. There have been shifting migration trends, too. In the early 1990s, the Slovak Republic was losing its citizens to the Czech Republic. This trend ceased after 1994 when Slovakia started gaining population from abroad and increasingly so, from the East. Most migration is temporary and circular with migrants returning after short stays in Slovakia. The number of those who actually ask for Slovak citizenship changes with domestic and international events, circumstances and legislation. The following tables and graphs show the numbers of successful applicants who acquired Slovak citizenship. Table 9.1 Number of persons who acquired citizenship of the Slovak Republic, 1993-2008 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Czech citizens 64,834 20,612 1,379 575 416 399 849 3,903 175 805 942 2,262 2,439 120 155 91 Other citizens 1,550 1,393 910 768 1,519 535 417 623 1,362 3,539 3,100 1,508 539 930 1,235 555 Total 66,384 22,005 2,289 1,343 1,935 934 1,266 4,526 1,537 4,344 4,042 3,770 2,978 1,050 1,390 646 Source: Ministry of the Interior, Slovak Republic As can be seen from Table 9.1, in 1993 and 1994, the vast majority of those who acquired Slovak nationality were Czech nationals. Due to the possibility of choosing citizenship in 1993, the proportion of Czech THE SLOVAK QUESTION AND THE SLOVAK ANSWER More than 54,000 foreigners have applied for asylum in Slovakia since 1992. However, as of the end of 2008, asylum status was granted to only 632 of them. This tendency makes Slovakia a country with one of the lowest rates of refugee recogni- tion in Europe. The year 2004 recorded the highest number of appli- cants, increasingly coming from countries such as India, Russia (particu- larly Chechnya), Pakistan and China. Since 2004, there has been a steady decline in the number of applications for asylum, mirroring larger migra- tion trends within Europe and changes in Slovak asylum policies as well as the 2007 entry of the Slovak Republic into the Schengen area.39 296 DAGMAR KUSA´ Figure 9.1 Czechs and other foreign nationals who acquired citizenship of the Slovak Republic, 1995-2008 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Czech citizens Other citizens Source: Ministry of the Interior, Slovak Republic Source: Ministry of the Interior, Slovak Republic Table 9.2 Refugees and asylum seekers in the Slovak Republic, 1995-2008 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Asylum applications 359 415 645 506 1,320 1,556 8,151 9,734 10,358 11,395 3,549 2,849 2,642 909 Persons granted refugee status 80 72 69 53 26 11 18 20 11 15 25 8 14 22 Refugees granted Slovak citizenship 0 4 14 22 2 0 11 59 42 20 2 5 18 4 Source: Ministry of the Interior, Slovak Republic 9.5 Conclusions The evolution of policies relating to the definition, granting and with- drawal of citizenship in Central Europe was closely tied to turbulent events on the international and regional political scene. More than in the West, the ideals and practices of citizenship were marked by strug- gles for national self-determination, as well as power struggles between the small neighbouring states squeezed in between the warring super- powers during the Cold War period. THE SLOVAK QUESTION AND THE SLOVAK ANSWER 297 Slovak national development had not run its course in the period be- fore 1948. The Slovaks had not achieved a truly independent statehood and were not content to be submerged in a centralised Czechoslovak state after the Second World War. The Slovak Question emerged as a dominant issue at several turning points in history. It impacted on citi- zenship policies within the common state of Czechs and Slovaks in 1968, when the Slovaks received the gift of federation from the invad- ing Soviet troops, and then again after 1989, when it led to the Velvet Divorce between the two nations. Citizenship practices as well as the understanding of what citizen- ship entails and should entail were murky due to frequent changes in policies prior to 1989, due to their ad hoc nature and inconsistencies in the first years of the post-communist regime, as well as because of the tumultuous political scene in Slovakia and new challenges result- ing from Slovak independence in 1993. Slovak citizenship policies were strongly shaped by international in- fluences, especially by pressures from the European Union and bind- ing treaties with the Council of Europe. On the other hand, they also reacted to the heated, historically and emotionally charged political de- bates on the status of Hungarians living abroad and the possibility of their acquiring dual citizenship in Hungary. Central European reality shows us how closely citizenship and identity are intertwined and how easily they are misused for political machinations that further the ego- istic agendas of parties and leaders. Citizenship policies are being gradually simplified and fitted to the new migratory trends that result from membership in the EU. Central European neighbours have not quite yet abandoned nationalist appeals and contentious policies that seek easy enemies to rally supporters. At the same time, they have to quickly figure out how to absorb inflows from parts of the world very different from theirs. Sets regulations for re- acquisition of Czech or Slovak citizenship by emigrants or others who were deprived of Czech or Slovak citizenship prior to 1989 www.centrum.usd.cas.cz (in Czech) 1953 Act No. 34/1953 Coll. on the Acquisition of Czechoslovak Citizenship by Particular Persons www.zbierka.sk (in Slovak) www.zbierka.sk (in Slovak) 2007 Act No. 344/2007 Coll. Amending and Supplementing the Act No. 40/1993 on Citizenship of the Slovak Republic 2005 Act. No. 474/2005 Coll. on Slovaks Living Abroad and on Amendments and Additions to Certain Laws 2005 Act No. 265/2005 on State Citizenship of the Slovak Republic 9.5 Conclusions All these develop- ments occur in the context of an enlarging European Union with the common citizenship of the Union linking the nationality policies of its Member States to each other. Chronological list of citizenship-related legislation in Czechoslovakia/the Slovak Republic Date Document Content Source 1945 President’s Constitutional Decree No. 33/1945 Coll. Concerning Czechoslovak Citizenship of Persons of German and Hungarian Ethnicity Deprives most ethnic Germans and Hungarians of Czechoslovak citizenship http://sudetengermans. freeyellow.com Chronological list of citizenship-related legislation in Czechoslovakia/the Slovak Republic 298 DAGMAR KUSA´ Date Document Content Source 1948 Act No. 245/1948 on the Citizenship of Persons of Hungarian Ethnicity Returns Czechoslovak citizenship to ethnic Hungarians who were Czechoslovak citizens on 1 November 1938 and were not subject to the ‘voluntary exchange of population’ between Slovakia and Hungary in 1946 www.centrum.usd.cas.cz (in Slovak) 1949 Act No. 194/1949 on the Acquisition and Loss of Czechoslovak Citizenship, amended by the Act No. 72/1958 Modifying the Regulations on the Acquisition and Loss of Czechoslovak Citizenship Is adopted as the new citizenship code after the communist coup d’e´tat in February 1948 www.portal.gov.cz (in Czech) 1952 Act No. 59/1952 on Contracting Marriage with a Foreigner Stipulates a requirement to obtain permission from the Ministry of the Interior to marry a person of non Czechoslovak citizenship www.lexdata.cz (in Czech) 1953 Act No. 34/1953 Coll. on the Acquisition of Czechoslovak Citizenship by Particular Persons Returns Czechoslovak citizenship to ethnic Germans who were deprived of it by the Presidential Constitutional Decree No. 33/1945 and are permanent residents of the Czechoslovak Republic www.centrum.usd.cas.cz (in Czech) 1968 Constitutional Act No. 143/ 1968 Coll. on the Czechoslovak Federation Transforms centralised Czechoslovakia into a federation of two entities: the Czech Republic and the Slovak Republic 1968 Act No. 165/1968 Coll. on Acquisition and Loss of Czech and Slovak Citizenship Provides a framework for the introduction of republic-level (Czech and Slovak) citizenship 1968 Act No. 206/1968 Coll. of Slovak National Council on Acquisition and Loss of Citizenship of the Slovak Socialist Republic Introduces republic-level Slovak citizenship 1990 Act No. 88/1990 Coll. Amending Regulations on Acquisition and Loss of Czechoslovak Citizenship Sets regulations for re- acquisition of Czech or Slovak citizenship by emigrants or others who www.zbierka.sk (in Slovak) Source www.centrum.usd.cas.cz (in Slovak) Date Document THE SLOVAK QUESTION AND THE SLOVAK ANSWER www.unhcr.sk (in Slovak) Notes 1 The author and the editors thank Lucia Mokra´ for her research contributions on legal and statistical developments. 1 The author and the editors thank Lucia Mokra´ for her research contributions on legal and statistical developments. 2 Nationality in this context is not a synonym for citizenship, but refers to membership in an ethnic nation. The idea of a Czechoslovak nation did not take root – it was neither popular with Czech and Slovak political representatives nor with the general population and was eventually abandoned in favour of separate Czech and Slovak nationalities. 3 For a detailed history of census taking and practices, see Kertzer & Arel 2002. 4 For a more detailed description of the development in the Czech part of Czechoslovakia see Barsˇova´ in this volume. 5 The Presidential Decree exempted from loss of citizenship those citizens of German and Hungarian ethnicity who had joined in the fight for liberation or were victims of Nazi persecution. The legislation also established a possibility to apply for the re- granting of Czechoslovak citizenship (a policy called ‘Re-Slovakisation in Slovakia’) within six months after the Decree entered into force. 6 For decades, the topic of the transfers of ethnic Hungarians was taboo in Slovak literature. The few texts that were written were from the pen of Hungarian authors in Slovakia – Zolta´n Fa´bry’s The Accused Speaks Out (written in 1946) was published in the 1960s, and in 1982 Ka´lma´n Janics’s Czechoslovak Policy and the Hungarian Minority, 1945-1948 was published in the US in a small edition of a few hundred co- pies. After 1989, the topic was begrudgingly picked up. The most comprehensive analysis and documentation was published by Vadkerty (2002). 7 The voluntary part was secured by leaflets promising return of Czechoslovak citizenship in return for being recruited as agricultural labourers. Leaflets also reiterated that this was the very last chance for Hungarians to reacquire Czecho- slovak citizenship. p 8 The Czechoslovak census of 1947 records 390,000 Hungarians in Slovakia, the 1961 census records 518,782 (data from Kocsis & Kocsis-Hodosi 1998). 9 See also Barsˇova´ in this volume for the same pieces of legislation from a Czech perspective. 10 Children born of mixed marriages, where one parent was a Czechoslovak citizen and the other was the citizen of the Soviet Union, Poland or Hungary, represented an exception. THE SLOVAK QUESTION AND THE SLOVAK ANSWER 299 Date Document Content Source 1991 Constitution of the Slovak Republic Contains the provision that ‘no one shall be deprived of his or her citizenship against his or her will’ and the Bill of Fundamental Rights and Freedoms, including the ‘right to choose one’s nationality’ www.government.gov.sk (in Slovak); www.legislationline.org (excerpts) 1993 Act No. 40/1993 Coll. on Citizenship of the Slovak Republic Enters into force as the new citizenship code in the Slovak Republic after the dissolution of Czechoslovakia www.coe.int; www.minv.sk (in Slovak) 1997 Act No. 70/1997 Coll. On Expatriate Slovaks and Changing and Complementing Some Laws Defines rights and benefits of Slovaks living abroad www.gszs.sk (in Slovak) 2002 Act No. 480/2002 Coll. on Asylum, amended by Act No. 1/2005 Coll. Regulates asylum acquisition procedure www.unhcr.sk (in Slovak) 2005 Act No. 265/2005 on State Citizenship of the Slovak Republic Regulates acquisition and loss of Slovak citizenship; details the requirements and procedure for the acquisition of Slovak citizenship; nullifies Act No. 194/1949 in its amended forms and Act No. 206/1968 in its amended forms 2005 Act. No. 474/2005 Coll. on Slovaks Living Abroad and on Amendments and Additions to Certain Laws Establishes the Office for Slovaks Living Abroad and regulates the competencies of the state administration regarding state support for Slovaks living abroad www.gszs.sk (in Slovak) 2007 Act No. 344/2007 Coll. Amending and Supplementing the Act No. 40/1993 on Citizenship of the Slovak Republic Introduces a longer waiting period for the acquisition of citizenship (eight consecutive years of permanent residence before applying instead of the previous five) and stricter control of l f lf l f www.zbierka.sk (in Slovak) 300 DAGMAR KUSA´ Date Document Content Source 2007 Act No. 502/2007 Coll. on Citizenship of the Slovak Republic Consolidates existing legislation on citizenship (Act. No. 40/1993, Act No. 70/1997 on Slovaks living abroad, Act No. 36/2005 on family, Act No. 265/ 2005 and Act No. 344/ 2007) www.zbierka.sk (in Slovak) THE SLOVAK QUESTION AND THE SLOVAK ANSWER 301 11 Act No. 34/1953 Coll. Concerning the Acquisition of Czechoslovak Citizenship by Particular Persons and Act No. 245/1948 on the Nationality of Hungarian Nationals. 11 Act No. 34/1953 Coll. Concerning the Acquisition of Czechoslovak Citizenship by Particular Persons and Act No. 245/1948 on the Nationality of Hungarian Nationals. 12 Stipulated by art. 6 of the Act on Czechoslovak Citizenship. 13 This provision was defined by Act No. 86/1950 of the Penal Code. The penalty included the loss of citizenship rights, expulsion from the army, and forfeiture of property. Act No. 63/1965 abrogated this penalty and the next codification of the Czechoslovak Penal Law did not include this kind of penalty. 13 This provision was defined by Act No. 86/1950 of the Penal Code. The penalty included the loss of citizenship rights, expulsion from the army, and forfeiture of property. Act No. 63/1965 abrogated this penalty and the next codification of the Czechoslovak Penal Law did not include this kind of penalty. p y 14 Most socialist states had concluded bilateral agreements that excluded dual citizenship among them. 14 Most socialist states had concluded bilateral agreements that excluded dual citizenship among them. 15 See also Barsˇova´ in this volume. 15 See also Barsˇova´ in this volume. 16 A child whose parents were Slovak citizens acquired Slovak citizenship. If one of them was Slovak and the other Czech, and the child was born in the Slovak territory, then the child acquired Slovak citizenship. If the child was born abroad, it acquired the mother’s citizenship. Parents could also agree on the child’s citizenship via a statement until six months after birth. 16 A child whose parents were Slovak citizens acquired Slovak citizenship. If one of them was Slovak and the other Czech, and the child was born in the Slovak territory, then the child acquired Slovak citizenship. If the child was born abroad, it acquired the mother’s citizenship. Parents could also agree on the child’s citizenship via a statement until six months after birth. 17 ‘Pragocentrism’ was a term used by the Slovak leaders to denote the tendency of the Czech representation to rule the country from a strong unitary centre, Prague. Slovak elites have had qualms with Pragocentrism ever since the creation of the first republic in 1918. 18 This claimed heritage is a controversial and complex one. Notes In those cases, citizenship was determined by an agreement of the parents at the time of inscription in the book of births. In cases where an agreement wasn’t reached, the child acquired the citizenship of the parent in the state of birth. If the child was born in the territory of a third state, it acquired citizenship of the state on whose territory the child’s parents had resided before they went abroad. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER Though perhaps only the Slovak National Party would fully claim the legacy of the Slovak Republic of the war period, together with the persona of its president, Jozef Tiso, responsible for sweeping anti-Semitic measures, all parties and most leaders do recognise at least its partial validity as the first form of official Slovak statehood. 19 For a description of the developments in the Czech Republic see Barsˇova´ in this volume. 20 ‘Conditions for Gaining Slovak Citizenship Will Tighten’, The Slovak Information Press Agency, 26 June 2007. 21 Art. 5 of Act No. 40/1993 Coll. on Citizenship of the Slovak Republic. 21 Art. 5 of Act No. 40/1993 Coll. on Citizenship of the Slovak Republic. 22 Art. 7 of Act No. 40/1993 Coll. on Citizenship of the Slovak Republic as amended by Act No. 344/2007 Coll. 23 Art. 7 of Act No. 40/1993 Coll. on Citizenship of the Slovak Republic as amended by Act No. 344/2007 Coll. 23 Art. 7 of Act No. 40/1993 Coll. on Citizenship of the Slovak Republic as amended by Act No. 344/2007 Coll. 24 Art. 8a, sect. 9 of Act No. 40/1993. 25 See European Roma Rights Center report ‘Personal Documents and the Threat to the Exercise of Fundamental Rights Among Roma in Former Yugoslavia’, www.errc.org, retrieved in May 2006. 25 See European Roma Rights Center report ‘Personal Documents and the Threat to the Exercise of Fundamental Rights Among Roma in Former Yugoslavia’, www.errc.org, retrieved in May 2006. 26 See also Barsˇova´ on Czech citizenship in the present volume. 26 See also Barsˇova´ on Czech citizenship in the present volume. 27 Embassy of the Czech Republic in Slovakia, www.mzv.cz. 27 Embassy of the Czech Republic in Slovakia, www.mzv.cz. 28 Among the main objections was the charge of ethnic discrimination concerning access to the benefits of the law. The Status Law is also territorially limited in implementation to certain neighbouring countries where the Hungarian minority is numerous and where the standard of living is not higher than within Hungary itself. Austria was therefore not included among the countries where the Status Law was to be implemented. ˇ 28 Among the main objections was the charge of ethnic discrimination concerning access to the benefits of the law. DAGMAR KUSA´ 32 Only 37.5 per cent of registered voters participated in the referendum. 51.5 per cent of the voters were in favour of dual citizenship, 48.5 per cent against. 50 per cent of eligible voters have to participate for a referendum to be valid in Hungary (or an equivalent of over 25 per cent of all eligible voters must select the same answer on the referendum). Source: ‘Neplatne´ madˇarske´ referendum o dvojitom obcˇianstve [Invalid Hungarian Referendum on Double Citizenship], BBC Slovak.com, 6 December 2004. www.bbc.co.uk. 33 Peter Stahl, ’Madˇari hlasuju´ o dvojitom obcˇianstve’ [Hungarians Vote on Double Citizenship], Hospoda´rske noviny [daily newspaper], 3 December 2004. http://hnonline.sk. 34 The SNS sued Miklo´s Duray many more times afterwards for treason, libel, damaging the name of the Republic, and more. Each charge was dismissed by the courts. SNS leader Jan Slota called the representatives of the Hungarian minority ‘radioactive extremists’ (Slota: ‘Politici z SMK su´ ra´dioaktı´vni extre´misti’ [Politicians from the Party of Hungarian Coalition are Radioactive Extremists], 6 June 2005, www.sns.sk). Shortly before the parliamentary elections of June 2006 SNS popularity climbed to almost 10 per cent in public opinion polls. In the June 2006 elections, the populist left-leaning party SMER-SD came out on top with 29 per cent of the votes. SNS came in third with almost 12 per cent of the votes. The former leader of the government coalition SDKU´ received 18 per cent of the votes (Source: SITA [Slovak Press Agency], 18 June 2006). 35 Arts. 5 and 6 of the Act No. 70/1997 Coll. on Expatriate Slovaks and Changing and Complementing Some Laws. 35 Arts. 5 and 6 of the Act No. 70/1997 Coll. on Expatriate Slovaks and Changing and Complementing Some Laws. 36 Act No. 474/2005 Coll. on Slovaks Living Abroad and on Amendments and Additions to Certain Laws. 36 Act No. 474/2005 Coll. on Slovaks Living Abroad and on Amendments and Additions to Certain Laws. 37 Art. 5 of the Act No. 474/2005 Coll. on Slovaks Living Abroad and on Amendments and Additions to Certain Laws. 38 Ondrej Sˇtefanko, ‘Slovenska´ republika a zahranicˇnı´ (dolnozemskı´) Slova´ci’ [Slovak Republic and Foreign (Hungarian) Slovaks],Cˇ esky´ a slovensky´ svet [Czech and Slovak World], www.svet.czsk.net, accessed in May 2006. 39 ‘Pocˇet zˇiadatel’ov o azyl na Slovensku vy´razne klesol’ [Number of applicants for asylum has gone significantly down in Slovakia], Sme, 1 March 2009. www.sme.sk. THE SLOVAK QUESTION AND THE SLOVAK ANSWER The Status Law is also territorially limited in implementation to certain neighbouring countries where the Hungarian minority is numerous and where the standard of living is not higher than within Hungary itself. Austria was therefore not included among the countries where the Status Law was to be implemented. 29 I. Stupnˇan, ‘Schva´lili dohodu s Madˇarskom’ [Agreement with Hungary Approved], SME, 12 December 2003. 29 I. Stupnˇan, ‘Schva´lili dohodu s Madˇarskom’ [Agreement with Hungary Approved] SME, 12 December 2003. 30 The interview with Frantisˇek Miklosˇko was conducted by the author in Bratislava on 13 June 2003. 31 The interview with La´szlo´ Nagy was conducted by the author in Bratislava on 18 June 2003. 302 DAGMAR KUSA´ Bibliography Drgonec, J. (1997), Za´kladne´ pra´va a slobody podlˇa U´ stavy SR. Bratislava: MANZ. ˇ ˇ ˇ Cˇ erny´, J. & V. Cˇervenka (1963), Sta´tnı´ obcˇanstvı´ Cˇ SSR. Prague: ORBIS. & M. Valasˇek (1996), Cˇ eske´ sta´tnı´ obcˇanstvı´. Prague Cˇ erny´, J. & M. Valasˇek (1996), Cˇ eske´ sta´tnı´ obcˇanstvı´. Prague: LINDE. ˇ Fa´bry, Z. (1994), Obzˇalovany´ prehovorı´: Dokumenty z dejı´n Madˇarov na Slovensku. Bratisla- va: Kalligram. Hrusˇovsky´, P. (2003), Face of a Country; Address of the President of the National Council of the Slovak Republic Pavol Hrusˇovsky´ on the Occasion of the 10th Anniversary of the Slovak Republic, 1st January 2003. Bratislava: Vydavatelˇstvo Michala Vasˇka. Janics, K. (1982), Czechoslovak Policy and the Hungarian Minority, 1945-1948. New York: Columbia University Press. Jurova´, A. (2002), ‘Historicky´ vy´voj ro´mskych osa´d na Slovensku a problematika vlast- nı´ckych vztˇahov k poˆde (‘‘nelega´lne osady’’)’, Cˇ lovek a spolocˇnostˇ 4. www.saske.sk. ˇ ˇ ˇ Kaplan, K. (1993), ‘Prˇedmluva’, in Sˇ. Sˇutaj (ed.), Akcia Juh, Odsun Madˇarov zo Slovenska do Cˇ iech v roku 1949. Prague: Sesˇity U´ stavu pro soudobe´ deˇjiny. Kertzer, D. & D. Arel (eds.) (2002), Census and Identity; The Politics of Race, Ethnicity, and Language in National Censuses. Cambridge: Cambridge University Press. THE SLOVAK QUESTION AND THE SLOVAK ANSWER THE SLOVAK QUESTION AND THE SLOVAK ANSWER 303 Klı´ma, K. (2002), U´ stavnı´ pra´vo. Prague: FINIDR Klı´ma, K. (2002), U´ stavnı´ pra´vo. Prague: FINIDR. 80), Vy´voj u´zemia Slovenska a utva´ranie jeho hranı´c Klimko, J. (1980), Vy´voj u´zemia Slovenska a utva´ranie jeho hranı´c. Bratislava: Obzor. Klimko, J. (1980), Vy´voj u´zemia Slovenska a utva´ran Kocsis, K. & E. Kocsis-Hodosi (1998), Hungarian Minorities in Carpathian Basin. Buda- pest: Simon Publications. Kusa´, D. (2005), ‘Historical Trauma in Ethnic Identity: the Years of Homelessness of the Hungarian Minority in Post-War Slovakia’, in E. Breuning, J. Lewis & G. Pritchard (eds.), Power and the People; A Social History of Central European Politics, 1945-56, 130- 149. Manchester: Manchester University Press. 49. Manchester: Manchester University Press. Lukes, I. (1995), ‘Czechs and Slovaks: The Failure to Find a Decent Past’, Cultural Survi- val Quarterly 2: 19-24. ˇ Matousˇek, S. & S. Zdobinsky´ (1984), Sˇta´tne obcˇianstvo socialisticky´ch krajı´n. Bratislava: Obzor. Nemcova´, K. (1992), Continuity and Change in Slovakia. Bratislava: Slovak Archive of So- cial Data SASD. Ocˇovsky´, Sˇ. (1992), ‘Interpreta´cia sˇtatisticky´ch u´dajov o na´rodnostiach na Slovensku’, in J. Plichtova´ (ed.), Minority v politike, Kultu´rne a jazykove´ pra´va, 82-91. Bratislava: Cˇesko- slovensky´ vy´bor Euro´pskej kultu´rnej nada´cie. y y p j j Paul, E. L. (1998), ‘Czech Teschen – Silesia and the Controversial Czechoslovak Census of 1921’, The Polish Review 43 (2), 161-171. Peroutka, F. (1991), Budova´nı´ sta´tu. Prague: Lidove´ noviny. Plichtova´, J. (ed.) (1992), Minority v politike, Kultu´rne a jazykove´ pra´va. Bratislava: Cˇ esko- slovensky´ vy´bor Euro´pskej kultu´rnej nada´cie. ˇ Posluch, M. & Lˇ. Cibulˇka (2003), Sˇta´tne pra´vo SR. Sˇamorı´n: Heure´ka. Sack, R. D. (1986), Human Territoriality: Its Theory and History. Cambridge: Cambridge University Press. ˇ Sva´k, J. & Lˇ. Cibulˇka. (1999), U´ stavne´ pra´vo SR. Bratislava: APZ SR. ˇ Svetonˇ, J. (1970), Vy´voj obyvatelˇstva na Slovensku. Bratislava: Epocha. Ther, P. & A. Siljak (eds.) (2001), Redrawing Nations; Ethnic Cleansing in East-Central Eur- ope, 1944-1948. Lanham, Maryland: Rowman and Littlefield Publishers, Inc. Tichy´, L., R. Arnold, P. Svoboda, R. Kra´l & J. Zema´nek. (1999), Evropske´ pra´vo. Prague: C. H. Beck. Vadkerty, K. (2002), Madˇarska´ ota´zka v Cˇ eskoslovensku, 1945-1948; Dekre´ty prezidenta Bene- sˇa a ich doˆsledky na deporta´cie a reslovakiza´ciu. Bratislava: Kalligram. ˇ Zdobinsky´, S. & S. Matousˇek. (1985), Sta´tnı´ pra´vo Cˇ SSR. Prague: Panorama. Zeman, Z. A. B. (1994), ‘The Four Austrian Censuses and Their Political Consequences’, in M. Cornwall (ed.), The Last Years of Austria-Hungary, 31-39. Exeter: University of Exeter Press. 10 From civic to ethnic community? The evolution of Slovenian citizenship Felicita Medved This chapter focuses on drzˇavljanstvo of the Republic of Slovenia, i.e. on citizenship or nationality as a legal bond between a person and a so- vereign state. After tracing the history of citizenship in the territory of present day Slovenia, it gives a brief description of the evolution of the Slovenian citizenship legislation, both in terms of the initial determina- tion of its citizenry at the inception of the state in June 1991 and the rules governing the acquisition and loss of citizenship. In fifteen years of statehood the legal regime on citizenship has undergone several changes. The Constitutional Law on citizenship was supplemented and changed five times, with the first supplement already adopted in De- cember 1991 and the latest amendments made in November 2006. These developments have, on the one hand, implied an opening to- wards certain groups, either in response to international standards or for national interests. On the other hand, they have slowly supplanted the civic conception of citizenship that governed the initial determina- tion of Slovenian citizenry in 1991 with a concept of nation as a com- munity of descent. 10.1.1 History of citizenship up to 1991 10.1.1 History of citizenship up to 1991 Citizenship legislation in the territory of Slovenia first evolved within the framework of the Habsburg Empire. The 1811 Austrian Civil Code, which established a link between unified citizenship status and civil rights and other regulations concerning citizenship, operated in the Slovenian lands until the collapse of the monarchy, except in Pre- kmurje, where Hungarian citizenship law was in force after 1879. In close relation to citizenship, the right of domicile in municipalities (do- movinska pravica, Heimatrecht), as a form of local citizenship, which gives rights to unconditional residence and poverty relief, was regu- lated on similar principles in both parts of the Austro-Hungarian mon- archy in the second half of the nineteenth century (Radmelicˇ 1994: 207; Kacˇ & Krisch 1999: 607-613). 306 FELICITA MEDVED FELICITA MEDVED On 1 December 1918 most of the Slovene lands, the Croat lands and Bosnia and Herzegovina joined Serbia and Montenegro to form the Kingdom of Serbs, Croats and Slovenes (SHS), later to be named the Kingdom of Yugoslavia. The Saint-Germain-en-Laye Peace Treaty, which came into force in July 1920, and the Trianon Treaty, which came into force one year later, established that a person who had a right of domicile outside of Austria and Hungary from then on ac- quired the citizenship of one of the successor states. The Saint-Ger- main Treaty postulated, inter alia, that such persons could opt for the citizenship of that successor state in which they once had domicile or the successor state where the majority was of their ‘race’ or spoke their language. However, not everyone who had domicile (pertinenza) in the Slovenian Littoral and part of Carniola that thereafter belonged to Italy automatically acquired Italian citizenship. Those who were not born there or acquired domicile after 24 May 1915 or once had domicile in this territory could opt for Italian nationality. On 25 November 1920 the provincial government of Slovenia issued the executive regulations to the Treaty on the acquisition and loss of Yugoslav citizenship by op- tion and request.1 The option was based on previous domicile or na- tionality, i.e. ethnicity. According to the Rapallo Treaty between the Kingdom of SHS and Italy of 12 November 1920, Yugoslavia provided a one-year right of option for Italian citizenship for ethnic Italians in the from then on Yugoslav territory (Kos 1994). 10.1.1 History of citizenship up to 1991 At the level of Yugoslav internal legislation, the 1928 Citizenship Act2 introduced a unified citizenship, primarily based on ius sanguinis a patre and the principle of a single citizenship. In the early 1930s, pro- visions of Austrian and Hungarian regulations concerning the right to domicile were replaced by membership of a municipality. p y p p y In the Slovenian Littoral, Italian citizenship legislation was in force from 7 June 1923 until mid-September 1947. Italy did not apply any special regulations concerning citizenship in the occupied territory dur- ing the Second World War, whereas the German and Hungarian occu- pying forces granted citizenship to certain groups of people by regula- tion and law respectively, which were subsequently nullified (Radmelicˇ 1994: 222-223). The post-war regulation of Yugoslav citizenship started on 28 August 1945 before the final organisation of the second Yugoslavia was clear.3 The following persons became Yugoslav citizens: 1) all those who, on the date of the enforcement of the Act, were citizens under the then va- lid 1928 Act; 2) persons who had domicile in one of the municipalities in the territory, which according to international treaties became part of Yugoslavia; and 3) persons who belonged to one of the Yugoslav na- tions and resided in its territory without right to domicile, unless they decided to emigrate or to opt for their previous citizenship. An excep- SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 307 tion to this regulation was added in 1948, excluding from citizenry with a retroactive effect those persons of German ethnicity who were abroad and were Yugoslav citizens as of 6 April 1941, having domicile in one of the municipal communities and were, according to art. 35a disloyal ‘to the national and state interests of the nations of Yugoslavia during and before the war.’4 Another Act adopted in 1945 (and nulli- fied in 1962) concerned officers of the former Yugoslav army who did not wish to return to Yugoslavia and members of various military for- mations who served occupying forces and escaped abroad. They lost ci- tizenship ex lege, followed by the sequestration of their property.5 p g y q p p y According to the Paris Treaty with Italy which came into force in September 1947 persons who had permanent residence on 10 June 1940 in the territory that became Yugoslavia lost Italian citizenship. 10.1.1 History of citizenship up to 1991 As obliged by the Treaty, Yugoslavia adopted a special Act on the citizen- ship of these persons in December 1947.6 The Italian-speaking popula- tion had a one-year option for Italian citizenship and Yugoslavia could demand emigration of these persons within one year of the date of the option. In 1947, an option for Yugoslav citizenship was also given to those whose citizenship issue was not solved by the Treaty, i.e. to some 100,000 emigrants from the Littoral to Yugoslavia or other countries before June 1940, who ethnically belonged to one of the Yugoslav na- tions. The Paris Treaty also established the Free Territory of Trieste, a project that lasted seven years until it was divided up between Italy and Yugoslavia by the 1954 London Memorandum of Understanding. The latter did not regulate citizenship directly, but gave guarantees for the unhindered return of persons who had formerly held domicile rights in the territories under Yugoslav or Italian administration, which the Yugoslav law interprets as a qualified option.7 Remaining unsolved questions were settled by the 1975 Osimo agreements, which con- firmed that both states regulate citizenship and provided the possibility of migration for members of minorities (Kos 1994).8 Yugoslav citizenship was unified and excluded other citizenship. Ac- quisition of citizenship remained based on ius sanguinis. A victorious revolutionary communist and national spirit of the immediate post-war period was expressed in legal provisions concerning naturalisation for members of Yugoslav nations and those foreign citizens who actively cooperated in the national liberation struggle, on the one hand, and ex- clusion and deprivation of citizenship for certain ethnic groups or mili- tary formations who really or supposedly worked against Yugoslav in- terests, on the other. The 1964 reform, following the new constitution, abolished loss of citizenship on grounds of absence (as in previous Austrian and Yugoslav legal arrangements), relaxed naturalisation of expatriates (emigrants) and abolished the oath of loyalty upon admis- sion. An odd characteristic of Yugoslav legislation was that in the areas 308 FELICITA MEDVED FELICITA MEDVED which did not pose a threat to the regime, such as the equality of spouses, introduced in 1945, gender equality and the position of min- ors the legislator was already progressive during the period when inter- national standards were only in the making. 10.1.1 History of citizenship up to 1991 Yugoslavia was also party to certain multilateral treaties concerning citizenship such as the Con- vention Relating to the Status of Stateless Persons of 1954, the Interna- tional Convention on the Nationality of Married Women of 1957, the Covenant on Civil and Political Rights of 1966, the International Con- vention on the Elimination of all Forms of Racial Discrimination of 1966, the Convention on the Elimination of All Forms of Discrimina- tion against Women of 1979 and the Convention on the Rights of the Child of 1989.9 9 9 To better understand the problems related to succession in the field of citizenship it is important to emphasise that Yugoslavia was a feder- al state with a so-called mixed system of citizenship. Jurisdiction to adopt citizenship legislation existed at two levels simultaneously, at the level of the federal state and at the level of the constituent federal units, i.e. republics. From the point of view of international public and private law, the primary citizenship was Yugoslav (Kos 1996a). Internally, how- ever, all Yugoslav citizens also had republic-level citizenship.10 Chan- ging the place of residence to another republic or abroad did not affect the republic-level citizenship. Access to another republic-level citizen- ship was relatively easy though. At first it was conditional on three years of residence, but already in 1946 one year of residence sufficed. In the 1960s a simple declaration was enough for a change of repub- lic-level citizenship, reflecting a high level of centralised decision mak- ing.11 The 1974 Constitution, however, brought decentralisation of power. According to the 1976 Citizenship Act of the Socialist Republic of Slovenia,12 citizens of other republics received citizenship of Slove- nia upon application if they had permanent residence in Slovenia. Re- sidents from other republics, however, had the same rights as Slove- nian citizens, except for those reserved only for citizens of the republic, such as voting rights. 10.1.2 Succession and initial determination of citizens of the new state Since the developments of the late 1980s and early 1990s showed that it would not be possible to reach a consensual agreement on some other organisational form for Yugoslavia or on succession, the Republic of Slovenia unilaterally declared its independence on 25 June 1991. Slo- venia had no historical heritage of independent statehood or concept of political membership beyond republic-level citizenship within the for- mer federation to fall back on. In that respect, Slovenia differs from some states which came into being following the break-up of former SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 309 federations, such as the USSR. Notably Estonia and Latvia restored their citizenship laws of half a century earlier, emphasising state conti- nuity broken by ‘lost’ or ‘occupied’ sovereignty (see Jaerve and Kru¯ma in this volume). Some other new states adopted a ‘zero-option’ policy, granting their citizenship to all people actually residing in the republic either at the time of independence or at the moment the new citizen- ship law was passed. This policy was more acceptable in those states where the proportion of the ‘titular’ ethnic population was very high (Medved 1996; Ziemele 2001; Mole 2001). At the international level, citizenship in the context of state succes- sion is addressed by binding and non-binding international instru- ments, such as the 1961 UN Convention on the Reduction of Stateless- ness and the 1978 Vienna Convention on Succession of States in Respect of Treaties, containing large principles but lacking comprehen- sive regulations.13 The primary concern of the international coverage of law on citizenship in cases of succession remains focused on reduction of dual citizenship and the avoidance of statelessness and deals less with the initial determination of citizens, which is not a concern of the established (old) states. Although there has been substantial develop- ment in human rights law, laws concerning the acquisition or loss of citizenship continue to be primarily considered a sovereign prerogative of the state.14 In this context, Slovenia regulated citizenship issues through the Citizenship Act adopted within the scope of the legislation relating to Slovenia’s gaining of independence. The constitution was adopted six months later, on 23 December 1991, and does not regulate citizenship, but leaves it to the law. Since then, the citizenship law has gone through several changes. 10.1.2 Succession and initial determination of citizens of the new state The first supplement had already been adopted in December 1991, followed by further changes in 1992, 1994, 200215 and most recently in 2006.16 Conceptually, the 1991 Act con- tains two main categories. The first category includes provisions of a transitional nature, which refer to the initial collective and automatic determination of the citizens of the new state, complemented by provi- sions governing the option for Slovenian citizenship.17 The second ca- tegory regulates the acquisition and loss of citizenship of a standard (permanent) nature. (p ) As regards the initial overall determination of citizenship, the basic principle is the continuity of previous citizenship upon state succes- sion. Art. 39 stipulates that any person, who held citizenship of Slove- nia and of Yugoslavia according to existing valid regulations, was con- sidered ex lege to be a citizen of Slovenia on the day when the Act came into force. This provision established the continuity with the previous legal order, meaning that all laws and regulations which due to various legal orders were in force in the territory of Slovenia in the past, in- 310 FELICITA MEDVED FELICITA MEDVED cluding international agreements, are applied within the framework of this provision. The period in which a person was born determines which regulations apply for ascertaining citizenship. g pp y g p The primary rule of the initial determination of citizens was comple- mented with the optional acquisition of Slovenian citizenship for citi- zens of other former Yugoslavian republics who had permanent residence in Slovenia on the day of the Plebiscite for the Independence and Autonomy of Slovenia on 23 December 1990, and who actually lived in Slovenia. These two cumulative conditions determined what was con- sidered the genuine link with Slovenia: the permanent residence con- nected with social, economic and certain political rights and the actual living there expressing the criterion of integration, which in practice meant that the person had to reside in Slovenia, not only have a formal residence there (Mesojedec-Pervinsˇek 1999: 656-659; Medved 2005: 467). In dimensions of time ‘actual living’ was established by the Su- preme Court to be at least the period between 23 December 1990 and the date of issuance of a final decision on citizenship. 10.1.2 Succession and initial determination of citizens of the new state As for the con- tent of this notion, which is not legally defined, administrative court practice did not interpret it to mean continuous physical presence but also considered living activities in a certain territory, such as where a person earns a living, resides and fulfils obligations to the state to qua- lify as such (Policˇ 1993). The December 1991 supplement on art. 40 specified a further re- striction, stating that the person’s application is to be turned down if that person has committed a criminal offence directed against the Re- public of Slovenia since Slovenian independence or if the petitioner is considered to form a threat to public order, the security and defence of the state.18 The legal period for the submission of the application was six months and expired on 25 December 1991. More than 174,000 per- sons, or 8.7 per cent of the total population, of which around 30 per cent were born in Slovenia, applied for citizenship on the basis of art. 40 and 171,125 became Slovenian citizens. The registration of the former republican citizenship was not carried out very thoroughly and some persons who firmly believed themselves to be Slovenian citizens were not considered as such and could not prove their former republican citizenship in order to acquire Slovenian citizenship. To address this problem two corrections were made in 1994, concerning the recognition and declaration of Slovenian citizen- ship. Art. 39a stipulates that a person is considered a Slovenian citizen if he or she was registered as a permanent resident on 23 December 1990 and has permanently and actually lived in Slovenia since that date. However, this only applies if the person in question would have acquired the citizenship of Slovenia according to the previous legal or- SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 311 der. On the other hand, according to the new art. 41, persons younger than 23 and older than eighteen years who were born in Slovenia can declare themselves Slovenian citizens if one of their parents was a citi- zen of Slovenia at the time of their birth, but the parents later agreed on adopting the citizenship of another republic. Registered permanent residency posed a problem for those immi- grants who were not registered, but had a long-time factual residence in Slovenia. 10.1.2 Succession and initial determination of citizens of the new state They could not apply for Slovenian citizenship since they were not legally considered residents.19 The problem of permanent re- sidency also arose for those who were registered, but did not apply for or did not acquire Slovenian citizenship. Becoming aliens, they had to apply for residency status irrespective of how long they had been resi- dent. The Aliens Act20 did not contain any special provisions for this group of people.21 It only provided that with respect to the said persons provisions of the Law should start to apply two months after the expiry of the time within which they could apply for Slovenian citizenship or on the date of issuance of a final decision on citizenship. On 26 Febru- ary 1992, when the Aliens Act started to apply to these persons, admin- istrative authorities transferred those who did not apply for residency status from the permanent population register to the foreigners record without any decision or notification addressed to those concerned to in- form them of their new legal position.22 This secret ‘erasure’ became known to the public only much later and the exact numbers of those affected remain unknown. The state admits that 18,305 persons had been deprived of their legal residence. In spite of several appeals by the Ombudsman for human rights,23 non-governmental organisations and some individuals, it was only in 1999 that the Constitutional Court found that the Aliens Act had failed to regulate the transition of the le- gal status of this group of people to the status of foreigners.24 The Constitutional Court decided that the error should be corrected by the legislator within six months which resulted in the Settling of the Status of Citizens of Other SFRY Successor States in the Republic of Slovenia Act.25 However, in 2003, the Constitutional Court also found this regu- lation unconstitutional and ordered the Ministry of the Interior to im- mediately issue decisions to retroactively return the status of perma- nent residence to those who already had had their status changed. Moreover, it asked the legislator to pass a new law within six months, clarifying the criteria for those who, in the period between 1992 and 2003, left Slovenia for shorter or longer periods.26 The polarisation of the political scene as well as public opinion led to various interpreta- tions of the Constitutional Court decision. 10.1.2 Succession and initial determination of citizens of the new state This resulted in a number of initiatives for referenda, supported by right-wing parties, as well as in the preparation of two separate acts. After the adoption of the so- called ‘technical law’ in October 2003, opposition parties succeeded in 312 FELICITA MEDVED FELICITA MEDVED calling a referendum on 4 April 2004. The voter turnout was less than a third of the 1.6 million electorate, and the Act was rejected by almost 95 per cent. This development succeeded in thwarting the adoption of any law to comply with the decisions of the Constitutional Court.27 The current right-centre government is now trying to prepare a special Constitutional Law. In the meantime, in order to settle the position of some of the peo- ple who could not or did not wish to apply for Slovenian citizenship in 1991, or whose applications were rejected and who subsequently be- came aliens or were even ‘erased’, the Citizenship Act was amended in 2002. The new ‘transitional and final provisions’ facilitated acquisition of Slovenian citizenship for citizens of other republics of the former Yugoslavia who were registered as permanent residents on 23 Decem- ber 1990 and who have been living in Slovenia continuously from that day. Duration of residence, personal, family, economic, social and other ties with Slovenia, as well as the consequences a denial of citizenship might have caused, were also taken into consideration. The deadline for a free application expired on 29 November 2003, with 1,676 per- sons being naturalised under this provision. Apart from the two main categories – initial determination of citizen- ship and optional naturalisation – the Citizenship Act contained a third category of transitional provisions that were of compensatory or restitu- tional nature. These provided for reacquisition of citizenship, which was, according to art. 41, made possible for those who were deprived of Yu- goslav citizenship and Slovenian citizenship on the basis of the 1945/ 46 federal law on the deprivation of citizenship or on the grounds of absence.28 They and their children could acquire Slovenian citizenship if they filed an application within one year of the enforcement of the Act. Since most of these people were living abroad, the application peri- od was prolonged to two years in 1992. At the same time, a new art. 10.1.2 Succession and initial determination of citizens of the new state 13a in the section concerning exceptional naturalisation stipulated that, notwithstanding the conditions for regular naturalisation, an adult may obtain Slovenian citizenship if he or she is of Slovenian descent through at least one parent and if his or her citizenship in the Republic of Slovenia has ceased due to release, renunciation or deprivation or because the person had not acquired Slovenian citizenship due to his- torical circumstances. The article also granted the government the right to give a preliminary opinion on the applications. Due to this extensive discretion and, inter alia, the violation of the principle of equality be- fore the law, arts. 41 and 13a were nullified in 1993.29 SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 313 10.2 Basic principles of acquisition and loss of Slovenian citizenship The characteristics of current legislation are the principle of ius sangui- nis and only limited application of ius soli, the prevention of stateless- ness, gender equality in acquisition of citizenship, equality of parents in deciding the citizenship of their minor children, equality of children born in wedlock with children born out of wedlock, will of the person concerned in the process of acquisition and loss of citizenship and pro- tection of personal data. Further principles are the relative tolerance of multiple citizenship and the validity of Slovenian citizenship in these cases, meaning that a dual or multiple citizen is treated as a citizen of the Republic of Slovenia, while in the territory of Slovenia, unless otherwise stated by an international agreement. Foreign citizens may acquire Slovenian citizenship by naturalisation on the basis of resi- dence or of family ties or because of special interests of the state.30 Fa- cilitated naturalisation is provided for immigrant children born and raised in Slovenia and for Slovene emigrants and their descendants. Discretionary power is provided for in all cases of naturalisation, how- ever, it may only be exercised if the reasons, including the proof there- of, are recorded in the written decision.31 10.2.1 Acquisition of citizenship Slovenian citizenship is acquired by descent, by birth in the territory of Slovenia, by naturalisation (through application) and in compliance with international agreement (which is applicable only in cases where borders have changed). Under the ius sanguinis principle there are two modes of acquiring Slovenian citizenship: ex lege and by registration. The registration has a constitutive character and retroactive effect (ex tunc). At birth, a natural person obtains Slovenian citizenship ex lege: i) when both parents are Slovenian citizens, ii) when the child is born in Slovenia and at least one parent is a Slovenian citizen (in the latter case the acquisition of the citizenship ex lege is combined with the territorial principle)32 and iii) when the child is born abroad and one of the par- ents is a Slovenian citizen while the other parent is unknown, of non- determined citizenship or stateless. Children born abroad with one parent of Slovenian citizenship at the time of the child’s birth can acquire Slovenian citizenship by registra- tion. Registration can be initiated within eighteen years after birth by the Slovenian parent without the consent of the other parent or if a minor is a ward of his or her guardian, who must be a Slovenian citi- zen.33 As of 1994, children over fourteen years of age have to give their 314 FELICITA MEDVED FELICITA MEDVED consent. Those over the age of eighteen can acquire Slovenian citizen- ship based on a personal declaration for registration. The age limit for this procedure was extended from 23 to 36 years of age in 2002. The November 2006 Act amending the Citizenship of the Republic of Slo- venia Act, further clarifies the procedure and adds the condition that those who register their Slovenian citizenship should not previously have lost it due to release, renunciation or deprivation after they reached majority. Acquisition of citizenship by adoption follows the principle of citizen- ship by descent when at least one of the adoptive parents is a Slovenian citizen. An adoptee foreigner older than fourteen years has to give his or her consent. Ius soli applies for a foundling or a newborn infant in the territory of Slovenia with no known parentage or if the parents are of unknown citizenship or stateless. If it is discovered prior to the child reaching the age of eighteen that the parents are foreign citizens, then Slovenian citizenship shall cease at the parents’ request. 10.2.1 Acquisition of citizenship This imprecisely defined status is clarified in the 2006 amendments as describing those people who have either a temporary or permanent residence permit, which in practice prolongs the waiting period for naturalisation. In addition, the applicant should not have had his or her residence in Slovenia curtailed. Further requirements are that the person does not constitute a threat to public order or the security and defence of Slovenia, has fulfilled his or her tax obligations and has a guaranteed permanent source of in- come.35 In fact, the latest amendments state that the applicant is re- quired to have such means of subsistence as will guarantee material and social security to the applicant and persons he or she has an obli- gation to support i.e. a basic minimum income for each person. More- over, the law demands a clean criminal record, meaning, inter alia, that the applicant should not have served a prison sentence of more than three months or have been sentenced to a conditional prison term of more than one year.36 The applicant will also be obliged to take an oath of respect for the free democratic constitutional order of Slovenia, which replaces the requirement to sign a declaration of consent to the legal order of the Republic of Slovenia introduced in 2002. Finally, there is the required knowledge of the Slovene language, which has changed substantially. In the early 1990s, it sufficed that the person could communicate. In 1994, an obligatory examination was instituted. Many people failed the examination even though they had been edu- cated in Slovenia. Currently, an obligatory examination at an elemen- tary level is required unless the applicant went to school or acquired education at a higher or at a university level in Slovenia or is over 60 years of age and has actually lived in the country for fifteen years or, as was added in 2006, has acquired an elementary or secondary educa- tion in the Slovenian language in a neighbouring country where there are autochthonous Slovene minorities. Exceptions are made for illiter- ates and for health reasons. Facilitated naturalisation reflects specific interests of the state and more recently, the will of the state to better comply with the standards of the 1997 European Convention on Nationality. 10.2.1 Acquisition of citizenship Those who acquire Slovenian citizenship under above described principles are regarded as citizens of the Republic of Slovenia by birth. Foreign citizens may acquire Slovenian citizenship by regular, facili- tated and exceptional naturalisation. Those who acquire Slovenian citizenship under above described principles are regarded as citizens of the Republic of Slovenia by birth. Foreign citizens may acquire Slovenian citizenship by regular facili- g y Foreign citizens may acquire Slovenian citizenship by regular, facili- tated and exceptional naturalisation. The conditions that must be fulfilled for regular naturalisation are very strict. The applicant has to submit a release from current citizen- ship or a proof that such a release will be granted if he or she acquires Slovenian citizenship unless the applicant is stateless or can submit evidence that his or her citizenship is cancelled by naturalisation by the law of his or her state of origin or that such a release was not decided upon by this state in a reasonable period of two years.34 In cases where applicants cannot present proof of expatriation, e.g. be- cause the voluntary acquisition of a foreign citizenship is considered an act of disloyalty, the declaration by an applicant that he or she will renounce his or her current citizenship if granted Slovenian citizen- ship suffices. However, the applicant usually has to present proof of ex- patriation before he or she can be naturalised. This may lead to tem- porary statelessness which can become permanent if after release from the previous citizenship an applicant is no longer eligible for naturali- sation, e.g. due to loss of means of subsistence or a prison penalty. Since the authorities have to check if other conditions are still fulfilled after the prescribed period within which an applicant must present proof of release, the 2006 amendments specify that only those condi- tions that can be verified administratively will suffice. The condition of a release from current citizenship is waived for citizens of those EU Member States where reciprocity exists. SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 315 A second condition is that the applicant must have lived in Slovenia for ten years, of which the five years prior to the application must be without interruption, and, as added in 2002, the person should have the status of foreigner. 10.2.1 Acquisition of citizenship This mode of naturali- sation affects particular groups of persons: Slovenian emigrants and their descendants, foreigners married to Slovenian citizens, minors and since 2002 persons with refugee status, stateless persons and those born in Slovenia and living there since their birth. To these groups of persons, the 2006 amendments added foreigners who have concluded their university education in Slovenia. Exemptions from certain require- ments are provided for these groups of applicants, in particular regard- 316 FELICITA MEDVED FELICITA MEDVED ing the release from current citizenship and the required duration of re- sidency with a foreign status in Slovenia. For example, an individual of Slovenian descent or a foreign spouse of a Slovenian citizen can become a Slovenian citizen after one year of uninterrupted residence. However, the 2006 amendments show that these two groups of persons are not treated equally. While the generational criterion (up to the third genera- tion for direct descent) for descendants of Slovenian emigrants was ex- tended up to the fourth generation, the period of marriage before a for- eign spouse of a Slovenian citizen is eligible to apply for naturalisation was prolonged from two to three years in order to dissuade marriages of convenience. For those who have lost Slovenian citizenship in accor- dance with the present Act or prior Acts valid in the territory of Slove- nia, the residence requirement is limited to six months. Acknowledged refugees and stateless persons may be naturalised after five years of ac- tual and uninterrupted residence in the country. For persons born in Slovenia who have lived there since birth (mainly citizens of successor states of the SFRY), personal, family, economic, social and other con- nections with Slovenia as well as the consequences a denial of naturali- sation may cause are taken into consideration. Foreigners who have concluded their university education in Slovenia will be eligible to ap- ply for naturalisation after seven years of residence. For all these cases, release from current citizenship is not necessarily required. A minor acquires Slovenian citizenship upon the request of one or both naturalised parents if the child has lived with that parent in Slove- nia for at least one year prior to the application. If the child is born in Slovenia, Slovenian citizenship can be acquired before the age of one year. 10.2.1 Acquisition of citizenship Citizenship may also be granted to a child having no parents or whose parents have lost their parental rights or functional capacity and who has lived in Slovenia since birth on the grounds of a petition by the guardian who is a Slovenian citizen and who lives with the child. The Ministry for Family and Social Affairs has to confirm that the ac- quisition of citizenship is for the benefit of the child. In all of the above cases the consent of the child above the age of fourteen is also neces- sary.37 In the case of adoption, where there is no such relation between the adoptive parent and adoptee as between parents and children, a child not older than eight years, living permanently in Slovenia, can ac- quire citizenship upon the request of the adoptive parents. In cases of exceptional naturalisation, the interests of the state for ex- ample in the field of culture, economy, science, sport, and human rights are decisive and must be confirmed by the government. A per- son qualifying for exceptional naturalisation may remain a double or multiple citizen, but has to actually live in Slovenia without interrup- tion for at least one year with a foreigner’s status before applying for ci- tizenship. The latter condition does not have to be fulfilled when his or SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 317 her naturalisation benefits the state for national reasons, i.e. when the person is of Slovene ethnicity. The 2006 amendments clarify the con- ditions for exceptional naturalisation of persons of Slovene descent, in- cluding persons belonging to Slovene minorities in neighbouring countries. Neither residence in Slovenia nor other conditions such as material and social security or fulfilled tax obligations in a foreign country are required in these cases. 10.2.2 Loss of citizenship A Slovenian citizen cannot lose his or her citizenship by mere opera- tion of law. There are five ways to lose one’s Slovenian citizenship: re- lease, renunciation, deprivation and loss of citizenship through interna- tional agreements, with the latter only being applied to cases involving changes to state borders. Citizenship can also be lost by the nullifica- tion of naturalisation. Release is the regular way of losing citizenship by application. It is the right of any Slovenian citizen who fulfils the stipulated conditions, such as actual residence abroad and proof that he or she will be granted a foreign citizenship. Release has to be approved by a public authority, but discretionary power is limited to specific reasons such as national security and national interests, reciprocity or other reasons de- rived from relations with a foreign country. Renunciation is a qualified option for dual citizens, meaning that such a person has the right to renounce Slovenian citizenship. It is ac- corded to individuals up to 25 years of age, born in a foreign country, residing there and holding a foreign citizenship. Other conditions have not been foreseen. The Ministry of the Interior has no discretionary power and may issue a decree stating that Slovenian citizenship of such a person ceased on the day that such a statement of renunciation was filed. Minors, up to the age of eighteen years, enjoy a substantially higher degree of protection regarding the release from and renuncia- tion of citizenship, compared to the acquisition of citizenship. The con- sent of both parents is required, regardless of their citizenship. In a case of dispute, the Ministry for Family and Social Affairs decides in the best interests of the child. Furthermore, children older than four- teen years must give their personal consent. Deprivation of citizenship is the only type of citizenship loss that the state may initiate. A Slovenian citizen, actually residing in a foreign country and in possession of a foreign citizenship may be deprived of citizenship if it is ascertained that this person’s activities are contrary to the international and other interests of the Republic of Slovenia. Proof of the existing conditions must be given in the decree on the depriva- 318 FELICITA MEDVED FELICITA MEDVED tion of citizenship, which may be issued by exception in the absence of the party concerned. 10.2.2 Loss of citizenship Cancellation of a decree on naturalisation may occur if it is discovered that naturalisation was granted based on false declarations or deliberate concealment of essential facts or circumstances on the side of the indi- vidual in question. It may also be nullified if the person acquired citi- zenship on the grounds of a foreign state’s guarantee that the person’s foreign citizenship will cease to exist if the person acquires the Slove- nian citizenship and evidence of the loss of the previous citizenship has not been submitted within the prescribed period. Nullification is not possible if such a person would become stateless. 10.2.3 Dual and multiple citizenship In general, Slovenian legislation is relatively tolerant of plural citizen- ship on both entry and exit sides. The ius sanguinis and gender equal- ity principles contribute to dual citizenship for citizens by birth, both in Slovenia and abroad, since ius sanguinis transmission of Slovenian citizenship is not limited to the first or second generation or by any other requirements. Multiple citizenship is even possible for adopted persons. Acquisition of the citizenship of another country does not mean that the Slovenian citizenship is automatically forfeit, neither is release from current citizenship required for certain groups that qualify for facilitated and exceptional naturalisation, nor in cases of regular naturalisation where expatriation would have harsh consequences. The number of dual and multiple citizens is unknown. In June 1991, there were 15,000 registered dual citizens residing abroad (Kon- cˇina 1992). After independence the number of dual and multiple citi- zens substantially increased, both in the country and abroad. In 2005, it was estimated that around 60,000 Slovenes permanently residing abroad had Slovenian citizenship. Slovenia grants substantial political rights to citizens abroad, including franchise in local, parliamentary and presidential elections. The number of dual citizens in Slovenia is much larger and is mainly the consequence of specific historical cir- cumstances in which the new state was created and was partially de- pendent on the citizenship legislation of other countries, notably Italy. The transitional provisions regulating the option for Slovenian citi- zenship did not touch upon dual citizenship and it is estimated that al- most all people from other republics of the former Yugoslavia are dual citizens. In 1991, it was also objectively impossible to make this type of naturalisation conditional on a release from current citizenship. The outcome of the Yugoslav crisis was unknown and the possibility of a bi- lateral or multilateral regulation of citizenship did not bear fruit. The break-up of Yugoslavia did not lead to de iure statelessness, since all SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 319 successor states applied the principle of continuity of former republic- level citizenship (Kos 1996b; Mesojedec-Pervinsˇek 1999: 655). Never- theless, the interest in Slovenian citizenship was much higher than ex- pected in 1991 when the authorities estimated that approximately 80,000 persons would apply for Slovenian citizenship (Mesojedec-Per- vinsˇek 1997: 32-34). The reasons for such a response are various and have so far not been well researched. 10.2.3 Dual and multiple citizenship Public discussions emphasise uti- litarian motives, in particular the possibility to purchase socially owned housing which was only open to Slovenian citizens. Suspicions that holders of dual citizenship may be disloyal and that they pose a poten- tial threat to state security led to a change in the political and public mood and to legislative attacks on this status, mainly supported by the Slovene National Party and the Peoples’ Party in the period from 1993 to 1996. While the liberal democratic government also proposed the abolishment of dual citizenship in 1993, some other proposals openly called for the retroactive nullification of all decrees under art. 40. In 1995, there was even an official initiative for a referendum on the is- sue, which was only stopped by the Constitutional Court38 (Cerar 1995; Dujic´ 1996; Medved 2005: 470-474). 10.2.4 Jurisdiction and procedures Up to the end of 2006, the Ministry of the Interior had jurisdiction over naturalisation and loss of citizenship. Following the concept of ter- ritorial de-concentration of state administration, the 2006 amend- ments to the Citizenship Act transfer this competence from the Minis- try of the Interior to local administrative units. Only cases of excep- tional naturalisation remain under the jurisdiction of the Ministry of the Interior. Moreover, the Ministry still has a ‘controlling’ role in the obligatory revision procedure for decisions on naturalisation and loss of citizenship as well as documents related to the release from prior ci- tizenship. In this procedure the Ministry can either confirm decisions made at the local level or make a new decision. A period of nine months is envisaged for the transfer of decision making. Legislative competence lies with the government, which specifies the requirements for regular naturalisation regarding, inter alia, residence, income and threat to public order, security and defence of the state, and defines the criteria for the naturalisation on the grounds of na- tional interest and for the refusal of release from citizenship. A deci- sion by the Ministry may be appealed and is open to judicial review (see Policˇ 1997a, 1997b). Fees are relatively low (30,000-35,000 SIT, i.e. 125-150 euros). Local administrative units have the authority to establish and register citizenship.39 Record keeping of citizenship is done in compliance with 320 FELICITA MEDVED FELICITA MEDVED the Births, Deaths and Marriages Registry Act at the register of births.40 The Ministry of the Interior keeps the central citizenship reg- ister. Since this register does not constitute a separate database but is part of the permanent population register, the 2006 amendments pro- vide that the Ministry of the Interior and local administrative units shall keep a register of persons who acquired citizenship by naturalisa- tion and those who lost Slovenian citizenship. This register shall be computerised and connected to the registers of foreigners and of births, deaths and marriages. Personal data from this register may be used by the employees of internal affairs when performing their duties defined by law and can be forwarded to other users only if these are authorised by law or upon the consent or request of the individual to whom they relate. 10.2.4 Jurisdiction and procedures The Ministry of the Interior can forward personal data of an individual to other states under the condition of reciprocity only if such data are used for clearly defined purposes (such as settling citizenship issues or realisation of penal proceedings) and that in that state personal data protection applies also to foreigners. Slovenian citi- zenship can be proven by attestation or any other public document of citizenship issued by an agency with the authority for administering the official register in which the citizenship of the person is entered, or by the administrative unit where the person permanently resides. Ac- cording to the latest change, any administrative unit can issue attesta- tions of citizenship. SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 321 fruit before the next parliamentary elections. Current political debates have thus shifted the focus to societal integration of naturalised ‘for- eigners’ and political participation and representation for these ‘new minorities’. This debate is further enhanced by the realities of more re- cent immigration and reforms necessary as a result of joining the Eur- opean Union in May 2004. p y 4 In the pre-accession period the eurocompatibility was influenced more by international trends, such as the 1997 European Convention on Nationality of which Slovenia is not a party, than by indirect pres- sure from the EU. This applies in particular to the amendments in 2002, refining and relaxing access to citizenship for recognised refu- gees, stateless persons and second- and third-generation immigrants. On the other hand, conditions for naturalisation have been maintained and tightened. Since 2002, applicants must have the status of foreign- er, as explained above. This status is an eligibility criterion that may be waived only in some exceptional cases of naturalisation. Further changes concern the question of loyalty. In 2002, the declaration of agreement with the legal order of Slovenia was introduced, which in 2006 was supplanted by an oath of loyalty. In July 2005, the govern- ment also further specified national interest as a reason for exceptional cases of naturalisation, in other words, criteria for cultural i.e. ethnic- affinity based naturalisation of Slovenes living abroad. The Govern- mental Office for Slovenes around the world may offer an opinion on the applicant, which has led to criticism and protests from members of the Slovene diaspora. In line with this protest, the political discussion focused on legisla- tion regulating relations between Slovenia and Slovenians abroad, in particular concerning the legal position of autochthonous minorities living in neighbouring countries and emigrants and their descendants with or without Slovenian citizenship. In April 2006, the National As- sembly passed the Republic of Slovenia and Slovenians Abroad Act.41 The Act is based on art. 10.3 Current political debates Until recently, the citizenship agenda was dominated by the heritage of the dissolution of Yugoslavia. Subsequently, the citizenship legislation went through a series of adjustments related to the admission of citi- zens of other successor states of the former Yugoslavia. As has already been pointed out above, the issue of plural citizenship prevailed in the mid-1990s. After unsuccessful legislative attempts to abolish dual citi- zenship there is an acceptance of plural citizenship for this group of people as a reflection of the historical experience. Since the late 1990s, the political scene has been dominated by the issue of the ‘erased’. Un- til now, there have only been partial solutions to resolve the problems of this group of people, either by regulating their status as foreigners or enabling them to naturalise. Moreover, despite the more recent criti- cism by the Commissioner for Human Rights at the Council of Europe and by the Commission against Racism and Intolerance (ECRI) of the Council of Europe, it is not expected that further attempts, including the current government’s attempt to introduce a special Constitutional Law, which requires a two-thirds parliamentary majority, will bear any SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 5 of the Constitution, the 1996 Resolution on the Position of Autochthonous Slovene Minorities in Neighbouring Countries and the Related Tasks of State and Other Institutions in the Republic of Slovenia42 and the 2002 Resolution on Relations with Slo- venes Abroad.43 The objectives of this legislation are to arrange rela- tions of the ‘homeland’ with Slovene diasporas in order to strengthen national identity and consciousness and promote mutual ties in the fields of culture, care for the Slovene language, education and science, sports, economy and regional cooperation. It introduces a new status of a Slovene without Slovenian citizenship, regulates its acquisition and loss and provides certain advantages to its beneficiaries. Acquisition of this status would primarily depend on descent, activity in Slovenian organi- sations abroad and active ties with the ‘homeland’. The Governmental 322 FELICITA MEDVED FELICITA MEDVED Office for Slovenes around the world will be responsible for issuing this status. When in Slovenia, the holders of this status will enjoy pre- ferential enrolment at institutions of higher education, equal access to research projects and public cultural goods, such as libraries or ar- chives, as well as equal property rights. They will also enjoy priority in employment over other third-country nationals. Until now, no one has applied for, let alone acquired, this ‘quasi-citizenship’ status. The Act also supports the return of Slovenian expatriates and their children and also provides for repatriation, meaning immigration of Slovenes, organised and financed by Slovenia in cases when there is, according to the assessment of the Ministry for Foreign Affairs, a severe crisis, political or otherwise, in the states where they reside, and if their repa- triation contributes to the development of the ‘homeland’. The Council for Slovenians Abroad and the Council for Slovenians in Neighbouring Countries will function as permanent advisory bodies. The councils are headed by the Prime Minister, who appoints their members, composed of representatives of state bodies, institutions, political organisations and civil society organisations from Slovenia and abroad.44 There are certain parallels between the Slovenians Abroad Act and the famous and controversial 2001 Hungarian Status Law, the 1997 Law on Expatriate Slovaks and the 1999-2001 failed Polish move to in- stall a similar law (see Liebich, Kova´cs & To´th, Kusa´ and Go´rny & Pud- zianowska in this volume). SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? However, the Slovenian Government claims that the Slovenian law cannot be equated with the Hungarian Status Law since it does not interfere with the competences of other EU Mem- ber States or the free movement of workers, nor does it establish Iden- tity Cards valid in the territory of any other EU Member State. y y y The most recent political discussion concerned the future develop- ment of citizenship legislation. A working group was set up to analyse current citizenship legislation and its implementation and prepare changes to the Citizenship Act. Due to the complexity of current citi- zenship legislation there was a tentative suggestion for an overall revi- sion, but this was hardly to be expected. In fact, when in July 2006 the Act amending the Citizenship of the Republic of Slovenia Act was pro- posed, the government claimed that it would not change the aims and principles of the existing legislation, but it focused instead on require- ments for naturalisation and some practical aspects, such as clarifica- tions of imprecisely defined provisions, and on harmonisation with other legislation, specifically concerning immigration and record keep- ing. Nevertheless, the Act was not adopted in a short parliamentary pro- cedure as initially proposed by the centre-right government but caused extensive discussion, particularly regarding the 89-word long oath of loyalty, suggested by the Ministry of Justice. While the Liberal-Demo- SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 323 crats proposed that the text of the oath be simplified, the Social Demo- crats, another opposition party, argued that the text confuses the con- cepts of ‘state’ and ‘homeland’ as the latter is not a legal concept and that taking an oath of loyalty ‘to my new homeland’ shames a civilised and modern society and is reminiscent of nineteenth century patrio- tism. Moreover, the Liberal Democrats opposed the transfer of the ju- risdiction over naturalisation and loss of citizenship from the Ministry of the Interior to local administrative units, which in the government’s view is the main novelty of the amended Act. They argued that such an arrangement could lead to arbitrary decisions in spite of the revision procedure by the Ministry. While none of the political parties opposed relaxed naturalisation for ethnic Slovenes, the Liberal Democrats criti- cised that conditions, such as residence in Slovenia or material and so- cial security, are waived in these cases. SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? The Act amending the Citizenship of the Republic of Slovenia Act was passed by the National Assembly on 24 November 2006. Since the National Council did not veto the adopted Act in the prescribed se- ven-day period, the Act came into force at the end of the year 2006. 10.4 Statistics At the end of 2008, there were 208,484 naturalised citizens or ap- proximately one-tenth of the total population of Slovenia. Roughly 81 per cent of all naturalised persons acquired citizenship according to the optional provisions in the immediate post-independence period, with the corrective provision of 2002 contributing to less than 1 per cent. The great majority (98.7 per cent) of naturalised citizens origi- nated in other successor states of the Social Federal Republic of Yugo- slavia, of these 46 per cent from Bosnia and Herzegovina, meanwhile only 1.3 per cent from other countries. When considering naturalisa- tion by standard provisions the share of the latter rises to 11 per cent. These were mainly citizens from Western European countries and overseas OECD-states. For example, 2,159 were citizens of EU States or Switzerland, among them 1,024 of Italy and 510 of Germany. These are followed by previous citizens of the Russian Federation (281) and of Ukraine (237). A quarter of all those naturalised according to standard provisions acquired Slovenian citizenship by fulfilling all conditions. Almost 58 per cent of those were naturalised according to facilitated procedure. Exceptional naturalisations present a rather large 17 per cent share. Over 80 per cent of facilitated naturalisations refer to extension of ci- tizenship to family members, i.e. to spouses and minor children. Eth- nic-affinity based naturalisations are also significant. 324 FELICITA MEDVED Table 10.1 Admission to Slovenian citizenship based on supplementary and corrective initial determination rules (art. 40, 1991, art. 19, 2002) and on standard provisions for naturalisation by country of origin, 25 June 1991-31 December 2008 Country of origin Art. 40, 1991 Art. 4,960 12 263 432 365 307 888 423 311 249 352 227 350 304 217 103 90 28 39 Source: Ministry of the Interior The development of release from citizenship is relatively modest, show- ing an increase until 1996 and since then a steady decrease to a low figure of 28 and 39 in 2007 and 2008, respectively; 103 persons alto- gether were not released from Slovenian citizenship until the end of 2005. Table 10.5 Exceptional naturalisations in Slovenia per year, 1991-2008 Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Granted 6,227 47 45 159 218 391 150 628 571 444 274 245 716 446 344 244 464 210 631 Not granted 1,185 3 19 68 36 88 66 14 88 35 27 32 130 57 16 506 n/a n/a n/a Note: n/a = not available Source: Ministry of the Interior Table 10.5 Exceptional naturalisations in Slovenia per year, 1991-2008 Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Granted 6,227 47 45 159 218 391 150 628 571 444 274 245 716 446 344 244 464 210 631 Not granted 1,185 3 19 68 36 88 66 14 88 35 27 32 130 57 16 506 n/a n/a n/a Note: n/a = not available Source: Ministry of the Interior Since the peak in 2002, there has been a decreasing trend of excep- tional naturalisations per year. On the one hand, this is the effect of the 2002 supplements to the Citizenship Act whereby second- and third-generation immigrants can be granted citizenship according to a facilitated procedure. On the other hand, a strikingly high number of refusals for naturalisation in 2005, mainly ethnic Slovenes living abroad, can be attributed to the government’s redefinition of national interest in citizenship acquisition. This is expected to drop again in the coming years due to the changes in 2006 concerning the naturalisa- tion of ethnic Slovenes. Nevertheless, a substantial rise in citizenship acquisition was recorded in the election year 2008. 10.4 Statistics 19, 2002 Standard provisions Total % Total % Total % Bosnia & Herzegovina 78,918 46.12 825 46.93 16,714 46.96 Croatia 58,531 34.20 319 18.15 7,194 20.21 Serbia & Montenegro 28,537 16.68 540 30.72 6,041 16.97 Macedonia 5,150 3.00 51 2.90 1,617 4.54 Other countries 23 1.30 4,029 11.32 Total 171,136 100 1,758 100 35,595 100 Source: Ministry of the Interior45 Table 10.2 Regular and facilitated naturalisations by groups of persons in Slovenia, 1991-2008 Regular naturali- sation Facilitated naturalisation Minors Spouses Slovenes by descent Born in Slovenia* Recognised refugees* Reacqui- sition* Stateless person* Educated in Slovenia** Total 9,547 9,691 8,108 1,789 68 25 21 5 8 21,870 From 2002 From 2006 Source: Ministry of the Interior Table 10.3 Regular and facilitated naturalisations in Slovenia per year, 1991-2008 Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Granted 30,931 303 320 2,539 1,233 1,781 926 544 2,747 1,893 2,102 1,101 2,092 2,860 2,989 2,440 2,660 1,287 1,114 *Including art. 19, 2002 Source: Ministry of the Interior The numbers naturalised annually reflect changes in legislation with a low of 303 persons in 1991 and a high of 2,989 in 2004. The increas- ing trend after 2001 can mainly be attributed to the corrective measure added in 2002 to incorporate those whose status was not adequately regulated in 1991. The decreasing number in 2005 indicates that this problem is diminishing. Table 10.4 Reasons for granting exceptional naturalisations in Slovenia National interest Born in Slovenia Culture Health sector Sport Education/ science Economy IT sector Interest of the state Religion Defence Tourism/ traffic 4,884 605 180 157 108 107 99 63 6 13 2 3 Source: Ministry of the Interior Table 10.4 Reasons for granting exceptional naturalisations in Slovenia SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 325 Ethnic affinity is the dominant ground of national interest for excep- tional naturalisations, with birth in Slovenia representing the second largest interest. All other state interests play only a secondary role, comprising a modest 12 per cent share. 10.5 Conclusions As a new state, Slovenia went through a process of initial determina- tion of its citizenry. The question of the initial ‘body’ of citizens and 326 FELICITA MEDVED FELICITA MEDVED simultaneously of legal integration of the majority of ‘non-ethnic’ Slovenes was resolved early in the process of independence and inter- national recognition, and without great controversy. Several factors con- tributed to this development. Firstly, although the establishment of Slo- venia as a nation-state can be considered as a product of the so-called eastern type of ethno-cultural nationalism, asserting the right to self- determination and self-governance of the Slovenian ‘nation’, the initial policy of citizenship rather supported democratic statehood over ‘na- tionhood’. Citizenship was defined in territorial terms, close to ‘zero- option’ policies, in order to ensure an even jurisdiction over the terri- tory and people within the boundaries of the new state. By adopting such an approach Slovenia could exercise ‘effective governance’, which supported its claim for international recognition, in combination with other elements of external conditionality attached to international re- cognition, notably democracy and respect for minorities. This meant that although some political groups had favoured, at this juncture, a more restrictive definition of citizenry and consequently of polity based primarily on ‘ethnic’ criteria, the timing would have worked against it. What mattered was the very fact of instituting an autonomous citizen- ship, a highly visible claim to external sovereignty. Secondly, such an approach afforded all those affected by state succession the possibility of participating in the establishment of Slovenia, reflecting confidence in a harmonious relationship between ‘titular’ nation and ‘other’ citi- zens. The promise given to permanent residents from other former Yu- goslav republics that they would receive the Slovenian citizenship, if they so wished, was seen as fulfilled.46 In order to satisfy e´migre´ communities, which largely supported the independence process and to remedy injustices caused by deprivation of citizenship under the previous regime, they were granted preferential treatment regarding naturalisation. What might initially have appeared as a progressive principle of membership based on a civic conception, which could serve as a refer- ence point for the evolving statehood and an opportunity for defining national identity by embracing the multiethnic reality, took an ambigu- ous turn after independence was achieved. There were attempts to abolish dual citizenship for people from other Yugoslavian successor states and only reluctantly was it eventually tolerated. 10.5 Conclusions Furthermore, some of those who did not apply for or were not granted Slovenian citi- zenship were deprived of their legal residence. At the same time, how- ever, citizenship policy and supplementary or changed provisions on naturalisation functioned as instruments for regulating the status of immigrants and citizens of other Yugoslavian successor states whose status had not adequately been regulated in 1991. In this process, the SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 327 judiciary, in particular the Constitutional Court, played an important role. At the same time, Slovenia responded to international standards in the field by introducing facilitated naturalisation for certain categories of persons such as second- and third-generation immigrants or recog- nised refugees. Nevertheless, state interests in naturalisation prevail over those of the individual. The concept of a nation as a community of descent means that the principle of ius sanguinis prevails in defin- ing those entitled to citizenship at birth, that ethnic criteria play a ma- jor role in naturalisation procedures and that Slovenia is attempting to establish a special connection with Slovenes residing abroad. It also supports a notion of imagined community by, for example, explicit re- quirement of proficiency in the Slovenian language for naturalisation. Furthermore, even naturalised citizens are often seen as foreigners in most areas of public life. Current debates point to a need for a stronger public sense of citizenship in the democratic polity, but do not suggest any substantial change of the basic philosophy guiding citizenship pol- icy nor – after the recent amendments – a comprehensive legislative re- form. 10.6 Epilogue In April 2007, less than half a year after the most recent amendments, the National Council of the Republic of Slovenia proposed a bill amending the Citizenship Act of Slovenia. The National Council is the 40-member ‘upper chamber’ of the parliament, representing social, economic, professional and local/territorial interests. It is designed to neutralise the influence of political parties that are involved in legisla- tive processes, primarily through the National Assembly. The bill was initiated by Marjan Maucˇec, the representative of local interests in the National Council and a member of the Slovenian People’s Party (SLS). He proposed that persons who were over 25 years of age in 1991 should have an opportunity to register as Slovenian citizens by perso- nal declaration until the age of 45, instead of 36, which was the result of a 2002 amendment. Moreover there was a proposal to further relax the conditions for the exceptional naturalisation of persons of Slove- nian descent, although the 2006 amendments had already facilitated naturalisation for this particular group. The 2007 proposal foresaw that ancestors of persons who applied for this type of naturalisation did not have to originate from the current territory of the Republic of Slovenia. In the discussion held at the National Assembly’s Committee of Inter- ior Affairs, Public Administration and Justice, it became clear that members of the Slovenian diaspora in Argentina, Australia, Brazil and 328 FELICITA MEDVED FELICITA MEDVED Canada had initiated the proposed amendments. They had been ‘pro- mised’ by some Slovenian politicians that these amendments would be accepted. Nevertheless, the proposal was rejected by the Committee, with the Minister of the Interior arguing that the age prescribed for re- gistration was already very high compared to some other states and that the exceptional naturalisation of persons who had at least one par- ent who held Slovenian citizenship should remain limited to those whose parents were citizens by descent and not by naturalisation. The Liberal Democrats (LDS) expressed concern that this argument might imply a differentiation between citizenship acquired by descent and ci- tizenship acquired by naturalisation. Chronological list of citizenship-related legislation in Yugoslavia/Slovenia Date Document Content Source 1811/1867 Austrian Civil Code (arts. 28-32) Regulates the citizenship status of citizens living in Slovenian territories placed under Austrian rule within the Austro- Hungarian Empire 1879 Law on Hungarian Citizenship (art. www.sluzbenilist.co.rs (in Serbo-Croatian) Law on Citizenship of Persons in the Territory Annexed by Yugoslavia according to the Peace Treaty with Italy (No. 104/1947) www.sluzbenilist.co.rs (in Serbo-Croatian) 10.6 Epilogue ‘L’) Regulates the citizenship status of citizens living in Prekmurje placed under Hungarian rule within the Austro-Hungarian Empire 1920/1921 Executive regulations to the Saint-Germain-en- Laye Peace Treaty (No. 147/1920; No. 122/1921) Regulates the acquisition and loss of Yugoslav citizenship by option and request 1920 Treaty of Rapallo Provides a one-year right of option for Italian citizenship for ethnic Italians residing in the former Italian territory that became part of Yugoslavia 1923 Italian citizenship legislation Regulates citizenship in the Littoral according to the operative Italian citizenship legislation from 7 June 1923 until mid-September 1947 1928 Citizenship Act of the Kingdom of Serbs, Croats and Slovenes (No. 254/1928) Introduces a unified citizenship throughout the Kingdom Chronological list of citizenship-related legislation in Yugoslavia/Slovenia www.sluzbenilist.co.rs (in Serbo-Croatian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 329 Date Document Content Source 1941 German and Hungarian citizenship legislation Regulates the citizenship ofcertaingroupsofpeople in the German and Hungarian occupied territory during the Second World War 1945/ 1946 Citizenship Act of the Democratic Federative Yugoslavia (DFY) (No. 64/1945; No. 54/ 1946) Defines the initial citizenry of the newly formed Yugoslavia; establishes Yugoslav federal citizenship and republic-level citizenship of the six constituent republics; is confirmed and amended after the adoption of the 1946 Constitution of the Federal People’s Republic of Yugoslavia (FPRY) www.sluzbenilist.co.rs (in Serbo-Croatian) 1945/ 1946 Law on the Deprivation of Citizenship for Officers and Non- Commissioned Officers of the Former Yugoslav Army Who Do Not Want to Return to the Homeland, and for the Members of the Military Who Have Served for the Enemy and Have Defected Abroad (No. 64/1945; No. 86/1946) Deprives Yugoslav citizenship of those military personnel of the former army of the Kingdom of Yugoslavia whodidnotwant toreturn to the ‘new’ Yugoslavia, those serving in military formations loyal to the occupying forces and those who left Yugoslavia at the end of the Second World War; is revoked in 1962 (No. 22/1962) www.sluzbenilist.co.rs (in Serbo-Croatian) 1947 Law on Citizenship of Persons in the Territory Annexed by Yugoslavia according to the Peace Treaty with Italy (No. 104/1947) Determines that people residing in territories annexed by Yugoslavia on 10 June 1940 lose their Italian citizenship and acquire Yugoslav citizenship; provides a one-year period for ethnic Italians to opt for Italian citizenship and a possibility for the Slav population from the contested borderland region between Yugoslavia and Italy to opt for Yugoslav citizenship www.sluzbenilist.co.rs (in Serbo-Croatian) FELICITA MEDVED 330 Date Document Content S 1948/1997 Amendment and Revision of the Law on Citizenship (No. 105/ 1948); Constitutional Court Decision: U-I-23/97 Excludes from the Yugoslav citizenry, with retroactive effect, all citizens of German ethnicity residing abroad on the basis of their ‘disloyal conduct toward the national and state interests of the peoples of FPRY’ during and before the war; is confirmed as constitutional in 1997 when applied to procedures concerning the ascertainment of citizenship w ( h ( E 1950 Citizenship Act of the People’s Republic of Slovenia (No. Citizenship Act of the Socialist Republic of Slovenia (No. 11/1965) Law on Citizenship of the Socialist Federal Republic of Yugoslavia (No. 58/1976) www.sluzbenilist.co.rs (in Serbo-Croatian) Citizenship Act of the Socialist Republic of Slovenia (No. 23/1976) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 20/1950) Defines ius sanguinis as the basic principle for acquisition of Slovenian citizenship; specifies conditions for a newborn child to acquire Slovenian citizenship if the parents hold different republic-level citizenships, depending on their residency 1964 Law on Yugoslav Citizenship (No. 38/1964; No. 42/ 1964) Enacted after the 1963 Yugoslav Constitution; regulates the relationship between the federal and republic-level citizenships; provides for a united Yugoslav citizenship; regulates that only a federal citizen can have republican citizenship and that republican citizenship is lost with the loss of federal citizenship w ( 1965 Citizenship Act of the Socialist Republic of Slovenia (No. 11/1965) Confirms ius sanguinis as the main criterion for automatic acquisition of Slovenian citizenship; unlike the 1950 Law, stipulates that both parents have to agree on the child’s citizenship if they hold different www.sluzbenilist.co.rs (in Serbo-Croatian); http://odlocitve.us-rs.si (in Slovenian and English) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? Act Amending the Citizenship of the Republic of Slovenia Act (No. 38/1992) www.2.gov.si (in Slovenian); www.coe.int www.dz-rs.si (in Slovenian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 331 Date Document Content Source republic-level citizenships; determines that any SFRY citizen has a right to choose his or her republic-level citizenship and is thus free to choose the citizenship of any republic without being born or residing there; abolishes the loss of citizenship on grounds of absence; relaxes the naturalisation of expatriates (emigrants); abolishes the oath of loyalty upon admission 1976 Citizenship Act of the Socialist Republic of Slovenia (No. 23/1976) Confirms ius sanguinis as the main criterion for automatically acquiring Slovenian citizenship; specifies conditions of citizenship acquisition in cases in which the parents do not agree or do not sign a statement in the first two months following the birth of their child; regulates acquisition of citizenship for citizens of other republics who have permanent residence in Slovenia 1976 Law on Citizenship of the Socialist Federal Republic of Yugoslavia (No. 58/1976) Is adopted after the 1974 Yugoslav Constitution; repeats the provisions from the 1964 Act on the relationship between the various federal and republic-level citizenships; adds an article on how to resolve possible disputes caused by the republican laws on citizenship www.sluzbenilist.co.rs (in Serbo-Croatian) 1976 1976 FELICITA MEDVED 332 Date Document Content Source 1991 Citizenship of the Republic of Slovenia Act (No. 1/1991-I) Constitutes the initial body of citizens and basic principles of acquisition and loss of citizenship; provides for optional naturalisation of residents from other republics of SFRY (art. 40) and reacquisition of citizenship for those who were deprived of it on the basis of prior federal law (art. 41) in the transitional period http://e-uprava.gov.si (in Slovenian); www.legislationline.org; www.coe.int 1991 Act Amending the Citizenship of the Republic of Slovenia Act (No. 30/1991-I) Supplements art. 40 and stipulates that the person’s application for citizenship is to be turned down if that person is considered to form a threat to public order, security or defence of the state or has committed a criminal offence directed against the Republic of Slovenia www2.gov.si (in Slovenian and English); www.legislationline.org; www.coe.int 1991 Constitution of the Republic of Slovenia (No. 33/91-I; No. 33I/1991-I; No. 42/1997; No. 66/ 2000; No. 24/2003; No. 69/2004; No. 69/2004; No. 69/2004; No. 68/ 2006) Postulates in art. 12 that citizenship shall be regulated by law and in art. 1991 Constitution of the Republic of Slovenia (No. 33/91-I; No. 33I/1991-I; No. 42/1997; No. 66/ 2000; No. 24/2003; No. 69/2004; No. 69/2004; No. 69/2004; No. 68/ 2006) Act Amending the Citizenship of the Republic of Slovenia Act (No. 30/1991-I) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 5 (2) that Slovenes not holding Slovene citizenship may enjoy special rights and privileges in Slovenia, the nature and extent of which shall be regulated by law www.dz-rs.si (in Slovenian) 1992 Act Amending the Citizenship of the Republic of Slovenia Act (No. 38/1992) Regulates naturalisation for persons of Slovenian descent through at least one parent if they lost their Slovenian citizenship due to release, renunciation or deprivation or if they have not acquired Slovenian citizenship due to historical circumstances (art. 13.a) www.2.gov.si (in Slovenian); www.coe.int http://e-uprava.gov.si (in Slovenian); www.legislationline.org; www.coe.int SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? www2.gov.si (in Slovenian); www.unhcr.org Decree on Criteria for Establishing the Compliance with Specified Conditions for Acquiring the Citizenship of the Republic of Slovenia (No. 47/1994) http://odlocitve.us-rs.si (in Slovenian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 333 Date Document Content Source 1992 Constitutional Court Decision repealing Articles 41 and 13.a of the Citizenship of Slovenia Act (No. 61/1992; Constitutional Court Decision U-I-69/92-30) http://odlocitve.us-rs.s (in Slovenian) 1992 Constitutional Court Decision repealing Article 28 of the Citizenship of Slovenia Act (No. 61/1992; Constitutional Court Decision U-I-98/91-21) Provides that the reasons for a discretionary decision must be stated in such a way that it is evident whether the administrative authority in exercising its discretionary power of decision has acted ‘within the limits of authorisation and in accordance with the intention of granting such an authorisation’ http://odlocitve.us-rs.s (in Slovenian) 1994 Act amending the Citizenship of the Republic of Slovenia Act (No. 13/1994) Provides for the recognition and declaration of citizenship for residents from other former Yugoslav republics; extends the age limit for citizenship by registration; tightens the naturalisation criteria, specifically as to language proficiency; introduces consent of minors older than fourteen www2.gov.si (in Slovenian); www.unhcr.org 1994 Decree on Criteria for Establishing the Compliance with Specified Conditions for Acquiring the Citizenship of the Republic of Slovenia (No. 47/1994) http://odlocitve.us-rs.si (in Slovenian) 1994 FELICITA MEDVED 334 Date Document Content Source 1996 Resolution on the Position of Autochthonous Slovene Minorities in Neighbouring Countries and the Related Tasks of State and Other Institutions in the Republic of Slovenia (No. 35/1996) Envisages various forms of aid for these minorities, taking into account their specific needs and interests and providing them with concrete aid in the cultural, language, informative, economic and financial areas www.uszs.gov.si (in Slovenian) 1999 The Settling of the Status of Citizens of Other SFRY Successor States in the Republic of Slovenia Act (No. 61/1999; No. 54/ 2000; Constitutional Court Decision U-I-295/ 99-13, No. 64/2001) Regulates the acquisition of permanent residence for citizens of other SFRY successor states registered as permanent residents in the territory of Slovenia on 23 December 1990 or residing in Slovenia since 25 June 1991 www.uradni-list.si (in Slovenian); www.legislationline.org 2002 Act amending the Citizenship of the Republic of Slovenia Act (No. 96/2002) Extends the age limit for acquiring citizenship by registration; adds stricter criteria for regular naturalisation, such as the status of foreigner; relaxes naturalisation for refugees, stateless persons and second- and third-generation immigrants www.uradni-list.si (in Slovenian) 2002 Resolution on the Relations with Slovenes Abroad (No. http://odlocitve.us-rs.si (in Slovenian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 335 Date Document Content Source 2006 Relations between the Republic of Slovenia and Slovenians abroad Act (No. 43/2006) Introduces the status of Slovenes without Slovenian citizenship www.uradni-list.si (in Slovenian) 2006 Act amending the Citizenship of the Republic of Slovenia Act (No. 127/2006) Introduces stricter requirements for regular naturalisation (material and social security, clean criminal record, oath of loyalty) and naturalisation of spouses of Slovenian nationals; redefines groups of persons eligible for facilitated and exceptional naturalisation www.dz-rs.si (in Slovenian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 7/2002) Envisages cooperation with Slovenes abroad in fields of culture, language, information, economy, science and foreign policy; declares interest in repatriation, a status of Slovenes without citizenship and citizenship and voting rights for citizens abroad www.uradni-list.si (in Slovenian) 2002 Constitutional Court Decision: U-I-246/02-28 (No. 36/2003) Finds the Settling of the Status of Citizens of Other SFRY Successor States in the Republic of Slovenia Act unconstitutional http://odlocitve.us-rs.si (in Slovenian) SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 12 Official Gazette of the Socialist Republic of Slovenia, 23/1976. Notes 336 FELICITA MEDVED FELICITA MEDVED 13 Moreover, most of these instruments such as the European Convention on Nationality, which was opened for signature on 6 November 1997 and entered into force on 1 March 2000, and the relevant provisions of the 1999 draft articles on the Nationality of Natural Persons in Relation to the Succession of States, were drafted after the changes that had reshaped the European political landscape at the end of the twentieth century. The former contains a chapter on state succession which focuses on principles and general rules but does not provide for specific rules which states should respect in cases of state succession. 14 In this regard, it must also be noted that the European Union does not consider nationality matters to be in its sphere of competence. 14 In this regard, it must also be noted that the European Union does not consider nationality matters to be in its sphere of competence. 14 In this regard, it must also be noted that the European Union does not consider nationality matters to be in its sphere of competence. 15 Official Gazette of the Republic of Slovenia, 1/1991-I. Amendments and Supplements to this Act were published in the Official Gazette of the Republic of Slovenia, 30/1991-I, 38/1992, 13/1994 and 96/2002. The officially revised text was published in the Offi- cial Gazette of the Republic of Slovenia, 7/2003. www.mnz.gov.si. 15 Official Gazette of the Republic of Slovenia, 1/1991-I. Amendments and Supplements to this Act were published in the Official Gazette of the Republic of Slovenia, 30/1991-I, 38/1992, 13/1994 and 96/2002. The officially revised text was published in the Offi- cial Gazette of the Republic of Slovenia, 7/2003. www.mnz.gov.si. 16 The Act amending the Citizenship of the Republic of Slovenia Act was adopted by the National Assembly on 24 November 2006. Official Gazette of the Republic of Slove- nia, 127/2006. 16 The Act amending the Citizenship of the Republic of Slovenia Act was adopted by the National Assembly on 24 November 2006. Official Gazette of the Republic of Slove- nia, 127/2006. 7/ 17 For these concepts also, see Barsˇova´ in this volume. 7 17 For these concepts also, see Barsˇova´ in this volume. 18 In 1999 the Constitutional Court repealed the paragraph related to the public order risk. Constitutional Court Decision, U-I-89/99 of 10 June 1999. Notes 1 Official Gazette of the Provincial Government for Slovenia, 147/1920 and 122/1921. 2 Official Gazette of the Kingdom of Serbs, Croats and Slovenes (SHS), 254/1928. 3 Official Gazette of the Democratic Republic of Yugoslavia (DRY), 64/1945; Official Gaz- ette of the Federal People’s Republic of Yugoslavia (FPRY), 54/1946, 90/1946, 88/1948 and 105/1948. 4 Official Gazette of the FPRY, 105/1948. In 1997, the Constitutional Court of the Re- public of Slovenia found that the use of this provision is not unconstitutional in pro- cedures concerning the ascertainment of citizenship. Constitutional Court Decision, U-I-23/93 of 20 March 1997. 5 Official Gazette of the DRY, 64/1945; Official Gazette of the FPRY, 86/1946 and 22/ 1962. 5 Official Gazette of the DRY, 64/1945; Official Gazette of the FPRY, 86/1946 and 22/ 1962. 6 Official Gazette of the FPRY, 104/1947. 6 Official Gazette of the FPRY, 104/1947. 7 The Memorandum includes a special statute that guarantees for both sides the rights of minorities. It is the first international document that regulates the protection of the Slovene ethnic minority (‘Yugoslav ethnic group’) in Italy – for the Trieste region. 7 The Memorandum includes a special statute that guarantees for both sides the rights of minorities. It is the first international document that regulates the protection of the Slovene ethnic minority (‘Yugoslav ethnic group’) in Italy – for the Trieste region. 8 See also Slovenia, Italy, White Book on Diplomatic Relations published in 1996 by the Ministry of Foreign Affairs of the Republic of Slovenia. 9 Official Gazette of the FPRY, 9-96/1959, 7-115/58; Official Gazette of the Socialist Feder- al Republic of Yugoslavia (SFRY), 7-35/1971, 31-448/1967, 11-48/1981 and 15-65/1990. Slovenia is a party to these instruments by succession. 10 In this chapter, the term ‘republic-level citizenship’ is used to denote the membership in constituting entities of the federal state. The term ‘citizenship’ is used to indicate membership of a sovereign state. In the Slovenian language and legal terminology, drzˇavljanstvo is used for both legal concepts. 11 Official Gazette of the SFRY, 38/64. 11 Official Gazette of the SFRY, 38/64. 12 Official Gazette of the Socialist Republic of Slovenia, 23/1976. Notes 19 That immigrants from other republics did not register their permanent residence was partly due to the fact that they did not know of this possibility or simply did not care; partly it can be attributed to the concept of migration registration and registration of permanent residency in the former state. Slovenia was the sole republic of the SFRY which registered in- and out-migration. 20 Official Gazette of the Republic of Slovenia, 1/1991-I, 44/1997 and 50/1998 – Constitu- tional Court Decisions. 20 Official Gazette of the Republic of Slovenia, 1/1991-I, 44/1997 and 50/1998 – Constitu- tional Court Decisions. 21 Under the then valid Aliens Act they could obtain a one-year temporary residence permit and after three years of uninterrupted residence a permit for permanent residence. Later this condition was prolonged from three to eight years. Cf. the controversial 1993 Estonian law on aliens, which declared that anybody living in Estonia without Estonian citizenship, which had no legal status in Estonian law in 1992/1993, would have to apply for residency status. The Council of Europe experts criticised that the status of those already resident in Estonia was equated with that of non-citizens not currently resident there (see Day & Shaw 2003; also see Jaerve in this volume). 22 Only upon the request of applicants themselves did administrative authorities issue a certificate of removal from the register. 22 Only upon the request of applicants themselves did administrative authorities issue a certificate of removal from the register. 23 The Ombudsman in his first yearly report of 1995 refers to the so-called ‘aliens sur place’ (zatecˇeni tujci) using a label which resembles the term ‘refugees sur place’. 24 Constitutional Court Decision, U-I-284/94 of 4 February 1999. 24 Constitutional Court Decision, U-I-284/94 of 4 February 1999. azette of the Republic of Slovenia, 61/1999 and 64/2 26 Constitutional Court Decision, U-I-246/02-28 of 3 April 2003. 26 Constitutional Court Decision, U-I-246/02-28 of 3 April 2003. 27 The Ministry of the Interior, however, issued decisions on residency from 26 February 1992 to those ‘erased’, who already had permanent residence permits. 4,107 such decisions were issued until mid-January 2005, while 8,470 were still in the procedure. 27 The Ministry of the Interior, however, issued decisions on residency from 26 February 1992 to those ‘erased’, who already had permanent residence permits. 4,107 such decisions were issued until mid-January 2005, while 8,470 were still in the procedure. SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 337 30 Decree on Criteria for Establishing the Compliance with Specified Conditions for Acquiring the Citizenship of the Republic of Slovenia through Naturalisation, Official Gazette of the Republic of Slovenia, 47/1994. 30 Decree on Criteria for Establishing the Compliance with Specified Conditions for Acquiring the Citizenship of the Republic of Slovenia through Naturalisation, Official Gazette of the Republic of Slovenia, 47/1994. f p f 47/ 994 31 Constitutional Court Decision, U-I-98/91 of 10 December 1992. 31 Constitutional Court Decision, U-I-98/91 of 10 December 1992. 32 Under the principle of equality of children born in wedlock and children born out of wedlock a child of a foreign mother is a Slovenian citizen if the fatherhood of a Slovenian citizen is acknowledged, declared or otherwise established. The legal effect of fatherhood is retroactive and as such affects the citizenship of the child. 32 Under the principle of equality of children born in wedlock and children born out of wedlock a child of a foreign mother is a Slovenian citizen if the fatherhood of a Slovenian citizen is acknowledged, declared or otherwise established. The legal effect of fatherhood is retroactive and as such affects the citizenship of the child. 33 The registration is not necessary if the child would otherwise become stateless or if the child moves to Slovenia, together with the Slovenian parent, before he or she is eighteen years old. 33 The registration is not necessary if the child would otherwise become stateless or if the child moves to Slovenia, together with the Slovenian parent, before he or she is eighteen years old. 34 Before 1994 an applicant did not have to submit this evidence. 34 Before 1994 an applicant did not have to submit this evidence. 34 994 pp 35 The condition of a guaranteed residence was dropped in 2002. 36 Before the 2006 amendments the requirements did not include conditional prison sentences. Moreover, the accepted period of imprisonment was decreased from a maximum of one year to three months. 36 Before the 2006 amendments the requirements did not include conditional prison sentences. Moreover, the accepted period of imprisonment was decreased from a maximum of one year to three months. 37 See also Constitutional Court Decision, U-I-124/94-8 of 9 February 1995. http://odlocitve.us-rs.si. Notes 28 1,278 Slovenes were deprived of citizenship based on collective decisions by federal authorities, of which the individuals were never notified, and 67 due to absence. 28 1,278 Slovenes were deprived of citizenship based on collective decisions by federal authorities, of which the individuals were never notified, and 67 due to absence. 29 Constitutional Court Decision, U-I-69/92-30 of 10 December 1992. 29 Constitutional Court Decision, U-I-69/92-30 of 10 December 1992. SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? FELICITA MEDVED FELICITA MEDVED Bibliography Cerar, M. (1995), ‘Zakonodajni referendum o odvzemu drzˇavljanstva’, Pravna praksa 14 (19-20): 8-11. Day, S. & J. Shaw (2003), ‘The boundaries of suffrage and external conditionality: Estonia as an applicant member of the EU’, European Public Law 9: 211-236. Dujic´, S. (1996), ‘Zakonodajni referendum o odvzemu drzˇavljanstva’, Pravna praksa 15 (2): 22-27. Kacˇ, M. & M. Krisch (1999), ‘Pregled predpisov o drzˇavljanstvu 1918-1991’, in Slovensko javno pravo na prehodu v novo tisocˇletje, Dnevi javnega prava Portorozˇ 1999, 607-631. Ljubljana: Insˇtitut za javno upravo. Koncˇina, N. (1992), ‘Dvojno drzˇavljanstvo’, Pravna praksa 11 (25): 11-12. Kos, B. (1994), ‘Mednarodni sporazumi z Italijo in drzˇavljanstvo’, Pravna praksa 13 (16): 2-3. Kos, B. (1996a), ‘Primerjava zakonov o drzˇavljanstvu drzˇav, ki so nastale na ozemlju nek- danje SFRJ’, Pravna praksa 15 (22-23): XIII. Kos, B. (1996b), ‘Zakoni o drzˇavljanstvu v novih drzˇavah na obmocˇju bivsˇe SFRJ: postop- ki, prehodne in koncˇne dolocˇbe’, Pravna praksa 15 (7): I. Kos, B. (1996b), ‘Zakoni o drzˇavljanstvu v novih drzˇavah na obm ki, prehodne in koncˇne dolocˇbe’, Pravna praksa 15 (7): I. Medved, F. (1996), ‘Slovenias bevisbyrde’, Nordisk O¨ stforum 2: 91-104. Medved, F. (2001), ‘Interaction Between Citizenship and Integration’, 2nd European Con- ference on Nationality: Challenges to national and international law on citizenship at the beginning of the new millennium’, 23-39. Strasbourg: Council of Europe, Proceedings. www.coe.int. Medved, F. (2005), ‘Politicˇna integracija: drzˇavljanstvo, politicˇna participacija in reprezen- tacija’, in M. Komac & M. Medvesˇek (eds.), Percepcije slovenske integracijske politike, 453-558. Ljubljana: Institute for Ethnic Studies. Mesojedec-Pervinsˇek, A. (1997), Predpisi o drzˇavljanih in tujcih z uvodnimi pojasnili. Ljub- ljana: Cˇ Z Uradni list Republike Slovenije. Mesojedec-Pervinsˇek, A. (1999), ‘Ucˇinek mednarodnega prava v notranjem pravu Repub- like Slovenije na podocˇju drzˇavljanstva’, in Slovensko javno pravo na prehodu v novo tiso- cˇletje, Dnevi javnega prava Portorozˇ 1999, 647-661. Ljubljana: Insˇtitut za javno upravo. Mole, N. (2001), ‘Multiple nationality and the European Convention on Human Rights’, 2nd European Conference on Nationality: Challenges to national and international law on nationality at the beginning of the new millennium’, 129-147. Strasbourg: Council of Europe, Proceedings. www.coe.int. p g Policˇ, V. (1993), ‘Kdo ‘‘tukaj dejansko zˇivi’’?’, Pravna Praksa 12 (22): 16-17. Policˇ, V. (1997a), ‘Drzˇavljanstvo RS. Tri odlocˇitve VS RS, v katerih je bilo obravnavano: nevarnost za javni red drzˇave; nicˇnostni razlog; ugotovitev drzˇavljanstva RS’, Pravna praksa 16 (17-18): 15-17. Policˇ, V. (1997b), Statusna stanja cˇloveka in drzˇavljana v Republiki Sloveniji. 1. del (part one). SLOVENIA: FROM CIVIC TO ETHNIC COMMUNITY? 38 Constitutional Court Decision on the request for holding a referendum on Article 40 of the Citizenship Act of the Republic of Slovenia, U-I-266/95-8 of 20 November 1995, Official Gazette of the Republic of Slovenia, No. 69/1995. 39 Until 1995 this function was performed by municipalities and then transferred to local administrative units by the Act on the Takeover of State Functions Performed until 31 December 1994 by Municipal Bodies, Official Gazette of the Republic of Slove- nia, 29/1995, 44/1996 – Constitutional Court Decision. 40 Official Gazette of the Republic of Slovenia, 37/2003 and 39/2006. The officially conso- lidated text was published in the Official Gazette of the Republic of Slovenia, 59/2006. 41 Official Gazette of the Republic of Slovenia, 44/2006. 42 Official Gazette of the Republic of Slovenia, 35/1996. 43 Official Gazette of the Republic of Slovenia, 7/2002. 44 The Council for Slovenians in Neighbouring Countries is composed of six representatives of the Slovenian minority in Austria, four from Italy and two from Hungary and Croatia. The Council for Slovenians Abroad consists of four representatives of Slovenians living in European states, three from South America and North America respectively, two from Australia and one from Slovenians residing in other countries around the world. 45 The Statistical Office of the Republic of Slovenia does not gather and analyse data on naturalisation. Tables in this section draw on data provided by the Ministry of the Interior. 46 This promise was given by all of the political parties and in the Letter of Good Intent (Official Gazette of the Republic Slovenia, 40/1990) adopted by the Slovenian Assem- bly prior to the plebiscite on the autonomy and independence on which all perma- nent residents could vote. Furthermore, the Constitutional Act Implementing the Ba- sic Constitutional Charter on the Independence and Sovereignty of the Republic of Slovenia (Official Gazette of the Republic Slovenia, 1/1991-I) in art. 13 specially provided that citizens of other republics having permanent residence registered in Slovenia on the day of the plebiscite and who actually lived there should have equal rights and duties as Slovenian citizens during the transitional period with regards to the acquisi- tion of Slovenian citizenship. Questions concerning the correct interpretation of this document, however, have arisen specifically concerning the implementation of the Aliens Act. 338 FELICITA MEDVED Bibliography Ljubljana: Center Marketing Int. Radmelicˇ, B. (1994), ‘Pridobitev drzˇavljanstva na ozemlju Slovenije od obdobja Avstro- Ogrske do konca druge svetovne vojne’, Javna uprava 30 (2): 207-227. Rakocˇevic´, S. (2002), ‘‘‘Izbrisani’’ prebivalci Slovenije, urejanje pravnega statusa drzˇavlja- nov drugih drzˇav naslednic nekdanje SFRJ v Republiki Sloveniji’, Pravna praksa 21 (32): 8-9. Ziemele, I. (2001), ‘General aspects of nationality and human rights in relation to state succession’, 2nd European Conference on Nationality: Challenges to national and interna- tional law on nationality at the beginning of the new millennium’, 149-171. Strasbourg: Council of Europe, Proceedings. www.coe.int. 11.1.1 Citizenship in federal Yugoslavia (1945-1991) The citizenship laws in the Croatian lands (Croatia, Slavonia and Dal- matia) that preceded the formation of the ‘first’ Yugoslavia in 1918, date back to the second half of the nineteenth century. The 1879 Law on Hungarian Citizenship (art. L) was applied in Croatia and Slavonia, whereas the laws on Austrian citizenship (arts. 28-32) based on the 1811 (1867) Austrian Civil Code were applied in Dalmatia. In 1918, fol- lowing the founding of the Kingdom of Serbs, Croats and Slovenes (re- named the Kingdom of Yugoslavia in 1929), the citizenship issues that arose due to the break up of the Austro-Hungarian Empire and the subsequent creation of new states, were largely settled by the post-First World War peace treaties signed by the new Kingdom with neighbour- ing countries.6 Yugoslavia only enacted its own law on citizenship in 1928, a law that established a single Yugoslav citizenship. 9 g g p On 28 August 1945, the recently liberated ‘new’ Yugoslavia, a state that resurrected itself as a federation on the political map of Europe after the collapse of the Kingdom of Yugoslavia in April 1941, enacted the Law on Citizenship of Democratic Federal Yugoslavia.7 Art. 35 of this law provided that everyone who had been a Yugoslav citizen on 28 August 1945 under the 1928 Citizenship Act of the Kingdom of Yugo- slavia would become a citizen in Democratic Federal Yugoslavia. Yugo- slav citizenship was primarily based on the principle of descent (ius sanguinis; see Medvedovic´ 1998: 27-29; Jovanovic´ 1977: 22; Tepic & Basic 1969: xxxvi). Since it was often impossible to prove former Yugo- slav citizenship due to the widespread destruction caused by the war, art. 25 declared that anyone belonging to one of the ‘peoples’ of Yugo- slavia (that is, to one of the South-Slavic ethnic groups), those born and raised in the territory of Yugoslavia and the permanent residents of the Federal People’s Republic of Yugoslavia (FPRY) would be consid- ered citizens of the FPRY. 11 Croatian citizenship: From ethnic engineering to inclusiveness Francesco Ragazzi and Igor Sˇtiks1 The politics of citizenship in post-Yugoslav Croatia are deeply marked by the political climate in which they emerged. The law on Croatian ci- tizenship was enacted on the day (8 October 1991) that the country’s declaration of independence from the Socialist Federal Republic of Yu- goslavia (SFRY) was enacted. The first decade of Croatia’s indepen- dence was burdened by the 1991-1995 war against Belgrade and mili- tary involvement in the war in neighbouring Bosnia-Herzegovina, and dominated by Franjo Tudjman’s overtly nationalist party HDZ2, which was in power between 1991 and 1999. p 99 999 The new citizenship legislation cannot, therefore, be analysed sepa- rately from the process of Yugoslavia’s disintegration. Almost all of Yu- goslavia’s successor states – with some variation according to their spe- cific context and at a different pace – used their founding documents, constitutions and citizenship laws as effective tools to accelerate na- tion-building and to ‘ethnically engineer’ their populations to the ad- vantage of the majority ethnic group. Croatia was no exception to this rule.3 In many ways, citizenship laws in Croatia were one of many in- struments used to create what could be defined as a ‘transnational na- tionalism’4, a nationalism that, by taking Croatian ethnicity as its core, aimed not only to homogenise the national population through the ex- clusion of non-Croats, but also to include all ethnic Croats in a single national group, regardless of their place or country of residence. The ci- tizenship laws proved a vital tool in the attempt to achieve this goal. This attempt at both deterritorialised inclusion and targeted exclusion was limited only by general international standards and norms related to citizenship laws that the Croatian government of the 1990s was ob- liged to respect.5 With the death of Franjo Tudjman in 1999 and the subsequent elec- toral defeat of the HDZ, the beginning of 2000 marked a sharp con- trast with the practices of the previous decade. Owing in part to demo- cratic changes within Croatian politics and Croatia’s bid for EU mem- bership, the implementation of the citizenship laws began to demonstrate more inclusiveness towards ethnic non-Croats, although the law on citizenship itself remained unchanged. 11 Croatian citizenship: From ethnic engineering to inclusiveness It is through an ex- amination of these political conflicts and debates and their historical FRANCESCO RAGAZZI AND IGOR SˇTIKS 340 context that we can best present the normative framework that regu- lates citizenship in Croatia today. 11.1.1 Citizenship in federal Yugoslavia (1945-1991) Enacted in 1948 was an unusually named law on deprivation of citizenship (which additionally entailed the con- fiscation of goods): the Law on the Deprivation of Citizenship for Offi- cers and Non-Commissioned Officers of the Former Yugoslav Army Who Do Not Want to Return to the Homeland, and for the Members of Military Forces Who Have Served the Enemy and Have Defected Abroad (this law was invalidated in 1962).8 Furthermore, in the same year the law on citizenship was revised in order to exclude from Yugo- slav citizenship all citizens of German ethnicity residing abroad on the CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 341 basis of their ‘disloyal conduct toward the national and state interests of the peoples of FPRY.9’ A special act related to Yugoslav citizenship – the Law on Citizenship of Persons Residing on the Territory Annexed to Yugoslavia According to the Peace Treaty with Italy10 – was adopted in 1947 following the Paris Peace Treaty between Yugoslavia and Italy. According to this Law, anyone who, as of 10 June 1940, had been a resident of the territories annexed by Yugoslavia would lose his or her Italian citizenship and ac- quired Yugoslav citizenship. Ethnic Italians had a one-year period to opt for Italian citizenship – in effect, to opt for whether they wanted to live in Yugoslavia or Italy. In addition, an equivalent offer of Yugoslav citizenship was made to the Slavic population from the contested bor- derland region between Yugoslavia and Italy. The citizenship of these groups was later defined by the Memorandum of Understanding be- tween the Government of Italy, the United Kingdom and Yugoslavia,11 which divided the Free Territory of Trieste (1947-1954) between Italy and Yugoslavia. Subsequently, their status was regulated by the 1975 Osimo Treaty between Italy and Yugoslavia (Medvedovic´ 1998: 32; Jova- novic´ 1977: 27-31; see also Medved in this volume). 977 7 3 ) Together with the law on Yugoslav federal citizenship, the republic- level citizenships of the constitutive republics were established.12 Yugo- slav citizens were allowed to have only one republic-level citizenship. This measure had very important practical and political consequences. According to the Voting Registers Act of 10 August 1946, only citizens of a particular republic had the right to vote in that republic. Citizens from other Yugoslav republics who happened to reside on the territory of that republic were not allowed to vote there. 11.1.1 Citizenship in federal Yugoslavia (1945-1991) Republican People’s As- semblies were supposed to be elected only by citizens of these repub- lics, although some republics, such as Croatia, would later allow both its citizens and residents to participate in elections of delegates for the Croatian Parliament (see Hondius 1968: 184). It is important to note that only republic-level registries of citizens existed in Yugoslavia be- tween 1945 and 1991.13 During the era of socialist Yugoslavia, three laws on Yugoslav citizenship were enacted (in 1945/1946, 1964 and 1976), following important constitutional changes in 1945 and 1946, 196314 and 1974. They defined the relationship between federal-level and republic-level citizenship. Art. 1, para. 2 of the 1945/46 Law on Yu- goslav citizenship stated that: ‘Every citizen of a people’s republic is si- multaneously a citizen of FPRY and every citizen of FPRY is principally a citizen of a people’s republic.’ The 1964 Law provided for a united Yu- goslav citizenship (art. 1), made republic-level citizenship conditional upon federal citizenship, and declared that the republic-level citizen- ship would be lost with the loss of federal citizenship (art. 2, para. 2).15 The 1976 Law on Yugoslav citizenship contained a similar provision FRANCESCO RAGAZZI AND IGOR SˇTIKS 342 and added an article (22) on how to resolve disputes caused by the re- publics’ laws on citizenship.16 These norms regulated the citizenship status of a child either according to the citizenship laws in force in the republic of which the child’s parents were citizens or, if the parents did not have the same citizenship, according to the citizenship laws of the republic where the child was born. The norms also offered an option for parents of different citizenships to agree on the citizenship of their child. If the parents could not agree, the child was granted a possibility of naturalising in the republic of his or her birth. Not surprisingly for the confederated structure that Yugoslavia progressively became after the late 1960s, these adopted norms show some similarities to the norms of international law in cases of legal collisions among sovereign states (Jovanovic 1977: 53). (J 977 53) The republic-level laws on citizenship were fashioned to harmonise with the federal law on citizenship, but in fact they varied from one re- public to the other. They were adopted in three waves: in 1950, in 1965 and in the period between 1975 and 1979. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 343 As shown above, citizenship in the socialist Yugoslavia was bifur- cated into a federal citizenship, on the one hand, and a republic-level citizenship, on the other hand. According to art. 249 of the last (1974) SFRY Constitution, citizens possessed a ‘single citizenship of SFRY’ and every citizen of a republic was ‘simultaneously’ a citizen of SFRY. The third line of the article offered an important right to all federal citi- zens: ‘a citizen of a republic on the territory of another republic has the same rights and obligations as the citizens of that republic.’ g g Yugoslav citizens were thus, in principle, able to choose their repub- lican citizenship depending on their residency or employment. Never- theless, since the republican citizenship was of no practical relevance, citizens usually did not change their republican citizenship status if they moved to another republic, and often they did not even register changes of residence. Internal Yugoslav migration established strong personal and family ties across republican borders, whilst economically motivated migrations and the resettlement of federal administration personnel resulted in a considerable number of individuals living out- side of their republic of origin. This in turn affected the balance be- tween ethnic groups in Yugoslav republics to a certain degree. From the moment of Yugoslavia’s dissolution, federal citizenship ceased to exist and the previously irrelevant republic-level citizenship became the main criterion for the initial determination of citizenship in the succes- sor states. The ‘internal’ Yugoslav migrants, residing in a republic whose citizenship they did not possess and to whose ethnic majority they did not belong, were the first to suffer the consequences of the new citizenship regimes. 11.1.1 Citizenship in federal Yugoslavia (1945-1991) In 1950, the Citizenship of the People’s Republic of Croatia17 provided that the basic principle for acquisition of Croatian republican citizenship was ius sanguinis. How- ever, if parents of a newborn child had different republican citizen- ships, the child could acquire Croatian citizenship if both parents agreed. If they did not agree and they had residence in Croatia, the child would automatically acquire Croatian citizenship. If the parents did not have residence in Croatia but the father had Croatian citizen- ship, the child would become a Croatian citizen as well. The 1965 Law on Citizenship of the Socialist Republic of Croatia brought some changes.18 Croatian citizenship was automatically granted if a child was born in Croatia and both parents had Croatian ci- tizenship. In all other cases, parents had to agree on the child’s citizen- ship. Nevertheless, the law offered a possibility to any SFRY citizen to opt for Croatian citizenship without being born or residing there and regardless of his or her ethnicity (UNHCR 1997: 16). In the 1977 Law on Citizenship of the Socialist Republic of Croatia19 we can observe some new changes related to the acquisition of Croa- tian citizenship. Ius sanguinis principle remained the automatic criter- ion for acquiring Croatian citizenship; if both parents were Croatian ci- tizens, the child would automatically become a Croatian citizen. How- ever, if only one parent was a Croatian citizen, the parents had to agree. In cases in which the parents did not agree or did not sign a statement within two months following the birth of their child, Croa- tian citizenship was automatically accorded to the child if the parents had permanent residence in Croatia. If the parents did not have perma- nent residence in Croatia, the child would acquire Croatian citizenship if his or her birth was registered in Croatia’s register of births.20 11.1.2 Croatian citizenship since 1991 11.1.2 Croatian citizenship since 1991 p 99 The Croatian declaration of independence of 25 June 1991 – which en- tered into force on 8 October 1991 after a three-month moratorium brokered by the international community – was based on the referen- dum on Croatian independence of 19 May 1991. Croatian citizens were essentially asked to vote – which they did in huge numbers – in favour of recognising the Republic of Croatia as a sovereign and independent state that guarantees the Serbs and members of other nationalities in C ti lt l t d ll i ht f iti 21 However, Croatian Serbs generally boycotted the referendum and even held their own to express their desire to remain part of Yugoslavia. However, Croatian Serbs generally boycotted the referendum and even held their own to express their desire to remain part of Yugoslavia. Six months previously, in December 1990, the new Croatian consti- tution had proclaimed ‘the Republic of Croatia as the national state of the Croatian people and the state of members of other nations and FRANCESCO RAGAZZI AND IGOR SˇTIKS 344 minorities who are its citizens.’ The new Constitution, adopted after the first democratic elections in Croatia, replaced the 1974 Constitution of the Socialist Republic of Croatia, which had defined Croatia ‘as a na- tional state of the Croatian people, state of the Serbian people in Croatia [emphasis added] and state of nationalities living on its territory’ (art. 1). Although the referendum question, together with the Constitution itself, mentioned the rights of ethnic minorities and their equal status in new Croatia, the constitutional definition of Croatia as primarily an ethnically Croatian state had a direct impact on the new citizenship law (which entered into force simultaneously on 8 October 1991).22 The law was conceived on the basis of two major principles: legal conti- nuity with citizenship of the Socialist Republic of Croatia and Croatian ethnicity (see Omejec 1998: 99). All holders of the former Croatian republican citizenship became ci- tizens of the new state ex lege (art. 30, para. 1). All other residents be- came aliens overnight, irrespective of how long they had resided in Croatia. Their naturalisation as Croatians was regulated by art. 8 of the Law on Croatian Citizenship. 11.1.2 Croatian citizenship since 1991 According to this article, in order to be naturalised a resident must have at least five years of registered resi- dence in Croatia, provided that the following conditions were met: that he or she had renounced a foreign citizenship or will submit proof that he or she will be released from a previous citizenship if admitted to Croatian citizenship; that he or she is proficient in the Croatian lan- guage and Latin script; that it can be concluded from the applicant’s conduct that he or she is attached to the legal system and customs of the Republic of Croatia; and finally, that he or she accepts the Croatian culture [emphases added].23 The law put those with less than five years of registered residence and those who were unable to prove that they had been released from foreign citizenship (i.e. previous republican citizenship)24 in a particu- larly difficult position. In a context in which Croatia was at war with the Yugoslav Federation (which initially consisted of the Republics of Serbia, Montenegro, Macedonia and Bosnia-Herzegovina and then pro- gressively shrank to just Serbia and Montenegro) it was virtually im- possible to satisfy this condition. Only aliens born in the territory, spouses, emigrants and those whose citizenship was of interest to Croatia did not have to prove release from their previous citizenship under the naturalisation procedure. However, the first two categories have to fulfil more requirements than the latter two. Moreover, all ap- plicants for naturalisation have to prove that they accept the Croatian legal system, customs and culture (see section 11.2.1.3 for further de- tails). Between 1991 and 1993 decisions on applications, which are made by the Ministry of Internal Affairs, were discretionary, since the Ministry was not obliged to state its reasons for refusing a request. In CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 345 1993 the Constitutional Court ordered the Ministry in charge to begin explaining its decisions.25 The ethnocentric features of the 1991 Citizenship Law were con- firmed again in the transitional provisions, determining the initial citi- zenry of Croatia and including a special mode of acquiring citizenship for ethnic Croats who were registered but did not possess Croatian re- public-level citizenship. They could acquire Croatian citizenship by de- claration, i.e. by issuing a written statement to the police that they con- sidered themselves Croatian citizens. 11.1.2 Croatian citizenship since 1991 11 facilitated the naturalisation of the Croatian eth- nic emigrants and their descendents, even if they did not satisfy the conditions stated in art. 8 regarding proficiency in the Croatian lan- guage. g g Since legal continuity with previous citizenship of the Socialist Re- public of Croatia was the determining factor for the establishment of the initial citizenry of the newly independent state, the Republican Re- gistrar’s Office was supposed to issue certificates on Croatian citizen- ship. Problems occurred, however, when an individual was registered but his or her republican citizenship was not Croatian (for instance, the father’s republican citizenship was sometimes used to determine the republican citizenship of a child), or if no republican citizenship was officially recorded. In the former cases, the persons were consid- ered aliens and had to apply for naturalisation, whereas the latter were sent to police agencies to have their citizenship determined or were al- lowed to register as Croatian citizens – according to art. 30, para. 2 – if they were able to prove Croat ethnic origins (UNHCR 1997). If they were not able to provide the necessary proof (or were simply of a differ- ent ethnicity), they were considered aliens by law. But how could some- one actually prove his or her Croat ethnic origins? Any official docu- ment released by SFRY or republican authorities in which a person de- clared himself to be ethnically Croat usually sufficed, but sometimes more unusual documents, such as Catholic Church certificates (among South Slavs born south of Slovenia being a Roman Catholic has been considered the strongest proof of someone’s ‘Croatness’) were also ac- cepted by state authorities.28 The citizenship status of Croatia’s Serb minority in the Krajina re- gion was particularly problematic.29 Given the fact that in 1991 the Croatian Serb militia, with the help of the Yugoslav federal army, took control of almost one-third of Croatia’s territory (mostly in the Krajina region but also in Central and Eastern Slavonia), the citizenship status of the ethnic Serb population living in these regions remained unre- solved for almost a decade. Croatian Serb refugees, who fled or were forced to leave Krajina during and after the Croatian military takeover in 1995 and found themselves in Serbia or Bosnia-Herzegovina (in the Serb entity), were in a particularly difficult situation. 11.1.2 Croatian citizenship since 1991 Once the police had checked whether the individual in question had fulfilled the above require- ments, he or she was then entered into the citizenship registry (see art. 30, para. 2).26 In 1993, the Croatian Constitutional Court rejected the petition filed by the Social Democratic Union demanding the removal of the entire art. 30 on the basis of the latter’s discrimination against non-Croats in the same position. The Court stated that the Croatian Ci- tizenship Law respected international law on statelessness and that it did not threaten to ‘leave a person without citizenship’, since all SFRY citizens had to have a republic-level citizenship. Furthermore, the Court stated that the Law itself did not explicitly revoke anyone’s citi- zenship (UNCHR 1997: 17). ( 997 7) Not only does the law lay down a specific procedure of acquiring Croatian nationality for residents of Croatian ethnicity in the article de- termining the initial citizenry of Croatia, it also offers facilitated natur- alisation to emigrants and their descendents, who accept the Croatian legal system, customs and culture (art. 12). Moreover, it paves the way for ethnic Croats without previous or current residence in Croatia to obtain Croatian nationality by declaration (art. 16). There is in fact a large population of Croats living abroad. Non-resident Croats can be classified in two categories. The first category is composed of the des- cendents of emigrants who left Croatian territory. This comprises the 1880-1914 migration, mainly to the Americas. About 600,000 Croats were believed to be living in North America by 1914 (Holjevac 1968: 23). This flow continued in the 1920s and 1930s. However, it also com- prises the post-Second World War emigration of about 300,000 people (Bilandzˇic´ 1985: 9) who fled Communist Yugoslavia, as well as an esti- mated number of 1,100,000 ‘gastarbeiter’ and their descendents27 who remained in their mainly European countries of destination (Baskin 1986: 27). The second ‘diaspora’ category is composed of Croats from Bosnia and Herzegovina, who represent about 16 per cent of the total population of that country. Despite the fact that they were one of the ‘constitutive peoples’ of the Republic of Bosnia and Herzegovina, they were considered potential Croatian citizens in ‘diaspora’. Indeed, art. 16 facilitated the naturalisation of ethnic Croats living in the ‘near abroad’ (former Yugoslav republics), especially for those in Bosnia-Her- 346 FRANCESCO RAGAZZI AND IGOR SˇTIKS zegovina, while art. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 347 With the death of Croatia’s President Franjo Tudjman and the subse- quent defeat of his party (HDZ) in the 2000 parliamentary elections, Croatia declared its willingness to quickly satisfy all the conditions ne- cessary for EU membership. As a consequence, the situation regarding citizenship policy has significantly improved. Although there have as yet been no changes in the text of its citizenship law, the administrative implementation reveals a greater degree of inclusiveness towards eth- nic non-Croats – without, however, withdrawing privileges offered to ethnic Croats outside the country. Today, Croatian Serb refugees face no significant obstacles in acquiring proof of Croatian citizenship, although some issues related to the citizenship policies of the 1990s remain unresolved.30 The EU stated that the return of these refugees to Croatia and the full restitution and reparation of their material goods was an important political condition for Croatia’s membership talks. 11.2.1 Acquisition of Croatian citizenship 11.2.1 Acquisition of Croatian citizenship The 1991 Law on Croatian Citizenship as amended in 1992 offers four modes of acquiring Croatian citizenship: 2. acquisition by birth on the territory of the Republic of Croatia; 3. acquisition through naturalisation; 4. acquisition through international treaties. The last of these is not explicitly discussed in the citizenship regula- tions, since the treaties themselves regulate the modalities for acquir- ing Croatian citizenship. The only treaty of interest here is the agree- ment on dual citizenship signed by Croatia and Bosnia-Herzegovina on 29 March 2007 but not yet ratified (see section 11.2.3 below). 11.1.2 Croatian citizenship since 1991 They were all leg- ally Croatian citizens but did not possess a certificate of Croatian citi- zenship (domovnica) and, therefore, could not claim all their rights as Croatian citizen. Up until the political changes in 2000, the Croatian authorities imposed numerous obstacles to prevent ethnic Serb refu- gees from acquiring valid documents (certificates on citizenship, pass- ports, etc.) testifying to their nationality – the goal being to make their return to Croatia and the restitution of their goods impossible (see re- port on Croatia in Imeri 2006: 129-131). 11.2.1.3 Acquisition by naturalisation 11.2.1.3 Acquisition by naturalisation Naturalisation has been pointedly used to grant Croatian citizenship to former citizens of SFRY who did not fulfil the criteria of art. 30 para. 1. and para. 2 that regulate the initial determination of the Croatian citi- zenry (see section 11.1.2 for further details). Following the description of Omejec (1998), Croatian citizenship legislation foresees two modes of acquiring Croatian citizenship through naturalisation: ‘regular’ and ‘facilitated’ naturalisation. It also considers the case of minors, and the case of individuals who can be ‘reintegrated’ into the Croatian citi- zenry. 11.2.1.2 Acquisition by birth on the territory q y y As is common practice in all European states, art. 7 adds a residual di- mension of ius soli in order to prevent statelessness: a child who was born or found on the territory of the Republic of Croatia acquires Croa- tian citizenship if both of his or her parents are unknown or of un- known citizenship or if they are stateless persons. However, the child loses his or her Croatian citizenship if by the time he or she is fourteen both of his or her parents are recognised as foreign citizens. 11.2.1.1 Acquisition by descent q y As regulated in art. 4 and art. 5, the principle of descent – ius sangui- nis, the dominant principle for the acquisition of Croatian citizenship – applies when (1) both parents are Croatian citizens at the time of a child’s birth, irrespective of the place of birth; (2) one parent is a Croa- tian citizen and the child is born in the Republic of Croatia; (3) one par- ent is a Croatian citizen and the other parent is stateless or of un- known citizenship and the child is born abroad; (4) one parent is a Croatian citizen and the other parent is a foreign citizen or of un- known citizenship and the child is born abroad, provided that the child, before turning eighteen, either (a) has been registered as a resi- FRANCESCO RAGAZZI AND IGOR SˇTIKS 348 dent in the territory of the Republic of Croatia or (b) has been regis- tered with Croatian authorities, abroad or in Croatia; (5) one parent is a Croatian citizen and the other parent is a foreign citizen or of un- known citizenship and the child is born abroad, even if the child does not comply with the above mentioned conditions, if he or she would otherwise be left stateless; finally (6) a stateless child, or a child of for- eign citizenship, has access to Croatian citizenship if he or she is adopted by Croatian citizens. There is no provision in the law specify- ing the number of generations that can benefit from the acquisition of citizenship by descent. Moreover, those born abroad who do not satisfy the above conditions can acquire citizenship through two other provi- sions: art. 11 (concerning emigrants) and art. 16 (concerning ethnic Croats who do not reside in the Republic of Croatia). 11.2.1.2 Acquisition by birth on the territory Facilitated naturalisation The procedure of facilitated naturalisation is used when, in spite of the fact that some of the conditions listed above are not fulfilled, there is an intention to admit an alien into the Croatian citizenry. There are several grounds on which an alien can be naturalised in this way: 1. Art. 9 allows for granting citizenship to aliens who were born in the Republic of Croatia, have had five years of residence prior to their application and for whom it can be concluded from their beha- viour that they have respected the legal order, the customs and the culture of the Republic of Croatia. Hence, this specific group of ap- plicants does not have to fulfil conditions 1, 2 and 4 of the regular naturalisation procedure.31 2. Art. 10 allows for the spouse of a Croatian citizen with permanent residency in the Republic of Croatia to obtain Croatian citizenship, provided that it can be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia. 3. Emigrants, their descendants and their spouses are similarly granted citizenship following art. 11, even if they do ‘not meet the prerequisites from art. 8, paragraph 1, points 1-4’. An emigrant is defined as a ‘person who has emigrated from Croatia with the in- tention to live permanently abroad’. There is no specification in the law as to the number of generations entitled to apply through art. 11. This opens a possibility to all emigrants and their descendents to acquire Croatian citizenship. Candidates have to show documents proving the emigration from the territory of the Republic of Croatia, and the connection to the original emigrant (through birth and marriage certificates). g ) 4. According to art. 12, any foreign citizen, as well as his or her spouse, can be granted Croatian citizenship by the competent min- istry if this is deemed to be in the interest of the Republic of Croatia (upon condition, as always, that it can be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia). 4. According to art. Regular naturalisation Regular naturalisation g In order to obtain Croatian citizenship, an alien is required to fulfil the following requirements contained in art. 8. The foreign national must: g In order to obtain Croatian citizenship, an alien is required to fulfil the following requirements contained in art. 8. The foreign national must: 1. be at least eighteen years old when submitting his or her request; 2. have renounced any foreign citizenship, or submit proof that he or she will be released from other citizenships; 3. have had registered residence in the territory of the Republic of Croatia for at least five years; 4. be familiar with the Croatian language and the Latin alphabet. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 349 5. Moreover, it must be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia. This particular provision (art. 8.1.5) of the law has been often used in the past by the Ministry of Interior (the Min- istry in charge of determining the validity of naturalisation applica- tions) to deny Croatian citizenship to ethnic non-Croats with long- term residence in Croatia. 5. Moreover, it must be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia. This particular provision (art. 8.1.5) of the law has been often used in the past by the Ministry of Interior (the Min- istry in charge of determining the validity of naturalisation applica- tions) to deny Croatian citizenship to ethnic non-Croats with long- term residence in Croatia. 5. Moreover, it must be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia. This particular provision (art. 8.1.5) of the law has been often used in the past by the Ministry of Interior (the Min- istry in charge of determining the validity of naturalisation applica- tions) to deny Croatian citizenship to ethnic non-Croats with long- term residence in Croatia. Acquisition by declaration Art. 16, quite problematically, grants citizenship to any ‘member of the Croatian people who does not have a place of residence in the Republic of Croatia […] if he or she meets the prerequisites of art. 8.1.5 of this Law and if he or she issues a written statement that he or she consid- ers himself or herself to be a Croatian citizen.’32 Reacquisition of Croatian citizenship q f p On top of the legal dispositions for the naturalisation of aliens, there are provisions for reacquisition of citizenship by former Croatian citi- zens who have lost it. There are two main possibilities for ‘reintegrat- ing’ people into the Croatian citizenry. According to art. 15, Croatian ci- tizenship can be granted again to an individual who had to renounce his or her Croatian citizenship for another citizenship in order to ‘con- duct a profession or a business’, even if he or she does not meet the prerequisites of art. 8.1.1-4. Another case concerns individuals who have lost their citizenship as minors. According to art. 23, children of Croatian citizens whose citizenship has been revoked (art. 20) or re- nounced by their parents while they were minors (art. 22) can regain citizenship if they reside for one year in the territory of the Republic of Croatia and issue a written statement stating that they consider them- selves Croatian citizens. Facilitated naturalisation 12, any foreign citizen, as well as his or her spouse, can be granted Croatian citizenship by the competent min- istry if this is deemed to be in the interest of the Republic of Croatia (upon condition, as always, that it can be concluded from his or her behaviour that he or she respects the legal order, the customs and the culture of the Republic of Croatia). FRANCESCO RAGAZZI AND IGOR SˇTIKS 350 f The Law on Croatian Citizenship does not allow minors to acquire Croatian citizenship independently of their parents. According to art. 13, there are three possibilities for a child to acquire Croatian citizen- ship through naturalisation: (1) if both parents acquire citizenship by naturalisation; (2) if only one parent acquires citizenship by naturalisa- tion and the child lives in the Republic of Croatia; or (3) if only one par- ent acquires citizenship by naturalisation and the other is a stateless person or a person of unknown citizenship and the child is living abroad. Finally, according to art. 14, a child adopted by a Croatian citi- zen with parental effect can be naturalised according to the facilitated procedure even if he or she does not meet the prerequisites defined in art. 8.1.1-4. 11.2.2.2 Renunciation d According to Omejec (1998: 122), the purpose of the right to renounce Croatian citizenship is to not restrict the freedom of choice of those in possession of dual citizenship. Croatian citizens have the right to re- nounce citizenship if they are over eighteen, have a foreign citizenship and reside abroad. The children of these citizens, if they are minors, are considered in the same way as their parents, although they can re- claim their lost citizenship, as explained above. 11.2.2.3 Lapse or withdrawal of citizenship The Croatian citizenship regulations do not specify cases in which citi- zenship is lost against the will of the person affected. Contrary to most countries, there are no provisions concerning, for example, treason or service in a foreign army. 11.2.2 Loss of Croatian citizenship According to art. 17, there are three principal ways in which Croatian citizenship can be terminated: (1) release,33 (2) renunciation or (3) through international treaties. The last of these, as is the case for the CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 351 acquisition of citizenship, is not explicitly discussed in citizenship reg- ulations for the above-mentioned reasons. 11.2.2.1 Release Release from citizenship by the state authorities, is regulated by art. 18 and art. 19. Release from citizenship cannot be obtained by a Croatian citizen who at the moment of the request is charged and prosecuted ex officio, or as long as he or she has not served his or her sentence. In ad- dition, the citizen must be at least eighteen years old, must have ful- filled his military service obligations, must have paid taxes and must have fulfilled any obligations to his or her spouse, parents and chil- dren. Moreover, proof of a foreign citizenship or evidence that the for- eign citizenship will be granted must also be submitted. The price of this procedure is, however, unusually high for an ordinary Croatian ci- tizen. It is currently fixed at 3,600 kunas, i.e. approximately 500 euros, which is just below the average monthly wage in Croatia.34 11.2.3 Dual citizenship The question of dual citizenship is rather ambiguously treated. Two ar- ticles are in fact in partial contradiction. On the one hand, art. 2 expli- citly states that Croatian citizens may have another citizenship, even if it is not recognised by the Republic of Croatia: ‘the citizen of the Re- public of Croatia who has foreign citizenship is, before the state autho- rities of the Republic of Croatia, to be considered a Croatian citizen ex- clusively’. On the other hand, art. 8 specifies that a foreign national who intends to acquire Croatian citizenship has to renounce his or her current citizenship (art. 8.1.2). In practice, members of the Croatian ‘diaspora’ in the US, Canada, Australia, Germany and other countries FRANCESCO RAGAZZI AND IGOR SˇTIKS 352 have been able to obtain Croatian citizenship quite easily and maintain their other citizenship. The same is true for citizens of Bosnia and Her- zegovina.35 11.3 Current political debates and planned changes Since its adoption in 1991, the Croatian Law on Citizenship has been heavily criticised, particularly by NGOs and international human rights agencies, as well as by some non-nationalist political parties, for its eth- nic overtones, open discrimination against ethnic non-Croats and Croa- tia’s policy of granting its citizenship to ethnic Croats abroad, particu- larly to those living in the ‘near abroad’ in Bosnia-Herzegovina. In spite of these occasional calls for changes to the text of the law, the 1991 Law, although amended, is still in force. Nevertheless, with the accel- eration of Croatia’s membership negotiations with the EU, changes to the law itself were announced by government officials in relation to Croatia’s adoption of the European Convention on Nationality. The Croatian Parliament (Hrvatski Sabor) was supposed to adopt the European Convention on Nationality in 2006. Art. 5 of the Convention explicitly forbids discrimination on ethnic, religious or racial grounds, and since the Council of Europe had already required Croatia to change its law on citizenship, it was made clear by the Croatian authorities that the law – especially the controversial points regarding unequal treatment of individuals of non-Croat ethnicity regarding the residency requirement – would be rewritten. If the Convention had been adopted, it would have been more difficult for ethnic Croats perma- nently residing outside Croatia to obtain citizenship without satisfying the usual requirements of current residence in Croatia. Some other provisions that discriminate against non-ethnic Croatian residents should likewise have been removed. However, the ruling conservative party (HDZ, Croatian Democratic Union) blocked the adoption of the Convention in the Sabor – even though the adoption of the Convention had actually been proposed by a government dominated by the HDZ – fearing it would automatically and detrimentally influence relations be- tween Croatia and the Croat ethnic diaspora, in particular Bosnia-Her- zegovina’s Croats. Interestingly, at almost the same time in 2006, Italy adopted a law granting Italian citizenship to a number of descendants of Italian ethnic origin who live in the Slovene and Croatian territories that were annexed by Italy in the inter-war period or during the Second World War. This move provoked a fierce reaction from both Slovenia and Croatia. 11.3 Current political debates and planned changes Some senior Croatian politicians (many from the HDZ) complained that Italy had deliberately created citizens with a ‘double loyalty’, clearly forgetting that granting Croatian citizenship to ethnic CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 353 Croats from Bosnia-Herzegovina – one of three constituent peoples in that country – resulted in precisely the same kind of ‘double loyalty’. 36 y p y y y It is certain that the HDZ had in mind a controversial Croatian elec- toral law that creates a special electoral constituency for the Croatian diaspora. The vast majority of votes in this constituency come from the Bosnian Croats, who vote predominantly for the HDZ and other na- tionalist parties. The outcome of recent parliamentary elections in Croatia (November 2007) clearly reveal all of the particularities of the current situation in Croatia. The HDZ eventually won the elections by a tiny margin thanks, in large part, to the votes from the diaspora con- stituency,37 the majority of which came from Bosnia-Herzegovina. This electoral unit had been boycotted by the largest opposition party, the Social-Democratic Party and other non-nationalist and left-leaning par- ties, which continue to demand changes in the electoral law. One can thus witness parallel attempts to, on the one hand, preserve the ethno- centric character of the state – most obviously by maintaining strong ties to and influence on Croatian ethnic population in Croatia’s ‘near abroad’ – and, on the other, to demonstrate a high degree of political inclusion of ethnic minorities in conformity with the democratic norms of the EU. After the 2007 parliamentary elections, and in har- mony with this new euro-compatible face of Croatia, one of the highest positions in the government was offered to a member of the largest ethnic Serb party in Croatia for the first time since 1991. 11.4 Statistical developments It is very difficult to obtain comprehensive statistical data related to citi- zenship matters in independent Croatia. After several enquiries direc- ted at the Croatian Ministry of Interior, the authors received the follow- ing response in June 2008: In the period between 2002 and 2007, 53,095 requests for ac- quisition of Croatian citizenship were favourably resolved, and 11,321 requests were denied. During the same period, the re- quests for acquisition of Croatian citizenship submitted by 7,057 persons were suspended or rejected. In the period between 2002 and 2007, 53,095 requests for ac- quisition of Croatian citizenship were favourably resolved, and 11,321 requests were denied. During the same period, the re- quests for acquisition of Croatian citizenship submitted by 7,057 persons were suspended or rejected. We would like to underline that we are communicating the above-cited data with reservations, and that we cannot be held responsible for their accuracy. We do not have the technical facil- ities necessary to generate exact statistical data. In the above- mentioned period, there were no unified parameters for the sta- tistical treatment of data related to the acquisition of Croatian ci- tizenship. As for the period between 1991 and 2001, we are un- FRANCESCO RAGAZZI AND IGOR SˇTIKS 354 able to communicate the required data. During this period, no statistical data was produced in relation to the resolved requests for acquisition of Croatian citizenship, because there was no adequate informational system for this database. It is difficult to assess the reliability of this statement. On the one hand, the first years of independence in the Republic of Croatia were marked by war and considerable administrative reorganisation. This gives credence to the Ministry’s claims. On the other hand, the highly politicised question of Croatian citizenship, as well as the ‘ethnic engi- neering’ aspect of the citizenship policies are obviously something that the Croatian government does not want to see quantified. The authors find the statement that between 1991 and 2001 (ten years!) there was ‘no adequate informational system for this database’ surprising. The fact that another author (Omejec 1998) was able to obtain some data for that period and that these data were quoted in an UNHCR report (1997), confirms that some statistical data must be available. 11.5 Conclusions The Croatian case confirms that the dominant paradigm of ethnic citi- zenship has not been radically challenged in the Balkans, except in those countries (Bosnia-Herzegovina, Kosovo and, to a large extent, Macedonia) that are under direct international supervision and where the UN and the EU have strong civilian, police and military missions. Since 2000, however, we have generally witnessed a greater degree of inclusiveness and less discrimination on ethnic grounds, as well as an increased sensitivity to the political aspirations of ethnic minorities (most clearly in the EU candidate countries, Macedonia and Croatia). Nonetheless, in countries such as Slovenia, Croatia and Serbia, where the EU is not in a position to directly control lawmakers or the beha- viour of the state apparatus, the pressure coming from Brussels is mostly concentrated not on eventual changes in citizenship legislation, but rather on the administrative practice and political life of the coun- tries in question. In order to satisfy the political conditions for EU membership, Croa- tia is demonstrating – even in the behaviour of its leading conservative politicians – more political inclusiveness towards the Serb minority and, in general, is acting as a democratic state that does not discrimi- nate on an ethnic basis (as was the case during the 1990s). Neverthe- less, it continues to do everything it can to preserve the strong ties it has established with its diaspora (again, primarily in Bosnia-Herzegovi- na) and here Croatian citizenship granted to ethnic Croats abroad plays a crucial role. The diaspora voting machine, based mainly in the Croat- populated Western Herzegovina, has been repeatedly used by the main Croatian right-wing party (HDZ) at the time of elections as a political chip in Croatian internal politics. Nevertheless, we can conclude that, beyond electoral campaigns, Croatia’s bid for EU membership relegates the question of Croatian ethnic diaspora from the political sphere to the spheres of educational, cultural and social ties. Since Croatia seems to be on a fast track to joining the EU, it is im- portant to point out that Croatia’s membership will automatically create more than 500,000 EU citizens permanently residing in a non-EU country. The Croatian policy of granting citizenship to ethnic Croats in Bosnia-Herzegovina will thus indirectly affect all other EU Member States as well. 11.4 Statistical developments In the period from October 8, 1991, to June 30, 1995, the Minis- try of Internal Affairs of RC [Republic of Croatia] resolved 557,379 requests for determination of Croatian citizenship ac- cording to art. 30 para. 1 LCC [Law on Croatian Citizenship]. Ac- cording to the analysis, these cases mostly involved citizens of the former SFRY with a residence in SRC [Socialist Republic of Croatia] who were not registered in the citizens’ registry of SRC or were registered after February 29, 1978 yet did not have a citi- zenship recorded in the ‘republican citizenship’ section, and their citizenship was disputable. There is no data regarding how many of the 557,379 requests submitted on the grounds of art. 30 para. 1 LCC were denied. In the same period (October, 8, 1991 – June, 3, 1995) the MIA of RC resolved 394,910 requests for subsequent registration on the grounds of art. 30 para. 2 LCC. These cases mostly involved ethnically Croatian individuals who were not citizens of SRC, but who had a residence on its territory and submitted a statement that they considered them- selves to be Croatian. Therefore, in the period from October 8, 1991, to June 30, 1995, 952,331 procedures for determination of Croatian citizenship were executed on the basis of art. 30 LCC (Omejec 1998: 116).38 CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 355 www.sluzbenilist.co.rs (in Serbo-Croatian) www.sluzbenilist.co.rs (in Serbo-Croatian) 1946 Citizenship for Officers and Non-Commissioned Officers of the Former Yugoslav Army Who do not Want to Return to the Homeland, and for the Members of Military Forces Who Have Served for the Enemy and Have Defected Abroad (No. 64/1945; No. 86/ 1946) 1964 Law on Yugoslav Citizenship (No. 38/1964; No. 42/1964) 1950 Citizenship of the People’s Republic of Croatia (No. 23/1950) 1948 Amendment and Revision of the Law on Citizenship (No. 105/1948) www.sluzbenilist.co.rs (in Serbo-Croatian) www.sluzbenilist.co.rs (in Serbo-Croatian) Date Document 1947 Law on Citizenship of Persons Residing in the Territory Annexed by Yugoslavia according to the Peace Treaty with Italy (No. 104/1947) 11.5 Conclusions To sum up, the case of Croatia demonstrates how sticks and carrots employed by the EU could alter relations between a nationalising state and its internal minorities as well as between a kin state and its ethnic diaspora in the ‘near abroad’. At the same time, it shows how the latter relations can be preserved – even if they remain politically dormant – within the institutional framework of the EU. We thus witness parallel 356 FRANCESCO RAGAZZI AND IGOR SˇTIKS attempts to integrate a country into the supranational institutions of the EU, democratise its political life and clearly show political and so- cial inclusiveness towards ethnic minorities, but also to maintain a transnational ethnic community by using ethno-centric citizenship laws. Chronological list of citizenship-related legislation in Yugoslavia/Croatia Date Document Content Source 1811/ 1867 Austrian Civil Code (arts. 28-32) Regulates the citizenship status of citizens living in Dalmatia placed under the Austrian rule within the Austro-Hungarian Empire 1879 Law on Hungarian Citizenship (Article ‘L’) Regulates the citizenship status of citizens living in Croatia and Slavonia placed under Hungarian rule within the Austro- Hungarian Empire 1928 Law on Citizenship of the Kingdom of Yugoslavia Establishes a single Yugoslav citizenship throughout the Kingdom 1945/ 1946 Law on Citizenship of the Democratic Federal Yugoslavia (No. 64/1945; No. 54/1946) Defines the initial citizenry of the newly formed Yugo- slavia; establishes Yugo- slav federal citizenship and republic-level citizen- ship of the six constituent republics; is confirmed and amended after the adoption of the 1946 Con- stitution of the Federal People’s Republic of Yugo- slavia (54/1946) www.sluzbenilist.co.rs (in Serbo-Croatian) 1945/ 1946 Law on Deprivation of Citizenship for Officers and Non-Commissioned Officers of the Former Yugoslav Army Who do not Want to Return to the Homeland, and for the Members of Military Forces Who Have Served for the Enemy and Have Defected Abroad (No. 64/1945; No. 86/ 1946) Deprives Yugoslav citizenship of those military personnel of the former army of the Kingdom of Yugoslavia who did not want to return to the ‘new’ Yugoslavia and those serving in military formations loyal to the occupying forces who left Yugoslavia at the end of the Second World War; is revoked in 1962 (No. 22/ 1962) www.sluzbenilist.co.rs (in Serbo-Croatian) Source 1977 Law on Citizenship of the Socialist Republic of Croatia (No. 32/1977) 1991 Law on Croatian Citizenship (No. 53/1991) www.nn.hr (in Croatian); www.legislationline.org (as amended in 1992) 1976 Law on Citizenship of the Socialist Federal Republic of Yugoslavia (No. 58/1976) www.sluzbenilist.co.rs (in Serbo-Croatian) CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 357 Date Document Content Source 1947 Law on Citizenship of Persons Residing in the Territory Annexed by Yugoslavia according to the Peace Treaty with Italy (No. 104/1947) Determines that persons residing in territories annexed by Yugoslavia on 10 June 1940 are to lose their Italian citizenship and acquire Yugoslav citizenship; provides a one-year period for ethnic Italians to opt for Italian citizenship and a possibility for the Slav population from the contested borderland region between Yugoslavia and Italy to opt for Yugoslav citizenship www.slu (in Serb 1948 Amendment and Revision of the Law on Citizenship (No. 105/1948) Excludes from the Yugoslav citizenry all citizens of German ethnicity residing abroad on the basis of their ‘disloyal conduct toward the national and state interests of the peoples of FPRY’ www.slu (in Serb 1950 Citizenship of the People’s Republic of Croatia (No. 23/1950) Defines ius sanguinis as the basic principle for acquisition of Croatian citizenship; specifies conditions for a newborn child to acquire Croatian citizenship if the parents hold different republic- level citizenships, depending on their residency 1964 Law on Yugoslav Citizenship (No. 38/1964; No. 42/1964) Enacted after the 1963 Yugoslav Constitution; regulates the relationship between the federal and republic-level citizenships; provides for a united Yugoslav citizenship; regulates that only a federal citizen can hold republican citizenship, and that the republican citizenship is lost with the loss of the federal citizenship www.slu (in Serb FRANCESCO RAGAZZI AND IGOR SˇTIKS 358 Date Document Content Source 1965 Law on Citizenship of the Socialist Republic of Croatia (No. 13/1965) Confirms ius sanguinis as the main criterion for automatically acquiring Croatian citizenship; unlike the 1950 Law, stipulates that both parents have to agree on the child’s citizenship if they hold different republic-level citizenships; guarantees that any SFRY citizen has a right to choose his or her republic-level citizenship and is thus free to choose citizenship of any republic without being born or residing there 1976 Law on Citizenship of the Socialist Federal Republic of Yugoslavia (No. 58/1976) Is adopted after the 1974 Yugoslav constitution; repeats the provisions from the 1964 Law on the relationship among the federal and republic-level citizenships; adds an article on how to resolve possible disputes caused by the republican laws on citizenship www.sluzbenilist. (in Serbo-Croatia 1977 Law on Citizenship of the Socialist Republic of Croatia (No. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 359 Date Document Content Source 1992 Law on Modifications and Amendments of the Law on Croatian Citizenship (No. 28/1992) Corrects the text of the 1991 Law; amends inconsistencies and legally problematic provisions; specifies the administrative procedures www.nn.hr (in Croatian) 1993 Decree of the Constitutional Court of the Republic of Croatia U-I- 206/1992, U-I-207/1992, U-I-209/1992, U-I-222/ 1992 of 8 December 1993 (No. 113/93) Orders the Ministry of Interior to justify its decisions in case of the refusal of requests made through the regular naturalisation procedure (art. 8) www.nn.hr (in Croatian) Notes 1 The authors thank Iris Goldner for her useful comments. 2 Hrvatska Demokratska Zajednica – Croatian Democratic Union. 2 Hrvatska Demokratska Zajednica – Croatian Democratic Union. 3 For a detailed analysis of the citizenship legislation and practices in other former Yugoslav states since 1991, see Sˇtiks 2006. For a study of Slovenia’s citizenship legislation, see also Medved in this volume. 4 For more on Croatia’s particular brand of ‘transnational nationalism’, see Ragazzi 2009; for the notion of transnational nationalism, see Basch, Glick-Schiller & Szanton Blanc 1995 and Kastoryano 2006. 4 For more on Croatia’s particular brand of ‘transnational nationalism’, see Ragazzi 2009; for the notion of transnational nationalism, see Basch, Glick-Schiller & Szanton Blanc 1995 and Kastoryano 2006. 995 y 5 International law itself does not question the right of sovereign states to enact their own nationality policy. However, international law, declarations and treaties do seek to impose certain rules and thereby influence the behaviour of states when it comes to citizenship and nationality legislation and administrative practice. Art. 15 of the 1948 Universal Declaration of Human Rights states that ‘everyone has the right to a nationality’ and that ‘no one shall be arbitrarily deprived of his nationality nor denied the right to change his nationality.’ The European Commission for Democracy through Law (also known as the Venice Commission), the Council of Europe’s advisory body for constitutional issues, adopted The Declaration on Consequences of State Succession for the Nationality of Natural Persons in September 1996. It states that, besides respecting the principle that every person has a right to a nationality and the general prevention of statelessness, states should ‘respect, as far as possible, the will of the person concerned.’ It also repeats that ‘in all cases of State succession, the successor State shall grant its nationality to all nationals of the predecessor State residing permanently on the transferred territory.’ In a similar fashion, art. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 32/1977) Confirms ius sanguinis as the main criterion for automatically acquiring Croatian citizenship; specifies conditions of citizenship acquisition in the case in which the parents do not agree or do not sign a statement during two months following the birth of their child 1991 Law on Croatian Citizenship (No. 53/1991) Defines the citizenship of the Republic of Croatia; determines the initial body of citizens according to the principles of legal continuity and Croatian ethnicity; defines all modes of acquiring and losing nationality after independence www.nn.hr (in Cr www.legislationlin (as amended in 1 Date Document 1965 Law on Citizenship of the Socialist Republic of Croatia (No. 13/1965) Notes 18 of the 1997 European Convention on Nationality, prepared by the Council of Europe, declares that, in the case of succession, states should take into account ‘the genuine and effective link of the person concerned with the State’ and ‘the habitual residence of the person concerned at the time of State succession.’ 6 6 The most important treaties for citizenship issues in the Croatian lands were the Peace Treaty of St.-Germain-en-Laye with the Republic of Austria, signed on 10 September 1919, and the Trianon Peace Treaty with Hungary, signed on 4 June 1920. 6 The most important treaties for citizenship issues in the Croatian lands were the Peace Treaty of St.-Germain-en-Laye with the Republic of Austria, signed on 10 September 1919, and the Trianon Peace Treaty with Hungary, signed on 4 June 1920. 360 FRANCESCO RAGAZZI AND IGOR SˇTIKS FRANCESCO RAGAZZI AND IGOR SˇTIKS 7 Official Gazette of Democratic Federal Yugoslavia 64/1945. The law was confirmed and amended on 5 July 1946 (see Official Gazette of the Federal People’s Republic of Yugosla- via (FPRY) 54/1946). The law was further amended and revised in 1947 (see Official Gazette of the FPRY 104/1947) and twice in 1948 (see Official Gazette of the FPRY 88 and 105/1948). 7 Official Gazette of Democratic Federal Yugoslavia 64/1945. The law was confirmed and amended on 5 July 1946 (see Official Gazette of the Federal People’s Republic of Yugosla- via (FPRY) 54/1946). The law was further amended and revised in 1947 (see Official Gazette of the FPRY 104/1947) and twice in 1948 (see Official Gazette of the FPRY 88 and 105/1948). 8 Official Gazette of the FPRY 86/1948 and 22/1962. 8 Official Gazette of the FPRY 86/1948 and 22/1962. 9 Official Gazette of the FPRY 105/1948. 10 Official Gazette of the FPRY 104/1947. 11 Official Gazette of the FPRY Supplement No. 6/1954. 11 Official Gazette of the FPRY Supplement No. 6/1954. 12 This was not the case in two other socialist multinational federations. Republic-level citizenship was established in Czechoslovakia only in 1969 and the first Soviet republic that enacted its own citizenship law was Lithuania in November 1989. 12 This was not the case in two other socialist multinational federations. Republic-level citizenship was established in Czechoslovakia only in 1969 and the first Soviet republic that enacted its own citizenship law was Lithuania in November 1989. p p 9 9 13 The fact that only republic-level registers existed at the moment of Yugoslavia’s break-up would prove to be very important, because all Yugoslav republics would adopt a policy of legal continuity between previous republic-level citizenship and citizenship of the new state. Only those granted Yugoslav citizenship at a Yugoslav embassy who were residing abroad were not included in the republic-level registers. Once they established their residence in Yugoslavia, they were also entered into the register of the republic in which they resided. 14 In the 1963 Constitution, the FPRY was renamed the Socialist Federal Republic of Yugoslavia (SFRY). 14 In the 1963 Constitution, the FPRY was renamed the Socialist Federal Republic of Yugoslavia (SFRY). 15 Official Gazette of the Socialist Federal Republic of Yugoslavia (SFRY) 38/1964 (cor- rected version in 42/1964). FRANCESCO RAGAZZI AND IGOR SˇTIKS 15 Official Gazette of the Socialist Federal Republic of Yugoslavia (SFRY) 38/1964 (cor- rected version in 42/1964). 16 Official Gazette of the SFRY 58/1976. 17 Official Gazette of the People’s Republic of Croatia 23/1950. 18 Official Gazette of the Socialist Republic of Croatia 13/1965. 19 Official Gazette of the Socialist Republic of Croatia 32/1977. 20 Obviously, between 1965 and 1977 the automatic acquisition of republic-level citizenship was not a rule if only one parent had Croatian citizenship, even if a child was born in Croatia (on changes in the Croatian law on republic-level citizenship and administrative practices between 1950 and 1991, see the report on Croatia in UNHCR 1997). 1 Official Gazette of the Republic of Croatia 21/199 22 Official Gazette of the Republic of Croatia 53/91; modifications and amendments in Of- ficial Gazette of the Republic of Croatia 28/92. These amendments were mainly correc- tions of inconsistencies in the law, or legal clarifications of its provisions, which were written and adopted hastily in the context of Croatia’s declaration of independence from SFRY and its open conflict with Belgrade. Some changes were obviously made after complaints were received from the ground about the implementation of the law. An important amendment was that the renunciation of foreign citizenship required for naturalisation was eased (see note 24). 22 Official Gazette of the Republic of Croatia 53/91; modifications and amendments in Of- ficial Gazette of the Republic of Croatia 28/92. These amendments were mainly correc- tions of inconsistencies in the law, or legal clarifications of its provisions, which were written and adopted hastily in the context of Croatia’s declaration of independence from SFRY and its open conflict with Belgrade. Some changes were obviously made after complaints were received from the ground about the implementation of the law. An important amendment was that the renunciation of foreign citizenship required for naturalisation was eased (see note 24). 23 These ‘conditions’ were imposed on ethnic non-Croats coming from other Yugoslav republics. They also provided a basis for the Ministry in charge to refuse Croatian citizenship to certain individuals, non-Croats from other republics, but also to some Croatian Serbs (see 11.2.1.3 for further details). 23 These ‘conditions’ were imposed on ethnic non-Croats coming from other Yugoslav republics. FRANCESCO RAGAZZI AND IGOR SˇTIKS This was certainly a somewhat delicate matter since a large percentage of ‘Croatian’ family names are shared by Serbs and other South Slavic groups (see also the report on Croatia in Imeri 2006: 127). 28 If a person did not declare himself or herself an ethnic Croat in official documents such as a birth certificate or a marriage certificate, or if a person had declared ethnicity as Yugoslav and/or was born in a so-called ‘mixed marriage’, the state authorities (the Ministry of the Interior) established a person’s membership of the Croatian people by using Catholic Church certificates (if available) and even passed judgement on the ‘Croatness’ of a person’s family name. This was certainly a somewhat delicate matter since a large percentage of ‘Croatian’ family names are shared by Serbs and other South Slavic groups (see also the report on Croatia in Imeri 2006: 127). 7) 29 A significant number of the Croatian Serbs continued to live in territory controlled by the Croatian authorities. They managed to regulate their status either smoothly (i.e. as holders of the former Croatian republican citizenship they were automatically registered into the new registries of citizens), or in some cases, with considerable difficulties. Numerous reports testify to cases of violations of their right to Croatian citizenship in the 1990s. See, for instance, reports on the issue published in Dika, Helton and Omejec 1998 and also the report on Croatia in Imeri 2006. 30 For more details on the present situation and descriptions of some concrete cases, see the report on Croatia published in Imeri (2006: 97-123). The report points out that, for instance, the status of persons of non-Croat ethnic origin who were permanent residents of Croatia before the 1991 Law on citizenship still awaits regulation. 31 This article was modified by art. 4 of the Law on Modifications and Amendments of the Law on Croatian Citizenship (Official Gazette of the Republic of Croatia 28/1992), deleting the five years of permanent residence requirement demanded in the first version of the law. As the law stood in 1991, those born in the country who could ap- ply for facilitated naturalisation had to fulfil a longer residence requirement than those applying for regular naturalisation. To obtain permanent residence one first has to prove five years of temporary residence, according to the new Law on Aliens. FRANCESCO RAGAZZI AND IGOR SˇTIKS They also provided a basis for the Ministry in charge to refuse Croatian citizenship to certain individuals, non-Croats from other republics, but also to some Croatian Serbs (see 11.2.1.3 for further details). 24 The 1992 amendments, however, facilitated access to Croatian citizenship for those who, for various reasons, are unable to obtain release from their previous citizenship. Following these amendments, applicants have to state that they will renounce their previous citizenship, if granted Croatian citizenship. 24 The 1992 amendments, however, facilitated access to Croatian citizenship for those who, for various reasons, are unable to obtain release from their previous citizenship. Following these amendments, applicants have to state that they will renounce their previous citizenship, if granted Croatian citizenship. 25 Official Gazette of the Republic of Croatia 113/1993. 26 The 1991 Law additionally required ten years of residence for this group, which was not in accordance with art. 8 and art. 16 and was therefore corrected in the 26 The 1991 Law additionally required ten years of residence for this group, which was not in accordance with art. 8 and art. 16 and was therefore corrected in the 26 The 1991 Law additionally required ten years of residence for this group, which was not in accordance with art. 8 and art. 16 and was therefore corrected in the CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 361 amendments adopted only seven months later on 8 May 1992. Applicants merely had to prove that they were registered as residents (see Official Gazette of the Republic of Croatia 28/1992). amendments adopted only seven months later on 8 May 1992. Applicants merely had to prove that they were registered as residents (see Official Gazette of the Republic of Croatia 28/1992). 27 Guestworkers, or ‘workers temporarily employed abroad’, according to the official Yugoslav terminology. 27 Guestworkers, or ‘workers temporarily employed abroad’, according to the official Yugoslav terminology. 28 If a person did not declare himself or herself an ethnic Croat in official documents such as a birth certificate or a marriage certificate, or if a person had declared ethnicity as Yugoslav and/or was born in a so-called ‘mixed marriage’, the state authorities (the Ministry of the Interior) established a person’s membership of the Croatian people by using Catholic Church certificates (if available) and even passed judgement on the ‘Croatness’ of a person’s family name. FRANCESCO RAGAZZI AND IGOR SˇTIKS This provision was enacted for the first time during the 1995 elections, during which the ruling party, the HDZ, decided that the seats attributed to the ‘diaspora’ should represent 10 per cent of the representatives, namely twelve seats. After many debates, this was changed in 1999 and the seats were apportioned according to voter turnout. This secured only six seats for the ‘diaspora’ vote in the 2000 and 2007 elections. 38 The UNHCR report quotes the same data, but adds that ‘a total of 412,137 requests were submitted to the Ministry of Internal Affairs between 8 October 1991 and 31 December 1995 under article 30 paragraph 2.’ This slightly changed the total number of processed requests through art. 30, paras. 1 and 2, available in Omejec (1998) and fixed it at 969,553 at the end of 1995 (UNHCR 1997: 16). Indeed, Jasna Omejec is quoted as a co-author of the national report on Croatia published in UNHCR. Although published in 1998, her own article was obviously written before the UNHCR report published in 1997. 38 The UNHCR report quotes the same data, but adds that ‘a total of 412,137 requests were submitted to the Ministry of Internal Affairs between 8 October 1991 and 31 December 1995 under article 30 paragraph 2.’ This slightly changed the total number of processed requests through art. 30, paras. 1 and 2, available in Omejec (1998) and fixed it at 969,553 at the end of 1995 (UNHCR 1997: 16). Indeed, Jasna Omejec is quoted as a co-author of the national report on Croatia published in UNHCR. Although published in 1998, her own article was obviously written before the UNHCR report published in 1997. 38 The UNHCR report quotes the same data, but adds that ‘a total of 412,137 requests were submitted to the Ministry of Internal Affairs between 8 October 1991 and 31 December 1995 under article 30 paragraph 2.’ This slightly changed the total number of processed requests through art. 30, paras. 1 and 2, available in Omejec (1998) and fixed it at 969,553 at the end of 1995 (UNHCR 1997: 16). Indeed, Jasna Omejec is quoted as a co-author of the national report on Croatia published in UNHCR. Although published in 1998, her own article was obviously written before the UNHCR report published in 1997. FRANCESCO RAGAZZI AND IGOR SˇTIKS In practice this would have thus meant that a person born in the country had to prove ten years of residence in the country, whereas those applying for regular natur- alisation only had to prove five. However, the modification does not imply that those born in the country do not have to fulfil any residence requirement; they now have to prove five years of registered residence just as those applying for regular naturali- sation. It is important here not to confuse this modification with another important modification of residence requirements introduced by art. 13 of the same amend- ments, which deleted the ten years residence required in art. 30 para. 2 of the 1991 Law defining a specific procedure of acquiring Croatian nationality by declaration for ethnic Croats (see note 26). The authors would like to thank Iris Goldner for this clarification. 32 It also adds that ‘the statement from paragraph 1 of this Article shall be given before the competent authority or before the diplomatic or consular office of the Republic of Croatia abroad’. 32 It also adds that ‘the statement from paragraph 1 of this Article shall be given before the competent authority or before the diplomatic or consular office of the Republic of Croatia abroad’. 33 ‘Revocation’ in the text of the law. 34 For more information on the procedure, see: www.mup.hr. The average net salary in 2007 was 4,841 kuna per month (see Central Bureau of Statistics, Statistical informa- tion 2008. www.dzs.hr). 362 FRANCESCO RAGAZZI AND IGOR SˇTIKS 35 Art. 4 of the Law on Citizenship of Bosnia-Herzegovina allows Bosnian citizens to hold a citizenship of another country provided there is a bilateral agreement. The question of the dual (Croatian and Bosnian) citizenship of many citizens of Bosnia- Herzegovina was regulated by the agreement on dual citizenship signed by Croatia and Bosnia-Herzegovina on 29 March 2007. The ratification is still pending. 35 Art. 4 of the Law on Citizenship of Bosnia-Herzegovina allows Bosnian citizens to hold a citizenship of another country provided there is a bilateral agreement. The question of the dual (Croatian and Bosnian) citizenship of many citizens of Bosnia- Herzegovina was regulated by the agreement on dual citizenship signed by Croatia and Bosnia-Herzegovina on 29 March 2007. The ratification is still pending. 36 See ‘Talijani u RH i Hrvati u BiH nemaju ista prava?!’ [Italians in Croatia and Croats in Bosnia-Herzegovina Do Not Have Equal Rights?!], www.tportal.hr, 10 March 2006. 36 See ‘Talijani u RH i Hrvati u BiH nemaju ista prava?!’ [Italians in Croatia and Croats in Bosnia-Herzegovina Do Not Have Equal Rights?!], www.tportal.hr, 10 March 2006. 37 Art. 45 of the 1990 Constitution granted Croats abroad the right to vote. This provision was enacted for the first time during the 1995 elections, during which the ruling party, the HDZ, decided that the seats attributed to the ‘diaspora’ should represent 10 per cent of the representatives, namely twelve seats. After many debates, this was changed in 1999 and the seats were apportioned according to voter turnout. This secured only six seats for the ‘diaspora’ vote in the 2000 and 2007 elections. 37 Art. 45 of the 1990 Constitution granted Croats abroad the right to vote. This provision was enacted for the first time during the 1995 elections, during which the ruling party, the HDZ, decided that the seats attributed to the ‘diaspora’ should represent 10 per cent of the representatives, namely twelve seats. After many debates, this was changed in 1999 and the seats were apportioned according to voter turnout. This secured only six seats for the ‘diaspora’ vote in the 2000 and 2007 elections. 37 Art. 45 of the 1990 Constitution granted Croats abroad the right to vote. Bibliography Basch, L. G., N. Glick-Schiller & C. Szanton Blanc (1995), Nations Unbound: Transna- tional Projects, Postcolonial Predicaments, and Deterritorialized Nation States. Amster- dam: Gordon and Breach. Baskin, M. A. (1986), Political Innovation and Policy Implementation in Yugoslavia: The Case of Worker Migration Abroad. Ph.D. dissertation. University of Michigan, Ann Ar- bor. Bilandzˇic´, D. (1985), Historija Socijalisticˇke Federativne Republike Jugoslavije: Glavni Procesi 1918-1985 [History of the SFRY: Main Processes 1918-1985]. Zagreb: Sˇkolska knjiga. Dika, M., A.C. Helton & J. Omejec (eds.) (1998), ‘The Citizenship Status of Citizens of the Former SFR Yugoslavia after its Dissolution’, Croatian Critical Law Review 3 (1-2): 1-259. Holjevac, V. (1968), Hrvati Izvan Domovine [Croats outside of the Homeland]. Zagreb: Matica Hrvatska. Hondius, F.W. (1968), The Yugoslav Community of Nations. The Hague: Mouton. Imeri, Sh. (ed.) (2006), Rule of Law in the Countries of the Former SFR Yugoslavia and Al- bania: Between Theory and Practice. Gostivar: Association for Democratic Initiatives. Jovanovic´, S.Ð. (1977), Drzˇavljanstvo Socijalisticˇke Federative Republike Jugoslavije [Citizen ship of the Socialist Federal Republic of Yugoslavia]. Belgrade: Sluzˇbeni List SFRJ. Kastoryano, R. (2006), ‘Vers un nationalisme transnational. Rede´finir la nati nalisme et le territoire’, Revue franc¸aise de science politique 4 (56): 533-555. Medvedovic´, D. (1998), ‘Federal and Republican Citizenship in the Former SFR Yugosla- via at the Time of its Dissolution’, Croatian Critical Law Review 3 (1-2): 21-56. Omejec, J. (1998), ‘Initial Citizenry of the Republic of Croatia at the Time of the Dissolu- tion of Legal Ties with the SFRY, and Acquisition and Termination of Croatian Citi- zenship’, Croatian Critical Law Review 3 (1-2): 99-128. CROATIAN CITIZENSHIP: FROM ETHNIC ENGINEERING TO INCLUSIVENESS 363 Ragazzi, F. (2009), ‘The Croatian “Diaspora Politics” of the 1990s: Nationalism Un- bound?’ in U. Brunnbauer (ed.), Transnational Societies, Transterritorial Politics. Migra- tions from the (Post)Yugoslav Area. New York: Palgrave (forthcoming). Sˇtiks, I. (2006), ‘Nationality and Citizenship in the Former Yugoslavia: From Disintegra- tion to the European Integration’, South East European and Black Sea Studies, 6 (4): 483-500. Tepic´, Ð. & I. Basˇic´ (eds.) (1969), Zbirka propisa o drzˇavljanstvu [The Collection of the Ci- tizenship Regulations]. Zagreb: Narodne novine. UNHCR, Regional Bureau for Europe (1997), Citizenship and Prevention of Statelessness Linked to the Disintegration of the Socialist Federal Republic of Yugoslavia. European Ser- ies 3 (1; June 1997). Geneva: UNHCR. 12 Malta’s citizenship law: Evolution and current regime1 Eugene Buttigieg Malta’s legal regime on citizenship is relatively young as it came into being on the day of Malta’s acquisition of independence from British rule in 1964. Throughout these years, however, particularly over the past two decades, it has undergone extensive alterations marking changes in the governing principles. This chapter first traces the evolu- tion of the citizenship laws during these years, noting the important policy changes, their possible causes and implications. It then explores the different modes of acquisition and loss of citizenship under the current regime. Finally, statistical data is produced to highlight the ex- tent to which persons seeking to acquire or reacquire Maltese citizen- ship by registration or naturalisation benefited from these legislative changes, apart from the non-quantifiable number of persons who through these legislative changes acquired or reacquired citizenship automatically. 12.1 Historical background Malta was a British colony from 1800 until 21 September 1964 when it acquired independence from British rule. All persons born in Malta during this period were automatically British subjects according to Brit- ish law. It was thus only on Independence Day, 21 September 1964, that Malta acquired its first provisions conferring and regulating Mal- tese citizenship. The Constitution of Malta, that entered into force on Independence Day, contained provisions conferring Maltese citizenship that were typical of independence constitutions drafted by the British for their colonies. The Constitution contained a section, chapter III, on citizenship, that conferred Maltese citizenship automatically on all per- sons who were born in Malta and were citizens of the United Kingdom and Colonies before 21 September 1964, provided that one of the par- ents was also born in Malta; thus a combined application of the ius soli and ius sanguinis principles. This was necessary to avoid imposing Maltese citizenship on children born in Malta to British military per- sonnel families and British nationals stationed in Malta, while Malta was a British colony. Persons born abroad also acquired Maltese citi- 368 EUGENE BUTTIGIEG EUGENE BUTTIGIEG zenship on 21 September 1964 provided the father and a paternal grandparent were both born in Malta. On the other hand, persons born on or after the date of indepen- dence acquired Maltese citizenship by mere birth in Malta irrespective of whether or not any of their parents were born in Malta; in other words on the strength of the ius soli principle only. In practice, this meant that children born of foreign parents in Malta acquired Maltese citizenship by birth even if they were not of Maltese descent. p y y Chapter III of the Constitution also established that a Maltese citizen should have no other citizenship. Adults in possession of another citi- zenship had to renounce it by 21 September 1967. If a minor who was a Maltese citizen possessed any other citizenship, upon reaching his or her eighteenth birthday, he or she would have had to renounce any other citizenship within a year if he or she wished to retain Maltese ci- tizenship. 12.1.1 The 1989 amendments Although throughout the years various amendments were made to all these laws, necessitated inter alia by Malta’s transformation into a re- public on 13 December 1974, the first major reform in the citizenship laws took place in August 1989 when chapter III of the Constitution, the Maltese Citizenship Act and the Immigration Act (via Acts XXIII, XXIV and XXV of 1989 respectively) were radically amended to indi- cate a clear change of policy regarding citizenship by (i) making an ex- ception to the prohibition against dual citizenship for emigrants born in Malta and who had spent at least six years abroad2 – this had signifi- cant implications as, especially in the 1950s and 1960s, well over 100,000 Maltese citizens (more than one fourth of the current popula- tion) had emigrated to countries such as the United Kingdom, Austra- lia, Canada and the United States to seek employment and thereby ob- tained a second citizenship (ii) shifting to a rule based more on ius sanguinis than on ius soli (iii) allowing Maltese mothers to transmit their citizenship to their children born abroad (iv) granting the same rights to foreign husbands of citizens of Malta as foreign wives of citi- zens of Malta by allowing them to be registered as citizens of Malta and (v) reintroducing acquisition of citizenship by adoption. This change in policy was due to the influence of changing interna- tional trends favouring ius sanguinis over ius soli and a greater inter- national acceptance of dual and multiple citizenship as well as the in- creasing recognition at the international level of the need to safeguard gender equality in the citizenship laws. Malta has always participated actively in international fora and endorsed international instruments in this field and has moulded its policy accordingly. Moreover, the govern- ment had been elected in 1987 on the strength of an electoral mandate that included the promise to allow expatriates to regain their lost citi- zenship retrospectively by acquiring dual citizenship and that citizen- ship laws would guarantee gender equality. A number of overseas asso- ciations representing expatriates also exerted pressure for this conces- sion to expatriates to be extended to further generations. g As a result of these amendments, Maltese emigrants were now al- lowed to hold dual citizenship. Art. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 369 12.1 Historical background Moreover, Maltese adults who acquired the citizenship of any other country would have automatically forfeited Maltese citizen- ship while foreigners who acquired Maltese citizenship by registration or naturalisation would have had to renounce any other citizenship held by them within six months from registration or three months from naturalisation. Not unlike the general policy worldwide at the time, in the case of children born abroad, the question whether the child would acquire Maltese citizenship or not depended on whether it was the father or the mother who possessed Maltese citizenship at the time of birth. If the father was Maltese (by birth in Malta, by registration, or by natura- lisation) though not the mother, the child would acquire Maltese citi- zenship but if the father was non-Maltese even though the mother was Maltese the child would not acquire Maltese citizenship. Thus, a Mal- tese mother could not transmit her citizenship to her child born out- side Malta unless she was unmarried. Likewise, consistent with the in- ternational trend at the time, while the foreign wife of a Maltese citizen was entitled to become a citizen of Malta by registration, a foreign hus- band of a Maltese citizen was not. The first law that complemented the Constitution on citizenship matters was the Maltese Citizenship Act (chapter 188 of the Laws of Malta) that was enacted the following year in 1965. This regulated in particular the acquisition of Maltese citizenship by registration and nat- uralisation. The law was prejudiced in favour of Commonwealth citi- zens as the latter could acquire Maltese citizenship by registration after five years of residence in Malta, while other foreigners required six years of residence in Malta to acquire Maltese citizenship by naturalisa- tion. The next development in this field was the enactment of the Im- migration Act (chapter 217 of the Laws of Malta) in 1970 that laid down rules providing for the control of immigration into Malta. 12.1.1 The 1989 amendments 27(3) of the Constitution was amended to enable Maltese emigrants born in Malta to hold dual citi- zenship, provided of course that the country of which they were citi- zens recognised the concept of dual citizenship. This applied retrospec- tively. A Maltese citizen born in Malta who, as the law stood at the time, had automatically lost his Maltese citizenship upon emigrating and acquiring the citizenship of the country to which he had emi- grated, would now be deemed never to have lost his Maltese citizen- ship, provided he had spent at least six years in that country. Thus, his 370 EUGENE BUTTIGIEG dual citizenship would be backdated to the date when he acquired the foreign citizenship. This also affected children born of a Maltese emi- grant father who had lost his Maltese nationality because he had ac- quired another nationality. Since the dual nationality would be back- dated so that the father is deemed never to have lost his Maltese citi- zenship, children who were born of fathers who had ‘lost’ their Maltese citizenship at the time of their birth and who were therefore deemed not to be Maltese citizens, also acquired Maltese citizenship with this amendment, effective from their date of birth, once their fathers were reinstated in their previous status as citizens of Malta. p It should be noted that under the current legislation, only Maltese persons habitually resident in Malta have voting rights in national gen- eral elections and voting does not take place abroad in Malta’s embas- sies or consulates. So this extension of citizenship to expatriates does not signify any right to participate in the process of democratic self- determination of the country. As stated above, under the Constitution, anyone born in Malta auto- matically became a citizen of Malta by mere birth in the country. How- ever, with the 1989 amendments to the Constitution this has changed, as these amendments limit such acquisition by adding the ius sangui- nis to the ius soli criterion in establishing that, as from the coming into force of these amendments on 1 August 1989, a person born in Malta will acquire Maltese citizenship only if at least one of the parents is a citizen of Malta or was born in Malta and emigrated and enjoys freedom of movement in Malta in terms of art. 44 of the Constitution. 12.1.1 The 1989 amendments The amendments also removed gender inequality in two respects: (i) in relation to Maltese mothers of children born abroad and (ii) with re- spect to foreign men married to Maltese women. p g Prior to 1989 Maltese citizenship was transmitted to the children only if the father was a Maltese citizen. However, with these amend- ments it now suffices that either of the parents is a Maltese citizen (by birth in Malta, by registration, or by naturalisation). The Maltese mother just as the Maltese father may now transmit citizenship to her children born abroad. Before the 1989 amendments to the Constitution, while a foreign woman married to a citizen of Malta or to someone who became a citi- zen of Malta was entitled to acquire Maltese citizenship by registration, a foreign husband of a female Maltese citizen was not. This was there- fore discriminatory against foreign husbands as compared to foreign wives. The amendments extended this right to foreign husbands of Maltese citizens so that they are now on a par with foreign wives of Maltese citizens. Moreover, this right now extends even to the widow or widower of a person who was a citizen of Malta at the time of death MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 371 or would have been on 21 September 1964 had he or she lived till that day. Another significant change in policy is related to the acquisition of citizenship by adoption. Until 1976 it was possible under Maltese law to acquire Maltese citizenship through adoption, i.e. a person lawfully adopted by a citizen of Malta would acquire Maltese citizenship by that adoption. This was no longer possible following a legislative amend- ment on 1 January 1977. In 1989, the amendments to the Constitution reintroduced this facility for the acquisition of citizenship through adoption, subject to the proviso that the child adopted must be under ten years of age on the date of adoption. y g p In 1989, the distinction made in the Maltese Citizenship Act be- tween Commonwealth citizens and other foreigners for the acquisition of Maltese citizenship by residence in Malta, a remnant of British in- fluence, was abolished, so now any person may be naturalised as a citi- zen of Malta if he or she has resided in Malta for at least five years. 12.1.1 The 1989 amendments The 1989 amendments to the Maltese Citizenship Act also extended naturalisation to any person who, being descended from a person born in Malta, is a citizen of a country other than the one in which he or she resides and whose access to the country of which he or she is a citi- zen is restricted. 12.1.2 The 2000 amendments In 2000, further changes were made to the citizenship laws (via Acts III and IV of 2000) building on and fine-tuning the 1989 amend- ments, in particular by now completing the shift in policy towards dual and multiple citizenship.3 One major legislative change was designed to dissuade marriages of convenience whereby foreigners were marry- ing Maltese citizens simply to acquire the benefits of Maltese citizen- ship since, according to the prevailing law, marriage with a Maltese citi- zen immediately entitled the foreign spouse to apply for Maltese citi- zenship. The detailed provisions on citizenship in chapter III of the Constitu- tion were transferred to the Maltese Citizenship Act that thereby be- came the main law regulating citizenship while the Constitution now only contains the general principles on citizenship in art. 22. Dual citizenship that was hitherto permitted only exceptionally in the case of Maltese emigrants has now become the rule, following the amendments of 2000, as Maltese citizens are now allowed to hold dual or even multiple citizenship.4 Thus, as from the entry into force of the amendments on 10 February 2000, Maltese citizens who acquire an- other citizenship do not lose their Maltese citizenship. Moreover, since minors holding another citizenship only lost their Maltese citizenship 372 EUGENE BUTTIGIEG if they did not renounce the foreign citizenship by their nineteenth birthday, all citizens of Malta having another citizenship who were minors on that date or had not reached their nineteenth birthday by that date were able to retain both citizenships after their nineteenth birthday. Likewise, foreigners who acquire Maltese citizenship by natur- alisation or registration are no longer required to renounce their other citizenships. p Not only was there this complete shift in policy in favour of multiple citizenship but these provisions were made applicable retrospectively to persons born in Malta or abroad and who had Maltese citizenship by birth or descent but had lost this citizenship when they acquired an- other citizenship, provided they had resided outside Malta for an aggre- gate period of at least six years. 12.1.2 The 2000 amendments In such cases, they would be deemed never to have lost their Maltese citizenship; with this provision they re- gained their lost citizenship automatically.5 On the other hand, those who had lost their Maltese citizenship because they had acquired an- other citizenship before this date but had not resided abroad for such an aggregate period of time or their Maltese citizenship had been ac- quired by registration or naturalisation not by birth or descent may re- gain Maltese citizenship only by registration (and so not automati- cally).6 Irrespective of where they are currently residing they may sub- mit an application to be registered as citizens of Malta. Building on the reform of 1989 that had extended citizenship to chil- dren born to Maltese mothers abroad, the law was further changed to entitle such children born between 21 September 1964 (date of inde- pendence) and 1 August 1989 (date of coming into force of the 1989 amendments) to be registered as Maltese citizens, irrespective of whether or not they reside or resided in Malta while allowing them to retain their other citizenship. As stated above, with a view to discouraging marriages of conveni- ence, the law was amended to provide that foreigners married to Mal- tese citizens may apply for Maltese citizenship on the strength of their marriage only if they have been married for at least five years and no longer immediately following the marriage.7 Another legislative change related to the position of foundlings. Un- til 2000, a new-born infant found abandoned in Malta was deemed to have been born in Malta but could not acquire Maltese citizenship as long as the identity and nationality of the parents remained unknown. As stated above, since 1989 it has become an essential pre-requisite for Maltese citizenship that at least one of the parents is a citizen of Malta. So this meant the child would be stateless. But in 2000 the Maltese Ci- tizenship Act was amended to the effect that, notwithstanding that the nationality of the parents was unknown, such a child would be deemed MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 373 to be a citizen of Malta until his or her right to any other citizenship is established.8 Malta’s accession to the European Union in 2004 did not necessitate nor lead to any changes in the country’s laws and policies on citizen- ship. 12.2.1 Modes of acquisition of citizenship Acquisition by ius soli and/or ius sanguinis9 q y / g Every person born in Malta before the date of independence (21 Septem- ber 1964), who until then was a citizen of the United Kingdom and Colonies and either of whose parents was born in Malta, automatically acquired Maltese citizenship on the date of independence. Moreover, even a person born outside Malta before the date of independence auto- matically acquired Maltese citizenship on the date of independence if he or she was a citizen of the United Kingdom and Colonies until the date of independence and his or her father and a paternal grandparent were both born in Malta. On the other hand, for those who were born in Malta on or after the date of independence but before 1 August 1989, the mere fact of being born in Malta was enough to entitle that person to automatically ac- quire Maltese citizenship at birth. The only exception is in the case of someone born in Malta during this period where both parents are non- Maltese with the father enjoying diplomatic immunity. Those born out- side Malta during this period acquired citizenship at birth only if at the time of birth the father10 was a citizen of Malta whether by birth in Malta, by registration or by naturalisation. However, following the 1989 amendments, for persons born on or after 1 August 1989, birth in Malta no longer sufficed to entitle the per- son to acquire Maltese citizenship at birth: one of the parents must also have been a citizen of Malta at the time of his or her birth. For those born outside Malta on or after 1 August 1989 citizenship is also acquired automatically at birth if, at the date of birth, one of the par- ents was a citizen of Malta whether by birth in Malta, by registration or by naturalisation. Thus, the essential requirement now is descent, not birth on Maltese territory. An exception to this rule is made in the case of newborn infants found abandoned anywhere in Malta who would as a result be state- less. Such infants are deemed to have been born in Malta and are con- sidered citizens of Malta, even though the identity and citizenship of the parents are unknown, until such time as their right to any other ci- tizenship is established. 12.2.1 Modes of acquisition of citizenship 374 EUGENE BUTTIGIEG A person who became a citizen of Malta on 21 September 1964 or at birth but subsequently lost this citizenship, having acquired or retained the citizenship of another country, reacquired citizenship automatically and retrospectively following the entry into force of the amendments of 2000 on 10 February 2000, that removed the prohibition of dual and multiple citizenship for Maltese citizens, if he or she resided outside Malta for an aggregate period of at least six years. By virtue of these amendments they are deemed retrospectively to never have lost their Maltese citizenship.11 A person who became a citizen of Malta on 21 September 1964 or at birth but subsequently lost this citizenship, having acquired or retained Acquisition by adoption12 q y p Since 1 August 1989, Maltese citizenship may also be acquired automa- tically by adoption when a person is lawfully adopted (under Maltese law) on or after this date with one of the adopting parents being a citi- zen of Malta at the time of adoption, provided that the person adopted is under ten years of age on the date of adoption. For persons whose adoptions took place prior to this date but after 31 December 1976, adoption did not automatically lead to acquisition of Maltese citizenship even if the adopters were citizens of Malta. This was because during this period adoptions were considered by law as without effect as far as Maltese citizenship is concerned. Persons adopted during these years would have to apply to be naturalised as citizens of Malta, a mode of acquisition that is discussed below. Although the granting of citizenship in these cases is subject to the dis- cretion of the minister responsible for matters related to Maltese citi- zenship (hereinafter ‘the minister’), it has, since 1987, been generally granted on humanitarian grounds as a matter of policy. Adoptions that took place before 1 January 1977 did lead to auto- matic acquisition of Maltese citizenship by the adopted person on adoption but, in the case of a joint adoption, as in the case of any other birth outside Malta at the time, it had to be shown that at least the male adopter was a citizen of Malta. It would not have sufficed if only the female adopter were a citizen of Malta. Spousal transfer of citizenship13 Spousal transfer of citizenship13 A non-Maltese person married to a citizen of Malta may, after five years of marriage, acquire Maltese citizenship by applying to be registered as a citizen of Malta, provided the spouses are still married and living to- gether (if the Maltese spouse is still alive) at the time the application for citizenship is made. However, if the couple were to separate de iure or de facto after five years of marriage the foreign spouse may still ap- ply for Maltese citizenship provided the spouses had lived together dur- ing those five years of marriage. Moreover, if the Maltese spouse dies before the fifth year of marriage, the foreign spouse may still apply for MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 375 Maltese citizenship following the lapse of the fifth year from the date of marriage, provided that up to the time of death the spouses were liv- ing together. Citizenship may also be acquired, if, although at the time of mar- riage both spouses were non-Maltese, subsequently one of the spouses acquires Maltese citizenship through some other mode of acquisition. The other spouse would now be entitled, subject to the conditions mentioned above, to apply to be registered as a citizen of Malta on the strength of the marriage. A foreign spouse is entitled to apply to be registered as a citizen of Malta even where the marriage took place before the date of indepen- dence so that at the time of marriage neither of the spouses was a citi- zen of Malta, if on independence the other spouse either (i) became, or would have become were it not for his or her death, a citizen of Malta on the date of independence or (ii) became a citizen of Malta after in- dependence. Acquisition by naturalisation15 Any person, including stateless persons, may apply to acquire Maltese citizenship by naturalisation if he or she has resided in Malta during the year immediately preceding the date of application and for a further aggregate period of at least four years over the past seven years immediately preceding the date of application, provided he or she has an adequate knowledge of the Maltese or English language, is of good character and is deemed to be a suitable citizen of Malta. In practice, however, unless the applicant is of Maltese descent, as described below, the Department for Citizenship and Expatriate Affairs follows a strict policy of granting naturalisation only where the appli- cant has resided in Malta for quite a number of years and has children born in Malta. Every case is dealt with on its own merits and the Minis- ter enjoys a non-reviewable discretion as explained below; but while in the past residence alone would not have been a ground for naturalisa- tion, today the general policy is to consider favourably requests for nat- uralisation by residents who have been residing in Malta for a substan- tial number of years and have formed a family here. Income and prop- erty are not determining factors. Nor is any exception made to this long-term residence rule for labour migrants. However, no residence conditions apply where the applicant was born abroad of a father that was likewise born abroad but the paternal grandfather and great-grandparent were both born in Malta. In such cases the person born abroad may apply for naturalisation merely on the strength of his or her Maltese descent. It should be noted, though, that the policy is that applications under this category would normally be accepted only if the applicant resides in Malta. Likewise, no residence conditions apply where the applicant had been a citizen of Malta by birth before he or she emigrated from Malta and ceased to be a citizen of Malta or if he or she had emigrated before the date of independence and failed to obtain Maltese citizenship on independence merely because he or she had ceased to be a citizen of the United Kingdom and Colonies on the date of independence. There have been few applications under this category as most persons that fall under this category already enjoy dual citizenship. Acquisition by registration14 Apart from the special case of spousal transfer of citizenship, there are other instances where a person may acquire Maltese citizenship by re- gistration. Former citizens who, having lost their citizenship before 2000 be- cause of the possession or acquisition of another citizenship as pre- scribed by the law prevailing at the time, fail to qualify for automatic reacquisition of this citizenship either because they had not spent the requisite six years abroad or because they were formerly citizens of Malta by registration or naturalisation and not by birth, may neverthe- less apply to be registered as citizens of Malta. y g Furthermore, an emigrant who was formerly a citizen of Malta by birth or descent but ceased to be a citizen of Malta after emigrating may also reacquire citizenship by registration if he or she returns to Malta and takes up permanent residence. Likewise, persons born outside Malta before 1 August 1989 who are not citizens of Malta because their mother rather than their father was a citizen of Malta by birth, registration or naturalisation, may also ac- quire Maltese citizenship by registration. Citizenship is acquired by registration only if the applicant takes an oath of allegiance to the country and in some instances, such as in the case of the spousal transfer of citizenship, provided the granting of citi- zenship to the applicant is not contrary to the public interest. With this mode of acquisition, citizenship takes effect from the date of registra- tion and not retrospectively. 376 EUGENE BUTTIGIEG Acquisition by naturalisation15 Again, no residence conditions apply to persons who prove descent from a person born in Malta and who are citizens of a country other than the country of their residence and who are denied access to the country of which they are citizens. They may apply to acquire Maltese citizenship by naturalisation merely on the strength of their Maltese descent. However, there have been few instances of naturalisation un- der this category because not many persons would qualify under it, as it requires the applicant to produce all the birth and marriage certifi- cates starting from his or her own birth up to the ancestor who was MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 377 born in Malta. If the link is broken or cannot be proven by documen- tary evidence or if the birth certificate of the ancestor born in Malta cannot be traced, the application for citizenship would not be success- ful. Persons in this category are usually persons of Maltese descent re- siding in North African countries who may generally encounter great difficulties to trace the documents in these countries that would prove this descent. Special rules apply for those who are and have always been stateless but were born in Malta of parents who are not citizens of Malta. In such cases the person is entitled to naturalisation as a citizen of Malta only if he or she has been ordinarily resident in Malta for a period of five years up to the date of his or her application and has not been con- victed in any country of an offence against the security of the state or sentenced to a punishment depriving personal liberty for a term of not less than five years. If the stateless person was not born in Malta but either of his or her parents was a citizen of Malta at the date of his or her birth, he or she is entitled to naturalisation as citizen of Malta only if he or she has been ordinarily resident in Malta for a period of three years up to the date of his or her application and has not been convicted in any coun- try of an offence against the security of the state. So once again, where Maltese descent can be shown, the conditions for naturalisation are less stringent than where only the connection by birth on Maltese terri- tory can be proved. As in the case of citizenship by registration, where citizenship is ac- quired by naturalisation, it takes effect from the date upon which the applicant was naturalised.16 All applications are made to the Minister and there is no right of appeal against the decision of the Minister on any such application nor is such a decision subject to review in any court.17 However, in the Cabinet Citizenship Guidelines that were is- sued in 1987, it is stated that all applications for citizenship by the fol- lowing persons are given favourable consideration: a. former citizens of Malta; b. children born abroad of returned migrants; c. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME foreign citizens born in Malta to a parent who is a citizen of Malta; d. children born to parents who were non-Maltese but who later ac- quired Maltese citizenship; and e. those born abroad but of Maltese descent. It is stated that, on the other hand, applications from those who do not fall under any one of these categories will only be given favourable con- sideration if there are humanitarian grounds.18 Since the drawing up of these guidelines in 1987, significant changes have been made to the Maltese Citizenship Act in 1989 and 378 EUGENE BUTTIGIEG 2000, as shown above. Hence those falling into categories (a) and (b) have practically all been re-instated as Maltese citizens or are now Mal- tese citizens automatically in view of the dual citizenship amendments to the law. Moreover, following these amendments, persons falling un- der category (c) may re-acquire Maltese citizenship simply by registra- tion. Though refugees in Malta are granted some rights they have no right to Maltese citizenship nor are there any provisions in the law that facil- itate the granting of citizenship to refugees.19 g g g Since, as stated above, the law prescribes that one of the conditions for naturalisation is that there should be evidence of the applicant’s good character and suitability for citizenship, apart from being sup- ported by documents attesting to the applicant’s place of residence, birth and Maltese descent, the application in question must also be sponsored by persons that are deemed trustworthy (such as lawyers, notaries, magistrates, judges, members of parliament, police officers, medical practitioners, parish priests, etc.) who, having had occasion to assess the applicant in the course of exercising their profession or voca- tion, are thereby able to vouch for his or her integrity. As in the case of citizenship by registration, the applicant is required to take an oath of allegiance to the country before he or she may be naturalised. 12.2.2 Modes of loss of citizenship Acquisition or retention of another citizenship no longer leads to the denial or forfeiture of Maltese citizenship as dual and multiple citizen- ship is now fully acknowledged by Maltese law.20 This also means that in the case of mixed marriages, the children can acquire the citizenship of both parents. The only ways in which citizenship may be lost are de- tailed below. Renunciation of citizenship21 Deprivation of citizenship acquired by registration or naturalisation22 Deprivation of citizenship acquired by registration or naturalisation22 A citizen of Malta who acquired his or her citizenship by registration or naturalisation may be deprived of this citizenship by order of the Minister if the Minister is satisfied that: a. the registration or naturalisation was obtained by means of fraud false representation or the concealment of any material fact; or a. the registration or naturalisation was obtained by means of fraud, false representation or the concealment of any material fact; or b. the citizen has shown himself or herself by act or speech to be dis- loyal or disaffected towards the President or the government of Mal- ta; or c. the citizen has, during any war in which Malta was engaged, unlaw- fully traded or communicated with an enemy or been engaged in or associated with any business that was to his or her knowledge car- ried on in such a manner as to assist an enemy in that war; or d. the citizen has within seven years after becoming naturalised or being registered as a citizen of Malta, been sentenced in any coun- try to a punishment depriving personal liberty for a term of not less than twelve months; or e. the citizen has been ordinarily resident in foreign countries for a continuous period of seven years and during this time has neither been at any time in the service of Malta or of an international orga- nisation of which the government of Malta was a member nor given notice in writing to the Minister of his or her intention to retain ci- tizenship of Malta. However, in all of these cases a person shall be deprived of his or her citizenship only if the Minister is satisfied that it is not conducive to the public good that the person should retain his or her citizenship and in the case referred to in (d) above only if it appears to the Minis- ter that that person would not thereupon become stateless. Before the Minister issues an order depriving a person of his or her citizenship, the person concerned must be given notice in writing in- forming him or her of the ground on which the order will be issued and of his or her right to an inquiry. Deprivation of citizenship acquired by registration or naturalisation22 If the person requests an inquiry the Minister will have to refer the case to a committee of inquiry ap- pointed by the Minister but presided over by a person with judicial ex- perience. Renunciation of citizenship21 Any citizen of Malta who is also a national of another country may re- nounce citizenship by making a declaration to this effect and upon re- gistration of this declaration he or she would cease to be a citizen of Malta. It is a condition for renunciation that the Maltese citizen should also be a national of another country so that acceptance of the renun- ciation would not lead to the person becoming stateless. Such renun- ciation may be refused if it is made during any war in which Malta is engaged or if in the opinion of the Minister it would otherwise be con- trary to public policy. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 379 12.3 Statistical developments 8 – – 177 139 109 64 37 25 29 21 25 Minor children of Maltese descent – – – – – – – – – 5 62 Persons of Maltese des- cent – – – – – – – – – 27 163 Total 111 79 512 1,062 684 496 514 490 406 460 594 Source: Department for Citizenship & Expatriate Affairs Figure 12.1 Number of naturalisations and registrations in Malta, 1990-2008 0 200 400 600 800 1,000 1,200 1,400 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Registration Naturalisation Source: Department for Citizenship & Expatriate Affairs Table 12.1 Acquisition of Maltese nationality by registration for 1998-2008 according to the grounds for registration Acquisition by registration 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 By virtue of marriage via art. 4 or art. 6 107 75 162 682 354 240 267 197 197 228 160 Resettling permanently in Malta after having emigrated and ceased to be citizens of Malta via art. 4(4) 4 4 – – – – – – – – – Being children born abroad to female citizens of Malta via art. 5(2)(a) – – 173 241 221 192 210 268 180 179 184 Being former citizens of Malta via art. 12.3 Statistical developments These legislative amendments, particularly the shift to dual and multi- ple nationality, and the 2007 amendments described below (see section 12.4) are reflected in the statistical developments in the period 1990- 2008. Statistics from the Department for Citizenship & Expatriate Af- 380 EUGENE BUTTIGIEG Table 12.1 Acquisition of Maltese nationality by registration for 1998-2008 according to the grounds for registration Acquisition by registration 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 By virtue of marriage via art. 4 or art. 6 107 75 162 682 354 240 267 197 197 228 160 Resettling permanently in Malta after having emigrated and ceased to be citizens of Malta via art. 4(4) 4 4 – – – – – – – – – Being children born abroad to female citizens of Malta via art. 5(2)(a) – – 173 241 221 192 210 268 180 179 184 Being former citizens of Malta via art. 8 – – 177 139 109 64 37 25 29 21 25 Minor children of Maltese descent – – – – – – – – – 5 62 Persons of Maltese des- cent – – – – – – – – – 27 163 Total 111 79 512 1,062 684 496 514 490 406 460 594 Source: Department for Citizenship & Expatriate Affairs Figure 12.1 Number of naturalisations and registrations in Malta, 1990-2008 0 200 400 600 800 1,000 1,200 1,400 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Registration Naturalisation Source: Department for Citizenship & Expatriate Affairs Table 12.1 Acquisition of Maltese nationality by registration for 1998-2008 according to the grounds for registration Acquisition by registration 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 By virtue of marriage via art. 4 or art. 6 107 75 162 682 354 240 267 197 197 228 160 Resettling permanently in Malta after having emigrated and ceased to be citizens of Malta via art. 4(4) 4 4 – – – – – – – – – Being children born abroad to female citizens of Malta via art. 5(2)(a) – – 173 241 221 192 210 268 180 179 184 Being former citizens of Malta via art. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 381 fairs for the years 1990-2008 show that the number of citizenships ac- quired by registration rose sharply from the year 2000 onwards (see Figure 12.1, which also shows that the change in policy regarding dual and multiple citizenship in 2000 had a greater effect on registrations than naturalisations). From an annual average of 111 in the 1990s the number of registrations shot up to 512 in 2000 and 1,062 in 2001 and has remained in the region of 500 a year ever since. g 5 y The figure of 1,062 in 2001 remains the highest figure ever recorded for citizenship registrations in Malta. This increase is attributed by the National Statistics Office (NSO) in the Demographic Review 2001 to the removal of the prohibition against dual and multiple nationality by the legislative amendments that entered into force in February 2000. The figure remained high in 2002 when 684 registrations were recorded.23 This might be attributed to the fact that until 2002 Australian law pro- hibited dual and multiple nationality24 and this prevented the many Maltese emigrants residing in Australia from taking advantage of the changes in the Maltese legislation in 2000 and registering for Maltese citizenship. When Australia changed its law on 4 April 2002 and re- moved the prohibition, this resulted in a surge of registrations in 2002 by persons who were now able to retain both Maltese and Australian ci- tizenship.25 In 2003 and 2004, the number of registrations decreased to 496 and 514, respectively,26 and remained at this level up to 2007. The 2007 amendments, described in section 12.4. below, have again served to trigger an increase in citizenship registrations, though not to the level of the 2000 amendments since they affect more remote gen- erations, raising the number of registrations to 594 in 2008. Table 12.1 gives a breakdown of the figures for acquisition by regis- tration for 1998-2008 according to the grounds for registration and shows that it was mostly (i) foreign spouses; (ii) children born abroad to female citizens of Malta; and (iii) former citizens of Malta who had lost their citizenship because of the possession or acquisition of an- other citizenship that took advantage of the change in policy regarding dual and multiple citizenship to acquire citizenship by registration. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME The last two grounds, ‘minor children of Maltese descent’ and ‘persons of Maltese descent’ are the new grounds introduced by the 2007 amendments. 12.3 Statistical developments 8 – – 177 139 109 64 37 25 29 21 25 Minor children of Maltese descent – – – – – – – – – 5 62 Persons of Maltese des- cent – – – – – – – – – 27 163 Total 111 79 512 1,062 684 496 514 490 406 460 594 Source: Department for Citizenship & Expatriate Affairs Table 12.1 Acquisition of Maltese nationality by registration for 1998-2008 according to the grounds for registration Figure 12.1 Number of naturalisations and registrations in Malta, 1990-2008 0 200 400 600 800 1,000 1,200 1,400 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Registration Naturalisation Source: Department for Citizenship & Expatriate Affairs Figure 12.1 Number of naturalisations and registrations in Malta, 1990-2008 0 200 400 600 800 1,000 1,200 1,400 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Registration Naturalisation Source: Department for Citizenship & Expatriate Affairs Source: Department for Citizenship & Expatriate Affairs Source: Department for Citizenship & Expatriate Affairs 12.4 Recent reforms In 2007 important amendments to the Maltese Citizenship Act were passed by the House of Representatives (Act X of 2007) bringing three significant changes to Malta’s citizenship law. 382 EUGENE BUTTIGIEG First, in response to repeated requests by the Maltese diaspora, dual citizenship has been extended to second and subsequent generations of Maltese born outside Malta and living abroad. The 1989 amend- ments to the Act had introduced dual citizenship for those who had been Maltese citizens by birth in Malta but who had emigrated and gi- ven up their Maltese citizenship, in order to enable them to reacquire Maltese citizenship. The 2000 amendments went a step further by ex- tending dual citizenship to those who had been Maltese citizens by descent (i.e. because at least one of their parents was born in Malta) rather than by birth in Malta and who had subsequently lost this citi- zenship upon acquisition of citizenship in another country. This made it possible for children of Maltese emigrants born abroad to acquire or retain Maltese citizenship provided one of their parents had been born in Malta. However, this still excluded the offspring of these children, i.e. second and subsequent generations, from citizenship, because in their case, neither parent, though Maltese, had been born in Malta. The 2007 amendments extend dual citizenship to these generations, as the ius soli requirement was moved even further up the line of an- cestry; provided that somewhere along the applicant’s direct line of Maltese ancestry there is an ascendant who was born in Malta of a par- ent likewise born in Malta, applicants would be entitled to Maltese citi- zenship even though neither of their parents had been born in Malta. Following the entry into force of the 2007 amendments on 1 August 2007 (LN 178 of 2007), art. 5 of the Citizenship Act now provides that those born outside Malta on or after the date of independence are en- titled to apply for registration as citizens of Malta if they prove (through documentary evidence such as birth, marriage and death cer- tificates) that they are the direct descendants of a Maltese ascendant born in Malta of a parent likewise born in Malta. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 383 eration and subsequent-generation Maltese are not as attached to Malta as first-generation Maltese.27 The second amendment, which came into force on 6 July 2007, makes an exception to the rule that a spouse of a citizen of Malta may acquire citizenship only five years after the marriage. A new provision in art. 10 provides that the Prime Minister may, by means of a notice published in the Government Gazette, authorise the Minister to grant a certificate of naturalisation to the spouse of any citizen of Malta with- out any residence requirements when either the spouse or the said citi- zen has rendered ‘exceptional services’ to Malta or humanity. p y A third amendment that entered into force on 1 August 2007 recti- fied an anomaly in the Act. Hitherto, those who were born outside Mal- ta before the date of independence were deemed to have automatically acquired Maltese citizenship on the date of independence only if they were citizens of the United Kingdom and Colonies up to that date and their father and a paternal grandparent were both born in Malta. Con- versely, those with a non-Maltese father whose mother and a maternal grandparent had both been born in Malta could acquire citizenship only by satisfying a five-year residency requirement. The amendments now redress this anomaly by introducing new provisions in art. 3 that remove this residency requirement and entitle such persons irrespec- tive of any residency period to be registered as Maltese citizens because of their mother’s and a maternal grandparent’s birth in Malta. More- over, the first amendment concerning dual citizenship for second and subsequent generations described above also benefits those born abroad before the date of independence who prove that they are des- cendants in the direct line of an ascendant born in Malta with a parent likewise born in Malta. Apart from these legislative amendments, a policy decision has re- cently been taken that benefits a small category of foreigners who, while themselves not qualifying for Maltese citizenship in terms of the Maltese Citizenship Act, have children who are Maltese citizens be- cause they were born in Malta before 1989, when the law as it stood then conferred citizenship on them, notwithstanding that they were not of Maltese descent. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME Maltese citizenship has been extended on the basis of a policy decision to these parents if they have been residing in Malta for fifteen years.28 12.4 Recent reforms However, if the appli- cant has parents, grandparents and other ancestors who are still alive and are also direct descendants themselves in this sense, they would also have to apply for Maltese citizenship under the new provision, as otherwise the Maltese citizenship link would have been broken down the line. If any of these ascendants entitled to Maltese citizenship died before 1 August 2007 or if a parent dies within three years from this date without applying for citizenship, he or she would still be deemed to have acquired citizenship for the purposes of this provision and so the line of Maltese citizenship throughout the generations would not be considered broken (i.e. a three-year grace period). It has been reported that since the 1989 amendments, the Citizen- ship Department has confirmed the right to dual citizenship for 12,250 applicants, a number that is expected to increase following this 2007 amendment, though not to the same extent, as obviously second-gen- 12.5 Conclusions The main motive for the acknowledgement of dual and multiple citi- zenship was to do justice to the thousands of Maltese citizens who had lost their citizenship when, due to economic circumstances, they had 384 EUGENE BUTTIGIEG been forced to emigrate to seek work overseas and thereby acquired for- eign citizenship. By extending citizenship to the third generation, the link to the diaspora has been extended beyond former Maltese citizens. Not only have Maltese diaspora regained their legal ties to their or their ancestors’ homeland but, following Malta’s accession to the European Union, they may now also partake of the benefits of European citizen- ship. Throughout the years, through its participation in international fora debating nationality issues Malta has regularly reviewed and re- vised its nationality policies in line with evolving concepts so that gen- der inequalities and other forms of discrimination prevailing in the law have now been mostly redressed bringing the legal regime in line with international trends. However, although Malta has signed, though not yet ratified, the European Convention on Nationality, the Maltese Citizenship Act has yet to fully embrace the principle of non-discrimination between its na- tionals incorporated in art. 5(2) of the Convention as the provisions on deprivation of citizenship in art. 14(2) of the Act discriminate against persons who acquired citizenship by registration or naturalisation. Maybe this is one reason why Malta has yet to ratify the Convention that it signed on 29 October 2003, though it should be noted that art. 5(2) of the Convention does not have a mandatory effect but only con- stitutes a ‘declaration of intent’ by the signatories.29 This discrimina- tory issue has not been the subject of any public debate or controversy in Malta. Following accession to the European Union, although accession it- self did not necessitate changes in Maltese citizenship laws as the laws were already in consonance with internationally accepted norms, the fact that Maltese citizenship now automatically confers European citi- zenship rights on holders of Maltese citizenship means that Maltese authorities must now consider the wider implications of any policy changes relating to the acquisition and loss of citizenship, particularly in relation to its immigration policy. 12.5 Conclusions Chronological list of citizenship-related legislation in Malta Date Document Content Source 1964 Constitution of Malta (chapter III) Contains provisions conferring and regulating Maltese citizenship based on a combination of the ius soli and ius sanguinis principles and prohibiting dual or multiple citizenship http://docs.justice.gov.mt Chronological list of citizenship-related legislation in Malta Date Document Content Source 1964 Constitution of Malta (chapter III) Contains provisions conferring and regulating Maltese citizenship based on a combination of the ius soli and ius sanguinis principles and prohibiting dual or multiple citizenship http://docs.justice.gov.mt Chronological list of citizenship-related legislation in Malta 2000 Act III amending the Constitution of Malta and Act IV amending the Maltese Citizenship Act Source 1964 Constitution of Malta (chapter III) http://docs.justice.gov.mt MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 385 Date Document Content Source 1965 Maltese Citizenship Act, chapter 188 of the Laws of Malta Introduces more detailed provisions and regulates in particular the acquisition of Maltese citizenship by registration and naturalisation http://docs.justice.gov.mt 1974 Act LVIII amending the Maltese Citizenship Act and the Constitution of Malta Amendments necessitated by Malta’s transformation into a republic http://docs.justice.gov.mt 1975 Act XXXI amending the Maltese Citizenship Act Slightly amends provisions on naturalisation http://docs.justice.gov.mt 1977 Act IX amending the Maltese Citizenship Act Prohibits acquisition of citizenship by adoption http://docs.justice.gov.mt 1989 Act XXIII amending the Constitution of Malta and Act XXIV amending the Maltese Citizenship Act Introduces an exception to the prohibition against dual citizenship for expatriates; shifts to a rule based more on ius sanguinis than on ius soli by providing that mere birth in Malta would no longer suffice to confer citizenship at birth but must be accompanied by Maltese descent of at least one of the parents; amends some of the provisions that were resulting in gender inequality; reintroduces acquisition of citizenship by adoption; removes the distinction between Commonwealth citizens and other foreigners for naturalisation purposes; extends the grounds for naturalisation http://docs.justice.gov.mt 2000 Act III amending the Constitution of Malta and Act IV amending the Maltese Citizenship Act Allows dual and multiple citizenship; introduces restrictions in the provisions on spousal transfer of citizenship to discourage marriages of i t f th http://docs.justice.gov.mt particular the acquisition of Maltese citizenship by registration and naturalisation Amendments necessitated by Malta’s transformation into a republic http://docs.justice.gov.mt Slightly amends provisions on naturalisation http://docs.justice.gov.mt Prohibits acquisition of citizenship by adoption http://docs.justice.gov.mt Introduces an exception to the prohibition against dual citizenship for expatriates; shifts to a rule based more on ius sanguinis than on ius soli by providing that mere birth in Malta would no longer suffice to confer citizenship at birth but must be accompanied by Maltese descent of at least one of the parents; amends some of the provisions that were http://docs.justice.gov.mt 386 EUGENE BUTTIGIEG Date Document Content Source that thereby becomes the main law regulating citizenship while the Constitution now only contains the general principles on citizenship; makes further amendments to redress gender inequality; introduces provisions to improve the position of foundlings 2007 Act X amending the Maltese Citizenship Act Introduces the right to dual or multiple citizenship for second- and subsequent- generation Maltese born outside Malta and living abroad; introduces an exception to the five-year rule for spouses of Maltese citizens who render ‘exceptional services’ to Malta or humanity; extends citizenship by registration to persons born abroad before 1964 of a non-Maltese father if their mother and a maternal grandparent were both born in Malta http://docs.justice.gov.mt Date Document 2007 Act X amending the Maltese Citizenship Act p 2 Hitherto only minors were allowed to have dual citizenship until their nineteenth birthday. 3 These legislative changes were preceded by the White Paper on Proposed Legislation to Amend the Citizenship and Immigration Laws, Office of the Prime Minister (OPM), 10 August 1998 that explained the proposed amendments. http://docs.justice.gov.mt 4 Art. 22 of the Constitution and art. 7 of the Maltese Citizenship Act. Notes 1 The author would like to thank Joseph Mizzi, Director at the Department for Citizenship and Expatriate Affairs, and Joseph Treeby Ward, former Director, for their valuable information and helpful comments on earlier drafts. p 2 Hitherto only minors were allowed to have dual citizenship until their nineteenth birthday. 3 These legislative changes were preceded by the White Paper on Proposed Legislation to Amend the Citizenship and Immigration Laws, Office of the Prime Minister (OPM), 10 August 1998 that explained the proposed amendments. 4 Art. 22 of the Constitution and art. 7 of the Maltese Citizenship Act. 5 Maltese Citizenship Act, art. 9. 7 Ibid., arts. 4 and 6. 8 Ibid., art. 5. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 387 9 Ibid., arts. 3-5, 17. 9 Ibid., arts. 3-5, 17. 9 3 5 7 10 Except in the case of illegitimate children where the national status of the mother becomes relevant – ibid. art 17. 11 Ibid., art. 9. 11 Ibid., art. 9. 12 Ibid., art. 17. 12 Ibid., art. 17. 13 Ibid., arts. 4 and 6. 13 Ibid., arts. 4 and 6. 14 Ibid., arts. 8 and 9. 14 Ibid., arts. 8 and 9. 15 Ibid., art. 10. 16 Ibid., art. 12. 17 Ibid., art. 19. 7 9 18 These guidelines currently appear on the website of the relevant ministry, the Ministry for Justice and Home Affairs, www.mjha.gov.mt. 18 These guidelines currently appear on the website of the relevant ministry, the Ministry for Justice and Home Affairs, www.mjha.gov.mt. 19 However, recently a government minister (Minister for the Family and Social Solidarity, Dolores Cristina, as reported in The Times of 18 June 2005 on p. 19) an- nounced that the government is considering a change in policy in this regard in fa- vour of granting citizenship to refugees who have been living in Malta for ten years so as to enable them to integrate better into society. So far, however, there has been no official change in policy on these lines. 19 However, recently a government minister (Minister for the Family and Social Solidarity, Dolores Cristina, as reported in The Times of 18 June 2005 on p. 19) an- nounced that the government is considering a change in policy in this regard in fa- vour of granting citizenship to refugees who have been living in Malta for ten years so as to enable them to integrate better into society. So far, however, there has been no official change in policy on these lines. 20 Maltese Citizenship Act, art. 7 and art. 22(2) of the Constitution. 20 Maltese Citizenship Act, art. 7 and art. 22(2) of the Constitution. 21 Maltese Citizenship Act, art. 13. 22 Ibid., art. 14. 23 Demographic Review 2002, National Statistical Office (NSO) 2003. 24 Art. 17 of Australia’s law on citizenship did not allow dual citizenship so that citizens of Australia would lose citizenship if they acquired another citizenship voluntarily through registration. 24 Art. 17 of Australia’s law on citizenship did not allow dual citizenship so that citizens of Australia would lose citizenship if they acquired another citizenship voluntarily through registration. MALTA’S CITIZENSHIP LAW: EVOLUTION AND CURRENT REGIME 25 Ministry for Justice and Home Affairs, Annual Report 2001-2002. 25 Ministry for Justice and Home Affairs, Annual Report 2001-2002. 26 Demographic Review 2003, NSO 2004. 27 Home Affairs Minister Borg, House of Representatives, sitting of 13 February 2007 and press conference of 15 May 2007. However, for the second and subsequent generation Maltese living in a non-EU country, such as Australia, the US and Canada, and the much smaller communities still living in Egypt and Tunisia, there is the added attraction that Maltese citizenship brings with it the right to travel, study, live and work in any EU country. 27 Home Affairs Minister Borg, House of Representatives, sitting of 13 February 2007 and press conference of 15 May 2007. However, for the second and subsequent generation Maltese living in a non-EU country, such as Australia, the US and Canada, and the much smaller communities still living in Egypt and Tunisia, there is the added attraction that Maltese citizenship brings with it the right to travel, study, live and work in any EU country. 28 Home Affairs Minister Borg, House of Representatives, sitting of 13 February 2007. 28 Home Affairs Minister Borg, House of Representatives, sitting of 13 February 2007. 29 Explanatory Report to the European Convention on Nationality, point 45. 13 Nationality and citizenship in Cyprus since 1945: Communal citizenship, gendered nationality and the adventures of a post-colonial subject in a divided country Cypriot policymakers still hope that the policy of accession to the 390 NICOS TRIMIKLINIOTIS EU will eventually act as a catalyst in the effort to find a settlement, but in the immediate aftermath of the referenda the two sides were di- vided about how to proceed (Hannay 2005; Palley 2005).3 The election of Demetris Christofias as President of the Republic of Cyprus in Feb- ruary 2008 has given new impetus to solving the Cyprus problem. Di- rect negotiations between Christofias and Talat, the two community leaders, began in September 2008. EU will eventually act as a catalyst in the effort to find a settlement, but in the immediate aftermath of the referenda the two sides were di- vided about how to proceed (Hannay 2005; Palley 2005).3 The election of Demetris Christofias as President of the Republic of Cyprus in Feb- ruary 2008 has given new impetus to solving the Cyprus problem. Di- rect negotiations between Christofias and Talat, the two community leaders, began in September 2008. To evaluate the question of nationality and citizenship, one is forced to view the ever-present ‘Cyprus problem’ in the historical and politico- social context of the island and the wider troubled region of the near Middle East. While the ‘Cyprus problem’ persists and the de facto divide continues, the politics of ‘citizenship’ has not been ‘frozen’ in time. Ci- tizenship has played a central role in political discourse, both during and following the referendum on the UN plan in April 2004. The par- ticular construction of the Cyprus Republic was such that the struggle for legitimacy was elevated to the primary struggle for control of the state. In this conflict the two communal leaderships – the Greek-Cy- priots and the Turkish-Cypriots – sought to materialise their ‘national aspirations’: For Greek-Cypriots the aim for Enosis (union with Greece) and for the Turkish-Cypriots the goal of Taksim (partition) would con- tinue post-independence. The very concept of citizenship was not only ethnically/communally defined by the Constitution, but it was also a sharply divisive issue between the Greeks and Turks, acquiring strong ethnic and nationalistic overtones (see Tornaritis 1982; Chrysostomides 2000; Trimikliniotis 2000). 13 Nationality and citizenship in Cyprus since 1945: Communal citizenship, gendered nationality and the adventures of a post-colonial subject in a divided country Nicos Trimikliniotis Mapping out the complex historical, structural, politico-legal and cul- tural setting that has generated a specific mode of nationality in the context of Cyprus is no easy task. In fact, we cannot speak of a nation- ality policy as such; such a policy has never been formally declared or publicly discussed, save for times in which the media hysterically criti- cised the granting of nationality.1 It is, however, possible to deduce a policy from the practices since independence (Trimikliniotis 2005). p y p p ( 5) In an area of 9,251 km,2 the total population of Cyprus is around 754,800, of whom 666,800 are Greek-Cypriots (living in the Cyprus Republic-controlled area). Upon independence in 1960, Turkish- Cypriots constituted 18 per cent of the population, whilst the smaller ‘religious groups’, as referred to in the Constitution, consisting of Ar- menians, Latins, Maronites and ‘others’ (such as Roma), constituted 3.2 per cent of the population. It is the third largest island in the Medi- terranean; its geographical position, in the far eastern part of the Medi- terranean Sea, historically adjoining Europe, Asia and Africa has been both a blessing and a curse. Invaders and occupiers for centuries sought to subordinate it for strategic reasons, followed by British colo- nial rule. It became an independent Republic in 1960. In the post-colonial years, there was inter-communal strife and constant foreign interven- tion of one kind or another, until 1974 when a coup by the Greek junta and EOKA B2 was used as a pretext for an invasion by the Turkish army and the subsequent division of the island (Hitchens 1997; Atta- lides 1979). Turkey still occupies 34 per cent of the territory, whilst 200,000 remain displaced and 80,000 Turkish-Cypriots remain in the northern, occupied territories. Attempts to resolve the Cyprus problem have not been successful. Following the overwhelming rejection of the UN plan to resolve the problem by the Greek-Cypriots and the over- whelming endorsement on the 24 April 2004 by the Turkish-Cypriots, Cyprus has entered the EU with the Cyprus problem in a state of lim- bo. 13.1 History of nationality policy since 1945 13.1.1 The national subject under the colonial spell: ‘Modernising’ the millet system, divide and rule and the rise of irredentist nationalism Following the opening of the Suez Canal in 1864, the UK persuaded the Ottomans to cede Cyprus to the UK.4 The British colonialists took over from the Ottoman rulers by order of Council on 7 October 1878. They immediately embarked on a programme of ‘modernisation’ from above and from outside by introducing an administrative system super- seding Ottoman law with English law. Britain formally annexed Cyprus in 1914, following Turkey’s support for Germany in the First World War; in 1923, under the Treaty of Lausanne, Turkey formally relin- quished all its claims to Cyprus and it became a Crown Colony in 1925. In the historical setting prior to the modern era,5 ‘identity’ was not based on ‘ethnicity’: The notion of ‘citizenship’ did not exist under Ot- toman rule outside the millet system. This implied that the Ottomans CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 391 basically recognised the religious leaders of the flock and were co-oper- ating with them in the administration (Katsiaounis 1996; Kyrris 1980).6 With the annexation of Cyprus by Britain, Cypriots became ‘na- tives of the colony’, but the essential characteristics of the Ottoman millet system, a system that was based on communal organisation and leadership along the lines of faith, were the bases upon which the faith-groups would be ‘modernised’ as ethnic communities. Hence, the Muslim community and Christian-orthodox community millets were gradually ‘modernised’ by the British administrator. There was a trans- formation of the quasi-medieval community elites into ‘ethno-commu- nal’ elites: on the one hand, the traditional religious leader of the Chris- tian Orthodox flock, the archbishop, became the leader of the Greek community and, on the other, the old Ottoman administrators, who re- presented the fusion of the political and religious order of the sulta- nate-caliphate at local level, were transformed into the new political lea- dership of the Turkish community. The Cypriot ‘natives of the colony’ were thus gradually ethnocised. Nevertheless, the leaders of the autoce- phalous Greek-orthodox Church retained their ‘ethnarchic role’ (i.e. po- litical leadership of the flock), despite the serious challenge from the mass secular movement AKEL (the Progressive Party for the Working People) from the 1940s onwards (Katsiaounis 2007). 13.1 History of nationality policy since 1945 Moreover, the old Ottoman administrators were eventually transformed into the Kemalist elite, following the rise of Mustafa Kemal to power in the Turkish Re- public (which succeeded the Ottoman empire).7 13.1.3 The ‘national’ rift: Collision and division between Greek-Cypriots and Turkish-Cypriots (1963-1974) In 1963, following a Greek-Cypriot proposal for amendment of the Constitution, the Turkish-Cypriot political leadership ‘withdrew’ from the government. Since then, the administration of the Republic has been carried out by the Greek-Cypriots. Inter-communal strife ensued until 1967. In 1964, the Supreme Court ruled that the functioning of the government must continue on the basis of the ‘law of necessity’, or, better yet, the ‘doctrine of necessity’, in spite of the constitutional deficiencies created by the Turkish-Cypriot withdrawal from the admin- istration.9 The short life of the consociation did not manage to gener- ate a strong enough inter-communal or trans-communal citizenship. This brief period of peaceful inter-communal political co-existence was tentative; we cannot therefore speak of a ‘nationality policy’ as such, above and beyond the politics of the Cyprus conflict and the separate national aspirations of Greek and Turkish Cypriots, who continued to work towards Enosis and Taksim respectively, even after independence. Although de iure the Republic continued to exist as a single interna- tional entity, in practice there were two de facto regimes in the enclaves, each group controlled, one for the Greek-Cypriots and one for the Turk- ish Cypriots – a situation aptly called ‘the first partition’ by one scholar (Droussiotis 2005). The fierce fighting between 1963 and 1967 was fol- lowed by efforts to reconciliation until 1974, but these efforts failed. 13.1.2 Moments of (in)dependence: Ethno-communal citizenship and the nationalising of legally divided subjects (1959-1963) 13.1.2 Moments of (in)dependence: Ethno-communal citizenship and the nationalising of legally divided subjects (1959-1963) The establishment of the Cyprus Republic marks an important devel- opment in the history of Cyprus, as the island became an independent republic for the first time since antiquity, albeit in a limited way (see Attalides 1979; Faustmann 1999). The anti-colonial struggle had started in the 1930s.8 The four-year armed campaign by the Greek-Cy- priot EOKA (1955-59) for Enosis and the Turkish-Cypriot response for Taksim brought about a regime of ‘supervised’ independence by three foreign ‘guarantor’ nations (UK, Turkey and Greece). The Cyprus Con- stitution, adopted under the Zurich-London Accord of 1959, contains a rigorous bi-communalism, whereby the two ‘communities’, Greek-Cy- priots, who made up 78 per cent of the population, and Turkish-Cy- priots, who accounted for 18 per cent of the population, share power in a consociational system. Citizenship is strictly ethno-communally di- vided. There are also three other minority groups who have the consti- tutionally recognised status of ‘religious groups’: the Maronites, the Ar- menians and the Latins. In addition, there is a small Roma commu- 392 NICOS TRIMIKLINIOTIS nity, registered mostly as part of the Turkish-Cypriot community, but never recognised as a minority group (Trimikliniotis & Demetriou 2009). 13.1.3 The ‘national’ rift: Collision and division between Greek-Cypriots and Turkish-Cypriots (1963-1974) CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 393 Greek-Cypriots, who lived in the reduced territory of the internationally recognised Republic of Cyprus, and that of the Turkish-Cypriots, who lived under an unrecognised regime, the ‘Turkish Republic of Northern Cyprus’, which relied heavily on Turkey to maintain it. Turkish-Cypriots are entitled to citizenship/nationality of the Cyprus Republic and tens of thousands obtained a passport. However, the vast majority did not have access to the authorities and was not allowed to cross over to the ‘other side’ by the occupying regime; up to April 2003 there were few opportunities for ordinary Greek-Cypriots and Turkish-Cypriots to meet; while Greek-Cypriots did not have access to the occupied terri- tories, Turkish-Cypriots were not allowed by the regime in the north to enter the area controlled by the Republic. Greek-Cypriots, who lived in the reduced territory of the internationally recognised Republic of Cyprus, and that of the Turkish-Cypriots, who lived under an unrecognised regime, the ‘Turkish Republic of Northern Cyprus’, which relied heavily on Turkey to maintain it. Turkish-Cypriots are entitled to citizenship/nationality of the Cyprus Republic and tens of thousands obtained a passport. However, the vast majority did not have access to the authorities and was not allowed to cross over to the ‘other side’ by the occupying regime; up to April 2003 there were few opportunities for ordinary Greek-Cypriots and Turkish-Cypriots to meet; while Greek-Cypriots did not have access to the occupied terri- tories, Turkish-Cypriots were not allowed by the regime in the north to enter the area controlled by the Republic. The period between 1974 and 2003 was characterised by the at- tempts of the break-away regime to consolidate partitionism in Cyprus (Dodd 1993). In spite of the efforts to reach an agreement on a solu- tion based on the ‘High Level Agreements’ of 1977 and 197910 the Turkish side continued its route towards separatism. The ‘Turkish Re- public of Northern Cyprus’ (TRNC), a regime recognised only by Tur- key, was declared. The constitution of the TRNC provides for an ethno-religious-based nationality and citizenship to a large extent reproducing the provisions of the Cyprus Republic (Dodd 1993). However, TRNC nationals cannot make use of the nationality of an unrecognised state. Hence, many Turkish-Cypriots sought passports from Turkey (see Kadirbeyoglu in this volume) and the Republic of Cyprus – particularly after accession to the EU. 13.1.4 The de facto partition: 1974-2003 following the invasion and occupation Since 1974 the northern part of Cyprus, some 35 per cent of its terri- tory, has been under Turkish occupation and outside the control of the Cyprus government. Some 100 Greek-Cypriots inhabit the northern territory, whilst only a few hundred Turkish-Cypriots continue to live in the government-controlled south (ECRI 2001, 2006; Kyle 1997) How- ever, since the end of May 2003 the regime in the occupied territories has allowed Turkish-Cypriots to visit the Republic-controlled south on the condition that they return before midnight and the Greek-Cypriots to visit the north, on the condition of passport inspection and with re- strictions on their stay. During this 30-year period the de facto partition meant that in effect there were two separate ‘stories’ about nationality: the story of the CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY In the late 1990s, the TRNC leadership attempted to crimi- nalise the access to the passport of the Republic of Cyprus, but such ef- forts were subsequently abandoned as the numbers of Turkish-Cypriots seeking passports grew and there was a reversal of this policy once the Annan Plan (version 1) was first introduced in late 2002. In fact, many Turkish-Cypriot politicians now criticised the authorities of the Repub- lic of Cyprus for failing to respond quickly enough to ensure the swift and full provisions of access to citizenship, passports and the public goods that are available to the nationals of the Republic of Cyprus. g y During the post-1974 period the Republic of Cyprus attempted to re- inforce its legitimacy claiming that Turkish-Cypriot citizens enjoy full and equal rights under the Republic’s Constitution, such as general ci- vil liberties and the rights provided by the European Court of Human Rights (ECHR) as well as other human rights, save for those provi- sions, that have resulted from (a) the ‘abandoning’ of the governmental posts in 1963-1964 and (b) the consequences of the Turkish invasion. The ‘doctrine of necessity’ would apply to allow for the effective func- tioning of the state, whilst the relevant provisions of the Constitution would be temporarily suspended, pending a political settlement (for 394 NICOS TRIMIKLINIOTIS more on this, see Chrysostomides 2000; Loizou 2001). However, Turk- ish-Cypriot citizens of the Republic had been denied their electoral rights since 1964, a matter that was found to be in violation of the Eur- opean Convention on Human Rights,11 save for the European Parlia- ment elections in 2004. A new law was passed to at least partially re- medy the situation before the parliamentary elections in May 2006. y p y y All of the Republic of Cyprus governments have maintained that Turkish-Cypriots are entitled to full citizenship rights and the national- ity of the Republic. The children of Cypriots who now reside in the oc- cupied territories or abroad and were born after 1974 are entitled to na- tionality (as with Greek-Cypriots and ‘others’). The bureaucratic ele- ments involved are due to the non-recognition of any documentation such as e.g. birth certificates from the TRNC.12 The policy regarding the treatment of Turkish-Cypriots, who are Cyprus Republic nationals, is rather contradictory. This reflects the complexity of the Cyprus con- flict and the constant conflict for legitimacy and recognition. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY Inevitably, ‘the discourse of recognition’ (Constantinou & Papadakis 2002) spilled- over into nationality politics, making a mess of the official policy of ‘rapprochement’. Ultimately, the consequences of the situation resulted in failing to properly treat ordinary Turkish-Cypriots as ‘strategic allies’, in the context of independence from the Turkish-Cypriots’ nationalistic leadership, who are perceived as ‘mere pawns of Ankara’. Even today, the Republic of Cyprus seems to be failing to address certain basic matters: Since Turkish is an official language of the Repub- lic, allowing Turkish-Cypriots to communicate with government offi- cials in their own language and making the laws, regulations and forms available in Turkish is a matter that could have been resolved, without much difficulty, and would protect the Republic from claims of discrimination and unconstitutionality (Trimikliniotis & Demetriou 2008). Moreover, the enjoyment of all rights, including the right to property (of those Turkish-Cypriots who fled their homes in 1963, 1967 and 1974), could have been handled with greater sensitivity and care, so that the Turkish-Cypriots, who are Cypriot citizens, would feel more welcome. At the same, time one has to appreciate the context, particularly the massive displacement of 100,000 Greek-Cypriots from the north, many of whom are housed in Turkish-Cypriot properties. 13.1.5 New issues for nationality/citizenship policies In the 1990s and early 2000, a number of key issues opening up the issue of citizenship and nationality and requiring a declared and con- sistent policy emerged. First, the arrival of migrant workers, who today make up 15 per cent of the total working population of the island, is a significant factor al- CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 395 tering the ethnic make up of the population. Although the initial de- sign was that they be ‘temporary’, they seem to be a permanent feature of Cypriot society (Matsis & Charalambous 1993; Trimikliniotis 1999; Trimikliniotis & Pantelides 2003; Trimikliniotis & Demetriou 2007). 3 7) Second, the arrival of Turkish-Cypriot Roma from the poorer (occu- pied) north in the south between 1999 and 2002 created a panic that they were being ‘flooded’ with ‘gypsies’. In spite of the fact that we are dealing with a group of Cypriots, who moved to the south, the reaction of the authorities, the media and the public at large displayed a hostile attitude as if they were undesirable ‘alien citizens’. Studies indicate that there is wide-spread resentment by the local Greek-Cypriot residents to the Turkish-speaking Roma coming to their neighbourhood in Limas- sol and ‘causing trouble’. There is evidence of discrimination against Roma in the Republic (Spyrou 2003; Trimikliniotis 2003; Trimiklinio- tis & Demetriou 2009), as they are generally viewed with suspicion by Greek-Cypriots, but also by Turkish-Cypriots. The arrival of large num- bers in the south was greeted with fear and suspicion,13 particularly when the then Minister of Justice and Public Order alleged that they may well be ‘Turkish spies’,14 whilst the Minister of the Interior as- sured Greek-Cypriots that the authorities ‘shall take care to move them to an area that is far away from any place where any people live’, in re- sponse to the racially motivated fears of local Greek-Cypriot residents.15 The socio-economic position of this generally destitute group renders them particularly vulnerable and dependent on welfare; the rights that derive from their citizenship status were thus mediated by the way var- ious state authorities approached them (e.g. their lifestyle and harass- ment means that many do not have the necessary documents for claiming nationality such as birth certificates, identity cards, etc.). 13.1.5 New issues for nationality/citizenship policies Hence the failure to take into account the socio-economic conditions of the Roma may result in the denial of the right to obtain a passport, as was found in cases investigated by the Cyprus Ombudsman.16 Third, the opening of the ‘borders’ which allowed many thousands of Turkish-Cypriots to visit the south were generally greeted by both Turkish-Cypriots and Roma residing in the south with relief and optimism.17 However, there was a tense atmosphere generated in the run up and aftermath of the referendum on the Annan plan to reunite the island on the 24 April 2004, the rejection of which by the Greek- Cypriots has given rise to nationalist sentiments in the south (see Had- jidemetriou 2006). The political atmosphere has drastically changed since the presidential election in February 2008 and the beginning of new negotiations to resolve the problem. The fourth issue concerns the children of settlers who are married with Turkish-Cypriots. This is a highly controversial issue as it brings out the conflict over the nature of the Cyprus problem: the Turkish pol- 396 NICOS TRIMIKLINIOTIS icy of colonising the north seems to be a major obstacle to a solution. There is a misguided conflation of the internationally condemned pol- icy by an aggressor country, with the fact that we are also dealing with some basic rights and humanitarian issues relating to the rights of children and individuals who marry, found families and continue with their lives. The granting of nationality rights to children and spouses of Turkish-Cypriots is a major political issue which has increasingly ta- ken up the headlines and is discussed in the last section of this chap- ter. Moreover, the condemnation of a war crime (colonisation) must not be conflated and confused with issues regarding the conditions of sojourn and living conditions of poor undocumented workers, who are primarily present to be exploited as cheap foreign labour. y g Finally, the issue of gender has become an important issue as re- gards citizenship. The position of women in the processes of nation- building and nationalism raises the crucial question of a gendered Cy- priot nationality, in what one scholar referred to as ‘the one remaining bastion of male superiority in the present territorially divided state’ (Anthias 1989: 150). This last ‘bastion’ was formally abolished with an amendment of the citizenship law in 1999 (No. 13.1.5 New issues for nationality/citizenship policies 65/99), which intro- duced entitlement to citizenship for descendants of a Cypriot mother and a non-Cypriot father. The apparent reluctance of Cypriot policy- makers to amend the citizenship law, allegedly due to the concern about upsetting the state of affairs as it existed prior to 1974, cannot stand closer examination. After all, there have been seven amendments to the citizenship law prior to the amendment No. 65/99. It is appar- ent that the issue of gender equality had not been a particularly high political priority. Besides, in the patriarchal order of things, the role of Cypriot women as ‘symbolic reproducers of the nation’, particularly in the context of ‘national liberation’, as transmitters of ‘the cultural stuff’, required that potential association and reproduction of women with men outside the ethnic group be strictly controlled (Anthias 1989: 151). CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 397 their Turkish-Cypriot counterparts. However, this bureaucratic measure which attempts to force on people the issue of ‘recognition’ has become part of the ‘struggle for legitimacy and recognition’ between the two po- litical regimes, even though it is up to states and international organi- sations to recognise them. Cross-boundary contacts and interaction opened up new possibilities for nationality policy, as the barbed-wire at last became penetrable. The fluidity of the situation allows greater scope for citizens’ initiatives aim- ing at reunification (see Demetriou 2006, 2007) and has opened up the debate on reconciliation in Cyprus (Kadir 2007; Sitas, Latif & Loi- zou 2007; Trimikliniotis 2007). The current measures cannot be a sub- stitute for a settlement; it is an awkward state of limbo, whereby the ‘nationals’ are divided along ethnic lines, even though all Turkish-Cy- priots are entitled to citizenship in the Republic of Cyprus and many thousands have actually acquired citizenship and passports. The con- tact since 2003 has created a pattern whereby a consistent number of persons cross over for work, leisure or other activities, estimated at about 20 per cent of the population.18 The Third ECRI Report on Cy- prus notes that a large number of Turkish-Cypriots has been issued with Cyprus passports (35,000), identity cards (60,000) and birth certi- ficates (75,000), all of which are relevant figures as far as Cypriot citi- zenship is concerned (ECRI 2006: para. 78). Interestingly, according to the Demographic Survey Report (PIO 2006: 12), the population of Cy- prus is estimated at 854,300 at the end of 2005 (compared to 837,300 at the end of the previous year), of whom 766,400 live in the terri- tories under the control of the Republic. Turkish-Cypriots are said to be 87,000 persons, Greek-Cypriots 656,000 and foreign citizens 110,000. The same report estimates, on the basis of data from Turkish Cypriot sources, that about 58,000 Turkish Cypriots have emigrated since 1974. The number of ‘illegal settlers from Turkey’ is said to be ‘most probably in the range of 150-160 thousand, which is estimated on in- formation of significant19 arrivals of Turks in the occupied area’ (PIO 2006: 11). The study by Hatay (2005) shows significantly lower figures for settlers and higher numbers for Turkish-Cypriots. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY The population issue remains a hotly contested issue, not only between the two com- munities, but also within the Turkish-Cypriot community (see Hatay 2008; Faiz 2008). 13.1.6 The rise of trans-communal subjectivity: Challenging the ethno- communal boundaries On 23 April 2003 there was a sudden decision by the authorities of the unrecognised TRNC, to partially lift the ban on freedom of movement. This has taken most by surprise (Demetriou 2007), as the TRNC was abandoning the long-term vigorous opposition to Greek-Cypriot and Turkish-Cypriot contacts. The Turkish-Cypriot leadership allowed the possibility for a course of action the peace and rapprochement move- ment had been advocating for years; yet the move was certainly a sur- prise. The issue of ‘passport control’ between the check points became an issue of tension between Greek-Cypriot politicians and media and 13.2 Modes of acquisition and loss of citizenship Following the annexation of Cyprus by the UK,20 all Ottoman citizens who were born in or normally resided in Cyprus became British sub- jects.21 From that day on the basic law regarding the granting of na- 398 NICOS TRIMIKLINIOTIS tionality in Cyprus was the British Nationality and Status of Aliens Act of 1914 and later the 1948 British Nationality Act. Post-independence art. 198 of the Constitution of the Cyprus Republic, and Annex D of the Treaty of Establishment, which was annexed to the Constitution, regulated the initial determination of the citizenry and the granting of citizenship/nationality. Annex D was implemented with independence, as required by art. 195, which provides for the general principle of in- ternational law that all residents of the former colonial territory would automatically become citizens of the Republic (Tornaritis 1982: 35; Loi- zou 2001: 441).22 Art. 198.1(b) provided that: any person born in Cyprus, on or after the date of the Constitu- tion coming into force, shall become a citizen of the Republic if on that date his father has become a citizen of the Republic or would but for his death have become such a citizen under the provisions of Annex D of the Treaty of Establishment. any person born in Cyprus, on or after the date of the Constitu- tion coming into force, shall become a citizen of the Republic if on that date his father has become a citizen of the Republic or would but for his death have become such a citizen under the provisions of Annex D of the Treaty of Establishment. This was the case until the enactment of the main Law on Citizenship in 1967.23 In 2002, a new Law on the Population Data Archives No. 141(I)/2002 unified all provisions regarding the archiving of births and deaths, registration of residents, registration of constituent voters and the registration of citizens. It also introduced special provisions for the issuing of passports and travel documents and refugee identity cards to refugees. The new Law has so far been amended four times; however, none of these changes affected the acquisition and loss of citizen- ship.24 Together with Annex D this law currently regulates the acquisi- tion and loss of Cypriot citizenship. Cypriot policy-makers have followed the ‘mixed’ principle that com- bines ius soli and ius sanguinis (Tornaritis 1982: 38-39). 13.2 Modes of acquisition and loss of citizenship However, ius sanguinis is far more important in the regulations than ius soli, as Cy- priot descent is the primary criterion for acquisition of citizenship as will be shown further down. Citizenship can be acquired automatically, via registration and naturalisation, but at the core of citizenship policy re- mains the notion that all persons of Cypriot descent are entitled to apply. 13.2.1 Acquisition by descent A person born in Cyprus or abroad on or after 16 August 1960 auto- matically acquires Cypriot citizenship provided that at the time of his or her birth either of the parents was a citizen of the Republic or, in the case that the parent(s) were deceased at the time of his or her birth, either of them was entitled to acquire citizenship had he or she not been deceased. In cases of permanent residents abroad, this provision CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 399 is not applicable unless the child’s birth is registered in the prescribed manner.25 Moreover, there are two exceptions to this general rule: Firstly, the current law provides that children born to parents, one of whom unlawfully entered or resides in the Republic, do not automati- cally become citizens of Cyprus even if the other parent holds or would have been entitled to Cypriot citizenship. They can become citizens only following a decision of the Council of Ministers.26 This amend- ment was apparently directed against Turkish nationals who settled in the north at a time when it was deemed politically ‘necessary’ or ‘expe- dient’ by policymakers. However, it is obviously discriminatory against persons who have Turkish-Cypriot descent from one parent and is con- trary to the Constitution and international obligations of the Republic. y g p Whether children of Turkish nationals should be granted Cypriot ci- tizenship is a hot political issue and there are conflicting accounts of what categories of persons are affected. Media reports and right-wing politicians seem to concur that the issue at stake is the granting of citi- zenship to children who have one Cypriot parent and another who is a settler. However, ministry officials claim that persons falling under this category are invariably granted nationality, albeit in a manner that does not cause strong reactions.27 In any case, making a child’s nationality conditional on the status of ‘legality’ or ‘illegality’ of the parents, or even worse of one of the two parents, not only violates the rights of children, as provided for in the UN Convention for the Rights of the Child, but also constitutes discrimination against the children who are victimised by the political situation and whom the Republic has an ob- ligation to protect and respect. Due to the lack of transparency, it is not possible to assess the implementation of this law. 13.2.1 Acquisition by descent The Third ECRI Re- port on Cyprus (2006: 8) notes that the Cyprus Ombudsman is cur- rently investigating ‘the conformity of this procedure with national and international standards’. Moreover, it notes that ‘citizenship has been granted by this procedure to children whose Cypriot parent was a Turk- ish Cypriot and whose other parent was a citizen of Turkey’; however, it also states that ‘decisions to grant nationality have resulted in intoler- ant and xenophobic attitudes in public debate’. Secondly, sect. 109(3) of law 141(I)/2002 expressly prescribes that the above provisions for acquisition of citizenship do not come into force in cases where a person is born in Cyprus or abroad between 16 August 1960 and 11 June 1999, if his or her claim is based solely on his or her mother’s citizenship, or the fact that she was entitled to citi- zenship of the Republic. However, the law stipulates that the person (or if the person is a minor, his or her father or mother) may submit an application to the minister to be registered as a citizen of Cyprus. The Equality Body of Cyprus examined a complaint claiming discrimi- nation on the grounds of sex/gender and nationality (and indirectly 400 NICOS TRIMIKLINIOTIS ethnic or racial origin) for descendants of women of Cypriot origin born between 16 August 1960 and 11 June 1999.28 The Equality Body (the Ombudsman in its capacity as the Equality and Anti-discrimina- tion Body) considered that the said provision was indeed discrimina- tory; however, in a rather obscure decision, it refused to take any further action, due to the ‘transitory nature of the provision, to counter the situation and the expectations that had formed up to 1999 on the basis of the regimen of acquiring citizenship’.29 In any case, they are entitled to obtain nationality via registration. y g Another mode of acquisition (sect. 109(3)) is provided for persons born on or after 16 August 1960 and who are of Cypriot origin, i.e. descendants of a person who: p a. became a British citizen on the basis of the Cyprus (Annexation) Order-in-Council between 1914 and 1943; or a. became a British citizen on the basis of the Cyprus (Annexation) O d i C il b t d b. was born in Cyprus between 5 November 1914 and 16 August 1960 during which time his or her parents were ordinarily resident in Cyprus. 13.2.1 Acquisition by descent These persons are entitled to be registered as citizens provided that they are adults and of sound mind,30 apply to the minister31 via the de- signated means and provide an official confirmation of loyalty to the Republic, according to the format provided in the Second Table an- nexed to the law.32 13.2.2 Acquisition via registration The following persons are entitled to be registered as Cypriot citizens upon application to the relevant Minister: 1. Citizens of the United Kingdom and Colonies or a country of the Commonwealth,33 who are of Cypriot descent,34 provided that they: L ordinarily reside in Cyprus and/or resided for a continuous peri- od of twelve months in Cyprus or a shorter period that the min- ister may accept under special circumstances of any specific case, immediately before the date of the submission of their applica- tion, or are serving in the civil or public service; L are of good character; L intend to remain in the Republic, or depending on the circum- stances, continue serving in the civil or public service (sub-sect. 110(1)); and L sign an official confirmation of loyalty to the Republic. 2. Spouses or widowers/widows of persons who were citizens of the Republic, or spouses of persons who, had they not been deceased, would have become or would have the right to become citizens of the Republic, provided that they: CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 401 L ordinarily reside in Cyprus and/or resided there for a period not less than three years;35 L ordinarily reside in Cyprus and/or resided there for a period not less than three years;35 L ordinarily reside in Cyprus and/or resided there for a period not less than three years;35 L are of good character; L intend to remain in the Republic, or depending on the circum- stances, continue serving in the civil or public service of the Re- public or the educational service of the Republic or the Police Force of the Republic even after registration as citizens of the Re- public (subsect. 110(2)); and p ( ( )) L sign an official confirmation of loyalty to the Republic. 3. Underage children of any citizen. In this case the application for ci- tizenship has to be submitted by the parent or the guardian of the child. A person who has renounced his or her citizenship of the Republic or has been deprived of it may not be registered as citizen of the Republic according to sect. 110, but may still be registered with the approval of the minister (subsect. 110(4)). Persons who have been registered under this section become citizens of the Republic from the date of their re- gistration (subsect. 110(5)). 13.2.2 Acquisition via registration This provision places Cypriot descent at the core of the right to ac- quire citizenship; spouses and under-age children who are resident in Cyprus can apply but their application is treated as dependent on the person of Cypriot origin. Moreover, there is an issue of how the rights of spouses and dependents are implemented. In fact, the practice of the immigration authorities to deport migrants who have been living in Cyprus for several years continued in spite of criticism from legal circles, human rights NGOs, from the Ombudsman and from the Commissioner for Legislation.36 Within a time span of only a few weeks, the Court cancelled deportation orders on numerous in- stances.37 13.2.3 Acquisition via naturalisation (πολιτογράφηση) A non-Cypriot who resides lawfully in the Republic may acquire citi- zenship via discretionary naturalisation if he or she fulfils all of the fol- lowing conditions formulated in Table 3 annexed to the law (subsect. 111): ) a. he or she has lawfully resided in the Republic of Cyprus for the en- tire duration of twelve months immediately preceding the date of application; b. over and above the twelve months referred to above, an additional continuous period of seven years in the period immediately prior to this, the applicant must have ordinarily resided in the Republic, or have been serving in the civil or public service of the Republic, or a 402 NICOS TRIMIKLINIOTIS bit of both, for periods amounting in total to no less than four years; bit of both, for periods amounting in total to no less than four years; c. he or she is of good character; and c. he or she is of good character; and d. he or she intends to reside in the Republic. d. he or she intends to reside in the Republic. The law also provides for acquisition of citizenship via naturalisation for students, visitors, self-employed persons, athletes and coaches, do- mestic workers, nurses and employees who reside in Cyprus with the sole aim of working there as well as spouses, children or other depen- dent persons. The prerequisites are that they must have ordinarily re- sided in the Republic for at least seven years and one year in the period immediately prior to the application their stay must be ‘continuous’.38 There are also exceptional situations where citizenship may be granted.39 One must bear in mind that all of the above are based on the discre- tion of the Council of Ministers and the Minister of the Interior. More- over, given that there has been a policy that migrant worker permits cannot be extended beyond four years, the chance of acquiring citizen- ship for these groups is rather slim, unless they are married to a Cy- priot or are granted leave to stay on other exceptional grounds. Cypriot authorities are very reluctant to grant citizenship to migrants. The Cy- prus government failed to transpose Directive 203/109/EC by 23 Janu- ary 2006. 13.2.3 Acquisition via naturalisation (πολιτογράφηση) The law was passed in February 2007; following criticism by NGOs and strong trade union opposition, the restrictive criteria origin- ally foreseen for granting long-term migrants this special status, which included proficiency in Greek language, history and civilisation, were eventually dropped by the parliament. The naturalisation procedure has been criticised in the Second ECRI Report on Cyprus as the conditions apparently ‘leave a wide margin of discretion to the Naturalisation Department as concerns decisions to grant citizenship’; moreover the same Report claims that ‘there have been complaints that these decisions are sometimes discriminatory’ (ECRI 2001: 9). The same practice was criticised by the Third ECRI Report (2006: 8), which also notes that ‘decisions are still excessively discretional and restrictive’ but that ‘this is reflected not only in the use made of public order considerations, but also in the application of resi- dency and language requirements’. The ‘Cyprus problem’ is often quoted as a ‘national priority’ and is invoked by Greek-Cypriot authorities as the reason for their reluctance to open up citizenship rules so as not to alter the demography, particu- larly in the context of the Turkish policy of settlement in the occupied northern territories. However, this does not withstand close scrutinity as numerous amendments were made to facilitate various population policies that benefit what is perceived as ‘the Greek-Cypriot interest’. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 403 Several decisions by the Ombudsman have criticised a number of practices of the Population Data Archives regarding the process of granting citizenship. In particular, criticism is directed at the restrictive approach of the Director of the Population Data Archives as regards the acquisition of citizenship via registration and naturalisation; parti- cularly critical are the decisions regarding the rejection of applications for citizenship based on marriage with Cypriots.40 Moreover, the deci- sions also highlight considerable delays in the processing of the appli- cations, prejudice based on the religion of the applicant and the exer- cising of administrative discretion in the interpretation of the regula- tion that excludes those who have entered the country illegally from acquiring citizenship.41 Overall, the implementation of the rules on naturalisation and with the wide margin of discretion provided for by the legislation, is an is- sue of concern regarding the fairness of these policies. 13.2.3 Acquisition via naturalisation (πολιτογράφηση) There is little encouragement and information for persons entitled to be naturalised and there are bureaucratic obstructions that make the application for naturalisation unattractive and cumbersome. One can explain this pol- icy as a mixture of the colonial legacy and the keenness of the authori- ties to hold on to their ‘sovereignty’ concerning entry, sojourn, resi- dence and citizenship, particularly as the protracted Cyprus conflict is often invoked as a pretext. The consequence is a restrictive regime that requires reform if it is to observe international law standards on the subject. 13.2.4 Renunciation and deprivation of citizenship Any adult citizen of sound mind who is also a citizen of another state may renounce his or her citizenship by submitting a confirmation of renunciation, and the minister will take the appropriate action for the registration of such confirmation (sect. 112). Deprivation of citizenship is possible, only for citizens who acquired citizenship via registration or naturalisation, via an Order of the Coun- cil of Ministers (sect. 113) under the following circumstances: a. When it is established that the registration or certification of citi- zenship was obtained by deceit, false pretences or concealment of a material fact (subsect. 113(2)). b. If the Council of Ministers (subsect. 113(3)) is satisfied that: L through deeds or words this person has demonstrated a lack of loyalty to the laws of the Republic;42 or If the Council of Ministers (subsect. 113(3)) is satisfied that: ( 3(3)) L through deeds or words this person has demonstrated a lack of loyalty to the laws of the Republic;42 or L in a war fought by the Republic this person was illegally involved in an exchange with the enemy, has contacted the enemy or was in any way involved in any operation in which he knowingly as- sisted the enemy; or 404 NICOS TRIMIKLINIOTIS L within five years from naturalisation, he or she is convicted in any country of a crime carrying a sentence of one year or more. c. If the Council of Ministers (subsect. 113(4)) is satisfied that the nat- uralised citizen has ordinarily resided in foreign countries for a con- tinuous period of seven years. The Council of Ministers cannot deprive a person of citizenship unless it is satisfied that it is not in the public interest that the said person re- mains a citizen of the Republic (sub-sect. 113(5)). It is apparent that the above is contrary to art. 5 of the 1997 Eur- opean Convention on Nationality, which Cyprus is yet to sign. In fact, the Second and Third ECRI Reports on Cyprus recommend that Cy- prus signs and ratifies this Convention. In any case, there is a com- plaint before the Equality and Anti-discrimination Body arguing that the above provision is contrary to the general prohibition of discrimina- tion as laid down in art. 1 of Protocol 12 to the European Convention on Human Rights, which has been ratified by the Republic of Cyprus. 13.2.4 Renunciation and deprivation of citizenship But they can acquire citizen- ship by discretionary naturalisation, providing that they fulfil the re- quired qualifications. 2. Children born in Cyprus to migrants who do not hold Cypriot citi- zenship or have a right to acquire it are not entitled to citizenship. 2. Children born in Cyprus to migrants who do not hold Cypriot citi- zenship or have a right to acquire it are not entitled to citizenship. 2. Children born in Cyprus to migrants who do not hold Cypriot citi- zenship or have a right to acquire it are not entitled to citizenship. 13.2.4 Renunciation and deprivation of citizenship It is apparent that the decisive element in the granting of citizenship is Cypriot descent which is combined with birth to form the various ca- tegories of rights provided. First, we can identify the following cate- gories of persons of Cypriot descent: 1. Greek-Cypriots (and the three religious groups) born in the area controlled by the Republic of Cyprus: this category is not really an issue as citizenship is granted automatically. 2. In principle, the same ought to apply to Turkish-Cypriots born in Cyprus and to children who have at least one Cypriot parent. Turk- ish-Cypriots born in the occupied territories are automatically en- titled to citizenship provided that they submit documents of their parents or grandparents issued by the Republic of Cyprus or the co- lonial authorities (TRNC documents are not recognised). However, in practice, art. 109 of the Law referred to above may result in a more discretionary regime for persons, one of whose parents is a Turkish national, even if they reside in the area under control of the Republic. 3. Persons of Cypriot origin born abroad, who have one Cypriot par- ent, are entitled to citizenship. 4. Persons of Cypriot origin born abroad between 16 August 1960 and 11 June 1999 and whose entitlement to Cypriot citizenship is solely based on their mother being Cypriot (or being entitled to Cypriot ci- tizenship) are not entitled to citizenship. They may, however, apply to acquire citizenship via registration. 5. Children born in Cyprus to non-Cypriot migrants who legally en- tered and reside in Cyprus and have acquired or would have been CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 405 entitled to acquire Cypriot citizenship via naturalisation are entitled to citizenship. ‘Collateral’ policies have been developed to use tax incentives and na- tional service ‘discount’ for men (six months if under 26 and three months if over 26 instead of the 25 months of normal national service) to attract Greek-Cypriots from abroad to live in Cyprus. y y Second, those who are not of Cypriot origin can only acquire citizen- ship via naturalisation or registration. Therefore, 1. Non-Cypriots who legally entered and reside in Cyprus are not en- titled to acquire Cypriot citizenship. But they can acquire citizen- ship by discretionary naturalisation, providing that they fulfil the re- quired qualifications. 1. Non-Cypriots who legally entered and reside in Cyprus are not en- titled to acquire Cypriot citizenship. 13.3.1 Europeanisation There is little doubt that the language of ‘Europe’ has become domi- nant in Cyprus as there is an orientation of political discourse and rhetoric towards Europe as a reference point.43 The question is whether the process of Europeanisation has touched upon citizenship and na- tionality. One issue is European citizenship itself, which affects the di- vided Cypriot citizenship. A number of key issues relate to the right to the free movement of workers in the EU; for instance, the fact that Cy- prus has not yet regulated same-sex marriages and extra-marital rela- tionships has resulted in various forms of discrimination against les- bian, gay, bisexual and transsexual Union citizens, thus constituting ef- fective obstacles to free movement. Another problem is the regulation of the ‘Green line’ that divides the country (see Trimikliniotis 2008). European Citizenship has different aspects relevant to the potential for transformation of the citizenship/nationality issue in Cyprus. First it may provide an all encompassing identity that has the potential to overcome the ethnic divide between Greek-Cypriots and Turkish-Cy- priots. It is argued that ‘shared cultural experience’ between Greek-Cy- priots and Turkish-Cypriots – many times suppressed by nationalists in the past ‘in order to focus on ethnic differences’ – could become a new focus as there are common aspects of identity that can unite the two communities. According to this optimistic view, EU membership may emphasise the shared culture and help in finding a solution to the Cy- prus problem (Botswain 1996: 94). Moreover, EU Citizenship may 406 NICOS TRIMIKLINIOTIS have a positive impact on human rights as the EU is expected to act as a guarantor of rights, such as the freedom of movement, settlement and ownership of land as provided in the Treaty of Rome and in line with the ‘acquis communautaire’. ‘Citizenship’ would underpin rights (communal/individual) thus assisting in creating a better climate of trust and security through the European Court of Justice, the European Court of Human Rights, the Council of Europe and the EU in gener- al.44 The European Conventions of the Council of Europe and other in- ternational instruments for ‘minority rights’ (Thornberry 1994), although technically outside the acquis, could arguably be a useful me- chanism from which Turkish-Cypriots stand to gain;45 however, Turk- ish-Cypriots are not a ‘minority’ but a ‘community’ in a consociation re- gime. g Moreover, matters are, in practice, far more complicated. 13.3.1 Europeanisation Since the rejection of the UN plan in April 2004, the Europeanisation issues have not acted as a constructive force: the issue of EU accession has be- come yet another point of contestation between Greek-Cypriots and Turkish-Cypriots and the question of what kind of future ‘European so- lution’ there will be for the Cyprus problem, is becoming a dominant question. Inevitably, the questions of citizenship have been more or less put on hold as they are subordinate to the solution of the Cyprus problem. It is, however, highly likely to return in the near future as it remains one of the key issues in the Cyprus problem. 13.3.2 Reunification, partition and settlers: Nationality turns into a hot political issue This is perhaps the greater challenge in the adventures of national- ity in Cyprus. We have already referred to some of the issues as re- gards the period 1974-2004 and the challenges of migration. How- ever, the central question arises out of the latest efforts to resolve the Cyprus problem, which resulted in the UN plan known as ‘the Annan Plan’. The issue of who is entitled to nationality is a hot political issue. In the northern territories the policy of Turkey is to ‘replace’ Turkish-Cy- priot e´migre´s with Turkish settlers from the mainland or to distort the demographic balance of the Cyprus population by giving TRNC nation- ality to a large number of settlers.46 In the area under the control of the Republic of Cyprus it is estimated that there are between 15,000 to 20,000 Pontian Greeks from the former Soviet Union, a few of whom were granted nationality, after residing for a period of seven years in Cyprus.47 The demographic study conducted by the Ministry of the In- terior in 2000 found that there were 10,000-12,000 Greek Pontians re- siding in Cyprus at the time. However, there have been changes since; CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 407 in any case, Pontian organisations claim to represent 40,000-45,000 Greek-Pontians residing in Cyprus. The UN proposal for resolution contains specific provisions over the number of settlers who would be granted nationality. This has proven to be a particularly sore point for the Greek-Cypriots, who eventually rejected the plan. In fact, it is widely believed that one of the reasons the Greek-Cypriots voted ‘NO’ to the plan was the fear over the ‘large numbers’ of settlers who would eventually be allowed to remain.48 Nevertheless, these provisions were seen by Greek-Cypriots as proble- matic in that they were alleged to allow for a ‘perpetual inflow of set- tlers’, in spite of the 5 per cent cap for any future migration from Tur- key and Greece.49 y In the ‘main articles’ of the Foundation Agreement of the Annan plan (art. 3) there is reference to ‘a single Cypriot citizenship’ regulated under federal law as well as the ‘internal constituent state citizenship status’ to be enjoyed by ‘all Cypriot citizens’; moreover, the plan lays out a set of complicated rules about preserving the ‘identity’ (see ap- pendix 1). 13.3.2 Reunification, partition and settlers: Nationality turns into a hot political issue The acquisition of citizenship is regulated by an agreed con- stitutional law which essentially deals with the issue of settlers from Turkey. Moreover the plan envisages a federal law on ‘aliens and immi- gration’ (Foundation Agreement, Attachment 5, Law 1) as well as a fed- eral law for international protection and the implementation of the Geneva Convention Relating to the Status of Refugees and the 1967 Protocol on the Status of Refugees (Foundation Agreement, Attach- ment 5, Law 2) which, in the event of a settlement, would replace the current laws on immigration and refugees. The plan was rejected by the Greek-Cypriots, but remains relevant to current and possible future negotiations as the most comprehensive plan ever to be negotiated, and is therefore a valuable source of ideas on the formation of a bizonal, bicommunal federation (see Trimiklinio- tis 2009). In the absence of a solution, prior to the referendum and soon after, a number of public debates erupted that centred on the question of nationality policy. The question of moving towards an effec- tive right to nationality by providing passports for the Republic of Cy- prus has been relevant particularly since accession. For the Greek-Cy- priot post-referendum political arena, an issue that became a hot politi- cal issue was the question of granting the right of nationality to children of Turkish-Cypriots who married Turkish settlers. Right-wing media attacked the cabinet decision to grant nationality rights to 703 persons one of whose parents was a Turkish settler.50 The government was forced to go on the defensive with the Minister of the Interior claiming that ‘the legislation does not allow the granting of nationality, either to settlers or an alien from another country, who has entered the Republic illegally.’51 The media as well as some members of the coali- 408 NICOS TRIMIKLINIOTIS tion partners52 stated that because ‘invasion, colonisation and changing the demographic character of a country’ is a ‘war crime’, granting na- tionality to the offspring of colonisers is never justified. In fact, there are allegations that there is an unofficial moratorium on the subject to freeze the applications of children of settlers married to Cypriots; a practice that has been criticised by human rights organisations.53 The current situation in Cyprus leaves the nationality policy regard- ing this category of persons in a state of limbo. 13.3.2 Reunification, partition and settlers: Nationality turns into a hot political issue In practice, pending a resolution of the problem, the Cyprus problem will always predominate and colour the nationality policy. The greatest challenge for Cypriot pol- icymakers is to adopt a nationality and citizenship policy that enhances the possibility for reunification and thus does not consolidate and in- directly officially endorse partition. 13.4 Statistical developments since 1985: The ‘politics of numbers’ and the ‘numbers game’ Table 13.1 shows that the anomaly of the Cyprus problem has had a massive impact on the numbers of citizenship acquisitions. The largest figure involves Turkish-Cypriots. Most of these acquired citizenship after 23 April 2003 when the checkpoints were opened – although Turkish-Cypriots had a right to Cypriot citizenship before that date, they could not make use of this right due to the war and the de facto partition since 1974. Between 1995 and 3 March 2009, 101,778 Turk- ish-Cypriots have acquired birth certificates of the Republic of Cyprus; 83,372 have acquired identity cards and 54,595 passports. Moreover, the numbers of applications for citizenship more than doubled since Cy- Table 13.1 Acquisitions of Cypriot citizenship by modes, 1985-3 March 2009 Mode of acquisition Number 1 Naturalisation 5,395 2 Spousal transfer of citizenship 12,824 3 Transfer to children whose parents obtained citizenship 5,160 4 Registrations of persons of Cypriot descent 2,250 5 Persons who acquired citizenship on grounds of origin, e.g. expatriate Cypriots 23,932 6 Turkish-Cypriots who acquired passports of the Republic of Cyprus since 1995 54,595 7 Turkish-Cypriots who acquired identity cards of the Republic of Cyprus since 1995 83,372 8 Turkish-Cypriots who acquired birth certificates of the Republic of Cyprus since 1995 101,778 Sources: Personal Communication of the Civil Registry Migration Department and the Population Data Archives, 3 March 2009 Table 13.1 Acquisitions of Cypriot citizenship by modes, 1985-3 March 2009 CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 409 prus acceded to the EU and there is a backlog of some hundreds of ap- plications pending. The second largest group of people granted Cypriot citizenship were Cypriot by descent or by origin (2,250 for category 4 and 23,932 for ca- tegory 5). This figure reflects the favourable conditions for return mi- gration to Cyprus, which has enjoyed a steady improvement of the liv- ing standards and relative stability despite the conflict and the continu- ing division. Moreover, it illustrates the importance of the ties with Cyprus and the ethnic connections amongst the Cypriot diasporic com- munities abroad. Finally, it may reflect the relative success of various population policies aimed at encouraging expatriates to return to Cy- prus. However, it is difficult to verify how far these incentives influ- enced the decision to migrate to Cyprus. It has to be noted that this ca- tegory includes an unknown number of Turkish-Cypriots. 13.4 Statistical developments since 1985: The ‘politics of numbers’ and the ‘numbers game’ The third largest category comprises of those who acquire citizen- ship by marriage, a total of 12,824 persons. Research shows that the large majority of these are foreign women married to Cypriot men (ra- tio 7:1) and that there is a preference for certain nationalities (Fulias- Souroulla 2008). The next largest group are naturalised persons (category 1). The number of naturalisations is rather small considering that that there are 138,000 non-Cypriots residing in Cyprus,54 which includes how- ever over 70,000 EU citizens.55 The number of non-Cypriot natura- lised migrants is even smaller since an unknown share of those natura- lised are persons of Cypriot origin born in the Commonwealth prior to 1960 and who could only acquire Cypriot citizenship by naturalisation as well as those who renounced or were deprived of Cypriot citizenship to acquire another citizenship (e.g. German). In any case, about half of those who acquire citizenship by naturalisation are Greek Pontians re- siding in Cyprus: it is estimated that about 400 Greek Pontians were granted Cypriot citizenship in 2004 and about 500 in 2005.56 13.5 Conclusions The mechanics of acquisition, renunciation and deprivation of citizen- ship in the Republic of Cyprus revolves around Cypriot descent: per- sons of Cypriot origin are basically entitled to citizenship, whilst per- sons of non-Cypriot descent may be allowed to apply if they have re- sided in Cyprus for seven years to acquire citizenship via registration and naturalisation mechanisms. The reference of one of the very few Cypriot legal scholars dealing with the subject, Criton Tornaritis (1982: 39), that Cyprus has adopted a ‘mixed principle combining ius soli and ius sanguinis’ is not very helpful as Cypriot descent forms the core. 410 NICOS TRIMIKLINIOTIS Although, we cannot locate a declared policy on citizenship/national- ity as such in the Republic of Cyprus, what we do find instead is a practice that derives from the long-standing Cyprus conflict as well as international developments such as accession to the EU, economic de- velopment and migration, and to some extent changing attitudes, parti- cularly as regards the question of gender. Other factors are also of rele- vance, such as population control, economic and welfare issues, social policy, etc. As for the unrecognised Turkish Republic of Northern Cy- prus, the issue of citizenship is totally subsumed in its own ‘struggle for recognition’ and it is a mirror image of the country it broke away from and yet can never escape from, the Republic of Cyprus. y p p yp In the context of Cyprus, nationality policy is inevitably subordinated to the unique historical conjunctures that perpetuate the island’s pro- tracted ethno-national conflict. In fact, the question of nationality goes to the heart of the existence of the country’s very own ‘nation-state dia- lectic’ (see Trimikliniotis 2000, 2005): the challenge for a citizenship that manages to transcend the ethno-national conflict and the ethno- communal divide is perhaps the greatest challenge of all for this coun- try’s European aspirations for a re-united and peaceful future. The ne- gotiations between the two community leaders resumed in September 2008 and have raised hopes for a resolution of the dispute in a man- ner that will reunite the island on the basis of a bi-zoned, bi-communal federation with a single sovereignty, territory and nationality. This solu- tion would radically transform the nationality issues as we have known them. 13.5 Conclusions Chronological list of citizenship-related legislation in the Republic of Cyprus Date Document Content Source 1960 Constitution of the Republic of Cyprus Regulates initial determination of Cypriot citizenry after independence (Annex D); regulates acquisition of citizenship (art. 198) www.kypros.org; www.legislationline.org 1967 Citizenship Law No. 43/ 1967 Regulates acquisition and deprivation of citizenship; defines person’s descent on his or her father’s side as basic criterion for citizenship acquisition www.legislationline.org; www.coe.int 1972 Citizenship (Amendment) Law No. 1/1972 Extends the minister’s discretion regarding deprivation or renunciation of citizenship, if the www.coe.int Chronological list of citizenship-related legislation in the Republic of Cyprus Date Document Content Source 1996 Citizenship (Amendment) Law No. 58(I)/1996 CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 411 Date Document Content Source minister is of the opinion that the aim of such a declaration is to avoid military service or criminal prosecution 1983 Citizenship (Amendment) Law No. 74/1983 Extends entitlement to citizenship to persons born in Cyprus prior to independence and whose father was of Cypriot descent; deleted subsect. 4 (d), which entitles ‘persons born in Cyprus, who are not entitled by birth to acquire any other citizenship’, to Cypriot citizenship www.coe.int 1996 Citizenship (Amendment) Law No. 19(I)/1996 Extends the right to citizenship via registration to persons whose father was a British subject on the basis of the Annexation of Cyprus Orders of Council 1914-1943 or was born in Cyprus between 1914-1960 1996 Citizenship (Amendment) Law No. 58(I)/1996 Regulates the naturalisation procedure for persons of non-Cypriot descent residing and working in Cyprus (conditions included: nine years of residence out of the previous thirteen years, plus twelve months of continuous residence immediately prior to application) 1996 Citizenship (Amendment) Law No. 70(I)/1996 Introduces facilitated naturalisation for reasons of public interest irrespective of residence rules 1997 Citizenship (Amendment) Law No. 50(I)/1997 Extends the right to apply for naturalisation to spouses, children or other dependent persons 1998 Citizenship (Amendment) Law No. 102(I)/1998 Deletes a section in the Second Table of the main law which empowered the Council of Ministers to use discretion for extending 412 NICOS TRIMIKLINIOTIS Date Document citizenship to persons of Cypriot descent 1998 Citizenship (Amendment) Law No. 105(I)/1998 Empowers the minister to grant citizenship to spouses or widows/ widowers married to a Cypriot for at least two years 1999 Citizenship (Amendment) Law No. 65(I)/1999 Extends the right to citizenship to any person of Cypriot descent (i.e. regardless of whether the father or the mother is Cypriot); makes automatic acquisition of citizenship conditional on lawful entry and stay in the Republic (effectively this covers children one of whose parents is a Turkish settler) 1999 Citizenship (Amendment) Law No. 128(I)/1999 Deletes the subsection empowering the Minister of the Interior to register the wives or widows of Cypriots as citizens provided that he or she is satisfied that they meet the required conditions 2001 Citizenship (Amendment) Law No. 168(I)/2001 Extends the period of marriage before spouses or widows/widowers can acquire citizenship by registration to at least three years 2002 Population Data Archives Law No. 1999 Citizenship (Amendment) Law No. 128(I)/1999 www.cyprus-un-plan.org 2004 Annan Plan: Foundation Agreement www.cyprus-un-plan.org CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 413 Notes 1 This occurred when the government decided to grant citizenship to children of Turkish-Cypriots married to settlers in 2004 and 2005. 2 This was an illegal terrorist organisation launched allegedly to campaign for Enosis, i.e. union with Greece; it carried out bombings, murders of civilians and tried several times to assassinate President Makarios (Droussiotis 1994). 3 These are two contrasting approaches regarding the refenda on 24 July 2004 and they have implications on how to proceed if a solution is to be found. 4 In return for protection from the expansionist aims of Russia and an annual payment to Turkey of the sum of £12,000. 5 The ‘modernisation’ began before the British arrived in Cyprus; however, it was intensified with the arrival of the British colonists at the end of the nineteenth and the beginning of the twentieth century (see Katsiounis 1996). 6 Such were the privileges granted to the Cypriot Greek Orthodox Church of Cyprus that the archbishop of Cyprus had direct recognition from the Sultan, as Ethnarchic leader, the millet bashi. 7 The beginning of the twentieth century saw a conflict between the ‘traditionalists’ and the ‘modernists’ in the Turkish-Cypriot community; a battle that was decisively won by the modernists (Anagnostopoulou 2004). 8 In the 1940s, the Left had risen and competed with the church for leadership of the anti-colonial movement (Katsiaounis 2000). By the mid 1950s the church re- established its authority with EOKA. EOKA (Ethniki Organosis Kyprion Agoniston – National Organisation of Cypriot Fighters) was the Greek-Cypriot nationalist organi- sation which started a guerrilla campaign against British colonial rule aimed at self- determination and union with Greece (Enosis). The political leadership of EOKA was the church. 9 The case was Attorney General of the Republic v Mustafa Ibrahim and Others (1964), Cyprus Law Reports 195 (see also Nedjati 1970; Loizou 2001). 10 These agreements set the basis for a bi-communal and bi-zonal federal Republic following the invasion. 11 See Aziz v. Republic of Cyprus, ECHR, Application No. 69949/01. The full text of the judgement is available on the website of the European Court of Human Rights: www.echr.coe.int. 12 Hence the requirements to produce documents relating to birth of their Cypriot parents prior to 1974. 13 M. Hadjicosta, ‘Fears over gypsy influx’, The Cyprus Weekly, 13-19 April 2001. www.domresearchcenter.com. 14 J. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 141(I)/2002 Unifies the Citizenship Law with various other population issues, such as archives, elections, registration, identity cards, passports and deaths, into one law called Population Data Archives Law No. 141 (I)/2002 2004 Annan Plan: Foundation Agreement Comprehensively restructures population issues including citizenship w CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY Notes Matthews, ‘More gypsies crossing from north as Koshis warns about spies’, The Cy- prus Mail, 3 April 2001. www.domresearchcenter.com. 15 Apparently, the Minister of the Interior at the time, Mr. C. Christodoulou, now Governor of the Central Bank, said that he would not reveal the options discussed, because, ‘in this country, when it comes to illegal immigrants or gypsies (moving into an area), everyone reacts’. See ‘Our reaction to gypsies raises some awkward questions’, The Cyprus Mail, 10 April 2001. www.domresearchcenter.com. q yp p 16 A Turkish-Cypriot woman filed a complaint because her application to be registered in the Republic’s Citizens Record was rejected, on the basis that the birth of her mother had not been recorded in the Republic’s archives. The complainant’s mother had been born to Roma parents who failed to register her birth. It was also noted that the complainant was inconvenienced for several months due to ill advice by government officers as to the procedure with regard to her registration. In addition, she complained about the rejection of her application to enrol her child in school 414 NICOS TRIMIKLINIOTIS because the child did not have a birth certificate from the Republic. Following the Commissioner’s report on the matter, her child was finally enrolled in school. because the child did not have a birth certificate from the Republic. Following the Commissioner’s report on the matter, her child was finally enrolled in school. 17 They thought that they could no longer be singled out, targeted and harassed and there was a general feeling of optimism and rapprochement (Trimikliniotis 2003). 18 Research by the college of Tourism in April 2004 is indicative. 19 The term ‘significant’ is not explained in the Demographic Report of 2005. 20 The Cyprus (Annexation) Order-in-Council 1914, No. 1629 Statutory Rules and Orders Rev. (1948), vol. V, pp. 577-578. The order can also be found in Cyprus, (1920), Handbook No. 65 in the series, London: HMSO. 21 The Cyprus (Annexation) Order-in-Council 1917, No. 1374 S.R & O. and The Cyprus (Annexation) Order-in-Council 1914, No.1629 S.R & O. Rev. (1948), vol. V, pp. 578- 582. 22 The provisions of Annex D are quite detailed governing different categories of persons and set out the basic structure of citizenship acquisition that was to follow also in the subsequent legislation on the subject (Tornaritis 1982: 33-41). 23 Law No. 43/67, as amended by Laws No. Notes 1/72, 74/83, 19(I)/96, 58(I)/96, 70(I)/96, 50(I)/97, 102(I)/98, 105(I)/98, 65(I)/99, 128(I)/99, 168(I)/2001. 24 With laws No. 65(I)/2003, 76(I)/2003, 62(I)/2004 and 13(I)/2006. 25 Sects. 109(1) and (2) of Law No. 141(I)/2002 provide for the procedure and the appropriate forms. In cases where the applicant is under age, the application can be made by a parent. 26 Art. 109 Population Data Archives Law No. 141(I)/2002. This clause was first introduced by Law 65(I)/1999 that came into force on 11 June 1999. 27 This information was provided by the officer of the Population Data Archives of the Ministry of the Interior, Christiana Ketteni, on 15 December 2006. She stated that the standard practice of the Council of Ministers is to approve ten to fifteen applications each time there is a meeting of the Council of Ministers. Moreover, she claimed that the people affected by the Council of Ministers’ discretion are ‘persons who have a Cypriot grandparent’, but it remained unclear how this category could fall under art. 109. 28 It was alleged that discrimination is ongoing as the specific provision has resulted in the perpetual and future discrimination of this category of persons and their descendants since the principle of anti-discrimination is not only momentarily applied, but it is also forward looking. It is likely that this provision is in violation of the laws against discrimination and, in particular, Law No.142(I)/2004, which transposes the anti-discrimination acquis and more importantly Protocol 12. See File No. 62/2005 of the Ombudsman’s Report. 28 It was alleged that discrimination is ongoing as the specific provision has resulted in the perpetual and future discrimination of this category of persons and their descendants since the principle of anti-discrimination is not only momentarily applied, but it is also forward looking. It is likely that this provision is in violation of the laws against discrimination and, in particular, Law No.142(I)/2004, which transposes the anti-discrimination acquis and more importantly Protocol 12. See File No. 62/2005 of the Ombudsman’s Report. 29 See File No. 62/2005 of the Ombudsman’s Report. 29 See File No. 62/2005 of the Ombudsman’s Report. 30 The Greek text refers to ‘πλήρης ικανότητα’, which literally translated means ‘full ability’, but it must be construed as meaning of ‘sound mind’, which was the old British formulation. Notes 30 The Greek text refers to ‘πλήρης ικανότητα’, which literally translated means ‘full ability’, but it must be construed as meaning of ‘sound mind’, which was the old British formulation. 32 A number of Tables are annexed to the Law. The First Table specifies the fees for issuance of passports; the Second Table includes the format of making a formal oath of allegiance to the Republic of Cyprus; the Third Table describes the conditions for naturalisation. 32 A number of Tables are annexed to the Law. The First Table specifies the fees for issuance of passports; the Second Table includes the format of making a formal oath of allegiance to the Republic of Cyprus; the Third Table describes the conditions for naturalisation. 33 For subsect. 110, ‘a country of the Commonwealth’ includes every country excluding the Republic of Cyprus, on the date of entering into force of the Law, which is a member of the British Commonwealth and includes the Republic of Ireland and any other country that has been declared by an Order of the Council of Ministers as a Commonwealth Country for the purposes of this section. 33 For subsect. 110, ‘a country of the Commonwealth’ includes every country excluding the Republic of Cyprus, on the date of entering into force of the Law, which is a member of the British Commonwealth and includes the Republic of Ireland and any other country that has been declared by an Order of the Council of Ministers as a Commonwealth Country for the purposes of this section. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY The deportation order was cancelled by the Court on 13 January 2006 based on the argument that the immigration authorities should have taken into account the fact that the appellant was married to a Polish (and therefore EU) citizen. 37 Some indicative cases are the following: Lali Jashiashvili & Costas Hadjithoma v. The Ministry of the Interior and the Immigration Officer and Nebojsa Micovic v. The Republic of Cyprus through the Chief Immigration Officer, where the Supreme Court cancelled the deportation order against nationals living with their families and working in Cyprus since 1998. Another case involved the deportation order issued against the Pakistani national Mahmoud Adil when his asylum application was rejected. The deportation order was cancelled by the Court on 13 January 2006 based on the argument that the immigration authorities should have taken into account the fact that the appellant was married to a Polish (and therefore EU) citizen. 38 Introduced by amendment 58(1)/1996. 38 Introduced by amendment 58(1)/1996. 39 Introduced by amendment 70(1)/1996. 39 Introduced by amendment 70(1)/1996. 40 See relevant Ombudsman Reports, Files No. 2599/2005, 1958/2005, 2059/2005, 2368/2005, 2599/2005, 2780/2005. 41 See Ombudsman Report, File No. 727/2006. 41 See Ombudsman Report, File No. 727/2006. 42 The Greek term used is νομιμοφροσύνη. 42 The Greek term used is νομιμοφροσύνη. 43 One scholar termed this as ‘the Europeanisation of political thinking’ (Theophylactou 1995: 121), whilst another scholar interpreted this as the embracing of a ‘Euro-centric ideology’ by the Greek-Cypriot political elite (Argyrou 1996: 43). 44 It is sometimes assumed that possible ‘weaknesses’ in the settlement would gradually be somehow eliminated by the operation of the acquis and via access to the European Court of Justice and the European Court of Human Rights. 44 It is sometimes assumed that possible ‘weaknesses’ in the settlement would gradually be somehow eliminated by the operation of the acquis and via access to the European Court of Justice and the European Court of Human Rights. 45 Minority rights for ‘old’ ethnic minorities have a significantly long tradition of protection under various treaties and authorities, even from the last century, though these were very restricted and at the whim of the great powers (Hannum 1996: 50- 74). However, art. 8 of the European Convention on Human Rights guarantees the right to private and family life (which has been interpreted as to include ethnic identity) and art. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 415 34 For the purposes of subsect. 110, a person of Cypriot descent is defined as any person born in Cyprus and whose parents ordinarily resided in Cyprus at the time of his or her birth and includes every person that descends from these persons. 34 For the purposes of subsect. 110, a person of Cypriot descent is defined as any person born in Cyprus and whose parents ordinarily resided in Cyprus at the time of his or her birth and includes every person that descends from these persons. 35 There are also specific provisions allowing the minister to take measures after less than three years, but it is restricted to a minimum of two years. Also, for the purposes of this subsection ‘ordinary residence’ requires at least six months stay in Cyprus but in any case the total residence in Cyprus during the preceding three years prior to submission of the application must not be less than two years. 35 There are also specific provisions allowing the minister to take measures after less than three years, but it is restricted to a minimum of two years. Also, for the purposes of this subsection ‘ordinary residence’ requires at least six months stay in Cyprus but in any case the total residence in Cyprus during the preceding three years prior to submission of the application must not be less than two years. 36 L. Leonidou, ‘Authorities in the dock over treatment of immigrants’, The Cyprus Mail, 15 January 2006. 36 L. Leonidou, ‘Authorities in the dock over treatment of immigrants’, The Cyprus Mail, 15 January 2006. 36 L. Leonidou, ‘Authorities in the dock over treatment of immigrants’, The Cyprus Mail, 15 January 2006. 37 Some indicative cases are the following: Lali Jashiashvili & Costas Hadjithoma v. The Ministry of the Interior and the Immigration Officer and Nebojsa Micovic v. The Republic of Cyprus through the Chief Immigration Officer, where the Supreme Court cancelled the deportation order against nationals living with their families and working in Cyprus since 1998. Another case involved the deportation order issued against the Pakistani national Mahmoud Adil when his asylum application was rejected. CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY regularly referred to the Pontians as the alternative to ‘an Afro-Asian’ new minority (see Trimikliniotis 1999). ( 999) 48 Obviously there was scare mongering and exaggeration by the Greek-Cypriot ‘No campaign’ about the figures and misinformation about the actual provisions. Palley (2005) has a chapter devoted to the subject and puts forward the case for the Greek- Cypriot side and the reasons for the Greek-Cypriot rejection as regards this issue. ( 999) 48 Obviously there was scare mongering and exaggeration by the Greek-Cypriot ‘No campaign’ about the figures and misinformation about the actual provisions. Palley (2005) has a chapter devoted to the subject and puts forward the case for the Greek- Cypriot side and the reasons for the Greek-Cypriot rejection as regards this issue. 48 Obviously there was scare mongering and exaggeration by the Greek-Cypriot ‘No campaign’ about the figures and misinformation about the actual provisions. Palley (2005) has a chapter devoted to the subject and puts forward the case for the Greek- Cypriot side and the reasons for the Greek-Cypriot rejection as regards this issue. 49 The provisions were depicted by Greek-Cypriot anti-Annan critics as rewarding the policy of colonisation. However, this is a highly complex issue which requires a detailed analysis and a resolution that bears in mind the principles of justice and international law, as well as the humanitarian, the individual rights and the personal dimensions of the problem. 50 See G. Psyllides, ‘Citizenship for settler children: Christou hits back’, Cyprus Mail, 1 July 2004. 50 See G. Psyllides, ‘Citizenship for settler children: Christou hits back’, Cyprus Mail, 1 July 2004. 50 See G. Psyllides, ‘Citizenship for settler children: Christou hits back’, Cyprus Mail, 1 July 2004. 51 Minister Andreas Christou quoted in Politis, 7 June 2004. Also see the explanations of the legal regulations in section 2.1 of this chapter. 51 Minister Andreas Christou quoted in Politis, 7 June 2004. Also see the explanations of the legal regulations in section 2.1 of this chapter. 52 See Cyprus Mail, 1 July 2004. Palley (2005) deals with the legal and political issues of the settlers. Also see Hannay (2005). 52 See Cyprus Mail, 1 July 2004. Palley (2005) deals with the legal and political issues of the settlers. Also see Hannay (2005). CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 53 In a press release dated 2 July 2004, the human rights NGO ‘KISA’ (Action for Equality, Support and Anti-racism) expressed concern over the intolerant and racist attitudes developing around the issue of granting nationality to these children. 54 Data provided by the Civil Registry Migration Department and the Population Data Archives on 3 March 2009. I would like to thank Andros Ktorides for his help in obtaining the data and for his lucid analysis. Of course, the argumentation and conclusions are mine. 55 This figure was provided by the Civil Registry Migration Department and the Population Data Archives (3 March 2009). However, different numbers are produced by the Ministry of Labour and Social Insurance: according to their data only 49,639 EU citizens were residing in Cyprus in July 2008 (see Total Aliens and Europeans Data 2008, www.mlsi.gov.cy). 56 This figure was provided by Christiana Ketteni, an official of the Population Data Archives, Ministry of the Interior who was asked to comment on the categories, figures and the underlying policies (15 December 2006). CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 9 guarantees ‘the right to freedom of thought, conscience and religion’. More specifically, art. 27 of the Covenant on Civil and Political Rights refers to the rights of ‘ethnic, religious or cultural minorities’ to ‘enjoy their own culture, to profess and practice their own religion, or use their own language’, but these are set to be extended in other areas of freedom (Hannum 1996: 62-63). However, the European ‘regime’ on ethnic minority groups’ protection, is problematic, as there is a distinct lack of enforcement mechanisms. These rights are heavily dependent upon the nation-states for implementation; in any case the mechanisms for implementation are very weak if not irrelevant (Hannum 1996). 45 Minority rights for ‘old’ ethnic minorities have a significantly long tradition of protection under various treaties and authorities, even from the last century, though these were very restricted and at the whim of the great powers (Hannum 1996: 50- 74). However, art. 8 of the European Convention on Human Rights guarantees the right to private and family life (which has been interpreted as to include ethnic identity) and art. 9 guarantees ‘the right to freedom of thought, conscience and religion’. More specifically, art. 27 of the Covenant on Civil and Political Rights refers to the rights of ‘ethnic, religious or cultural minorities’ to ‘enjoy their own culture, to profess and practice their own religion, or use their own language’, but these are set to be extended in other areas of freedom (Hannum 1996: 62-63). However, the European ‘regime’ on ethnic minority groups’ protection, is problematic, as there is a distinct lack of enforcement mechanisms. These rights are heavily dependent upon the nation-states for implementation; in any case the mechanisms for implementation are very weak if not irrelevant (Hannum 1996). 46 The veteran Turkish-Cypriot leader has often been quoted saying: ‘A Turk leaves, another Turk comes’. 46 The veteran Turkish-Cypriot leader has often been quoted saying: ‘A Turk leaves another Turk comes’. 47 It appears that in the days of the collapse of the USSR, Greek-Cypriot policy-makers toyed with the idea of bringing to Cyprus Greek-Pontians rather than other migrants, due to their ethnic origin, in part to unofficially and quietly ‘redress’ the Turkish settler policy. Officially this was never admitted by right-wingers, and nationalists 416 NICOS TRIMIKLINIOTIS regularly referred to the Pontians as the alternative to ‘an Afro-Asian’ new minority (see Trimikliniotis 1999). Bibliography Anagnostopoulou, S. (2004), Τουρκικός εκσυγχρονισμός – Ισλάμ και Τουρκοκύπριοι στη δαι- δαλώδη διαδρομή του κεμαλισμού [Turkish Modernisation. Islam and the Turkish- Cypriots on their Dedalus-like Route to Kemalism]. Athens: Vivliorama. y Anthias, F. (1989), ‘Women and nationalism in Cyprus’, in N. Yuval-Davis & F. Anthias (eds.), Woman, Nation-State, 150-167. London: Macmillan. Argyrou, V. (1996), Tradition and Modernity: The Symbolic Class Struggle of the Cypriot Wedding. Cambridge: Cambridge University Press. g g g y Attalides, M. (1979), Cyprus Nationalism and International Politics. Edinburgh: Q Press. Botswain, T. (1996), ‘Perceptions of Cyprus as European’, in J. Charalambous, M. Sarafis & E. Timini (eds.), Cyprus and the European Union. A Challenge, 93-119. London: Uni- versity of North London. Chrysostomides, K. (2000), The Republic of Cyprus. A Study in International Law. London: Martinus Nijhoff Publishers. Constantinou, M.C. & Y. Papadakis (2001), ‘The Cypriot State(s) in situ: cross ethnic con- tact and the discourse of recognition’, in T. Dier (ed.), The European Union and the Cy- CYPRUS: POSTCOLONIAL CITIZENSHIP IN A DIVIDED COUNTRY 417 prus Conflict. Modern Conflict, Post-Modern Union, 73-97. Manchester: Manchester University Press. prus Conflict. Modern Conflict, Post-Modern Union, 73-97. Manchester: Manchester University Press. y Demetriou, O. (2006), ‘Freedom Square: The unspoken reunification of a divided city’, HAGAR Studies in Culture, Polity and Identities 7 (1): 55-77. y Demetriou, O. (2006), ‘Freedom Square: The unspoken reunification of a divided city’, HAGAR Studies in Culture, Polity and Identities 7 (1): 55-77. Demetriou, O. (2007), ‘To cross or not to cross? Subjectivization and the abs Cyprus’, Journal of the Royal Anthropological Institute (N.S.) 13: 987-1006. Demetriou, O. (2007), ‘To cross or not to cross? Subjectivization and the absent state in C ’ J l f th R l A th l i l I tit t (N S ) 8 6 Cyprus’, Journal of the Royal Anthropological Institute (N.S.) 13: 987-1006. Dodd, C.H. (1993), ‘From Federated State to Republic 1975-84’, in C. H. Dodd (ed.), The Political Social and Economic Development of Northern Cyprus, 103-135. Cambridgeshire: The Eothen Press. Droussiotis, M. (1994). Από το Εθνικό Μέτωπο στην ΕΟΚΑ Β [From the National Front to EOKA B]. Nicosia: Alfadi. Droussiotis, M. (2005), Η Πρώτη Διχοτόμηση, Κύπρος 1963-1964 [The First partition, Cy- prus 1963-1964]. Nicosia: Alfadi. ECRI (2001), Second Report on Cyprus. European Commission against Racism and Intol- erance. Strasbourg: Council of Europe. ECRI (2006), Third Report on Cyprus. European Commission against Racism and Intoler- ance. Bibliography The UN Secretary-General’s Mission of Good Offices in Cyprus 1999-2004. Oxford: HART Publishing. PIO (2006), Demographic Survey Report of 2005. Statistics Department. Nicosia: Republic of Cyprus. Sitas, A., D. Latif & N. Loizou (2007), Prospects of Reconciliation, Coexistence and Forgive- ness in Cyprus. A Research Report. PRIO Report 4/2007. Oslo: International Peace Re- search Institute (PRIO). www.prio.no. Spyrou, S. (2003), Educational Needs of Turkish-speaking Children in Limassol. Report for UNOPS. Nicosia. Theophylactou, D.A. (1995), Security, Identity and Nation Building. Cyprus and the Eur- opean Union in Comparative Perspective. Avebury: Ashgate. Thornberry, P. (1994), ‘International and European Standards on Minority Rights’, in H. Miall (ed.), Minority Rights in Europe. The Scope for a Transnational Regime, 13-29. London: Pinter. Tornaritis, C. (1982), Το Πολιτειακό Δίκαιο της Κύπριακής Δημοκρατίας [The Law Relating to the Government of the Republic of Cyprus]. Nicosia: Cyprus Research Centre. Trimikliniotis, N. (1999), ‘New Migration and Racism in Cyprus: The Racialisation of Mi- grant Workers’, in F. Anthias & G. Lazaridis (eds.), Into the Margins: Migration and Exclusion in Southern Europe, 139-179. Aldershot: Ashgate. Trimikliniotis, N. (2000), The Role of State Processes in the Production and Resolution of ‘Ethnic’ and ‘National’ Conflict: the Case of Cyprus. PhD thesis. University of Green- wich. Trimikliniotis, N. (2003), Discriminated Voices. Cyprus Report. www.multietn.uu.se. Trimikliniotis, N. (2005), ‘Πέραν του έθνους-κράτους: η δυνατότητα για μια ομοσπονδιακή, πολυεθνοτική ιδιότητα του πολίτη’ [Beyond the nation-state: the potential for a federal multiethnic citizenship], in N. Trimikliniotis (ed.) Το Πορτοκαλί της Κύπρου [The Or- ange Colour of Cyprus], 167-244. Athens: Nisos. Trimikliniotis, N. (2006), ‘A Communist’s Post-modern Power Dilemma: One Step Back, Two Steps Forward, “Soft No” and Hard Choices’, The Cyprus Review 18 (1): 37-86. Trimikliniotis, N. (2006), A Communist s Post modern Power Dilemma: One Step Back, Two Steps Forward, “Soft No” and Hard Choices’, The Cyprus Review 18 (1): 37-86. Trimikliniotis, N. (2007), ‘Reconciliation and Social Action in Cyprus: Citizens’ Inertia and the Protracted State of Limbo’, The Cyprus Review 19 (1): 123-160. Trimikliniotis, N. (2008), Cyprus Report 2007 of Network on the Free Movement of Workers within the European Union. Odysseus Academic Network coordinated by the Institute for European Studies Brussels. Trimikliniotis, N. (2009), ‘Annan V: Rethinking the Viability of the Constitutional Ar- rangement and its Future Importance’, in H. Faustmann & A. Varnava (eds.), The Failure to Reunify Cyprus: The Annan Plan, the Referendums of 2004 and the Aftermath. London: Tauris (forthcoming). Bibliography Strasbourg: Council of Europe. Faiz, M. (2008), ‘The Population Issue in North Cyprus’, The Cyprus Review 20 (2): 175- 187. Faustmann, H. (1999), Divide and Quit? British Colonial Policy in Cyprus 1878-1960. In- cluding a special Survey of the Transitional Period: February 1959-August 1960. Mann- heim: Mateo. Fulias-Souroulla, M. (2008), ‘Marriage and Migration: Greek Cypriot Representations and Attitudes towards Inter-societal Marriage’, The Cyprus Review 20 (2): 117-143. Hadjidemetriou, T. (2006) Το Δημοψήφισμα της 24ης Aπριλίου 2004 και η Λύση του Κυπρια- κού [The Referendum of 24 April 2004 and the Solution to the Cyprus Problem]. Athens: Papazisis. Hannay, D. (2005), Cyprus: The Search for a Solution. London: I. B. Tauris. Hannum, H. (1996), Autonomy, Sovereignty and Self-determination. The Accommodation of Conflicting Rights. Philadelphia: University of Pennsylvania Press. Hatay, M. (2006), Beyond Numbers. An Inquiry into the Political Integration of the Turkish ‘Settlers’ in Northern Cyprus. PRIO Report 4/2005. Oslo: International Peace Research Institute (PRIO). Hatay, M. (2008), ‘The Problem of Pigeons: Xenophobia and a Rhetoric of the “Local” in North Cyprus’, The Cyprus Review 20 (2): 145-171. Hitchens, C. (1997), Hostage to History. Cyprus From the Ottomans to Kissinger. 2nd edi- tion. London: Verso. Kadir, D. (2007), ‘Truth and Reconciliation on Cyprus will be Possible, when…’, The Cy- prus Review 19 (1): 163-169. Katsiaounis, R. (1996), Labour, Society and Politics in Cyprus during the Second Half of the Nineteenth Century. Nicosia: Cyprus Research Centre. Katsiaounis, R. (2007), ‘Cyprus 1931-1959: The Politics of the Anti-colonial Movement’, Επετηρίδα 33: 441-469. Kyle, K. (1997), Cyprus: In Search for Peace. Minority Rights Group International Report. London: Minority Rights Group. Kyrris, C. (1980), Peaceful Coexistence. Nicosia: Public Information Office. Loizou, A. N. (2001), Σύνταγμα Κυπριακής Δημοκρατίας [The Constitution of the Republic of Cyprus]. Nicosia: Kaila Printing. Matsis, S. & A. Charalambous (1993), ‘The Demand and Supply Dimensions of the La- bour Market: The Issue of Foreign Labour’, in E.I. Demetriades, N.F. Khoury & S. Matsis (1996), Labour Utilization and Income Distribution in Cyprus, 23-54. Nicosia: Department of Statistics and Research, Ministry of Finance. 418 NICOS TRIMIKLINIOTIS Nedjati, Z. (1970), Cyprus Administrative Law. Nicosia. Nedjati, Z. (1970), Cyprus Administrative Law. Nicosia. Ombudsman (2005), Επίτροπος Διοικήσεως, Ετήσια Έκθεση 2004 [Annual Report 2004]. Nicosia: Cyprus Republic Printing Office. y g Palley, C. (2005), An International Relations Debacle. The UN Secretary-General’s Mission of Palley, C. (2005), An International Relations Debacle. 14 Changing conceptions of citizenship in Turkey1 Zeynep Kadirbeyoglu International migration and globalisation are factors which affect citi- zenship practices throughout the world. Increasing tolerance of multi- ple citizenship is, amongst other things, one of the results of these trends. This chapter analyses the Citizenship Law in Turkey and argues that the most important changes in the law were made to accommo- date the needs and wishes of the emigrants who – even up to the third generation – maintain vibrant ties with their home countries. The chapter starts with the history of citizenship in Turkey. The following section outlines the amendments to the current law that regulates the acquisition and loss of citizenship. Subsequently, the main forms of ac- quisition and loss of citizenship in Turkey are mapped out. A final sec- tion looks at the statistics of people acquiring and losing citizenship in Turkey. Bibliography Trimikliniotis, N. & C. Demetriou (2007), ‘Cyprus’, in A. Triandafyllidou & R. Gropas (eds.), European Immigration: A sourcebook, 45-58. Aldershot: Ashgate. Trimikliniotis, N. & C. Demetriou (2008), ‘Evaluating the Anti-discrimination Law in the Republic of Cyprus: A Critical Reflection’, The Cyprus Review 20 (2): 79-116. Trimikliniotis, N. & C. Demetriou (2009), “The Cypriot Roma and the Failure of Educa- tion: Anti-Discrimination and Multiculturalism as a Post-accession Challenge”, in N. Coureas & A. Varnava (eds.), The Minorities of Cyprus: Development Patterns and the Identity of the Internal Exclusion. Cambridge: Cambridge Scholars Publishing (forth- coming). Trimikliniotis, N. & P. Pantelides (2003), ‘Mapping Discrimination in Cyprus: Ethnic Dis- crimination in the Labour Market’, The Cyprus Review 15 (1): 121-148. 14.1 History of Turkish citizenship law 14.1.1 From the Ottoman Empire to the founding of the Republic 14.1.1 From the Ottoman Empire to the founding of the Republic An analysis of the history of Turkish citizenship should begin with the last period of the Ottoman Empire. Whereas, prior to the 1869 Otto- man Citizenship Law (Tabiyet-i Osmaniye Kanunu), the subjects of the Ottoman Empire were divided along religious lines, the new law recog- nised all residents of the Ottoman territories as nationals of the Empire. It was based on the ius sanguinis principle, but allowed for non-Ottoman children born in the Ottoman territories to apply for citizenship in the Empire when they reached adulthood (I˙c¸duygu, C¸olak & Soyarık 1999). The first constitution of the Republic of Turkey (1924) granted Turk- ish nationality to all residents of the Republic irrespective of race or re- ligion. The nationality law of the Republic was accepted in 1928 and, like its Ottoman predecessor, it was based on ius sanguinis but was complemented by a territorial understanding (I˙c¸duygu et al. 1999: 193). Aybay (2001: 45) argues that behind this decision was the desire to extend Turkish nationality to as many people as possible.2 420 ZEYNEP KADIRBEYOGLU I˙c¸duygu et al. (1999: 195), for example, argue that the notion of na- tionality was not defined solely in terms of ethnic background since the new Turkish nationality was ‘open to non-Turkish Muslim groups […] so long as they were willing to assimilate culturally and linguisti- cally into the Turkish culture.’ However, the analysis of groups that were given the right to settle in Turkey reveals that in practice the abil- ity to enjoy full citizenship rights was related to ethnicity and religion (Kiris¸c¸i 2000: 1). Specifically, in accordance with the Law on Settlement adopted in 1934,3 Turkey provided refugee and immigrant status to groups such as Muslim Bosnians, Albanians, Circassians, Tatars, etc., but declined to accept the settlement of groups such as Christian Orthodox Gagauz Turks and Shi’a Azeris. This policy effectively pre-screened those apply- ing for citizenship and helped Sunnis settle in Turkey, in spite of offi- cial statements that only those of Turkish descent and culture would be so favoured (Kiris¸c¸i 2000).4 At the beginning of the twentieth century, Anatolia (Asia Minor) was a heterogeneous piece of land and was home to Rum (an Orthodox Christian Greek speaking group), Armenian, Kurdish, Jewish, Circas- sian, Laz and some other ethnic or religious groups. 14.1.1 From the Ottoman Empire to the founding of the Republic The spread of na- tionalism from Western Europe, its birthplace, to the Ottoman lands led to conflicts and to the disappearance of heterogeneity by way of the forced migration of Armenians during the First World War and the po- pulation exchange with Greece in 1923. During the War of Indepen- dence there was a clear reference to the multicultural nature of Anato- lia. However, after the Sheikh Said uprising of 1925,5 there was no longer any reference made to the ‘peoples of Turkey’ and thus all citi- zens of Turkey were expected to adopt Turkish identity (Ergil 2000: 125). This was a fabricated umbrella identity and was instituted through education and cultural policies but carried the name of one of the ethnic groups (the Turks). The group which was not willing to identify with this were the Kurds. Their struggle for autonomy, and sometimes secession, led to a battle between the PKK (Kurdistan Work- ers Party) and the army. At the height of this armed conflict, the Presi- dent at the time, Suleyman Demirel, began a discussion on constitu- tional citizenship, which was intended to create a new common iden- tity (I˙c¸duygu et al. 1999: 192). However, these discussions were short- lived and did not lead to any policy changes. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 421 3.1 million Turkish citizens were living in Europe. Together with an- other 530,000 Turkish citizens living in other parts of the world, Tur- key’s emigrant population numbers an approximate 3.6 million (TCCSGB 2005). In order to understand the economic significance of these emigrants for Turkey, we should first examine the initial goals of the process of la- bour force exportation to Western European countries. According to Sayarı (1986) the main goals included fighting the rising unemploy- ment within Turkey and bolstering foreign exchange reserves in order to cover trade deficits. A secondary goal was to increase the skill level of workers who would, then, through remittances, be able to increase the level of investment in small and medium-sized companies in the emigrants’ home towns in Turkey (Sayarı 1986). The remittances were very important for Turkey. During the 1980s, 24 per cent of Turkey’s imports were covered by the cash remittances and foreign exchange de- posits of Turkish workers abroad (Kumcu 1989). Germany was the main destination for guest-workers from Turkey. Turkish workers in Germany were encouraged to maintain their ties to Turkey and not to undergo ‘Germanisation’7 so that a constant flow of remittances could be guaranteed (Hunn 2001). Migrants were encour- aged to remit their savings by means of special interest rates given to foreign currency saving accounts in Turkey and by certain privileges that were extended to emigrants who wished to import goods to Turkey (Sayarı 1986). Lately, in addition to remittances, direct investments by the second generation of Turkish emigrants, especially in the textiles industry, are increasing in importance (Faist 1998: 213). In addition to the economic investment, it is expected that Turkey will enjoy political benefits thanks to the migrants living in Western Europe. The lobbying potential of migrants living in European countries has been seen as an asset by governments in Turkey.8 The realisation that Turkish workers are not temporary guests in their host countries has led to significant amendments to the citizen- ship law in Turkey. The motives of politicians and bureaucrats have been shaped by the demands of emigrants who faced problems related to military service, property ownership, and lack of political rights in their countries of immigration. A fairly organised and quasi-official process was used to communicate the needs of citizens living abroad to the Turkish officials. 14.1.2 The impact of Turkish emigration to Western Europe The current law that regulates the acquisition and loss of Turkish citi- zenship was put into effect in 1964.6 This period also marks the begin- ning of the migration of guest-workers to Western Europe. As of 2005, CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY The first amendment to the law took place in 1981 and legalised dual citizenship as long as the person acquiring a second nationality in- formed the government (Keyman & I˙c¸duygu 2003); otherwise, public authorities could withdraw his or her Turkish citizenship. Further- more, the amendment initiated gender equality in the transfer of citi- 422 ZEYNEP KADIRBEYOGLU zenship to children; as a result women can also transfer their citizen- ship to their children through ius sanguinis. The change in article 23/III of the Citizenship Law made it possible to release individuals from Turkish nationality if they wished to acquire another country’s citizenship.9 In subsequent years, many individuals who acquired a new citizenship reacquired their Turkish citizenship immediately after renouncing it. This was supported and encouraged by Turkish authorities and embassies. This method of circumventing German Citizenship Law – which prohibits dual citizenship – was leg- ally possible only until 2000. The pre-2000 law maintained only that the person naturalising in Germany should not have another national- ity. Yet, the new law made it possible for German officials to withdraw German citizenship from those who had taken up another citizenship following their naturalisation in Germany – hence those who had be- come dual citizens ‘illegally’.10 Based on this clause, the German Gov- ernment declared that 48,000 people of Turkish origin who had natur- alised in Germany since 2000 had lost their German nationality be- cause they had become ‘illegal dual citizens’.11 These people were to have their German nationality withdrawn but could stay in Germany as permanent residents and reapply for naturalisation there provided they were willing to renounce their Turkish nationality.12 This did not have a significant impact on the public debate in Turkey but was strongly opposed by Turkish associations in Germany. These associations blamed the Turkish Government for not responding even though they had encouraged these 48,000 people to reacquire Turkish nationality. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY Even though the spokesperson for the German Ministry of the Interior claimed that they had compiled a list of those who were ‘il- legal’ dual citizens from the records collected at borders and in govern- ment offices, there were claims that the Turkish authorities had sub- mitted the list because of threats that their EU application process would not be supported.13 There is evidence that the German regional authorities have been contacting those they suspect of holding two passports by mail and asking them whether they had acquired a second nationality. The results of these inquiries and bureaucratic confusion are yet to be seen. The 1981 change was debated in a secret session by the National Se- curity Council because it was initiated by the Ulusu Government, which was established following the military coup.14 The amendment also facilitated the processes for stripping individuals of their citizen- ship.15 The clause added to the law stated that those who are outside the borders of Turkey and who have been charged with endangering the internal or external security of the country will have their Turkish citizenship withdrawn unless they return within three months during regular periods and one month under emergency rule.16 CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 423 Following this coup, 227 people had their Turkish citizenship with- drawn by means of this clause. However, in February 1992, the Parlia- ment removed this clause after hearing arguments that the clause had permitted a violation of human rights.17 Those who wished were allowed to reacquire their citizenship and to have their property rein- stated or receive compensation for the value of confiscated property.18 p p p y Parliamentary debates on issues of citizenship and/or problems of Turkish citizens living abroad have not been restricted to amendments of the laws pertaining to citizenship. The events in Solingen, where five Turkish emigrants died as a result of an arson attack on their house, were debated in the Turkish Parliament on 8 June 1993. During these debates, the ANAP (centre right party) group spokesperson em- phasised the importance of having the right to vote in Germany. He claimed that there are individuals who, despite having lived in Ger- many for the last 30 years, are still denied the right to vote. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY Pink card holders were only denied the right to vote in local and national elections.24 Aybay states that the head of the TGD, 424 ZEYNEP KADIRBEYOGLU Hakkı Keskin, a very old friend of his, invited him to find a solution to citizenship-related problems faced by Turkish people living in Ger- many.25 He makes it quite clear that the main issue was how to devise a mechanism that would allow people living in Germany to acquire German citizenship without losing their rights in Turkey.26 This was the motivation behind the creation of the special non-citizen status.27 p During the parliamentary debates when this amendment was dis- cussed, the spokesperson of the ANAP group argued that this law was what all factions of Turkish emigrants in Germany had been demand- ing for years. He claimed that these emigrants wanted to have political rights in Germany and that this amendment would ease their difficul- ties in acquiring German citizenship. He also mentioned that Turkish emigrants would become a key electoral group in Germany, with some influence in the tight electoral competition between the two major par- ties.28 Another MP emphasised the benefits of this amendment by re- ferring to the possibility of Turkish people becoming elected represen- tatives in Germany and, therefore, politically strengthening the posi- tion of Turkey.29 Some MPs raised their concern about whether this amendment would enable the ‘Armenians, Jews, Rum, etc.’30 (who had renounced their Turkish citizenship in order to acquire another citizenship) to come back to Turkey and reclaim property that had been confiscated when they changed their citizenship. This is telling in that it demon- strates that the tolerance for dual citizenship and special rights for those who had renounced their citizenship was intended to apply ex- clusively to Turkish emigrants who had left the country under specific conditions; the amendment was never intended to include the minori- ties who left Turkey before 1981, and explicitly stated that the privileged non-citizen status would apply only to those who had acquired Turkish citizenship by birth and who had relinquished it by being granted per- mission by the Council of Ministers.31 This way of renouncing Turkish citizenship was made possible only after the amendments to the citi- zenship law in 1981. Despite good intentions, the special non-citizen status was criticised by groups who were dissatisfied with its implementation. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY According to this argument, the right to vote is the key to finding a long-term so- lution to the problems faced by Turkish persons residing in Germany. He claimed that under the current circumstances dual citizenship rights were of greater importance and the Turkish Government ought to propose that Germany put this issue on its agenda.19 The SHP (centre-left party) group spokesperson claimed that in addi- tion to the security aspects surrounding the Solingen events, political and legal issues should also be debated. He stated that obtaining equal rights in the political, economic and social spheres by obtaining Ger- man citizenship would not automatically prevent these attacks, but that extreme right parties would be more cautious about taking an anti-im- migration stance as immigrants would form part of the electorate. His argument was that as long as Germany banned dual citizenship, the goal of the Turkish State should be to encourage emigrants to natura- lise in Germany while maintaining their rights in Turkey.20 y g g y Following this logic, the amendment to the Turkish Citizenship Law in 1995 instituted the so-called ‘pink card’ or the privileged non-citizen status.21 In the statement giving reasons for this amendment, the gov- ernment stressed the fact that it was a result, among other factors, of the actions of countries that refused to accept multiple citizenship. The proposal for this amendment was drafted by Rona Aybay (a pro- minent law professor specialising on citizenship issues) after he had at- tended meetings in Germany at the invitation of the Tu¨rkische Ge- meinde in Deutschland (TGD).22 Once accepted in 1995, the amend- ment created a privileged non-citizen status. This status permits holders of a pink card23 to reside, to acquire property, to be eligible for inheritance, to operate businesses and to work in Turkey like any citi- zen of Turkey. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 425 nounce their citizenship through new legislation. This would enable Turkish citizens to enjoy dual citizenship through an exception in the new German Law which states that in cases where the country of ori- gin does not permit its citizens to relinquish their original citizenship, Germany might allow dual citizenship. This instance shows how the demands of immigrant organisations have changed depending on the situation in Germany. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY The TGD or- ganised a summit in July 2000 and produced a declaration pertaining to the problems and expectations of the Turkish citizens living in Ger- many. The declaration stated that there were many problems in the practical use of the pink card in Turkey as the bureaucracy was not in- formed about it. Therefore, people who had renounced their Turkish ci- tizenship were facing problems in their interactions with the bureau- cracy in Turkey. During the same summit there was a call for Turkey to stop releas- ing its citizens and to make it impossible for Turkish citizens to re- 14.1.3 Policies towards historic Turkish groups abroad Some Bulgarian Turks, who had not been able to natura- lise in Turkey, were sent back to Bulgaria towards the end of the 426 ZEYNEP KADIRBEYOGLU 1990s.36 Special laws were enacted in order to regulate the settlement of other groups known to have ethnic Turkish origin such as Afghan immigrants and Ahiska Turks who migrated from Russia.37 There are not many organised immigrant groups in Turkey able to place significant pressure on the government. Two of the few immi- grant groups that made it to the media, for instance, were the Network of Foreign Spouses and Muslim immigrants such as Bulgarian Turks. The Network of Foreign Spouses referred to ideals of fairness and de- manded more rights for individuals who are foreigners in Turkey.38 The pragmatic nature of the debates on citizenship and the reactive policy style hinders the politicisation of, and reciprocation of tolerance towards, immigrants in Turkey.39 In other words, if values that under- lie the promotion of dual citizenship for Turkish emigrants were brought into the public sphere, they could lead to demands of recipro- city for immigrants in Turkey. 14.1.3 Policies towards historic Turkish groups abroad Emigrants were not the only group who influenced the amendments to the citizenship law in Turkey. The disintegration of the USSR and the increasing numbers of arranged marriages in Turkey alerted authori- ties and the amendment in 2003 requires spouses to wait for three years before spousal transfer of nationality is possible.32 The second amendment that same year made it possible for citizens of Northern Cyprus to easily acquire Turkish citizenship (see Trimikliniotis in this volume).33 In 2003, a total of 2,403 Cypriots acquired Turkish citizen- ship.34 The latest amendment was passed in 2004 and concerned a minor issue relating to the pink card. As can be seen from the amendments that were outlined above, apart from the one attempting to prevent arranged marriages, there is no debate about immigrants in Turkey. The focus has been on emi- grants from Turkey who live in Western Europe. Politicians in Turkey feel little need to respond to immigrant issues because these are not yet politicised, which is a common feature of countries that have only recently begun receiving economic immigrants. Prior to the 1980s, immigrants accepted to Turkey have been predo- minantly from among peoples considered to be ‘of Turkish descent and culture’ and they were settled using the Law on Settlement.35 The Law on Settlement allowed for two types of migration to Turkey: those who were settled by the state and those who settled themselves (Dog˘anay no date). According to Dog˘anay this law was considered insufficient during the last two decades and it was amended to accommodate those forced to migrate to Turkey from Bulgaria in 1989 (see Smilov & Jileva in this volume). Many of the Bulgarian Turks who arrived with the first wave of migration in 1989 were granted Turkish nationality. When these migrants could reacquire their Bulgarian nationality and pass- ports in 2000 (hence become dual citizens), Turkish politicians encour- aged them to vote in the elections in Bulgaria in order to strengthen the political party representing ethnic Turks and play a positive role in establishing cooperation between two countries on the way to EU membership. 14.2 Modes of acquisition and loss of Turkish citizenship The law currently regulating the acquisition and loss of Turkish citizen- ship was put into effect in 196440 and was amended as described in the previous section. There are three broad principles through which Turkish citizenship can be acquired or lost: change of status can be brought about ex lege, by a decision of the authorities and through op- tion. 14.2.2 Acquisition of nationality through the decision of authorities There are three types of acquisition within this category. The first is the regular mechanism through which naturalisation takes place and is regulated by art. 6 of the Law. The conditions for application are the following. The person should: g p a. be an adult (eighteen years or older); b. have five years of residence in Turkey; c. have decided to settle in Turkey; d. have good moral conduct; e. not have a threatening illness; f. speak sufficient Turkish; g. have a job or revenue to support himself or herself and dependents. a. be an adult (eighteen years or older); b. have five years of residence in Turkey; c. have decided to settle in Turkey; d. have good moral conduct; e. not have a threatening illness; f. speak sufficient Turkish; g. have a job or revenue to support himself or herself and dependents. The second mechanism, exceptional acquisition, can apply to the fol- lowing categories of persons without enforcing requirements b) and c): the adult children of those who have lost Turkish citizenship, those who are married to a Turkish citizen and their adult children, those who are of Turkish descent, their spouse and their adult children, those who are residents of Turkey with the intention of marrying a Turkish citizen and those who have or will serve Turkey as industrialists, scien- tists or artists (achievement-based acquisition of nationality). The third path, which is reacquisition, applies to all those who have renounced their Turkish citizenship in the past for various reasons. In all three types of acquisition the procedure for naturalisation is lengthy and goes through the Ministry of Internal Affairs and the Prime Minis- ter. The decision to grant citizenship is given by the Council of Minis- ters. 14.2.1 Ex lege acquisition of citizenship The acquisition of citizenship for children of Turkish mothers or fathers is automatic whether the child is born in Turkey or abroad. This rule is clearly based on ius sanguinis. Children of non-Turkish ci- tizens born in Turkey become Turkish citizens automatically if they cannot acquire the citizenship of their parents (the ius soli exception). Marriage to Turkish citizens does not automatically transfer citizen- ship. There is a waiting period of three years after which the spouse can acquire Turkish citizenship by option. However, those who lose their original citizenship due to marriage automatically become Turk- ish citizens. Turkish citizenship is extended to children of women who marry a Turkish citizen, if the child’s father is dead, unknown or state- less or if the mother has custody over the child. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 427 14.2.3 Acquisition through option Children who lost their Turkish citizenship when their parents re- nounced their citizenship can choose to reacquire their citizenship upon reaching adulthood. As mentioned above, foreign spouses also can acquire their partner’s Turkish nationality by option three years after the marriage. There is no residency requirement for the naturali- sation of spouses as long as they remain married. Children who lost their Turkish citizenship when their parents re- nounced their citizenship can choose to reacquire their citizenship upon reaching adulthood. As mentioned above, foreign spouses also can acquire their partner’s Turkish nationality by option three years after the marriage. There is no residency requirement for the naturali- sation of spouses as long as they remain married. 14.2.6 Loss through option This mode of loss applies to children who acquired Turkish citizenship when their mothers naturalised in Turkey. They can renounce their Turkish citizenship within a year of reaching adulthood as long as this does not result in statelessness. Furthermore, women who acquired Turkish citizenship upon marriage can renounce it upon divorce. 14.2.5 Loss through a decision of the authorities The first method through which Turkish citizenship can be lost is to re- nounce it (i.e. to ask for a permission to exit). This path of loss is mostly used by citizens who wish to naturalise in countries that do not accept dual citizenship. The release from citizenship may be granted by the Ministry of the Interior by declaration if certain conditions are satisfied. The procedures do not permit renunciation if it results in sta- telessness. The second method is the nullification of Turkish citizenship for peo- ple who have acquired it in the last five years and who have submitted false information in their application. The third method is the withdra- wal of Turkish citizenship from individuals because of specific actions, such as working against the interests of Turkey in a foreign country de- spite warnings, acquiring another citizenship without informing the Turkish authorities, working for a foreign state which is at war with Turkey, not responding to a call to military service for three months and residing abroad for more than seven years and not showing any in- terest in maintaining ties with Turkey. 14.2.4 Loss of citizenship ex lege This is valid only for women who wish, upon marriage, to automati- cally receive the foreign citizenship of their husbands. Although Turk- ish nationality law calls this a loss by law, it is in fact an optional loss 428 ZEYNEP KADIRBEYOGLU since it occurs only if there is a declaration by the individual to the re- levant authorities. / Source: General Directorate of Population and Citizenship, Ankara / Source: General Directorate of Population and Citizenship, Ankara p p Note: n/a = not available 14.3 Statistics Table 14.2 Acquisition of Turkish citizenship through a decision of the authorities, 1990-2008 Year Regular Acquisition Exceptional acquisition Reacquisition Total 1990 119 785 n/a 904 1991 1,172 475 n/a 1,647 1992 888 452 n/a 1,340 1993 634 439 n/a 1,073 1994 949 467 n/a 1,416 1995 1,229 710 n/a 1,939 1996 955 3,927 n/a 4,882 2000 633 736 13,004 14,373 2001 1,161 3,917 28,317 33,395 2002 745 14,564 8,330 23,639 2003 1,236 12,938 3,040 17,214 2004 1,276 6,434 1,999 9,709 2005 816 4,650 864 6,330 2006 987 2,161 2,006 5,154 2007 718 1,358 979 3,055 2008 824 2,383 1,348 4,555 Note: n/a = not available Source: General Directorate of Population and Citizenship, Ankara Table 14.2 Acquisition of Turkish citizenship through a decision of the authorities, 1990-2008 Table 14.2 Acquisition of Turkish citizenship through a decision of the authorities, 1990 430 ZEYNEP KADIRBEYOGLU The statistics provided by the General Directorate of Population and Citi- zenship reveal that in the category of regular acquisition by a decision of the authorities, 60 per cent were Greek heimatloss41 in 1991 whereas 9 per cent were Iranian citizens. Between 2000 and 2003, approximately 50 per cent of this same category were Bulgarians. Between 1990 and 1993, the majority of those who acquired Turkish citizenship on excep- tional grounds had previously held Iraqi citizenship (31 per cent for 1990, 32 for 1991, 23 for 1992 and 34 per cent for 1993). The largest group within this category were Bulgarians (they constituted 82 per cent of the total exceptional acquisition in 2002 and 84 per cent in 2003). p q 4 p 3) Table 14.3 shows the statistics on the numbers of withdrawals of Turkish citizenship (the third method explained in section 2.5 above). It should be noted that within the group of people who lost their Turk- ish nationality between 2000 and 2005 there is no case of loss result- ing from failure to reside in the country during the preceding seven years. The majority of people whose citizenship was withdrawn were those who did not return to the country to fulfil their military service despite being called up by the authorities – for instance, out of 1,920 people who lost their Turkish citizenship in 2000, 1,868 were in this category. 14.3 Statistics In this section, I will undertake a preliminary analysis of the statistics on the acquisition and loss of citizenship. The statistics on acquisitions through the law are shown in Table 14.1. The data for the years 1997- 1999 are missing yet it is possible to conclude that following the disin- tegration of the Soviet Bloc there has been a steady rise in the number of women who acquired Turkish citizenship through spousal transfer. Consequently, the change in 2003 of the law on spousal transfer of citi- zenship led to a sharp decline in numbers in the following year. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 429 Table 14.1 Automatic acquisition of Turkish citizenship, 1990-2008 Year Through mother or father Through adoption Through ius soli Through marriage Total 1990 187 – 5 491 683 1991 118 – 7 1,067 1,192 1992 339 – 7 1,057 1,403 1993 344 – 9 1,380 1,733 1994 434 – 25 1,590 2,049 1995 290 – 25 1,148 1,463 1996 104 – 3 933 1,040 2000 259 1 41 5,384 5,685 2001 230 n/a 57 7,630 7,917 2002 231 n/a 52 8,416 8,699 2003 659 n/a n/a 6,912 7,571 2004 885 n/a n/a 528 1,413 2005 598 n/a n/a 1,261 1,859 2006 507 n/a n/a 1,798 2,305 2007 422 n/a n/a 2,721 3,143 2008 342 n/a n/a 3,820 4,162 Note: n/a = not available Source: General Directorate of Population and Citizenship, Ankara Table 14.1 Automatic acquisition of Turkish citizenship, 1990-2008 The statistics on acquisition through the decision of authorities are shown in Table 14.2 below. Note: n/a = not available The General Directorate of Population and Citizenship reported there was no record for this category for the years 2006-2008. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 431 in this category cannot really be explained with the available data or in- formation. The only possibility is the sensitisation of the authorities as a result of events that led to the withdrawal of the Turkish citizenship of a member of parliament who had sworn allegiance to the US by be- coming a citizen there prior to the elections in Turkey. g p y Statistics on loss of citizenship are also published for those who have subsequently reacquired their Turkish citizenship (see Table 14.4 be- low). Up until 2002, individuals who renounced their Turkish citizen- ship could easily reacquire their original citizenship following naturali- sation in Germany. However, the realisation that a new law can lead to nullification of their German citizenship if it is discovered that they have reacquired their original citizenship has led to a sharp drop in the number of individuals who have reacquired Turkish citizenship there- after. Table 14.4 Previous loss of citizenship by those who have reacquired Turkish citizenship accord- ing to three main categories, 2000-2004 Reason for Loss 2000 2001 2002 2003 2004 Permission to exit 12,635 27,576 8,027 2,874 1,828 Inappropriate conduct 29 71 58 85 121 Loss by option 340 670 245 81 50 Total 13,004 28,317 8,330 3,040 1,999 Note: The General Directorate of Population and Citizenship reported no data for the years 2005-2008 Source: General Directorate of Population and Citizenship, Ankara Table 14.4 Previous loss of citizenship by those who have reacquired Turkish citizenship accord- ing to three main categories, 2000-2004 Table 14.4 Previous loss of citizenship by those who have reacquired Turkish citizenship accord- 14.3 Statistics This figure is 2,689 out of 2,735 in 2001, 2,193 out of 2,316 in 2002, 5,077 out of 5,489 in 2003, 1,975 out of 2,367 and 178 out of 464 in 2005. The number of Turkish citizens whose nationality was withdrawn because they did not inform the Turkish authorities that they were ac- quiring another citizenship increased between 2000 and 2005. The numbers are 42 for 2000, 24 for 2001, 81 for 2002, 272 for 2003, 246 for 2004 and 242 for 2005. The application of this rule is random at best since there are many people in this situation who have maintained their Turkish citizenship for many years. The increase in the numbers Table 14.3 Loss of Turkish citizenship by a decision of the authorities, 2000-2008 Year Withdrawal of citizenship 2000 1,920 2001 2,735 2002 2,316 2003 5,489 2004 2,367 2005 464 2006 n/a 2007 n/a 2008 n/a able 14.3 Loss of Turkish citizenship by a decision of the authorities, 2000-2008 Table 14.3 Loss of Turkish citizenship by a decision of the authorities, 2000-2008 Year Withdrawal of citizenship / The General Directorate of Population and Citizenship reported there was no record for this category for the years 2006-2008. g y y Source: General Directorate of Population and Citizenship, Ankara 14.4 Conclusions The findings suggest that maintaining vibrant economic links with citi- zens living abroad (especially those living in Germany) has been a con- stant concern for Turkish governments despite the severe neglect for the social problems faced by these groups. The research results show that there are a number of organisations and actors, especially within Germany, that pressure the policymakers in Turkey to accommodate their need to integrate into their host country without having to relin- quish their rights to land ownership and inheritance in Turkey. The main amendments to the Law on Citizenship in Turkey were made as a result of the realisation that the guest-workers were in fact perma- nent residents in their host countries. The most interesting finding is the interaction between the Turkish and German governments and the attempts of the former to formulate legislation based on the develop- ments in Germany. 432 ZEYNEP KADIRBEYOGLU Turkish governments have demonstrated a willingness to address the practical problems faced by the Turkish people living abroad. In many cases the intentions were sincere even though official actions to solve the problems were either slow or non-existent. However, this in- ability did not stem from apathy towards the real problems or the aim of strategically using the issue for political gain. It was rather the result of a general lack of political incentives, as those living abroad who still possess the right to vote in Turkey cannot practically do so unless they return to Turkey during elections.42 As outlined in the sections above, there is a very pragmatic debate concerning citizenship in Turkey. The principles of citizenship acquisi- tion and loss are seldom discussed and immigrants have not been a real concern of policymakers, either because they are not mobilised or because the issue is not politicised. Foreigners, like Bulgarian Turks or those coming from Central Asia, are not considered part of these im- migrant groups since, in most cases, they acquired Turkish citizenship based on their cultural, linguistic and religious backgrounds. g g g There are many cases of immigrants who find ways to work in Tur- key and leave the country every three months (this applies to many Bulgarian Turks who do not have citizenship). Many foreigners who do not need a visa to enter Turkey are employed in Turkey illegally. Even some Western European citizens who reside in Turkey without a work permit resort to this method. 14.4 Conclusions Very few of these immigrant groups have organised and begun trying to pressure the Turkish state. Bru¨cke, a German-Turkish bridging organisation, and the Association of Foreign Wives are exceptions. Hence, if in the next five to ten years immigra- tion issues become more important and appear in the public sphere we might begin to see more pressure applied to Turkey.43 14.5 Epilogue The history of Turkish Citizenship Law, which was recounted above, shows that there have been numerous changes to the law since 1964. Most of these changes regarded two issues: they either addressed the interests of emigrants from Turkey or adapted the Turkish regulations to international standards. As a consequence of these amendments, the law has been transformed into a patchwork full of inconsistencies, which is one of the reasons why the Turkish government decided to propose a new Nationality Law. Moreover, this new law aims to harmo- nise the Turkish regulations with those of the European Convention on Nationality (Tiryakioglu 2006). Hence, the new law mainly attempts to reduce inconsistencies. Moreover, there are also three substantive changes to the acquisition CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 433 and loss of citizenship. The most important change concerns the with- drawal of citizenship from males who have not served in the military and those citizens who have acquired another country’s citizenship without informing the authorities. The law, as it currently stands, sti- pulates that these two types of actions are punishable by the withdra- wal of Turkish citizenship. The new law eliminates this possibility and restores the citizenship of those who have lost their citizenship as a re- sult of these clauses. The second major change, which will be imple- mented by 2010, will abolish preferential treatment of those applicants for naturalisation who are of Turkish origin by applying a five-year resi- dency requirement for all applicants. The third substantive change is the three-year residency requirement for those who wish to reacquire their Turkish citizenship following loss through renunciation or with- drawal as a result of inappropriate conduct. The new law on citizenship was accepted in the parliament on 29 May 2009 (No. 5901/2009 on Turkish Citizenship). Finally, the Turkish government recently adopted a new law that fa- cilitates voting by Turkish citizens abroad. Even though Turkish citi- zens residing abroad were able vote in Turkey’s general elections, there was no practical method for doing so other than the setting up of ballot boxes at the borders for emigrants who travelled to and from Turkey. This situation changed with Law No. 5749 (adopted on 13 March 2008), which clarifies the methods through which Turkish citizens liv- ing abroad can vote in general elections, presidential elections, and re- ferenda in Turkey. There are now four different ways to vote: by regular mail, at the borders (during a 75-day period prior to the election date as practised during previous elections), at the consulates abroad (over a period of 45 days prior to the election date), and electronically (over a period of 30 days prior to the election date). The Constitutional Court cancelled the possibility of voting through mail ballots because it vio- lates the secrecy of voting. Chronological list of citizenship-related legislation in Turkey Date Document Content Source 1869 Ottoman Nationality Regulation Recognises all residents of the Ottoman territories as nationals of the Empire 1924 Constitution of the Republic of Turkey Grants Turkish nationality to all residents of the Republic irrespective of race or religion 1928 Law No. 1992 Law No. 3808/1992 amending Law No. 2383/ 1981 CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 1312/1928: Turkish Citizenship Act Based on ius sanguinis but complemented by a territorial understanding Chronological list of citizenship-related legislation in Turkey Date Document Content Source 1869 Ottoman Nationality Regulation Recognises all residents of the Ottoman territories as nationals of the Empire 1924 Constitution of the Republic of Turkey Grants Turkish nationality to all residents of the Republic irrespective of race or religion 1928 Law No. 1312/1928: Turkish Citizenship Act Based on ius sanguinis but complemented by a territorial understanding Chronological list of citizenship-related legislation in Turkey ZEYNEP KADIRBEYOGLU 434 Date Document Content Source 1934 Law No. 2510/1934 on Settlement Provides refugee and immigrant status to groups such as Muslim Bosnians, Albanians, Circassians, Tatars, etc. www.ifc.org 1961 Constitution of the Republic of Turkey Renewed following the coup; states that children born to Turkish mothers or fathers are Turkish and that it is not possible to revoke the citizenship of individuals unless they have been disloyal to the country; determines that children born to Turkish mothers and foreign fathers acquire citizenship based on the citizenship law www.legislationline.org; www.hri.org 1964 Law No. 403/1964: Turkish Citizenship Act Based on the principles outlined in art. 54 of the 1961 Constitution www.coe.int 1981 Law No. 2383/1981 amending Law No. 403/ 1964 Legalises dual citizenship provided that the person acquiring a second nationality informs the government www.legislationline.org 1982 Constitution of the Republic of Turkey (as amended in 1987, 1995, 2001) Keeps the same principles regarding citizenship as the 1961 Constitution www.tbmm.gov.tr (in Turkish) 1989 Law No. 3540/1989 amending Law No. 403/ 1964 Amends two articles of the law regulating the process of acquisition of Turkish citizenship, specifically regarding the procedure for conditional naturalisation; determines that persons who fail to fulfil a requirement within two years following naturalisation are likely to lose their citizenship 1992 Law No. 3808/1992 amending Law No. 2383/ 1981 Removes the clause stating that those who are outside the borders of Turkey and who have been charged with endangering the internal or external security of the country will be stripped of Turkish citizenship unless they return within three Date Document Content Source 1934 Law No. 2510/1934 on Settlement Provides refugee and immigrant status to groups such as Muslim Bosnians, Albanians, Circassians, Tatars, etc. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY www.ifc.org 1961 Constitution of the Republic of Turkey Renewed following the coup; states that children born to Turkish mothers or fathers are Turkish and that it is not possible to revoke the citizenship of individuals unless they have been disloyal to the country; determines that children born to Turkish mothers and foreign fathers acquire citizenship based on the citizenship law www.legislationline.org; www.hri.org 1964 Law No. 403/1964: Turkish Citizenship Act Based on the principles outlined in art. 54 of the 1961 Constitution www.coe.int 1981 Law No. 2383/1981 amending Law No. 403/ 1964 Legalises dual citizenship provided that the person acquiring a second nationality informs the government www.legislationline.org 1982 Constitution of the Republic of Turkey (as amended in 1987, 1995, 2001) Keeps the same principles regarding citizenship as the 1961 Constitution www.tbmm.gov.tr (in Turkish) www.legislationline.org; www.hri.org p citizenship law 1964 Law No. 403/1964: Turkish Citizenship Act Based on the princip outlined in art. 54 of 1961 Constitution 1981 Law No. 2383/1981 amending Law No. 403/ 1964 Legalises dual citize provided that the pe acquiring a second nationality informs t government 1982 Constitution of the Republic of Turkey (as amended in 1987, 1995, 2001) Keeps the same prin regarding citizenship 1961 Constitution 1989 Law No. 3540/1989 amending Law No. 403/ 1964 Amends two articles law regulating the p of acquisition of Tur citizenship, specifica regarding the proced conditional naturalis determines that pers who fail to fulfil a requirement within t years following naturalisation are lik lose their citizenship 1992 Law No. 3808/1992 amending Law No. 2383/ 1981 Removes the clause that those who are o the borders of T rke 1964 Law No. 403/1964: Turkish Citizenship Act 1981 Law No. 2383/1981 amending Law No. 403/ 1964 1982 Constitution of the Republic of Turkey (as amended in 1987, 1995, 2001) 1989 Law No. 3540/1989 amending Law No. 403/ 1964 1964 Law No. 403/1964: Turkish Citizenship Act 1982 Constitution of the Republic of Turkey (as amended in 1987, 1995, 2001) 1989 Law No. 3540/1989 amending Law No. 1 The author wishes to thank Esra Derle, Selc¸uk Ug˘uz and O¨ zlem Atikcan for their help in facilitating access to resources. CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 5901 Turkish Citizenship Law Eliminates the possibility to withdraw citizenship from males who have not served in the military and those citizens who acquired another country’s citizenship without informing the authorities CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 403/ 1964 Amends two articles of the law regulating the process of acquisition of Turkish citizenship, specifically regarding the procedure for conditional naturalisation; determines that persons who fail to fulfil a requirement within two years following naturalisation are likely to lose their citizenship / Removes the clause stating that those who are outside the borders of Turkey and who have been charged with endangering the internal or external security of the country will be stripped of Turkish citizenship unless they return within three 1989 Law No. 3540/1989 amending Law No. 403/ 1964 Amends two articles of the law regulating the process of acquisition of Turkish citizenship, specifically regarding the procedure for conditional naturalisation; determines that persons who fail to fulfil a requirement within two years following naturalisation are likely to lose their citizenship 1992 Law No. 3808/1992 amending Law No. 2383/ 1981 Removes the clause stating that those who are outside the borders of Turkey and who have been charged with endangering the internal or external security of the country will be stripped of Turkish citizenship unless they return within three 2008 Law No. 5749 amending Law No. 298/1961 on Elections and Voter Registration CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 435 Date Document Content Source months during regular periods and one month under emergency rule 1995 Law No. 4112/1995 amending Law No. 403/ 1964 Institutes the privileged non-citizen status (also known as the ‘Pink Card’) www.legislationline.org 1999 Law No. 4465/1999 Ratifies an agreement between the Turkish Republic and the Republic of Northern Cyprus on facilitating the naturalisation of Cypriots in Turkey 2003 Law No. 4866/2003 amending Law No. 403/ 1964 Introduces a waiting period of three years for acquisition of citizenship by spouses 2003 Law No. 4862/2003 amending Law No. 403/ 1964 Introduces facilitated acquisition of Turkish citizenship for citizens of Northern Cyprus 2004 Law No. 5203/2004 amending Law No. 403/ 1964 Clarifies the rights linked to the privileged non-citizen status; states that holders retain social insurance rights but lose voting rights, the right to be elected and the right to be employed in the civil service 2008 Law No. 5749 amending Law No. 298/1961 on Elections and Voter Registration Allows Turkish citizens living abroad to vote by mail (repealed by the Constitutional Court), electronically, at the border or at consulates 2009 Law No. 5901 Turkish Citizenship Law Eliminates the possibility to withdraw citizenship from males who have not served in the military and those citizens who acquired another country’s citizenship without informing the authorities Date Document Content S months during regular periods and one month under emergency rule 1995 Law No. 4112/1995 amending Law No. 403/ 1964 Institutes the privileged non-citizen status (also known as the ‘Pink Card’) w 1999 Law No. 4465/1999 Ratifies an agreement between the Turkish Republic and the Republic of Northern Cyprus on facilitating the naturalisation of Cypriots in Turkey 2003 Law No. 4866/2003 amending Law No. 403/ 1964 Introduces a waiting period of three years for acquisition of citizenship by spouses 2003 Law No. 4862/2003 amending Law No. 403/ 1964 Introduces facilitated acquisition of Turkish citizenship for citizens of Northern Cyprus 2004 Law No. 5203/2004 amending Law No. 403/ 1964 Clarifies the rights linked to the privileged non-citizen status; states that holders retain social insurance rights but lose voting rights, the right to be elected and the right to be employed in the civil service 2008 Law No. 5749 amending Law No. 298/1961 on Elections and Voter Registration Allows Turkish citizens living abroad to vote by mail (repealed by the Constitutional Court), electronically, at the border or at consulates 2009 Law No. www.legislationline.org Notes O¨ zdemir, ‘Ankara-Berlin Kıskacında: C¸ifte Vatandas¸lık Gerc¸eg˘i’ [Caught between Ankara and Berlin: the Truth about Dual Citizenship], Evrensel [daily newspaper], 26 January 2005. 14 After the military coup Bu¨lend Ulusu was given the responsibility of forming a technocratic government (www.tbmm.gov.tr). Until the Advisory Council was formed the National Security Council (NSC) sanctioned all decisions of the government. The members of the NSC were the four generals and one admiral who staged the coup. The minutes of the 13 February 1981 meeting of the National Security Council (38th Meeting, Volume 1, 1981) indicate that the members of the Council voted in favour of debating all amendments related to Turkish Citizenship Law in a secret session. The debate lasted for approximately two hours. 15 Cumhuriyet, 15 February 1981. 15 Cumhuriyet, 15 February 1981. 15 Cumhuriyet, 15 February 1981. 6 Law No. 2383/1981 amending Law No. 403/1964 on Turkish Citizenship. 16 Law No. 2383/1981 amending Law No. 403/1964 on Turkish Citizenship. 16 Law No. 2383/1981 amending Law No. 403/1964 on Turkish Citizenship. 17 Law No. 3808/1992 amending Law No. 2383/1981. In between these two amend- ments there is Law No. 3540/1989, which amended two articles of the law regulating the process of acquisition of Turkish citizenship. 17 Law No. 3808/1992 amending Law No. 2383/1981. In between these two amend- ments there is Law No. 3540/1989, which amended two articles of the law regulating the process of acquisition of Turkish citizenship. 18 Parliamentary Minutes, 27 May 1992, Period 19, Legislative Year 1, Volume 12, 53-55. 18 Parliamentary Minutes, 27 May 1992, Period 19, Legislative Year 1, Volume 12, 53-55. 19 Parliamentary Minutes, 8 June 1993, Period 19, Legislative Year 2, Volume 36, 189- 19 Parliamentary Minutes, 8 June 1993, Period 19, Legislative Year 2, Volume 36, 189- 192. 19 Parliamentary Minutes, 8 June 1993, Period 19, Legislative Year 2, Volume 36, 189- 192. 20 Ibid., 203-206. 21 Law No. 4112/1995 amending Law No. 403/1964 on Turkish Citizenship. 22 The Turkish Immigrants Union (later to become Almanya Tu¨rk Toplumu – TGD) was established in 1985. It is an umbrella association with around 200 members, including the German Turkish Academics Association Union, German Turkish Students Association Union and various occupational organisations. TGD promotes the interests of the Turkish population of Germany vis-a`-vis both the German and 22 The Turkish Immigrants Union (later to become Almanya Tu¨rk Toplumu – TGD) was established in 1985. Notes 436 ZEYNEP KADIRBEYOGLU 2 It should not be forgotten that this was taking place in the context of sharp declines in the size of the population of Anatolia as a result of the First World War. 2 It should not be forgotten that this was taking place in the context of sharp declines in the size of the population of Anatolia as a result of the First World War. 3 Law No. 2510/1934 on Settlement. 3 Law No. 2510/1934 on Settlement. 4 Sunni Islam, which is considered to be the mainstream, differs from Shi’a Islam. 4 Sunni Islam, which is considered to be the mainstream, differs from Shi’a Islam. 5 The Sheikh Said uprising was one of the first important rebellions against the state. The Sheikh gathered support on the basis of tribal and religious allegiance, and hence the insurgency was not exclusively one of Kurdish nationalism (Robins 1993: 660). 6 Law No. 403/1964 on Turkish Citizenship. 7 Turkish authorities were counselling emigrants not to lose their socio-cultural identity and to maintain ties with Turkey. Germanisation, according to this perspective, would distance emigrants from Turkey. 8 Parliamentary Minutes, 7 June 1995, Period 19, Legislative Year 4, Volume 88, 89- 109. 9 Law No. 2383/1981 on Turkish Citizenship. 9 Law No. 2383/1981 on Turkish Citizenship. 10 German Citizenship Law, art. 25. The only exceptions to the strict ban on dual citizenship are those who have a second passport from a European Union country and those who have applied for permission. 10 German Citizenship Law, art. 25. The only exceptions to the strict ban on dual citizenship are those who have a second passport from a European Union country and those who have applied for permission. 11 Y. O¨ zdemir, ‘Ankara-Berlin Kıskacında: C¸ifte Vatandas¸lık Gerc¸eg˘i’ [Caught between Ankara and Berlin: the Truth about Dual Citizenship], Evrensel [daily newspaper], 26 January 2005. This move came at a critical juncture in German politics whereby expelling these citizens impacted on the number of voters. According to one estimate, approximately 20,000 out of 600,000 German-Turkish voters were dis- enfranchised in the general elections of 2005 (Deutsche Welle, 17 September 2005, www.dw-world.de). 12 Radikal [daily newspaper], 11 February 2005. ¨ 12 Radikal [daily newspaper], 11 February 2005. ¨ 13 Y. O¨ zdemir, ‘Ankara-Berlin Kıskacında: C¸ifte Vatandas¸lık Gerc¸eg˘i’ [Caught between Ankara and Berlin: the Truth about Dual Citizenship], Evrensel [daily newspaper], 26 January 2005. 13 Y. Notes It is an umbrella association with around 200 members, including the German Turkish Academics Association Union, German Turkish Students Association Union and various occupational organisations. TGD promotes the interests of the Turkish population of Germany vis-a`-vis both the German and CHANGING CONCEPTIONS OF CITIZENSHIP IN TURKEY 437 the Turkish governments, attempts to influence public opinion, and to secure rights through legislative changes (www.tgd.de). the Turkish governments, attempts to influence public opinion, and to secure rights through legislative changes (www.tgd.de). 23 The pink card is the document given to the people who have the special non-citizen status. 23 The pink card is the document given to the people who have the special non-citizen status. 24 Law No. 4112/1995 amending Law No. 403/1964 on Turkish Citizenship 25 Interview with Rona Aybay, 20 August 2002. 26 People who have acquired Turkish citizenship by means other than birth do not have the right to a pink card. 27 Parliamentary Minutes, 8 June 1993, Period 19, Legislative Year 2, Volume 36, 203- 206. 28 Parliamentary Minutes, 7 June 1995, Period 19, Legislative Year 4, Volume 88, 89- 90. 30 Speaker of the RP (Refah Partisi – religious right wing party) group (Parliamentary Minutes, 7 June 1995, Period 19, Legislative Year 4, Volume 88, 103). Many other MPs voiced their concern on this issue as well. 31 Art. 29 of Law No. 4112/1995 amending Law No. 403/1964 on Turkish Citizenship. This provision is against the principle of non-discrimination between citizens by birth and by naturalisation incorporated in the 1997 European Convention on Nationality. Turkey has not signed this Convention. 32 Law No. 4866/2003 amending Law No. 403/1964 on Turkish Citizenship. 33 Law No. 4862/2003 amending Law No. 403/1964 on Turkish Citizenship. The citizens of the Turkish Republic of Northern Cyprus (TRNC) have always enjoyed preferential treatment in Turkey. Law No. 4465/1999 further strengthened this by attempting to provide TRNC citizens with all the social and economic rights of Turkish citizens except voting rights. Since TRNC is not a recognised state (except by Turkey) TRNC citizens could travel abroad only with a Turkish passport (except for the UK and USA which recognised the TRNC passport as an identity card and issued visas for TRNC citizens on a blank page and not the passport itself). TRNC citizens could obtain a Turkish passport without becoming a citizen of Turkey. ZEYNEP KADIRBEYOGLU 40 Law No. 403/1964 on Turkish Citizenship. 40 Law No. 403/1964 on Turkish Citizenship. 41 The term Greek Heimatloss is used to refer to those Greek citizens of Western Thrace (of Turkish origin) who were expelled from Greek citizenship. 41 The term Greek Heimatloss is used to refer to those Greek citizens of Western Thrace (of Turkish origin) who were expelled from Greek citizenship. 42 Voting during general elections in Turkey has been a widely debated issue. Legally it is possible for Turkish people living abroad to vote during elections from the country where they reside. However, due to practical problems, such as setting up ballot boxes in other countries and the insecurity of mail ballots, this has never been practised. Fuat Boztepe, who is the head of the department in charge of workers abroad at the Ministry of Labour, stated that the greatest problem occurs in countries where there are a significant number of workers and the host country does not allow ballot boxes to be put in public spaces. Given the number of people who could vote, setting up ballot boxes only in the consulates and embassies does not provide a solution (interview with Fuat Boztepe, Head of the Department of External Relations and Services for Workers Abroad at the Turkish Ministry of Labour and Social Security, 14 May 2003). 43 Ahmet I˙c¸duygu, Bilkent University, Department of Political Science, confirmed this possibility (interview: 15 May 2003). 43 Ahmet I˙c¸duygu, Bilkent University, Department of Political Science, confirmed this possibility (interview: 15 May 2003). Notes They also had the right to be dual citizens and Law No. 4465/1999 states that there shall be a fast- track process for the citizenship applications of those TRNC citizens who want to acquire the citizenship of the Republic of Turkey. Dual citizenship has also existed for those Turkish citizens who settled in the TRNC. Those with five years residence are granted TRNC citizenship provided that they fulfil certain conditions (Law No. 25/1993 TRNC Nationality Law). Yet the TRNC Council of Ministers can also grant TRNC citizenship on a discretionary basis. The TRNC government was accused of such discretionary behaviour prior to the 2003 elections in order to influence the election results (Hylland 2003). 25/1993 TRNC Nationality Law). Yet the TRNC Council of Ministers can also grant TRNC citizenship on a discretionary basis. The TRNC government was accused of such discretionary behaviour prior to the 2003 elections in order to influence the election results (Hylland 2003). 34 Data used in this paper related to citizenship in Turkey were provided by the General Directorate of Population and Citizenship, Ankara. 35 Law No. 2510/1934 on Settlement. The Council of Ministers was in charge of determining which groups were considered to be of Turkish descent. Groups such as Pomacks, Roma and Albanians have also been settled in Turkey by being assigned this status (S¸ahin no date). 36 ‘Sofya’da bir Kurultay’, Milliyet [daily newspaper], 16 July 2000; ‘Soydas¸a Green Card’, Milliyet, 4 March 1997; ’Menderes: ‘‘C¸ifte Vatandas¸lık Kolaylas¸tırılsın’’’, Milli- yet, 24 February 1997. 37 Law No. 2641/1982 and Law No. 3835/1992 respectively. 38 The majority of the women in this association were Germans and they did not want to naturalise in Turkey because they would lose their German citizenship. 38 The majority of the women in this association were Germans and they did not want to naturalise in Turkey because they would lose their German citizenship. 39 For a classification of policy styles, see Richardson (1982). 39 For a classification of policy styles, see Richardson (1982). 438 ZEYNEP KADIRBEYOGLU Bibliography Aybay, R. (2001), Vatandas¸lık Hukuku [Citizenship Law]. I˙stanbul: Aybay Yayınları. Dog˘anay, F. (no date), Tu¨rkiye’ye Go¨c¸men Olarak Gelenlerin Yerles¸imi [The Settlement of Migrants to Turkey]. www.balgoc.org.tr. Ergil, D. (2000), ‘The Kurdish Question in Turkey’, Journal of Democracy 11 (3): 122-135. Faist, T. (1998), ‘Transnational Social Spaces out of International Migration: Evolution, Significance and Future Prospects’, Archives Europe´ennes de Sociologie 39 (2): 213-247. Hunn, K. (2001), ‘‘‘Alamanya, Alamanya, Tu¨rk gibi I˙sc¸i Bulamanya… Alamanya Alamanya, Tu¨rkten Aptal Bulamanya’’ Labour Migration from Turkey to the Federal Republic of Germany in the Years of Official Labour Recruitment (1961-1973)’, German-Turkish Summer Institute Working Paper 4. Hylland, A. (2003), Northern Cyprus Parliamentary Elections. www.humanrights.uio.no. ˙ Hylland, A. (2003), Northern Cyprus Parliamentary Elections. www.humanrights.uio.no. I˙c¸duygu, A., Y. C¸olak & N. Soyarık (1999), ‘What is the Matter with Citizenship? A Turk- ish Debate’, Middle Eastern Studies 35 (4): 187-208. I˙c¸duygu, A., Y. C¸olak & N. Soyarık (1999), ‘What is the M ish Debate’, Middle Eastern Studies 35 (4): 187-208. Keyman, E. F. & A. I˙c¸duygu (2003), ‘Globalization, Migration and Citizenship: The Case of Turkey’, in E. Kofman & G. Youngs (eds.), Globalization: Theory and Practice, 193- 206. London: Continuum. Kiris¸c¸i, K. (2000), ‘Disaggregating Turkish Citizenship and Immigration Practices,’ Mid- dle Eastern Studies 36 (3): 1-22. Kumcu, M. E. (1989), ‘The Savings Behavior of Migrant Workers: Turkish Workers in W. Germany,’ Journal of Development Economics 30: 273-286. y J f p 3 73 Richardson, J. J. (ed.) (1982), Policy Styles in Western Europe. Boston: Allen and Unwin. Robins, P. (1993), ‘The Overlord State: Turkish Policy and the Kurdish Issue’, Interna- tional Affairs 69 (4): 657-676. S¸ahin, Z. (no date), Tu¨rkiye’ye Yo¨nelik Dıs¸ Go¨c¸teki Deg˘is¸im Ve Su¨reklilik [The change and continuity in the migration towards Turkey]. www.stradigma.com. Sayarı, S. (1986), ‘Migration Policies of Sending Countries: Perspectives on the Turkish Experience’, Annals of the American Academy of Political and Social Science 485: 87-97. TCCSGB (TC C¸alıs¸ma ve Sosyal Gu¨venlik Bakanlıg˘ı) (2005), Bulletin 5 (2). ve Sosyal Gu¨venlik Bakanlıg˘ı) (2005), Bulletin 5 (2 Tiryakioglu, B. (2006) ‘Multiple Citizenship and its Consequences in Turkish Law’, An- kara Law Review 3(1): 1-16. ‘A call to kinship’? Citizenship and migration in the new Member States and the accession countries of the EU Wiebke Sievers When the black British poet John Agard (2000) warns his readers to ‘remember the ship in citizenship’, he is implying that citizenship is not a fixed institution but something that should be adaptable to changes in the population. Agard explicitly refers to black immigration to Britain. In fact, his poem entitled ‘Remember the Ship’ was first published in 1998, 50 years after the SS Empire Windrush arrived at Tilbury, an event that has come to epitomise the beginning of black im- migration to Britain. Commemorating this event, Agard demands: ‘citi- zenship shall be / a call / to kinship / that knows / no boundary / of skin’ (Agard 2000: 259). Of course, the poem implies a wider under- standing of citizenship than what has been discussed in the present vo- lume, including specific questions that address equal participation in the community. Nevertheless, if we only look at citizenship regulations, it is obvious that these have changed dramatically in recent years and that immigration has played a major role in these changes, albeit not always in the way Agard envisages. Thus, Britain has used citizenship regulations in order to reduce the immigration of blacks, in particular, from its former colonies when it introduced a new nationality law in 1981 (see Dummett 2006: 568-570). Moreover, the NATAC study has shown that many recent changes in citizenship laws of the fifteen pre- 2004 EU Member States were in response to immigration, even if there is no single clear trend. While some countries, including Austria, Denmark, France, Greece, the Netherlands and the UK raised their re- quirements for naturalisation, others, such as Belgium and Portugal, have liberalised access to their nationalities for immigrants and their descendants (see Baubo¨ck, Ersbøll, Groenendijk & Waldrauch 2006: 23-25). This is probably the most striking difference between the citizenship regulations in the fifteen ‘old’ Member States and those in the twelve new EU Member States and the two accession countries discussed in this book. Most of these are only just now starting to become countries of immigration. Yet, migration was also a very important issue con- cerning their citizenship regulations over the past two decades, albeit 440 WIEBKE SIEVERS WIEBKE SIEVERS in different ways. These specific links between citizenship and migra- tion shall be discussed in more detail in this summary. ‘A call to kinship’? Citizenship and migration in the new Member States and the accession countries of the EU The first sec- tion will analyse policies of initial citizenship determination, which have often implied discrimination against long-term resident popula- tions and their descendants, in particular those in Estonia and Latvia but also those in the former Yugoslavia. Subsequently, I will look at the legal regulations that specifically target emigrants, and the interactions between these and the citizenship regulations in the emigrants’ current countries of residence. Finally, I will focus on various naturalisation regulations and their implications for the new immigrants who began to settle in these countries in the 1990s. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia The Czech Republic, Slovakia and the former Yugoslav republics decided to base their criteria for citizenship on the existing republic-le- vel citizenship introduced in Yugoslavia just after the Second World War, and in Czechoslovakia in response to the Slovak national move- ment of the late 1960s. These existing regulations stipulated that all ci- tizens of Yugoslavia and Czechoslovakia automatically were citizens of their respective states and of one of the federal republics within this entity. Although it was very easy to change the republic-level citizen- ship in both states, internal migrants usually did not bother because there was (almost) no benefit from such a change. As a consequence, 65,000 Slovaks applied for Czech republic-level citizenship in 1992 when Slovakian threats to break away became a realistic possibility. De- spite this run on applications, many Czechs and Slovaks ended up with the citizenship of the state in which they were not residing and, in some cases, where they had not even been granted permanent resi- dence. Moreover, although each state gave the citizens of the respective other state the option to apply for its citizenship, there is some indica- tion that it was particularly difficult for many Roma who had moved from the Slovak to the Czech part of Czechoslovakia after the Second World War to fulfil some of the requirements. It took about a decade and several legislative corrections to sort out these and similar pro- blems (see Barsˇova´ and Kusa´ in this volume). ( ) The situation was comparable in Slovenia, where citizens of the other Yugoslav republics, registered as residents in Slovenia on the day of the referendum for independence (23 December 1990), were granted the option to apply for Slovenian citizenship within six months after independence, i.e. by 25 December 1991. A total of 171,125 per- sons (or about 8 per cent of the total population at the time) were granted Slovenian citizenship under this regulation. Despite the fact that this was a more inclusive approach to the initial determination of citizenry than in the other Yugoslav successor states, it nevertheless ex- cluded those long-term residents who had never bothered to register their residence in Slovenia, which was not rare since Slovenia was the only one of the Yugoslav republics that recorded in- and out-migration and there was no immediate benefit to registering. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia All of the newly formed states have to figure out how to determine who their citizens are. The majority of new states solve this puzzle by declaring those born or residing in their territory their citizens (see Brubaker 1992: 277). However, in order to prevent both the imposition of citizenship onto citizens of other states born or living in their coun- tries and creating stateless persons abroad, emerging states usually dis- tinguish between those who automatically become their citizens on the day of their establishment and those who can apply for citizenship within a certain period of time after that date. In Cyprus, for example, residence in the country was the decisive element in this process. Only those who had a special connection to the country1 and had ordinarily resided on the island of Cyprus for any period of time in the five years before the day of independence automatically became citizens on that day, whereas those of Cypriot origin who were not residing in the country could apply for citizenship. In Malta, by contrast, birth in the country was considered more important. Only those citizens of the United Kingdom and the colonies who were born in Malta and one of whose parents was also born there, as well as those born outside Malta whose father and one of whose paternal grandparents were born in Malta, automatically became citizens of Malta. Those born in Malta of parents who were not born there had to apply for citizenship. In addi- tion, both Cyprus and Malta made provisions for those who were natur- alised under former regulations and women married to citizens of their countries (see Trimikliniotis and Buttigieg in this volume). While Cyprus’ and Malta’s initial determinations of who their citi- zens are do not seem to have created too many problems, the situation was quite different in the post-communist states that were established ‘A CALL TO KINSHIP’? 441 or re-established in the 1990s. The regulations that determined the in- itial citizenry in these countries, which resulted from partitioning or secession, turned former internal migrants into international migrants. Their legal statuses became unclear in countries like the Czech Repub- lic, Slovakia and Slovenia, difficult in Croatia and extremely proble- matic in Estonia and Latvia. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia Moreover, those who had registered their residence in Slovenia but did not apply for, nor acquire, Slovenian citizenship became aliens and had to apply for residency status even if they had resided in the country for years. If, 442 WIEBKE SIEVERS WIEBKE SIEVERS for various reasons – including the lack of information, the difficulty of obtaining documentation in the period of Yugoslavia’s break-up, con- fusing procedures, etc. – they had not applied for residency by the day when the Aliens Act became law (23 February 1992), they were auto- matically transferred from the register of residents to the aliens record without even being notified of this change in their status, which later became known as erasure. This erasure meant that they lost all of their civil, social and political rights that had been granted to them under the Yugoslav legal framework, such as the rights to work, healthcare and social benefits. The government admitted that it had deprived more than 18,000 persons of their residency status. However, this fig- ure probably excludes those affected who left Slovenia before the mat- ter became publicly known. Despite pressure from several NGOs and the ombudsman for human rights, who pointed out this problem in his first report in 1995, it was only in 1999 that the Constitutional Court ordered the legislature to eradicate this mistake. For various rea- sons, the legislature has yet to follow up on this directive. However, 7,000 of those affected by this act have since been granted Slovenian citizenship under the 1999 Act Settling the Status of Citizens from other Yugoslav Successor States before it was declared unconstitutional in 2003. The same constitutional court decision ordered the Slovenian state to reissue permanent residence permits to those deleted from the population register whose status had yet to be determined at the time. Finally, this group was granted the right to apply for facilitated natura- lisation by 23 November of the same year. Another 1,767 persons ac- quired Slovenian citizenship under this provision (see Medved in this volume; Sˇtiks 2006: 490-491). Like Slovenia, Croatia decided to follow the principle of legal conti- nuity. Hence, all citizens of the Yugoslav republic of Croatia automati- cally became citizens of the new state when it became independent on 8 October 1991. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia Unlike Slovenia, however, Croatia did not grant citi- zens of other Yugoslav republics/successor states residing in Croatian territory the right to opt for Croatian citizenship. As a consequence, these residents, who had sometimes been living in Croatia for years or were even born there, automatically became aliens on the day of inde- pendence, thereby losing all citizenship rights they had held under the Yugoslav legal framework. This most likely affected a much larger group of persons than the erasure in Slovenia, which had similar im- plications, but the exact figures of those excluded from Croatian citi- zenship by this action remain unknown. Only a specific group among these was granted privileged access to Croatian citizenship in the tran- sitional provisions regulating the initial determination of who was Croatian: namely, just resident ethnic Croats. They could acquire citi- zenship by issuing a written statement to the police that they consid- ‘A CALL TO KINSHIP’? 443 ered themselves Croatian citizens. All other residents who had become foreigners and who wanted to acquire the citizenship of their state of residence had to apply for naturalisation. The naturalisation procedure, in line with the general spirit of the 1991 Law, required abiding by Croatia’s legal system, customs and culture. This gave the Ministry of Interior wide discretion in rejecting applications, which, according to several reports, seems to have resulted in discrimination against all non-ethnic Croats but in particular against Serbs. Administrative prac- tice has become less discriminatory since Franjo Tudjman’s death and subsequent political changes. However, the Croatian government has yet to adopt a new and less discriminatory law (see Ragazzi & Sˇtiks in this volume; Human Rights Watch 1995). The initial determination of citizenship was most problematic in Es- tonia and Latvia because both states had created a large number of sta- teless persons in the process. Originally founded in 1920, the three Baltic states of Latvia, Estonia and Lithuania were first occupied by the Soviet Union in 1940 and, after a short period of German occupation, they became part of the Soviet Empire in 1944. During the period of Soviet annexation, which was also a period characterised by rapid in- dustrialisation, especially in Latvia and Estonia, a large number of Rus- sian workers moved to these two countries. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia When the Baltic states re- gained their independence in 1990, these internal migrants ‘secure in their Soviet citizenship’ became ‘international migrants of contested le- gitimacy and uncertain membership’ (see Brubaker 1992: 269). In principle, these three Baltic states followed the notion of state continuity, which means that they reinstalled the 1940 institutions and laws, including their respective citizenship legislation. However, Lithuania had already enacted a new nationality law in 1989, using the authority in nationality matters granted by the Soviet authorities dur- ing the final days of the Soviet Union. This law was the outcome of ne- gotiations between Lithuanian nationalist forces, which intended to re- install the pre-1940 citizenship laws, and the Soviet government, which opposed the exclusion of Russians and Poles who lived in Lithuanian territory. Departing from the pre-1940 citizenship laws, it only regarded those pre-1940 nationals and permanent residents and their children and grandchildren who resided in Lithuania and did not hold another citizenship as citizens. Hence, emigrants and expatriates were excluded, a decision that Lithuania has been at pains to change ever since, as will be shown in the following section. At the same time, all of those persons who had held permanent resident status in Lithua- nia for at least two years prior to the adoption of the law and who had a legal source of income in the country, could declare their intention to become nationals within two years following enactment of the law. This meant that Lithuania gave immigrants who had arrived during 444 WIEBKE SIEVERS WIEBKE SIEVERS the Soviet period the possibility of acquiring Lithuanian nationality. About 90 per cent of all non-Lithuanians seized this opportunity de- spite the fact that they had to renounce their USSR citizenship in the process, which at that time was not an easy option (see Kru¯ma in this volume; Budryte 2005: 149ff). y 5 49 ) Unlike Lithuania, Latvia and Estonia decided to reestablish their pre- 1940 citizenry in 1991 and 1992, respectively, by restoring the national- ity laws that were in force prior to their annexation. According to these regulations, all of those who were Estonian nationals prior to 16 June 1940 and Latvian nationals on 17 June 1940 and their descendants had a right to Estonian and Latvian nationalities, respectively, regardless of whether they resided in the country or not. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia By contrast, those who ar- rived after these dates and had lived in these countries ever since, had to go through the normal naturalisation procedures regardless of the amount of time they had spent in their country of residence. As a con- sequence, about one-third of the populations residing in these two countries, again, mostly Russian immigrants who arrived as workers during the time of Soviet occupation, became aliens or, even worse, sta- teless, since the state of their nationality, the Soviet Union, had ceased to exist. This also meant that they had no right to participate in refer- enda on their constitutions, nor could they vote or run in the first elec- tions, plus they were prohibited from seeking a number of public posi- tions, all of which enhanced their exclusion (see Kru¯ma and Ja¨rve in this volume). The differing approaches towards the Russian long-term residents in these three countries have often been explained by the fact that the numbers were much higher in Latvia and Estonia than in Lithuania. It is true that the share of the titular ethnic group declined from 90 to 61 per cent in Estonia and from 77 to 52 per cent in Latvia between 1939 and 1989 (these are self-ascriptions gathered in the censuses). Based on these figures, both countries argued that a strict stance on national- ity was necessary in order to guarantee the survival of their ethnic na- tions and cultures. However, a large percentage of the non-titular eth- nic groups in Latvia automatically acquired citizenship in 1991 since they were (descendants of) persons who were citizens or permanent re- sidents in 1940 (see Aasland & Fløtten 2001: 1025). This explains why the percentage of aliens and stateless was lower in Latvia than in Esto- nia in 1993 (28 vs. 32 per cent). This ratio changed drastically in subsequent years, as the naturalisa- tion policies were very different in the two countries, with the require- ments being far stricter in Latvia than in Estonia. The Estonian law adopted in 1992 required two years of permanent residence prior to ap- plication (as of 30 March 1990) and one year after an application had been filed, as well as a language exam. More than 87,000 persons were ‘A CALL TO KINSHIP’? 445 naturalised under these conditions between 1992 and 1995, when a new Citizenship Act came into law. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia The 1995 Act increased the re- quired period of permanent residence to five years prior to, and one year after, application, introduced a test of applicants’ knowledge of the Estonian Constitution and the Citizenship Act and upgraded the lan- guage exam. As a consequence, the naturalisation figures dropped sharply, decreasing from more than 22,000 who were granted citizen- ship in 1996 (under the 1992 Law) to 8,000 naturalised in 1997 (un- der the 1995 Law). Despite international criticism, especially while it prepared for its accession to the EU, and several attempts to encourage naturalisation, the naturalisation figures have remained low and have only slightly increased since Estonia’s EU accession in 2004. Neverthe- less, the share of stateless persons decreased from 32 to 9 per cent be- tween 1992 and 2008, albeit not all of this was due to their being nat- uralised. While a similar share of the stateless left the country, a smal- ler share acquired Russian citizenship but remained in Estonia. However, the numbers of stateless persons applying for Russian citi- zenship has increased dramatically since the Estonian government decided to relocate a Soviet war memorial in April 2007, an event which not only initiated riots by mostly Russian-speaking youths but also considerably increased Estonians’ mistrust of all Russian speakers. Moreover, there is some danger that older cohorts will continue to re- main stateless, since the language exam prevents them from naturalis- ing (see Ja¨rve in this volume). In Latvia, the naturalisation requirements were far stricter from the outset. The law, which was restored in 1991 and in force until 1994, imposed sixteen years of residence among other requirements. Thus, it is not surprising that not a single person was naturalised under these conditions. The first proposals for a new Citizenship Act remained very strict. They foresaw first applications for citizenship for the year 2000, limiting the numbers to 0.1 per cent of the previous year’s total num- ber of Latvian nationals. In practice, this would have meant that under these regulations about 1,000 stateless persons could have become nat- uralised in 2000, a ridiculously small number considering that Latvia had 673,398 stateless persons and foreigners in 1993. While the parlia- ment adopted this proposal, the president never signed it. This was due to international pressure, mainly from the Council of Europe, which threatened not to admit Latvia if the law was adopted. 1 Excluding long-term residents and their descendants: Initial determination of citizenship in Estonia, Latvia, Slovenia and Croatia It took an- other year before a new Citizenship Law was enacted in 1994. Although this law was more liberal with regard to the naturalisation re- quirements than the first proposal (five years of permanent residence and a language exam, among other requirements), it retained the so- called window system which limited the number of persons allowed to 446 WIEBKE SIEVERS WIEBKE SIEVERS naturalise per year. This system was adopted since the legislators ex- pected a run on Latvian citizenship. However, the expected run on Latvian citizenship never materialised. In fact, the number of applications was so low and the pressure from abroad so high that the law was amended again in 1995. These amend- ments did not affect the naturalisation requirements but addressed the problem of the large number of stateless persons residing in Latvia from two different angles. Firstly, they added a new category to those regarded as Latvian nationals: those who were permanently residing in Latvia, were duly registered and had completed their educations in a school where Latvian is at least one of the languages of instruction. This category has been interpreted to include some former USSR na- tionals. Secondly, they introduced a special non-citizen status for for- mer USSR citizens who had become stateless, which guaranteed the holders of this status almost all of the same rights that nationals have except for electoral rights at the national level. After these amendments were enacted, the numbers of applications for naturalisation further decreased (from 4,543 in 1995 to 2,627 in 1996), with the disinterest in Latvian citizenship being attributed to the fact that persons were happy with the rights they had thus far been granted.2 It is only after the window system was abolished in 1998 that the numbers of applica- tions for Latvian citizenship increased significantly. Nevertheless, the number of non-citizens and foreign nationals remains high in Latvia, constituting 20 per cent of the total population in 2008 – the same as in 2005 (compared to 28 per cent in 1993). Moreover, only about half of the former non-citizens and foreigners have thus far acquired Lat- vian citizenship, while the rest have either become citizen of another country or have left the country (see Kru¯ma in this volume). Reaching out to emigrants and expatriates: Restitution laws, dual nationality and status laws 2 Apart from the decision made by Estonia and Latvia to deny a large group of residents the right to opt for their citizenships, the most de- bated citizenship issues in the countries under discussion in this vo- lume were most probably Romania’s decision to restore citizenship to those living in the former territories of Greater Romania and Hun- gary’s decision to grant kin minorities in the former Hungarian terri- tories a quasi-citizenship status. This was mainly because these laws were perceived as infringing upon the territorial sovereignty of neigh- bouring states, which unleashed an international debate. However, Ro- mania and Hungary were not the only two countries that reached out to former citizens and ethnic kin beyond their borders. Most of the ‘A CALL TO KINSHIP’? 447 countries under discussion in this book introduced similar policies, al- beit mainly addressing emigrants and their descendants, whose coun- tries of residence are distant, large and powerful enough not to feel im- mediately threatened by these regulations. In the following sections, I will focus on these regulations, which roughly fall into three categories: post-communist restitution laws, special regulations that allowed ex- patriates to reacquire or maintain their original citizenship and quasi- citizenship regulations that have granted a number of social rights to ethnic kin residing in other countries. Restitution laws Restitution laws have, in principle, been meant to right communist (or older) wrongs. Many post-communist countries adopted such laws but they often only had symbolic value, which should nevertheless not be underestimated. Czechoslovakia first adopted such legislation in 1990 and the Czech Republic renewed it in 1999 (since the first regulation expired in 1993). Although the introductory phrases of this law imply that it aims to ‘assuage the legacy of certain wrongs that occurred in the period 1948 to 1989’, the law applies, in practice, to all those who lost Czechoslovak citizenship in the communist period. Moreover, although Czech law prohibits dual nationality (expressly exempting only Slovaks from this rule), the restitution law did not require appli- cants to renounce any other nationality. Nevertheless, the number of those who applied for Czech citizenship under this law has remained relatively low probably because the law was only introduced after the date for the application for restitution of property expired (the right to property restitution was limited to Czechoslovak citizens). Another ex- ample of this restitution law, which mainly had symbolic relevance, is the Polish right to repatriation, which was first introduced in the 1990s and then reformulated in a separate Repatriation Act adopted in 2000. This Act is addressed to ethnic Poles who were residing ‘in the East […] due to deportations, exile and other ethnically motivated forms of persecution’. Again, this law applies to all ethnic Poles residing in the defined areas since they did not have to provide proof of deporta- tion or persecution – which would probably also have been difficult. Nevertheless, it only attracts a small number of repatriates (see Liebich in this volume). Restitution laws have, in principle, been meant to right communist (or older) wrongs. Many post-communist countries adopted such laws but they often only had symbolic value, which should nevertheless not be underestimated. Czechoslovakia first adopted such legislation in 1990 and the Czech Republic renewed it in 1999 (since the first regulation expired in 1993). Although the introductory phrases of this law imply that it aims to ‘assuage the legacy of certain wrongs that occurred in the period 1948 to 1989’, the law applies, in practice, to all those who lost Czechoslovak citizenship in the communist period. Moreover, although Czech law prohibits dual nationality (expressly exempting only Slovaks from this rule), the restitution law did not require appli- cants to renounce any other nationality. Restitution laws Nevertheless, the number of those who applied for Czech citizenship under this law has remained relatively low probably because the law was only introduced after the date for the application for restitution of property expired (the right to property restitution was limited to Czechoslovak citizens). Another ex- ample of this restitution law, which mainly had symbolic relevance, is the Polish right to repatriation, which was first introduced in the 1990s and then reformulated in a separate Repatriation Act adopted in 2000. This Act is addressed to ethnic Poles who were residing ‘in the East […] due to deportations, exile and other ethnically motivated forms of persecution’. Again, this law applies to all ethnic Poles residing in the defined areas since they did not have to provide proof of deporta- tion or persecution – which would probably also have been difficult. Nevertheless, it only attracts a small number of repatriates (see Liebich in this volume). Unlike the Czech and Polish laws, the Bulgarian restitution law adopted in 1990 not only had symbolic value but also had a real im- pact. This law served to restore citizenship to the more than 300,000 members of the Turkish minority who were deprived of this status and forced to resettle in Turkey in the final stages of the communist regime after years of ethnic repression. A large number of these refugees had, 448 WIEBKE SIEVERS WIEBKE SIEVERS in the meantime, been granted Turkish citizenship and some of them had also actually remained in Turkey. But the law restoring their Bul- garian citizenship did not demand the renunciation of other citizen- ships nor did it require resettlement to Bulgaria. However, the resent- ment against the former communist regime in Bulgaria, which found expression in the restitution law, has since been supplanted by ethnic nationalism. As a consequence, the openness of the citizenship law, in particular to those residing in Turkey, has come under criticism. The debate was sparked off by the fact that all Bulgarian nationals, regard- less of their place of residence, have the right to vote. Mobilised by the MRF, the Bulgarian party historically associated with the Turkish min- ority, Bulgarians residing in Turkey have made extensive use of this right. This situation was first criticised as undemocratic during the par- liamentary elections of 1994. Restitution laws Protests escalated prior to the European parliamentary elections in 2007, in advance of which the radical na- tionalist party Ataka suggested changing the citizenship laws so that only ethnic Bulgarians could hold dual nationality. While this sugges- tion was not accepted in Bulgaria, it is a normal practice in many coun- tries as will be shown below (see Smilov & Jileva in this volume). ( J ) Restitution assumed a wider meaning in Romania, Latvia and Esto- nia. Romania’s restitution policies started on a note that was similar to Polish and Czech laws. A decree adopted in Romania as early as 1989 not only guaranteed repatriation to all Romanian citizens residing abroad but also granted former Romanian citizens residing abroad the right to reacquire Romanian citizenship by repatriating without requir- ing them to renounce their other nationality. This implicit toleration of dual nationality was a novelty in Romanian law at the time. A second decree issued in 1990 further extended the group of former citizens who were allowed to reacquire Romanian nationality by also waiving the residency requirement. However, it was only with the adoption of the new citizenship law in 1991 that restitution acquired something more than mere symbolic significance in Romania. This law explicitly included a third group of former Romanian citizens now granted the right to reacquire citizenship, namely those who resided in the terri- tories of Bessarabia and Northern Bukovina, which belonged to Roma- nia before they were occupied by the Soviet Union in 1940-1941 and annexed in 1944. Like the Baltic states, the Romanian government ar- gued that this annexation had been illegal. While this particular law did not address migrants, it encouraged migration between Moldova, Romania and other EU countries. Moreover, several EU agencies warned that this policy of citizenship restitution could become a way of circumventing EU immigration controls when Romania acceded to the EU in 2007. This was one of the reasons why the Romanian govern- ment brought the naturalisation process to a halt and introduced more ‘A CALL TO KINSHIP’? 449 requirements for the reacquisition of citizenship for this particular group of people. While at least 100,000 largely Moldovan citizens reac- quired their citizenship in the first decade after the introduction of the law, this number dropped to fewer than 3,000 reacquisitions between 2001 and 2007. Restitution laws Since the nationality law was amended in September 2007 the number of nationality restitutions has increased. More than 3,807 restitutions were granted within a year. However, between 500,000 and 900,000 applications by Moldovans alone are said to be awaiting a decision (see Iordachi in this volume). g ( ) A similarly broad understanding of restitution was applied in Esto- nia and Latvia; it restored nationality to all pre-1940 citizens and their descendants even if they had emigrated after that date. Moreover, while both countries in principle prohibit dual nationality, their regulations are often unclear and sometimes even openly tolerate dual nationality where emigrants are concerned. Thus, the Estonian regulations state that Estonians can be deprived of their nationality if they acquire the nationality of another state; this provision, nevertheless, does not apply to those who acquired Estonian nationality by birth. It therefore re- mains unclear whether 1940 nationals by birth and their descendants are still considered nationals even if they hold another nationality. Moreover, the state has been quite tolerant of dual nationality in cases of resumption by those who lost Estonian nationality as minors (see Ja¨rve in this volume). The restored nationality law in Latvia, by con- trast, still explicitly demanded the renunciation of other nationalities for emigrants and their descendants. Only one group was excluded from this requirement in the transitional regulations of the restored law: refugees and those who were deported between 1940 and 1990 could register as citizens until 1 July 1995. The new nationality law adopted in 1994 extended this exemption from the general prohibition of dual nationality to all 1940 nationals and their descendants. Under this law, only those who had acquired a new nationality after Latvia’s independence were required to renounce that nationality. In 2007, the Constitutional Court officially confirmed Latvia’s unofficial policy of prohibiting dual nationality only in cases of naturalisation. Since then, there have been several discussions about also extending dual national- ity to those who have recently left the country (see Kru¯ma in this vo- lume). 2.2 Other special regulations allowing emigrants to acquire citizenship A second group of countries openly reached out to emigrants and ex- patriates by adopting special regulations in order to renew or maintain ties with these particular groups. As mentioned above, this holds true for Lithuania. Lithuania was the only one of the three Baltic states that 450 WIEBKE SIEVERS WIEBKE SIEVERS had expressly excluded expatriates from its initial citizenry in 1989 by making permanent residence in the country a requirement for the ac- quisition of nationality for 1940 nationals and their descendants. More- over, the law did not provide for dual nationality, primarily because the Lithuanian nationalist forces did not want Lithuanian citizens to keep their USSR citizenship. Both of these rules were amended in the course of the 1990s in response to pressure from expatriate commu- nities and criticism from the Council of Europe. However, Russian speakers were also interested in dual citizenship. These opposing forces might explain the special development of the Lithuanian citizen- ship regulations in the course of the 1990s. The first new citizenship law adopted after independence lifted the residence requirement for 1940 nationals as well as their children and grandchildren. Regarding the prohibition of dual nationality, early exceptions had already been introduced when the 1989 Law was amended in 1991. In 1993, those who had been deported from Lithuania or had left during the occupa- tion were allowed to recover their nationality without having to re- nounce another one. In 1994, the Constitutional Court declared that 1940 nationals and their descendants were the only group exempted from losing Lithuanian nationality when acquiring another nationality. Based on this ruling, the nationality law was again amended in 1995. Since then, all 1940 nationals and their children have been considered nationals unless they had returned to their ethnic homeland (according to this rule an ethnic Pole who had relocated to Poland after 1940 would not be allowed to keep both citizenships). Further pressure from expatriate groups led to an extension of this regulation to grandchil- dren and great-grandchildren of 1940 nationals in the new citizenship law that was enacted in 2003.3 However, all of these special regulations on dual citizenship for expatriates were declared unconstitutional by the Constitutional Court in 2006. The Court argued that the Constitu- tion allowed dual nationality only in exceptional cases. 2.2 Other special regulations allowing emigrants to acquire citizenship How this relates to its earlier judgement regarding 1940 nationals and their descen- dants remains unclear (Kru¯ma in this volume; Budryte 2005: 152ff.). Other countries used similar methods in order to renew or maintain links to expatriate communities in the 1980s and 1990s. In 1989, Mal- ta allowed emigrants born in Malta and who had spent at least six years abroad to hold dual nationality.4 A large number of Maltese citizens had left Malta for the UK, Australia, Canada and the US in the 1950s and 1960s. Following the previous regulations, they automatically lost their Maltese citizenship when acquiring the citizenship of their new country of residence. The 1989 amendments applied retroactively, i.e. Maltese emigrants who had lost their Maltese citizenship when they ac- quired the citizenship of their country of residence were now deemed to have never lost it. In 2007, Malta extended the right to dual nation- 451 ‘A CALL TO KINSHIP’? ality to second and subsequent generations residing abroad. Unlike Lithuania, Malta proceeded to make dual citizenship the rule in 2000. This means that foreigners applying for Maltese citizenship no longer have to renounce their other nationalities (see Buttigieg in this vo- lume). ) Of course, it takes two states for these regulations to have any im- pact. If the emigrants’ state of residence prohibits dual nationality, a change of law in the country of origin will not be enough of an incen- tive for former citizens to reacquire their citizenship. The Maltese changes are a good case in point: while in the 1990s only about 200 persons annually reacquired their Maltese nationality by registration, this number increased to more than 1,000 in 2002 when Australia be- gan allowing dual nationality. How many former Maltese citizens auto- matically reacquired their Maltese citizenship because of this legal change in their country of residence is unknown (see Buttigieg in this volume). However, there is no automatic guarantee that the legal regulations of other states are respected, as has been made evident in the Turkish case. Turkey introduced toleration of dual citizenship in 1981 with a view to maintaining links with Turkish workers who have moved to Western Europe, particularly Germany, since the 1960s. 2.2 Other special regulations allowing emigrants to acquire citizenship Moreover, the Turkish state encouraged former Turkish nationals to reacquire their Turkish nationality after having renounced it in order to acquire Ger- man and Austrian nationality, for example, although these two states prohibited dual nationality acquired through naturalisation. While this was known in the countries of residence, the German state had no le- gal basis that would have allowed it to stop this practice, mainly be- cause the German constitution did not permit withdrawing German nationality from German citizens residing in the country; this was in response to the Third Reich’s massive denaturalisation of German Jews. This changed in 2000 when Germany introduced a new nation- ality law, which also granted the German state the right to withdraw German nationality from those who had acquired another nationality without the authorities’ consent. More than 40,000 German-Turkish dual nationals lost their German nationality as a result of this new reg- ulation (see Kadirbeyoglu in this volume). Quasi-citizenship regulations for expatriates 2.3 Several countries in our sample adopted laws that provided co-ethnics with a form of quasi-citizenship by granting them a set of rights usual- ly reserved to citizens, including privileged access to education and em- ployment. The most important and most debated of these was the in- troduction of the so-called Status Law in Hungary in 2001. Granting 452 WIEBKE SIEVERS WIEBKE SIEVERS quasi-citizenship to ethnic Hungarians residing in neighbouring coun- tries, this law was met with heavy criticism (until 2003 when the most debated provisions were deleted), particularly in Slovakia, despite the fact that Slovakia had already introduced a similar law in 1997. So had Bulgaria in 2000, Slovenia in 2006 and Poland in 2007 (see Kova´cs & To´th, Kusa´, Medved, Go´rny & Pudzianowska and Smilov & Jileva in this volume). ) While all of these countries introduced a form of quasi-citizenship in order to establish and maintain links with co-ethnics abroad holding the citizenship of their country of residence, Turkey introduced a simi- lar regulation in order to encourage citizens residing abroad to acquire the nationality of their respective country of residence. When several Turkish emigrants were killed in xenophobic arson attacks in Mo¨lln in 1992 and Solingen in 1993, the Turkish government decided to encou- rage its nationals abroad to acquire the nationality of their country of residence, arguing that a large Turkish electorate would at least prevent parties from openly mobilising against immigrants. The main reason for Turkish nationals not acquiring German nationality was the fact that they had to renounce their Turkish nationality and thus the right to property and inheritance in Turkey. That is why the Turkish govern- ment introduced a quasi-citizenship status in 1995, which has become known as the ‘pink card’ (reflecting the colour of the document given to such status holders). This status grants former Turkish nationals all the rights of citizens except the right to vote. While the numbers of Turkish citizens granted German citizenship has increased signifi- cantly since the introduction of the pink card, the number of applica- tions for the pink card has remained low. This implies that other fac- tors were responsible for the increase in naturalisations (see Kadirbeyo- glu in this volume; Caglar 2004; the statistics compiled by Harald Waldrauch for the NATAC project as published on www.imiscoe.org). Quasi-citizenship regulations for expatriates All of the states discussed here have reached out to their expatriates and kin minorities in the last two decades. As the following section will show, this attitude contrasts strongly with the lack of attention paid to immigrants as potential future citizens. 3 Excluding recent immigrants: Naturalisation requirements 3 As mentioned above, the countries discussed in this book have only re- cently begun to attract immigration. As a consequence, these new im- migrants have not really become an issue in their respective citizenship regulations. Granted, all of them have regulations for the acquisition of citizenship by naturalisation, the requirements of which are in princi- ple similar to those in the fifteen pre-2004 EU Member States. All of ‘A CALL TO KINSHIP’? 453 the countries under discussion here require a specific period of resi- dence in the country, ranging from five (in nine countries) to ten years (in Lithuania and Slovenia). However, unlike many of the ‘old’ Member States, six of the new Member States do not count all of the years from the date of entry but rather count from the date when the respective person was granted a residency permit, which certainly increases the required period of residency significantly. Moreover, all of these coun- tries demand a clean criminal record, thirteen out of the fourteen countries require command of the national language, ten demand proof of some kind of legal income, seven demand that the applicants swear an oath of loyalty and three conduct a test on the country’s con- stitution (also see Dumbravaˇ 2007: 462 for the new Member States; Waldrauch 2006: 134-159 for the fifteen pre-2004 Member States). 34 59 p 4 ) Only rarely are there any clear indications that specific changes in the citizenship laws are motivated by recent immigration. One example is Romania’s tightening of its naturalisation requirements in 1999 and 2003. In 1999, the Romanian government not only increased the resi- dence requirement from five to seven years, but also introduced tests on an applicant’s knowledge of the Romanian language, elementary norms of Romanian culture and civilisation. In 2003, the residence re- quirement was further increased to eight years (but reduced to five years for foreign spouses of Romanian nationals). Moreover, the law now also demands knowledge of the national anthem and a declaration of loyalty. This marked increase in requirements, particularly in the cultural domain, is not unlike recent developments in West European countries such as Denmark, Germany, the Netherlands and the UK. y Malta, Cyprus and Poland have the lowest number of legal require- ments. 3 Excluding recent immigrants: Naturalisation requirements For example, Poland – the only post-communist country which has not introduced a new citizenship law since the fall of the Iron Cur- tain – officially demands only five years of permanent residence, which in practice comes to at least ten years of residence in the country. How- ever, this does not mean that these are the most open countries but rather that their authorities exercise broad discretion in deciding who can become a citizen. Again, Poland is an interesting case in point: ap- plications for naturalisation in Poland have to include additional infor- mation on the parents’ nationality, sources of income, the applicants’ most recent job, knowledge of the Polish language and services ren- dered to Poland (or Polish diaspora organisations, etc.). However, the citizenship law does not contain any requirements that explain why these documents are necessary to make a decision on an applicant. Nor are there clear rules for their evaluation. Practice indicates that not only Poland but also Cyprus and Malta select future citizens based on their ancestry (see Buttigieg, Trimikliniotis and Go´rny & Pudzianowska in this volume). 454 WIEBKE SIEVERS WIEBKE SIEVERS Moreover, even in those countries where the requirements are very similar to those in the pre-2004 EU Member States, the focus is rarely on new immigration, which becomes obvious when we look at regula- tions that address specific groups. Apart from the three countries men- tioned above where descent is important in practice although not men- tioned in the law, five countries offer facilitated naturalisation to co-eth- nics by reducing the residence requirement from ten years to one in the case of Slovenia (see Medved in this volume), from eight years to at least one year for ethnic Hungarian descendants of former citizens (see Kova´cs & To´th in this volume) and from five years to no residence requirement at all in Bulgaria, Croatia and Turkey (see Iordachi, Ragazzi & Sˇtiks and Kadirbeyoglu in this volume). Hence, eight of the fourteen countries apply ethnic preferences in one way or another. There are only two other groups of foreigners who enjoy preferential treatment in a large number of these countries: those regarded as being of interest to the country because of their special merits and the spouses of nationals. 4 Conclusions This chapter’s discussion of migration and citizenship legislations in the new Member States, Croatia and Turkey has shown that the trends in these fourteen countries are comparable. Returning to Agard’s ear- lier quotation we might be able to summarise that the main trend has been ‘a call to kinship’, although this call has not been addressed to im- migrants but to kin minorities and former citizens in neighbouring states and to emigrants and expatriate communities in more distant countries. Furthermore, long-term residents were often subject to se- vere discrimination in the process of the establishment and re-estab- lishment of states after the end of the communist period. Since immi- gration is a comparatively recent phenomenon in all of the countries under discussion, these new immigrants have not really become an is- sue with regard to citizenship regulations. In that respect, the citizen- ship legislations in the twelve new Member States, Croatia and Turkey differ markedly from those in the pre-2004 EU Member States, where most of the recent changes in citizenship laws were motivated by im- migration. However, as Dumbravaˇ (2007) rightly points out, we should not fall into the trap of describing these differences in terms of restrictive East- ern European laws and liberal Western European laws (for a different opinion on this matter, see Howard 2009). Firstly, several of the pre- 2004 Member States also have regulations that facilitate citizenship ac- quisition for expatriates and co-ethnics, including Germany, Greece, Italy, Ireland, Spain and Portugal. Both the Western European and the above-described Eastern European regulations can, in principle, be used to enter any other Member State, since citizenship in one EU Member State automatically implies EU citizenship. There is, for ex- ample, some indication that Argentine citizens acquire Italian citizen- ship to be able to live and work in Spain. Hence, European concerns about national citizenship regimes are not only raised by Eastern Eur- opean policies but also by analoguous Western European regulations. Secondly, as pointed out above, citizenship in Western Europe has moved in opposite directions in recent years, with some formerly liber- al countries, such as Denmark or the Netherlands, dramatically raising their naturalisation requirements (see Baubo¨ck, Ersbøll, Groenendijk & Waldrauch 2006: 23-25). Thirdly, there are specific trends that are si- milar in all of these countries. For instance, dual citizenship has be- come more accepted throughout the European Union. 3 Excluding recent immigrants: Naturalisation requirements However, in their most recent amendments, and in order to prevent marriages of convenience, Slovenia and Malta have already raised the number of years that spouses are required to reside in the country be- fore they can naturalise (see Medved and Buttigieg in this volume). Special regulations for other groups of immigrants are rare: Bulgaria, the Czech Republic, Slovenia and Hungary have reduced residence re- quirements for refugees (see Smilov & Jileva, Barsˇova´, Medved and Ko- va´cs & To´th in this volume), while Croatia, Slovenia, Lithuania and Hungary have special regulations for foreigners born in their countries, but these usually address very specific groups, such as residents from other former Yugoslav republics in Slovenia and Croatia (see Ragazzi & Sˇtiks, Medved, Kru¯ma and Kova´cs & To´th in this volume). In the Czech Republic, all foreigners born within its borders are treated somewhat more favourably than other foreigners, as they do not necessarily need to meet the five-year residency requirement to qualify for naturalisa- tion. The Czech Republic is also the only country that has made an at- tempt to introduce special regulations for second-generation foreigners born in the Czech Republic of immigrant parents, but the legislative process was hampered by the parliamentary elections of 2006. A new draft bill containing regulations for this particular group is expected to be presented in 2009 (see Barsˇova´ in this volume and Dumbravaˇ 2007: 459). ‘A CALL TO KINSHIP’? 455 Notes 1 British subjects, according to the Cyprus (Annexation) Orders, and persons born in Cyprus on or after 5 November 1914, as well as descendants in the male line of both of these groups (see annex D to the Treaty concerning the Establishment of the Republic of Cyprus, sect. 2, para. 2, available at www.fco.gov.uk). 1 British subjects, according to the Cyprus (Annexation) Orders, and persons born in Cyprus on or after 5 November 1914, as well as descendants in the male line of both of these groups (see annex D to the Treaty concerning the Establishment of the Republic of Cyprus, sect. 2, para. 2, available at www.fco.gov.uk). 2 The most important reasons for the lack of interest in naturalisation were that people did not know Latvian, that they wanted to avoid military service and that it was easier for non-citizens than for Latvians to receive a Russian visa. The third point has since been further simplified; in 2008, Russia decided to grant visa-free travel to former USSR citizens who reside in Estonia and Latvia and have not acquired another citizenship (see Kru¯ma in this volume). 3 Grandchildren of 1940 nationals also had a right to nationality under the previous law but were required to renounce other nationalities when they wanted to exert that right. B f h d l i ll d h h i li il 3 Grandchildren of 1940 nationals also had a right to nationality under the previous law but were required to renounce other nationalities when they wanted to exert that right. 4 Before that date only minors were allowed to have more than one nationality until their nineteenth birthdays. 3 Grandchildren of 1940 nationals also had a right to nationality under the previous law but were required to renounce other nationalities when they wanted to exert that right. 4 Before that date only minors were allowed to have more than one nationality until their nineteenth birthdays. 4 Before that date only minors were allowed to have more than one nationality until their nineteenth birthdays. 4 Conclusions However, the reasons for the growing tolerance of dual nationality differ; in Western Europe this is mostly linked to facilitating naturalisation for immi- grants, while in the twelve new Member States and two accession countries under discussion tolerance of dual citizenship is mainly re- 456 WIEBKE SIEVERS WIEBKE SIEVERS served for co-ethnics and emigrants residing abroad. As a consequence, there has been a tendency to regard only the former as liberal, although the latter reforms also liberalise access to citizenship and may lead to a general acceptance of dual citizenship in the long run, as the case of Malta shows. 1 British subjects, according to the Cyprus (Annexation) Orders, and persons born in Cyprus on or after 5 November 1914, as well as descendants in the male line of both of these groups (see annex D to the Treaty concerning the Establishment of the Republic of Cyprus, sect. 2, para. 2, available at www.fco.gov.uk). Bibliography Aasland, A. & T. Fløtten (2001), ‘Ethnicity and Social Exclusion in Estonia and Latvia’, Europe-Asia Studies 53 (7): 1023-1049. Agard, J. (2000), ‘Remember the Ship’, in J. Procter (ed.), Writing Black Britain 1948- 1998. An interdisciplinary anthology, 258-259. Manchester: Manchester University Press. Baubo¨ck, R., E. Ersbøll, K. Groenendijk & H. Waldrauch (2006), ‘Introduction’, in R. Baubo¨ck, E. Ersbøll, K. Groenendijk & H. Waldrauch (eds.), Acquisition and Loss of Nationality: Policies and Trends in 15 European Countries. Vol. 1: Comparative Analyses, 15-34. Amsterdam: Amsterdam University Press. 15-34. Amsterdam: Amsterdam University Press. Brubaker, W. R. (1992), ‘Citizenship Struggles in the Soviet Successor States’, Interna- tional Migration Review 26 (2): 269-291. Budryte, D. (2005), Taming Nationalism? Political Community Building in the Post-Soviet Baltic States. Aldershot: Ashgate. Caglar, A. (2004), ‘“Citizenship Light”: Transnational Ties, Multiple Rules of Member- ship, and the “Pink Card”’, in J. Friedmann & S. Randeria (eds.), Worlds on the Move: Globalisation, Migration and Cultural Security, 273-291.Taurus: London. Dumbravaˇ, C. (2007), ‘Citizenship regulations in Eastern Europe. Acquisition of citizen- ship at birth and through regular naturalization in sixteen postcommunist countries’, CEU Political Science Journal 2 (4): 450-472. Dummett, A. (2006), ‘United Kingdom’, in R. Baubo¨ck, E. Ersbøll, K. Groenendijk & H. Waldrauch (eds.), Acquisition and Loss of Nationality. Policies and Trends in 15 European States. Vol. 2: Country Analyses, 551-585. Amsterdam: Amsterdam University Press. ‘A CALL TO KINSHIP’? 457 Howard, M. (2009), The Politics of Citizenship in Europe. Cambridge: Cambridge Univer- sity Press (forthcoming). Howard, M. (2009), The Politics of Citizenship in Europe. Cambridge: Cambridge Univer- sity Press (forthcoming). y ( g) Human Rights Watch (1995), Civil and Political Rights in Croatia. www.hrw.org. ˇ Sˇtiks, I. (2006), ‘Nationality and Citizenship in the Former Yugoslavia: From Disintegra- tion to European Integration’, in Southeast European and Black Sea Studies 6 (4): 483- 500. Waldrauch, H. (2006), ‘Acquisition of nationality’, in R. Baubo¨ck, E. Ersbøll, K. Groenen- dijk & H. Waldrauch (eds.), Acquisition and Loss of Nationality. Policies and Trends in 15 European Countries. Vol. 1: Comparative Analyses, 121-182. Amsterdam: Amsterdam University Press. List of contributors Andrea Barsˇova´, Government Office, Human Rights Department, Prague barsova@seznam.cz Rainer Baubo¨ck, Department of Political and Social Sciences, European University Institute, Florence rainer.baubock@eui.eu Eugene Buttigieg, Department of European and Comparative Law, Faculty of Laws, University of Malta eugene.buttigieg@um.edu.mt Agata Go´rny, Centre of Migration Research, University of Warsaw agata@gorny.edu.pl Constantin Iordachi, Department of History, Central European University, Budapest iordachinc@ceu.hu Priit Ja¨rve, Independent researcher, Vilnius, Lithuania jaerve@yahoo.com Elena Jileva, Centre for Political and Constitutional Studies, Madrid ejileva@cepc.es Zeynep Kadirbeyoglu, Political Science Department, Bogazici University, Istanbul zeynep.kadirbeyoglu@mail.mcgill.ca Ma´ria Kova´cs, Nationalism Studies Program, Central European University, Budapest kovacsma@ceu.hu Rainer Baubo¨ck, Department of Political and Social Sciences, European University Institute, Florence rainer.baubock@eui.eu Priit Ja¨rve, Independent researcher, Vilnius, Lithuania jaerve@yahoo.com Elena Jileva, Centre for Political and Constitutional Studies, Madrid ejileva@cepc.es Zeynep Kadirbeyoglu, Political Science Department, Bogazici University, Istanbul zeynep.kadirbeyoglu@mail.mcgill.ca Ma´ria Kova´cs, Nationalism Studies Program, Central European University, Budapest kovacsma@ceu.hu CITIZENSHIP POLICIES IN THE NEW EUROPE 460 Kristı¯ne Kru¯ma, Riga Graduate School of Law Kristine.Kruma@rgsl.edu.lv Dagmar Kusa´, International Center for Conciliation, Boston and EUROCLIO, The Hague dagmarkusa@gmail.com Andre Liebich, Graduate Institute of International and Development Studies, Geneva andre.liebich@graduateinstitute.ch Felicita Medved, Independent researcher, Brezovica pri Ljubljani felicita.medved@siol.net Bernhard Perchinig, Institute for Urban and Regional Research, Austrian Academy of Sciences, Vienna bernhard.perchinig@oeaw.ac.at Dorota Pudzianowska, Faculty of Law, Warsaw University d.pudzianowska@uw.edu.pl Francesco Ragazzi, School of Oriental and African Studies, London francesco.ragazzi@sciences-po.org Wiebke Sievers, Commission for Migration and Integration Research, Austrian Academy of Sciences, Vienna wiebke.sievers@oeaw.ac.at Daniel Smilov, University of Sofia and Centre for Liberal Strategies, Sofia daniel@cls-sofia.org Igor Sˇtiks, Institut d’Etudes Politiques de Paris, Paris and Northwestern University, Chicago/Evanston igorstiks@yahoo.com Judit To´th, Faculty of Law, Department of Constitutional Law, University of Szeged skula@juris.u-szeged.hu Nicos Trimikliniotis, Centre for the Study of Migration, Inter-ethnic & Labour Relations, University of Nicosia, Cyprus nicostrim@logosnet.cy.net Bernhard Perchinig, Institute for Urban and Regional Research, Austrian Academy of Sciences, Vienna bernhard.perchinig@oeaw.ac.at Judit To´th, Faculty of Law, Department of Constitutional Law, University of Szeged skula@juris.u-szeged.hu Nicos Trimikliniotis, Centre for the Study of Migration, Inter-ethnic & Labour Relations, University of Nicosia, Cyprus nicostrim@logosnet.cy.net Other IMISCOE titles IMISCOE Research IMISCOE Research Rinus Penninx, Maria Berger, Karen Kraal, Eds. The Dynamics of International Migration and Settlement in Europe: A State of the Art 2006 (ISBN 978 90 5356 866 8) (originally appearing in IMISCOE Joint Studies) Rinus Penninx, Maria Berger, Karen Kraal, Eds. The Dynamics of International Migration and Settlement in Europe: A State of the Art 2006 (ISBN 978 90 5356 866 8) (originally appearing in IMISCOE Joint Studies) Leo Lucassen, David Feldman, Jochen Oltmer, Eds. Paths of Integration: Migrants in Western Europe (1880-2004) 2006 (ISBN 978 90 5356 883 5) Rainer Baubo¨ck, Eva Ersbøll, Kees Groenendijk, Harald Waldrauch, Eds. Acquisition and Loss of Nationality: Policies and Trends in 15 European Countries, Volume 1: Comparative Analyses 2006 (ISBN 978 90 5356 920 7) Rainer Baubo¨ck, Eva Ersbøll, Kees Groenendijk, Harald Waldrauch, Eds. Acquisition and Loss of Nationality: Policies and Trends in 15 European Countries, Volume 2: Country Analyses 2006 (ISBN 978 90 5356 921 4) Rainer Baubo¨ck, Bernhard Perchinig, Wiebke Sievers, Eds. Citizenship Policies in the New Europe 2007 (ISBN 978 90 5356 922 1) Veit Bader Secularism or Democracy? Associational Governance of Religious Diversity 2007 (ISBN 978 90 5356 999 3) Holger Kolb & Henrik Egbert, Eds. Migrants and Markets: Perspectives from Economics and the Other Social Sciences 2008 (ISBN 978 90 5356 684 8) Ralph Grillo, Ed. The Family in Question: Immigrant and Ethnic Minorities in Multicultural Europe 2008 (ISBN 978 90 5356 869 9) Rainer Baubo¨ck, Bernhard Perchinig, Wiebke Sievers, Eds. Citizenship Policies in the New Europe 2007 (ISBN 978 90 5356 922 1) Corrado Bonifazi, Marek Oko´lski, Jeannette Schoorl, Patrick Simon, Eds. International Migration in Europe: New Trends and New Methods of Analysis 2008 (ISBN 978 90 5356 894 1) Maurice Crul, Liesbeth Heering, Eds. The Position of the Turkish and Moroccan Second Generation in Amsterdam and Rotterdam: The TIES Study in the Netherlands 2008 (ISBN 978 90 8964 061 1) Marlou Schrover, Joanne van der Leun, Leo Lucassen, Chris Quispel, Eds. Illegal Migration and Gender in a Global and Historical Perspective 2008 (ISBN 978 90 8964 047 5) Gianluca P. Parolin Citizenship in the Arab World: Kin, Religion and Nation-State 2009 (ISBN 978 90 8964 045 1) Gianluca P. Parolin Citizenship in the Arab World: Kin, Religion and Nation-State 2009 (ISBN 978 90 8964 045 1) IMISCOE Reports Rainer Baubo¨ck, Ed. Migration and Citizenship: Legal Status, Rights and Political Participation 2006 (ISBN 978 90 5356 888 0) Michael Jandl, Ed. Innovative Concepts for Alternative Migration Policies: Ten Innovative Approaches to the Challenges of Migration in the 21st Century 2007 (ISBN 978 90 5356 990 0) Jeroen Doomernik, Michael Jandl, Eds. Modes of Migration Regulation and Control in Europe 2008 (ISBN 978 90 5356 689 3) Michael Jandl, Ed. Innovative Concepts for Alternative Migration Policies: Ten Innovative Approaches to the Challenges of Migration in the 21st Century 2007 (ISBN 978 90 5356 990 0) Michael Jandl, Ed. Innovative Concepts for Alternative Migration Policies: Ten Innovative Approaches to the Challenges of Migration in the 21st Century 2007 (ISBN 978 90 5356 990 0) Jeroen Doomernik, Michael Jandl, Eds. Modes of Migration Regulation and Control in Europe Michael Jandl, Christina Hollomey, Sandra Gendera, Anna Stepien, Veronika Bilger Migration and Irregular Work in Austria. A Case Study of the Structure and Dynamics of Irregular Foreign Employment in Europe at the Beginning of the 21st Century 2008 (ISBN 978 90 8964 053 6) Heinz Fassmann, Ursula Reeger, Wiebke Sievers, Eds. Statistics and Reality: Concepts and Measurements of Migration in Europe 2009 (ISBN 978 90 8964 052 9) IMISCOE Dissertations IMISCOE Dissertations Panos Arion Hatziprokopiou Globalisation, Migration and Socio-Economic Change in Contemporary Greece: Processes of Social Incorporation of Balkan Immigrants in Thessaloniki 2006 (ISBN 978 90 5356 873 6) Floris Vermeulen The Immigrant Organising Process: Turkish Organisations in Amsterdam and Berlin and Surinamese Organisations in Amsterdam, 1960-2000 2006 (ISBN 978 90 5356 875 0) Floris Vermeulen The Immigrant Organising Process: Turkish Organisations in Amsterdam and Berlin and Surinamese Organisations in Amsterdam, 1960-2000 2006 (ISBN 978 90 5356 875 0) Anastasia Christou Narratives of Place, Culture and Identity: Second-Generation Greek-Americans Return ‘Home’ 2006 (ISBN 978 90 5356 878 1) Anastasia Christou Narratives of Place, Culture and Identity: Second-Generation Greek-Americans Return ‘Home’ 2006 (ISBN 978 90 5356 878 1) Katja Rusˇinovic´ Dynamic Entrepreneurship: First and Second-Generation Immigrant Entrepreneurs in Dutch Cities 2006 (ISBN 978 90 5356 972 6) Inge Van Nieuwenhuyze Getting by in Europe’s Urban Labour Markets: Senegambian Migrants’ Strategies for Survival, Documentation and Mobility 2009 (ISBN 978 90 8964 050 5) Nayla Moukarbel Sri Lankan Housemaids in Lebanon: A Case of ‘Symbolic Violence’ and ‘Every Day Forms of Resistance’ 2009 (ISBN 978 90 8964 051 2) Nayla Moukarbel Sri Lankan Housemaids in Lebanon: A Case of ‘Symbolic Violence’ and ‘Every Day Forms of Resistance’ 2009 (ISBN 978 90 8964 051 2) Nayla Moukarbel Sri Lankan Housemaids in Lebanon: A Case of ‘Symbolic Violence’ and ‘Every Day Forms of Resistance’ 2009 (ISBN 978 90 8964 051 2) Je´roˆme Jamin L’imaginaire du complot: Discours d’extreˆme droite en France et aux Etats-Unis 2009 (ISBN 978 90 8964 048 2) John Davies ‘My Name Is Not Natasha’: How Albanian Women in France Use Trafficking to Overcome Social Exclusion (1998-2001) 2009 (ISBN 978 90 5356 707 4)
https://openalex.org/W1916507221	https://www.scielo.br/j/rbcdh/a/v88QKChQhCWR5hC3M8trT8m/?lang=pt&format=pdf	Portuguese	null	Atividades físicas e barreiras referidas por adolescentes atendidos num serviço de saúde. DOI: 10.5007/1980-0037.2011v13n3p163	Revista Brasileira de Cineantropometria & Desempenho Humano	2,011	cc-by	4,897	Physical activities and barriers reported by adolescents attending a health service Physical activities and barriers reported by adolescents attending a health service 1 1 Leandro Martin Totaro Garcia Mauro Fisberg 1 1 Leandro Martin Totaro Garcia Mauro Fisberg Resumo – O estudo caracterizou a atividade física e as barreiras referidas dos adolescen- tes atendidos pelo Centro de Atendimento e Apoio ao Adolescente (São Paulo/SP), em seu ambulatório de Educação Física. Trata-se de um estudo exploratório, com dados das anamneses de 118 adolescentes com idade de 10 a 19 anos, atendidos entre abril de 2005 e junho de 2008. Analisaram-se, segundo gênero e faixa etária: participação em atividades físicas de lazer e na Educação Física escolar, atividades físicas preferidas e barreiras para a prática da atividade física preferida. Descreveram-se os dados por meio de frequências, comparadas por meio do Teste Exato de Fisher. Relataram gostar de atividades físicas 93,2% dos adolescentes, mas 50,8% não praticavam atividades físicas no lazer. Houve maior nú- mero de relatos de não participação na Educação Física escolar entre os adolescentes mais velhos, em ambos os gêneros. Brincadeiras e jogos coletivos foram as atividades preferidas, independentemente de gênero e idade. Falta de suporte social ou ambiental foi o motivo mais importante para não praticar a atividade física preferida. Os resultados sustentam a importância de um serviço para adolescentes que promova, oriente e suporte um estilo de vida mais ativo. Palavras-chave: Serviços de saúde para adolescentes; Atividade física. Abstract – This study characterized the physical activity and barriers reported by adolescents attending the Physical Education service of the Adolescent Care and Support Center, São Paulo, Brazil. An exploratory study was conducted using anamnesis data from 118 adolescents aged 10 to 19 years seen between April 2005 and June 2008. The following aspects were analyzed according to gender and age group: participation in leisure-time physical activities and physical education classes, physical activity preferences, and barriers to preferred physical activity. Data are reported as frequencies and were compared by Fisher’s exact test. Enjoying physical activities was reported by 93.2% of the adolescents, whereas 50.8% did not perform any physical activity during their leisure time. The lack of participation in school physical education classes predo- minated among older adolescents of both genders. Games and team games were the preferred activities, irrespective of gender or age. The lack of company or friends and the lack of places were the most frequently reported barriers to preferred physical activity. 1 Universidade Federal de São Paulo. Departamento de Pediatria. Especialida- des Pediátricas. Centro de Atendimento e Apoio ao Adolescente. São Paulo, SP, Brasil. ARTIGO original DOI: 10.5007/1980-0037.2011v13n3p163 Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169 PROCEDIMENTOS METODOLÓGICOS Trata-se de um estudo exploratório, realizado por meio de levantamento dos dados contidos nas anamneses dos adolescentes atendidos pelo ambula- tório de Educação Física pertencente ao Centro de Atendimento e Apoio ao Adolescente (CAAA), do Departamento de Pediatria da Universidade Federal de São Paulo, situado na cidade de São Paulo/SP. Apesar disso, constata-se que uma parcela im- portante dos adolescentes brasileiros não pratica ati- vidades físicas regularmente4,5 e que determinantes biológicos, comportamentais, familiares, sociocul- turais, econômicos e ambientais podem contribuir para este fato4,6,7. Por ser um problema também em vários outros países, diversas estratégias, visando à promoção da prática de atividades físicas, já foram implantadas e, em 2002, os Centers for Disease Control and Prevention (CDC), por meio de sua força-tarefa Community Preventive Services, publicou recomendações baseadas em evidências sobre formas de intervenção para elevar os níveis de atividades física de indivíduos e comumidades8. O CAAA é constituído por uma equipe mul- tidisciplinar formada por médicos, nutricionistas, profissionais de educação física, psicólogos, psico- pedagogos, fonoaudiólogos e dentistas que atendem gratuitamente pessoas com idade entre 10 e 19 anos, principalmente, paulistanas. Os adolescentes foram encaminhados ao ambulatório de Educação Física pelos demais profissionais do Centro sempre que, em seus atendimentos, avaliassem ser necessária a orien- tação ou a prescrição de atividades físicas, seja por demanda do paciente ou do profissional que o atendeu. Tendo como base essas recomendações, o Centro de Atendimento e Apoio ao Adolescente (CAAA), pertencente ao Departamento de Pediatria da Uni- versidade Federal de São Paulo, atua em seu ambu- latório de Educação Física promovendo um estilo de vida ativo por meio de orientações, prescrições, acompanhamento e suporte individualizados para a mudança de comportamento, de acordo com as necessidades, dificuldades, oportunidades e gostos de cada adolescente. Investimentos e intervenções que ajudem adolescentes a superar as barreiras e aumentem a sua predisposição para a prática de atividades físicas, como o atendimento ambulatorial em Educação Física do CAAA, são justificáveis e devem ser incentivados9. No entanto, para ser uma intervenção bem-sucedida, é necessário que contenha informações que possibilitem aos profissionais envolvidos elaborar soluções mais bem direcionadas e adequadas à população que atende10. O investigador principal revisou, em julho de 2008, as anamneses de todos os pacientes enca- minhados ao ambulatório, entre abril de 2005 e junho de 2008, período no qual se utilizou o mesmo modelo de anamnese e protocolo de atendimento. Physical activities and barriers reported by adolescents attending a health service The results highlight the importance of a health service program for adolescents that promotes, guides, and supports a more active lifestyle. Recebido em 02/09/10 Revisado em 02/11/10 Aprovado em 03/11/10 Recebido em 02/09/10 Revisado em 02/11/10 Aprovado em 03/11/10 Key words: Adolescent health services; Physical activity. CC Licença: Creative Commom Garcia & Fisberg Atividades físicas em jovens no serviço de saúde INTRODUÇÃO como objetivo identificar as características dos ado- lescentes atendidos pelo ambulatório de Educação Física do CAAA do Departamento de Pediatria da Universidade Federal de São Paulo, quanto à prática de atividades físicas, especificamente com relação à participação em atividades físicas de lazer e nas aulas de Educação Física escolar, atividades fí- sicas preferidas e barreiras percebidas para a prática. Sabe-se que a prática de atividades físicas na ado- lescência é parte integrante e necessária para a prevenção de diversos agravos à saúde, além de ser importante para o pleno desenvolvimento e exercer efeitos psicossociais positivos1. Além disso, a prá- tica regular de atividades físicas nesta faixa etária está relacionada com maior prática desse tipo de atividades durante a idade adulta2,3, tendo grande impacto na saúde individual e coletiva. RESULTADOS Entre abril de 2005 e junho de 2008 (39 meses) chegaram ao ambulatório de Educação Física do CAAA 118 adolescentes, sendo 67 moças (56,8%) e 51 rapazes (43,2%). A média de idade dos jovens foi de 14,6 anos (dp=2,3), variando de 10,1 a 19,0 anos. Nas moças, a média de idade foi de 14,5 anos (dp=2,3); nos rapazes, foi de 13,9 anos (dp=2,3). As respostas para as perguntas, “Qual a sua ati- vidade física preferida?” e “Qual o principal motivo para você não praticar a atividade física preferida?” foram abertas, sendo todas catalogadas, agrupadas por semelhança e cada categoria rotulada, sendo todo o processo realizado pelo investigador principal. As respostas para a segunda pergunta foram categoriza- das em barreiras: situacionais – indisponibilidade de suporte social ou ambiental; de recursos – motivos fi- nanceiros ou falta de materiais; e pessoal – problemas médicos ou indisposição do indivíduo para a prática. Na Tabela 1, estão descritas a prática de ativi- dades físicas e as barreiras referidas dos adolescentes atendidos. Apesar de 93,2% dos adolescentes relata- rem gostar de atividades físicas, aproximadamente, metade (50,8%) não praticava qualquer atividade física no lazer. Somente 35 adolescentes (29,7%) praticavam sua atividade física preferida. As brinca- deiras e jogos coletivos foram as atividades preferidas pelos adolescentes, independentemente do gênero. Com relação às barreiras, tanto para as moças como para os rapazes a falta de suporte social ou ambiental Para análise estatística, foi utilizado o software Statistical Package for the Social Sciences®, versão 15.0 para Windows (SPSS Inc., Chicago, Illinois). As variáveis foram descritas por meio de frequências. Além disso, a análise descritiva da idade também foi realizada por meio de média aritmética e desvio-padrão (dp). R B Ci D h H 2011 13(3) 163 169 165 Tabela 1. Prática de atividades físicas e barreiras referidas entre os adolescentes atendidos pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente. São Paulo/SP, Brasil. 2005-2008. PROCEDIMENTOS METODOLÓGICOS Os atendimentos no ambulatório de Educação Fí- sica foram realizados por profissionais de educação física que estavam cursando ou haviam cursado a Especialização em Adolescência oferecida pelo Centro. Todos foram previamente treinados para o atendimento e atuavam sob supervisão de um profissional especialista mais experiente. Foram coletados os dados referentes ao pri- meiro atendimento realizado pelo ambulatório de Educação Física aos jovens. As variáveis analisadas foram: idade (10 a 14 anos; 15 a 19 anos), gênero (masculino; feminino), participação nas aulas de Educação Física escolar (sim; não), prática de ativi- dade física de lazer (sim; não), gostar de atividades físicas (sim; não), atividade física preferida (brin- cadeiras e jogos coletivos; aulas de dança, lutas ou natação; musculação ou ginástica; outros; não tem preferência/não citou) e principal barreira para pra- ticar a atividade física preferida (situacional; falta Por ser voltado ao aconselhamento e prescrição de atividades físicas, torna-se fundamental conhe- cer o perfil de prática deste tipo de atividades entre os adolescentes que chegam a este tipo de ambula- tório, assim como as dificuldades que encontram para aderir a este hábito. Soma-se a isso, o fato de haver certa escassez de dados na literatura sobre as características dos adolescentes atendidos pelos ser- viços de saúde brasileiros, inclusive sobre a prática de atividades físicas. Sendo assim, este estudo teve 164 de recursos; pessoal; não soube responder). Todas as informações coletadas foram respondidas oral- mente pelo paciente durante o atendimento, exceto a idade, calculada a partir da data de atendimento e de nascimento do adolescente. Para a comparação das frequências entre os gêneros e faixas etárias, foi utilizado o Teste Exato de Fisher, para o qual se adotou nível de significância de 5% (p≤0,05). O estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de São Paulo, sob o protocolo 0639/08, de 30 de maio de 2008 e está de acordo com a Resolução 196/96 do Conselho Nacional de Saúde. A idade foi calculada pelo software Microsoft® Office Excel® 2007 por meio da seguinte fórmula: Idade = (data do atendimento - data de nascimento) / 365. As datas foram apresentadas na forma dia/ mês/ano. Após o cálculo, os adolescentes foram categorizados em duas faixas etárias: de 10,0 a 14,9 anos e de 15,0 a 19,9 anos. Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169 RESULTADOS Variável Total Moças Rapazes n % n % n % p* Participação na Educação Física escolar 118 67 51 0,53 Sim 89 75,4 49 73,1 40 78,4 Não 29 24,6 18 26,9 11 21,6 Prática de atividade física no lazer 118 67 51 0,35 Sim 58 49,2 30 44,8 28 54,9 Não 60 50,8 37 55,2 23 45,1 Gosta de atividade física 118 67 51 0,29 Sim 110 93,2 64 95,5 46 90,2 Não 8 6,8 3 4,5 5 9,8 Atividade física preferida 118 67 51 0,23 Brincadeiras e jogos coletivos 61 51,7 33 49,3 28 54,9 Aulas de dança, lutas ou natação 23 19,5 13 19,4 10 19,6 Musculação ou ginástica 11 9,3 9 13,4 2 3,9 Outros 8 6,8 6 9,0 2 3,9 Não tem preferência/não citou 15 12,7 6 9,0 9 17,6 Principal barreira 83 67 34 0,39 Situacional 37 31,4 20 40,8 17 50,0 Falta de recursos 11 9,3 9 18,4 2 5,9 Pessoal 10 8,5 7 14,3 3 8,8 Não soube responder 25 21,2 13 26,5 12 35,3 * Valor-p do Teste Exato de Fisher entre gêneros. Tabela 1. Prática de atividades físicas e barreiras referidas entre os adolescentes atendidos pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente. São Paulo/SP, Brasil. 2005-2008. Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169 165 Atividades físicas em jovens no serviço de saúde Garcia & Fisberg Garcia & Fisberg não soube responder qual era a principal barreira para a prática de sua atividade física preferida. foi o mais importante motivo para a não praticar a atividade física preferida, seguido da falta de recursos financeiros ou materiais e motivos pessoais. Ressalta- -se, também, que um em cada cinco adolescentes Os dados também foram analisados após os jovens serem estratificados por faixa etária, dentro de cada gê- Tabela 2. Prática de atividades físicas e barreiras percebidas por moças atendidas pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente, segundo faixa etária. São Paulo/SP, Brasil. 2005-2008. RESULTADOS Variável 10 a 14 anos 15 a 19 anos n % n % p* Participação na Educação Física escolar 43 24 <0,001 Sim 40 93,0 9 37,5 Não 3 7,0 15 62,5 Prática de atividade física no lazer 43 24 0,45 Sim 21 48,8 9 37,5 Não 22 51,2 15 62,5 Gosta de atividade física 43 24 0,04 Sim 43 100,0 21 87,5 Não - - 3 12,5 Atividade física preferida 43 24 0,03 Brincadeiras e jogos coletivos 26 60,5 7 29,2 Aulas de dança, lutas ou natação 9 20,9 4 16,7 Musculação ou ginástica 3 7,0 6 25,0 Outros 3 7,0 3 12,5 Não tem preferência/não citou 2 4,7 4 16,7 Principal barreira 29 20 0,36 Situacional 12 41,4 8 40,0 Falta de recursos 3 10,3 6 30,0 Pessoal 5 17,2 2 10,0 Não soube responder 9 31,0 4 20,0 * Valor-p do Teste Exato de Fisher entre faixas etárias. Tabela 3. Prática de atividades físicas e barreiras percebidas por rapazes atendidos pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente segundo faixa etária São Paulo/SP Brasil 2005 2008 Tabela 2. Prática de atividades físicas e barreiras percebidas por moças atendidas pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente, segundo faixa etária. São Paulo/SP, Brasil. 2005-2008. Tabela 3. Prática de atividades físicas e barreiras percebidas por rapazes atendidos pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente, segundo faixa etária. São Paulo/SP, Brasil. 2005-2008. Tabela 3. Prática de atividades físicas e barreiras percebidas por rapazes atendidos pelo ambulatório de Educação Física do Centro de Atendimento e Apoio ao Adolescente, segundo faixa etária. São Paulo/SP, Brasil. 2005-2008. DISCUSSÃO Este estudo analisou dados do primeiro atendimento realizado aos 118 adolescentes que chegaram, entre abril de 2005 e junho de 2008, ao ambulatório de Educação Física inserido no CAAA, com o intuito de obter orientações e suporte para a prática de atividades físicas. Aproximadamente, 50% dos jovens relataram não praticar qualquer atividade física no lazer. Apesar de não ter havido associação com gênero ou faixa etária, percebe-se que as prevalências de prática de atividades físicas no lazer tendem a ser maiores nos rapazes e nos mais novos. Pesquisas vêm demonstran- do que, de fato, maior proporção de rapazes realiza atividades físicas de lazer, quando comparados às moças, o mesmo ocorrendo com os adolescentes mais novos em comparação com os mais velhos, sendo este fenômeno mais acentuado entre as moças20,21. Com relação à participação na Educação Física escolar, um quarto dos jovens referiu não realizar as aulas, sendo que moças e rapazes mais velhos relataram com maior frequência não participar, quando comparados aos mais novos. Resultado semelhante foi observado em estudo realizado com 5.028 escolares catarinenses de 15 a 19 anos, no qual 42,2% não participavam das aulas de Educação Física, sendo que esta proporção também aumen- tava entre os adolescentes mais velhos11. Dados de um levantamento realizado pelo Instituto Brasileiro de Geografia e Estatística com 60.973 adolescentes da 9a série (89% com idade entre 13 e 15 anos) de escolas privadas e públicas das capitais do país, apontam que 20% dos alunos não fizeram aula de Educação Física na semana anterior à pesquisa12. ç Além disso, menos de um terço dos adolescen- tes referiu praticar sua atividade física preferida. Entre aqueles que não a praticavam, as barreiras situacionais foram as mais citadas como sendo o principal motivo para não praticá-la, independente- mente de gênero ou idade. Esta categoria representa a falta de suporte social ou ambiental, principal- mente de companhia de amigos e de locais para a prática. Com relação à companhia para praticar as atividades físicas, diversos estudos suportam a ideia de que há maior envolvimento dos adolescentes quando seus pares também participam22-24. Van Der Horst et al. realizaram uma revisão sobre os fatores relacionados com a prática de atividade física dos adolescentes, na qual se observou que o suporte de amigos está associado a este comportamento23. RESULTADOS 166 p , g , Variável 10 a 14 anos 15 a 19 anos n % n % P* Participação na Educação Física escolar 33 18 0,04 Sim 29 87,9 11 61,1 Não 4 12,1 7 38,9 Prática de atividade física no lazer 33 18 0,77 Sim 19 57,6 9 50,0 Não 14 42,2 9 50,0 Gosta de atividade física 33 18 0,33 Sim 31 93,9 15 83,3 Não 2 6,1 3 16,7 Atividade física preferida 33 18 0,22 Brincadeiras e jogos coletivos 20 60,6 8 44,4 Aulas de dança, lutas ou natação 5 15,2 5 27,8 Musculação ou ginástica - - 2 11,1 Outros 2 6,1 - - Não tem preferência/não citou 6 18,2 3 16,7 Principal barreira 22 12 0,13 Situacional 12 54,5 5 41,7 Falta de recursos - - 2 16,7 Pessoal 1 4,5 2 16,7 Não soube responder 9 40,9 3 25,0 * Valor-p do Teste Exato de Fisher entre faixas etárias. 166 nero (Tabelas 2 e 3). A não participação na Educação Física escolar foi mais prevalente entre os adolescentes de maior idade, em ambos os gêneros. Houve predo- minância das brincadeiras e jogos coletivos como as atividades físicas preferidas, tanto nas moças como nos rapazes. Entre as moças, com o aumento da idade houve redução das que relataram gostar de atividades físicas. Independentemente da faixa etária e do gênero, a principal barreira para a prática da atividade física preferida permaneceu sendo a situacional (indisponi- bilidade de suporte social ou ambiental). Ao comparar as moças e rapazes de mesma faixa etária não foram encontradas diferenças (p≥0,20). Mais de 90% dos adolescentes relataram gostar de praticar atividades físicas. Neste sentido, diver- sos estudos têm apontado que o fato de gostar de atividades físicas atua como importante mediador e preditor na adoção deste comportamento em adolescentes14-16. Entretanto, um número maior de adolescentes mais velhos, principalmente moças, parecem não gostar de praticar atividades físicas quando comparados aos mais novos. Pouco se sabe sobre a associação entre ida- de e o prazer em praticar atividades físicas. Este resultado pode estar associado às diferenças no desenvolvimento puberal que se espera encontrar entre as faixas etárias, já que este fenômeno altera drasticamente a autopercepção e o autoconceito dos adolescentes, podendo haver redução de au- toestima e piora da autoimagem17,18. Ashford et al. argumentam que pouco bem-estar psicológico também está associado com menos prazer com a prática de atividades físicas em jovens19. DISCUSSÃO Wold e Hendry comentam que a influência de amigos pode ocorrer de três formas: os adolescentes se influenciarem mutuamente para iniciarem uma atividade física; um adolescente pode iniciar uma atividade física pelo fato de seu amigo já ser ativo; e a formação de um vínculo de amizade entre Acredita-se que a Educação Física escolar seja um momento importante, às vezes o único no ambiente escolar, para a promoção da prática de atividades físicas13. Gordon-Larsen et al. demons- traram que a maior frequência semanal nessas aulas aumenta a chance dos jovens alcançarem as recomendações de prática de atividades físicas de intensidade moderada a vigorosa suficiente para influenciar positivamente a saúde7. Desta forma, é importante que os profissionais que atuam no am- bulatório identifiquem os motivos individuais para a não participação nas aulas de Educação Física, orientem e ofereçam suporte para o envolvimento na Educação Física escolar como parte da estratégia de adoção da prática de atividades físicas, princi- palmente, entre as moças mais velhas. 167 Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169 Atividades físicas em jovens no serviço de saúde Garcia & Fisberg adolescentes que se envolveram na mesma ativi- dade física24. Duncan et al. referem que o suporte social entre os jovens também ocorre em relação a aspectos emocionais (encorajamento) e instru- mentais (compartilhamento de equipamentos)22. Estas afirmações ganham ainda mais força quando se observa que, de fato, as brincadeiras e jogos coletivos foram citados como as atividades físicas preferidas da maioria dos adolescentes atendidos. específica e sua extrapolação deve ser feita com cautela. Além disso, por tratar-se de um estudo de desenho transversal, não é possível apontar rela- ções de causalidade nas associações encontradas. Por outro lado, este trabalho buscou contribuir com informações sobre a prática de atividades físicas e as barreiras relatadas por adolescentes que buscam os serviços de saúde a fim de obter orientação e suporte individualizados para esta prática, haja vista a escassez de dados deste gênero na literatura. O acesso a locais adequados de prática de ati- vidades físicas foi outra barreira importante para os sujeitos estudados. Uma pesquisa realizada na cidade de Curitiba/PR, com 1.718 adolescentes, encontrou que não praticar atividades físicas em parques estava associada, em ambos os gêneros, à percepção de falta de espaços para ser fisicamente ativo, de atividades para escolher e de equipamentos adequados, após controlar idade, nível socioeconômico, atividade física habitual e distância até o parque referidos pelos jovens25. CONCLUSÕES Em síntese, quanto à prática de atividades físicas dos adolescentes atendidos pelo ambulatório de Educação Física do CAAA, entre abril de 2005 e junho de 2008, aproximadamente, metade não praticava qualquer atividade física de lazer, apesar da maioria afirmar gostar deste tipo de atividade. Estas informações sustentam a importância de um serviço que promova, oriente, suporte e acompanhe um estilo de vida mais ativo dentro de um local que vise à atenção integral à saúde de adolescentes. Além disso, a participação nas aulas de Edu- cação Física escolar foi maior entre as moças e os rapazes de 10 a 14 anos de idade. Devido a sua im- portância como disciplina escolar, a possibilidade de contribuir com o volume semanal de atividades físicas e com informações e estímulos que determi- narão a posição dos jovens com relação à prática de atividades físicas, deve-se incentivar a participação nas aulas de Educação Física, principalmente, entre os adolescentes mais velhos, utilizando-a, inclusive, como estratégia de mudança de atitude quanto este tipo de atividade.i Ainda com relação às barreiras, 21,2% dos adoles- centes não souberam responder o que os impedia de praticar sua atividade física preferida. Isso se deve ao fato de haver não somente fatores externos atuando sobre o comportamento, mas também internos, que podem ser de difícil percepção ou nomeação pelos jovens. Entre eles, a autoeficácia é o mais bem docu- mentado na literatura, sendo forte e positivamente associada à prática de atividades físicas23,28,29. Jovens que se sentem confiantes com relação a sua capaci- dade de serem fisicamente ativos percebem menos barreiras e são menos influenciados por elas14. Outros constructos psicológicos, que podem ser de difícil percepção, também parecem estar associados à prá- tica de atividades físicas, como atitude favorável para a prática e orientação/motivação para uma meta23. É Por fim, a falta de companhia de amigos e de locais para prática parecem ser as barreiras exter- nas mais importantes para a população atendida, sendo necessário, também, melhor acesso a fatores internos que possam mediar a prática de ativida- des físicas. Ter conhecimento das necessidades, dificuldades, oportunidades e gostos dos jovens que buscam este tipo de ajuda deve servir como subsídio para que os profissionais envolvidos neste tipo de serviço desenvolvam materiais, protocolos e estratégias adequados para obter mais sucesso em seus atendimentos. DISCUSSÃO Sabe-se, também, que melhores condições socioeconômicas podem facilitar o acesso às instituições organizadas de prática de atividades físicas, como academias e clubes26, além de possi- bilitar moradia em locais mais seguros e mais bem urbanizados, fatores também relacionados com maior prática de atividades físicas6,7. De fato, grande parte das famílias atendidas pelo CAAA apresenta perfil socioeconômico que está associado a maiores chances de residir em locais menos seguros, mal urbanizados e com falta de espaços gratuitos ou com custos acessíveis para a prática de atividades físicas, organizadas ou não. Essa é uma característica comum entre as pessoas atendidas pelo Sistema Único de Saúde27, ao qual o CAAA está vinculado. REFERÊNCIAS BIBLIOGRÁFICAS 1. Physical Activity Guidelines Advisory Committee. Physical Activity Guidelines Advisory Committee report, 2008. Washington, Estados Unidos: U.S. De- partment of Health and Human Services, 2008. É necessário apontar alguns limites deste estu- do. Primeiramente, as informações coletadas e os resultados apresentados referem-se a uma população É necessário apontar alguns limites deste estu- do. Primeiramente, as informações coletadas e os resultados apresentados referem-se a uma população 168 2. Alves JGB, Montenegro FMU, Oliveira FA, Alves RV. Prática de esportes durante a adolescência e atividade física de lazer na vida adulta. Rev Bras Med Esporte 2005;11(5):291-4. 17. Brooks-Gunn J, Reiter EO. The role of pubertal pro- cesses. In: Feldman SS, Elliott GR, organizadores. At the threshold: the developing adolescent. Cambridge, Estados Unidos: Harvard University Press, 1990. p.16-53. 3. Azevedo MR, Araujo CL, Silva MC, Hallal PC. Tra- cking of physical activity from adolescence to adul- thood: a population-based study. Rev Saude Publica 2007;41(1):69-75. 18. Murdey ID, Cameron N, Biddle SJH, Marshall SJ, Gore- ly T. Pubertal development and sedentary behaviour during adolescence. Ann Hum Biol 2004;31(1):75-86. 19. Ashford B, Biddle S, Goudas M. Participation in community sports centres: motives and predictors of enjoyment. J Sports Sci 1993;11(3):249-56. 4. Hallal PC, Bertoldi AD, Goncalves H, Victora CG. Prevalence of sedentary lifestyle and associated factors in adolescents 10 to 12 years of age. Cad Saude Publica 2006;22(6):1277-87. 20. Bastos JP, Araujo CL, Hallal PC. Prevalence of insuf- ficient physical activity and associated factors in Brazil- ian adolescents. J Phys Act Health 2008;5(6):777-94. 5. Souza GS, Duarte MFS. Estágios de mudança de comportamento relacionados à atividade física em adolescentes. Rev Bras Med Esporte 2005;11(2):104-8. 21. Nelson MC, Neumark-Stzainer D, Hannan PJ, Sirard JR, Story M. Longitudinal and secular trends in physi- cal activity and sedentary behavior during adolescence. Pediatrics 2006;118(6):E1627-34. 6. Bracco MM, Colugnati FAB, Pratt M, Taddei JAAC. Modelo hierárquico multivariado da inatividade física em crianças de escolas públicas. J Pediatr (Rio J) 2006;82(4):302-7. 22. Duncan SC, Duncan TE, Strycker LA. Sources and types of social support in youth physical activity. Health Psychol 2005;24(1):3-10. 7. Gordon-Larsen P, McMurray RG, Popkin BM. Deter- minants of adolescent physical activity and inactivity patterns. Pediatrics 2000;105(6):e83. 23. Van der Horst K, Paw M, Twisk JWR, Van Mechelen W. A brief review on correlates of physical activity and sedentariness in youth. Med Sci Sports Exerc 2007;39(8):1241-50. 8. REFERÊNCIAS BIBLIOGRÁFICAS Kahn EB, Ramsey LT, Brownson RC, Heath GW, Howze EH, Powell KE et al. The effectiveness of in- terventions to increase physical activity - a systematic review. Am J Prev Med 2002;22(4):73-108. 24. Wold B, Hendry L. Social and environmental factors associated with physical activity in young people. In: Biddle S, Sallis J, Cavill N, organizadores. Young and active? Young people and health-enhancing physical activity: evidence and implications. Londres, Inglat- erra: Health Education Authority, 1998. p.119-32. 9. Fernandes R, Junior I, Cardoso J, Ronque EV, Loch M, Oliveira A. Association between regular participation in sports and leisure time behaviors in Brazilian ado- lescents: a cross-sectional study. BMC Public Health 2008;8(1):329. 25. Reis RS, Hino AA, Florindo AA, Añez CR, Domingues MR. Association between physical activity in parks and perceived environment: a study with adolescents. J Phys Act Health 2009;6(4):503-9. 10. World Health Organization. Evaluation in health promotion: principles and perspectives. Copenhagen, Dinamarca: WHO Regional Office for Europe, 2001. 11. Silva KS, Nahas MV, Peres KG, Lopes AS. Fatores as- sociados à atividade física, comportamento sedentário e participação na educação física em estudantes do ensino médio em Santa Catarina, Brasil. Cad Saude Publica 2009;25(10):2187-200. 26. Oehlschlaeger MHK, Pinheiro RT, Horta B, Gelatti C, San’Tana P. Prevalência e fatores associados ao sedentarismo em adolescentes de área urbana. Rev Saude Publica 2004;38(2):157-63. 27. Ribeiro MCSA, Barata RB, Almeida MF, Silva ZP. Per- fil sociodemográfico e padrão de utilização de serviços de saúde para usuários e não-usuários do SUS - PNAD 2003. Cien Saude Colet 2006;11(4):1011-22. 12. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar 2009. Rio de Janeiro: Instituto Brasileiro de Geografia e Estatística, 2009. 13. Active Living Research. Active education: physical education, physical activity and academic performance. Princeton (EUA), 2007. Disponível em: <http://www. activelivingresearch.org/alr/alr/files/Active_Ed.pdf> [2010 mai 16]. 28. Dishman RK, Motl RW, Sallis JF, Dunn AL, Birnbaum AS, Welk GJ et al. Self-management strategies medi- ate self-efficacy and physical activity. Am J Prev Med 2005;29(1):10-8. 29. Sallis JF, Prochaska JJ, Taylor WC. A review of cor- relates of physical activity of children and adolescents. Med Sci Sports Exerc 2000;32(5):963-75. 14. Dishman RK, Motl RW, Saunders R, Felton G, Ward DS, Dowda M et al. Enjoyment mediates effects of a school-based physical-activity intervention. Med Sci Sports Exerc 2005;37(3):478-87. 15. Paxton RJ, Estabrooks PA, Dzewaltowski D. Endereço para correspondência Leandro Martin Totaro Garcia. Rua Belchior de Azevedo, 222, bloco 2, ap. 34. Vila Leopoldina. São Paulo, SP. CEP: 05089-030. Email: leandromtg@gmail.com Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169 REFERÊNCIAS BIBLIOGRÁFICAS Attraction to physical activity mediates the relationship between perceived competence and physical activity in youth. Res Q Exerc Sport 2004;75(1):107-11. 16. Wenthe PJ, Janz KF, Levy SM. Gender similarities and differences in factors associated with adolescent moderate-vigorous physical activity. Pediatr Exerc Sci 2009;21(3):291-304. Vila Leopoldina. São Paulo, SP. CEP: 05089-030. Email: leandromtg@gmail.com 169 Rev Bras Cineantropom Desempenho Hum 2011, 13(3):163-169
https://openalex.org/W4366346888	https://www.researchsquare.com/article/rs-2632946/latest.pdf	English	null	Estimating the crashworthiness performances of Collision Boxes Using ANN	Research Square (Research Square)	2,023	cc-by	7,494	Estimating the crashworthiness performances of Collision Boxes Using ANN Ömer ADANUR (  oadanur@subu.edu.tr ) sakarya University of Applied Sciences https://orcid.org/0000-0001-5591-9661 Research Article Keywords: Arti¦cial neural network, Crashbox, Thin-walled structures, Crashworthiness, Energy absorption Posted Date: April 18th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2632946/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Artificial neural network, Crashbox, Thin-walled structures, Crashworthiness, Energy absorption Keywords: Artificial neural network, Crashbox, Thin-walled structures, Crashworthiness, Energy absorption Abstract: Studies on the development of energy absorbing systems that minimize vehicle chassis damage in traffic accidents are increasing day by day. Many designs have been made in the studies on crash boxes used to absorb the energy released in the event of an accident. The specific energy absorption/ energy/FP crashworthiness values used to determine the efficiency of the designed crash boxes in this study were estimated using the Artifical Neural Network (ANN). The input layer of the ANN model consists of materials (DP600, DP800 and DP1000), thickness of materials (between 0.8 and 2.2 mm) and initial speed (10m/s, 12m/s and 16 m/s). In the ANN model, 42 different models were created by changing the different training function (training, trainlm and trainrp), transfer function (tansig and logsig) and the number of neurons in the hidden layer (between 9 and 33). R2 and RSME methods were used to evaluate the efficiency of ANN models. The training function was found to be highly accurate (R2: 0.99999 and RSME: 0.314727E-05) when the training function was “Trainlm” and the number of neurons in the hidden layer was 33. The training and testing results of the ANN model show that ANN can be used to estimate the specific energy absorption/energy/PCH value of crash boxes. 1 Introduction Many components in automobiles are classified as energy absorbers in the event of a crash. The two main energy absorbing structures in automobiles are the bumper and crash box structures. The purpose of these crash boxes is to prevent the transfer of the energy of the collision impact to the passengers in the vehicle by absorbing the kinetic energy of the crash collisions, which occur as very large energy in a very short time, as much as possible. In other words, it is desired that during an accident, the negative acceleration of the vehicle is ensured within safe limits and the kinetic energy is absorbed by plastic and elastic deformation. It is desirable that the crash boxes designed for this purpose are deformed in the event of an accident. If the impact energy cannot be absorbed sufficiently, the passengers may be exposed to intense energy, resulting in undesirable situations such as injury and death. The design of crash boxes is therefore an ongoing topic. As a crash box, steel and aluminum are the most commonly used materials. In the case of aluminum use, approximately 25% of the weight can be saved. Therefore, aluminum material is used as an alternative to steel materials in crash box design. In crash box design, it is important to reduce the impact energy and force to levels that the human body can withstand. The human body can withstand a load of approximately 50 kN as impact resistance [1] . There are different studies carried out to absorb the energy generated at the time of the accident. Some of these studies aim to increase the energy absorption capacity by producing different geometries with design changes in crash boxes and bumpers, allowing them to deform more during crashes[2] while some studies aim to increase the energy absorption capacity of the material used [3], and to increase the energy absorption performance by applying metal foam inside the crash boxes. Nia et al. investigated the energy absorption capacity of crash boxes with different cross-section geometries both experimentally and by finite element method. According to the results obtained, the highest energy absorption capacity was realized in the cylindrical design, and the energy absorption rates decreased as the number of vertices decreased for polygonal structures. 1 Introduction It has been stated that increasing the sheet thickness from 1 mm to 1.5 mm did not make any difference in energy absorption, maximum and average forces [4]. Altın et al. evaluated the effect of profile thickness, apex angle, foam density and foam diameter/height of the crash box used. Mranwhile, the energy absorption capacity of empty and aluminum foam filled tubes was tested under quasi-static test conditions. It has been found that the lateral form was more efficient than the axial form in terms of both energy absorption capacity and impact force effectiveness in filling the crash box with foam [5]. In another study conducted by Sohn et al. to absorb the energy that occurs during crashes in vehicles, Hydroformed (shaped by liquid pressure) buffers were used and the collision energy was tried to be absorbed linearly. According to the results obtained, it was reported that good crashworthiness was obtained [6]. Lin Yuan et al. designed and analyzed a series of origami crash boxes with square, rectangular and polygonal cross-sections with a conical shape [7]. Quasi-static crushing responses of polystyrene foam filled Al tubes were investigated using experimental and numerical tests by L. Aktay et al., and it was found that there was a 50% increase in the sea values of the tubes [8]. E. Acar investigated the effect of axisymmetric indentations on impact performance on conical thin-walled pipes and designed multi-cell thin-walled structures with different concepts and carried out optimization studies [9]. Xu provided a parametric analysis and multi-objective optimization of circular columns with functionally graded thickness (FGT) [10]. Yang researched and formulated the multi-objective optimization design (MOD) of empty and foam-filled thin-walled square columns under oblique impact with both no variation in load angle and variation [11]. Tarlochan conducted a numerical investigation of the axial and oblique crush responses of a thin-walled, ductile metallic alloy (mild steel A36) with various cross-section profiles [12]. Zhang and Zhang investigated the crushworthiness of corner elements with various thickness configurations produced on a wire electro-erosion machine (WEDM) of square specimens and conducted experimental work, numerical simulations and theoretical analysis to estimate the axial crush resistance [13]. 1 Introduction Costas compared the frontal crashworthiness capabilities of carbon-fiber reinforced polymers, glass-fiber reinforced polyamide, polyethylene terephthalate foam and cork conglomerates in combination with cold- formed steel polygonal tubes using quasi-static and dynamic numerical simulations, and found that the best result was obtained in the combination of glass fiber reinforced polyamide padding and steel box [14]. Gedikli compared the crushworthiness performance of TWTs subjected to axial impact load with monolithic tubes, and investigated the effects of tube thickness and tube length on the dynamic behavior of tubes [15]. The impact behavior of multi-cell tubes was extensively studied by Fang under both axial and oblique loads, and it was found that energy absorption (EA) and peak force (Fmax) increased in direct proportion to the number of cells [16]. Yin proposed a new type of energy-absorbing structure named as bionic thin-walled structure (BTS) based on the structural characteristics of the horsetail. Next, the bending behavior of six types of BTSs with different cross-section configurations was studied. Numerical results showed that cell number, inner wall diameter and wall thickness of BTSs had a remarkable effect on their crushworthiness [17]. Wei proposed a new scheme for modeling and predicting the processes of frontal vehicle-barrier crashes. It defined a piecewise model structure to represent the vehicle's accelerations and showed good agreement with FE simulations in three different cases. A piecewise model structure was defined to represent the accelerations of the vehicle and good agreement was obtained with the finite element (FE) simulations in three different cases [18]. Similarly, Marzbanrad conducted the design and analysis of the front bumper beam and compared the most important parameters, including material, thickness, shape and impact condition, to improve the crash-resistance design of the car in a low-speed impact [19]. In this study, a representative model of thin-walled structures that provide energy absorption by folding from certain parts due to trigger mechanisms was designed. Then, using the finite element method (FEM), the expilit dynamic crash scenario was simulated with different thickness and material properties. Crashworthiness properties were obtained from the performance indicator values obtained from the simulation results of the models. ANN model was created using the data set obtained from the simulations and specific energy absorption/ energy/ FP values were predicted. ANN models with three neurons in the input layer (material, thickness of material and initial speed), one hidden layer and one neuron in the output layer were created. 1 Introduction The prediction accuracy of the ANN models was statistically compared, and the usability of specific energy absorption/ energy/FP value in estimation in crash boxes of ANN is discussed. Thus, a faster alternative model is proposed for the optimization of the designed crash box. 2.1 Calculating of crashworthiness performance 𝐶𝐸= 𝐹𝑀 𝐹𝑃 (5) 𝐶𝐸= 𝐹𝑀 𝐹𝑃 (5) Used as a way to determine the risk of head and neck injury, this indicator can also be used to see how much force will be transmitted to other materials [22]. Used as a way to determine the risk of head and neck injury, this indicator can also be used to see how much force will be transmitted to other materials [22]. CE is inversely proportional to the size of the peak in the stress-strain plot. Larger peaks offer the least protection by reducing the CE to 0, while smaller peaks increase the CE to a maximum of 1 to offer the most protection [23]. Fig. 1. In the force-displacement curve crashworthiness indicators. Fig. 1. In the force-displacement curve crashworthiness indicators. 2.1 Calculating of crashworthiness performance Crashworthiness indicators such as energy absorption total (ET), specific energy absorption (SEA), peak force (FP), mean crushing force (FM) and crush efficiency (CE) are widely used to calculate the crush performance of thin-walled structures. The energy absorption total (ET) can be calculated by integrating the force (F) with respect to displacement (d) as follows: (1) ET = ∫𝐹(𝑥)𝑑𝑥 𝑑 0 Furthermore, the total energy absorbed (ET) can be determined by means of the software (LS-DYNA etc.) by loading the force-displacement curve. The region in under the force-displacement curve divided by the (y) line as shown in Fig. 1 is the total energy absorption region of the structure. Furthermore, the end of this region is the beginning of region where the force is transferred directly to the opposite side [20]. Specific energy absorbtion (SEA) is an important indicator for comparing the energy absorption capacity of thin-walled structures. In order to increase the effectiveness of energy absorbers, it is important to have a high specific energy absorption (SEA), which converts crush energy per unit mass into strain energy through deformation. The SEA value is determined by the ratio of the total energy absorbed (ET) to mass (M) as follows [21]: (2) Peak force (FP) is the peak force point carried out to the structure that absorbs energy along the distance (d) as in the Eq. 3 An excessive FP value, which causes shock waves to be transferred to the opposite side, negatively affects energy absorption. The thin-walled structure with frictional system presented in this study can be attained to decreasing this effect. FP = 0 ≤ ma𝑥 F(x) ≤ d (3) (3) FP = 0 ≤ ma𝑥 F(x) ≤ d Mean crushing force (FM) is the ratio of the total energy absorbed (ET) to the distance (d). FM which is the average of the force-displacement curve can be calculated as follows: Mean crushing force (FM) is the ratio of the total energy absorbed (ET) to the distance (d). FM which is the average of the force-displacement curve can be calculated as follows: FM = 1 𝑑 ∫𝐹(𝑥)𝑑𝑥 𝑑 0 (4) FM = 1 𝑑 ∫𝐹(𝑥)𝑑𝑥 𝑑 0 FM = 1 𝑑 ∫𝐹(𝑥)𝑑𝑥 𝑑 0 (4) The crush efficiency (CE) parameter is defined as the ratio of the main crush force (FM) to the peak force (FP) (Fig.1). 2.2 Material Properties Vehicle collision process often contain a high degree of deformation. DP-steel high strength steels (HSS) are frequently preferred in automotive applications owing to energy absorbing capability to decrease damage in accidents. Compared to conventional steels, due to the superior combination of structural components such as ductility and strength are particularly suitable for thin-walled structures. DP 600 and DP 800 material properties (Fig. 2.) are used in this study as they are effective in the dynamic impact performance of automotive parts. Furthermore, density (𝜌), poisson ratio (𝜈), young's Modulus (E), yield stress (𝜎𝑦) and ultimate stress (𝜎u) of DP steels are as in the Table 1. Table 1 Mechanical properties of DP Steels utilized in the simulations [24, 25]. Material 𝜌 (kg/mm3) 𝜈 E (GPa) 𝜎𝑦 (MPa) 𝜎u(MPa) DP 600 DP 800 DP 1000 7.8×10-6 7.8×10-6 7.8×10-6 0.3 0.3 0.3 202 206 206 380 460 680 635 860 1034 Table 1 Mechanical properties of DP Steels utilized in the simulations [24, 25]. 2.3 Model design of thin-walled structures Unlike conventional thin-walled structures previously modeled, a system that absorbs energy by frictional force is considered. In order to determine the function of frictional force in energy absorption, two configurations were developed, one classically and the other absorbing energy by generating friction force, as shown in Fig. 3. The configuration absorbing energy by friction force is operated with the principle of interpenetrating of multi-parts when an axial impact load is enforced. During the collapse of the structure, friction force is generated by the resistance applied by the crush beads on the walls of the parts to each other. In order to increase the resistance of the friction force on the walls of the finite element models, the crush beads are positioned in opposite directions. In addition, the crush beads lead to progressive collapse of foldings. The length and width of these thin-walled structures are designed with appropriate tolerances and dimensions in terms of the standard manufacturing process. Additionally in the all analyses remains constant at 235 mm and 110 mm, respectively (Fig. 2). Fig. 2 Dimensions of thin-walled structure subjected to axial impact Fig. 2 Dimensions of thin-walled structure subjected to axial impact 2.4.1 FEA Surrogate models In this study, combinations of surrogate models with friction and without friction using mechanical properties of Dp600-DP800 steel materials with different wall thicknesses are analysed to measure the effect of friction force on energy absorption. The numbering of the surrogate models for managing analyzes is as follows: Code D10T1V10, parametrizes to a surragote model with a thickness of 1 mm, using DP1000 steel material properties. In addition, the code D8MT1.2 represent to a surragote model with a thickness of 1.2 mm, manufactured from multi parts, using the DP800 steel material properties. 2.4.2 Finite element simulations In order to investigate the effect of friction force on of thin-walled structures, finite element models are designed to utilize in simulations. The non-linear Explicit dynamic crushing simulations of models are performed in accordance with collision standards. Finite element modeling and boundary conditions of interpenetrating thin-walled structure is illustrated in Fig. 4. In order to avoid deformation, “MAT_RIGIT_(TITLE)(020)” code was used in the modeling of Dynamic Rigid Wall and Fixed Rigid Wall. In addition, thin-walled tubes were modeled with the code ‘‘MAT_PIECEWISE_LINEAR_PLASTICITY_ (TITLE)(024)’'. The model was widely used in crush simulations, exhibiting linear elastic-plastic behavior because of strain rate sensitivity and flexibility. Approach of linear plasticity requires the least amount of material model development because of stress- strain curves are interpolated linearly. The interpenetrating tubes are modellened The Belytschko-Lin-Tsay to reduced mathematical integration of shell elements compared to other method [26]. code Three different types of definitions were employed for contact algorithms. algorithm contact “Automatic node to surface” between thin-walled tubes and rigid walls is adopted. The contact algorithm ''Automatic single surface'' is utilizied to describe the contact within self-walls of the tubes. In addition, the "Automatic surface to surface" contact algorithm was characterized in order to prevent boundary infractions on the interfaces during to generate friction force by the interpenetrating of parts. The static and dynamic coefficients of friction in contacts were accepted to be 0.3 and 0.2, respectively [9]. The shell element size of 2 mm2 was optimise for modeling thin-walled tubes as Fig. 3. Fig. 3 3d modeling of crashbox with Finite Element Method Fig. 3 3d modeling of crashbox with Finite Element Method 2.5 ANN Model architecture selection Artificial neural network (ANN) is an information processing technology inspired by the information processing system of the human brain. ANN is a digital modeling of neuron cells like a simple nervous system and the syniptic link structure established between these cells [27]. Information is processed within neurons and transmitted to the other neuron by connection weights. Thus, the resulting network has the ability to learn, memorize and transform the relationship between data into a mathematical model. ANN has the ability to solve complex problems quickly and efficiently by distributing information over neurons and performing parallel processing. Connections between neurons transmit signals, and each connection has a weight value. To obtain the network output of each neuron, it is assumed that the neuron obtains its network input by passing it through an activation function [28]. Although ANN is an algorithm based on training and learning, it is an innovative method used in many fields such as computer science, medicine and control systems [29]. In the ANN model, information is given externally to the input neurons determined according to the nature of the problem. These inputs are multiplied by the weights determined by the algorithm and summed. Then they were passed through the transfer function and transferred to the hidden layer. By repeating the same operations, the data is propagated to the output layer (Fig. 4.) [29]. The general structure of the ANN is given in Figure 4.a, and the input, output and hidden layer structure adapted to the study are given in Figure 4b. Fig. 4 ANN basic elements and the input/output layer of the model. Fig. 4 ANN basic elements and the input/output layer of the model. Fig. 4 ANN basic elements and the input/output layer of the model. ANN network structure includes five basic elements: input values (Xn), weights (Wn), bias (b), summation function (Σ), activation function (f(Σ)) and output (y). The learning ability of an artificial neural depends on the appropriate adjustment of weights within the selected algorithm. The output of neuron output is calculated by equation Eq. (1): (6) 𝑦𝑗= 𝑓(∑𝑊𝑗,𝑖+ 𝑏𝑗 𝑛 𝑖=1 ) (6) The advantages of ANN are its ability to model nonlinear relationships, its ability to learn and generalize, its adaptability to different problems, its ability to produce accurate outputs even if variable interactions are not fully understood, and its fault tolerance. 2.5 ANN Model architecture selection One of the practical features of ANN is that it allows linear or nonlinear modeling of ANN due to the preferred activation function for artificial nerve cells that make up ANN. It also reveals hidden relationships in the data structure using ANN input data [30]. The architecture of the ANN model affects the information flow of the model, its general structure, and thus its predictive ability [31]. Feedforward neural networks, which is a multi-layer perceptron model, is the most widely used ANN architecture, where information begins to propagate from the input layer to the network and moves in only one direction towards the output layer [32]. The reason why the multi-layer perceptron model is widely used is that it gives good results in modeling nonlinear systems [33]. This architecture consists of three layers: input layer, hidden layer and output layer [34]. The number of neurons in the input and output layers depends on the nature of the problem. In this study, the input layer has three neurons (material type, material thickness, and initial speed), and the output layer has one neuron (specific energy absorption-SEA). The predictive ability of the ANN is directly affected by the number of hidden layers and the number of neurons in these layers. However, the number of hidden layers and the number of neurons in these layers are determined by trial and error [35]. The ANN model used in this study had feedforward/ multi-layer perceptron characteristic, mean square error (MSE) was used as error criteria, and “tansig” was used as output layer transfer function. For the optimization of the ANN model, the hidden layer and number of neoron in the hidden layer, transfer function in the hidden layer and training function were changed. The algorithms and functions used in the ANN model are given in Table 2. 2.5 ANN Model architecture selection Table 2 ANN Modelde kullanılan algoritma/fonksiyonların tanımları Algorithm/Function Description Train Algorithm Trainlm (LM) Levenberg - Marquardt Trainrp (RP) Resilient Back propagation Traingda (GDA) Gradient descent with adaptive learning rate backpropagation Transfer function Tansig Hyperbolic tangent sigmoid transfer function Purelin Linear Transfer Function Table 2 ANN Modelde kullanılan algoritma/fonksiyonların tanımları Algorithm/Function Description Train Algorithm Trainlm (LM) Levenberg - Marquardt Trainrp (RP) Resilient Back propagation Traingda (GDA) Gradient descent with adaptive learning rate backpropagation Transfer function Tansig Hyperbolic tangent sigmoid transfer function Purelin Linear Transfer Function Table 2 ANN Modelde kullanılan algoritma/fonksiyonların tanımları Logsig Log-Sigmoid Function As a result of the crashbox tests conducted using SEM, the SAE, Energy (Kj) and Pick Crush Force (Kn) values required for the ANN model were obtained. The material, material thickness, and initial speed were used in the input layer developed using 33 data sets, and the specific energy absorption (SEA) value was used in the output layer. Firstly, the prediction ability of the SAE value of the ANN model developed was tested. Then, the ability to predict the Energy (Kj) and Pick Crush Force (Kn) values of the same model was examined. ANN models were evaluated statistically. R-square (R2) and root mean square error (RMSE) were used for evaluation (Eq. 7-8) [36]. 𝑅2 = 1 − ∑ (𝑘exp(𝑖)−𝑘𝐴𝑁𝑁(𝑖)) 2 𝑁 𝑖=1 ∑ (𝑘exp(𝑖)) 2 𝑁 𝑖=1 (7) RMSE = √1 𝑁− ∑ \|𝑘exp(𝑖) −𝑘𝐴𝑁𝑁(𝑖)\| 2 𝑁 𝑖=1 (8) 𝑅2 = 1 − ∑ (𝑘exp(𝑖)−𝑘𝐴𝑁𝑁(𝑖)) 2 𝑁 𝑖=1 ∑ (𝑘exp(𝑖)) 2 𝑁 𝑖=1 RMSE = √1 𝑁− ∑ \|𝑘exp(𝑖) −𝑘𝐴𝑁𝑁(𝑖)\| 2 𝑁 𝑖=1 𝑅2 = 1 − ∑ (𝑘exp(𝑖)−𝑘𝐴𝑁𝑁(𝑖)) 2 𝑁 𝑖=1 ∑ (𝑘exp(𝑖)) 2 𝑁 𝑖=1 RMSE = √1 𝑁− ∑ \|𝑘exp(𝑖) −𝑘𝐴𝑁𝑁(𝑖)\| 2 𝑁 𝑖=1 𝑅2 = 1 − ∑ (𝑘exp(𝑖)−𝑘𝐴𝑁𝑁(𝑖)) 2 𝑁 𝑖=1 ∑ (𝑘exp(𝑖)) 2 𝑁 𝑖=1 RMSE = √1 𝑁− ∑ \|𝑘exp(𝑖) −𝑘𝐴𝑁𝑁(𝑖)\| 2 𝑁 𝑖=1 (7) (8) 3 Results And Discussion In the study, dynamic crash tests were carried out using FEM in different materials, thickness and velocity values. Specific energy absorption (SEA), Pick crush force (Kn) and energy (Kj) values were calculated according to the FEM results. The data set prepared as a result of the FEM analysis was used to train and test the ANN model with three neurons (material, thickness, and initial speed) in the input layer and a single neuron (SEA) model in the output layer. In the ANN model, the training algorithm, the number of neurons in the hidden layer and the transfer function were changed. Training results were evaluated using R2 and RSME. 3.1 FEM results of crasbox The analysis results of the created models were found using the FEM method. Simulations were carried out at different material, initial velocity and thickness values. It was determined that all models that underwent plastic deformation reached the maximum energy absorption capacity along the 190 mm deformation distance (d). The most efficient representative model was obtained as a result of the optimization study made by considering the performance indicator data in the deformation process. It should be noted that the model with low FP performance indicator values along with high SEA and CE values exhibits the most efficient crashworthiness characteristics. It is understood from Table 3 that the model with code D10T1.0V10 shows the best performance with 13,771 kJ/kg, 118,300 kN and 0.476% of SEA, FP and CE indicator values, respectively. Figure 5 shows instantaneous force-displacement and plastic deformation behavior graphs of the model coded D10T1.0V10. Points a, b, c, and d marked on the force-deformation curve show the instantaneous deformation of the model. It has been determined that the first fold starts from the near part of the model, followed by successive folds stacked on the first fold, and stable buckling lobes are formed inwards at the edges and outwards at the corners. It is seen that an efficient energy absorption occurs due to the gradual collapsing behavior during dynamic crushing. The folds caused by the impact contribute to the absorption of negative energy and shock waves caused by the crash. The protrusions on the side surfaces of the thin-walled structure help to increase energy absorption by triggering folding. Fig. 5 Gradual collapse of the classically energy-absorbing model code D6ST1.5 under axial impact loading conditions. Fig. 5 Gradual collapse of the classically energy-absorbing model code D6ST1.5 under axial impact loading conditions. Table 3 Crashworthiness performance values of axial impact loaded models. 3.1 FEM results of crasbox SEA (kJ/kg) ET (kJ) FP (kN) FM (kN) CE (%) M (kg) d (mm) D6T0.8V10 D6T1.0V10 D6T1.1V10 D6T1.2V12 D6T1.3V12 D6T1.4V12 D6T1.5V12 D6T1.6V12 D6T1.8V12 D6T2.0V16 D6T2.2V16 D8T0.8V10 D8T1.0V10 D8T1.1V10 D8T1.2V12 D8T1.3V12 D8T1.4V12 D8T1.5V12 D8T1.6V12 D8T1.8V12 D8T2.0V16 D8T2.2V16 D10T0.8V10 D10T1.0V10 D10T1.1V10 D10T1.2V12 D10T1.3V12 D10T1.4V12 D10T1.5V12 D10T1.6V12 D10T1.8V12 D10T2.0V16 D10T2.2V16 5,968 7,282 7,286 8,409 8,361 8,954 9,231 9,435 9,640 10,645 11,059 7,113 8,038 9,287 9,903 10,597 11,101 11,282 11,532 12,374 12,903 14,000 10,635 13,771 12,882 13,220 13,871 13,107 13,350 14,222 15,694 15,839 17,729 3,700 5,680 6,193 7,820 8,361 9,760 10,800 11,700 13,400 16,500 18,800 4,410 6,270 7,894 9,210 10,597 12,100 13,200 14,300 17,200 20,000 23,800 6,594 10,741 10,950 12,295 13,871 14,287 15,620 17,635 21,814 24,551 30,140 51,000 70,600 77,710 86,400 94,129 104,000 115,000 126,000 146,000 177,000 199,000 64,000 83,500 95,168 107,000 117,584 130,000 142,000 156,000 183,000 220,000 239,000 89,780 118,095 134,106 149,569 164,269 183,818 200,210 220,354 252,228 291,336 330,109 40.942 51.099 56.021 48.220 63.351 69.110 44.136 61.047 70.613 57.016 73.534 40.942 51.099 56.021 48.220 63.351 69.110 44.136 61.047 70.613 57.016 73.534 40.942 51.099 56.021 48.220 63.351 69.110 44.136 61.047 70.613 57.016 73.534 19,372 29,738 32,424 40,942 43,775 51,099 56,545 61,257 70,157 86,387 98,429 23,089 32,827 41,330 48,220 55,482 63,351 69,110 74,869 90,052 104,712 124,607 34,524 56,236 57,330 64,372 72,623 74,801 81,780 92,330 114,209 128,539 157,801 0,380 0,421 0,417 0,474 0,465 0,491 0,492 0,486 0,481 0,488 0,495 0,361 0,393 0,434 0,451 0,472 0,487 0,487 0,480 0,492 0,476 0,521 0,385 0,476 0,427 0,430 0,442 0,407 0,408 0,419 0,453 0,441 0,478 0,620 0,780 0,850 0,930 1,000 1,090 1,170 1,240 1,390 1,550 1,700 0,620 0,780 0,850 0,930 1,000 1,090 1,170 1,240 1,390 1,550 1,700 0,620 0,780 0,850 0,930 1,000 1,090 1,170 1,240 1,390 1,550 1,700 3.2 Optimization of ANN Model The optimization of the ANN model created for the prediction of the SEA value in the dynamic crash test was found by trial and error method. In the study, 42 different ANN models were created using trainlm, (Levenberg-Marquardt), trainrp (resilient back propagation) and traingda (gradient descent with adaptive learning rate backpropagation) training algorithms, tansig, logsig and purelin transfer functions, and the number of neurons between 9 and 33 in the hidden layer. The characteristics of the created ANN models are given in Table 4. Table 4 Characteristics of the ANN model. 3.2 Optimization of ANN Model No Noron number in HL Training Function Transfer Function (HL) Transfer Function (OL) Performance R2 RMSE 1 9 Traingda Tansig Purelin 0.994626 0.009051547 2 9 Trainlm Tansig Purelin 0.99999 3.14526E-05 3 9 Trainrp Tansig Purelin 0.999068 0.003480069 4 9 Traingda Logsig Purelin 0.991248 0.010638659 5 9 Trainlm Logsig Purelin 0.999992 0.000331619 6 9 Trainrp Logsig Purelin 0.999364 0.002876098 7 13 Traingda Tansig Purelin 0.992555 0.009803145 8 13 Trainlm Tansig Purelin 0.9999 3.05072E-05 9 13 Trainrp Tansig Purelin 0.998938 0.003714363 10 13 Traingda Logsig Purelin 0.935873 0.027825538 11 13 Trainlm Logsig Purelin 0.999999 0.000104172 12 13 Trainrp Logsig Purelin 0.998987 0.003629192 13 17 Traingda Tansig Purelin 0.988429 0.012211178 14 17 Trainlm Tansig Purelin 0.9999 3.15064E-05 15 17 Trainrp Tansig Purelin 0.999525 0.002486019 16 17 Traingda Logsig Purelin 0.987278 0.01271354 17 17 Trainlm Logsig Purelin 0.99999 2.71671E-05 18 17 Trainrp Logsig Purelin 0.998535 0.00436276 19 21 Traingda Tansig Purelin 0.988079 0.01234052 20 21 Trainlm Tansig Purelin 0.999999 3.14071E-05 21 21 Trainrp Tansig Purelin 0.999335 0.002941349 22 21 Traingda Logsig Purelin 0.99586 0.007294165 23 21 Trainlm Logsig Purelin 0.999999 3.15259E-05 24 21 Trainrp Logsig Purelin 0.999385 0.002827397 25 25 Traingda Tansig Purelin 0.989578 0.011666764 26 25 Trainlm Tansig Purelin 0.999999 3.14122E-05 27 25 Trainrp Tansig Purelin 0.9994 0.002793099 28 25 Traingda Logsig Purelin 0.994167 0.008713765 29 25 Trainlm Logsig Purelin 0.994167 0.008713765 30 25 Trainrp Logsig Purelin 0.999263 0.003095465 31 29 Traingda Tansig Purelin 0.97043 0.019552122 32 29 Trainlm Tansig Purelin 0.999999 3.13068E-05 33 29 Trainrp Tansig Purelin 0.999176 0.003273435 34 29 Traingda Logsig Purelin 0.984266 0.014161505 35 29 Trainlm Logsig Purelin 0.99999 3.13291E-05 36 29 Trainrp Logsig Purelin 0.99932 0.002974011 37 33 Traingda Tansig Purelin 0.992671 0.009749605 38 33 Trainlm Tansig Purelin 0.99999 0.314727E-05 39 33 Trainrp Tansig Purelin 0.999599 0.002283573 40 33 Traingda Logsig Purelin 0.99932 0.002974011 41 33 Trainlm Logsig Purelin 0.99999 3.10644E-05 42 33 Trainrp Logsig Purelin 0.999189 0.003246884 Statistical evaluation of ANN models according to their different characteristics is given in Fig. 7. Fig. 7. (a) shows the change in R2, and (b) shows the change in the RMSE values. In determining the most appropriate ANN model for the solution of the problem, it is desired that the RMSE value is close to zero and the R2 value is close to 1 (and R2 <1) [37]. The Trainingda training algorithm performed poorly according to the training results. 3.2 Optimization of ANN Model The models with the lowest performance for this training algorithm were models 10 and 31. Trainlm had the best prediction performance among training algorithms. The results of the Trainlm training algorithm were found to be close to each other, which indicates that it is not affected by the transfer function or number of neurons in the hidden layer. Statistical evaluation of ANN models according to their different characteristics is given in Fig. 7. Fig. 7. (a) shows the change in R2, and (b) shows the change in the RMSE values. In determining the most appropriate ANN model for the solution of the problem, it is desired that the RMSE value is close to zero and the R2 value is close to 1 (and R2 <1) [37]. The Trainingda training algorithm performed poorly according to the training results. The models with the lowest performance for this training algorithm were models 10 and 31. Trainlm had the best prediction performance among training algorithms. The results of the Trainlm training algorithm were found to be close to each other, which indicates that it is not affected by the transfer function or number of neurons in the hidden layer. In all three training algorithms, the best results were obtained when the number of neurons in the hidden layer was 33. It is known that with the increase in the number of neurons, more effective learning occurs between the input and output values up to a certain threshold [38]. In this study, a nonlinear relationship was observed between the increase in the number of neurons and the prediction rate. According to the best performance evaluation, the optimum values of ANN models are given in Table 5. Fig. 6 Variation of RMSE (a) and R2 (b) at ANN Models for training data. Table 5 The characteristics of the best ANN model for training algorithm. Characteristics Trainlm Traingda Traingrp mber of hidden layer 1 1 1 Fig. 6 Variation of RMSE (a) and R2 (b) at ANN Models for training data. Table 5 The characteristics of the best ANN model for training algorithm. 3.2 Optimization of ANN Model Characteristics Trainlm Traingda Traingrp Number of hidden layer 1 1 1 Number of neurons hidden layers 33 33 33 Number of iterations 5000 5000 5000 Learning rate 0.001 0.001 0.001 RMSE 3.14727E-06 0.002974011 0.002284 R2 0.9999 0.99932 0.9996 Transfer function in Hidden Layer Tansig Logsig Tansig Table 5 The characteristics of the best ANN model for training algorithm. Table 5 The characteristics of the best ANN model for training algorithm. Characteristics Trainlm Traingda Traingrp Number of hidden layer 1 1 1 Number of neurons hidden layers 33 33 33 Number of iterations 5000 5000 5000 Learning rate 0.001 0.001 0.001 RMSE 3.14727E-06 0.002974011 0.002284 R2 0.9999 0.99932 0.9996 Transfer function in Hidden Layer Tansig Logsig Tansig ANN Model Figure 8 shows the training results and FEM values of the three models that best predicted the SEA value in the dynamic crash test (trainlm, trainrp and trainingda, respectively). Figure 8 shows the training results and FEM values of the three models that best predicted the SEA value in the dynamic crash test (trainlm, trainrp and trainingda, respectively). As the correlation coefficient (R) gets closer to 1, prediction and the values given to the ANN network get closer to each other, which means that the data is predicted accurately. In predicting the SEA value, the training correlation was obtained with 99.994%, 99.674% and 94.908% accuracy, respectively. Studies in crash boxes include many different parameters. Each parameter affects the complexity of the problem and thus the predictive quality of the ANN. Therefore, a database should be created from the studies and the ANN model should be integrated. Figure 7 FEM and ANN predicted values for train data. (a) Model 38 (b) Model 39 (c) Model 40. Figure 7 FEM and ANN predicted values for train data. (a) Model 38 (b) Model 39 (c) Model 40. 3.3 Selection of ANN numbers of epochs Another parameter that affects ANN prediction performance is the epoch number. Increasing the epoch number increases the accuracy of ANN estimates, but has no effect after a certain threshold value [39]. Increasing the epoch number also extends the solution time. Therefore, determining the optimum epoch number also provides optimization of the solution time. Figure 3 shows the effect of epoch number on prediction of SEA value using ANN in crashbox dynamic crash test. The statistical performances of R2 and RMSE are plotted as a function of the epoch number. The epoch number was changed as 100, 500, 1000, 2500 and 5000. A significant improvement was noticeable in R2 when the epoch number was increased from 100 to 500. It was seen that the performance increase stopped when the epoch number was bove 500. In the RMSE value, the performance increase continued up to the 2500 epoch number. Accordingly, the optimum epoch number should be determined as 1000 according to R2 performance and 2500 according to RMSE performance. Figure 8 Effect of epoch's number on ANN prediction performance. Figure 8 Effect of epoch's number on ANN prediction performance. 4 Conclusion In this study, the crashworthiness characteristics of thin-walled structures with trigger mechanisms were compared by the FEM method using material (DP600, DP800 and DP1000), thickness of material (between 0.8 and 2.2) and initial speed (10m/s, 12m/s and 16m/s). It was seen that the model with code D10T1.0V10 exhibited the best performance values with 13,771 kJ/kg, 118,300 kN and 0.476% SEA, FP and CE values, respectively. ANN model was developed using the obtained data sets. The input layer of the ANN model consists of three neurons, material, thickness of material and initial speed, and the output layer consists of a single neuron representing the energy absorption/ energy/PCH value that determines the characteristic of crash box. To find the best prediction model, 42 models were developed using two different training functions (trainda, trainlm and trainrp), two different transfer functions (tansig and logsig), and different numbers of neurons in the hidden layer (between 9 and 33). As a result of the study, the following results were obtained: It was seen that the prediction results with the highest accuracy rate was obtained with the training function “trainlm”, and the number of neurons in the hidden layer and the transfer function affected the prediction results less. In all three training algorithms, the best results were obtained when the number of neurons in the hidden layer was 33. In the prediction of the SEA value, 99.994%, 99.674% and 94.908% accuracy rates was obtained as a result of the training correlation, respectively. It was determined according to R2 performance that epoch number should be 1000, and it should be 2500 according to RMSE performance. Although there was no change in the prediction performance of the ANN model over this number of iterations, the solution time increased significantly. The ANN model, which performed best in predicting the SEA value (training function: trainlm, number of neurons in the hidden layer: 33 and transfer functions: tansig/purelin) was also trained for the estimation of the energy and PCH values. The training results were highly accurate in terms of energy and PCH. ANN and FEM results were found to be compatible in the test data. According to the R2 performance results, the ANN test results for SAE, energy and PCH were determined as 96.7604%, 96.0027% and 99.2444%, respectively. 3.4 ANN training performance (for energy/PCH) and test performance The ANN model which performed best in estimating the SEA value (trainlm, 33 noron and tansig/purelin) was also trained in the prediction of energy and PCH values (used in the evaluation of crash test results). The training results were as accurate as in SEA (Table 3). Table 6 Performance of Results for SEA, Energy and PCH. Output Noron Number of Epochs R2 RMSE SEA 2500 0.99999 0.314727E-05 Energy 2500 0.99999 3.10065E-05 PCH 2500 0.99999 3.15771E-05 Table 6 Performance of Results for SEA, Energy and PCH. In Figure 4 presented , the SAE (a), energy (b) ve PCH (c) values obtained by FEM and ANN predictions. The fact that the FEM and ANN prediction values are close to each other shows that the model predicts with high accuracy. R2 performance ratings for SAE, energy and PCH were determined as 96.7604%, 96.0027% and 99.2444%, respectively. Accordingly, The PCH value is predicted by the ANN with high accuracy. When all the graphs are examined together, it is seen that the prediction results are compatible with the FEM results, although there are small deviations between the small FEM and ANN values. Figure 9 Comparison of FEM and predicted ANN a) SEA, b) Energy, c) PCH Figure 9 Comparison of FEM and predicted ANN a) SEA, b) Energy, c) PCH References 1. Marzbanrad, J., A. Abdollahpoor, and B. Mashadi, Effects of the triggering of circular aluminum tubes on crashworthiness. International Journal of Crashworthiness, 2009. 14(6): p. 591-599. 2. Tabacu, S., Axial crushing of circular structures with rectangular multi-cell insert. Thin-Walled Structures, 2015. 95: p. 297-309. 3. Davoodi, M.M., S.M. Sapuan, and R. Yunus, Conceptual design of a polymer composite automotive bumper energy absorber. Materials & Design, 2008. 29(7): p. 1447-1452. 4. Nia, A.A. and J.H. Hamedani, Comparative analysis of energy absorption and deformations of thin walled tubes with various section geometries. Thin-Walled Structures, 2010. 48(12): p. 946-954. 5. Altin, M., E. Acar, and M.A. Guler, Foam filling options for crashworthiness optimization of thin- walled multi-tubular circular columns. Thin-Walled Structures, 2018. 131: p. 309-323. 6. Sohn, S.M., et al., Evaluation of the crash energy absorption of hydroformed bumper stays. Journal of Materials Processing Technology, 2007. 187: p. 283-286. 7. Yuan, L., et al., Quasi-static impact of origami crash boxes with various profiles. Thin-Walled Structures, 2019. 141: p. 435-446. 8. Aktay, L., A.K. Toksoy, and M. Guden, Quasi-static axial crushing of extruded polystyrene foam- filled thin-walled aluminum tubes: Experimental and numerical analysis. Materials & Design, 2006. 27(7): p. 556-565. 9. Acar, E., et al., Multi-objective crashworthiness optimization of tapered thin-walled tubes with axisymmetric indentations. Thin-Walled Structures, 2011. 49(1): p. 94-105. 10 X F X E h i t i l ffi i f b b th h d d thi k t t 10. Xu, F.X., Enhancing material efficiency of energy absorbers through graded thickness structures. Thin-Walled Structures, 2015. 97: p. 250-265. 11. Yang, S. and C. Qi, Multiobjective optimization for empty and foam-filled square columns under oblique impact loading. International Journal of Impact Engineering, 2013. 54: p. 177-191. 12. Tarlochan, F., et al., Design of thin wall structures for energy absorption applications: Enhancement of crashworthiness due to axial and oblique impact forces. Thin-Walled Structures, 2013. 71: p. 7- 17. 13. Zhang, X. and H. Zhang, Crush resistance of square tubes with various thickness configurations. International Journal of Mechanical Sciences, 2016. 107: p. 58-68. 14. Costas, M., et al., Static and dynamic axial crushing analysis of car frontal impact hybrid absorbers. International Journal of Impact Engineering, 2013. 62: p. 166-181. 15. Gedikli, H., Numerical investigation of axial crushing behavior of a tailor welded tube. Materials & Design, 2013. 44: p. 587-595. 16. 4 Conclusion When the results are evaluated, it has been determined that ANN will be an effective tool in evaluating the performance of the ANN for different parameters in crash boxes. Studies in crash boxes include many different parameters. Each parameter will change the difficulty of the problem and thus the predictive quality of the ANN. Therefore, larger data sets should be experimented by increasing the input parameters. References References Fang, J.G., et al., On design of multi-cell tubes under axial and oblique impact loads. Thin-Walled Structures, 2015. 95: p. 115-126. 17. Yin, H.F., et al., Crushing analysis and multi-objective optimization design for bionic thin-walled structure. Materials & Design, 2015. 87: p. 825-834. 18. Wei, Z.L., K.G. Robbersmyr, and H.R. Karimi, Data-based modeling and estimation of vehicle crash processes in frontal fixed-barrier crashes. Journal of the Franklin Institute-Engineering and Applied Mathematics, 2017. 354(12): p. 4896-4912. pp p 19. Marzbanrad, J., M. Alijanpour, and M.S. Kiasat, Design and analysis of an automotive bumper beam in low-speed frontal crashes. Thin-Walled Structures, 2009. 47(8-9): p. 902-911. 20. Qureshi, O.M. and E. Bertocchi, Crash behavior of thin-Walled box beams with complex sinusoidal relief patterns. Thin-Walled Structures, 2012. 53: p. 217-223. 21. Wang, G.F., et al., Crashworthiness design and impact tests of aluminum foam-filled crash boxes. Thin-Walled Structures, 2022. 180. 22. Panirani, P.N. and J. Ghanbari, Design and optimization of bio-inspired thin-walled structures for energy absorption applications. International Journal of Crashworthiness, 2022. 23. Reddy, T.J., Y.V.D. Rao, and V. Narayanamurthy, Thin-walled structural configurations for enhanced crashworthiness. International Journal of Crashworthiness, 2018. 23(1): p. 57-73. 24. Santos, R.O., et al., Damage identification parameters of dual-phase 600-800 steels based on experimental void analysis and finite element simulations. Journal of Materials Research and Technology-Jmr&T, 2019. 8(1): p. 644-659. gy p 25. Wang, J.F., et al., Effect of energy input on the microstructure and properties of butt joints in DP1000 steel laser welding. Materials & Design, 2016. 90: p. 642-649. 26. Wu, S.Z., et al., On design of multi-cell thin-wall structures for crashworthiness. International Journal of Impact Engineering, 2016. 88: p. 102-117. 27. Davim, J.P., V.N. Gaitonde, and S.R. Karnik, Investigations into the effect of cutting conditions on surface roughness in turning of free machining steel by ANN models. Journal of Materials Processing Technology, 2008. 205(1-3): p. 16-23. Hamzacebi, C., Forecasting of Turkey's net electricity energy consumption on secto Energy Policy, 2007. 35(3): p. 2009-2016. 29. Nayak, R., L.C. Jain, and B.K.H. Ting, Artificial neural networks in biomedical engineering: A review. Computational Mechanics, Vols 1 and 2, Proceedings, 2001: p. 887-892. 30. Kaya, G.U. and Z. Sarac, The Usage of Artificial Neural Network As Post Processing Algorithm In Digital Holography. 2018 26th Signal Processing and Communications Applications Conference (Siu), 2018. 31. References Maier, H.R., et al., Methods used for the development of neural networks for the prediction of water resource variables in river systems: Current status and future directions. Environmental Modelling & Software, 2010. 25(8): p. 891-909. p 32. Razavi, S. and B.A. Tolson, A New Formulation for Feedforward Neural Networks. Ieee Transactions on Neural Networks, 2011. 22(10): p. 1588-1598. İ 33. DİZDAR, E.N., O.J.A.P.-J.o.E. KOÇAR, and Science, Evaluation of Risks of OSH Management System with Artificial Neural Networks 2018. 6(3): p. 73-83. Ö 34. Selimefendigil, F. and H.F. Öztop, Numerical study and pod-based prediction of natural convection in a ferrofluids–filled triangular cavity with generalized neural networks. Numerical Heat Transfer, Part A: Applications, 2015. 67(10): p. 1136-1161. 35. Kocar, O., et al., Prediction and Control of Product Ouality in Deep Drawing Process: Design and Implementation of an intelligent System Using Knowledge Technologies. 2012: p. 1323-1326. 36. Çolak, A.B., A novel comparative analysis between the experimental and numeric methods on viscosity of zirconium oxide nanofluid: Developing optimal artificial neural network and new mathematical model. Powder Technology, 2021. 381: p. 338-351. 37. Shariati, M., et al., Assessment of longstanding effects of fly ash and silica fume on the compressive strength of concrete using extreme learning machine and artificial neural network. Journal of Advanced Engineering and Computation, 2021. 5(1): p. 50-74. 38. Parashar, N., et al., An artificial neural network approach for the prediction of dynamic viscosity of MXene-palm oil nanofluid using experimental data. Journal of Thermal Analysis and Calorimetry, 2021. 144: p. 1175-1186. 39. Maknickienė, N., A.V. Rutkauskas, and A. Maknickas, Investigation of financial market prediction by recurrent neural network. Innovative Technologies for Science, Business and Education, 2011. 2(11): p. 3-8.
https://openalex.org/W4286322158	https://zenodo.org/records/6874420/files/A%20Machine%20Learning%20Approach%20for%20Tracking%20and%20Predicting%20Student%20Performance%20in%20Degree%20Programs.pdf	English	null	A Machine Learning Approach for Tracking and Predicting Student Performance in Degree Programs	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,202	I. INTRODUCTION Analyzing a vast amount of data to provide usable summaries can be a difficult undertaking for humans. Grading students' academic performance is a more difficult and time-consuming task that will help instructors keep track of students' progress.. For all educational institutes, improving the quality of education in order to improve student performance is more vital. Predicting student outcomes in advance is advantageous; knowing this ahead of time will affect student performance. Predicting student success within a degree program, on the other hand, is quite different and presents different problems. Zhang et al. [4] conducted a study on current teaching learning backgrounds using Technology Mediated Learning (TML). The DA (Dynamic Assessment) method has a bigger influence on teaching and learning approaches. This study solves two research gaps on how DA influences academic achievement in students. For starters, pre- and post-test assessments have been utilised to focus previous research on DA. It is vital to understand the influence of DA based on computer for projecting students' performance over time and to employ information technology to do so. Second, because many students benefit from remote TML support, solutions are being developed based on TML assessments. This system has a few flaws, such as a small class size and the inability to specify external parts. Students can have vastly diverse backgrounds and areas of interest, resulting in varied course selections and sequences. Students from other places, on the other hand, might study the same course. One of the most difficult components of designing an effective predictor is finding out how to deal with heterogeneous student data due to varied areas and interests, because forecasting student performance in a certain course is predicated on the student's previous performance in other courses. Similarly, in-course tests that are supposed to be the same for all students are frequently used to predict students' performance in classes. Predicting student performance with high accuracy is more important since it aids in the identifying of pupils who are academically underperforming. The delivery of better and higher education is the primary goal of all educational institutions. The methodology for analysing student performance was created by Chew Li Sa et al. This model focuses on creating a system that analyses student performance. A Machine Learning Approach for Tracking and Predicting Student Performance in Degree Programs Ashtami Prasad Department of Computer Application Amal Jyothi College of Engineering Kanjirappally, India ashtamiprasad2022a@mca.ajce.in Shelly Shiju George Department of Computer Application Amal Jyothi College of Engineering Kanjirappally, India shellyshijugeorge@amaljyothi.ac.in Abstract — Predicting students' future performance based on their current academic records is important for ensuring essential pedagogical interventions and assuring students' prompt and efficient graduation. Although there is a considerable literature on using data-driven methodologies to anticipate student performance whether solving problems or studying for classes, forecasting student performance is still a challenge. when finishing degrees is far less researched. Existing techniques have mostly used elements linked to academic performance and family financial assets, whereas features related to family expenditures and personal information of students have been largely overlooked. The purpose of this study is to acquire data from students in various classrooms in order to analyze the aforementioned feature sets. Furthermore, students' performance would suffer as a result of their parents' negligence, therefore parents must keep a constant eye on their children's progress. These variables are beneficial in the process of making key decisions in educational institutions. Tripathi et al. [2] suggested a study that uses a naive bayes technique to analyse student performance forecasts. In terms of accuracy and overall execution time, the proposed model's results are compared to those of existing systems. According to this study, forecasting students' performance using prediction analysis for complex datasets is a more difficult task. The new model was proved to be more accurate than the previous model and to take less time to execute. Keywords— Academic records, predicting. Ching-Chieh Kiu [3] created a model for predicting academic success in students. The authors undertake research to see if student background, coursework success, and social activities have an impact on performance prediction. In predicting student achievement and identifying poor individuals, the author emphasises the importance of a student's past as well as their social activities. Early prediction by the model helps students do better in school and teachers improve the teaching learning process through inventiveness in the teaching process, which improves student performance. II. LITERATURE REVIEW Kasthuriarachchi and S. Liyanage [1] Developed a system that correlates with students' attendance, as well as innovative strategies for encouraging students to improve their attendance and reporting on their success in the classroom. Academicians, educators, and parents will be able to make better decisions as a result of this. In addition, the institute's administration provides financial assistance to students from low-income households through scholarships. I. INTRODUCTION For the study, the author considers the course "TMC1013 System Analysis and Design." Various data mining techniques, such as classification algorithms, are employed to ensure the accuracy of performance forecasts throughout the course. The system's key benefit is that it assists teachers and academicians in analysing student performance and identifying poor pupils who may fail the course. DOI: 10.5281/zenodo.6874420 ISBN: 978-93-5607-317-3@2022 MCA, Amal Jyothi College of Engineering Kanjirappally, Kottayam Proceedings of the National Conference on Emerging Computer Applications (NCECA)-2022 Vol 4 Issue 1 Proceedings of the National Conference on Emerging Computer Applications (NCECA)-2022 Vol 4 Issue 1 453 III. METHODOLOGY Removing Stop words – Stop words are removed from the datasets to tidy them up. These are the ones that pop up the most and aren't really important in the text. This can be done to improvethe model's accuracy. 2. Removing Stop words – Stop words are removed from the datasets to tidy them up. These are the ones that pop up the most and aren't really important in the text. This can be done to improvethe model's accuracy. 5. Find the final grade by frequency of going out. 5. Find the final grade by frequency of going out. 5. Find the final grade by frequency of going out. 3. Splitting of Data – The data is divided into training and testing datasets after it has been cleaned. After that, the data chosen for training totrain our model is used. 3. Splitting of Data – The data is divided into training and testing datasets after it has been cleaned. After that, the data chosen for training totrain our model is used. Fig 6: C. Machine Learning- Here machine learning is used to analyze and predict the students performance. C. Machine Learning- Here machine learning is used to analyze and predict the students performance. C. Machine Learning- Here machine learning is used to analyze and predict the students performance. DOI: 10.5281/zenodo.6874420 ISBN: 978-93-5607-317-3@2022 MCA, Amal Jyothi College of Engineering Kanjirappally, Kottayam III. METHODOLOGY The goal of this work is to develop a Machine Learning model for determining student performance.. In this system, the PyCharm is used that makes it easy to set up and access in python. Proceedings of the National Conference on Emerging Computer Applications (NCECA)-2022 V l 4 I 1 454 , Fig 1: Students performance prediction The steps that require to be followed are: 1. Data Gathering 2. Data Preparation 3. Model Building 4. Analyzing 5. Predicting 6. Result A. Data Gathering: The user has two datasets for student data, which they must integrate into one. Fig 2: 2. Read Datasets Fig 3: 3. Combine the datasets Fig 4: 4. Calculate the Grade Fig 5: 5. Find the final grade by frequency of going out. Fig 2: Fig 1: Students performance prediction The steps that require to be followed are: Fig 1: Students performance prediction The steps that require to be followed are: Fig 1: Students performance prediction The steps that require to be followed are: 2. Read Datasets Fig 3: 3. Combine the datasets p q 1. Data Gathering 2. Data Preparation 3. Model Building 4. Analyzing 5. Predicting 6. Result A. Data Gathering: The user has two datasets for student data which they must integrate into one 1. Data Gathering 2. Data Preparation 3. Model Building 4. Analyzing 5. Predicting 6. Result A. Data Gathering: The user has two datasets for student A. Data Gathering: The user has two datasets for student data, which they must integrate into one. B. Data Preparation: This is the first stage in any machine learning algorithm. This procedure entails data. Cleaning, data transformation, and data reduction are all things that may be done with data. All of this is done to improve the data's effectiveness. The data can be analysed to improve the accuracy of our model. In order for the categorization to be correct. 4. Calculate the Grade Fig 5: 4. Calculate the Grade Fig 5: 1. Cleaning Data – First, the data is cleansed. Users are eliminating the unnecessary entries in this area. The dataset is also possible cleaned up by removing the noisy data. 2. Removing Stop words – Stop words are removed from the datasets to tidy them up. These are the ones that pop up the most and aren't really important in the text. This can be done to improvethe model's accuracy. 2. IV. IMPLEMENTATION The model building is the main step in the Students performance analysis and prediction. The model building is the main step in the Students performance analysis and prediction. 1. First step is open a Pycharm project then, Import the packages that are necessary. 1. First step is open a Pycharm project then, Import the packages that are necessary. Fig 6: Fig 6: Proceedings of the National Conference on Emerging Computer Applications (NCECA)-2022 Vol.4, Issue.1 455 Vol.4, Issue.1 6. Find Final Grade By Desire to Receive Higher Education Fig 7: Fig 11: 7. Find the accuracy and model scores of two algorithm. 6. Find Final Grade By Desire to Receive Higher Education Fig 7: Fig 11: VI. CONCLUSION Fig 8: Fig 9: A new technique for predicting students' future performance in degree programmes based on prior academic achievement is offered in this study. Performance both now and in the past. A latent component model-based course clustering method was developed to discover acceptable courses for building base predictors. An ensemble-based progressive prediction architecture was created to incorporate students' evolving performance into the prediction. These data-driven strategies can be used in conjunction with other pedagogical methods to assess students' performance and provide useful information for academic advisors in recommending the number of students who should continue their studies and implementing pedagogical intervention measures as needed. This work will have an impact on the curriculum design of degree programmes as well as education policy planning in general. 7. Find the accuracy and model scores of two algorithm. 6. Find Final Grade By Desire to Receive Higher Education Fig 7: Fig 8: Fig 9: 7. Find the accuracy and model scores of two algorithm. 6. Find Final Grade By Desire to Receive Higher Education Fig 7: Fig 8: 7. Find the accuracy and model scores of two algorithm. Fig 11: Fig 11: VI. CONCLUSION A new technique for predicting students' future performance in degree programmes based on prior academic achievement is offered in this study. Performance both now and in the past. A latent component model-based course clustering method was developed to discover acceptable courses for building base predictors. An ensemble-based progressive prediction architecture was created to incorporate students' evolving performance into the prediction. These data-driven strategies can be used in conjunction with other pedagogical methods to assess students' performance and provide useful information for academic advisors in recommending the number of students who should continue their studies and implementing pedagogical intervention measures as needed. This work will have an impact on the curriculum design of degree programmes as well as education policy planning in general. Fig 8: Fig 9: VII. REFERENCES DOI: 10.5281/zenodo.6874420 ISBN: 978-93-5607-317-3@2022 MCA, Amal Jyothi College of Engineering Kanjirappally, Kottayam V. RESULT V. RESULT In this work two algorithms are used, that is logistic regression and decision tree. Using these algorithms the accuracy and model scores can be determined . [1].Kasthuriarachchi, K. T. S., & Liyanage, S. R. (2019),“Predicting Students’ Academic Performance Using Utility Based Educational Data Mining”, Frontier Computing, pp. 29–39. Fig 10: Then result shows that the Students performance andpredict the number of students goes to future studies. [2].A. Tripathi, S. Yadav and R. Rajan (2019), "Naive Bayes Classification Model for the Student Performance Prediction," 2nd International Conference on Intelligent Computing, Instrumentation. [3].C. Kiu (2018), "Data Mining Analysis on Student’s Academic Performance through Exploration of Student’s Background andSocial Activities," Fourth International Conference on Advances in Computing, Communication & Automation (ICACCA), Subang Jaya, Malaysia, pp. 1- 5. [4]. Zhang, R.-C., Lai, H.-M., Cheng, P.-W., & Chen, C.-P. (2017), “Longitudinal effect of a computer-based graduated prompting assessment on students’ academic performance”, Computers & Education, , 110, pp. 181–194 Fig 10: Fig 10: [5]. C. L. Sa, D. H. b. Abang Ibrahim, E. Dahliana Hossain and M. bin Hossin (2014), "Student performance analysis system (SPAS)," The5th International Conference on Information and Communication Technology for The Muslim(ICT4M), Kuching, pp. 1-6. Then result shows that the Students performance andpredict the number of students goes to future studies. Then result shows that the Students performance andpredict the number of students goes to future studies.
https://openalex.org/W2900615759	https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1140&context=pss_facpub	English	null	Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia	bioRxiv (Cold Spring Harbor Laboratory)	2,018	public-domain	18,862	Genome-Wide Association Studies for Yield-Related Traits in Soft Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Wheat Grown in Virginia Red Winter Wheat Grown in Virginia Part of the Genetics and Genomics Commons, Plant Sciences Commons, and the Soil Science Commons rt of the Genetics and Genomics Commons, Plant Sciences Commons, and the Soil Scienc ons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. University of Kentucky University of Kentucky UKnowledge UKnowledge Plant and Soil Sciences Faculty Publications Plant and Soil Sciences 2-22-2019 Genome-Wide Association Studies for Yield-Related Traits in Soft Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Wheat Grown in Virginia Red Winter Wheat Grown in Virginia Brian P. Ward North Carolina State University Gina Brown-Guedira USDA Agricultural Research Service Frederic L. Kolb University of Illinois at Urbana-Champaign David A. Van Sanford University of Kentucky, dvs@uky.edu Priyanka Tyagi North Carolina State University See next page for additional authors Follow this and additional works at: https://uknowledge.uky.edu/pss_facpub Part of the Genetics and Genomics Commons, Plant Sciences Commons, and the Soil Science Commons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. Repository Citation Repository Citation Ward, Brian P.; Brown-Guedira, Gina; Kolb, Frederic L.; Van Sanford, David A.; Tyagi, Priyanka; Sneller, Clay H.; and Griffey, Carl A., "Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Wheat Grown in Virginia" (2019). Plant and Soil Sciences Faculty Publications. 140. https://uknowledge.uky.edu/pss_facpub/140 University of Kentucky University of Kentucky UKnowledge UKnowledge Plant and Soil Sciences Plant and Soil Sciences Faculty Publications Plant and Soil Sciences Faculty Publications Plant and Soil Sciences Faculty Publications This Article is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and Soil Sciences Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Notes/Citation Information Notes/Citation Information Published in PLOS ONE, v. 14, no. 2, e0208217, p. 1-28. Published in PLOS ONE, v. 14, no. 2, e0208217, p. 1-28. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Repository Citation Repository Citation Ward, Brian P.; Brown-Guedira, Gina; Kolb, Frederic L.; Van Sanford, David A.; Tyagi, Priyanka; Sneller, Clay H.; and Griffey, Carl A., "Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Wheat Grown in Virginia" (2019). Plant and Soil Sciences Faculty Publications. 140. https://uknowledge.uky.edu/pss_facpub/140 This Article is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and Soil Sciences Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Genome-Wide Association Studies for Yield-Related Traits in Soft Red Winter Wheat Grown in Virginia Wheat Grown in Virginia Digital Object Identifier (DOI) https://doi.org/10.1371/journal.pone.0208217 Brian P. Ward, Gina Brown-Guedira, Frederic L. Kolb, David A. Van Sanford, Priyanka Tyagi, Clay H. Sneller, and Carl A. Griffey Authors Authors Brian P. Ward, Gina Brown-Guedira, Frederic L. Kolb, David A. Van Sanford, Priyanka Tyagi, Clay H. Sneller, and Carl A. Griffey This article is available at UKnowledge: https://uknowledge.uky.edu/pss_facpub/140 This article is available at UKnowledge: https://uknowledge.uky.edu/pss_facpub/140 RESEARCH ARTICLE Abstract Citation: Ward BP, Brown-Guedira G, Kolb FL, Van Sanford DA, Tyagi P, Sneller CH, et al. (2019) Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia. PLoS ONE 14(2): e0208217. https://doi.org/ 10.1371/journal.pone.0208217 Grain yield is a trait of paramount importance in the breeding of all cereals. In wheat (Triti- cum aestivum L.), yield has steadily increased since the Green Revolution, though the cur- rent rate of increase is not forecasted to keep pace with demand due to growing world population and increasing affluence. While several genome-wide association studies (GWAS) on yield and related component traits have been performed in wheat, the previous lack of a reference genome has made comparisons between studies difficult. In this study, a GWAS for yield and yield-related traits was carried out on a population of 322 soft red winter wheat lines across a total of four rain-fed environments in the state of Virginia using single- nucleotide polymorphism (SNP) marker data generated by a genotyping-by-sequencing (GBS) protocol. Two separate mixed linear models were used to identify significant marker- trait associations (MTAs). The first was a single-locus model utilizing a leave-one-chromo- some-out approach to estimating kinship. The second was a sub-setting kinship estimation multi-locus method (FarmCPU). The single-locus model identified nine significant MTAs for various yield-related traits, while the FarmCPU model identified 74 significant MTAs. The availability of the wheat reference genome allowed for the description of MTAs in terms of both genetic and physical positions, and enabled more extensive post-GWAS characteriza- tion of significant MTAs. The results indicate a number of promising candidate genes con- tributing to grain yield, including an ortholog of the rice aberrant panicle organization (APO1) protein and a gibberellin oxidase protein (GA2ox-A1) affecting the trait grains per square meter, an ortholog of the Arabidopsis thaliana mother of flowering time and terminal flower- ing 1 (MFT) gene affecting the trait seeds per square meter, and a B2 heat stress response protein affecting the trait seeds per head. Editor: Guihua Bai, USDA, UNITED STATES Received: November 8, 2018 Accepted: February 5, 2019 Published: February 22, 2019 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Brian P. WardID1¤, Gina Brown-Guedira2, Frederic L. Kolb3, David A. Van Sanford4, Priyanka Tyagi5, Clay H. Sneller6, Carl A. Griffey1 Brian P. WardID1¤, Gina Brown-Guedira2, Frederic L. Kolb3, David A. Van Sanford4, Priyanka Tyagi5, Clay H. Sneller6, Carl A. Griffey1 1 Department Of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America, 2 Eastern Regional Small Grains Genotyping Laboratory, USDA-ARS, Raleigh, North Carolina, United States of America, 3 Department of Crop Sciences, University of Illinois, Urbana, Illinois, United States of America, 4 Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky, United States of America, 5 Department of Crop and Soil Sciences, North Carolina State University, Raleigh, North Carolina, United States of America, 6 Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Department of Crop and Soil Sciences, North Carolina State University, Raleigh, North Carolina, United States of America * bpward2@ncsu.edu ¤ Current address: Department of Crop and Soil Sciences, North Carolina State University, Raleigh, North Carolina, United States of America * bpward2@ncsu.edu Introduction (Wheat Coordinated Agricultural Project [WheatCAP]) from the USDA National Institute of Food and Agriculture: https://www.triticeaecap.org/ . BPW and CAG received funding from Virginia Agricultural Council Grant 617: http://www.vdacs. virginia.gov/boards-virginia-agricultural-council. shtml, and from the Virginia Small Grains Board (no grant number available): http://www. virginiagrains.com/leadership/va-small-grains- board/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Worldwide, wheat has the fourth-highest production of all crops, with a net production value that is second-highest of any crop [1]. In addition, wheat maintains the highest global har- vested acreage of any crop [2]. To keep pace with an increasing world population and changes in diets due to increasing affluence, worldwide cereal production will have to increase by an estimated 50% over the period ending in 2050, requiring continuing genetic gains in yield potential of approximately 1.1% per year [3]. Sharma, et al. [4] estimated a historical average increase in grain yields in spring wheat of 0.65% per year when analyzing data across 15 years and 919 environments, while a recent study involving winter wheat in the Eastern United States estimated yearly increases in grain yield between 0.56% and 1.41%, depending upon environment [5]. Competing interests: The authors have declared that no competing interests exist. Improvements to yield via direct selection are hampered by its highly quantitative and poly- genic nature. Selection based on different yield component traits and crop physiology theory may offer additional avenues for increasing genetic gain in yield while avoiding yield plateaus [6]. However, relationships between traits must be taken into account, as there are many nega- tive correlations between related traits, such as the well-documented negative correlation between the number of grains m-2 and average grain size [7]. A wide body of literature suggests that wheat is primarily sink-limited with respect to production of photosynthetic assimilates (reviewed in [8]). Supporting this theory, several studies have suggested that maximizing the number of seeds per unit area is critical for avoiding assimilate sink limitations and maximiz- ing yield (reviewed in [9]). The two possible routes for increasing seeds per unit area are to: 1) increase the average number of seeds per head, and/or 2) increase the number of heads per unit area. Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Abstract Data Availability Statement: All genotypic, phenotypic, and covariate data used in the study may be downloaded from https://doi.org/10.6084/ m9.figshare.7700012. Funding: CHS and GBG received funding from the Agriculture and Food Research Initiative Competitive Grants 2011-68002-30029 (Tritaceae Coordinated Agricultural Project [TCAP]) from the USDA National Institute of Food and Agriculture: https://www.triticeaecap.org/. GBG received funding from the Agriculture and Food Research Initiative Competitive Grant 2017-67007-25939 1 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Germplasm selection Germplasm selection was performed as detailed in Ward et al., 2019 [35]. The study was con- ducted over two years, and included a total of 329 genotypes (S1 Table). Of these, 41 genotypes were tested across both years. Of the remaining genotypes, half (144) were tested only in the first year, and the other half (144) were tested only in the second year. Within each year, geno- types were sourced from breeding programs in Illinois (31), Kentucky (30), Missouri (2), and Virginia (122). Five checks were included in the study: ‘Bess’, ‘Branson’, IL00-8530, ‘Roane’, and ‘Shirley’. With the exception of checks and several older cultivars, the majority of geno- types were either F4 or F5 filial generation. Introduction Increases in yield will require both the production of greater numbers of grains, and an increase in the availability of photosynthates to prevent a corresponding drop in kernel sizes due to the compensation between these two traits [10]. Several previous studies have focused on increasing photosynthate availability via alterations to canopy light interception. Reynolds et al. [9] note that the amount of light interception is already nearly maximized in many wheat cultivars during the period after canopy closure and prior to leaf senescence. This leaves traits increasing the duration of light interception, e.g. faster canopy establishment through increased early-season vigor or later senescence via the “stay-green” trait [11], as appealing avenues for increasing photosynthate production. In winter wheat, several genes are known to exert major effects on traits involved with seed production per unit area and photosynthesis duration. The time required for plants to flower and ultimately reach maturation is affected by multiple homeologous copies of the VRN ver- nalization requirement genes and the PPD photoperiod response genes [12]. Multiple major- effect quantitative trait loci (QTLs) affecting grain weight have also been identified, including TaSus2-2B [13], the TaGW2 homeologous genes [14], and cell wall invertase (Cwi) genes [15]. However, it is likely that many genes affecting yield-related traits have yet to be identified. Genome-wide association studies (GWAS) and linkage mapping are the two predominate methods employed in plant breeding for associating phenotypic variation with underlying genetic variation. GWAS offers higher resolution due to many more ancestral gene recombi- nations within the testing panel, as opposed to only one or a few meiotic recombinations in a linkage mapping population. However, allele frequency is a primary factor limiting power in GWAS studies; marker-trait associations (MTAs) will be difficult or impossible to detect if causal variants are rare within the testing population [16]. GWAS methods utilizing mixed lin- ear models have become standard methodology for their ability to guard against false positives due to population structure or kinship among genotypes [17]. Subsequent mixed model imple- mentations have utilized multiple methods to increase power to detect loci of smaller effect. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 2 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia The multi-locus mixed model fits loci of large effect as covariates, allowing for the detection of more marker trait associations with smaller effects [18]. Introduction Recent methods including FaS- T-LMM-Select [19] and SUPER [20] have been developed which increase power by first per- forming a preliminary GWAS to identify the SNPs which are highly associated with the trait of interest, and subsequently using only this subset of SNPs when estimating kinship among lines in a second GWAS. Finally, the programs FarmCPU [21] and BLINK [22] combine the advan- tages of multi-locus mixed models with subsetting kinship estimation. Relatively few GWAS analyses for yield and yield-related traits have been conducted in wheat, and of these, fewer still have been conducted in winter wheat germplasm [23–30]. Asso- ciation studies have been more common in spring wheat, though the majority of these have been candidate-gene studies, with genome-wide studies being more limited [31]. Finally, it has been more common to perform GWAS in crop species using assembled diversity panels, rather than elite germplasm in current use by breeding programs [32]. GWAS in elite germ- plasm is typically limited to the identification of smaller-effect MTAs, as MTAs of major effect will have likely already become fixed within the mapping population [33]. Nevertheless, GWAS using panels of elite germplasm remain useful due to their higher relevance to the pro- cess of cultivar development [32]. One limitation of previous wheat GWAS studies was the lack of a reference genome, making comparisons between studies’ findings difficult. The recent publication of a full chromosome-anchored wheat genome assembly should allow for better curation of MTAs uncovered by GWAS and linkage mapping studies. The present study sought to perform GWAS in a panel of elite soft winter wheat lines sourced from breeding programs in the Eastern and Midwestern United States. Genotyping was performed via genotyping-by-sequencing (GBS), using genetic maps and anchoring SNPs to the Chinese Spring reference sequence International Wheat Genome Sequencing Consor- tium (IWGSC) RefSeq v1.0 [34], allowing for determination of both physical and genetic posi- tions for identified MTAs. In addition, targeted genotypic assays were used to interrogate loci which are known to exert major effects on many traits of agronomic importance. Phenotyping Experimental design and data collection. Experimental design, field management prac- tices, and phenotypic data collection were performed as detailed in Ward et al., 2019 [35]. Briefly, the experiment was planted in a total of four environments (two locations in two years) in the 2013–14 and 2014–15 winter wheat growing seasons. Within each year, trials were planted at Kentland Farm near Blacksburg, Virginia (Guernsey/Hayter silt loams, 37.1965˚ N, 80.5718˚ W, 531 m elevation) and the Eastern Virginia Agricultural Research and Extension Center in Warsaw, Virginia (Kempsville sandy loam, 37.9879˚ N, 76.7770˚ W, 40 m elevation). A generalized randomized complete block design (GRCBD) with two replications PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 3 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia was used in each environment. Data was collected on 14 traits, including flag leaf stay green duration (FLSG), grains per square meter (GSQM), grain weight (GW), heading date (HD), plant height (HT), physiological maturity date (MAT), normalized-difference vegetation index (NDVI) at Zadok’s growth stage 25 [36], whole-grain protein content (PROT), seeds per head (SPH); spikes per square meter (SSQM), whole-grain starch content (STARCH), thousand kernel weight (TKW), test weight (TWT), and grain yield (YLD). S2 Table summarizes all phe- notypic traits, and lists their abbreviations, units of measure, and trait ontologies. Spatial corrections. For each individual trait/environment combination, an ad-hoc cor- rection for field heterogeneity was performed using two-dimensional tensor product penalized B-splines [37], implemented in a mixed model framework in the R [38] package ‘SpATS’ [39]. The package default parameters were used to fit cubic splines with quadratic penalization func- tions in both row and column dimensions. For a particular environment with plots arranged in m rows and n columns, the number of knots used to fit splines was set to (bmc/2) −1 and (bnc/2) −1, respectively. Fitted values generated by the SpATS model were used for subsequent modeling of phenotypes across environments, as described in the section below. Heritability for each trait/environment combination was estimated using the method of Cullis, et al. [40]: h2 g ¼ 1 Att 2s2 G ð1Þ ð1Þ Where the generalized heritability (h2 g) is a function of the average pairwise prediction error between pairs of genotypes within an environment (Att), and the genotypic variance (s2 G). Phenotyping Her- itability estimates were calculated from the models fit by the SpATS package, and were com- pared against the heritability estimates generated by a baseline model: Yi ¼ m þ Gi þ εi ð2Þ ð2Þ Where phenotypic response (Yi) is a function of the within-environment mean (μ), the fixed effect of the ith genotype (Gi), and residual error (εi). Where phenotypic response (Yi) is a function of the within-environment mean (μ), the fixed effect of the ith genotype (Gi), and residual error (εi). Modelling of phenotypes across environments. Each location/year combination was considered as a unique environment in order to model phenotypic response across environ- ments. For each trait, the following random effects ANOVA model was fit in R using the ‘lme4’ package [41]: Yijk ¼ m þ Gi þ Ej þ RkðEjÞ þ GEij þ εijk# ð3Þ ð3Þ Where the phenotypic response (Yijk) is a function of the overall mean (μ) and the random effects of the ith genotype (Gi), the kth replication (Rk) nested within the jth environment (Ej), the genotype-environment interaction (GEij) and the residual error (εijk). Genotypic best-lin- ear unbiased predictors (BLUPs) were calculated for use as the phenotypic input for the subse- quent GWAS analyses. Genotyping Genotyping-by-sequencing. Genomic DNA was isolated from fresh green seedling leaf tissue using an LGC Genomics Oktopure robotic extraction platform with sbeadex magnetic microparticle reagent kits. Genotyping-by-sequencing was performed using an Illumina HiSeq 2500 following a double digest of genomic DNA using the restriction enzymes PstI and MseI, using the protocol of Poland et al., 2012 [42]. SNP calling was performed using TAS- SEL-GBS v5.2.43 [43,44]. The Burrows-Wheeler Aligner [45] v0.7.17-r1188 was used to align Illumina-generated short reads to the Chinese Spring IWGSC RefSeq v1.0 wheat reference PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 4 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia sequence. The raw genotypic data was filtered to retain only biallelic SNPs, and to remove SNPs with missing data frequencies > 50%, mean sequencing depth < 2, heterozygous call frequencies > 15%, or minor allele frequency < 5%. SNPs that aligned to unmapped contigs were removed. Genotypes containing > 85% missing data were removed. Missing data was then imputed using Beagle v4.1 with default settings [46]. After imputation, the genotypic data was filtered a second time to remove SNPs with minor allele frequency < 5% or heterozygous call frequency < 15%. PLINK 1.9 [47] was used to remove all but one SNP in groups of SNPs in perfect linkage disequilibrium (LD; r2 = 1). After filtering, 29,949 SNPs and 322 genotypes remained for further analysis. Assays for polymorphisms of major effect. Several genes and polymorphisms of major effect were assayed using LGC Genomics KASPar SNP assays. All included assays are listed with primer sequences in S3A Table. Briefly, they included assays for the 1RS:1AL and 1RS:1BL translocations from rye (Secale cereal L.), polymorphisms within the Ppd-A1, Ppd-B1, and Ppd-D1 photoperiod sensitivity genes located on chromosomes 2A, 2B, and 2B respec- tively, the Rht-B1 and Rht-D1 dwarfing genes located on chromosomes 4B and 4D respectively, and polymorphisms of the Vrn-A1 and Vrn-B1 genes located on chromosomes 5A and 5B. p y p g KASP assays for polymorphisms within exon 4 and exon 7 of Vrn-A1 were included in this study. The molecular mechanism behind Vrn-A1’s effects on vernalization requirement remains contested. Chen et al. Genotyping [48] proposed that a SNP occurring in exon 4 of Vrn-A1 was the causal locus differentiating between the Vrn-A1a short vernalization requirement allele present in the cultivar Jagger and the Vrn-A1b long vernalization requirement allele present in the cultivar ‘2174’. Dı´az et al. [49] later proposed that the differences between Vrn-A1a and Vrn-A1b vernalization requirements are due to Vrn-A1 copy number variations. Li et al. [50] subsequently reported that although Jagger contains two copies of Vrn-A1, whereas ‘2174’ con- tains three copies of the gene, differences in vernalization requirements between the two culti- vars are in fact due to structural differences, particularly due to a SNP located in exon 7, converting the 180th amino acid residue from alanine in Vrn-A1a to valine in Vrn-A1b. Finally, Kippes et al. [51] proposed that the effects of Vrn-A1 arise from the product of gene VRN-D4 disrupting the binding of the RNA-binding repressor TaGRP2 to Vrn-A1. The KASP assays for Vrn-A1 used in this study have been shown to be suggestive, but not perfect predictors of vernalization requirement due to Vrn-A1 alleles. In addition, assays for the Sr36 stem rust (Puccinia graminis Pers.) resistance gene, and the sucrose-synthase gene TaSus2-2B, which affects kernel weight, were included. Gene Sr36 is located on a 2G:2B alien translocation originating from Triticum timopheevi [Zhuk.] Zhuk. (AmAmGG) [52,53]. Gene TaSus2-2B is located on the short arm of chromosome 2B, and is one of the three sucrose synthase Sus2 orthologs located on chromosomes 2A, 2B, and 2D. Two common haplotypes for TaSus2-2B include Hap-H (high seed weight) residing on the 2G:2B translocation, and Hap-L (low seed weight) [13]. While there is no evidence to suggest that TaSus2-2B was inherited from T. timopheevi, Cabrera et al. found that TaSus2-2B alleles were in perfect LD with alleles of the microsatellite marker Xwmc477 [54], which itself was pre- viously found to be in perfect LD with Sr36 [55]. Genome-wide association analysis For each trait, single-locus mixed linear model genome-wide association analyses were per- formed with the Genome-Wide Complex Trait Analysis (GCTA) software [60], using a leave- one-chromosome-out (LOCO) method in which a separate genetic relationship matrix (GRM) is estimated from SNP data for each chromosome. Specifically, the LOCO approach entails excluding all SNPs located on the chromosome of the SNP undergoing testing when estimat- ing the GRM. Adjusted p-values for each SNP were calculated using the method of Benjamini and Hochberg [61], and SNPs with adjusted p-values below 0.05 were considered significant. g [ ] j p g In addition, GWAS was performed for each trait using the Fixed and Random Model Cir- culating Probability Unification (FarmCPU) model [21], using the R package ‘FarmCPUpp’ [62]. The same significance threshold applied in the single-locus tests was used for the Farm- CPU results. To enhance our confidence in significant MTAs identified by FarmCPU, we implemented a bootstrapping method utilized by Wallace, et al. [63], in which 10% of the phe- notypic observations were randomly replaced with missing data for a total of 100 runs of the model. Subsequently, for each trait the resample model inclusion probability (RMIP) [64] was calculated for each SNP by determining the fraction of bootstraps in which its adjusted p-value exceeded the significance threshold. The value 0.1 was chosen as a lower threshold for the RMIP as it coincided with the point of inflection in the RMIP density curves (data not shown). For each model, the first four principal components were included to model population struc- ture, based upon visual examination of the scree plot and cumulative sum plots for variance explained by each PC (S1 Fig). Approximate genetic positions were calculated for each MTA using a set of PstI-MspI GBS markers generated from 88 doubled haploid lines derived from the synthetic W7894 × Opata M85 (SynOp) cross [65], aligned to the Chinese Spring IWGSC RefSeq v1.0 wheat reference sequence [34]. The R package ‘MonoPoly’ [66] was used to fit a monotonically increasing spline to each chromosome of the SynOp genetic map, allowing for the estimation of genetic positions corresponding to MTA physical positions. Population structure and linkage disequilibrium Prior to performing GWAS, population structure was examined via principle component anal- ysis (PCA) of the filtered and imputed genotypic data using the SNPRelate package [56] in R. Linkage disequilibrium (r2) was estimated between all intrachromosomal pairs of SNPs up to a physical distance of 10Mb using PLINK 1.9. The pairwise r2 estimates were then sorted into bins from 102 to 107 base pairs, with an exponent interval size of 0.1. The mean r2 value was PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 5 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia calculated for each bin, and then plotted against physical distance, with a second-degree locally-weighted scatterplot smoothing (LOESS) curve fit to the data [57]. Separate LOESS curves were fit to LD data from each genome, and from each chromosome. Mean r2 values equal to or greater than 0.2 were considered significant LD. In addition, LD was calculated between the KASP Sr36 assay and all other SNPs present on chromosome 2B. Finally, the fixa- tion index (FST) was calculated for each SNP using Weir and Cockerham’s estimator [58], with two subpopulations defined by the presence or absence of Sr36 as defined by the KASP assay results. All FST-related calculations were performed using VCFtools v0.1.17 [59]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Trait heritability and correlations The use of spatially-corrected data tended to drastically increase generalized heritability within each environment. The trait GW had the lowest mean generalized heritability, averag- ing 0.2 across environments for raw, uncorrected data, and 0.33 after performing spatial cor- rections (Table 1). The trait TKW had the highest mean generalized heritability when using uncorrected data ðh2 G ¼ 0:95Þ, though heritability was slightly decreased when using spatially- corrected data ðh2 G ¼ 0:9Þ. When spatially-corrected data was used, TWT had the highest aver- age heritability ðh2 G ¼ 0:95Þ. YLD had a moderate mean, across-environment heritability of 0.57 when calculated using uncorrected data, though this increased to 0.81 when using spa- tially-corrected data. Pearson correlation coefficients were calculated for each pair of traits using the phenotypic BLUPs (Table 2). Phenological traits (HD, FLSG, and MAT) all displayed a high degree of intercorrelation. This was also generally the case for traits relating to grain size and density per unit area (TKW, GSQM, SSQM, and SPH). For instance, the traits GSQM and TKW demonstrated a strong negative correlation (-0.66), as predicted due to trait com- pensation effects. Of these four traits, the weakest correlation was between TKW and SPH (-0.24). Critically, only weak correlation was observed between traits relating to grain size/ density and phenological traits, suggesting that these two classes of traits could be improved independently. YLD was most highly correlated with the traits GW and MAT (positive), and Table 1. Trait descriptive statistics and generalized heritability estimates calculated using raw plot values and spatially-adjusted values. Candidate genes and translation effects Haplotype blocks surrounding each significant MTA were identified using the method of Gabriel et al. [67] by running the—blocks command in PLINK 1.9. Some significant MTAs did not reside within any larger haplotype block, while some haplotype blocks contained mul- tiple significant MTAs. Subsequently, all genes overlapping significant MTAs and associated haplotype blocks were identified using the IWGSC v1.1 RefSeq annotation [34] with functional annotations from the IWGSC v1.0 annotation. In addition, Ensembl identifiers were retrieved for all genes overlapping with significant SNPs or haplotype blocks. A table of wheat genes with trEMBL or Swissprot-generated protein annotations in UniProt was downloaded and used to identify all wheat genes with predicted functions located within 1Mb of each signifi- cant MTA. The Ensembl Variant Effect Predictor [68] was then used to classify significant SNPs as being either intergenic, intronic, exonic, or upstream/downstream proximal variants. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 6 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia The predicted allele substitution effects of exonic SNPs on protein translation were classified as synonymous, missense, or nonsense. For gene-proximal SNPs, the distance to the closest gene was recorded. The predicted allele substitution effects of exonic SNPs on protein translation were classified as synonymous, missense, or nonsense. For gene-proximal SNPs, the distance to the closest gene was recorded. a FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT wet chemistry-validated whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield VA 2014; 15Bb Blacksburg, VA 2015; 15War Warsaw, VA 2015 14Bb Blacksburg, VA 2014; 14War Warsaw, VA 2014; 15Bb Blacksburg, VA 2015; 15War Warsaw, VA 2015 https://doi.org/10.1371/journal.pone.0208217.t001 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Table 2. Phenotypic correlations among traits calculated using the across-environment genotype BLUPs. FLSG GSQM GW HD HT MAT NDVI PROT SPH SSQM STARCH TKW TWT YLD FLSG 1 GSQM 0.01 1 GW 0.27 0.64 1 HD -0.43 0.14 -0.06 1 HT -0.18 -0.16 -0.21 0.15 1 MAT 0.18 0.26 0.19 0.71 -0.07 1 NDVI 0.05 0.05 0.08 0.12 0.13 0.10 1 PROT 0.07 -0.41 -0.39 -0.15 0.11 -0.21 0.15 1 SPH 0 0.48 0.40 0.12 0.02 0.21 -0.20 -0.39 1 SSQM 0.02 0.53 0.24 0.02 -0.18 0.05 0.25 -0.01 -0.48 1 STARCH 0.20 0.30 0.32 -0.04 -0.23 0.19 -0.12 -0.62 0.29 0.01 1 TKW 0.25 -0.66 0.14 -0.24 -0.01 -0.13 0.01 0.15 -0.24 -0.43 -0.07 1 TWT 0.04 -0.22 -0.19 -0.28 0.18 -0.31 -0.08 0.25 -0.20 -0.02 -0.25 0.10 1 YLD 0.32 0.36 0.58 0.18 -0.08 0.48 0.13 -0.45 0.33 0.03 0.41 0.11 -0.20 1 FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT wet chemistry-validated whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield Correlation significant at the 0.01 level Table 2. Phenotypic correlations among traits calculated using the across-environment genotype BLUPs. FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT wet chemistry-validated whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield Correlation significant at the 0.01 level grain protein (negative). The negative correlation between yield and grain protein content has been well documented in the past (e.g. [69–71]). grain protein (negative). The negative correlation between yield and grain protein content has been well documented in the past (e.g. [69–71]). Trait heritability and correlations Descriptive Statistics Generalized Heritability Raw Plot Values Spatially-Adjusted Values Trait a Units min mean max SD 14Bb 14War 15Bb 15War mean 14Bb 14War 15Bb 15War mean FLSG days 21.0 28.7 38.0 2.76 0.26 0.80 0.75 0.55 0.59 0.55 0.83 0.76 0.75 0.72 GSQM Grains m-2 8460 1.85E+04 3.13E+04 3277 0.62 0.50 0.51 0.58 0.55 0.71 0.51 0.54 0.67 0.61 GW g dwt m-1 row 47.68 96.64 157.9 16.53 0.19 0.14 0.44 0.05 0.20 0.43 0.16 0.47 0.25 0.33 HD Julian days (Jan1) 121 128 136 3.21 0.75 0.94 0.95 0.96 0.90 0.82 0.95 0.96 0.97 0.92 HT cm 59 85 119 9.3 0.85 0.85 0.84 0.86 0.85 0.92 0.88 0.88 0.87 0.89 MAT Julian days (Jan1) 151 159 171 4.72 0.83 0.87 0.86 0.76 0.83 0.89 0.91 0.89 0.9 0.90 NDVI - 0.26 0.54 0.75 0.08 0.06 0.39 0.41 0.41 0.32 0.70 0.75 0.68 0.64 0.69 PROT % 9.67 12.3 16.0 1.01 0.66 0.38 0.63 0.74 0.60 0.67 0.40 0.71 0.78 0.64 SPH count 8.54 21.9 33.3 3.04 0.86 0.82 0.77 0.81 0.82 0.85 0.82 0.75 0.84 0.82 SSQM Spikes m-2 459.3 853.0 1485 161.2 0.60 0.62 0.45 0.52 0.55 0.65 0.63 0.50 0.63 0.60 STARCH % 46.88 52.51 56.49 1.410 0.46 0.71 0.19 0.69 0.51 0.80 0.72 0.73 0.83 0.77 TKW grams 24.1 34.6 91.6 3.98 0.96 0.97 0.94 0.93 0.95 0.97 0.98 0.69 0.97 0.90 TWT g L-1 652.6 759.0 810.9 19.70 0.96 0.92 0.91 0.93 0.93 0.98 0.96 0.90 0.95 0.95 YLD kg ha-1 3579 6627 9053 1027 0.63 0.45 0.73 0.46 0.57 0.84 0.81 0.78 0.81 0.81 zed heritability estimates calculated using raw plot values and spatially-adjusted values. g, ; , ; g, ; , a FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT wet chemistry-validated whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT wet chemistry-validated whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield Correlation significant at the 0.01 level Polymorphisms of major effect The first and second principal components of the SNP matrix were plotted against each other, using (A) all SNPs that passed initial filtering parameters, and (B) a thinned subset of SNPs selected to be in approximate linkage equilibrium, so that no pair of SNPs displayed significant LD (r2 > 0.2). The percent of the total genotypic variance explained is listed on each axis. Genotypes are divided into two groups based on the presence of absence of the Sr36 stem rust resistance gene and underlying 2G:2B translocation as determined by KASP assay. https://doi.org/10.1371/journal.pone.0208217.g001 Fig 1. Biplots of genotypic principal components. The first and second principal components of the SNP matrix were plotted against each other, using (A) all SNPs that passed initial filtering parameters, and (B) a thinned subset of SNPs selected to be in approximate linkage equilibrium, so that no pair of SNPs displayed significant LD (r2 > 0.2). The percent of the total genotypic variance explained is listed on each axis. Genotypes are divided into two groups based on the presence of absence of the Sr36 stem rust resistance gene and underlying 2G:2B translocation as determined by KASP assay. https://doi.org/10.1371/journal.pone.0208217.g001 Fig 1. Biplots of genotypic principal components. The first and second principal component https://doi.org/10.1371/journal.pone.0208217.g001 https://doi.org/10.1371/journal.pone.0208217.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Polymorphisms of major effect An estimation of allele effects for the KASP markers assaying genes of known function revealed that the stem rust resistance gene Sr36 and the sucrose-synthase gene TaSus2-2B pro- duced many significant differences among genotypes for multiple traits (S3B Table). These two genes also consistently produced significant differences of similar magnitudes for the same traits. The presence of the Rht-B1b and Rht-D1b dwarfing alleles produced significant effects of opposite signs for many traits, including SSQM, TKW, and TWT. Effects were also of opposite signs for HT and YLD, though in both of these cases the effects of Rht-B1b were not significant. The Vrn-A1 exon 4 polymorphism and Vrn-B1 polymorphism each exerted significant effects on six traits, while the Vrn-A1 exon 7 polymorphism produced significant effects on three traits. However, these polymorphisms occurred at low frequencies (0.14, 0.03, and 0.07 for Vrn-A1 exon 4, Vrn-A1 exon7, and Vrn-B1 respectively). Of the three Ppd photo- period response genes assayed, Ppd-D1 produced significant effects on five traits, while also occurring at a high frequency (0.69), while Ppd-B1 produced significant effects on four traits, occurring at a lower frequency (0.26). Ppd-A1 occurred at a high frequency, but only produced a significant effect on the trait GW. Finally, the 1RS:1AL and 1RS:1BL translocations produced significant effects on five and four traits, respectively, but occurred at relatively low frequencies of 0.07 and 0.19. Despite these significant effects on phenotype, none of the loci of major effect assayed with KASP markers were classified as significant in the GWAS. Some potential reasons for these findings will be discussed below. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 8 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Fig 1. Biplots of genotypic principal components. The first and second principal components of the SNP matrix were plotted against each other, using (A) all SNPs that passed initial filtering parameters, and (B) a thinned subset of SNPs selected to be in approximate linkage equilibrium, so that no pair of SNPs displayed significant LD (r2 > 0.2). The percent of the total genotypic variance explained is listed on each axis. Genotypes are divided into two groups based on the presence of absence of the Sr36 stem rust resistance gene and underlying 2G:2B translocation as determined by KASP assay. https://doi.org/10.1371/journal.pone.0208217.g001 Fig 1. Biplots of genotypic principal components. Population structure and linkage disequilibrium Principle component analysis of the processed GBS SNP data revealed substantial admixture among genotypes, with the first principle component only explaining 7.84% of the total geno- typic variance (S1 Fig). Lines included in the panel formed two distinct clusters in the biplot of the first two principle components. These two clusters were largely delineated by the presence or absence of the 2G:2B translocation as determined by the Sr36 KASP assay (Fig 1A; similar results for TaSus2-2B not shown). In contrast, neither the 1BL:1RS nor the 1AL:1RS alien translocations produced any discernable clustering of genotypes (data not shown). When the SNP data was thinned to remove SNPs in high LD with each other (i.e. limiting the maximum pairwise LD between SNPs to r2 = 0.2), the population stratifying effects of Sr36 and the under- lying 2G:2B translocation were removed, and the first principle component explained 3.68% of variation (Fig 1B). Linkage disequilibrium decay plots demonstrated that LD decayed below significant levels (r2 < 0.2) at distances of approximately 1Mb (Fig 2). LD decay was highly similar between chromosomes, with the exception of chromosomes located on the D genome, which exhibited greater variation in LD decay patterns. This may simply be due to differences in SNP density among the genomes. As SNP density was much lower in the D genome (S2 Fig), many D genome chromosomes had far fewer pairs of SNPs in close proximity to each other. This likely inflated the variance of LD estimates at short distances. The genome-wide FST scan following the splitting of the overall panel based on the result of the Sr36 KASP assay demonstrated that the 2G:2B translocation formed a large block of high-FST SNPs spanning almost all of chromo- some 2B (Fig 3). In addition, an enrichment of high-FST SNPs on chromosomes 2A and 2D suggested misalignment of SNPs among the group 2 homeologous chromosomes. Further examination of FST values on chromosome 2B indicated a general linear relationship between FST and the value of r2 measured against the Sr36 KASP marker for most SNPs, with a minority of SNPs exhibiting high FST with little or no corresponding LD with the Sr36 KASP marker (Fig 4A). These SNPs were distributed throughout the chromosome, though SNP density PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 9 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Fig 2. Population structure and linkage disequilibrium Linkage disequilibrium by genome and chromosome. LOESS regressions of mean r2 between pairs of SNPs vs. physical distance, pooled for each genome (top row), and for each of the seven chromosomes present in the A, B, and D genomes (bottom row). https://doi.org/10.1371/journal.pone.0208217.g002 Fig 2. Linkage disequilibrium by genome and chromosome. LOESS regressions of mean r2 between pairs of SNPs vs. physical distance, pooled for each genome (top row), and for each of the seven chromosomes present in the A, B, and D genomes (bottom row). nome and chromosome. LOESS regressions of mean r2 between pairs of SNPs vs. physical distance, pooled for each genome (t osomes present in the A, B, and D genomes (bottom row). Fig 2. Linkage disequilibrium by genome and chromosome. LOESS regressions of mean r2 between pairs of SNPs vs. physical distance, pooled for each genome (top row), and for each of the seven chromosomes present in the A, B, and D genomes (bottom row). https://doi.org/10.1371/journal.pone.0208217.g002 https://doi.org/10.1371/journal.pone.0208217.g002 tended to be higher in telomeric regions. In addition, SNPs with T. timopheevi private alleles were far more abundant than SNPs with shared alleles. (Fig 4B). PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies In general, the single-locus model implemented in GCTA identified far fewer significant MTAs than FarmCPU. In total, GCTA identified 44 significant MTAs at the 0.05 FDR signifi- cance threshold. However, many of these were located within high-LD blocks and, therefore, clustered at the same putative underlying QTLs. Discounting those SNPs believed to co-local- ize to identical QTLs yielded nine unique MTAs for HD, MAT, SSQM, TKW, and TWT (Table 3). One SNP, located at 58,633,321bp on chromosome 7D, was pleiotropic for the traits HD and MAT, and hence the nine significant MTAs represented eight putative QTLs. In con- trast, FarmCPU identified 108 MTAs at the same significance threshold. Removing those SNPs with RMIPs below 0.1 yielded a total of 74 significant MTAs for the traits FLSG, GSQM, GW, HD, HT, MAT, NDVI, PROT, SPH, SSQM, STARCH, TKW, TWT, and YLD (Table 4). After filtering by the RMIP threshold, the remaining significant FarmCPU MTAs exhibited an approximate uniform distribution for RMIP values. A significant MTA affecting the trait TWT located on the long arm of 6A had the highest RMIP value of 0.99. Since FarmCPU fits significant MTAs as covariates based partially upon LD estimates, the MTAs identified by this model for a given trait all reside within different putative QTLs. However, several MTAs did PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 10 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Fig 3. Genome-wide FST scan. FST values were estimated for each SNP in the A genome (top row), B genome (middle row), and D genome (bottom row). The dashed line signifies the 99th percentile of FST values for all SNPs. https://doi.org/10.1371/journal.pone.0208217.g003 Fig 3. Genome-wide FST scan. FST values were estimated for each SNP in the A genome (top row), B genome (middle row), and D genome (bottom row). The dashed line signifies the 99th percentile of FST values for all SNPs. lues were estimated for each SNP in the A genome (top row), B genome (middle row), and D genome (bottom row). The dash values for all SNPs. Fig 3. Genome-wide FST scan. FST values were estimated for each SNP in the A genome (top row), B genome (middle row), and D genome (bottom row). The dashed line signifies the 99th percentile of FST values for all SNPs. https://doi.org/10.1371/journal.pone.0208217.g003 Fig 4. Triticum timopheevi private and shared alleles on chromosome 2B. (A) FST plotted against r2 with the KASP Sr36 assay for all SNPs on chromosome 2B. Two clusters of SNPs were manually differentiated—those with putative Triticum timopheevi private alleles for which FST and r2 values show a general linear relationship (blue), and those with putative Triticum timopheevi/Triticum aestivum shared alleles for which FST shows no relationship with r2 (red). Horizontal dashed line represents the 99th percentile of genome-wide FST values, excluding SNPs on chromosomes 2A, 2B, and 2D. (B) Density plots of both classes of SNPs across chromosome 2B. https://doi.org/10.1371/journal.pone.0208217.g004 Fig 4. Triticum timopheevi private and shared alleles on chromosome 2B. (A) FST plotted against r2 with the KASP Sr36 assay for all SNPs on chromosome 2B. Two clusters of SNPs were manually differentiated—those with putative Triticum timopheevi private alleles for which FST and r2 values show a general linear relationship (blue), and those with putative Triticum timopheevi/Triticum aestivum shared alleles for which FST shows no relationship with r2 (red). Horizontal dashed line represents the 99th percentile of genome-wide FST values, excluding SNPs on chromosomes 2A, 2B, and 2D. (B) Density plots of both classes of SNPs across chromosome 2B. Fig 4. Triticum timopheevi private and shared alleles on chromosome 2B. (A) FST plotted against r2 with the KASP Sr36 assay for all SNPs on chromosome 2B. Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Table 3. Significant marker-trait associations identified by the GCTA leave-one-chromosome-out method. Traita Chr Posb cMc Alleles MAF P-value Effect Units References HD 1B 50989662 52.8 G\A 0.29 1.00E-06 0.533 Julian days [72,73] HD 6A 150099119 109.5 A\T 0.17 1.52E-05 -0.507 Julian days [72] HD 6A 542256574 128.5 T\C 0.34 3.39E-05 -0.417 Julian days [74,75] HD 7D 58633321A 84.6 A\G 0.33 3.70E-10 -0.634 Julian days MAT 2B 3266917 0 C\A 0.15 2.35E-05 -0.527 Julian days MAT 7D 58633321A 84.6 A\G 0.33 5.71E-07 -0.495 Julian days SSQM 3A 6788503 12.5 C\T 0.46 1.56E-06 16.0 Spikes m-2 TKW 1A 40498783 68.0 G\T 0.09 1.62E-06 1.25 grams [75,76] TWT 1A 583992305 218.8 T\C 0.23 9.33E-07 -3.39 g L-1 Table 3. Significant marker-trait associations identified by the GCTA leave-one-chromosome-out method. Chr chromosome; Pos physical position; cM genetic position in centiMorgans; Alleles major allele listed first, minor allele second, favorable allele (if any) underline MAF minor allele frequency; Effect mean difference in trait value caused by substituting minor allele for major allele a HD heading date; MAT physiological maturity date; SSQM spikes per square meter; TKW thousand-kernel weight; TWT test weight b Superscript letters indicate QTLs with pleiotropic effects c Genetic position estimated from synthetic W7894 × Opata M85 GBS genetic map aligned to Chinese Spring IWGSC RefSeq v1.0 wheat reference sequence https //doi org/10 1371/jo rnal pone 0208217 t003 Chr chromosome; Pos physical position; cM genetic position in centiMorgans; Alleles major allele listed first, minor allele second, favorable allele (if any) underlined; MAF minor allele frequency; Effect mean difference in trait value caused by substituting minor allele for major allele a HD heading date; MAT physiological maturity date; SSQM spikes per square meter; TKW thousand-kernel weight; TWT test weight b Superscript letters indicate QTLs with pleiotropic effects p p Q p p c Genetic position estimated from synthetic W7894 × Opata M85 GBS genetic map aligned to Chinese Spring IWGSC RefSeq v1.0 wheat reference sequence occur for different traits associated with the same SNP, or else clustered in high-LD regions with pleiotropic effects, and hence the 74 total MTAs are represented by 67 putative QTLs, with five QTLs affecting multiple traits. Of the eight putative QTLs identified by GCTA, four were also identified by FarmCPU. Two clusters of SNPs were manually differentiated—those with putative Triticum timopheevi private alleles for which FST and r2 values show a general linear relationship (blue), and those with putative Triticum timopheevi/Triticum aestivum shared alleles for which FST shows no relationship with r2 (red). Horizontal dashed line represents the 99th percentile of genome-wide FST values, excluding SNPs on chromosomes 2A, 2B, and 2D. (B) Density plots of both classes of SNPs across chromosome 2B. 11 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 https://doi.org/10.1371/journal.pone.0208217.t003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 q y; y g j a HD heading date; MAT physiological maturity date; SSQM spikes per square meter; TKW thousand-kernel weight; TWT test weight b Superscript letters indicate QTLs with pleiotropic effects Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Table 4. Significant marker-trait associations identified by the FarmCPU algorithm. Examination of the Manhattan plots and uniform quantile- quantile plots of the p-values produced by the single-locus model (S1 File) and FarmCPU (S2 File) demonstrated adequate control of p-value inflation for most traits, with FarmCPU prop- erly detecting and correcting for LD between SNPs co-located on QTLs. MTAs identified by GCTA and FarmCPU were distributed sparsely among traits and chro- mosomes (Fig 5). The 1D, 3D, 4B, and 5D chromosomes did not contain significant MTAs for any trait. Chromosomes with high numbers of identified MTAs included: 1A, with two and nine MTAs identified by GCTA and FarmCPU, respectively; 6A, with two and six MTAs iden- tified by GCTA and FarmCPU; 7A, with nine MTAs identified by FarmCPU; and 7D, with two and seven MTAs identified by GCTA and FarmCPU. Traits with high numbers of MTAs identified included: FLSG, with seven MTAs identified by FarmCPU; HD, with four and seven MTAs identified by GCTA and FarmCPU respectively; and TKW, with one and nine MTAs identified by GCTA and FarmCPU. The trait with the lowest number of identified MTAs was NDVI, with two MTAs identified by FarmCPU. We attempted to compare our results against those of recent GWAS studies performed in wheat (references column in Tables 3 and 4). However, the lack of a reference genome in previous studies means that QTLs can only be compared based upon genetic positions. The use of multiple marker types and genetic maps across studies makes comparisons of QTLs difficult; while we were able to compare results based on approximate genetic positions, the evidence of prior discovery in Tables 3 and 4 should be regarded as tenuous. Nevertheless, after surveying past GWAS studies of yield, yield components, and phenological traits, we categorized 4 (44%) of the MTAs detected by GCTA and 20 (26%) of the MTAs detected by FarmCPU as previously-discovered. The traits with the most previously-identified MTAs were HD, MAT, HT, and TKW; the individual MTA that was most consistently identified in prior studies was located at 44,983,849bp (79.0cM) on chromosome 2B, affecting the trait TKW. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 12 / 28 Traita Chr Posb cMc Alleles MAF P-value Effect Units RMIP References FLSG 2A 664114222 101.7 G\C 0.26 4.30E-06 0.198 days 0.12 FLSG 3A 660434027 145.9 T\C 0.07 6.09E-09 0.553 days 0.61 FLSG 4D 389572522 82.9 G\A 0.12 3.37E-07 -0.278 days 0.19 FLSG 6A 13888173 26.4 A\C 0.06 3.16E-07 0.487 days 0.33 FLSG 7A 727690323 222.7 G\A 0.16 3.65E-06 -0.217 days 0.14 FLSG 7B 59821499 61.8 C\T 0.37 3.84E-07 0.229 days 0.12 FLSG 7B 64138826 62.4 A\G 0.17 7.28E-06 0.235 days 0.37 GSQM 1A 103223278 85.0 A\T 0.43 1.25E-08 -309 Grains m-2 0.37 GSQM 5B 396479359 104.4 C\T 0.46 5.71E-06 -246 Grains m-2 0.14 GSQM 7A 673460466A 151.6 C\T 0.46 1.89E-07 256 Grains m-2 0.72 GSQM 7B 444463299B 82.4 A\G 0.06 2.03E-06 -650 Grains m-2 0.25 GSQM 7D 117245582 131.0 T\G 0.08 3.38E-06 450 Grains m-2 0.17 GW 2A 768420387 162.2 G\C 0.38 9.84E-06 0.467 g dwt m-1 row 0.22 GW 3A 704973519 192.7 A\G 0.12 4.29E-06 0.441 g dwt m-1 row 0.16 GW 5B 34551165 61.2 C\T 0.38 7.62E-08 -0.494 g dwt m-1 row 0.31 GW 7A 55399749 61.5 A\G 0.17 8.56E-08 0.651 g dwt m-1 row 0.23 HD 1B 50989662 52.8 A\G 0.29 1.04E-09 0.438 Julian days 0.31 [72,73] HD 2D 35084672 46.8 G\A 0.12 1.30E-07 -0.415 Julian days 0.66 HD 5B 710175294 312.9 A\C 0.05 9.92E-07 0.797 Julian days 0.16 HD 6A 449693253C 112.4 C\T 0.34 2.01E-06 0.317 Julian days 0.58 [72] HD 7B 97678492 65.4 G\A 0.05 1.43E-06 -0.862 Julian days 0.38 [30] HD 7D 58633321D 84.6 A\G 0.32 3.00E-13 -0.517 Julian days 0.78 [72] HD 7D 553154931 173.3 C\T 0.4 1.57E-06 -0.314 Julian days 0.22 [73] HT 1A 13666754 29.4 T\G 0.06 4.91E-08 -1.83 cm 0.38 [77] HT 2A 764324892 158.4 T\A 0.22 5.36E-06 -1.05 cm 0.40 [78] HT 2D 181663205 100.4 C\A 0.49 1.13E-07 2.21 cm 0.58 HT 6B 706327428 160.6 A\G 0.07 5.73E-07 1.53 cm 0.31 [78] MAT 2A 7231537 3.0 G\C 0.4 2.92E-10 0.460 Julian days 0.36 MAT 2B 4475209 1.8 T\A 0.48 3.55E-08 -0.315 Julian days 0.25 MAT 3B 737436815 178.2 G\A 0.38 2.77E-09 -0.380 Julian days 0.73 MAT 7B 232267370 75.5 G\A 0.11 3.52E-07 -0.486 Julian days 0.17 MAT 7D 58633321D 84.6 A\G 0.32 1.27E-07 -0.364 Julian days 0.47 [72] NDVI 1A 251018060 88.4 C\T 0.07 8.85E-07 -0.00799 - 0.79 NDVI 7A 127596189 104.1 T\C 0.11 7.94E-08 -0.00481 - 0.11 PROT 1A 465502281 103.5 A\G 0.36 1.13E-06 0.0641 % 0.31 PROT 5A 594957276 119.2 A\G 0.11 1.13E-06 0.109 % 0.14 PROT 6A 72549494 92.2 G\A 0.34 3.83E-07 -0.0932 % 0.21 PROT 7A 169622110 111.9 C\A 0.07 8.50E-06 -0.114 % 0.12 SPH 1A 52268667E 75.1 G\A 0.2 4.53E-06 -0.449 count 0.31 SPH 1A 507879685 130.6 G\A 0.1 2.33E-06 0.571 count 0.20 SPH 3B 189122341 118.9 T\A 0.07 1.18E-05 0.715 count 0.34 [79] SPH 4A 672693794 151.3 A\C 0.22 8.87E-14 -0.830 count 0.32 SPH 4D 217349774 81.6 T\G 0.06 3.67E-09 -1.05 count 0.68 SPH 5B 617822981 227.0 C\A 0.22 9.20E-11 -0.917 count 0.52 SPH 7A 672644138A 151.0 A\G 0.4 9.39E-09 -0.476 count 0.96 [80] SSQM 1A 49502948E 73.8 T\C 0.11 4.33E-12 27.2 Spikes m-2 0.56 (Continued) Table 4. (Continued) Traita Chr Posb cMc Alleles MAF P-value Effect Units RMIP References SSQM 1A 372638734 92.9 T\C 0.07 7.38E-11 29.1 Spikes m-2 0.54 [73] SSQM 2A 40249083 50.0 A\T 0.06 4.07E-07 31.3 Spikes m-2 0.18 SSQM 3A 6788503 12.5 T\C 0.46 4.25E-11 15.8 Spikes m-2 0.35 SSQM 6A 466270386C 112.9 G\A 0.34 9.45E-06 -11.9 Spikes m-2 0.29 SSQM 7D 216134381 148.1 A\C 0.07 2.90E-06 -20.1 Spikes m-2 0.11 STARCH 1B 39382550 47.3 G\A 0.22 3.05E-07 -0.139 % 0.72 STARCH 2A 620961503 93.1 G\A 0.05 7.61E-06 -0.188 % 0.25 STARCH 2D 633668158 151.2 G\T 0.07 7.23E-07 0.177 % 0.22 STARCH 4A 122852500 59.0 A\G 0.05 1.43E-07 -0.241 % 0.31 STARCH 4A 581248758 94.3 G\A 0.05 9.61E-09 -0.315 % 0.31 STARCH 7A 196456932 113.6 T\G 0.05 6.42E-06 -0.185 % 0.59 TKW 2A 740238876 138.6 C\T 0.37 1.25E-05 0.513 grams 0.25 [81–84] TKW 2B 44983849 79.0 A\G 0.38 2.97E-07 -0.770 grams 0.12 [28,77,84,85] TKW 4A 379041624 64.6 A\C 0.06 1.23E-05 -1.17 grams 0.44 [85,86] TKW 6A 617591341 215.2 G\T 0.21 1.43E-08 0.703 grams 0.84 TKW 6B 687528310 133.5 C\G 0.48 3.45E-06 -0.562 grams 0.36 TKW 6D 467652883 184.8 C\T 0.33 1.19E-07 0.773 grams 0.95 TKW 7A 672856129A 151.2 C\T 0.46 1.83E-07 -0.542 grams 0.36 [80] TKW 7B 444463299B 82.4 A\G 0.06 5.85E-07 1.46 grams 0.51 [28] TKW 7D 197300822 147.6 G\T 0.09 1.33E-06 0.772 grams 0.33 [28] TWT 1A 583992305 218.8 C\T 0.23 1.69E-08 -2.67 g L-1 0.89 TWT 3A 477707675 104.2 A\G 0.05 3.65E-08 -5.40 g L-1 0.56 TWT 6A 614373502 208.5 A\G 0.34 6.58E-12 -3.14 g L-1 0.99 TWT 6B 12668423 8.5 G\A 0.34 2.81E-08 2.77 g L-1 0.42 YLD 2B 4638412 2.2 G\A 0.08 9.69E-06 118 kg ha-1 0.18 YLD 6A 355929298 111.9 G\C 0.08 1.36E-06 -110 kg ha-1 0.38 YLD 6B 656280645 110.7 C\T 0.14 9.23E-06 -75.7 kg ha-1 0.50 [80] YLD 7A 30243496 32.8 C\T 0.28 2.96E-07 -66.9 kg ha-1 0.29 [81,84] YLD 7D 66984783 93.8 C\A 0.31 6.41E-08 67.4 kg ha-1 0.71 Chr chromosome; Pos physical position; cM genetic position in centiMorgans; Alleles major allele listed first, minor allele second, favorable allele (if any) highlighted in bold; MAF minor allele frequency; Effect mean difference in trait value caused by substituting minor allele for major allele; RMIP resampling model inclusion probability probability a FLSG flag leaf stay green; GSQM grains per square meter; GW grain weight; HD heading date; HT plant height; MAT physiological maturity date; NDVI normalized- difference vegetation index at Zadok’s GS25; PROT whole-grain protein content; SPH seeds per head; SSQM spikes per square meter; STARCH whole-grain starch content; TKW thousand kernel weight; TWT test weight; YLD grain yield b Superscript letters indicate QTLs with pleiotropic effects c Genetic position estimated from synthetic W7894 × Opata M85 GBS genetic map aligned to Chinese Spring IWGSC RefSeq v1.0 wheat reference sequence Significant marker-trait associations identified by the FarmCPU algorithm. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 13 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Table 4. Chr chromosome; Pos physical position; cM genetic position in centiMorgans; Alleles major allele listed first, minor allele second, favorable allele (if any) highlighted in bold; MAF minor allele frequency; Effect mean difference in trait value caused by substituting minor allele for major allele; RMIP resampling model inclusion MTA haplotypes and translation effects The GCTA and FarmCPU analyses identified a total of 77 significant MTAs, 35 of which were located on haplotype blocks containing two or more SNPs (S4A Table). One haplotype block spanning a 2Mb segment on chromosome 7A contained three significant MTAs located at 672,644,138bp, 672,856,129bp, and 673,460,466bp. The set of SNPs involved in MTAs over- lapped 48 genes encoding 68 transcripts, and produced a total of 109 predicted transcriptional PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 14 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Fig 5. Distribution of MTAs across traits and chromosomes. The number of MTAs detected by the GCTA single- locus model (preceding slashes) and by the FarmCPU model (following slashes) are summed for each trait/ chromosome combination. Grey shading indicates that no MTAs were found for that particular trait/chromosome combination. Red shading is proportional to the total number of MTAs detected by both models. Marginal totals of MTAs are displayed for each trait and chromosome, with the grand total displayed in the lower right cell. https://doi.org/10.1371/journal.pone.0208217.g005 Fig 5. Distribution of MTAs across traits and chromosomes. The number of MTAs detected by the GCTA single- locus model (preceding slashes) and by the FarmCPU model (following slashes) are summed for each trait/ chromosome combination. Grey shading indicates that no MTAs were found for that particular trait/chromosome combination. Red shading is proportional to the total number of MTAs detected by both models. Marginal totals of MTAs are displayed for each trait and chromosome, with the grand total displayed in the lower right cell. https://doi.org/10.1371/journal.pone.0208217.g005 https://doi.org/10.1371/journal.pone.0208217.g005 consequences due to alternative splicing (S4C Table). The most severe predicted consequences were isolated and compiled for each SNP. A total of 56 of the SNPs involved in significant MTAs (73%) were intergenic, and of these intergenic SNPs, 15 were labeled as upstream or downstream proximal variants (i.e. within 5Kb of the start or end of a gene). The remaining 21 SNPs (27%) were located within genes, with 11 of these occurring in introns, one occurring in a 5’ untranslated region, two occurring in 3’ untranslated regions, and the remaining seven causing missense mutations within exons. MTA haplotypes and translation effects Putative functions for genes containing SNPs caus- ing missense substitutions include a disease resistance protein, a RNA binding protein, a ubi- quitin-family protein, a chloroplast membrane translocase, an aspartyl protease family protein, a glutamate receptor, and a Myb transcription factor. In addition, a total of 155 wheat genes of known function were located within the empirically-derived 1Mb radius of significant LD sur- rounding the set of significant MTAs (S4D Table). Discussion These MTAs were also not present in the FarmCPU results which were discarded due to low RMIP values, suggesting the possibility of false negatives in the set of significant MTAs returned by FarmCPU. While FarmCPU did indeed identify many more significant MTAs than the GCTA mixed linear model, its methodology is more complex and less transparent to the user, and hence we still recommend using a single-locus mixed linear model, or perhaps a simpler multi-locus mixed model [18] to compare results. In the present study, many previously-characterized loci of agronomic importance interro- gated with KASP-SNP assays had significant effects on multiple traits (S3B Table). Loci that significantly affected many traits included the Sr36 stem rust resistance gene and the TaSus2- 2B sucrose synthase gene (both assaying the presence/absence of the 2G:2B T. timopheevi translocation), the 1AL:1RS and 1BL:1RS translocations, the Ppd-B1 and Ppd-D1 photosensi- tivity genes, the Rht-B1 and Rht-D1 dwarfing genes, and polymorphisms within the Vrn-A1 vernalization gene. However, despite their significant effects on many traits, the KASP assays for these loci were not among the significant MTAs identified in the current study. Some potential explanations for these results are presented below. The two Rht dwarfing genes Rht-B1b and Rht-D1b mostly occurred in repulsion within the testing panel. Of the 322 lines included in the panel, 22 had neither the Rht-B1b nor the Rht- D1b dwarfing alleles, one line was heterozygous for both alleles, and one line was homozygous for both alleles, with the rest being homozygous for either one dwarfing allele or the other. Note that lines lacking both Rht-B1b and Rht-D1b may contain other dwarfing genes which were not assayed in this study. At face value, this high degree of repulsion led to the somewhat odd finding that the presence of the Rht-B1b allele increased height and decreased yield when allelic effects were calculated for each of the KASP assays individually. A subsequent ANOVA pooling together the two dwarfing alleles revealed that at an alpha level of 0.05, lines with either Rht-B1b or Rht-D1b were significantly shorter and yielded significantly higher than lines that were wild-type for both genes. Yield was significantly higher for lines with only Rht-D1b vs. those with only Rht-B1b, though height was not significantly different between lines with these dwarfing genes. (These analyses excluded lines that were heterozygous for either allele, and the single line that was homozygous for both alleles). Discussion The current study demonstrates that the recent availability of the wheat reference genome will greatly facilitate reliable QTL curation and comparisons between studies. The ability to describe MTAs in terms of both physical and genetic positions removes much of the ambiguity associated with earlier studies which relied solely on genetic positions to describe QTLs. The availability of a reference genome also enables more accurate QTL and haplotype boundary delineation and comparative genomics analyses. We were able to use positional information both to identify GBS SNPs that could potentially be used as substitutes for existing KASP SNP assays, and to identify candidate genes located near significant MTAs. Although it was difficult PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 15 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia to compare the QTLs we identified with those discovered in previous studies, we do believe that many of the QTLs we detected were previously-characterized. These QTLs mainly affected HD, MAT, HT, and TKW, perhaps reflecting the relative ease of measurement for these traits, and the very low measurement error inherent to TKW. This study evaluated both a traditional mixed linear model and the more-recently devel- oped FarmCPU algorithm for performing GWAS. While a thorough comparison of these two methods is beyond the scope of this article, some general recommendations will be discussed. The authors of FarmCPU suggested that it can increase the power to detect causative geno- type-phenotype associations over traditional mixed linear model methods while limiting the false-discovery rate and type I error rate [21]. A subsequent study compared single-locus mixed linear models, FarmCPU and the BayesCπ Bayesian model developed by Habier et al. [87], and found that all three offered comparable power for simple traits controlled by a few QTL, with FarmCPU offering enhanced power for moderately complex traits, and BayesCπ exhibiting the highest power for complex, highly polygenic traits [88]. Based upon our experiences, we recommend exercising some caution when using Farm- CPU. Our experiences support the practice of bootstrapping of results if computational resources allow; out of 108 total MTAs identified by FarmCPU, 34 (31.5%) had RMIP values below our selected threshold of 10%. Additionally, the traditional mixed linear model identi- fied four significant MTAs which were not identified by FarmCPU. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Discussion This perhaps serves as a good reminder of how population-specific epistasis can prevent the accurate estimation of many allele effects. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 16 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia While multi-locus GWAS models may partially ameliorate these problems in some cases, the careful choice of experimental design, such as the use of nested association mapping panels [89], is likely a better solution. In the present study, both the Sr36 and TaSus2-2B genes were highly confounded with the testing panel’s population structure. A SNP located at 44,983,849bp on chromosome 2B was in moderate LD with both Sr36 and TaSus2-2B (r2 = 0.24), and had the expected significant impact on TKW, given the known effects that TaSus2-2B exerts on kernel weight [13]. The 2G:2B translocation from T. timopheevi on which the Sr36 gene resides introduced a large alien haplotype into the panel germplasm. Cavanagh et al. [90] found that Sr36 was associated with a large segment of segregation distortion on chromosome 2B in seven different mapping populations. In the present study, splitting the population by the presence or absence of the Sr36 gene and subsequently performing a genome-wide FST scan (Fig 3) revealed a large high- LD block spanning most of chromosome 2B, containing many SNPs which could almost per- fectly differentiate between lines with and without the 2G:2B translocation. In addition, an enrichment in high-FST SNPs was observed on chromosomes 2A and 2D, likely due to SNP misalignments between chromosomes within this homeologous group. On chromosome 2B, there was a general positive linear trend between a SNP’s FST score and its LD with the Sr36 KASP marker. This was as expected, as the presence or absence of this marker was used to par- tition the subpopulations. However, a smaller subset of SNPs simultaneously exhibited high FST and an r2 value with the KASP Sr36 assay that approached zero. These SNPs were distrib- uted throughout the translocation (Fig 4). It is likely that SNPs with a high FST value that are also in high LD with the Sr36 KASP marker share a common private haplotype allele inherited from T. timopheevi, while those with a high FST are in low LD with the Sr36 KASP assay repre- sent a set of SNPs with shared alleles between T. aestivum and T. timopheevi, where recombi- nation rates are higher. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Discussion This high LD suggests that these GBS markers, or others in close proximity, could be used as suit- able indicators for the 1AL:1RS and 1BL:1RS translocations. Although further validation is required, ideally GBS markers, individually or in groups, could be used to ascertain the pres- ence or absence of these translocations without necessitating the use of the additional stand- alone assays that are currently performed. The effects of previously-characterized genes affecting phenological traits were likewise insignificant in the GWAS, likely due to a variety of factors. Polymorphisms of Vrn-A1 exon 7 and Vrn-B1 occurred at low frequencies of 0.03 and 0.07 within the testing panel, respectively, most likely precluding their detection in the GWAS. The Vrn-A1 exon 4 SNP had a higher minor allele frequency of 0.14, and was in moderate LD (r2 = 0.29) with one GBS SNP located at 594,957,276bp on chromosome 5A. The Vrn-A1 KASP assays used in this study may simply have not been predictive enough to adequately distinguish among different vernalization alleles present at the Vrn-A1 locus. The Ppd loci were likewise never identified as significant, most likely due to masking from the predominance of long-vernalizing genotypes in the panel. However, a BLAST analysis indicated that the GBS SNP located at 35,084,672bp on chromo- some 2D, which FarmCPU identified as significant for the trait HD, was located within approximately 1Mb of Ppd-D1. As previously mentioned, there was evidence for pleiotropic effects involving multiple SNPs and traits in both the GCTA and FarmCPU results (Tables 3 and 4). The sole region of pleiotropic effect identified by GCTA was a QTL located at approximately 58.5Mb (84.6 cM) on chromosome 7D, affecting the phenological traits HD and MAT. The haplotype block anal- ysis indicated that this QTL spanned a region of roughly 1.5Mb. This QTL was also identified by FarmCPU, and was one of the more consistently significant QTLs identified, with a RMIP value of 0.78. Many of the other pleiotropic QTLs identified by FarmCPU demonstrated trait compensation effects relating to grain number and grain weight. For example, the minor allele of a QTL located at approximately 444Mb (82.4cM) on chromosome 7B produced an increase in TKW and a decrease in GSQM, while the minor allele of a QTL at approximately 50Mb (75cM) on chromosome 1A produced an increase in SSQM, but a slight decrease in SPH. Discussion Despite the effects of the 2G:2B translocation in the current study, population structure was generally subdued, as evidenced by principal component plots of the LD-thinned dataset, where the first principal component explained only 3.68% of total varia- tion (Fig 1B). This finding is in line with those of previous studies examining population struc- ture in elite European winter wheat germplasm [91,92]. This suggests extensive past admixture among the lines included in the population, which is as expected given the frequent germplasm exchanges that are typical of public small grains breeding programs. A previous report by Sukumaran et al. [31] found much more pronounced population structure effects due to rye translocations, using a panel of elite spring germplasm which clus- tered into two distinct sub-populations explained by the presence or absence of the 1BL:1RS translocation. In addition, Sukumaran et al. found that the 1BL:1RS translocation explained significant differences among the two subpopulations for most of the traits included in their study (e.g. grain yield, grain number, grain weight, plant height, and several phenological traits). The 1AL:1RS and 1BL:1RS translocations have been associated with desirable disease and insect resistance traits, as well as drought and general environmental stress resistance. However, the 1BL:1RS translocation has been associated with lateness, and effects on yield due to these translocations may be manifested depending upon environment and genetic back- ground [93–95]. While both the 1AL:1RS and 1BL:1RS translocations did produce significant differences for many traits in this study (S3B Table), their contributions to population struc- ture were not noticeable in comparison to the effects of the 2G:2B translocation (data not shown). In addition, the indicator KASP markers for these translocations did not produce sig- nificant p-values in either the GCTA or FarmCPU GWAS. The 1AL:1RS translocation occurred at a very low frequency (0.07) within the tested germplasm, while the 1BL:1RS trans- location occurred at a frequency of 0.19. In the study of Sukumaran et al. [31], the 1BL:1RS translocation occurred at a frequency of 0.39. In the present study, the KASP markers for PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 17 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 1AL:1RS and 1BL:1RS were in high LD (r2 approximately 0.8) with the SNPs located at 103,223,278bp on chromosome 1A and 39,382,550bp on chromosome 1B respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Discussion The minor allele of a QTL on chromosome 7A at approximately 673Mb (151cM) caused a decrease in TKW, and an increase in GSQM. It also apparently produced slight decrease in SPH, though the effects of this locus will be discussed in greater detail below. Finally, a large region on chro- mosome 6A affected the relatively uncorrelated traits HD and SSQM. It is not clear whether this region is being affected by long-range LD, or simply physical linkage, as the distance between the two MTAs involved is approximately 16.5Mb. A simple pairwise LD analysis indi- cated that these SNPs were in moderate LD with each other, though the haplotype block analy- sis placed them in separate haplotypes. Due to the large number of significant findings, we limit further discussion of the GWAS results to several MTAs in close proximity to plausible candidate genes (S5 Table). The previ- ously-mentioned pleiotropic region on chromosome 7A affecting the traits TKW, SPH, and GSQM spanned a distance of approximately 1.5Mb, containing three MTAs. FarmCPU identi- fied one significant SNP within this haplotype with a RMIP value of 0.96. The 7A pleiotropic region contains a total of 22 genes, only three of which have listed functions in UniProt. How- ever, the IWGSC’s automated gene functional annotation process [34,96] had previously iden- tified one gene within this haplotype as a likely ortholog of the aberrant panicle organization 1 (APO1) protein, which plays an important role in panicle architecture and development in rice (Oryza sativa L.). The loss of function of APO1 results in rice plants which produce small pani- cles with a lower number of inflorescence branches and spikelets [97]. The minor alleles for PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 18 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia two of the MTAs in this region decreased TKW and increased GSQM. We also found that the minor allele of the third MTA in this region decreased SPH, which was against expectation. However, the high MAF of all three MTAs within this QTL (0.4 and 0.46) suggested the possi- bility that at least one was actually in trans linkage with the other two. Upon closer inspection of the SNP data, we discovered that this was indeed the case, with the SNP associated with SPH in trans configuration with the others. Discussion Therefore, we detected two primary haplotypes present at this QTL with frequencies close to 0.5: one increasing GSQM and SPH while decreasing TKW, and the other with the opposite effect. The MTAs for SPH and TKW are not in perfect LD; a total of 40 lines (12.4%) exhibited recombination between these SNPs. The SNP associated with TKW within the 7A QTL is predicted to cause a missense protein transla- tion effect within an aspartyl protease family protein, though it is not known whether this is relevant to the QTL’s phenotypic effects. The fact that FarmCPU fit different covariates for dif- ferent traits within this region raises the possibility that this QTL’s effects could be due to mul- tiple candidate genes, rather than a single gene such as APO1 exhibiting pleiotropic effects. However, we cannot at this time test this hypothesis. Chromosome 7B also contained a large number of putative candidate genes for multiple traits. A pectin esterase was located in close proximity (67Kb) with a MTA affecting FLSG located at 64Mb (62.4cM). Due to the ubiquity of pectin within plant cell walls, pectin esterases have been associated with a wide range of cellular processes (reviewed in [98]). Notably, pectin esterases have been found to play a role in fruit maturation in heat-stressed tomato plants [99], as well as the initiation of flowering in day lilies [100]. Another MTA on 7B located at 97Mb (65.4cM), affecting the trait HD, was within 485Kb of a MYB transcription factor. The MYB family of proteins function in a wide number of signal transduction pathways in plants. Gib- berellin-interacting MYB factors (GAMYBs) were first identified in barley (Hordeum vulgare L.), inducing the activation of alpha-amylase in the aleurone tissue of grains [101]. GAMYB factors were subsequently identified in wheat [102]. Currently, GAMYB factors have been putatively implicated in contributing to flowering initiation, though their role in this process is not well understood. Gocal et al. [103] suggested that a GAMYB protein in the grass species Lolium temulentum L. was an important component in signaling pathways for flower develop- ment. However, Kaneko et al. [104] later found that while rice GAMYBs were important in normal flower organ development, knockout mutants did not display any differences from wild-type plants in the timing of flower development. Therefore, while GAMYB factors may play a role in wheat floral development, their exact function in this process remains unknown. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Discussion A pleiotropic SNP located at approximately 444Mb (82.4cM) on chromosome 7B exerted large effects on the traits GSQM and TKW, and was intronic within an ortholog of an intracellular protein transporter identified in Medicago truncatula Gaertn. While this SNP is not associated with a haplotype block, it is located 170Kb from a glycosyltransferase gene. The glycosyltrans- ferases are a superfamily consisting of thousands of identified proteins. Notably, multiple fami- lies of glycosyltransferases have been associated with grain development in wheat [105]. On chromosome 7D, a SNP located at approximately 59Mb (84.6cM) was identified by both the single-locus and FarmCPU models, produced pleiotropic effects for both HD and MAT, had a large effect size magnitude of approximately 0.5 days, and produced a RMIP value of 0.78 for HD. Due to its exertion of effects with the same sign on both HD and MAT, it is likely that this locus could be involved with plant vernalization or photoperiod response. While this SNP is approximately 10Mb away from the VRN3 vernalization response/flowering time gene, a significant MTA located at 66,984,783bp on 7D affecting yield is located only 1.5Mb away from VRN3, and there is no significant LD between these two SNPs, making it likely that some other as yet undetermined causal agent is underlying this locus’ effects on heading and maturation dates. This SNP is intronic within an ortholog of the Escherichia coli PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 19 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha gene, which is involved in fatty acid synthesis via the synthesis of malonyl-CoA [106], though any connection between this function and vernalization response in plants remains unknown. Several other MTAs were in close proximity to candidate genes that have previously been functionally characterized in wheat. One MTA on chromosome 1A affecting the trait GSQM was located 409Kb away from the gibberellin oxidase gene TaGA2ox-A1. Gibberellins are an important family of plant hormones, which have many functions in plant growth and develop- mental processes, including a crucial role in grain volume increase in wheat [107]. The GA2ox family of oxygenases play a role in deactivating gibberrelins, and paralogs of many GA2ox genes have been identified in all three genomes of hexaploid wheat [108]. Discussion On Chromosome 3A, the GCTA and FarmCPU models both detected a MTA affecting the trait SSQM, located within 506Kb of the gene TaMFT. This gene, an ortholog of the Arabidopsis thaliana gene MFT, encodes a protein which functions as a key promoter of seed dormancy and suppressor of precocious seed germination during seed development [109]. Finally, one MTA located on chromosome 1A, affecting the trait SPH, is located within 76Kb of TaB2, a wheat ortholog of a protein first isolated from carrot (Daucus carota L.) [110]. In wheat, B2 proteins were found to function as heat stress response proteins, and are highly upregulated in developing seeds dur- ing and for several days following the application of heat stress [111]. Additionally, TaB2 was found to influence plant growth and development upon transformation into Arabidopsis thali- ana [112]. Taken together, these findings suggest a viable set of candidate genes and QTLs that may be exploited to increase yield in soft winter wheat breeding. The recent use of CRISPR-Cas9 genome editing systems in wheat (e.g. [113–116]) may allow for rapidly testing the functions of the candidate genes mentioned above without the need for expensive and time-consuming fine mapping and gene cloning. The finding of multiple MTAs affecting phenological traits suggests that there is still variation in the tested elite germplasm that can be exploited in breed- ing programs to fine-tune the timing of growth stages and grain fill duration, perhaps allowing for finer control of maturation date to maximize grain fill time while avoiding heat stress dur- ing kernel formation. The findings regarding traits relating to grain density per unit area and grain weight give cause for hope, while also suggesting strategies for ongoing germplasm improvement. Many MTAs for these traits appeared to entail no significant tradeoff between grain number and grain size. These included MTAs for GSQM on chromosomes 1A and 5B, MTAs for SSQM on chromosomes 1A, 2A, 3A and 7D, and MTAs for TKW on chromosomes 2A, 2B, 4A, 6A, 6B, 6D and 7D. Barring any epistatic effects, favorable alleles for these QTLs could gradually be combined within the tested elite germplasm pool. In contrast, several QTLs located on chromosomes 1A, 7A, and 7B exerted pleiotropic effects for these traits, with favor- able alleles in a trans configuration. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Discussion If these QTL represent tightly trans-linked genes, then they may eventually be exploited if the component genes are broken up by recombination over generations. However, QTLs that represent true pleiotropic effects of a single gene will likely not be useful for achieving genetic gain. Many of the MTAs for GSQM, TKW, and particularly SSQM had favorable minor alleles, suggesting either that breeders have unintentionally per- formed selections which favored the less-desirable allele, or that these favorable alleles initially occurred at low frequencies and remain rare due to a lack of selection pressure. While the availability of a reference genome allows for previously impossible follow-up analyses, this study also identifies a number of areas for continued improvement for GWAS experiments in wheat. For instance, the genome-wide FST scan based upon the presence or absence of Sr36 revealed multiple SNPs which were misaligned between the group 2 homeolo- gous chromosomes, and therefore non-allelic. In addition to the obvious problem of poten- tially identifying significant MTAs on the wrong chromosome, these SNPs complicate the PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 20 / 28 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia process of haplotype estimation, as they may artificially “break” patterns of strong LD within haplotype blocks. There is currently no simple solution to identify misaligned SNPs within GBS datasets, as discarding sequencing reads that don’t uniquely map to a single location may entail discarding a large portion of data. However, several methods for identifying non-allelic SNPs in polyploids are under development (e.g. [117,118]). Finally, although the availability of a reference genome has greatly aided the interpretation of GWAS studies in wheat, it should be noted that the causal variants underlying the majority of significant MTAs identified in this study remain unknown. As the amount of bioinformatics data available for wheat increases, this situation may improve through the increasing availability of gene expression data and gene ontology information, enabling the use of new techniques such as gene set analysis. Conclusions The significant MTAs reported in this study indicate that there is still genetic variation in the tested elite germplasm that may be exploited for yield gains. In particular, the combination of identified MTAs affecting traits relating to grains per unit area and phenological development offer promise for increasing the former while avoiding the penalizing effect of lower average grain weights. In addition, this study suggests that GBS markers can be used to capture much of the variance explained by previously-characterized polymorphisms of major effect. We made use of the first reference genome assembled for wheat, enabling the identification of MTAs based on both physical and genetic positions; it is hoped that the ability to anchor MTAs by physical position will lead to better curation of results and consistency across GWAS studies in the future. This study also identifies some potential targets for future in vitro studies to ascertain the biological functions of several candidate genes affecting yield-related traits in wheat. Future challenges will include the proper design of GBS or other genotyping assays to capture the effects of previously-characterized polymorphisms while simultaneously allowing for the discovery of novel polymorphisms affecting traits of interest, better identification of non-allelic SNPs which are misaligned between homeologous chromosomes, introgression of multiple favorable alleles into suitable genetic backgrounds, and more thorough characteriza- tion of gene functions to ease the identification of candidate genes following association analyses. Author Contributions Conceptualization: Brian P. Ward, Clay H. Sneller, Carl A. Griffey. Data curation: Brian P. Ward, Priyanka Tyagi. Formal analysis: Brian P. Ward, Priyanka Tyagi. Funding acquisition: Brian P. Ward, Gina Brown-Guedira, Clay H. Sneller, Carl A. Griffey. Investigation: Brian P. Ward, Carl A. Griffey. Methodology: Brian P Ward Carl A Griffey Conceptualization: Brian P. Ward, Clay H. Sneller, Carl A. Griffey. Data curation: Brian P. Ward, Priyanka Tyagi. Formal analysis: Brian P. Ward, Priyanka Tyagi. Methodology: Brian P. Ward, Carl A. Griffey. Project administration: Clay H. Sneller, Carl A. Griffey. Resources: Gina Brown-Guedira, Frederic L. Kolb, David A. Van Sanford, Clay H. Sneller, Carl A. Griffey. Supervision: Gina Brown-Guedira, Carl A. Griffey. Visualization: Brian P. Ward. Writing – original draft: Brian P. Ward. Writing – review & editing: Brian P. Ward, Gina Brown-Guedira, Carl A. Griffey. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 S1 Table. List of germplasm tested in the study. (XLSX) S2 Table. Description of traits examined in the study, with trait ontologies as described in http://www.planteome.org/. (XLSX) S3 Table. Design of KASP SNP assays for interrogating previously-characterized loci of major effect, and a summary of the allelic effects of these loci. (XLSX) S3 Table. Design of KASP SNP assays for interrogating previously-characterized loci of major effect, and a summary of the allelic effects of these loci. (XLSX) S3 Table. Design of KASP SNP assays for interrogating previously-characterized loci of major effect, and a summary of the allelic effects of these loci. (XLSX) S4 Table. List of genes overlapping significant SNPs and haplotype blocks, predicted pro- tein translation effects for all significant SNPs, and list of all wheat genes with annotations in UniProt occurring within 1Mb of significant MTAs. (XLSX) S4 Table. List of genes overlapping significant SNPs and haplotype blocks, predicted pro- tein translation effects for all significant SNPs, and list of all wheat genes with annotations in UniProt occurring within 1Mb of significant MTAs. (XLSX) S4 Table. List of genes overlapping significant SNPs and haplotype blocks, predicted pro- tein translation effects for all significant SNPs, and list of all wheat genes with annotations in UniProt occurring within 1Mb of significant MTAs. (XLSX) 21 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia S1 Fig. Individual and cumulative portions of variance explained by the first 25 principal components of the imputed genotypic data, prior to LD-based filtering. (TIFF) S2 Fig. Number of SNPs per chromosome following the application of all genotypic data filtering steps. (TIFF) S1 File. Manhattan and QQ plots for SNP p-values generated by the GCTA leave-one-chro- mosome-out (LOCO) mixed linear model GWAS. (PDF) S2 File. Manhattan and QQ plots for SNP p-values generated by the FarmCPU GWAS. (PDF) S2 Fig. Number of SNPs per chromosome following the application of all genotypic data filtering steps. (TIFF) S1 File. Manhattan and QQ plots for SNP p-values generated by the GCTA leave-one-chro- mosome-out (LOCO) mixed linear model GWAS. (PDF) S2 File. Manhattan and QQ plots for SNP p-values generated by the FarmCPU GWAS. (PDF) 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x 8. Borra´s L, Slafer GA, Otegui ME. Seed dry weight response to source-sink manipulations in wheat, maize and soybean: a quantitative reappraisal. Field Crops Res. 2004; 86: 131–146. https://doi.org/ 10.1016/j.fcr.2003.08.002 9. Reynolds MP, Pellegrineschi A, Skovmand B. Sink-limitation to yield and biomass: a summary of some investigations in spring wheat. Ann Appl Biol. 2005; 146: 39–49. https://doi.org/10.1111/j.1744- 7348.2005.03100.x 10. Slafer G, Calderini D, Miralles D. Yield components and compensation in wheat: opportunities for fur- ther increasing yield potential. Increasing yield potential in wheat: Breaking the Barriers. 1996. pp. 101–133. 11. Silva SA, de Carvalho FIF, Caetano V da R, de Oliveira AC, de Coimbra JLM, de Vasconcellos NJS, et al. Genetic basis of stay-green trait in bread wheat. J New Seeds. 2001; 2: 55–68. https://doi.org/10. 1300/J153v02n02_05 12. Guedira M, Xiong M, Hao YF, Johnson J, Harrison S, Marshall D, et al. Heading date QTL in winter wheat (Triticum aestivum L.) coincide with major developmental genes VERNALIZATION1 and PHO- TOPERIOD1. PLOS ONE. 2016; 11: e0154242. https://doi.org/10.1371/journal.pone.0154242 PMID: 27163605 13. Jiang Q, Hou J, Hao C, Wang L, Ge H, Dong Y, et al. The wheat (T. aestivum) sucrose synthase 2 gene (TaSus2) active in endosperm development is associated with yield traits. Funct Integr Geno- mics. 2011; 11: 49–61. https://doi.org/10.1007/s10142-010-0188-x PMID: 20821031 14. Su Z, Hao C, Wang L, Dong Y, Zhang X. Identification and development of a functional marker of TaGW2 associated with grain weight in bread wheat (Triticum aestivum L.). Theor Appl Genet. 2011; 122: 211–223. https://doi.org/10.1007/s00122-010-1437-z PMID: 20838758 15. Ma D, Yan J, He Z, Wu L, Xia X. Characterization of a cell wall invertase gene TaCwi-A1 on common wheat chromosome 2A and development of functional markers. Mol Breed. 2012; 29: 43–52. https:// doi.org/10.1007/s11032-010-9524-z 16. Myles S, Peiffer J, Brown PJ, Ersoz ES, Zhang Z, Costich DE, et al. Association mapping: critical con- siderations shift from genotyping to experimental design. Plant Cell Online. 2009; 21: 2194–2202. https://doi.org/10.1105/tpc.109.068437 PMID: 19654263 17. Yu JM, Pressoir G, Briggs WH, Bi IV, Yamasaki M, Doebley JF, et al. A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet. 2006; 38: 203– 208. https://doi.org/10.1038/ng1702 PMID: 16380716 18. Segura V, Vilhja´lmsson BJ, Platt A, Korte A, Seren U¨ , Long Q, et al. References 1. Food and Agriculture Organization of the UN. FAOSTAT Food and Agricultural Commodities Produc- tion [Internet]. 2013 [cited 30 Mar 2016]. http://faostat3.fao.org/ 2. USDA Foreign Agricultural Service. USDA Production, Supply and Distribution Online [Internet]. 2015 [cited 30 Mar 2016]. https://apps.fas.usda.gov/psdonline/ 3. Bruinsma J. The resources outlook: by how much do land, water and crop yields need to increase by 2050? Looking Ahead in World Food and Agriculture: Perspectives to 2050. Rome, Italy: Food and Agriculture Organization of the United Nations; 2011. pp. 233–278. 4. Sharma RC, Crossa J, Velu G, Huerta-Espino J, Vargas M, Payne TS, et al. Genetic gains for grain yield in CIMMYT spring bread wheat across international environments. Crop Sci. 2012; 52: 1522–1533. https://doi.org/10.2135/cropsci2011.12.0634 5. Green AJ, Berger G, Griffey CA, Pitman R, Thomason W, Balota M, et al. Genetic yield improvement in soft red winter wheat in the eastern United States from 1919 to 2009. Crop Sci. 2012; 52: 2097–2108. https://doi.org/10.2135/cropsci2012.01.0026 6. Donald CM. The breeding of crop ideotypes. Euphytica. 1968; 17: 385–403. https://doi.org/10.1007/ BF00056241 6. Donald CM. The breeding of crop ideotypes. Euphytica. 1968; 17: 385–403. https://doi.org/10.1007/ BF00056241 22 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x An efficient multi-locus mixed- model approach for genome-wide association studies in structured populations. Nat Genet. 2012; 44: 825–830. https://doi.org/10.1038/ng.2314 PMID: 22706313 19. Widmer C, Lippert C, Weissbrod O, Fusi N, Kadie C, Davidson R, et al. Further improvements to linear mixed models for genome-wide association studies. Sci Rep. 2014; 4: 6874. https://doi.org/10.1038/ srep06874 PMID: 25387525 20. Wang Q, Tian F, Pan Y, Buckler ES, Zhang Z. A SUPER powerful method for genome wide associa- tion study. PLOS ONE. 2014; 9: e107684. https://doi.org/10.1371/journal.pone.0107684 PMID: 25247812 21. Liu X, Huang M, Fan B, Buckler ES, Zhang Z. Iterative usage of fixed and random effect models for powerful and efficient genome-wide association studies. PLOS Genet. 2016; 12: e1005767. https:// doi.org/10.1371/journal.pgen.1005767 PMID: 26828793 22. Huang M, Liu X, Zhou Y, Summers RM, Zhang Z. BLINK: a package for the next level of genome-wide association studies with both individuals and markers in the millions. GigaScience. https://doi.org/10. 1093/gigascience/giy154 PMID: 30535326 23. Breseghello F, Sorrells ME. Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars. Genetics. 2006; 172: 1165–1177. https://doi.org/10.1534/genetics.105.044586 PMID: 16079235 24. Neumann K, Kobiljski B, Denčić S, Varshney RK, Bo¨rner A. Genome-wide association mapping: a case study in bread wheat (Triticum aestivum L.). Mol Breed. 2011; 27: 37–58. https://doi.org/10.1007/ s11032-010-9411-7 25. Dodig D, Zoric M, Kobiljski B, Savic J, Kandic V, Quarrie S, et al. Genetic and association mapping study of wheat agronomic traits under contrasting water regimes. Int J Mol Sci. 2012; 13: 6167–6188. https://doi.org/10.3390/ijms13056167 PMID: 22754357 23 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 26. Tadesse W, Ogbonnaya FC, Jighly A, Sanchez-Garcia M, Sohail Q, Rajaram S, et al. Genome-wide association mapping of yield and grain quality traits in winter wheat genotypes. PLOS ONE. 2015; 10: e0141339. https://doi.org/10.1371/journal.pone.0141339 PMID: 26496075 27. Lozada DN, Mason RE, Babar MA, Carver BF, Guedira G-B, Merrill K, et al. Association mapping reveals loci associated with multiple traits that affect grain yield and adaptation in soft winter wheat. Euphytica. 2017; 213: 222. https://doi.org/10.1007/s10681-017-2005-2 28. Zanke CD, Ling J, Plieske J, Kollers S, Ebmeyer E, Korzun V, et al. Analysis of main effect QTL for thousand grain weight in European winter wheat (Triticum aestivum L.) by genome-wide association mapping. Front Plant Sci. 2015; 6: 644. https://doi.org/10.3389/fpls.2015.00644 PMID: 26388877 29. 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Zanke CD, Ling J, Plieske J, Kollers S, Ebmeyer E, Korzun V, et al. Whole genome association map- ping of plant height in winter wheat (Triticum aestivum L.). PLOS ONE. 2014; 9: e113287. https://doi. org/10.1371/journal.pone.0113287 PMID: 25405621 30. Zanke C, Ling J, Plieske J, Kollers S, Ebmeyer E, Korzun V, et al. Genetic architecture of main effect QTL for heading date in European winter wheat. Plant Genet Genomics. 2014; 5: 217. https://doi.org/ 10.3389/fpls.2014.00217 PMID: 24904613 31. Sukumaran S, Dreisigacker S, Lopes M, Chavez P, Reynolds MP. Genome-wide association study for grain yield and related traits in an elite spring wheat population grown in temperate irrigated environ- ments. Theor Appl Genet. 2015; 128: 353–363. https://doi.org/10.1007/s00122-014-2435-3 PMID: 25490985 32. Spindel J, Begum H, Akdemir D, Virk P, Collard B, Redoña E, et al. Genomic selection and association mapping in rice (Oryza sativa): effect of trait genetic architecture, training population composition, marker number and statistical model on accuracy of rice genomic selection in elite, tropical rice breed- ing lines. PLOS Genet. 2015; 11: e1004982. https://doi.org/10.1371/journal.pgen.1004982 PMID: 25689273 33. Salvi S, Tuberosa R. The crop QTLome comes of age. Curr Opin Biotechnol. 2015; 32: 179–185. https://doi.org/10.1016/j.copbio.2015.01.001 PMID: 25614069 34. The International Wheat Genome Sequencing Consortium (IWGSC), IWGSC RefSeq principal investi- gators, Appels R, Eversole K, Feuillet C, Keller B, Rogers J, et al. Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science. 2018; 361: eaar7191. https://doi. org/10.1126/science.aar7191 PMID: 30115783 35. Ward BP, Brown-Guedira G, Tyagi P, Kolb FL, Van Sanford DA, Sneller CH, et al. Multi-environment and multi-trait genomic selection models in unbalanced early-generation wheat yield trials. Crop Sci. 2019. 36. Zadoks JC, Chang TT, Konzak CF. A decimal code for the growth stages of cereals. Weed Res. 1974; 14: 415–421. https://doi.org/10.1111/j.1365-3180.1974.tb01084.x 37. Eilers PHC, Marx BD. Multivariate calibration with temperature interaction using two-dimensional penalized signal regression. Chemom Intell Lab Syst. 2003; 66: 159–174. https://doi.org/10.1016/ S0169-7439(03)00029-7 38. R Core Team. R: A Language and Environment for Statistical Computing. [Internet]. R Foundation for Statistical Computing, Vienna, Austria; 2015. http://www.R-project.org/ 39. Rodrı´guez-A´ lvarez MX, Boer MP, van Eeuwijk FA, Eilers PHC. Correcting for spatial heterogeneity in plant breeding experiments with P-splines. Spat Stat. 2018; 23: 52–71. https://doi.org/10.1016/j. spasta.2017.10.003 40. Cullis BR, Smith AB, Coombes NE. On the design of early generation variety trials with correlated data. J Agric Biol Environ Stat. 2006; 11: 381. https://doi.org/10.1198/108571106X154443 41. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Bates D, Ma¨chler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw. 2015; 67. https://doi.org/10.18637/jss.v067.i01 42. Poland JA, Brown PJ, Sorrells ME, Jannink J-L. Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLOS ONE. 2012; 7: e32253. https://doi.org/10.1371/journal.pone.0032253 PMID: 22389690 43. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics. 2007; 23: 2633–2635. https://doi.org/10.1093/bioinformatics/btm308 PMID: 17586829 44. Glaubitz JC, Casstevens TM, Lu F, Harriman J, Elshire RJ, Sun Q, et al. TASSEL-GBS: a high capac- ity genotyping by sequencing analysis pipeline. PLOS ONE. 2014; 9: e90346. https://doi.org/10.1371/ journal.pone.0090346 PMID: 24587335 45. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformat- ics. 2009; 25: 1754–1760. https://doi.org/10.1093/bioinformatics/btp324 PMID: 19451168 24 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 46. Browning SR, Browning BL. Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering. Am J Hum Genet. 2007; 81: 1084–1097. https://doi.org/10.1086/521987 PMID: 17924348 47. Chang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ. Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience. 2015; 4: 7. https://doi.org/10.1186/s13742- 015-0047-8 PMID: 25722852 48. Chen Y, Carver BF, Wang S, Zhang F, Yan L. Genetic loci associated with stem elongation and winter dormancy release in wheat. Theor Appl Genet. 2009; 118: 881–889. https://doi.org/10.1007/s00122- 008-0946-5 PMID: 19130033 49. Dı´az A, Zikhali M, Turner AS, Isaac P, Laurie DA. Copy number variation affecting the Photoperiod-B1 and Vernalization-A1 genes is associated with altered flowering time in wheat (Triticum Aestivum). PLOS ONE. 2012; 7: e33234. https://doi.org/10.1371/journal.pone.0033234 PMID: 22457747 50. Li G, Yu M, Fang T, Cao S, Carver BF, Yan L. Vernalization requirement duration in winter wheat is controlled by TaVRN-A1 at the protein level. Plant J. 2013; 76: 742–753. https://doi.org/10.1111/tpj. 12326 PMID: 24033823 51. Kippes N, Debernardi JM, Vasquez-Gross HA, Akpinar BA, Budak H, Kato K, et al. Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia. Proc Natl Acad Sci. 2015; 112: E5401–E5410. https://doi.org/10.1073/pnas.1514883112 PMID: 26324889 52. Allard RW, Shands RG. 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Inheritance of resistance to stem rust and powdery mildew in cytologically sta- ble spring wheats derived from Triticum timopheevi. Phytopathology. 1954; 44: 266–274. 53. Nyquist WE. Monosomic analysis of stem rust resistance of a common wheat strain derived from Triti- cum timopheevi. Agron J. 1957; 49: 222–223. 54. Cabrera A, Guttieri M, Smith N, Souza E, Sturbaum A, Hua D, et al. Identification of milling and baking quality QTL in multiple soft wheat mapping populations. Theor Appl Genet. 2015; 128: 2227–2242. https://doi.org/10.1007/s00122-015-2580-3 PMID: 26188588 55. Tsilo TJ, Jin Y, Anderson JA. Diagnostic microsatellite markers for the detection of stem rust resis- tance gene Sr36 in diverse genetic backgrounds of wheat. Crop Sci. 2008; 48: 253–261. https://doi. org/10.2135/cropsci2007.04.0204 56. Zheng X, Levine D, Shen J, Gogarten SM, Laurie C, Weir BS. A high-performance computing toolset for relatedness and principal component analysis of SNP data. Bioinformatics. 2012; 28: 3326–3328. https://doi.org/10.1093/bioinformatics/bts606 PMID: 23060615 57. Cleveland WS. Robust locally weighted regression and smoothing scatterplots. J Am Stat Assoc. 1979; 74: 829–836. https://doi.org/10.1080/01621459.1979.10481038 58. Weir BS, Cockerham CC. Estimating F-statistics for the analysis of population structure. Evolution. 1984; 38: 1358–1370. https://doi.org/10.1111/j.1558-5646.1984.tb05657.x PMID: 28563791 59. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, et al. The variant call format and VCFtools. Bioinformatics. 2011; 27: 2156–2158. https://doi.org/10.1093/bioinformatics/btr330 PMID: 21653522 60. Yang J, Lee SH, Goddard ME, Visscher PM. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011; 88: 76–82. https://doi.org/10.1016/j.ajhg.2010.11.011 PMID: 21167468 61. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol. 1995; 57: 289–300. 62. Kusmec A, Schnable PS. FarmCPUpp: Efficient large-scale genomewide association studies. Plant Direct. 2018; 2: e00053. https://doi.org/10.1002/pld3.53 63. Wallace JG, Zhang X, Beyene Y, Semagn K, Olsen M, Prasanna BM, et al. Genome-wide association for plant height and flowering time across 15 tropical maize populations under managed drought stress and well-watered conditions in sub-Saharan Africa. Crop Sci. 2016; 56: 2365–2378. https://doi.org/10. 2135/cropsci2015.10.0632 64. Valdar W, Holmes CC, Mott R, Flint J. Mapping in structured populations by resample model averag- ing. Genetics. 2009; 182: 1263–1277. https://doi.org/10.1534/genetics.109.100727 PMID: 19474203 65. Sorrells ME, Gustafson JP, Somers D, Chao S, Benscher D, Guedira-Brown G, et al. Reconstruction of the Synthetic W7984 × Opata M85 wheat reference population. Genome. 2011; 54: 875–882. https://doi.org/10.1139/g11-054 PMID: 21999208 66. Murray K, Mu¨ller S, Turlach BA. 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Fast and flexible methods for monotone polynomial fitting. J Stat Com- put Simul. 2016; 86: 2946–2966. https://doi.org/10.1080/00949655.2016.1139582 67. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplo- type blocks in the human genome. Science. 2002; 296: 2225–2229. https://doi.org/10.1126/science. 1069424 PMID: 12029063 25 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 68. McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinforma Oxf Engl. 2010; 26: 2069–2070. https://doi.org/10.1093/bioinformatics/btq330 PMID: 20562413 69. Groos C, Robert N, Bervas E, Charmet G. Genetic analysis of grain protein-content, grain yield and thousand-kernel weight in bread wheat. Theor Appl Genet. 2003; 106: 1032–1040. https://doi.org/10. 1007/s00122-002-1111-1 PMID: 12671751 70. Cox MC, Qualset CO, Rains DW. Genetic variation for nitrogen assimilation and translocation in wheat. I. Dry matter and nitrogen accumulation. Crop Sci. 1985; 25: 430–435. https://doi.org/10.2135/ cropsci1985.0011183X002500030002x 71. Terman GL, Ramig RE, Dreier AF, Olson RA. Yield-protein relationships in wheat grain, as affected by nitrogen and water. Agron J. 1969; 61: 755–759. https://doi.org/10.2134/agronj1969. 00021962006100050031x 72. Turuspekov Y, Baibulatova A, Yermekbayev K, Tokhetova L, Chudinov V, Sereda G, et al. GWAS for plant growth stages and yield components in spring wheat (Triticum aestivum L.) harvested in three regions of Kazakhstan. BMC Plant Biol. 2017; 17: 190. https://doi.org/10.1186/s12870-017-1131-2 PMID: 29143598 73. Ain Q, Rasheed A, Anwar A, Mahmood T, Imtiaz M, Mahmood T, et al. Genome-wide association for grain yield under rainfed conditions in historical wheat cultivars from Pakistan. Front Plant Sci. 2015; 6: 743. https://doi.org/10.3389/fpls.2015.00743 PMID: 26442056 74. Sehgal D, Autrique E, Singh R, Ellis M, Singh S, Dreisigacker S. Identification of genomic regions for grain yield and yield stability and their epistatic interactions. Sci Rep. 2017; 7: 41578. https://doi.org/ 10.1038/srep41578 PMID: 28145508 75. Godoy J, Gizaw S, Chao S, Blake N, Carter A, Cuthbert R, et al. Genome-wide association study of agronomic traits in a spring-planted North American elite hard red spring wheat panel. CROP Sci. 2018; 58: 1838–1852. https://doi.org/10.2135/cropsci2017.07.0423 76. Ogbonnaya FC, Rasheed A, Okechukwu EC, Jighly A, Makdis F, Wuletaw T, et al. Genome-wide association study for agronomic and physiological traits in spring wheat evaluated in a range of heat prone environments. Theor Appl Genet. 2017; 130: 1819–1835. https://doi.org/10.1007/s00122-017- 2927-z PMID: 28577083 77. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Theor Appl Genet. 2014; 127: 791–807. https://doi.org/10.1007/s00122-013-2257-8 PMID: 24408378 87. Habier D, Fernando RL, Kizilkaya K, Garrick DJ. Extension of the bayesian alphabet for genomic selection. BMC Bioinformatics. 2011; 12: 186. https://doi.org/10.1186/1471-2105-12-186 PMID: 21605355 88. Miao C, Yang J, Schnable JC. Optimising the identification of causal variants across varying genetic architectures in crops. Plant Biotechnol J. 2018; 0: 1–13. https://doi.org/10.1111/pbi.13023 PMID: 30320953 89. Yu J, Holland JB, McMullen MD, Buckler ES. Genetic design and statistical power of nested associa- tion mapping in maize. Genetics. 2008; 178: 539–551. https://doi.org/10.1534/genetics.107.074245 PMID: 18202393 90. Cavanagh CR, Chao S, Wang S, Huang BE, Stephen S, Kiani S, et al. Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and culti- vars. Proc Natl Acad Sci. 2013; 110: 8057–8062. https://doi.org/10.1073/pnas.1217133110 PMID: 23630259 91. Reif JC, Gowda M, Maurer HP, Longin CFH, Korzun V, Ebmeyer E, et al. Association mapping for quality traits in soft winter wheat. Theor Appl Genet. 2011; 122: 961–970. https://doi.org/10.1007/ s00122-010-1502-7 PMID: 21153626 92. Wu¨rschum T, Langer SM, Longin CFH, Korzun V, Akhunov E, Ebmeyer E, et al. Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers. Theor Appl Genet. 2013; 126: 1477–1486. https://doi.org/10.1007/s00122-013-2065-1 PMID: 23429904 93. Moreno-Sevilla B, Baenziger PS, Peterson CJ, Graybosch RA, McVey DV. The 1BL/1RS transloca- tion: agronomic performance of F3-derived lines from a winter wheat cross. Crop Sci. 1995; 35: 1051–1055. https://doi.org/10.2135/cropsci1995.0011183X003500040022x 94. Singh RP, Huerta-Espino J, Rajaram S, Crossa J. Agronomic effects from chromosome translocations 7DL.7AG and 1BL.1RS in spring wheat. Crop Sci. 1998; 38: 27–33. https://doi.org/10.2135/ cropsci1998.0011183X003800010005x 95. Ehdaie B, Whitkus RW, Waines JG. Root biomass, water-use efficiency, and performance of wheat– rye translocations of chromosomes 1 and 2 in spring bread wheat ‘Pavon’. Crop Sci. 2003; 43: 710–717. 96. Hallab A, Klee K, Boecker F, Girish S, Schoof H. Automated Assignment of Human Readable Descrip- tions [Internet]. 2017. https://github.com/groupschoof/AHRD 97. Ikeda K, Ito M, Nagasawa N, Kyozuka J, Nagato Y. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate. Plant J. 2007; 51: 1030–1040. https://doi.org/10. 1111/j.1365-313X.2007.03200.x PMID: 17666027 98. Micheli F. Pectin methylesterases: Cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 2001; 6: 414–419. https://doi.org/10.1016/S1360-1385(01)02045-3 PMID: 11544130 99. Kagan-Zur V, Tieman DM, Marlow SJ, Handa AK. 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Soriano JM, Malosetti M, Rosello´ M, Sorrells ME, Royo C. Dissecting the old Mediterranean durum wheat genetic architecture for phenology, biomass and yield formation by association mapping and QTL meta-analysis. PLOS ONE. 2017; 12: e0178290. https://doi.org/10.1371/journal.pone.0178290 PMID: 28542488 78. Mengistu DK, Kidane YG, Catellani M, Frascaroli E, Fadda C, Pe ME, et al. High-density molecular characterization and association mapping in Ethiopian durum wheat landraces reveals high diversity and potential for wheat breeding. Plant Biotechnol J. 2016; 14: 1800–1812. https://doi.org/10.1111/ pbi.12538 PMID: 26853077 79. Guo J, Shi W, Zhang Z, Cheng J, Sun D, Yu J, et al. Association of yield-related traits in founder geno- types and derivatives of common wheat (Triticum aestivum L.). BMC Plant Biol. 2018; 18: 38. https:// doi.org/10.1186/s12870-018-1234-4 PMID: 29458339 80. Wang S-X, Zhu Y-L, Zhang D-X, Shao H, Liu P, Hu J-B, et al. Genome-wide association study for grain yield and related traits in elite wheat varieties and advanced lines using SNP markers. PLOS ONE. 2017; 12: e0188662. https://doi.org/10.1371/journal.pone.0188662 PMID: 29176820 81. Sukumaran S, Lopes M, Dreisigacker S, Reynolds M. Genetic analysis of multi-environmental spring wheat trials identifies genomic regions for locus-specific trade-offs for grain weight and grain number. Theor Appl Genet. 2018; 131: 985–998. https://doi.org/10.1007/s00122-017-3037-7 PMID: 29218375 82. Ma F, Xu Y, Ma Z, Li L, An D. Genome-wide association and validation of key loci for yield-related traits in wheat founder parent Xiaoyan 6. Mol Breed. 2018; 38. 83. Liu Y, Lin Y, Gao S, Li Z, Ma J, Deng M, et al. A genome-wide association study of 23 agronomic traits in Chinese wheat landraces. Plant J. 2017; 91: 861–873. https://doi.org/10.1111/tpj.13614 PMID: 28628238 84. Sukumaran S, Reynolds MP, Sansaloni C. Genome-wide association analyses identify QTL hotspots for yield and component traits in durum wheat grown under yield potential, drought, and heat stress environments. Front Plant Sci. 2018; 9. https://doi.org/10.3389/fpls.2018.00081 PMID: 29467776 85. Zhang J, Gizaw SA, Bossolini E, Hegarty J, Howell T, Carter AH, et al. Identification and validation of QTL for grain yield and plant water status under contrasting water treatments in fall-sown spring wheats. Theor Appl Genet. 2018; 131: 1741–1759. https://doi.org/10.1007/s00122-018-3111-9 PMID: 29767279 26 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 86. Edae EA, Byrne PF, Haley SD, Lopes MS, Reynolds MP. Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x Differential regulation of polygalacturonase and pec- tin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant Mol Biol. 1995; 29: 1101–1110. https://doi.org/10.1007/BF00020455 PMID: 8616211 100. Panavas T, Reid PD, Rubinstein B. Programmed cell death of daylily petals: activities of wall-based enzymes and effects of heat shock. Plant Physiol Biochem. 1998; 36: 379–388. https://doi.org/10. 1016/S0981-9428(98)80079-X 101. Gubler F, Kalla R, Roberts JK, Jacobsen JV. Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI α-amylase gene promoter. Plant Cell. 1995; 7: 1879–1891. https://doi.org/10.1105/tpc.7.11.1879 PMID: 8535141 102. Chen L, Nishizawa T, Higashitani A, Suge H, Wakui Y, Takeda K, et al. A variety of wheat tolerant to deep-seeding conditions: elongation of the first internode depends on the response to gibberellin and potassium. Plant Cell Environ. 2001; 24: 469–476. https://doi.org/10.1046/j.1365-3040.2001.00688.x 103. Gocal GFW, Poole AT, Gubler F, Watts RJ, Blundell C, King RW. Long-day up-regulation of a GAMYB gene during Lolium temulentum inflorescence formation. Plant Physiol. 1999; 119: 1271–1278. https:// doi.org/10.1104/pp.119.4.1271 PMID: 10198085 104. Kaneko M, Inukai Y, Ueguchi-Tanaka M, Itoh H, Izawa T, Kobayashi Y, et al. Loss-of-function muta- tions of the rice GAMYB gene impair α-amylase expression in aleurone and flower development. Plant Cell. 2004; 16: 33–44. https://doi.org/10.1105/tpc.017327 PMID: 14688295 27 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 Genome-wide association studies for yield-related traits in soft red winter wheat grown in Virginia 105. Sado P-E, Tessier D, Vasseur M, Elmorjani K, Guillon F, Saulnier L. Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses. Funct Integr Genomics. 2009; 9: 43–58. https://doi.org/10.1007/s10142-008-0100-0 PMID: 19005709 106. Freiberg C, Brunner NA, Schiffer G, Lampe T, Pohlmann J, Brands M, et al. Identification and charac- terization of the first class of potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activ- ity. J Biol Chem. 2004; 279: 26066–26073. https://doi.org/10.1074/jbc.M402989200 PMID: 15066985 107. Radley M. The development of wheat grain in relation to endogenous growth substances. J Exp Bot. 1976; 27: 1009–1021. https://doi.org/10.1093/jxb/27.5.1009 108. Pearce S, Huttly AK, Prosser IM, Li Y, Vaughan SP, Gallova B, et al. Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family. BMC Plant Biol. 2015; 15. https://doi.org/10.1186/s12870-015-0520-7 PMID: 26044828 109. Nakamura S, Abe F, Kawahigashi H, Nakazono K, Tagiri A, Matsumoto T, et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0208217 February 22, 2019 7. Slafer GA. Genetic basis of yield as viewed from a crop physiologist’s perspective. Ann Appl Biol. 2003; 142: 117–128. https://doi.org/10.1111/j.1744-7348.2003.tb00237.x A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination. Plant Cell. 2011; 23: 3215–3229. https://doi.org/10.1105/tpc.111.088492 PMID: 21896881 110. Schrader S, Kaldenhoff R, Richter G. Expression of novel genes during somatic embryogenesis of suspension-cultured carrot cells (Daucus carota). J Plant Physiol. 1997; 150: 63–68. https://doi.org/ 10.1016/S0176-1617(97)80182-4 111. Chauhan H, Khurana N, Tyagi AK, Khurana JP, Khurana P. Identification and characterization of high temperature stress responsive genes in bread wheat (Triticum aestivum L.) and their regulation at vari- ous stages of development. Plant Mol Biol. 2011; 75: 35–51. https://doi.org/10.1007/s11103-010- 9702-8 PMID: 20972607 112. Singh A, Khurana P. Molecular and functional characterization of a wheat B2 protein imparting adverse temperature tolerance and influencing plant growth. Front Plant Sci. 2016; 7. https://doi.org/10.3389/ fpls.2016.00642 PMID: 27242843 113. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc Lond. 2014; 9: 2395–410. http://dx.doi.org.prox.lib.ncsu.edu/10.1038/nprot.2014.157 114. Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun. 2016; 7: 12617. https://doi.org/10.1038/ncomms12617 PMID: 27558837 115. Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun. 2017; 8: 14261. https://doi. org/10.1038/ncomms14261 PMID: 28098143 116. Howells RM, Craze M, Bowden S, Wallington EJ. Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9. BMC Plant Biol. 2018; 18: 215. https://doi.org/10.1186/s12870-018-1433- z PMID: 30285624 117. Clevenger JP, Korani W, Ozias-Akins P, Jackson S. Haplotype-based genotyping in polyploids. Front Plant Sci. 2018; 9. https://doi.org/10.3389/fpls.2018.00564 PMID: 29755500 118. Clevenger JP, Ozias-Akins P. SWEEP: a tool for filtering high-quality SNPs in polyploid crops. G3 Bethesda Md. 2015; 5: 1797–1803. https://doi.org/10.1534/g3.115.019703 PMID: 26153076 28 / 28
https://openalex.org/W1986249381	https://www.scielo.br/j/bjorl/a/qxyw46wtzSH6XbX45mvdGkC/?lang=en&format=pdf	English	null	Unilateral hearing loss: benefits and satisfaction from the use of hearing aids	Brazilian Journal of Otorhinolaryngology	2,011	cc-by	5,567	1 Graduanda do 4° ano do Curso de Fonoaudiologia da Faculdade de Odontologia de Bauru, Universidade de São Paulo. Discente. 2 Mestranda do Programa de Pós Graduação em Fonoaudiologia da Faculdade de Odontologia de Bauru, Universidade de São Paulo. Fonoaudióloga da Clínica de Fonoaudiologia da Faculdade de Odontologia de Bauru, Universidade de São Paulo. 3 Doutora em Distúrbios da Comunicação Professora Doutora do Curso de Fonoaudiologia da Faculdade de Odontologia de Bauru, Universidade de São Paulo. Trabalho realizado na Clínica de Fonoaudiologia da Faculdade de Odontologia de Bauru, Universidade de São Paulo. Endereço para correspondência: Alameda Octávio Pinheiro Brisola 9-75 Vila Universitária Bauru SP 17012-901. Trabalho financiado pela FAPESP - processo no 2009/00768-0. Paper submitted to the BJORL-SGP (Publishing Management System – Brazilian Journal of Otorhinolaryngology) on April 27, 2010; and accepted on May 26, 2010. cod. 7044 Maria Renata José1, Patrícia Danieli Campos2, Maria Fernanda Capoani Garcia Monde Keywords: hearing aids, hearing loss, unilateral, questionnaires. Unilateral hearing loss: benefits and satisfaction from the use of hearing aids original article raz J Otorhinolaryngol. 011;77(2):221-8. BJORL .org Braz J Otorhinolaryngol. 2011;77(2):221-8. original article Abstract A A unilateral hearing loss is characterized by reduced hearing in one ear. The problems caused by sensory deprivation can be minimized with the use of hearing aids (HA). Aim: To analyze the correlation between the prescribed grain and the insertion gain difference and with the results obtained regarding the benefit and satisfaction with the use of hearing aids in unilateral hearing impaired patients. Materials and Methods: Prospective study with 15 subjects, mean age of 41.6 years, of both genders, users of hearing aids effectively. We used the International Questionnaire Results for hearing aids (International Outcome Inventory for Hearing Aids - IOI-HA), measured with a probe microphone. Results: The mean values in the analyses of the IOI-HA per item were positive and higher than four points. In relation to the objective measures, the frequencies in which we obtained the gain values which were closer to the target were: 1K Hz, 2K Hz and 500 Hz, respectively. Conclusion: The satisfaction of individuals using hearing aid unilaterally is not completely correlated to the prescribed gain, because even if the target is not being reached in some frequencies, the individuals were pleased as to the use of their hearing aids. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 221 characterized as a psychological process of dealing with the idea and the sensation of sound amplification, at the same time as it incorporates the device to your lifestyle. Satisfaction is built according to the subjective impressions of the individual. Thus, it becomes clear that, while there is no acceptance, there will never be satisfaction, just like not all acceptance and benefit associated with the device are enough parameters to guarantee satisfaction. While the benefits can be shown through objective tests, personal satisfaction is a very personal assessment of the value of the sound amplification device after a given time of use8. INTRODUCTION Hearing is one of the fundamental senses in life, playing a very important role in society, being considered the basis for human communication development. Indi- viduals with hearing impairment may suffer handicaps in their social, psychological and professional lives1. According to the 2002 Census (IBGE)2, 5.7 million Brazilians stated they had hearing impairment (HI). This number is probably much higher, because often times the problem is not perceived, or it is denied by the individuals. Abstract The non-acceptance does not lead to treatment, and this may worsen the frustration of not being able to hear and the individual isolates himself. It is possible to state that the checking procedures, as the functional gain and the insertion measures, are not enough to assess the individual’s satisfaction with the de- vice in daily communication tasks. There was a growing interest in the development of validation procedures which enabled to study the user’s benefit outside the clinical settings, making up self-assessment questionnaires9. Many are the causes which contribute to the in- crease in the number of hearing impaired individuals: presbycusis, hereditary diseases, metabolic disorders, use of ototoxic drugs, acoustic trauma, excess noise, different types of neoplasia, infections and vascular damage. Among the resulting effects, we stress anxiety, frustration, feeling of insecurity, emotional instability, depression, social pho- bia, a feeling of frustration and incapacity to guide oneself3. The self-assessment is a simple, fast and efficient procedure which enables the assessment of the indivi- dual in his process of ISAD’s adaptation. This procedure enables the comparison between different devices and/ or calibrations, as well as the assessment of the benefit achieved with the same ISAD along time, enabling the user to recognize the advantages provided by the ISAD in relation to the individual’s auditory difficulties and psychosocial disadvantages. Thus, by means of question- naires which enable the measuring and analysis of these auditory difficulties or that of the handicap, it is possible to optimize the time it takes for the person to adapt him/ herself to the amplification10. Unilateral hearing loss is characterized by hearing reduction in only one ear and it happens, predominantly among males4. In one study5 they found the main etiolo- gies to be mumps, ototoxicity, meningitis, NIHL, German measles, head injury and sensorineural unilateral hearing loss of unknown cause. The effects of unilateral hearing loss are lower than the ones caused by bilateral hearing loss, which can also cause problems. Under room noise, individuals with unilateral hearing loss find more difficulties than their normal hearing counterparts to understand speech, even when the best ear is positioned towards the source of speech. Moreover, the spatial location of the sound source is compromised6. There are numerous assessment instruments which have scores used to assess the level of individual satis- faction, even because there are numerous factors which influence different dimensions associated with the use of a sound amplification device8. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br MATERIALS AND METHODS Patient selection and assessment started after the Ethics Committee approved the study under process # 001/2009 and signed the Free and Informed Consent Form. - In order to obtain the REIG, the procedure was carried out in the following sequence: measuring response on the external ear without the ISAD Real Ear Unaided Response (REUR) followed by measuring the response with the ISAD in the external ear - Real Ear Aided Response (REAR) thus obtaining the Real Ear Insertion Gain (REIG) using the following calculation: REIG=REAR-REUR. At the end, we noticed that the REIG result reached the target rule, in other words, the calculated target considering the prescription rule formula: NAL-NL1 by previous selection of the equipment software. - In order to obtain the REIG, the procedure was carried out in the following sequence: measuring response on the external ear without the ISAD Real Ear Unaided Response (REUR) followed by measuring the response with the ISAD in the external ear - Real Ear Aided Response (REAR) thus obtaining the Real Ear Insertion Gain (REIG) using the following calculation: REIG=REAR-REUR. At the end, we noticed that the REIG result reached the target rule, in other words, the calculated target considering the prescription rule formula: NAL-NL1 by previous selection of the equipment software. This cross-sectional contemporary cohort study was carried out with 15 individuals with a mean age of 41.6 years, of both genders, (12 females and 3 males). Participant inclusion criteria were: Participant inclusion criteria were: - Age range: adult individuals (18 to 60 years) - Hearing loss: mixed unilateral or sensorineural of moderate, severe and profound levels; - Effective ISAD user for more than six (6) months. The HI degree was classified using the audiometric thresholds of the 500; 1,000; 2,000; 3,000 and 4,000 Hz frequencies: mild HI (mean between 26 and 40 dBHL), moderate HI (mean between 41 and 60 dBHL), severe HI (mean between 61 and 80 dBHL) and profound HI (mean higher than 81 dBHL), according with the WHO11. Measurements were carried out with stimuli of 50, 65 and 80 dBSPL of the modulated speech type16 because it is the stimulus which comes closer to the continuous speech discourse. In order to do the procedures, the extra resources such as feedback control and noise reduction were turned off from the ISAD’s programming so as to avoid its influence on the analyzed responses17. Abstract - the patient was seated at 50cm away from the sound speaker with the ears in a horizontal plane vis-à-vis the speaker and at 1.5m away from the walls of the room and at a 0º azimuth in relation to the speaker. Because of the scarcity of studies concerning uni- lateral hearing loss, the goal of this study is to objectively and subjectively measure the benefit and the satisfaction of the individuals using unilateral ISADs. - the insertion of the probe microphone in the external acoustic meatus placed at 27-30mm of depth in the external acoustic meatus, using the method in which the probe tube is placed at approximately 3mm from the tip of the ear mold. - the insertion of the probe microphone in the external acoustic meatus placed at 27-30mm of depth in the external acoustic meatus, using the method in which the probe tube is placed at approximately 3mm from the tip of the ear mold. RESULTS We scored the IOI-HA instrument of the 15 indivi- duals who answered the questionnaire. On Tables 1 and 2 we can find the answers associated with the daily use, limitations, satisfaction, restrictions, social activities and quality of life. The questionnaire was used as an individual inter- view in order to make sure all the questions would be answered and that the individual had fully understood the question. We created graphs so as to better see the results associated with the insertion gains in the frequencies of 500; 1,000; 2,000; 3,000 and 4000 Hz. During the checking process, we carried out mea- sures with a probe microphone according to indications from international protocols15. Thus, the data was collec- ted using the Unity (Siemens) equipment, following the criteria below: MATERIALS AND METHODS We used the International Outcome Inventory for Hearing Aids - IOI-HA developed as the product of an international workshop on self-assessment measures in auditory rehabilitation12,13. Currently, the IOI-HA questio- nnaire is included in the Hearing Aid Selection and Fitting Form (Ordinance SAS/MS # 587, of 10/07/2004)14. This questionnaire assesses seven domains deemed important for the successful use of a hearing aid (use, benefit, limi- tations in residual activity, satisfaction, impact on others and quality of life) (Attachment 1). We used description by mean and absolute values for the statistical study. Abstract The problems brought about by sensorial depriva- tion can be minimized with the use of an Individual Sound Amplification Device (ISAD) - hearing aid, which enables one to bring back the perception of speech sounds, besides environmental sounds, bringing about an improvement in communication skills1. In Brazil, some self-assessment questionnaires, the APHAB (Abbreviated Profile of Hearing Aid Benefit), the IOI-HA (International Outcome Inventory for Hearing Aids), the HHIE (Hearing Inventory for the Elderly) and the HHIA (Hearing Handicap Inventory for the Adults) among them, were translated and adapted to our reality in Brazil, investigating the degree of user satisfaction, the benefits obtained from using ISADs and the reduction in auditory capacity with the use of amplification, and others with the goal of comparing the benefit of different Technologies and checking the ISAD fitting by means of objective and subjective measures9. There are many factors which contribute to the successful use of amplification. Age, type and degree of the hearing loss, physical factors (ear size and manual dexterity), auditory processing skills, prior use of sound amplification device and hearing loss extension, which together, have a crucial role for accepting the amplification. Added to this, the perception of the auditory handicap, cost, personal expectations, satisfaction, performance and benefits may indicate whether or not we will have a happy and satisfied user of a sound amplification device7. The benefit has been traditionally assessed by means of objective data, in other words, the hearing loss nature and severity are defined as basis in the assessment of the thresholds obtained by means of equipment ca- libration in controlled environments. Thus, the hearing performance improvement enjoyed by individuals with Acceptance may be characterized in two ways: ei- ther the device is accepted or rejected; but it can also be 222 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 222 ISADs can be obtained by means of measurements such as the insertion gain which is understood as a benefit9. - the patient was seated at 50cm away from the sound speaker with the ears in a horizontal plane vis-à-vis the speaker and at 1.5m away from the walls of the room and at a 0º azimuth in relation to the speaker. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br DISCUSSION Distribution of the answers from each individual conce As to the residual limitation in activities, 40% of the individuals reported that they did not have any difficulties in daily hearing situations, 33.33% had little difficulties; and 20% reported still having moderate difficulties in these activities, even with the use of ISAD. These data tell us that the users of unilateral ISAD had improvements concerning the hearing difficulties they had had before the amplification. When asked about satisfaction concerning the use of the hearing aids, 60% of the individuals reported that it is really worth using the ISAD; and 40% reported that it is reasonably worth using them unilaterally. Table 2. Distribution of the answers from each individual concerning the different domains. Questions Individuals 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Daily use 2 5 5 5 5 4 4 5 4 5 5 5 5 4 5 Benefit 3 4 4 4 5 3 4 4 4 4 4 4 4 4 5 Limitations 4 5 4 5 5 1 3 5 5 4 4 3 5 3 4 Satisfaction 4 4 5 5 5 4 4 5 5 5 5 5 4 4 5 Restrictions 5 4 5 5 5 5 5 5 5 5 3 5 5 4 5 Social activities 1 4 5 5 5 3 5 5 5 5 4 3 5 2 5 Quality of life 3 4 4 4 5 4 2 4 3 2 5 4 4 1 4 The application of these procedures in the clini- cal routine requires knowledge of the ISAD technology, which is to be fit to a patient, and the tests chosen for application21, considering the necessary resources, must be selected respecting the particularities of each individual22. To check whether or not the hearing aid features were achieved is crucial to the fitting process18. Concerning the restriction towards daily activities after fitting the individual sound amplification device, 80% of the individuals reported that with the unilateral fitting of the devices, their hearing difficulties no longer affected their daily activities. As to the impact the hearing loss causes on people, 60% of the individuals reported that their hearing difficul- ties did not affect or bother other people; 13.33% reported it affected moderately their relations with other people. DISCUSSION There are numerous protocols available to use in the selection and fitting of a hearing aid, such as the one from Ordinance 587 from the Health Department14, Inter- national Society of Audiology18, Valente19, Matas & Iório20, with different types of procedure; however, they are not unanimous, just like it happened in the present study, there was a need to carry out an objective assessment as well as a subjective one with validated questionnaires and orientation at different stages of the hearing aid selection and fitting. - we inserted the threshold tonal audiometry data, obtained through air conduction and bone conduction in the software used for the probe microphone measures in a way as to generate the gain prescribed by the NAL-NL1 rule. - with the aim of making the probe tube acoustically transparent we did the calibration, positioning the tube in the horizontal plane at 30cm away from the speaker and near the reference microphone. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 223 they had hearing difficultie As to the residual lim individuals reported that the in daily hearing situations, and 20% reported still ha these activities, even with tell us that the users of unila concerning the hearing dif the amplification. When asked about s of the hearing aids, 60% of it is really worth using the I is reasonably worth using t Table 1. Distribution of the answers in each domain of the IOI-HA instrument. Item Mean Standard Deviation Daily use 4.53 0.83 Benefit 4.00 0.53 Limitations 4.00 1.13 Satisfaction 4.60 0.51 Restrictions 4.73 0.59 Social activities 4.13 1.30 Quality of life 3.53 1.13 Total 29.53 4.07 Table 2. Distribution of the answers from each individual concerning the different domains. Table 1. Distribution of the answers in each domain of the IOI-HA instrument. they had hearing difficulties. they had hearing difficulties. Table 1. Distribution of the answers in each domain of the IOI-HA instrument. Item Mean Standard Deviation Daily use 4.53 0.83 Benefit 4.00 0.53 Limitations 4.00 1.13 Satisfaction 4.60 0.51 Restrictions 4.73 0.59 Social activities 4.13 1.30 Quality of life 3.53 1.13 Total 29.53 4.07 Table 2. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br DISCUSSION - Target Response Obtained Response explanatory characteristic of the questionnaire, which does not require additional help to be answered13, in this study it was applied by the researcher in charge in order to make sure the individuals understood the questions and answers. Still concerning the IOI-HA, we may say that the mean values obtained in the analysis per item were po- sitive and higher than 4 points, keeping in mind that the maximum score per question is five. Consequently, the analysis of the summation of all the questions was also positive, indicating a good subjective result in ISAD fitting. Cox and Alexander15 also found a high score in the individuals they assessed in their study using the IOI-HA questionnaire, suggesting favorable attitude concerning their ISAD. They commented on the probable question- naire sensitiveness to detect individuals with a negative experience regarding the sound amplification. Chart 2. Values of the prescribed gain and the insertion gain at 1000 Hz. - Target Response Obtained Response Chart 3. Values of the prescribed gain and the insertion gain at 2000 Hz. - Target Response Obtained Response As far as the objective measures go, they noticed that the frequencies in which the gain obtained was closer to the target were: 1,000 HZ; 2,000 Hz and 500 HZ, respec- tively (Charts 2, 3 and 1), and in the frequencies of 3,000 HZ and 4,000 Hz (Charts 4 and 5), in average, half of the individuals did not reach the prescribed value. The only individuals who reached the target in all the frequencies assessed were: 4, 6, 8 and 15, and individuals 4, 8 and 15 had scores higher than 30 in the IOI-HA questionnaire, showing their satisfaction concerning the use of their ISAD, and individual 6 scored 24 (Table 2), showing that even reaching the prescribed gain, he was not entirely pleased with the use of a hearing aid. Contrary to individual 5, who even not reaching the target in all the frequencies asses- sed, had a maximum score in the IOI-HA questionnaire. Chart 3. Values of the prescribed gain and the insertion gain at 2000 Hz. - Target Response Obtained Response Chart 3. Values of the prescribed gain and the insertion gain at 2000 Hz. - Target Response Obtained Response Chart 4. Values of the prescribed gain and the insertion gain at 3000 Hz. - Target Response Obtained Response Chart 1. DISCUSSION In order to analyze the benefit and satisfaction, we used the IOI-HA questionnaire, and the use of validated questionnaires is a must in a gold standard protocol23 with the use of objective and subjective tests geared towards a good communication and proper quality of life for the individual with hearing impairment. And finally, when the individuals were asked about their quality of lives associated with the use of the sound amplification device, 53.33% reported that after fitting the hearing aid, they became happier with life; 13.33% repor- ted no changes as improvements in quality of life; 13.33% reported a little more happiness in with life after fitting the ISAD; and 13.33% reported a lot more happiness with life after they started using their hearing aids. This data depicts the importance of using these devices in order to enable a better quality of life for those individuals with unilateral hearing loss. As far as the use is concerned, 66.67% of the in- dividuals reported using the ISAD for more than 8 hours per day, and 26.67% reported uses between 4 and 8 hours per day, and it was possible to notice that all the patients effectively used their hearing aids. As far as the benefit is concerned, 73.34% reported that the hearing aid helped them much in the situations in which before they had major hearing difficulties; 13.33% of the individuals reported that the ISAD helped them moderately in the situations in which they had hearing difficulties; and 13.33% reported that the hearing aid helped them very much in the situations in which before IOI-HA was used for being a brief, encompassing type of measurement, accessible to different cultural and social factors for use and different types of comparisons12 in this study focusing on the satisfaction of unilateral sound amplification users. Nonetheless, despite this self- Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 224 Chart 2. Values of the prescribed gain and the insertion gain at 1000 Hz. - Target Response Obtained Response Chart 3. Values of the prescribed gain and the insertion gain at 2000 Hz. - Target Response Obtained Response Chart 4. Values of the prescribed gain and the insertion gain at 3000 Hz. - Target Response Obtained Response Chart 2. Values of the prescribed gain and the insertion gain at 1000 Hz. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br DISCUSSION Values of the prescribed gain and the insertion gain at 500 Hz. Target Response Obtained Response Chart 4. Values of the prescribed gain and the insertion gain at 3000 Hz. - Target Response Obtained Response Chart 1. Values of the prescribed gain and the insertion gain at 500 Hz. Target Response Obtained Response compromises the values programmed in the ISAD, since these can be different from the prescribed target value. In fact, the checking process is crucial, and it is possible to identify the performance of the amplification being provided - both an under-amplification, which may bring about losses in the amplification of speech sounds, as well as an over-amplification, which may cause discomfort and even worsening of the hearing loss24. We stress that those individuals who were pleased with the use of sound amplification, even not reaching the target, likely because one of the ears did not have normal hearing, since it is known that when the target is not reached, there is a loss for the hearing impaired con- cerning speech reception and understanding. This indicates that by not doing the checking with objective measures 225 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 225 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 225 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 225 Chart 5. Values of the prescribed gain and the insertion gain at 4000 Hz. - Target Response Obtained Response the recording of the ISAD performance. This is a powerful tool in the process of selecting and fitting these devices, providing objective data and essential information to the process, enabling greater precision in the adjustments and in the evaluation of the amplification characteristics received by the hearing impaired individual25. The inser- tion gain is little applied vis-à-vis its importance23 and, as a consequence, there are but a handful of publications, which is too few to provide for greater discussions, espe- cially associated with unilateral hearing loss. CONCLUSION Chart 5. Values of the prescribed gain and the insertion gain at 4000 Hz. - Target Response Obtained Response By means of the self-assessment questionnaire we report on the satisfaction of individuals with unilateral hearing aids, even when the gain necessary to overcome the difficulties brought about by hearing impairment is not reached. Speech and hearing therapy requires studies which may contribute to the fitting of a hearing aid in unilateral hearing loss cases, especially concerning the way with which the checking process is carried out and the use of objective measures in the centers certified by the National Policy for Hearing Health Care14. The satisfaction of those individuals users of unilate- ral hearing aids is not totally associated with the prescribed gain, even though this is an important characteristic for the effective fitting of a hearing aid. In terms of the insertion gain, its results are extre- mely versatile, and when well utilized, the method enables 1. Consider the time during which you used your hearing aid in the last two weeks. For how many hours did you use it during a normal day? REFERENCES 10. Hush JL, Hosford-Dunn H. Inventories of Self-Assessment Measure- ments of Hearing Aid Outcome. In: Sandlin RE. Hearing Aid Ampli- fication: Technical and Clinical Considerations. 2nd ed. San Diego, California: Singular Publishing Group; 2000p.489-556. 1. Magni C, Freiberger F, Tonn K. Avaliação do grau de satisfação entre os usuários de amplificação de tecnologia analógica e digital. Braz J Otorhinolaryngol.2005,71(5):650-7. 11. World Health Organization. Grades of Hearing Impairment, 2007. 12. Cox R, Hyde M, Gatehouse S, Noble W, Dillon H, Bentler R, et al. Optimal outcome measures research priorities and international cooperation. Ear Hear.2000;21:5-6. y g 2. Instituto Brasileiro de Geografia e Estatística (IBGE). Disponível em http://www.ibge.gov.br/home/presidencia/noticias. 2. Instituto Brasileiro de Geografia e Estatística (IBGE). Disponível em http://www.ibge.gov.br/home/presidencia/noticias. 3. Kochins S. Hearing aids positively improve your quality of life. In: Carmen R. The consumer handbook on hearing loss and hearing aids: a bridge to healing. 2nd ed. Sedona, Arizona: Auricle Ink Publishers; 2004,p.62-76. 3. Kochins S. Hearing aids positively improve your quality of life. In: Carmen R. The consumer handbook on hearing loss and hearing aids: a bridge to healing. 2nd ed. Sedona, Arizona: Auricle Ink Publishers; 2004,p.62-76. 13. Cox RM, Stephens D, Kramer SE. Translations of the International Out- come Inventory for Hearing Aids (IOI-HA). Int J Audiol.2002;41:3-26. 14. Ministério da Saúde. Portaria número. 2.073/GM de 28 de setembro de 2004. Institui a Política Nacional de Saúde Auditiva [acesso em 20/02/2010]. Disponível em http://dtr2001.saude.gov.br/sas/PORTA- RIAS/Port2004/GM/GM-2073.htm 4. Vartiainen EA, Karjalainen S. Prevalence and etiology of unilateral sensoryneural hearing impairrment in finnish childhood population. Int J Pediatr Otorhinolaryngol.1998;3(2):253-9. Int J Pediatr Otorhinolaryngol.1998;3(2):253-9. 15. American Academy of Audiology. Guidelines for the audiologic management of adult hearing impairment. United States: AAA, 2008. 44p. Available from: http://www.audiology.org/resources/ documentlibrary/Documents/haguidelines.pdf. Cited: 01 Set 2009. 5. Mariotto LDF, Alvarenga KF, Filho OAC. Avaliação vestibular na perda auditiva sensorioneural unilateral: estudo vecto-electronistagmográ- fico. Disturb Comun.2006,18(1):27-38. 6. Almeida K, Santos TMM. Seleção e adaptação de próteses auditivas em crianças. In: Almeida K, Iorio MCM. Próteses auditivas: fundamentos teóricos e aplicações clínicas. São Paulo: Lovise; 2003.p.357-80. 16. Scollie S, Sweewald R. Evaluation if eletroacustic test signals I: com- paration with amplified speech. Ear Hear.2002;23(5):447-87. 17. Oslen SO. Simulated real ear measurements of benefit from digital feedback suppression. Int J Audiol.2008;47:51-8. 7. Bongiovani R. Principles of Posfitting Rehabilitation. In: Sandlin RE. Hearing Aid Amplification: Technical and Clinical Considerations. 2nd Edition. San Diego, California: Singular Publishing Group; 2000.p.439- 66. 18. ATTACHMENT How much did your hearing problem affected you in your a ( ) Very much ( ) Much ( ) Moderately ( ) A little ( ) Not at all 6. Consider the last two weeks using the ISADs. How much did your hearing problems affect or bothered other people? ( ) Very much ( ) Much ( ) Moderately ( ) A little ( ) Not at all 7. Taking everything into account, how do you think your ISADs change your joy in living or your enjoyment of life? ( ) To worse, or less joy in living ( ) There was no change ( ) A little more joy in living ( ) Much joy in living ( ) Very much joy in living ( ) Very much Taking everything into account, how do you think your ISADs change your joy in living or your enjoyment of life 7. Taking everything into account, how do you think your ISADs change your joy in living or your enjoyment of life? ( ) To worse, or less joy in living ( ) Very much joy in living ATTACHMENT ATTACHMENT International Outcome Inventory for Hearing Aids (IOI-HA) ATTACHMENT International Outcome Inventory for Hearing Aids (IOI-HA) 1. Consider the time during which you used your hearing aid in the last two weeks. For how many hours did you use it during a normal day? 1. Consider the time during which you used your hearing aid in the last two weeks. For how many hours did y which you used your hearing aid in the last two weeks. For how many hours did you use it during a normal day? ( ) Did not use it ( ) Less than 1 hour per day ( ) Between 1 and 4 hours per day ( ) Between 4 and 8 hours per day ( ) More than 8 hours per day situation you would like to be able to hear better before and after obtaining the hearing aid. In the last two weeks elp you in this same situation? ( ) It did not help at all ( ) It helped a little ( ) It helped moderately ( ) It helped much ( ) It helped very much 3. Consider again the same situation in which you would like to be able to hear better before and after the ISAD. Which is the level of difficul- ty you still have in this same situation using the hearing aids? ( ) It did not help at all ( ) It helped a little ( ) It helped moderately ( ) It helped much ( ) It helped very much 3. Consider again the same situation in which you would like to be able to hear better before and after the ISAD. Which is the level of difficul- ty you still have in this same situation using the hearing aids? ( ) Very much difficulty ( ) Much difficulty ( ) Moderate difficulty ( ) No difficulty at all Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 226 ( ) It is not worth it ( ) It is a little worth it ( ) It is moderately worth it ( ) It is much worth it ( ) It is very much worth it ) y nk about your last week using the ISADs. How much did your hearing problem affected you in your activities? 5. Think about your last week using the ISADs. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 20. Matas CG, Iório MCM. Verificação e validação do processo de seleção e adaptação de próteses auditivas. In: Almeida K, Iório MCM. Prótese Auditiva: Fundamentos Teóricos & Aplicações clínicas. 2ª ed. São Paulo: Lovise; 2003.p.305-20. 23. Zandavalli MB, Christmann LS, Garcez VRC. Rotina de procedimen- tos utilizados na seleção e adaptação de aparelhos de amplificação sonora individual em centros auditivos na cidade de Porto Alegre, Brasil-RS. Rev Cefac.2009;11(1):106-15. 21. Alpiner JG, Mc Carthy PA. Rehabilitative evaluation of hearing im- paired adults. In: Alpiner JG, Mc Carthy PA. Rehabilitative Audiology. 3rd ed. Philadelphia: Lippincott Williams e Wilkins; 2000.p.305-31. 22. Cox RM. Waiting for evidence - based practice for your fittings? It’s here. Hear J.2004;57(8):10-7. 24. Marcoux A, Hansen M. Ensuring Accuracy of the Pediatric Hearing Aid Fitting. Trends In Amplification.2003;7(1):11-27 25. Costa MJ, Couto CM, Almeida K. A utilização das mensurações in situ na avaliação do desempenho das próteses auditivas. In: Próteses auditivas: fundamentos teóricos e aplicações clínicas. 2. ed. São Paulo: Lovise; 2003.p141-60 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br REFERENCES International Society of Audiology. Good practice guidance for adult hearing aid fittings and services - Background to the document and consultation. Disponível em: <from:http//www.isa-audiology.org/ members/pdf/GPG-ADAF.pdf>. Acesso em 28/10/2009. 8. Hosford-Dunn H, Hush JL. Acceptance Benefit and Satisfaction Measures of Hearing Aid User Attitudes. In: Sandlin RE. Hearing Aid Amplification: Technical and Clinical Considerations. 2nd ed. San Diego, California: Singular Publishing Group; 2000p.467-88. 19. Valente M. Guideline for audiologic management of the adult patient. Audiology Online. 2006. Disponível em: http://www.audiologyonline. com/articles/article_detail.asp?article_id=1716. Acesso em 23/11/2009. 9. Almeida K. Avaliação dos Resultados da Intervenção. In: Almeida K, Iorio MCM. Próteses Auditivas: Fundamentos Teóricos & Aplicações Clínicas. 2ª ed. São Paulo: Editora Lovise; 2003.p.335-52. Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 227 25. Costa MJ, Couto CM, Almeida K. A utilização das mensurações in situ na avaliação do desempenho das próteses auditivas. In: Próteses auditivas: fundamentos teóricos e aplicações clínicas. 2. ed. São Paulo: Lovise; 2003.p141-60 Brazilian Journal of Otorhinolaryngology 77 (2) March/April 2011 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 228
https://openalex.org/W4237326392	https://misesjournal.org.br/misesjournal/article/download/827/511	Portuguese	null	Sobre as Origens do Bitcoin:	Mises/Mises: Revista Interdisciplinar de Filosofia, Direito e Economia	2,016	cc-by	12,051	MISES: Revista Interdisciplinar de Filosofia, Direito e Economia ISSN 2318-0811 Volume IV, Número 1 (Edição 7) Janeiro-Junho 2016: 121-139 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia ISSN 2318-0811 Volume IV, Número 1 (Edição 7) Janeiro-Junho 2016: 121-139 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia ISSN 2318-0811 Volume IV, Número 1 (Edição 7) Janeiro-Junho 2016: 121-139 * Texto baseado na versão de 3 de novembro de 2014. Esta parte contém duas novas seções empíricas que propor- cionam interpretações de padrões de eventos por ano (Apêndice A) e um único gráfico de formação de preços em cinco anos (Apêndice B). Com base nisto, o primeiro padrão claro de utilização do Bitcoin como meio de troca é especificado, pelo autor, como não ocorrendo até 2011. Traduzido do inglês para o português por Claudio A. Téllez-Zepeda. ** Konrad S. Graf é autor de diversos artigos sobre a teoria monetária do Bitcoin e filosofia do Direito. Também é conferencista e trabalha como tradutor profissional. Twitter: @konradsgraf Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II* Konrad S. Graf Resumo: Nesta obra, o autor desenvolve um estudo econômico e histórico da origem do Bitcoin e de seu valor enquanto moeda. Em sua argumentação, o autor explora a relação entre a moeda digital Bitcoin e a Economia Austríaca, discutindo, em parti- cular, o Teorema da Regressão de Ludwig von Mises e sua conexão com a evolução do mercado monetário, bem como a abordagem evolutiva seminal de Carl Menger. Palavras-Chave: Criptomoedas. Teoria monetária. Teorema da Regressão. Evolução monetária. pi p Traduzido do inglês para o português por Claudio A. Téllez-Zepeda. VIII - 120.000 Anos de “Meros” Colecionáveis Provavelmente, o primeiro meio de tro- ca histórico, que apareceu muitas e muitas vezes no registro arqueológico e em muitas áreas geográficas diferentes, são os colares de contas esculpidas a partir de várias conchas marinhas. A primeira descoberta conhecida de tais contas longe do oceano foi na Algéria moderna e data de aproximadamente 120.000 anos atrás. Quando itens são encontrados a esta distância da fonte, é considerado um bom sinal de que tenham sido provavelmente obtidos no comércio1. Ocorre que tais colares de contas de conchas têm características que agora enten- demos, em retrospecto, serem características essenciais dos meios de troca e de armazena- mento de valor. São duráveis, divisíveis, por- táveis e, em certo grau, intercambiáveis (ape- sar de que diferentes cores e tipos poderiam naturalmente ter sido valorizadas de forma distinta). Contas podem ser desencordoadas, contadas e novamente encordoadas em dife- rentes configurações numéricas. Nick Szabo, em suas discussões sobre as origens da moeda, chamou esses tipos de pro- to-moedas de “colecionáveis”2. Não são úteis no que poderia ser considerado um sentido prático. São coisas que muitas pessoas gos- tam de ter porque gostam de colecioná-las e usá-las. São distintivas, raras e difíceis de ob- ter, duram por muito tempo e são altamente portáveis. Szabo também apontou para as ca- racterísticas de segurança de tais itens. Dado que podem ser usadas, um ladrão deve agar- rá-las diretamente de uma pessoa em vez de simplesmente apanhá-las silenciosamente, sem ser visto. Esses pequenos colecionáveis também podem ser facilmente enterrados ou escondidos de outras maneiras durante épo- cas complicadas e recuperados em algum mo- mento posterior indefinido. Assim, é fácil imaginar como trocas indi- retas utilizando essas contas poderiam come- çar gradualmente a se desenvolver e difundir. Evidências arqueológicas altamente sugesti- vas do comércio de longa distância usando colares de contas remontam a muitas dezenas de milhares de anos e são encontradas em di- versas áreas. Mais tarde, descobrimos que as pessoas também gostam de colecionar pedaços de metais brilhantes que igualmente não eram realmente bons para grande parte do que era “prático”. As pessoas as conformavam em decorações fáceis de usar e eventualmente em alguns outros itens, tais como recipien- tes ornamentais. Vendo em uma longa escala temporal, o emprego de metais preciosos na cunhagem é um desenvolvimento histórico bastante recente. 1 Matt Ridley desenvolve tal evidência no contexto de lidar com o caso muito maior de trocar um coisa por outra coisa diferente ao mesmo tempo – em oposição ao tipo de reciprocidade retardada de favores, que também é encontrada em outras espécies – como talvez algo que possa ser a única característica distintiva que apareceu pela primeira vez com o Homo sapiens sapiens (e não com os primos conhecidos mais próximos tais como o Homo heidelbergensis e o Homo neanderthalensis) e que tornou possível a composição da especialização e a acumulação de conhecimento (RIDLEY, Matt. The Rational Optimist: How Prosperity Evolves. New York: Harper, 2010. Esp. cap. 2.). On The Origins of Bitcoin: Stages of Monetary Evolution – Part II Abstract: In this work, the author develops an economic and historical study of the origin of the Bitcoin and of its value as money. In his argument, the author explores the relation between the Bitcoin digital currency and Austrian Economics. Particularly, he discusses Ludwig von Mises’ Regression Theorem and its connection to the evolution of the monetary market, as well as Carl Menger’s seminal evolutionary approach. Keywords: Cryptocurrencies. Monetary theory. Regression Theorem. Monetary Evolution. Classificação JEL: G10, B53, E14, E42. Classificação JEL: G10, B53, E14, E42. Classificação JEL: G10, B53, E14, E42. Classificação JEL: G10, B53, E14, E42. Konrad S. Graf é autor de diversos artigos sobre a teoria monetária do Bitcoin e filosofia do Direito. Também é conferencista e trabalha como tradutor profissional. Twitter: @konradsgraf Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 122 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II Não eram de fato “moeda” em um sen- tido moderno, mas eram utilizadas para rea- lizar algumas de suas mesmas funções: arma- zenar valor durante longos períodos, trans- feri-los entre gerações (tal como na ocasião de casamentos), utilizá-los em negociações maiores ou para ajudar a selar pactos e como reserva, permitindo comprar de grupos vizi- nhos diante de uma queda local na disponibi- lidade de alimentos. No entanto, todos esses usos teriam que ter sido descobertos gradual- mente após as contas já terem existido como ornamentos. Teriam que ter algum valor de não-troca antes de que pudessem começar a ser negociadas. 2 SZABO, Nick. Shelling Out: The Origins of Money (2002). Acessado em altruists.org/f40. MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 123 Enquadrando a questão fundamen- tal sobre as origens da moeda em 1892, Carl Enquadrando a questão fundamen- tal sobre as origens da moeda em 1892, Carl de ouro e prata (com porcentagens natural- mente variáveis de cada metal). Isto foi ante- rior ao desenvolvimento de métodos efetivos de refino. Os às vezes apregoados empregos industrial e eletrônico do ouro e da prata são bastante modernos e, portanto, inteiramente irrelevantes quanto à primeira emergência des- ses metais para fins de negociação monetária em vários lugares muitos séculos antes. Menger (1840-1921) questionou: Qual é a natureza desses pequenos discos ou documentos, os quais em si mesmos pare- cem não servir a um propósito útil e que, ape- sar disso, em contradição com o resto da experiência, passam de uma mão para ou- tra em trocas pelas mercadorias mais úteis, mais ainda, pelas quais todos ficam tão an- siosamente empenhados em entregar suas mercadorias4? Menger (1840-1921) questionou: Qual é a natureza desses pequenos discos ou documentos, os quais em si mesmos pare- cem não servir a um propósito útil e que, ape- sar disso, em contradição com o resto da experiência, passam de uma mão para ou- tra em trocas pelas mercadorias mais úteis, mais ainda, pelas quais todos ficam tão an- siosamente empenhados em entregar suas mercadorias4? g O Bitcoin, durante pelo menos seus pri- meiros um ou dois anos, poderia facilmente ter sido descrito como uma curiosidade estra- nha e inútil, sem valor prático. Somente uns poucos programadores e criptógrafos acredi- tavam que o Bitcoin poderia terminar se tor- nando útil para alguma coisa além de jogos em computadores. O Bitcoin foi uma ideia e então um experimento de ciência da compu- tação e criptografia. Não estava claro, anteci- padamente ou mesmo depois de ter começa- do, se funcionaria tecnicamente ou se decola- ria economicamente. Chegou após um longo rastro de experimentos fracassados. Porque não poderia se tornar apenas mais um experi- mento fracassado? 3 Meramente “existem” como relações especificadas entre diversas categorias distintas de dados criptográficos. No tema dos “colecionáveis” proto- monetários, é interessante observar que, enquanto o MtGox mudou para se tornar um câmbio de Bitcoin em 2010, foi fundado em 2009 como um câmbio de figurinhas virtuais utilizadas no jogo Magic: The 4 MENGER, Carl. On the Origins of Money. Auburn, AL: Ludwig von Mises Institute, 2009 [1892]. p. 12-13. Gathering Online – de onde vem seu nome acronímico. Este é o DNA moderno da empresa orientada a “colecionáveis”, se é que alguma vez houve alguma. VIII - 120.000 Anos de “Meros” Colecionáveis A cunhagem lídia, que ocor- reu por volta de 700 a.C., é tipicamente citada como a primeira cunhagem de um metal pre- cioso. Era feita de electrum, uma liga natural IX - Camadas Técnicas e Econômicas Sempre Presentes tâncias brilhantes que podiam ser moldadas em pequenos adornos e utilizadas como or- namento? Imagine o quão estranho isto pode ter parecido às pessoas para as quais era algo totalmente novo. Ao ouvir que algumas pes- soas na reunião anual de comércio poderiam aceitar esses metais (nem sequer joias, apenas pedaços brutos de metal), o que um pastor pensaria? O caso do Bitcoin também mostra como a linha entre a invenção e a descoberta é muito mais tênue do que aquilo que em geral se pen- sa. Em cada caso da evolução monetária inicial, há um aspecto técnico e econômico. Antes de que alguma coisa possa começar a funcionar nas trocas, deve primeiramente existir em al- guma forma minimamente negociável. Contas devem ser esculpidas, pedaços de metal raro devem ser desenterrados e limpos, e mais tar- de moldados ou mesmo refinados. “Preciso ver isto por mim mesmo! Você está me dizendo que as pessoas são tolas o suficiente para aceitar pequenos pedaços bri- lhantes de metal em troca de bens reais?” É claro que, em retrospecto, é mais fácil en- tendermos até onde isto pode levar. Tais metais são fortes em várias características monetárias que os economistas agora entendem concei- tualmente como aspectos que podem ajudar a facilitar as trocas. Ademais, assim como as con- tas, a usabilidade e a capacidade das joias para serem escondidas podem proporcionar uma certa segurança e vantagem quanto à pou- pança. Muitas mulheres indígenas, até os dias atuais, tendem a usar no corpo suas economias familiares de longo prazo. Entretanto, para aqueles que não possuem tal conhecimento, o quão estranho este novo mundo deve ter sido, aprender a trocar coisas úteis por bugigangas sem utilidade, vez após vez, geração após ge- ração, até que a prática tomou conta de forma mais ampla, até que as pessoas “compraram” a ideia bizarra de que esses materiais poderiam também ser “úteis” e valorizáveis, não somen- te como objetos bonitos usáveis, mas também como unidades dentro de um sistema social que facilita as trocas. Não é diferente com o Bitcoin. Muito antes de que as unidades pudessem começar a funcionar nas trocas, tiveram que ser trazi- das à existência de tal maneira que os atores humanos pudessem possivelmente reconhe- cê-las, começar a brincar com elas e, eventual- mente, começar a dar o salto de negociá-las em troca de bens e serviços “reais”. Uma maneira de os modernos abor- darem o desafio de entender como entradas de registro contábil digitais que nem mesmo compreendem poderiam ser “realmente” va- lorizadas é refletir sobre o quão estranhos os tipos anteriores de novos meios de troca de- vem ter parecido para as pessoas do passado distante a partir de suas perspectivas. Colares de contas são, acima de tudo, objetos não prá- ticos e opcionais. Comparados com a carne, tubérculos e frutas; comparados a lanças, rou- pas e ferramentas; colares de contas e o va- lor “real” devem ter parecido distantes. No entanto, com o tempo, muitas pessoas pare- ceram gostar de fabricá-los e colecioná-los se pudessem. Eram decorativos. As contas eram raras e difíceis de obter. Levava um longo tempo para esculpi-las. Duravam por muito tempo (por exemplo, ainda podemos desen- terrá-las, 120.000 anos depois). Demonstra- ram socialmente um certo poder de excesso de capacidade, precisamente por causa de sua impraticabilidade. Somente mais tarde, uma população mais ampla começou a tomar conhecimento acerca das possibilidades de usar bitcoins, aqueles objetos digitais escassos e, de outro modo, inúteis para trocá-los por bens e servi- ços “reais”. Mas, obviamente, já vimos versões de tudo isto anteriormente. Vimos isto acontecer com os colares de contas de dezenas de mi- lhares de anos atrás e vimos isto ocorrer com as pessoas colecionando e brincando com me- tais macios e brilhantes há muitos milhares de anos. Agora, vemos isto nas entradas digitais de registros contábeis que formam objetos di- gitais que, em certos sentidos, não existem na realidade3. Da mesma forma, pedaços brilhantes de prata, ouro ou electrum, na maior parte dos lugares e há muitos milhares de anos atrás, não eram o epítome da utilidade. A lã era quente, a carne poderia alimentar, um arado poderia melhorar a colheita. Armas poderiam vir a calhar. O que acontecia com essas subs- Gathering Online – de onde vem seu nome acronímico. Este é o DNA moderno da empresa orientada a “colecionáveis”, se é que alguma vez houve alguma. Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 124 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II IX - Camadas Técnicas e Econômicas Sempre Presentes A rede primeiro teve que ser imaginada e lançada. Teve que ser testada e refinada, desafiada e provada ao longo do tempo. Em cada instância de uma nova evolu- ção monetária, há uma fase durante a qual alguma forma utilizável das unidades que eventualmente serão negociadas primeiro vêm a existir. Isto ocorre na camada técnica. Somente então uma camada monetária pode possivelmente emergir, com a negociação dessas unidades desenvolvidas tecnicamente. Ambas as camadas, técnica e econômica, con- tinuam subsequentemente a evoluir e intera- gir, adotando formas tais como ferramentas melhores para esculpir contas, técnicas para o refino de metais, ou o lançamento do pró- ximo software de Bitcoin. Contudo, ainda é importante entender como as camadas técni- ca e econômica estão sempre presentes, e não somente com o Bitcoin. De fato, devido ao estado lastimável da educação econômica, a maioria das pessoas até hoje ainda não entende conceitualmente a magnitude dos ganhos mútuos que advêm das possibilidades mais amplas para o comér- cio e que são facilitadas pela moeda. Não en- tendem que, sem essas práticas distintamente humanas de trocar bem por bem empregando moeda, somente a menor fração da população corrente poderia almejar sobreviver. As camadas técnicas envolvidas na pro- dução e troca de unidades físicas de merca- dorias são, obviamente, totalmente diferentes das camadas técnicas para as unidades cripto- gráficas descentralizadas de moeda corrente. MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 125 Com o Bitcoin, o aspecto “sistema” é muito mais proeminente do que o aspecto “unidade” em comparação com as evoluções de mercado de meios de troca tangíveis an- teriores. O aspecto de sistema agora é literal- mente uma massiva rede de computadores – que já é a mais poderosa do planeta em poder de processamento bruto por uma margem muito ampla – uma rede de desenvolvimento de software totalmente voluntária, e incontá- veis cópias distribuídas do registro de transa- ções descentralizado e não falsificável da ca- deia de blocos. Da mesma maneira, as camadas técnicas en- volvidas nos sinais de fumaça são totalmente diferentes das camadas técnicas envolvidos nas interfaces de chat pela internet. Não obs- tante, ambos os tipos de métodos tecnológicos conseguiram satisfazer, em diferentes graus, os propósitos humanos de enviar mensagens à distância (a camada “econômica”). 5 Aqueles que estão familiarizados com o modelo integral de todos os quadrantes desenvolvido pelo filósofo americano Ken Wilber podem reconhecer sua aplicação parcial nesta seção e na anterior. Em um eixo, a divisão epistemológica dualista entre o interior (significado) e o exterior (matéria) é representada na divisão entre o econômico (baseado em ação no sentido misesiano) e o técnico. O outro eixo, a dualidade das perspectivas da unidade e do sistema, é representada IX - Camadas Técnicas e Econômicas Sempre Presentes O que pode ter levado dezenas de mi- lhares de anos, em cada lugar, para as con- tas, e séculos para os metais, pode agora estar levando alguns anos e meses para o Bitcoin. Nos casos do passado, a prática de utilizar itens particulares nas negociações se desen- volveu, em cada contexto geográfico, em li- nhas de tempo separadas. Hoje, entretanto, os contextos do comércio, da imitação e da des- coberta são definidos em uma escala global pela demografia dos usuários e pelas escolhas na internet, em vez da geografia local ou das particularidades das rotas de comércio terres- tres e marítimas. Fluxos instantâneos de no- tícias e comunicações globais, mídias sociais, publicidade e a completa familiaridade das pessoas modernas com o conceito geral de troca indireta de larga escala são todos, agora, catalisadores. Unidades de Bitcoin podem existir so- mente na cadeia de blocos e só podem ser transferidas de endereço(s) para endereço(s) dentro do contexto da rede Bitcoin6. Possuir um bitcoin em um dado momento pode tam- bém ser visto, portanto, como algo análogo a possuir cotas de associação no clube de ne- gócios baseado na cadeia de blocos. Todos os controladores correntes das unidades de bitcoins são capazes de usar tanto o sistema de negociação quanto as suas unidades para seus próprios propósitos e de acordo com seu grau de “valor de entrada” autosselecionado para o sistema. p No entanto, aderir a um meio de troca começando a utilizá-lo como tal tem sem- pre implicado em algo mais do que somente comprar alguns objetos fora de um contexto. Significa, ao menos implicitamente se não ex- plicitamente, demonstrar uma pequena mar- gem de suporte para a ideia de que este tipo de unidade é algo que pode funcionar como um meio de troca na sociedade. É devido ao processo de mais e mais pessoas, uma por 6 Apesar das chaves privadas poderem também ser fisicamente transferidas offline utilizando tokens (chaves eletrônicas) e cartões, a segurança e a confiabilidade de tais elementos permanece como altamente experimental. na seção corrente. Para um panorama relativamente acessível do modelo integral de todos os quadrantes e conceitos relacionais em um contexto mais amplo, ver: WILBER, Ken. The Marriage of Sense and Soul: Integrating Science and Religion. New York: Broadway Books, 1998. na seção corrente. Para um panorama relativamente acessível do modelo integral de todos os quadrantes e conceitos relacionais em um contexto mais amplo, ver: WILBER, Ken. The Marriage of Sense and Soul: Integrating Science and Religion. New York: Broadway Books, 1998. X - A Importância das Perspectivas do Sistema e da Unidade Qualquer sistema monetário não é so- mente uma coleção de unidades fora de con- texto, mas sim um sistema. Isto inclui uma rede de pessoas com um entendimento social semelhante a respeito dos usos comerciais das unidades relevantes. Objetos isolados não constituem um sistema de trocas5. Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 126 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 126 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II uma, tornarem esta afirmação do “sistema” mais ampla por estarem realmente aceitando, economizando, e gastando uma unidade, que isso começa a se desenvolver para além de um item meramente colecionável. É somente den- tro do contexto de um sistema ativo de trocas que este ciclo adicional de valorização pode se acumular. Isto é verdadeiro para todos os meios de troca possíveis enquanto crescem neste papel, independentemente das caracte- rísticas técnicas incidentais tais como tangibi- lidade contra intangibilidade. leiro) não aceitar o bem intermediário preten- dido (ovos). O componente de valor do meio- -de-troca de um bem que se torna utilizado de forma mais geral neste papel pode se mul- tiplicar quando outros comecem a aceitá-lo mais e mais no comércio porque também espe- ram que outros irão aceitá-lo em negociações subsequentes. Este ciclo de construção de va- lor pode ser circular e de autorreforço. Entre- tanto, nenhum ciclo pode começar em causa- ção circular. O teorema da regressão explica em termos abstratos o que deve estar presente para iniciar qualquer ciclo de rede autorrefor- çante - alguma outra valorização anterior ao início de sua utilização. Em casos históricos prévios, o aspecto unidade era mais óbvio e o aspecto de valor de entrada do sistema era perceptível somen- te de forma mais abstrata e indireta. Com o Bitcoin, entretanto, o aspecto de valor de entrada do sistema é mais óbvio e explícito, enquanto o aspecto unidade é perceptível so- mente mais abstrata e indiretamente. Não é mais possível, literalmente, perder o aspec- to sistema porque as unidades Bitcoin são compreensíveis somente dentro do contexto de um sistema descentralizado de registros contábeis que permite a primeira apropria- ção confiável (mineração), o armazenamento e a transferência. A realidade subjacente que fundamenta seu aspecto unidade é técnico e difícil de entender. 7 economicsofbitcoin.com. “Professor Walter Block is clueless about Bitcoin”. 22 de setembro de 2013. XI - Uma Ilustração Bem Documentada O que segue é de um comentário que deixou sob a postagem acima: No entanto, mesmo tais itens digitais de jogo podem também ser contados como bens de acordo com uma abordagem estritamente misesiana da economia, que estuda as ações como tais. Como um campo, a economia na tradição misesiana não pretende julgar de ma- neira objetiva se certas ações são “racionais” e outras não, se certas ações são “econômicas” ou “morais” e outras não, e assim por diante. Essas são tarefas reservadas para outros cam- pos, tais como a filosofia ou a psicologia, com diferentes metodologias e critérios. Bens que existem somente dentro de jogos são bens do ponto de vista daqueles que estão engajados em jogá-los e que gastam tempo e esforços para obtê-los8. p g Eu ainda estava esperando pela descoberta de soluções mais eficientes... Eu também es- tava pensando que bitcoin tinha uma chance em um milhão de poder inicializar para um valor que não fosse de brinquedo. Então não fiz download, nem garimpei naquela épo- ca... Então, apesar de ter inventado a função de mineração que o bitcoin utiliza e de ter gasto muito esforço de pesquisa ao longo de talvez 5 anos conectado e desconectado com outros, tais como Wei Dai, Hal Finney, Nick Szabo e um elenco de dúzias, inclusi- ve colaboradores anônimos (um dos quais pode bem ter sido Satoshi) para tentar des- cobrir como desenhar um sistema de ecash descentralizado utilizando o hashcash como uma função de mineração, não me dei con- ta, a não ser anos mais tarde, quando estava claramente óbvio que o Bitcoin já tinha sido inicializado há muito tempo. Todas as sementes e a produção inexis- tente em jogos de fazenda, podem argumentar alguns críticos, “na verdade” são apenas pu- nhados de uns e zeros em um computador. As- sim como são, alegam, os bitcoins. No entanto, os proponentes deste ponto de vista seriam ca- pazes de reconhecer que a diferença entre um e outro filme digital, ou entre um e outro arqui- vo de música, poderia, da mesma forma, ser reduzida a uns e zeros intercambiáveis? Nos primeiros dias, mesmo peritos nos campos relevantes, assim como qualquer ou- tra pessoa, poderiam facilmente ter visto a mineração de bitcoins como uma atividade razoavelmente sem sentido. XI - Uma Ilustração Bem Documentada As unidades Bitcoin não tiveram qual- quer valor de troca reconhecido por quase um ano após o início do funcionamento da rede Bitcoin em janeiro de 2009. Ademais, não ti- nham ainda nenhuma utilidade específica como meio de troca. Não facilitavam quais- quer negociações. Em vez disso, programa- dores e entusiastas da criptografia estavam rodando o programa, conectando-se à rede incipiente, testando o código e trabalhando para aprimorá-lo, garimpando e transmitindo bitcoins entre si por diversão e experimenta- ção. Em contraste, moedas de metal precio- so, apesar de também estarem totalmente imersas em um sistema social de trocas, des- tacaram-se desse sistema pela sua concretude e tangibilidade. Com a moeda de metal, ainda podemos ingenuamente imaginar que, de al- guma forma, mantinha o valor totalmente por si mesma. Entretanto, isto nunca foi verdade. O valor de troca é sempre dependente da dis- posição de um comprador para comprar. Um componente de valor de um meio de troca é, de forma semelhante, sempre dependente da expectativa implícita de aceitação futura por pelo menos uma outra parte. Uma unidade de negociação somente pode funcionar dentro de um sistema social de negociação. Peter Šurda tem reunido algumas evi- dências relatadas em primeira mão sobre este período inicial. Em uma postagem recente7, citou o participante inicial e desenvolvedor Mike Hearn, falando a partir de sua perspec- tiva como novo participante nos primeiros dias: Encontrei [Bitcoin] logo no início, quanto ninguém o estava usando, então ninguém, nenhuma troca, não havia nem sequer uma taxa de câmbio, então estavam apenas flu- tuando totalmente em um espaço abstrato. Dissemos acima que uma tentativa de troca indireta de bife por farinha falharia se a parte destinada para a segunda troca (o mo- MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 127 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 127 Você sabe o que era uma moeda? Bem, na realidade, nada. vos para os jogadores depois de terem gasto dezenas ou centenas de horas construindo um próspero negócio (não existente). Adam Back, o desenvolvedor de ele- mentos cruciais que foram utilizados como partes do sistema Bitcoin, era altamente céti- co durante este período inicial de desenvolvi- mento. 8 Ver meu artigo do dia 15 de setembro de 2013, “The labor, leisure, and happiness game: Psychology, praxeology, and ethics”, em konradsgraf.com. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro No dia 5 de outubro de 2009, nove meses após o início da rede Bitcoin, o primeiro pre- ço de oferta de Bitcoin registrado foi postado. Atingiu 13 bitcoins por um centavo, ou espe- cificamente, 1.309,03 Bitcoins por dólar, o que o autor da postagem calculou com base em seus custos de mineração variáveis9. ç Muitos sistemas estavam lá fora, espe- rando ter sucesso como algum tipo novo de dinheiro digital – e fracassando. Peritos não podiam antecipar universalmente que este novo tipo iria dar certo. Seu sucesso começou com uma nova combinação de ideias, mas seu sucesso precisava ser definido através da ex- perimentação e descoberta na prática. Se nos- so propósito tivesse sido negociar ou comprar bens ou serviços com Bitcoins em seus pri- meiros um ou dois anos, então eram totalmen- te inúteis. No entanto, não obstante já tinham gradualmente começado a desempenhar vá- rios papéis nas estruturas de ação de alguns experimentadores iniciais. ç Alguns meses mais tarde, no dia 22 de maio de 2010, ocorreu o que se acredita ter sido a primeira utilização de Bitcoin para comprar um bem de mercado “real” ao custo de 10.000 Bitcoins por $25 no valor de uma pi- zza10. Na realidade, uma outra parte facilitou uma compra de pizza aceitando Bitcoin e pa- gando pela pizza com um cartão de crédito. A fama da “transação Bitcoin da pizza”, ape- sar de ser um importante marco simbólico, mal constitui uma exceção à generalização de que o Bitcoin não era utilizado, à época, para comprar bens ou serviços. Em vez de simples- mente facilitar a compra de uma pizza como tal, isto pode ser visto mais como uma ocasião para vender 10.000 Bitcoins para alguém que pagou $25 usando o cartão de crédito e, quan- do isso aconteceu, os $25 foram direcionados para o fornecedor de pizza ao invés do vende- dor de Bitcoin. Este é o momento para o qual o com- ponente de origens do teorema da regressão tenta “regredir” de volta. Esta era uma época na qual os bitcoins tinham algum valor para algumas pessoas, mas sair e comprar alguma coisa com bitcoins – facilitar trocas com eles – não era algo que estivesse entre os usos dispo- níveis. XI - Uma Ilustração Bem Documentada Teria sido fácil para pessoas de fora descartá-la como algo não muito diferente, em princípio, de uma versão glorificada do jogo social online Farm- ville. A diferença foi que era como se alguns dos jogadores também estivessem conven- cidos de que tinham descoberto o futuro do verdadeiro cultivo. Isto apenas faz com que pareça ainda mais ridículo do que simples- mente admitir estar jogando um jogo. Tente dizer a alguém absorto em um filme de ação ou em um jogo do seu esporte preferido que “na realidade” seria a mesma coisa se mudasse para um vídeo da Hello Kitty ou para um jogo de algum esporte estrangeiro que detestasse. Agora, pegue as listas de re- produção de música pop de pré-adolescentes e dos concertos clássicos do vovô, troque-as e veja o que acontece. A realidade das distin- ções significativas entre “meros zeros e uns”, quando se trata de bens digitais, deve ter sido estabelecida de maneira convincente (se o ex- perimentador conseguir sobreviver para rela- tar os resultados). Nos jogos de fazenda, sabe-se que núme- ros incontáveis de pessoas plantam sementes “inutilmente”, utilizando seus computadores. Depois checam com ansiedade para colherem os cereais resultantes. Esses objetos não-reais podem se tornar completamente significati- Alguns dos primeiros mineradores/ex- perimentadores do Bitcoin, ao menos aqueles Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 128 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 128 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II que acreditavam que o sistema tinha futuro, acumularam bitcoins, somente em caso do seu valor vir a poder aumentar no futuro. Para os participantes, após começarem, às vezes pode ter sido mais fácil manter as moedas do que se livrar delas. Enquanto alguns as mantiveram seguras, não obstante, outros dos primeiros mineradores apagaram arquivos de carteira que davam acesso a grandes quantidades de bitcoins. Outras ainda consideraram que não valia a pena sequer começar. Estavam con- vencidos de que nada jamais poderia resultar, no futuro, dessas unidades. Cada um destes julgamentos foi distinto, valorações demons- tradas em ação. tóricas dos seus princípios teóricos o Bitcoin proporciona um exemplo documentado de livro didático. 10 Em Block 57035, ID de transação: blockchain.info/tx /49d2adb6e476fa46d8357babf78b1b501fd39e177ac7833 124b3f67b17c40c2a. 9 Ver o Apêndice B para um gráfico de preços de todos os tempos para cinco anos. 9 Ver o Apêndice B para um gráfico de preços de todos os tempos para cinco anos. 10 Em Block 57035, ID de transação: blockchain.info/tx /49d2adb6e476fa46d8357babf78b1b501fd39e177ac7833 124b3f67b17c40c2a. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro Naquela época, poderia ter sido mais fácil trocar diretamente um bife por farinha com o nosso vizinho ovo-vegetariano do que obter qualquer coisa que fosse em troca de Bitcoin. Como quer que seja caracterizado, o va- lor equivalente era de $25 por 10.000 bitcoins, ou quatro bitcoins por um centavo, um au- mento triplicado sobre o primeiro preço cota- do, meio ano antes. Esta foi uma transação de câmbio incipiente quase-indireta (e mal pode ser contada como tal) usando Bitcoin e prova- Esta condição, na qual havia algum va- lor para alguns atores, mas zero valor para trocas indiretas, estava prestes a começar a mudar. Apesar do teorema da regressão ser uma dedução lógica a partir das definições, para aqueles que gostam de ilustrações his- MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 129 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 129 velmente feita, em parte, em tom de brincadei- ra, 16 meses depois que a rede começou a exis- tir. Claramente, o Bitcoin não saltou simples- mente para a vida como uma moeda funcional. meios adequados para garantir o que lhes corresponde. Por isso, também é claro que nada poderia ter sido tão favorável à gênese de um meio de troca quanto a aceitação, por parte dos sujeitos econômicos mais pers- picazes e capazes, para seu próprio ganho econômico, e durante um período consi- derável de tempo, de bens eminentemente vendáveis preferencialmente em relação a todos os outros. Muitos meses mais tarde, no dia 17 de julho de 2010, a primeira operação de câmbio pública registrada no novo câmbio MtGox in- dicava um preço de $0,05, que aumentou para $0,06-$0,09 por bitcoin, variando durante os dias subsequentes. O que pode ser difícil de entender é que, devido à natureza digital das unidades de bitcoin, seus custos de transação próximos de zero e pontos extremamente for- tes em muitas das principais características valiosas para o uso de um meio de troca, o início de qualquer valor de troca, não importa o quão pequeno, pode ser suficiente para aju- dar a começar a negociar estas unidades. Esse início eventualmente ocorreu. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro Assim, a prática e o hábito certamente não contribuíram com pouco para fazer com que os bens, que eram mais vendáveis em qualquer momento, fossem aceitos não somen- te por muitos, mas finalmente por todos os su- jeitos econômicos em troca pelos seus bens me- nos vendáveis; não somente isso, mas para se- rem aceitos com a intenção inicial de trocá-los novamente. Os bens que dessa forma se torna- ram meios de troca de aceitação geral foram chamados pelos alemães de Geld, que vem de gelten, ou seja, pagar, executar, enquanto ou- tras nações derivaram suas designações para o dinheiro principalmente a partir da substância utilizada, a forma da moeda, ou mesmo a par- tir de certos tipos de moeda12. Assim que algumas transações tiveram início e as primeiras empresas começaram a aceitar Bitcoin em pagamento por bens e ser- viços “reais”, novas expectativas de um futu- ro componente de valor de meio de troca tam- bém poderiam começar a crescer – dentro dos grupos de usuários relevantes. Na medida em que mais pessoas testemunhavam como os estranhos pioneiros de fato utilizavam o sistema para interagir com coisas reais, o que anteriormente tinha sido somente um experi- mento começou a realizar tentativas de inte- ração com a economia de troca mais ampla. Isto somente se tornou inequívoco, no regis- tro histórico, em 201111. Bitcoins oferecem ao menos diversas vantagens competitivas que são atraentes so- bre outras opções: p 1. São uma unidade global inerentemente apolítica com relação a fronteiras. Parceiros comerciais potenciais diretos a longo prazo incluem, portanto, a totalidade da popula- ção humana, desde o barista da esquina até o produtor de grãos de café para envio dire- to do outro lado da esquina Somente umas poucas pessoas, e então mais umas poucas, tinham que dar passos marginais nesta direção, mais outras para eventualmente imitá-las, ano após ano. Carl Menger descreveu, em 1892, o processo que, portanto, teve início e que continua a acelerar até o presente: 2. São infinitamente duráveis no tempo e (quase) sem custos de transporte no espaço. Podem durar potencialmente para sempre (para qualquer propósito prático), com de- gradação zero. 11 Ver o Apêndice A para uma interpretação monetária- histórica dos padrões de eventos novos por ano. 12 MENGER. On the Origin of Money, p. 37. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro Tal como as contas e os me- tais que vieram antes – só que mais ainda p Não existe método melhor para esclarecer qualquer um a respeito de seus interesses econômicos do que fazer com que perceba o sucesso econômico daqueles que usam os – seu acesso pode ser facilmente realizado, ou mesmo apenas recordado, então podem ser escondidos durante tempos de ameaça. 3. Possuem características de escassez superiores a todas as demais alternativas 12 MENGER. On the Origin of Money, p. 37. Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 130 130 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II entram em jogo13. entram em jogo13. monetárias. A limitação de sua oferta está incorporada em sua definição. Esta aborda- gem é superior às limitações ancoradas mera- mente no fornecimento prático e variável de alguma substância química, tal como a prata. São ainda mais obviamente superiores em comparação com o histórico comprovado de maquinações inflacionárias por parte dos in- dicados políticos cuidadosamente seleciona- dos e encarregados do suprimento de moeda fiduciária. No entanto, não importa qual seja seu va- lor corrente em relação a outras moedas, pode continuar a desempenhar um papel como um método de custo quase zero para transmitir poder de compra à distância para qualquer transação dada. Os usuários que optam por serviços que convertem imediatamente para dentro e fora do Bitcoin em cada transação podem estar totalmente isentos de risco cam- bial, enquanto outros usuários optam por assumir tais riscos, conscientemente, na ex- pectativa da obtenção de ganhos de curto ou longo prazo. Um papel futuro possível para o Bitcoin como o que eu tenho definido como moeda (unidade de preços, de contabilidade e de cálculo econômico) teria que esperar por muito mais estabilidade de valor relativo em comparação com as opções concorrentes para essas funções. 4. Oferecem custos de transação que são qua- se infinitamente pequenos do ponto de vista dos usuários e isto vale para qualquer quanti- dade e a qualquer distância. 4. Oferecem custos de transação que são qua- se infinitamente pequenos do ponto de vista dos usuários e isto vale para qualquer quanti- dade e a qualquer distância. 5. Não podem ser falsificados, diluídos, ou multiplicados, ao contrário das notas de papel e das moedas metálicas. 5. Não podem ser falsificados, diluídos, ou multiplicados, ao contrário das notas de papel e das moedas metálicas. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro Com tal alinhamento totalmente sem precedentes de características monetárias (entre outras), qualquer valor de negociação de partida poderia ter sido suficiente para fazer com que essas unidades extraordi- nariamente bem-adaptadas começassem a adquirir um componente de valor de meio de troca entre alguns usuários. Sendo a pri- meira entre as criptomoedas correntes a dar esse salto, o Bitcoin tem permanecido muito à frente de moedas imitadoras posteriores com base na vantagem do pioneirismo, dos efeitos de rede e da velocidade agregada de hashing de rede, tornando-se, e permanecen- do por uma margem enorme, a mais “ven- dável” de todas as criptomoedas existentes. No entanto, também deve ser salientado que o crescimento do valor, em contraste com o declínio estável do valor da moeda fiduciá- ria, é uma vantagem separada da estabilidade per se. Algumas pessoas poderiam preferir ter a opção de manter alguns saldos em unidades que ganharam valor de forma instável, em contraste com os saldos de outras unidades que perderam valor progressivamente. 13 O Bitcoin é frequentemente criticado por suas amplas movimentações de preços. Isto é sobretudo uma consequência do Bitcoin ainda ser jovem e pequeno em escala, o que o torna suscetível aos altos e baixos do ciclo de sensibilização da mídia. Dito isto, o primeiro movimento dramático para cima e para baixo em 2011 também pode ser causalmente conectado à contínua imposição governamental de políticas de proibição. Sem elas, o aumento da demanda de possíveis participantes no então novo mercado online Silk Road de resistência à proibição teria sido muito mais modesto em comparação com os movimentos dos mercados ordinários, não assolados pelos resultados sempre estranhos e perigosos das políticas de proibição. Isto provavelmente explica o único efeito preço “montanha” ano após ano visto em 2011, em termos YoY. Ver meu artigo de 25 de julho de 2013, “Tiger cub growing up? Bitcoin weekly average closing price year over year”, em konradsgraf.com. O componente de valor de meio de tro- ca dos bitcoins é agora claro e dominante em retrospecto. Assim como um novo tipo de metal, desenterrado um dia há muitos milhares de anos, seu perfil de valor na so- ciedade emergiu gradualmente e aparente- mente a partir do nada. O seu valor de troca, em seguida, moveu-se por todo o caminho até... praticamente nada. 13 O Bitcoin é frequentemente criticado por suas amplas movimentações de preços. Isto é sobretudo uma consequência do Bitcoin ainda ser jovem e pequeno em escala, o que o torna suscetível aos altos e baixos do ciclo de sensibilização da mídia. Dito isto, o primeiro movimento dramático para cima e para baixo em 2011 também pode ser causalmente conectado à contínua imposição governamental de políticas de proibição. Sem elas, o aumento da demanda de possíveis participantes no então novo mercado online Silk Road de resistência à proibição teria sido muito mais modesto em comparação com os movimentos dos mercados ordinários, não assolados pelos resultados sempre estranhos e perigosos das políticas de proibição. Isto provavelmente explica o único efeito preço “montanha” ano após ano visto em 2011, em termos YoY. Ver meu artigo de 25 de julho de 2013, “Tiger cub growing up? Bitcoin weekly average closing price year over year”, em konradsgraf.com. XIII - Distinguindo os Componentes de Valor Iniciais tos de investimento financeiro de alto risco) com dinheiro, apenas no caso das unidades poderem vir a valer alguma coisa no futuro. Bitcoins podem ter funcionado em qualquer uma das pelo menos várias formas não mone- tárias possíveis: Os primeiros pioneiros do Bitcoin esta- vam tentando criar um sistema funcional de transmissão de valor que contivesse unidades que pudessem ser utilizadas como um meio de troca. Apesar dessa esperança, durante pelo menos 2009, e as evidências sugerem também em 2010, ainda não haviam conseguido. O Bit- coin não foi utilizado como um meio de troca. Quaisquer que fossem seus sonhos ou fantasias futuras, as operações de troca indireta, basica- mente, não foram facilitadas usando o Bitcoin14.i p 1. Um conjunto de objetos digitais que po- deriam vir a se tornar um meio de troca no futuro se a rede de alguma forma tivesse sucesso e se suas unidades eventualmente começassem a ser tomadas como meio de troca. Qualquer classe nova valorizável de bens é reconhecida e valorizada pela pri- meira vez por poucas pessoas, geralmen- te antes de que tenha qualquer utilidade “prática”. Muitos desses bens jamais atin- gem grandes resultados, mas outros deco- lam e são eventualmente valorizados de maneira ampla. As unidades de Bitcoin, no entanto, fize- ram seu caminho para dentro das estruturas de ação desses primeiros usuários de várias maneiras. Eram os objetos da ação, tanto dire- tamente quanto indiretamente, “empacotadas” com outros fins, assim como as metas de aper- feiçoamento do sistema, o desenvolvimento da mineração e a participação no projeto. Isso os torna bens (meios em direção a fins, em termos misesianos). 2. Um conjunto de objetos digitais que po- deriam ser enviados e recebidos para os demais participantes como uma forma de teste formal ou informal da rede, ou até mesmo como um tipo de interação social dentro do projeto. Muitas das interações do projeto ocorreram online, à distância e em fóruns, e, em alguns casos, os partici- pantes utilizavam pseudônimos. 2. Um conjunto de objetos digitais que po- deriam ser enviados e recebidos para os demais participantes como uma forma de teste formal ou informal da rede, ou até mesmo como um tipo de interação social dentro do projeto. Muitas das interações do projeto ocorreram online, à distância e em fóruns, e, em alguns casos, os partici- pantes utilizavam pseudônimos. 14 Uns poucos casos isolados não podem ser descartados, mas nenhum padrão de tal utilização é visível no registro histórico, uma situação que muda inequivocamente de 2011 em diante. 15 Isto era, no entanto, muito raro. Uma varredura ocasional através da cadeia de blocos de 2009 revela um grande número de blocos que não contêm nada além de uma transação de geração. XII – O Primeiro Surgimento de Preços de Bitcoin por Dinheiro Finalmente, construiu-se gradualmen- te ao longo dos últimos três ou quatro anos, com alguns saltos dramáticos ao longo do caminho, na medida em que círculos cada vez mais amplos de reconhecimento social MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 131 XIII - Distinguindo os Componentes de Valor Iniciais Em resumo, a mistura dos seguintes não- -componentes de valor de meio de troca das unidades de bitcoin existia para alguns usuá- rios em um período ao longo de 2009 e 2010, antes que o Bitcoin operasse desempenhando qualquer papel de meio de troca monetário que fosse. Isto ocorreu depois que o Bitcoin começou a existir e ganhar valor para alguns atores dentro de suas respectivas estruturas de fins e meios, mas antes que começasse a ter qualquer componente de valor para a sua utilização como facilitador de trocas indiretas por outros bens e serviços. 3. Os blocos estavam sendo garimpados e uma das finalidades disso era verificar as transações. Sem transações para verificar, os processos de teste e experimentação da rede teriam permanecido incompletos, propor- cionando uma razão para emitir transações em alguns pontos da rede, mesmo que ape- nas com o propósito de teste e experimen- tação15. 4. Em alguns pontos e para algumas pes- soas, as unidades de bitcoin poderiam ter funcionado como um conjunto de objetos digitais de brinquedo que poderiam ser coletadas incidentalmente ou mesmo com- petitivamente no decurso dos processos de experimentação acima. Isto poderia ser semelhante a como as pessoas naturalmen- te apreciam competir pela acumulação e As pessoas relevantes foram os pionei- ros que realmente participaram da rede Bit- coin, contribuindo para o aperfeiçoamento inicial do seu software e de outras tecnolo- gias. Mais tarde, alguns participantes adicio- nais compraram bitcoins (como instrumen- Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 132 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 132 e a liberdade da “censura financeira”; e es- forços educativos para explicar as vantagens econômicas gerais da disponibilidade de uma unidade de negociação de valor crescente para aqueles que desejam utilizar algo assim. comparação de diferentes quantidades de pontos ou moedas digitais nos jogos, inde- pendentemente de quaisquer valorizações de “mundo real”. Seria um bônus adicio- nal se um eventual valor de “mundo real” realmente pudesse surgir, como no item 1 acima. 5. A posse de maiores ou menores quanti- dades de bitcoins poderia ser considerada como símbolo de adesão e compromisso, ou como um dos sinais e artefatos de participa- ção. XIV - Registre Isto Aqui Também para Aqueles que São Loucos O processo pelo qual o valor do Bitcoin para utilização como meio de troca surgiu tem sido documentado e arquivado em regis- tros online e em memórias vivas. Os passos iniciais do Bitcoin servem como uma ilustra- ção do aspecto de origens do teorema da re- gressão que é semelhante, em princípio – em- bora, naturalmente, não na forma específica, configuração geográfica ou escala de tempo – às narrativas da evolução do mercado para a interpretação dos processos pelos quais sur- giram meios de troca tangíveis anteriores. 6. Uma forma de propriedade de ações no início do sistema de pagamentos (potencial) que transmitisse a possibilidade de parti- cipar de forma mais favorável no futuro e ajudar a promovê-lo a partir de uma fase inicial. Esta metafórica parcela de investi- mento em todo o sistema também cria um incentivo minúsculo e discreto para agir de uma maneira favorável para o bem-estar geral do sistema. Todos os primeiros parti- cipantes investiram seu tempo e esforço de forma individual e voluntária. Ou seja, em cada um dos casos que te- mos considerado, um punhado de “loucos” da humanidade16 estavam coletando e brin- cando com coisas que eram inúteis para qual- quer coisa que pudesse ter sido considerada um propósito geral “prático” nas fases origi- nas em questão, tais como contas de conchas, metais brilhantes, ou bitcoins. Eventualmen- te, porém, uma utilidade econômica adicional para estas meras quinquilharias – na realida- de, utilizá-las para facilitar as transações – foi posta em prática gradualmente e aos trancos e barrancos, com alguns resultados favoráveis. Esta prática então se difundiu através de al- guma combinação de imitação e redescoberta independente recorrente. Algumas dessas valorações das unida- des de bitcoin, não como um meio de troca, ou dela decorrentes, persistem até hoje entre alguns participantes do sistema. 16 Em referência ao icônico comercial da Apple de 1997, “Here’s to the crazy ones”. Em youtu.be/tjgtLSHhTPg. XIII - Distinguindo os Componentes de Valor Iniciais Possíveis interesses, não como meio de troca, poderiam facilmente incluir: interesse teórico e científico, desafio de programação, pesquisa sobre segurança, interesse ideoló- gico em avançar a liberdade de transações e interesse na possibilidade de criar uma moeda de valor crescente disponível pela primeira vez em um século para aqueles que desejavam ter a opção de utilizar algo assim. Motivações de adesão e promoção são comuns em empreendimentos humanos e estão presentes sempre que dizemos que alguém está “apoiando uma causa”. XIV - Registre Isto Aqui Também para Aqueles que São Loucos Incluem: re- tenção de longo prazo de saldos de Bitcoin como poupança (muitas vezes tratada pejora- tivamente como “especulação”); esforços para difundir o evangelho e apresentar outros ao Bitcoin em bases tanto individuais como ins- titucionais – inclusive dando pequenas quan- tidades de Bitcoin para novos usuários – o que amplia a rede de usuários e aplicações; esfor- ços para promover a liberdade das transações Tais experiências provavelmente encon- traram, em cada caso, sobrancelhas franzidas e descrença por parte dos observadores mais sóbrios, ainda não dispostos a trocar coisas “reais” por coisas tais como conchas, metais macios e brilhantes, ou, no caso atual... algo so- MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 133 as pessoas ainda tinham que adotá-lo e colo- cá-lo para este uso. Cada pessoa permanece inteiramente livre para ignorá-lo ou utilizá-lo de alguma forma e apenas na exata medida da participação voluntária de cada um. bre o que não fazem a menor ideia do que seja. A diferença é que, hoje, o processo de mais e mais pessoas estarem descobrindo como utili- zar os Bitcoin para facilitar as negociações está ocorrendo em todo o mundo e com a velocida- de da internet e do boca-a-boca. Os elementos de invenção intencional provavelmente ajudam a explicar por que o Bitcoin é superior a qualquer meio de troca conhecido em praticamente todas as caracte- rísticas significativas relevantes (a tangibilida- de sendo uma característica histórica inciden- tal, conforme tenho argumentado). Deveria uma superioridade competitiva esmagadora e de base ampla para um dado propósito ser considerada como um desqualificador para esse propósito? Isso seria de fato estranho. No entanto, alguém já familiarizado com os aspectos teóricos do surgimento da moeda poderia levar em consideração, e parece ter levado, essas mesmas percepções na tentati- va de moldar um bem melhor para ser usado nesse papel. Que o(s) inventor(es) e os primeiros de- senvolvedores do Bitcoin pretendessem que a rede fosse utilizada para este propósito pode ser confuso para algumas pessoas, à luz das caracterizações gerais de fenômenos de mer- cado como sendo “emergentes” e “o resulta- do da ação humana mas não do projeto hu- mano”. Isso pode lembrá-los, vagamente, da história totalmente diferente, da teoria estatal da moeda, da imposição da “fidúcia” por um governante. XIV - Registre Isto Aqui Também para Aqueles que São Loucos No entanto, Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 134 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 134 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II metral, quanto mais informadas as pessoas são a respeito dos fundamentos técnicos do Bitcoin, mais tendem a apreciá-lo. Enquanto isso, detratores casuais em geral parecem exi- bir pouca ou nenhuma compreensão técnica de como o sistema funciona. A emergência do Bitcoin como meio de troca foi original, tal como ocorreu, no passa- do, com as contas ou com os metais. Não foi derivativo, com valores monetários retoma- dos a partir de precursores tangíveis, como no caso da história das modernas notas em papel e entradas de contabilidade fiduciária digitais. O Bitcoin nos dá uma ilustração didática da afirmação praxiológica de Mises do teore- ma da regressão. Proporciona um caso de es- tudo empírico que tem sido documentado em nível de detalhamento nunca antes visto. Se assim o quiserem, pesquisadores do campo da história econômica ainda poderiam entre- vistar quase todas as pessoas que estiveram envolvidas em todos os estágios deste proces- so (com exceção dos sabiamente esquivos in- ventores), um projeto que Peter Šurda já tem avançado. Ao mesmo tempo, o Bitcoin é um caso novo. A sua escassez tem sido obtida sem tan- gibilidade. Não foi coletado na praia e escul- pido, também não foi desenterrado da encosta de uma montanha e limpo ou derretido. Sua escassez foi construída a partir dos meandros das inovações criptográficas e da ciência da computação, que foram remixadas e reunidas de uma forma nova e valiosa. O Bitcoin é re- tirado da encosta de uma montanha no crip- to-ciberespaço. O primeiro golpe de picareta neste minério nunca antes visto foi ouvido no dia 3 de janeiro de 2009. Nossos primeiros negociantes de colares de contas de milhares de anos atrás igualmen- te não mais dão entrevistas. O Bitcoin pode muito bem ser nosso caso histórico melhor documentado das primeiras origens de um meio de troca inteiramente novo no mercado, livre dos ditames de qualquer governante. Satoshi Nakamoto colocou esta rede em movimento no contexto de um enorme traba- lho e de inovações complexas sobrepostas por parte de muitos outros antes e desde o times- tamp inicial do bloco gênesis. XIV - Registre Isto Aqui Também para Aqueles que São Loucos Cabia ao teste do tempo descobrir se o que havia começado iria continuar. Cabia às livres escolhas do res- to da humanidade quem iria tomar estas uni- dades e começar a utilizá-las como um novo meio de troca. XIV - Registre Isto Aqui Também para Aqueles que São Loucos Pode até recordar alguns a res- peito de um complexo engodo no qual golpis- tas habilidosos empurram ações sem valor em seu último esquema. No entanto, o Bitcoin não possuía qual- quer status jurídico enquanto estava nascen- do. Isto o torna tão distante quanto possível de uma imposição central de “fidúcia” atra- vés de alguma designação oficial ou taxa de câmbio fixa. Mesmo anos após o seu lança- mento, os sistemas jurídicos convencionais estão apenas começando a tentar descobrir como categorizá-lo dentro dos esquemas bu- rocráticos de classificação existentes. Entretanto, nós ainda temos visto que permaneceu uma grande lacuna entre a imple- mentação dos elementos intencionais por trás do novo bem digital produzido tecnicamente e as fases graduais subsequentes, quando as pessoas começaram de maneira hesitante a utilizar este estranho novo bem para algumas de suas funções vislumbradas. Em outras pa- lavras, houve uma grande distância entre o lançamento da rede de código aberto e peer-to- -peer (camada técnica), e o momento em que as unidades de bitcoin começaram de fato a ser utilizadas no comércio (a camada econômica ou, mais precisamente, “cataláctica”). Os usuários não são obrigados a utili- zar o Bitcoin. As pessoas não têm sido nem mesmo pressionadas a usá-lo por vendedores espertos inventando histórias. Embora tenha havido alguns poucos golpistas de pirâmide e ladrões no mundo do Bitcoin, seus golpes não eram o Bitcoin, seus golpes eram seus golpes. Ocorreu que esses golpes foram denominados em Bitcoin nesses casos particulares, porém engodos semelhantes envolveram todos os ti- pos de outras unidades valorizáveis ao longo do tempo. Houve uma fase puramente técnica e ex- perimental em andamento bem antes de ter ocorrido qualquer captação da unidade/siste- ma para uso monetário na sociedade. Durante esta fase inicial, não estava claro, nem mesmo para especialistas internos, se o projeto teria sucesso tecnicamente ou se ganharia qualquer tração monetária. A obtenção eventual de um preço de $1 foi motivo para grande comemo- ração, na época, entre os primeiros entusiastas, juntamente com uma pequena obtenção de lu- cros por alguns garimpeiros pioneiros. O Bitcoin foi oferecido como uma inven- ção/descoberta de uma nova maneira de fa- zer algo muito antigo – transferir o controle do poder de compra de uma parte para ou- tra. Este método foi oferecido gratuitamente para toda a população humana. Apêndice A: Interpretação Monetária Por Ano Estes apêndices fornecem detalhes e análises adicionais sobre o histórico de pre- ços a longo prazo e a interpretação de dife- rentes fases históricas do desenvolvimento do Bitcoin. O Apêndice A desenvolve uma interpretação empírica ano a ano dos padrões de novos eventos do Bitcoin em termos dos estágios de evolução monetária. Em seguida, o Apêndice B desenvolve um único gráfico de preços para todos os tempos e explica a sele- ção dos dados. Não estava claro se, ou a extensão na qual alguém iria “comprar” tanto as unida- des quanto a ideia de utilizar o novo proto- colo de pagamento no qual se inserem. Isto é, provavelmente, muito mais semelhante do que pode inicialmente ser imaginado para os processos de pensamento de alguns caçado- res-coletores céticos ou primeiros agriculto- res respirando fundo e, finalmente, aceitando pela primeira vez alguns colares de contas ou peças de metais macios e brilhantes em troca de bens “reais”. Nesta base, 2011 parece fornecer o con- junto mais claro de marcadores empíricos para o primeiro aparecimento de um com- ponente de valor de meio de troca. O Bitcoin não teve um tal componente durante todo o ano de 2009 e durante a maior parte, senão Nenhum decreto ou comando faz com que as pessoas comecem a negociar em Bit- coin. A sua adoção também não resultou de alguma pressão de vendas inteligente tirando partido dos desinformados. Em contraste dia- MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 135 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 135 dos principais entusiastas. Não somente não há evidências de qualquer utilização como meio de troca, um valor de negociação discer- nível das unidades mal era evidente e se limi- tava a casos esparsos. No entanto, as unida- des passaram a existir e usuários começaram gradualmente a brincar com elas, a coletá-las e a colocá-las em uso dentro de estruturas de ação. a totalidade, do ano de 2010. Este é o com- ponente de valorização para a finalidade de facilitar trocas indiretas – em termos simples, oferecer Bitcoin em troca de bens e serviços. Em contraste com o período 2009-2010, este componente específico se desenvolveu de for- ma inequívoca ao longo de 2011, e tem estado se desenvolvendo a cada ano sucessivo. Apêndice A: Interpretação Monetária Por Ano Os preços semanais de referência de abertura anual e fechamento anual vêm ou da média ponderada semanal, ou de estimativas grosseiras utilizadas para construir o gráfico no Apêndice B (os detalhes estão explicados). O impulso chave das informações de eventos abaixo são derivadas das páginas History e Bitcoin First em Bitcoin.it/wiki, com informa- ções adicionais de artigos de notícias e comu- nicados de imprensa. Isto não pretende ser uma história completa, mas sim fornecer um conjunto suficiente de exemplos bem conheci- dos para ilustrar as etapas gerais. Bitcoin Ano 2 (2010) Primeira semana (média): $0,0036 Última semana (média): $0,27 Variação: +7400% Marcos Fevereiro: O Mercado de Bitcoin abre para negociação Maio: A transação Bitcoin da pizza Julho: O câmbio MtGox abre no Japão para transa- ções de Bitcoin Outono: Experimentação financeira anedótica com vendas de opções e de ações a descoberto de Bitcoin Caracterização Experimento técnico crescente, com negociações mais organizadas do Bitcoin como novo bem de investimento digital Bitcoin Ano 2 (2010) Primeira semana (média): $0,0036 Última semana (média): $0,27 Variação: +7400% Bitcoin Ano 1 (2009) Primeira semana: Sem preço. Última semana (média): $0,0006 (ASK) Variação: N/A Primeira semana: Sem preço. Última semana (média): $0,0006 (ASK) Variação: N/A Outono: Experimentação financeira anedótica com vendas de opções e de ações a descoberto de Bitcoin Outono: Experimentação financeira anedótica com vendas de opções e de ações a descoberto de Bitcoin Marcos Marcos Fevereiro: O Mercado de Bitcoin abre para negociação Fevereiro: O Mercado de Bitcoin abre para negociação Bitcoin Ano 1 (2009) Primeira semana: Sem preço. Última semana (média): $0,0006 (ASK) Variação: N/A Marcos Janeiro: Bloco gênesis, primeira transação Outubro: A New Liberty Standard começa a publicar o custo de produção com base nas cotações. Geral: Desenvolvimento do software e da rede. Caracterização Experimento técnico sem usos monetários em funcionamento Bitcoin Ano 1 (2009) 17bitcoin-otc.com; Registro de doação: 1F1dPZxdxVVigpGdsafnZ3cFBdMGDADFDe Bitcoin Ano 4 (2012) Janeiro: Doações de apoio ao sistema de negociação Bitcoin OTC IRC (a primeira em 28 de de- zembro de 2010)17. Três notas de cem trilhões de dólares do Zimbábue foram compradas no Bitcoin OTC por 4 bitcoins Primeira semana (média): $5,84 Última Semana (média): $13,37 Variação: +129% Primeira semana (média): $5,84 Última Semana (média): $13,37 Variação: +129% Marcos Outubro: A New Liberty Standard começa a publicar o custo de produção com base nas cotações. Caracterização Experimento técnico crescente, com negociações mais organizadas do Bitcoin como novo bem de investimento digital O Ano 2 viu o primeiro desenvolvimen- to de preços de mercado autênticos para com- prar e vender Bitcoin (além das cotações de custo de produção). Ainda havia pouca ou nenhuma utilização visível de Bitcoin para fa- cilitar a compra e venda de bens e serviços, embora o comércio de Bitcoin como um bem de investimento digital tenha progredido. Caracterização Experimento técnico sem usos monetários em funcionamento O Ano 1 foi uma fase de experimentações técnicas com possibilidades de êxito remotas e de longo prazo para o Bitcoin em seu papel pretendido como meio de troca, nas mentes 136 Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II viços, tanto online quanto pessoalmente. As possibilidades ainda eram bastante limitadas, mas era evidente que o Bitcoin estava sendo utilizado para pagar por bens e serviços, em oposição a ser transacionado somente como bem de investimento digital. Primeira semana (média): $0,31 Última semana (média): $4,26 Variação: +1274% Marcos Bitcoin Ano 4 (2012) Bitcoin Ano 5 (2013 - YTD) Bitcoin Ano 5 (2013 - YTD) Primeira semana (média): $13,11 Semana do dia 28 de outubro (média): $198,88 Variação: +1417% Primeira semana (média): $13,11 Semana do dia 28 de outubro (média): $198,88 Variação: +1417% Apêndice B: Gráfico da Formação de Preços em Cinco Anos O gráfico a seguir reflete, em uma ima- gem, toda a história da formação de preços do Bitcoin. A visualização destes dados em con- junto requer uma escala logarítmica, de modo que os números menores não desapareçam na linha de base. Em conformidade com uma perspectiva de longo prazo, são utilizados nú- meros semanais típicos ou, quando disponí- veis, médias ponderadas semanais. Isto ajuda a filtrar o ruído dos picos extremos de curto prazo e os vales (inícios de expansão dos ci- clos de negócios) de mania/pânico que são tão amados para utilização nos bordões da mídia pop. A reta azul horizontal que vai de janeiro a outubro de 2009 deve ser entendida como “ne- nhum preço de nenhum tipo”, apesar da escala logarítmica não permitir uma verdadeira linha zero (vista aqui como $0,0001). Marcos Fevereiro: Reddit e Mega começam a aceitar Bitcoin Março/Abril: Corrida para valor de entrada associada nos meios de comunicação com o auto-resgate (bail-in) do Chipre Março/Abril: Corrida para valor de entrada associada nos meios de comunicação com o auto-resgate (bail-in) do Chipre Maio: Anunciada a compra de cartões de pre- sentes Gyft através do BitPay. Lançado o mer- cado BitPremier de bens de luxo Setembro: BitPay anuncia que tem 10.000 clientes comerciais, mais de 10x YoY Outubro: O mercado Silk Road é derrubado. Queda curta e brusca da taxa de câmbio se- guido por um mês de recuperação, até 60% do nível pré-anúncio, acompanhado de uma onda midiática na China. Primeiros ATMs de Bitcoin enviados e sendo instalados para uso comercial Caracterização Caracterização Expande-se a utilização como meio de troca, de- senvolvendo-se a partir do ano anterior Caracterização Expande-se a utilização como meio de troca, de- senvolvendo-se a partir do ano anterior O Ano 5 testemunhou ciclos de atenção da mídia, movimentos de preço dramáticos, e incontáveis empresas iniciantes e expansões em todo o mundo, geralmente tendo como ob- jetivo tornar a utilização do Bitcoin mais fácil para círculos mais amplos de consumidores e comerciantes. Criados vários fundos de inves- timento e fundos anjo de Bitcoin. Conferências realizadas com frequência cada vez maior em todo o mundo. Um fluxo constante de novos itens continuou a indicar a ampliação das op- ções para a compra e venda de bens e serviços com Bitcoin. O Ano 4 testemunhou consolidação cons- tante, misturada com notícias negativas de es- cândalos esparsos e quebras de taxas de câm- bio ou da segurança de usuários. No entanto, um fluxo de novos itens indicou a expansão das opções para a compra e venda de bens e serviços com Bitcoin; seu componente de valor de meio de troca continuou a crescer. Marcos Geral: A página “Bitcoin First” continua a listar as histórias de supostas transações primeiras- -do-tipo (first-of-type) com Bitcoin. O Ano 3 foi o primeiro durante o qual o registro histórico mostra, de forma inequívo- ca, várias linhas de evidência de que o Bitcoin estava sendo usado para comprar bens e ser- MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 137 18 Isto toma o gráfico de três anos desenvolvido em meu artigo do dia 25 de julho de 2013, “Tiger cub growing up? Bitcoin weekly average closing prices year over year”, em konradsgraf.com e o estende 1) adicionando novos dados subsequentes e 2) extrapolando para o passado pré-MtGox. Caracterização Expande-se a utilização como meio de troca, desen- volvendo-se a partir dos dois anos anteriores Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II Sobre as Origens do Bitcoin: Estágios da Evolução Monetária – Parte II 138 site do NLS também inclui os preços do Bit- coin Market (refletidos na reta BtMk). Links diretos para os dados do Bitcoin Market pare- cem não estar mais acessíveis. A NLS ofereceu a venda de Bitcoin em blocos mínimos de valor iguais a um grama de ouro na época (em particular, unidades Pe- cunix). As taxas de venda da NLS foram des- critas como “a média da produção ajustada de Bitcoin por dia dividida pelos custos mé- dios de produção por dia”20. Não estou certo a respeito da medida em que os comprado- res tomaram NLS nesses preços ofertados ou da medida em que os preços indicados para o Bitcoin Market verdadeiramente refletiram transações concretas, ou em que volumes. Uma segunda versão deste gráfico, a se- guir, clarifica a associação das diferentes retas de dados com os anos do calendário18. As re- tas de declividade, do início ao fim de cada ano do calendário (ou o ponto de dados mais disponível) mostram como as retas de dados combinam-se para sugerir uma tendência his- tórica geral.i Como estimativas de custo de produção, os números históricos da NLS não constituem necessariamente os preços de mercado per se, dado que não está claro que negociações esta- vam realmente sendo realizadas. No entanto, na medida em que o Bitcoin foi comprado a esses preços de venda (ou a preços próximos), isto proporciona uma fonte de dados sistemá- ticos para esta fase inicial. Estes dados publica- dos de custos de produção também ajudaram a definir o contexto para o início dos desenvol- vimentos subsequentes de formação de preços. O gráfico combina diferentes fontes de dados para diferentes períodos de tempo. A reta Bstp-13 é o gráfico da média ponderada semanal para a câmbio Bitstamp em 2013. Abandonei os dados de câmbio MtGox dado que se tornaram claramente pontos fora da curva durante o ano de 2013. Gox-10 - Gox12 são os gráficos das médias ponderadas sema- nais para toda a história do MtGox a partir da sua negociação de abertura de $0,05 ao longo de 2012. Uma notícia anedótica recente diz que Kristoffer Koch comprou 5.000 bitcoins por 150 coroas norueguesas “em 2009”21. Caracterização Assumo NSL-09 - NSL-10 se referem aos preços de oferta de Bitcoin publicados no New Liberty Standard19 no final de 2009 e início de 2010. O (gold.org/investment/statistics/gold_price_chart/). Fi- nalmente, converti as cotações de Bitcoin/dólar resul- tantes para cotações de dólar/Bitcoin, para ajustar ao resto do gráfico. O gráfico mostra que os preços médios listados para o Bitcoin Market em média funcionam por debaixo da média da NLS dos preços de compra dos custos de produção durante este período. 20 Para 2009, a NLS explicou que a sua “taxa de câmbio foi calculada dividindo $1,00 pela quantidade média de eletricidade necessária para fazer funcionar um computador de CPU elevada durante um ano, 1331,5 kWh, multiplicada pelo custo médio da eletricidade residencial nos Estados Unidos para o ano anterior, $0,1136, dividido por 12 meses e dividido pelo número de bitcoins gerado pelo meu computador durante os últimos 30 dias”. 19 Citado da página para 2010: newlibertystandard.wi- kyfoundry.com/page/Exchange+Rate. Para os preços NLS de 2010, tomei os preços de oferta listados diaria- mente para Bitcoins por unidades de grama de ouro e estimei um preço típico aproximado para cada semana do calendário. Converti isto para termos de dólar ao preço do dólar por grama de ouro para essa semana utilizando os dados históricos do World Gold Council 21 21 Nenhuma notícia que pude encontrar especificou uma data de compra. Aqui está uma notícia como MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 139 MISES: Revista Interdisciplinar de Filosofia, Direito e Economia Konrad S. Graf 139 perimento. Os mineradores estavam garim- pando moedas (aqueles que suspeitavam que o experimento poderia ter êxito algum dia as estavam mantendo), os desenvolvedores estavam aprimorando o software e algumas transações experimentais – e mais tarde, nes- se mesmo ano, algumas transações de Bitcoin por dinheiro – estavam sendo realizadas, com preços em frações de centavos. que isto ocorreu provavelmente tarde se foi em 2009 e, em dezembro de 2009, isto equiva- lia a $26. O preço pago foi então equivalente a $0,0052 por bitcoin. Mais detalhes sobre tais negociações anedóticas poderiam suplemen- tar nosso conhecimento deste período inicial e as datas reais seriam úteis para tais dados. exemplo:news.com.au/money/money-matters/ norwegian-student-kristoffer-koch-buys-27-in- bitcoin-in-2009-now-he-owns-an-apartment/story- e6frfmd9-1226748159679. Caracterização Para referência, a “transação Bitcoin da piz- za” de maio de 2010 refletiu uma valoração do Bitcoin de $0,0025, abaixo do preço do Bit- coin Market que, naquela época, estava por volta de $0,0045 e também abaixo do preço da NLS, que estava em torno de $0,0055. Em suma, a faixa de preços, na época, ficava entre um quarto de centavo e meio centavo.i p No período 2009-2010, Bitcoins eram utilizados como objetos dentro das estruturas de meios/fins dos atores humanos, sem que essas ações implicassem na utilização do Bit- coin para facilitar trocas indiretas. Esta foi uma fase durante a qual as unidades de bitcoins es- tavam sendo descobertas como novos objetos de ação, tornando-as bens no sentido mais bá- sico – colecionáveis promissores (2009) ou ex- cepcionalmente bens de investimento digital de alto risco acerca dos quais poucas pessoas viam qualquer potencial (2010). Os bitcoins co- meçaram a funcionar de maneira discernível como meio de troca apenas em 2011. A reta NoEXV significa “nenhum valor de troca”. Isto representa um período de pelo menos nove meses para o qual não há nenhu- ma evidência visível de qualquer padrão de unidades de bitcoins sendo compradas e ven- didas, e muito menos utilizadas como meio de troca. Contudo, ao longo de 2009, a rede Bitcoin estava sendo operada como um ex-
W2802728270.txt	https://link.springer.com/content/pdf/10.1007/s10278-018-0073-z.pdf	en		DICOMweb™: Background and Application of the Web Standard for Medical Imaging	Journal of digital imaging	2,018	cc-by	3,994	Journal of Digital Imaging (2018) 31:321–326 https://doi.org/10.1007/s10278-018-0073-z DICOMweb™: Background and Application of the Web Standard for Medical Imaging Brad W. Genereaux 1 & Donald K. Dennison 2 & Kinson Ho 3 & Robert Horn 4 & Elliot Lewis Silver 3 & Kevin O’Donnell 5 & Charles E. Kahn Jr 6 Published online: 10 May 2018 # The Author(s) 2018 Abstract This paper describes why and how DICOM, the standard that has been the basis for medical imaging interoperability around the world for several decades, has been extended into a full web technology-based standard, DICOMweb. At the turn of the century, healthcare embraced information technology, which created new problems and new opportunities for the medical imaging industry; at the same time, web technologies matured and began serving other domains well. This paper describes DICOMweb, how it extended the DICOM standard, and how DICOMweb can be applied to problems facing healthcare applications to address workflow and the changing healthcare climate. Keywords DICOMweb . DICOM . REST . Imaging . Interoperability . Standards . WADO * Brad W. Genereaux brad.genereaux@agfa.com Donald K. Dennison don@dondennison.com Kinson Ho kinson.ho@mckesson.com Robert Horn rjhorniii@gmail.com Elliot Lewis Silver Elliot.Silver@mckesson.com Kevin O’Donnell kodonnell@MRU.MEDICAL.CANON Charles E. Kahn, Jr Charles.Kahn@uphs.upenn.edu 1 Agfa HealthCare, 375 Hagey Boulevard, Waterloo, ON N2L 6R5, Canada 2 Don K Dennison Solutions Inc., 205 Fern Cres, Waterloo, ON N2V 2P9, Canada 3 Change Healthcare, 10711 Cambie Road, Richmond, BC V6X 3G5, Canada 4 Fairhaven Technologies, Maynard, Massachusetts, USA 5 Canon Medical Research USA, Inc., 706 Deerpath Drive, Vernon Hills, IL 60061, USA 6 Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA Introduction Medical imaging plays a critical role as a diagnostic tool. Reynolds concluded back in 2003, Bwhile written descriptions of lesions and infectious processes may be detailed, the visual presentation of these conditions is much more accurate and effective for diagnosis and treatment^ [1]. With that in mind, Bimage enablement^ in healthcare software becomes paramount. Image enablement extends beyond inserting pictures into a health record; it includes the set of collaborative workflows that helps clinicians convey and interpret the patient’s condition in ways that text cannot. Because imaging is key to so many clinical workflows, if imaging exists, but is not accessible, clinicians may need to re-image the patient [2], which adds risk from ionizing radiation, creates unnecessary cost, causes delays in treatment, increases stress and discomfort for the patient and their families, and increases strain on hospital resources. In order to address these challenges, all applications that touch upon medical imaging—be they Picture Archive and Communication Systems (PACS), Electronic Medical Records (EMR), Electronic Health Records (EHR), Radiology Information Systems (RIS), Vendor Neutral Archives (VNA), imaging acquisition devices, network gateways, and proxies— must agree to communicate information in a standard way and in a standard format. The standard for medical imaging information is DICOM (Digital Imaging and Communication in Medicine). DICOM 322 was first released in 1985, has undergone many changes, and has been adopted worldwide. The demands upon networking technologies have increased, which has brought about an evolution. Higher bandwidth wireless connectivity is now possible. At the same time, the devices clinicians use has changed, from a limited use of dedicated workstations to expanded use coupled with mobile devices with limited bandwidth, limited resolution, and limited battery life. The way information is accessed has changed, from being limited within a hospital to being shared across the enterprise and between enterprises. These changes necessitated new security requirements and enterprise IT policies for DICOM. As a result, DICOM adopted new technologies beginning in 2003. DICOM introduced web technologies with the inclusion of WADO (Web Access to DICOM Persistent Objects). Those DICOM web services have evolved to become the standard called DICOMweb. J Digit Imaging (2018) 31:321–326 formatted as single or multi-frame objects, multidimensional volumes, or cine loops. These may be original or derived images and may be associated with annotations and other presentation settings. These objects can refer to related documents, such as requisitions, consent forms, and reports. Other non-image medical data, like ECG waveforms, ultrasound measurements, or radiation dose information, can also be stored using DICOM. For all objects, DICOM defines significant meta-data, for example, for patient identification and demographics, ordering information, acquisition parameters, and workflow context. This metadata is used to query, sort, route, display, and manage the images. In terms of an information model, DICOM defines a logical hierarchy, starting with the patient, as demonstrated in Fig. 1. & & Review The most basic imaging use case includes scheduling, acquiring, managing, processing, displaying, reporting on, and distributing images. A patient is scheduled for a diagnostic exam (e.g., a chest radiograph). A radiography technician uses an imaging device (e.g., an X-ray unit) to acquire images and transmit them to an archive (e.g., a PACS). A radiologist accesses these images, interprets them, and creates a report that is sent to the Radiology Information System (RIS) for distribution to the referring physician, sometimes alongside the images. During interpretation, radiologists may need to see prior imaging studies. Increasingly, such studies are available from other institutions, from jurisdictional, regional or national archives, from cloud-based image sharing solutions, or brought by patients or their families on a CD or DVD. Today, one may choose to store imaging information to a long-term archive, burn the images to a CD or DVD for the patient, share it through an EHR, render them on mobile devices (like tablets or phones), digitally reconstruct the images to form 3D visualization, and share them to portals (for ordering physicians or to patients themselves). DICOM is the standard for retrieving, storing, printing, and transmitting information in medical imaging, and includes both a file structure and communication protocol [3]. It is an international standard (ISO 12052) for medical imaging and related information. DICOM defines formats for images, waveforms, and derived structured data to provide the entire dataset necessary for clinical use. It enables imaging department workflow management, media exchange and printing, and it achieves this using service-based network protocols over TCP/IP and HTTP. DICOM defines how to store acquired images from modalities such as CT, MRI, X-Ray, Ultrasound, Angiography, PET, microscopes, cameras and ophthalmological devices. These image instances may be & & A patient represents the human or animal subject of medical imaging and has a collection of imaging studies. Each study represents an organized set of images for a particular diagnostic purpose and contains a collection of series. Each series represents a single acquisition event on a single machine and contains a collection of instances. Each instance represents a single data object which may be an image frame, a set of images frames, or a document (measurements or a report). Furthermore, even when distributed or accessed independently, each DICOM instance is fully usable since it is fully self-describing. Each instance contains a complete set of metadata, including: & & & Study-level attributes, such as the study unique identifier, the study description, and the patient demographics. Series-level attributes, such as the series unique identifier, the modality, and the body part imaged. Instance-level attributes, such as the instance unique identifier, the image resolution, and the table position where the image was acquired. Each attribute of the metadata has a defined data type and cardinality and is identified by a 32-bit tag—usually shown as two sets of four hexadecimal digits. For example, the tag (0010, 0010) identifies the data attribute containing the patient’s name, which has the datatype of a person name, and can occur at least once. Each DICOM instance is identified by a globally unique identifier (meaning, no two instances ever use the same identifier, worldwide). DICOM also defines non-image objects that are associated with images and studies—these include: & Key object selections (called KOS) identify one or more images intended for a specific purpose, such as representing something clinically significant. J Digit Imaging (2018) 31:321–326 323 Fig. 1 DICOM information model & & & Grayscale presentation states (called GSPS) describe how an individual image or a set of images can be annotated, rotated, cropped, and brightness and contrast adjusted, without modifying the underlying image instance. Structured reports (called SR) encode measurement data in a machine-readable way (e.g., radiation dose SR for ionizing radiation exposures) Encapsulation objects take non-DICOM files such as CDA [4] (clinical document architecture), PDFs, or textual reports and store them in the DICOM format so they can be stored and managed as part of an imaging study. As new imaging-related technologies emerge, such as multi-energy CT, or clinical 3D printing, new object types are defined. To understand how DICOMweb became part of the standard, it is important to understand the healthcare and technology ecosystems. Technology in general, and specifically in healthcare, had evolved and so the technologies used in medical imaging had to evolve as well to meet the needs of clinical systems utilizing imaging. Imaging is not the major focus for many healthcare systems, which thus may be reluctant to invest time and effort into understanding a Bniche^ protocol such as DICOM. There may be safety risks if systems are misconfigured or misused; for example, displaying an arm X-ray for an amputation surgery without the laterality markers specified in the DICOM metadata can lead to serious errors. There are many nuances associated with display of medical images, including tag morphing, lifecycle management, scaling, and dealing with huge data sets. In addition, given that Information Technology (IT) personnel are familiar with concepts such as image display and content upload over the web, and data parsing, historically, DICOM did not use the same technology commonly encountered for those applications. The DICOM file format is not an image format recognized by internet browsers (unlike JPEG, GIF, PNG, and others). Storing newly acquired images (such as, from a clinician’s smartphone for an ad-hoc acquisition) requires in-depth knowledge of both the acquisition parameters and DICOM rules to create the right tags and file format. Managing the image lifecycle (from acquisition, to ingestion, to storage) requires specialized software. Metadata extraction requires parsing binary data, as opposed to XML or JSON technologies. Scaling a system upward requires specialized knowledge of imaging systems, unlike growing a set of clustered nodes in a virtual cloud infrastructure. Storage and bandwidth/latency issues are magnified as image data sets grow into gigabytes and terabytes. There was a need to leverage new technologies in medical imaging, and the users and vendors in the medical imaging industry needed ways to take advantage of web technologies in ways that were compatible with their significant investments in existing infrastructure. Aside from the technology, the needs of clinicians and healthcare institutions changed over time too. Patientcentered care continues to evolve and it demands consistent access to data wherever it is stored (including other institutions) from anywhere the clinician happens to be (in the hospital, at home, in an airport terminal) and on whatever device the clinician is using (a desktop computer, a tablet, a telemedicine cart, a patient bedside terminal, a hallway-mounted computer, or a mobile phone). Hospital IT departments have evolved from individual departmental specialists into enterprise teams, marked with some associated loss of specialist knowledge. Application development paradigms have shifted toward service-oriented architectures. Security and privacy concerns have become much more prevalent, especially with moves to cloud services that have begun to reflect patient confidentiality regulations and protections. The era of Bpushing^ healthcare data from system to system has started to come to an end, and the era of Bpulling^ data on demand has begun. Furthermore, the principles of traditional radiological imaging are being supplanted by a broader concept of enterprise imaging with governance of all clinical images regardless of their origin. Enterprise imaging is Ba set of strategies, initiatives and workflows implemented across a healthcare enterprise to consistently and optimally capture, index, manage, store, distribute, view, exchange, and analyze all clinical imaging and multimedia content to enhance the electronic health record.^ [5] So, for both technology and non-technology reasons, a different approach had to be taken to address the needs of users and systems across the spectrum. 324 Starting in 2003, a web-enabled DICOM extension began to take shape. Initially called WADO, or Web Access to DICOM Persistent Objects, it defined a method to retrieve DICOM objects using HTTP, the same technology used on the web. Over time, other services were added to DICOM to augment the retrieval use case—this included search via QIDO-RS (Query based on ID for DICOM Objects) and upload via STOW-RS (Store over the Web). WADO was upgraded to WADO-RS to more closely follow the emerging RESTful style of web interfaces. Collectively, these technologies became known as DICOMweb. DICOMweb adds HTTP powered services to DICOM, modeled after the style of Representational State Transfer (REST). REST has become the dominant API style used by companies and market verticals all around the world [6]. REST enables systems that are scalable, fault-tolerant, recoverable, secure, and looselycoupled [7]. DICOMweb was designed to augment existing DICOM systems with web technologies. This is important because, given the penetration of DICOM-enabled systems all around the world by hundreds of companies producing trillions of DICOM images, it is not feasible to switch to a completely different standard without disruption. DICOMweb makes it possible to render DICOM instances into widely used consumer-friendly formats suitable for web browsers on both desktops and mobile devices, without requiring a mass conversion of the existing historic data or information model. DICOMweb preserves the historical DICOM information model and metadata rules while enabling access to this information in web formats. Through embracing web technologies, DICOMweb helps to enable communication of imaging data not only to systems within an institution but also to those outside. Further, it supports the security necessary to make this safe natively using the same industry standards that secure commercial activities like banking. Industry-standard security and acceleration appliances can be used to achieve greater performance, since they are optimized for industry-standard HTTP traffic. And, through using RESTful principles, DICOMweb allows for rapid development by software developers. It is possible to access basic DICOMweb retrieval services directly in a browser with no special tools needed; to access traditional DICOM services, often specialized software libraries were needed. Medical imaging over the web has undergone its own evolution, as well. This technology evolution is indicated by the suffix on the service names; BURI^ representing a Uniform Reference Identifier method, BWS^ representing a Web Service (or SOAP) method, and BRS^ representing a RESTful method. The original WADO service, now known as WADO-URI, was developed to provide retrieval of DICOM instances using query parameters on a single resource. For example, a WADO-URI service resource URL may look like Bhttps://myserver.com/wado?studyUID=2.16. 840.1.1.2.3&seriesUID=2.16.840.1.4.5.6&objectUID=2.16. J Digit Imaging (2018) 31:321–326 840.1.7.8.9^ where the resource is named Bwado^ and the parameters passed include BstudyUID^, BseriesUID^, and BinstanceUID^. This has since evolved into what is known as WADO-RS, which uses multiple resource concepts to provide retrieval services. For example, Bhttps://myserver. com/studies/2.16.840.1.1.2.3/series/2.16.840.1.4.5.6/ instances/2.16.840.1.7.8.9^ refers to a hierarchal relationship of resources—the 2.16.840.1.7.8.9 instance resource in the Binstances^ resource in the B2.16.840.1.4.5.6 series resource in the Bseries^ resource in the 2.16.840.1.1.2.3 study resource in the Bstudies^ resource. This more accurately reflects how the study contents are nested and provides the ability to request bundles of resources at the series or study level. DICOMweb also aligns with evolutions in other healthcare APIs; an example of this is HL7’s Fast Healthcare Interoperability Resources (FHIR) [8]. Reflecting evolving approaches to network communications, for a period DICOM also defined a web API called WADO-WS using the SOAP (Simple Object Access Protocol) specification, but this API has since been retired from the DICOM Standard [9]. During the same time period, an independent group began to work on a competing standards project, called MINT (Medical Imaging Network Transport) [10]. Many of the concepts from MINT were later re-worked and adopted into the development of the DICOMweb *-RS services, with input from the user and vendor communities alike. Discussion DICOMweb defines five core services that correspond to traditional DICOM operations—query, retrieve, store, workflow, and service discovery (see Fig. 2). Part 18 of the DICOM Standard defines these services using a common language and terminology. DICOMweb defines resources and operations on those resources using the style of REST on top of the HTTP standard. Studies, series, and instances, along with their identifiers, are all resources that Fig. 2 DICOMweb operations J Digit Imaging (2018) 31:321–326 can be retrieved (via GET operations) and created (via POST operations). Query operations, referred to as QIDO-RS (Query based on ID for DICOM Objects using RESTful Services), are invoked by requesting one of the generic resources (e.g., Bhttps:// myserver.com/studies/2.16.840.1.1.2.3/series/2.16.840.1.4.5. 6/instances^ requests the Binstances^ resource for a specific study and series). Additional request parameters are typically supplied to filter the results. For example, BGET http://server. com/studies/?00100010=DOE^JOHN^ will return all studies that belong to patients named John Doe. Responses can be returned in either XML or JSON format, depending on what the client requests and what the server supports. Retrieve operations, referred to as WADO-RS (Web Access to DICOM Persistent Objects using RESTful Services), are invoked by requesting one of the specific identifier resources (e.g., Bhttps://myserver.com/studies/2.16.840. 1.1.2.3/series/2.16.840.1.4.5.6/instances/2.16.840.1.7.8.9^ requests the instance with the unique identifier B2.16.840.1.7. 8.9^). Without further request parameters, this will return a full DICOM instance—a binary object that contain the metadata payload and pixel data. At an instance resource level, only one object is returned, but at the study or series resource level—for example, Bhttps://myserver.com/studies/2. 16.840.1.1.2.3/^—all of the instances in all of the series of that study are returned. Rendered images suitable for display in browsers can also be requested by specifying the media type, like Bimage/jpeg^ (a JPEG image) or Bimage/png^ (a PNG encoded image). Images can be requested to be a particular size or to be cropped to contain only a specific region of an image or to have a presentation state applied. Store operations, referred to as STOW-RS (Store over the Web using RESTful Services), are invoked to create instances by uploading instances to the Bstudy^ resource (e.g., Bhttps:// myserver.com/studies^) with pixel data and a properly formed set of metadata in either XML or JSON. A set of instances can be uploaded as well. In response, the server returns a receipt of what was processed, and what failed processing. With the store operation, an application is able to easily take a standard JPEG image (by a consumer camera or mobile phone, for example) and upload it to a DICOM archive with an HTTP POST with a few key metadata elements in XML. Workflow operations, referred to as UPS-RS (Unified Procedure Step using RESTful Services), are invoked by creating, claiming, and updating specific work item resources that store and communicate the state of a work item, its inputs and outputs. Systems can create work items for other systems to fulfill and monitor their progression through to completion. Furthermore, systems can subscribe to receive notifications whenever a work item is updated. The notifications use web sockets [11] (defined as part of the HTTP protocol). Service discovery operations are invoked by using the HTTP OPTIONS verb [12] on any resource. This allows client 325 systems to find out in a machine understandable way what DICOMweb services are available on a given server. This is particularly useful since not all systems implement all DICOMweb services. DICOMweb servers return a document that defines what the host server is capable of, using a description language called WADL (web application description language). For DICOM instances that are not in the patient-studyseries-instance data structure, there is also a non-patient object service. This includes, for example, color profiles used in the rendering of images, as well as CT protocols used when acquiring images. For the convenience of developers, these DICOMweb services can return metadata rendered using Extensible Markup Language (XML) [13], or JavaScript Notation (JSON) [14]. Both indicate the relevant DICOM tag, the data type, and the specific value of that field. Consider a study date of April 9, 2013 contained in DICOM attribute [0008,0020] which has a DATETIME value representation. Data that is unwieldy to return in a response (such as binary content) can be replaced in the response by a bulk data reference, which can be separately retrieved if the client needs to do so. Summary In summary, DICOMweb, and DICOM as a whole, represents medical imaging in a standard way allowing products around the world from hundreds of vendors to interoperate successfully in all sorts of use cases. DICOMweb enables collaborative use of medical imaging in medical use cases combining imaging and non-imaging data. When presenting a report of findings in a health application, there was traditionally just text, perhaps organized in sections. By leveraging DICOMweb, reports can contain links to original images, derived images, and supportive data. Using DICOMweb, those links allow the images to be retrieved in a displayable format and presented inside the health application. DICOMweb is now a stable standard. New applications of DICOMweb can enable next generation technologies, such as image analytics and machine learning, by lowering the barrier to entry to retrieve instances appropriate for analysis. By preserving the information model, it is entirely tractable to create a proxy system that could add DICOMweb interfaces to an existing DICOM product that has not implemented the DICOMweb components. Furthermore, DICOMweb enhancements are being added; for example, in 2017, new resources at the study, series and instances levels is being developed to return small representational images (e.g., Bthumbnails^) suitable for display in applications to aid clinicians in selecting the data they wish to display. 326 J Digit Imaging (2018) 31:321–326 DICOMweb provides methods that are common across every market vertical and sector. It provides the same API methodologies and strategies that are familiar to software developers. It lowers the barrier to entry of working with medical images by using familiar tools. DICOMweb enables patientcentric care by providing secured and protected modern webdriven architectures that are both backward-compatible and forward-thinking. Improving access to imaging and reducing the cost of interoperability makes imaging more ubiquitous; this in turn reduces delays and repeat examinations and enables clinicians to better collaborate with care teams locally and globally, ultimately improving access to quality care. 2. Acknowledgements The authors gratefully acknowledge the works and dedication of David Clunie, Chris Hafey, Jeroen Medema, Jim Philbin, Harry Solomon, Stephen Vastagh, and Jonathan Whitby. The authors also wish to acknowledge the efforts of standards bodies and associated organizations of DICOM, NEMA, MITA, RSNA, ACR, HL7, IHE, HIMSS, and SIIM. 7. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 10. 3. 4. 5. 6. 8. 9. 11. 12. References 1. Susan I. Reynolds. 2003. Imaging: New electronic tool for clinicians. AMIA Annu Symp Proc. Retrieved from http://www. ncbi.nlm.nih.gov/pmc/articles/PMC1480347/ 13. 14. Vest JR, Kaushal R, Silver MD, Hentel K, Kern LM. 2014. Health information exchange and the frequency of repeat medical imaging. Am J Manag Care 20(11 Spec No. 17):eSP16–eSP24. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/25811815 Wikipedia. 2017. DICOM. Retrieved from https://en.wikipedia.org/ wiki/DICOM DICOM. 2017. Imaging Reports using HL7 Clinical Document Architecture. Retrieved from http://dicom.nema.org/medical/ dicom/current/output/chtml/part20/chapter_1.html Christopher J. Roth, Louis M. Lannum, Kenneth R. Persons. 31 May 2016. A Foundation for Enterprise Imaging: HIMSS-SIIM Collaborative White Paper. Retrieved from http://link.springer. com/article/10.1007/s10278-016-9882-0 John Musser. May 5, 2011. Open APIs – State of the Market 2011. Retrieved from http://www.slideshare.net/jmusser/open-apis-stateof-the-market-2011 Fielding RT: Chapter 5: Representational state transfer (REST). Architectural Styles and the Design of Network-based Software Architectures. Irvine: University of California, 2000 HL7. 2017. FHIR Release 3 (STU). Retrieved from https://www. hl7.org/fhir/ DICOM Standards Committee. 2017. Retirement of WADO-WS. Retrieved from ftp://medical.nema.org/MEDICAL/Dicom/final/ sup198_ft2_retire_wado-ws.pdf MINT. March 14, 2014. Medical Imaging Network Transport Readme. Retrieved from https://github.com/medical-imagingnetwork-transport/medical-imaging-network-transport DICOM. 2017. OpenEventChannel. Retrieved from http:// dicom.nema.org/medical/dicom/current/output/chtml/part18/ sect_6.9.10.html DICOM. 2017. RS Capabilities Service. Retrieved from http:// dicom.nema.org/medical/dicom/current/output/chtml/part18/sect_ 6.8.html#sect_6.8.1.1.1 DICOM. 2017. Native DICOM Model. Retrieved from http:// dicom.nema.org/medical/dicom/current/output/chtml/part19/ chapter_A.html#sect_A.1 DICOM. 2017. DICOM JSON Model. Retrieved from http:// dicom.nema.org/medical/dicom/current/output/chtml/part18/ chapter_F.html
https://openalex.org/W3109582972	https://www.e3s-conferences.org/10.1051/e3sconf/202021302009/pdf	English	null	Prediction of water breakthrough time in horizontal Wells in edge water condensate gas reservoirs	E3S web of conferences	2,020	cc-by	3,126	Prediction of water breakthrough time in horizontal Wells in edge water condensate gas reservoirs Huang QuanHua1, Lin XingYu1 1Petroleum engineering school, Southwest petroleum university, Chengdu, Sichuan, 610500, china Huang QuanHua1, Lin XingYu1 1Petroleum engineering school, Southwest petroleum university, Chengdu, Sichuan, 610500, china Abstract: Horizontal Wells are often used to develop condensate gas reservoirs. When there is edge water in the gas reservoir, it will have a negative impact on the production of natural gas. Therefore, reasonable prediction of its water breakthrough time is of great significance for the efficient development of condensate gas reservoirs.At present, the prediction model of water breakthrough time in horizontal Wells of condensate gas reservoir is not perfect, and there are mainly problems such as incomplete consideration of retrograde condensate pollution and inaccurate determination of horizontal well seepage model. Based on the ellipsoidal horizontal well seepage model, considering the advance of edge water to the bottom of the well and condensate oil to formation, the advance of edge water is divided into two processes. The time when the first water molecule reaches the bottom of the well when the edge water tongue enters is deduced, that is, the time of edge water breakthrough in condensate gas reservoir.The calculation results show that the relative error of water breakthrough time considering retrograde condensate pollution is less than that without consideration, with a higher accuracy. The example error is less than 2%, which can be effectively applied to the development of edge water gas reservoir.  Corresponding author: cnlinxingyu@163.com 1 Introduction increases because the condensate occupies the pore volume, thus accelerating the advance of edge water. Therefore, although the retrograde condensate area of condensate gas reservoirs is limited, the influence of condensate oil on the water breakthrough time cannot be ignored. In this paper, a model for predicting water breakthrough time of edge water condensate gas reservoir is developed by taking into account such factors as gas flow degree ratio, bound water saturation, residual gas saturation, condensate oil saturation, distance between gas well and edge water, and horizontal well length. Edge water often causes water invasion to condensate gas reservoir, which affects gas reservoir recovery. At present, there are many studies on conventional gas reservoir models of edge water. In 1983, KUO[1] predicted the water breakthrough time of edge water and bottom water gas reservoirs based on the seepage law of fluids in porous media.In 2017, GUO[2] established a water breakthrough time prediction model for high sulfur gas reservoirs.In 2011, Wu Keliu [3] established a prediction model of water breakthrough time for edge water gas reservoirs considering retrograde condensate effect, which provided a theoretical basis for controlling water breakthrough time.In 2016, we [4] analyzed the water breakthrough time of bottom water gas reservoirs with baffles. In 2018, Ming Ruiqing [5-6] established a water breakthrough time prediction model for the edge water condensate gas reservoir, but the horizontal well seepage model was circular, which was not consistent with the actual situation.In 2019, we [7-8] established a horizontal gas well water breakthrough model with consideration of high- speed non-Darcy effect. The model adopted an ellipsoidal seepage model, which was successfully applied to the prediction of water breakthrough time of high-yielding horizontal Wells.In 2020, we [9] also established a prediction model for water breakthrough time in vertical Wells considering edge water condensate gas reservoirs. E3S Web of Conferences 213, 02009 (2020) ACIC 2020 E3S Web of Conferences 213, 02009 (2020) ACIC 2020 https://doi.org/10.1051/e3sconf/202021302009 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 3 The formulas (6) ( ) By deforming and integrating equation (6), the breakthrough time of water quality point from the initial gas-water interface to the bottom hole can be obtained: ( ) By deforming and integrating equation (6), the breakthrough time of water quality point from the initial gas-water interface to the bottom hole can be obtained: Based on the above seepage law, it is assumed that M is the water tongue thrust point, N is the central position of the horizontal perforation section, and the distance between M and N is R.On the basis of satisfying the basic seepage law of gas-water two-phase, the following assumptions are made for the derivation process :(1) the reservoir is equithick and homogeneous, and the water phase drives the gas phase by piston displacement.(2) Capillary force and gravity are not considered.(3) In the seepage process, the viscosity and density of gas and water are constant.(4) The three phase seepage of oil, gas and water conforms to Darcy's Law. 2 Model g w gw v v M  （5） g w gw v M  （5） g gw w w / / g K u M K u  （5） g gw w w / / g K u M K u  Where, Where, Mgw—Gas-water flow ratio, decimal;pg— Gas phase pressure, MPa;pw—Water phase pressure, MPa;r—The distance that the water quality point moves to the well, m;μg—Viscosity of gas phase, mPa· S;υg— Gas phase velocity, m/d;υw—Water phase velocity, m/d;Kg—Gas permeability ，mD ；Kw—Water permeability，mD；ρg—The density of the gas phase， g/cm3；Kgwi—Gas permeability under bounded water saturation Swi，mD；Kwgr—Water permeability under residual gas saturation Sgr，mD。 Figure 1 edge water propelling of horizontal wells In porous media, considering the influence of porosity, original irreducible water saturation and residual gas saturation, the distance that water quality point moves to the center of horizontal perforation section N in dt time is : Figure 1 edge water propelling of horizontal wells Figure 1 edge water propelling of horizontal wells   w wi gr d d / [ 1 ]     r v t S S 2 Model As shown in Fig. 1, the initial gas water boundary of edge water condensate gas reservoir is approximately treated as a straight line, which is recorded as y. A horizontal well producing at the distance o from Y, and the horizontal section length of the horizontal well was L. According to the relevant seepage mechanics theory, the ellipsoidal model is adopted as the horizontal well seepage model. And according to the existing research results of horizontal well water flooding in edge water reservoirs, water invasion in horizontal Wells is pushed forward by water tongue. Therefore, when the gas well begins to produce, the gas-water interface starts to push toward the bottom of the well, and the gas-water interface becomes surface Y'. At the same time, because of gas reservoir is condensate gas reservoir, when the pressure is lower than dew point pressure, formation of precipitation of Retrograde condensate pollution exists in condensate gas reservoirs, and the fluid flow rate near the well zone E3S Web of Conferences 213, 02009 (2020) ACIC 2020 https://doi.org/10.1051/e3sconf/202021302009 condensate oil. At the same time, there is a gas condensate advance interface, which is record as X, when bottom hole pressure is lower than dew point pressure, the interface from the bottom hole to the edge water area. Considering the horizontal seepage characteristics and the characteristics of the pressure in the horizontal section of the wellbore is the same, this model will be treated as interface x approximate to linear interface. 3 The formulas   bt1 wi gr g 1 a b gw a M t S S dr v       （7） Assume that the vent zone of a horizontal well is elliptic.The equipotential surface at point M on the front edge of water drive is also elliptic, the size of its long half axis is r+L/2 and the short half axis is r.It can be known that the seepage velocity of the gas phase can be expressed as: From interface Y to interface X, the time of edge water propulsion is tbt1, and from interface X to wellbore, the time of edge water propulsion is tbt2.Therefore, the water breakthrough time of edge water condensate gas reservoir can be expressed as: g g g (2 2 )    q B v r L h （8）（8） Where, qg—Gas well production，m3/d；Bg—The volume coefficient of natural gas, decimal；h—Reservoir thickness，m；L—Horizontal section length of horizontal well，m。 bt bt1 bt1 = + t t t （1）（1） According to the gas-water two-phase seepage law, considering the Darcy flow effect of gas, the gas-water two-phase motion equation can be written as follows: Substituting Equation (8) into Equation (7) can be obtained； g g g g d d p u v r k  （2） w w w w d d  p u v r k （3）   bt1 wi gr 1 1 2 2 a b b t S S dr A r L         （9）（2）（9）（3） g g gw q B A hM  。 Regardless of capillary force, the pressure gradient of gas phase and water phase is equal at the gas-water interface.There are: Where， Twice substitution integral of Equation (9) can be solved as follows： g w ' ' d d d d  y y p p r r （4）（10）   2 2 bt1 wi gr 2( ) ( 2 ) 1 [ ] a b a b L b bL t S S A            （）（10）（4） Equations (2) and (3) are substituted into Equation (4), and the units in the formula are unified according to the practical system units of the mine, so as to obtain the seepage velocity of the aqueous phase Equation (10) refers to the time when the edge water advance of horizontal well just affected the condensate oil region in the edge water gas reservoir. 4 Calculation Figure 2 Production performance data of gas well Figure 3 Advance distance of condensate blocking radius production days of the well is 698 days, the condensate meets the edge water, the distance of condensate advancing is 178.42m, and the influence days o condensate on edge water is 62.51 days. The prediction accuracy of water breakthrough time is improved by 4.43 when considering the influence of condensate oil, and the influence of condensate oil can not be ignored in the prediction of water breakthrough time of condensate ga reservoir. Since the condensate oil reduces the pore volume, i further improves the seepage velocity of water and gas in the near well zone, and accelerates the water breakthrough Figure 2 Production performance data of gas well Figure 2 Production performance data of gas well Figure 2 Production performance data of gas well Figure 3 Advance distance of condensate blocking radius production days of meets the edge w advancing is 178 condensate on edge accuracy of water b when considering th influence of conde prediction of water reservoir. Since the conde further improves the th ll Figure 2 Production pe Figure 3 Advance distance of condensate blocking radius Figure 2 Production performance data of gas well Figure 3 Advance distance of condensate blocking radius production days of the well is 698 days, the condensate meets the edge water, the distance of condensate advancing is 178.42m, and the influence days of condensate on edge water is 62.51 days. The prediction accuracy of water breakthrough time is improved by 4.43% when considering the influence of condensate oil, and the influence of condensate oil can not be ignored in the prediction of water breakthrough time of condensate gas reservoir. Since the condensate oil reduces the pore volume, it further improves the seepage velocity of water and gas in the near well zone, and accelerates the water breakthrough in condensate gas reservoir. Therefore, in the production, it is necessary to ensure that the bottom hole pressure is not too low to reduce the condensate precipitation rate for delaying the breakthrough time of edge water in gas wells. 3 The formulas 2 2 E3S Web of Conferences 213, 02009 (2020) E3S Web of Conferences 213, 02009 (2020) ACIC 2020 https://doi.org/10.1051/e3sconf/202021302009 4 Calculation   bt2 wi gr 0 g 1 b gw oc M t S S S dr v       （11）   bt2 wi gr 0 1 1 2 2 b oc t S S S dr A r L         (12) A side water vapor have 1 mouth production of horizontal well thermal fluid activity, reservoir thickness h=10m m, porosity φ=0.052, irreducible water saturation Swi=0.43, residual gas saturation Sgr=0.11, the critical condensate saturation Soc=0.1, the gas phase permeability under the irreducible water saturation Swi Kgwi=0.217mD, residual gas saturation water phase permeability under the Sgr Kwgr=0.364mD, gas phase permeability Kg=9.81mD, the density of the gas phase ρg=0.2829g/cm3, gas volume coefficient of Bg=0.00313m3/m3,Gas viscosity μg=0.0348mPa·s, water viscosity μw=0.39mPa·s.The horizontal section length of the horizontal wellL=480m, and the central position of the horizontal section is 450m from the initial gas-water boundary. See the average production before waterqg=560000m3/d.The daily gas and water production curves of this horizontal well are shown in Figure 2.As can be seen from Figure 2, the well was put into production on January 8, 2010. On June 10, 2012, the gas well began to see water, and then the production declined rapidly. The water breakthrough time was 884d.The schematic diagram of condensate blocking radius (FIG. 3) can be calculated according to the Muskat formula, and the linear regression can be used to reverse the time TBT1 when the condensate oil meets the edge water. According to the Muskat condensate saturation variation formula, the blocking radius to achieve critical flow saturation is derived: bt1 101 g g oc R q ZTyt b h S Kp    （13） = a b o  （14）（13）（14） Therefore, the formula of water breakthrough time in horizontal Wells of condensate gas reservoirs can be obtained:   2 bt wi gr 2 ( ) 2( ) = 1 [ ] 2 [ ] oc a b a b L t S S A b bL S A            （15）（15） In combination with equations (10), (13), (14) and (15), the water breakthrough time of horizontal Wells in condensate gas reservoirs can be solved. 4 Calculation At the same time, because the gas wells used in the case are high-yield horizontal wells, and the formation pressure is high, there will be start-up pressure gradient, water lock effect and oil lock effect in the process of fluid Since the condensate oil reduces the pore volume, it further improves the seepage velocity of water and gas in the near well zone, and accelerates the water breakthrough in condensate gas reservoir. Therefore, in the production, it is necessary to ensure that the bottom hole pressure is not too low to reduce the condensate precipitation rate for delaying the breakthrough time of edge water in gas wells. Figure 3 Advance distance of condensate blocking radius With the above parameters taken into account, the water breakthrough time of horizontal wells without considering reverse condensate pollution is 935 days, and the error is 5.77% compared with the actual situation. The results show that the water breakthrough time of horizontal well considering retrograde condensate pollution is 872d, and the error is 1.34% compared with the actual situation. According to the analysis, when the At the same time, because the gas wells used in the case are high-yield horizontal wells, and the formation pressure is high, there will be start-up pressure gradient, water lock effect and oil lock effect in the process of fluid 3 E3S Web of Conferences 213, 02009 (2020) https://doi.org/10.1051/e3sconf/202021302009 E3S Web of Conferences 213, 02009 (2020) ACIC 2020 Reservoirs, 05:1-7. Reservoirs, 05:1-7. seepage, which will increase the seepage resistance of water molecules in the pores, reduce the seepage velocity of water molecules in the pores, and delay the breakthrough time of edge water; in the condensate seepage area, the actual condensate saturation near the well is higher than the critical flow saturation, it will also increase the resistance to the movement of edge water. Therefore, there are still some errors in the breakthrough time of edge water calculated by this formula. 6. Ming R Q, He HQ, Hu QF, et al. (2018)A new prediction model for water breakthrough time in high- yielding Wells of edge-water condensate gas reservoir. Special Oil & Gas Reservoirs, 25(02):76-79. 7. Huang Q H, Lin XY，Tong K.(2018) An Approach to Predict Water Breakthrough Time for Horizontal Wells in Gas Reservoirs with Edge-Water in Consideration of Non-Darcy Flow. GMEE 2018. 8. 5 Conclusion By analyzing the advance of edge water in condensate gas reservoir and considering the retrograde condensate pollution, we establish a water breakthrough prediction model for horizontal wells in condensate gas reservoirs. The ellipsoidal seepage model of horizontal wells is adopted, and the gas water mobility ratio is used to convert the natural gas seepage velocity into the formation water seepage velocity, and the calculation formula of water breakthrough time is derived. This method can also calculate the time and distance when edge water and condensate meet, which is helpful to understand the actual performance of edge water breakthrough in condensate gas reservoir. The reliability of the formula is verified by case analysis, which can effectively improve the prediction accuracy of water breakthrough time. It is considered that the influence of condensate oil can not be ignored in the prediction of water breakthrough time in horizontal wells. 9. Huang Q H, Lin XY，Xu QL; et al. (2020) A New Method for Predicting Water Breakthrough Time in the Edge Water Condensate Gas Reservoir. IOP Conference Series: Earth and Environmental Science.526: 012126. Acknowledgments Supported by the National Major Science and Technology Project "Research on percolation Mechanism and Development Technology Policy of Thick Heterogeneous Gas Reservoirs" (No. :2016ZX05027-004-005). 4 Calculation Huang Q H, Lin XY，Tong K.(2019) Prediction of water breakthrough time in horizontal Wells of non- Darcy Seepage flow marginal water gas reservoirs, 31(01):147-152. Reference 1. KUO M C T. (1983) A simplified method for water coning predictions . SPE 12067. 2. Guo X, Wang P, Liu J, Song G et al. (2017)Gas-well water breakthrough time prediction model for high- sulfur gas reservoirs considering sulfur deposition. Journal of Petroleum Science and Engineering. 157: 999–1006 3. Wu K L, Li XF, Zhang GT, et al. (2011)A new method for prediction of water breakthrough time of edge- water condensate gas reservoir considering retrograde condensation .Science Technology and Engineering, 11(19):4574-4577. 4. Huang Q H, Lu Y, Fu YH, et al. (2016)An approach to predict water breakthrough time of gas reservoirs with bottom water in consideration of non-Darcy flow. Science Technology and Engineering, 14: 137-140. 5. Ming R Q, He HQ, Hu QF. (2018)A new method for prediction of water breakthrough time of bottom water condensate gas reservoir. Special Oil & Gas 4 4
https://openalex.org/W3000231128	https://hts.org.za/index.php/hts/article/download/5543/14394	German	null	Martin Luther en gebed	HTS teologiese studies	2,019	cc-by	9,797	HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 Page 1 of 7 Original Research Original Research Page 1 of 7 Page 1 of 7 Description:i Description: Natie van Wyk is participating in the research project, ‘Justice and Human Dignity. A Reformed perspective’, directed by Prof Wim Dreyer, Department of Church History and Church Polity, Faculty of Theology, University of Pretoria. Keywords: Martin Luther; Prayer; Second Petition; Sunday Rogate; Morning and evening prayers. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Inleiding Dit was gedurende die laaste dekades vir Protestantse teoloë baie moeilik om oor gebed te skryf – ten spyte van die feit dat godsdiens en gebed sinoniem is. Volgens Gerhard Ebeling (1975 [1973]:405–416) is moderne mense skaam om oor gebed te teologiseer, aangesien die gedagte van God in die lig van die Westerse lewenservarings uiters problematies geraak het. Die filosofiese godsdienskritiek het egter al die laaste twee eeue gebed geteiken as ’n saak wat nie met redelikheid en wetenskap versoenbaar is nie. In hierdie tye van onsekerheid en verwarring sal dit goed wees om Martin Luther se gebedsteologie vir die Afrikaanse leserspubliek daar te stel. Hy het waardevolle insigte in die Christelike gebed gehad, en lesers behoort sy bydrae tot die nadenke oor gebed insiggewend te vind. Martin Luther en gebed Martin Luther and prayer. For decades, not many theologians published on the theme of prayer. The philosophical critique on religion is one reason. A sensible thing to do in times of uncertainty and disorientation is to fall back on the advice of theologians of name who guided the church in the past. Martin Luther is one such theologian. He was a theologian of prayer. Prayer was a pivotal element in his understanding of spirituality. It was also a constitutive factor for his theology. In Luther, we find a respected and reliable teacher on Christian prayer. Luther himself prayed often in the privacy of his home and in public spaces. His life is an example of a praying Christian. He left behind many sermons and publications on prayer. The research on Luther’s theology of prayer is vast. Unfortunately, we have no publications on Luther and prayer in Afrikaans. This is hopefully the first of many to come. The article concentrates on Luther’s practical advice regarding prayer to congregants who joined the Reformation. The advice could also be useful to the South African community that is becoming more and more secularised. In the second part of the article, his theology of prayer is discussed and his Rogationtide sermons are emphasised. His introductions and theology on, especially, John 16 receive attention. Thirdly, as an example of his expositions on prayer, we look at the second petition of the Lord’s Prayer. The relationship between the kingdom and the church is explained. Project Leader: Wim Dreyer Project Number: 77370920 1.Die haarkapper, Peter Beskendorf, was ’n ouer vriend van Luther. Op Stil Saterdag in 1535 het hy in ’n twis met sy skoonseun betrokke geraak en het hom per ongeluk doodgesteek. Luther het in die hofsaak voor die ‘adjunk-regterpresident’ van Sakse, Franz Burkhard (1503–1560), vir hom voorspraak gemaak. Beskendorf se lewe is gespaar, maar hy is verban en het in Dessau asiel gevind. Luther bied pastorale ondersteuning aan Beskendorf deur hom van advies te bedien oor ’n lewe van gebed. Die eerste weergawe is twee keer uitgegee. Luther het spoedig daarna ’n uitgebreide weergawe geskryf wat agt keer gedruk is en deur Johann Freder in Latyn vertaal is. Soos ander traktate uit die oeuvre van sy stigtingsliteratuur, was ook hierdie een uit die gebedspraktyk in sy eie huishouding gevoed (vgl. inleiding van D. Korsch tot Luther 2012c [1535]:601). Die vestiging van ’n Protestantse gebedspraktyk Dit is ’n ‘werk’ wat beloon wil word. Derdens word dit deur die herhaling van baie woorde gekenmerk wat uiteindelik niks vir niemand beteken nie. Volgens Luther moet daar nadenkend en met min woorde omgegaan word. Mense moet besef wat hulle bid, en wat gebid word moet met erns gedoen word. In die lig hiervan, beveel Luther aan dat daar aan hand van die Onse Vader gebid moet word – maar die bidder moet verstaan wat gebid word. Onderrig oor die inhoud van die Onse Vader (en die Psalms) is daarom van uiterste belang. Sy advies dat daar vroeg in die oggend en in die aand vóór slaaptyd gebid moet word, is volledig in sy Klein Kategismus opgeneem. Sy voorgestelde gebede (WA 30, 320–326; 2012a [1529]:594) word vir kennisname aangebied: Hoe die huisvader sy mense moet leer om soggens en saans te bid. Soggens, wanneer jy uit die bed opstaan, moet jy die kruis maak [of ’n kruis om jou nek hang], en sê: God, Vader Seun en Heilige Gees het oor my die wag gehou. Daarna moet jy gekniel of staande die Geloofsbelydenis opsê en die Onse Vader bid. As jy wil, kan jy ook hierdie gebed byvoeg: Ek dank U, my hemelse Vader, deur Jesus Christus, u geliefde Seun, dat U my deur hierdie nag van alle skade en gevaar bewaar het, en bid U, dat U my ook deur die loop van hierdie dag sal bewaar van sonde en allerhande euwels, sodat al my doen en late, ja my hele lewe U sal geval. Ek gee my lewe en my siel en alles [wat ek het en besit] oor in u hande. U heilige engel is met my, en daarmee het die bose vyand geen houvas op my nie. Amen. Dan spring jy met vreugde aan die werk en sing miskien ’n lied soos die Tien Gebooie, of wat ook al in jou gedagtes mag opkom. Luther het tussen 1519 en 1535 talle adviese oor die gebedslewe gegee en geskryf. Uit hierdie adviese blyk dit dat Luther homself nie net as ‘eksegeet’ en ‘sistematiese teoloog’ beskou het nie, maar ook as ‘pastorale teoloog’. Sy pastorale advies aan mense was dikwels in die vorm van besinnings oor gebed. In 1535 het hy ’n pastorale advies aan die haarkapper, Peter Beskendorf,1 gelewer. Dates: How to cite this article: Van Wyk, I.W.C., 2019, ‘Martin Luther en gebed’, HTS Teologiese Studies/ Theological Studies 75(4), a5543. https://doi.org/ 10.4102/hts.v75i4.5543 Martin Luther se lewe was een van gebed. Hy het in sy ouerhuis geleer om gereeld te bid. Sy skoolloopbaan en studentetyd in Erfurt is gekenmerk deur deelname aan kerklike aktiwiteite wat in ’n groot mate met gebede te make gehad het. Vanselfsprekend was sy lewe in die klooster deur vaste gebedsure en -rituele gereguleer. As jong dosent in filosofie en latere professor in teologie, het hy voortgegaan om lang ure in gebed deur te bring. Dit is daarom nie vreemd dat hy reg deur sy hele loopbaan gereeld oor die tema gebed gepreek en geskryf het nie. Vandat hy in 1519 sekere Roomse gebedspraktyke krities verwerp het, het hy ook begin om ’n Reformatoriese gebedspraktyk en -teologie te ontwikkel. Hy het van die begin af besef dat sy primêre verpligting by die gemeentelede berus. Hiervan getuig sy Duitse uitleg van die Onse Vader vir die gewone lidmate (WA 2, 81–130; 1991b [1519]) en sy Gebedeboekie (WA 10.2, 331–406; 1907 [1522]). Teologiese besinnings oor gebed sou later vanselfsprekend nie agterweë bly nie. Volgens Lindberg (2016:417), is daar in die twintigste eeu ’n enorme hoeveelheid navorsing oor Luther en gebed gedoen (en tragies genoeg, niks in Afrikaans nie). Die resultate het in slagspreuke soos ‘Luther se teologie is ’n teologie van gebed’, ‘gebed is ’n kernelement in Luther se teologie’ en ‘Luther se teologie is geaktualiseerde gebed’ uitgemond. Dit is dus duidelik dat, indien daar tot die hart van sy teologie deurgedring wil word, ’n Afrikaanse Luther-resepsie deeglike aandag aan sy gebedsteologie moet gee. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. In hierdie artikel word daar eerstens aan Luther se advies vir die vestiging van ’n Protestantse gebedspraktyk aandag gegee. Tweedens word daar aan Luther se gebedsteologie aandag geskenk, Note: HTS 75th Anniversary Maake Masango Dedication. Note: HTS 75th Anniversary Maake Masango Dedication. Open Access http://www.hts.org.za Page 2 of 7 Original Research advies, skets ’n vollediger beeld van sy verstaan van gebed. Dates: Opsommend kan die volgende kortliks genoem word: ’n Mens behoort die oggend te begin en die aand met ’n gebed af te sluit; harde, getroue arbeid is ook ’n gebed, en wie hard werk, bid ook gereeld; daar moet gereeld gebid word, aangesien daar gereeld gesondig word; gebed word in vertroue op die belofte en in die naam van Jesus afgesluit; die Onse Vader moet gereeld gebid word, maar is nie die enigste gebed wat gebid moet word nie; kennis van die betekenis van die onderskeie bedes is essensieel vir ’n ernstige gebed; gebed is ’n saak van vreugde; wanneer daar gebid word, moet dit in erns en met konsentrasie gedoen word. Onbekend (meen ek) is dat Luther (2012c [1535]:612–630) ook aanbeveel dat daar aan hand van die dekaloog gebid kan word. Hiervoor kan vier riglyne gevolg word: Eerstens moet daar by elke gebod gevra word wat God van my verwag; tweedens moet God hiervoor gedank word; derdens moet daar aan hand van die spesifieke gebod skuld bely word; en vierdens kan aan die gebed méér inhoud gegee word op grond van meerdere kennis oor die spesifieke gebod. Deur dít te doen, sal die gebedslewe nie tot ’n eindelose herhaling van dieselfde gedagtes verstar nie. en derdens word daar eksemplaries na sy uitleg van die tweede bede van die Onse Vadergebed gekyk. Die rede vir hierdie keuse het te make met die konjunktuur wat die idee van die ‘koninkryk’ tans in kerk en teologie geniet. Die vestiging van ’n Protestantse gebedspraktyk In die inleidende gedeelte tot die Duitse uitleg van die Onse Vader vir die gewone lidmate (WA 2, 81–83; 1991b [1519]:204–207) het Luther ’n aantal praktiese adviese neergepen wat lidmate wat van die Roomse Kerk vervreemd geraak het, kon help met die vestiging van ’n gebedspraktyk. Sy (WA 2, 81, 11–16; 1991b [1519]) eerste advies is dat wanneer daar gebid word, daar: min woorde gebruik moet word, maar [dit moet gepaard gaan met] baie en diepe besinning. Hoe minder woorde gebruik word; des te beter is die gebed. Hoe meer woorde gebruik word; hoe slegter is die gebed. Min woorde en diep besinning is Christelik; baie woorde en min besinning is heidens. (bl. 204) Luther se tweede advies (WA 2, 81, 22–82, 29; 1991b [1519]:204–206) is dat gebed ‘in die gees’ gedoen moet word en nie as ’n blote ‘liggaamlike aksie’ afgehandel moet word nie. Luther het dit teen die Roomse gebedsrituele wat sonder denkende en sprekende erns, sinneloos, herhaal word. Ware gebed is ’n saak van erns en werklike verlange om met God deur Christus te kommunikeer. Die uiterlike gebedsritueel word gekenmerk deur die voorlees van skriftelike gebede deur ’n priester waarna byna niemand luister nie. Tweedens word die ritueel deur traagheid, lusteloosheid en geldgierigheid gekenmerk. Dit is ’n ‘werk’ wat beloon wil word. Derdens word dit deur die herhaling van baie woorde gekenmerk wat uiteindelik niks vir niemand beteken nie. Volgens Luther moet daar nadenkend en met min woorde omgegaan word. Mense moet besef wat hulle bid, en wat gebid word moet met erns gedoen word. In die lig hiervan, beveel Luther aan dat daar aan hand van die Onse Vader gebid moet word – maar die bidder moet verstaan wat gebid word. Onderrig oor die inhoud van die Onse Vader (en die Psalms) is daarom van uiterste belang. Luther se tweede advies (WA 2, 81, 22–82, 29; 1991b [1519]:204–206) is dat gebed ‘in die gees’ gedoen moet word en nie as ’n blote ‘liggaamlike aksie’ afgehandel moet word nie. Luther het dit teen die Roomse gebedsrituele wat sonder denkende en sprekende erns, sinneloos, herhaal word. Ware gebed is ’n saak van erns en werklike verlange om met God deur Christus te kommunikeer. Die uiterlike gebedsritueel word gekenmerk deur die voorlees van skriftelike gebede deur ’n priester waarna byna niemand luister nie. Tweedens word die ritueel deur traagheid, lusteloosheid en geldgierigheid gekenmerk. 2.Sondag Rogate vorm deel van die Paaskring. Die wortels van die 50 dae Paasfeesvierings gaan terug na die tweede eeu. Die liturgiese struktuur van die Paaskring was waarskynlik teen die vierde eeu gefinaliseer (vgl. Bieritz 2014 [1986]:229–247; Cobb 2004 [1992] en Connell 2006:158–178 vir inligting oor die ontstaansgeskiedenis). In hoogs-gesekulariseerde gebiede in Duitsland, staan hierdie Sondag al meer bekend as ‘Sendingsondag’. Die Sondag van gebed moet, volgens die inisieerders van die projek, op voorbidding vir die sendingprojekte onder die kerk-vervreemde Duitsers konsentreer (vgl. Senftleben 2019). Tóg word die nuwe uitgawe van Luther (2016 [2014]) se gebede-dagboek in groot oplaes in boekwinkels verkoop wat daarop dui dat duisende Duitsers nog steeds graag Luther se gebede wil lees en bid. Engelsberger (2013:59–63) en Goldschmidt (2018:184) konsentreer weer eerder op die krisis wat gebed vir moderne mense inhou. Die riglyne vir die erediens sowel as die aanbevole gebede (Goldschmidt 2018:180–184) het te make met die twyfel of geëmansipeerde mense vandag nog kan bid. Die twyfel oor die sinvolheid van gebed behoort, volgens hierdie twee Duitsers, aandag te geniet op hierdie Sondag. Teoloë van die Afrikaanse kerke kan hiermee identifiseer, aangesien dieselfde probleme in hierdie kerke ondervind word. Die teologie van gebed Talle van Luther se teologiese uitsprake oor gebed word in inleidende gedeeltes en preke gevind wat met die sesde Sondag van die Paaskring (Sondag Rogate) verband hou. Die vyfde Sondag van die Paaskring, Sondag Kantate [die Sondag van jubelende kerkmusiek], word deur die Sondag van gebed opgevolg.2 Op hierdie Sondag word die gemeente nie net vermaan om te bid nie, maar ook aangemoedig om te bid (rogate!), aangesien gebed ’n aktiwiteit is wat met die opgewekte en verhoogde Heer verband hou.3 Luther se gebedsteologie het hierdie liturgiese konteks altyd in gedagte gehou. In die voorwoord tot die uitleg van die Onse Vader in sy Groot Kategismus van 1529 (wat op 10 jaar se Rogate-prediking berus) vind ’n mens vier pilare waarbinne Luther die gebed bedink. Hierdie pilare (vgl. WA30(I), 193–198; 1991a [1529]:94–99) is die volgende: Gebed berus op die bevel (praeceptum) van Christus en gaan hand aan hand met Christus se belofte (promissio) van verhoring van gebede. Gebed ontspring uit die menslike noodsituasie (necessitas) en vind vertroosting in die sekerheid van verhoring (desideratio). In die inleidende gedeelte van sy Rogate-preek van 13 Mei 1520 vind ’n mens egter ’n omvangryker raamwerk vir sy denke oor die gebed. Hierdie raamwerk is in latere preke en traktate oor die gebed ligweg aangepas en verdiep sonder om van die oorspronklike skema afstand te doen. Volgens Luther (WA 4, 622–624; 1886 [1520]:622–624; 2018b [1540]) moet gebed (oratio) aan vyf kenmerke voldoen – andersins sal dit te tevergeefs wees: Eerstens, elke gebed word gebid met die belofte van God (promissio Dei) dat dit aangehoor en verhoor sal word. Die belofte spruit voort uit sy goedheid en sy betroubaarheid om sy woord te hou. Die belofte van God bevry die mens van die vermoede dat God ’n toornige God is. Die belofte van gebedsverhoring bevestig sy liefde en barmhartigheid. Die belofte-karakter van gebed bevry die mens ook van misplaaste selfvertroue en/of wantroue oor die eie waardigheid4 om te bid. Geestelike hoogmoed en/of 3.In die evangelie van Johannes is daar ’n eng band tussen die hemelvaart van Christus, gebed en gebedsverhoring. In Johannes 14:1–12 volg ’n hele gedeelte oor sy weggaan na die Vader toe – en dán, in verse 13 en 14, volg hierdie woorde oor gebed: Wat julle ook al in my Naam vra, sal ek doen, sodat die Vader deur die Seun verheerlik kan word. 4.Die mense van die sestiende eeu moes gesukkel het met minderwaardigheidsgevoelens voor God. Luther moes meermale lidmate en predikante oortuig dat hulle waardig genoeg is om tot God te bid. ’n Goeie voorbeeld is sy Kort troos-nota vir die Christene sodat hulle hul nie in sake die gebed laat mislei nie (WA 51, 455–456; 1991c Die dankgebed: Net só moet u ná die ete eerbiedig en met gevoude hande sê: ‘Ons dank die Here want Hy is vriendelik en sy goedheid duur tot in ewigheid. U gee aan alles wat lewe voedsel, U gee die vee voer, en ook aan die jong rawe wat daarna roep. U verlustig U nie in die krag van ’n perd, of die mag van ’n mens nie. Die Here het ’n welbehae in diegene wat Hom vrees en diegene wat op sy goedheid wag.’ Hierna volg die Onse Vader en die volgende gebed: ‘Ons dank U, Here God, Vader deur Jesus Christus, ons Here, vir al u weldade. U leef en regeer tot in ewigheid. Amen.’ Die vestiging van ’n Protestantse gebedspraktyk Hierdie advies (WA 38, 358–375; 2012c [1535]) staan bekend as ’n Eenvoudige wyse van gebed: Vir ’n goeie vriend. Luther (2012c [1535]:602–612) se praktiese aanwysings en riglyne vir die gebedslewe in hierdie Saans, wanneer jy bed toe gaan, moet jy weer die kruis maak en sê: Mag God, Vader, Seun en Heilige Gees oor my die wag hou. Amen. Daarna moet jy gekniel of staande die Geloofsbelydenis en die Onse Vader opsê. As jy wil, kan jy hierdie gebed ook bid: Ek dank U, my hemelse Vader, deur Jesus Christus, u geliefde Seun, dat U my hierdie dag deur u genade beskerm het, en bid U dat U al my sondes sal vergewe, [oral] waar ek onreg gepleeg het, en dat U my genadiglik deur die nag sal bewaar. Ek gee my liggaam en siel en alles [wat ek is en besit] oor in u hande. Mag u heilige engel met my wees, sodat die bose vyand geen houvas op my kan kry nie. Amen. En dan val jy om, om vrolik te slaap. Luther se gebedsonderrig was nie tot oggend- en aandgebede beperk nie. Ewe belangrik was vir hom (WA 30, 320-326; 2012a [1529]:596) gebede aan tafel: http://www.hts.org.za Page 3 of 7 Original Research Page 3 of 7 Hoe ’n huisvader sy mense moet leer om tafel- en dankgebede te doen. Tafelgebed: 3.In die evangelie van Johannes is daar ’n eng band tussen die hemelvaart van Christus, gebed en gebedsverhoring. In Johannes 14:1–12 volg ’n hele gedeelte oor sy weggaan na die Vader toe – en dán, in verse 13 en 14, volg hierdie woorde oor gebed: Wat julle ook al in my Naam vra, sal ek doen, sodat die Vader deur die Seun verheerlik kan word. As julle iets in my Naam vra, sal ek dit doen. Johannes 15:1–6 gaan oor die wingerdstok en die lote. Jesus sluit sy betoog in vers 7 af met die woorde: As julle in My bly en my woorde in julle, vra dan net wat julle wil hê, en julle sal dit kry. Hierna in Johannes 15:9–17 gaan dit oor die onderlinge liefde in sy afwesigheid. Hy sluit af met die woorde: So sal die Vader aan julle gee wat julle ook al in my Naam vra ... Julle moet mekaar liefhê! In Johannes 16:16–24 gaan dit weer oor sy weggaan na die Vader toe en weer sluit die gedeelte af met onderrig oor die gebed (vv. 23–24): Wat julle die Vader ook al in my Naam sal bid, sal Hy vir julle gee. Tot nou toe het julle nog nie in my Naam gebid nie. Bid en julle sal ontvang, sodat julle blydskap volkome kan wees. Gerhard Sauter (2015:173) is korrek as hy beweer dat die hemelvaartserediens jaarliks plaasvind omdat die kerk die rede vier waarom Jesus Christus as Here en as God se erbarming aangeroep word. Luther se teologie van Sondag Rogate Martin Luther het sedert 1520 gereeld (1886 [1520]; 1899 [1524]; 1912 [1538]; 2018b [1540] om enkele voorbeelde te noem) op hierdie Sondag oor Johannes 16 met toespitsing op verse 23b–24, gepreek: Die kinders en die res van die huishouding moet met gevoude hande en met volle eerbied voor die tafel gaan staan en sê: ‘Alle oë wag op U, Here, en U gee u kinders voedsel op die regte tyd. U maak u hand oop en versadig alles wat lewe in welgevalligheid.’ Welgevalligheid beteken hier: Alle diere ontvang soveel kos dat hulle vrolike en goeie dinge word, want besorgdheid en gierigheid verhinder sodanige welgevalligheid. Dit verseker Ek julle: Wat julle die Vader ook al in my Naam sal bid, sal Hy vir julle gee. Tot nou toe het julle nog nie in my Naam gebid nie. Bid, en julle sal ontvang, sodat julle blydskap volkome kan wees. Hierna volg die Onse Vader en die volgende gebed: ‘Here God, hemelse Vader, seën ons en hierdie gawes van u, wat ons uit u empatiese goedheid aanneem. Deur Jesus Christus onse Heer. Amen.’ Luther (1884 [1519]) se erns met die Rogate-preke, volg op sy kritiek in 1519 op die Roomse Kerk se afwatering van die gebedslewe op Sondag-Rogate en die eerste drie dae van die Rogate-week wat in daardie tyd as die drie dae van die kruis bekend gestaan het. Volgens Luther was die aanvanklike bedoeling goed, naamlik om die kruis selfs tydens die Paaskring prominensie te gee. Tydens die Rogate-week is daar gebid en die gebede is met prosessies aangevul waartydens kruise die dorpe en stede ingedra is. Eventueel het die prosessies die gebede verdring (vgl. Luther 2018b [1540]:538). Luther wou, as bybelse teoloog, die gebed, kort voor Hemelvaart en Pinkster, in die sentrum van die gemeentelike lewe ruk. Vanselfsprekend het Luther se bemoeienis met die gebed nie net met Roomse polemiek te make gehad nie, maar ook met sy eksegetiese insigte dat Jesus sy dissipels nie net vóór sy kruisiging nie, maar ook vóór sy hemelvaart oor die gebed onderrig het. Luther, wat Christus in die sentrum van teologiese nadenke geruk het, het dit as ’n noodwendigheid beskou om oor Christus se gebedsonderrig te besin. Die dankgebed: (footnote 4 continues...) [1540]:203–204). In hierdie nota uit 1540 bekla Luther die feit dat lidmate en predikante hulle van gebed weerhou, aangesien hulle vermoed dat hulle nie waardig genoeg is om te bid nie of dat hulle gebede op grond van hulle onwaardigheid nie verhoor sal word nie. Hy hou aan hulle agt argumente voor waarom elke gelowige waardig genoeg is om, veral in moeilike tye, self tot God te bid. Hulle is waardig omdat hulle deur God geskape is; deur sy Seun verlos is; deur die Heilige Gees geleer word; deur God self uitverkies is tot predikant en of lidmaat; op grond van God se wil moet ly; deur God bygestaan word in tye van beproewing; dat hulle dít alles mag glo; en dat hulle deur Christus nie aan die genadige Vaderhart hoef te twyfel nie. Die teologie van gebed As julle iets in my Naam vra, sal ek dit doen. Johannes 15:1–6 gaan oor die wingerdstok en die lote. Jesus sluit sy betoog in vers 7 af met die woorde: As julle in My bly en my woorde in julle, vra dan net wat julle wil hê, en julle sal dit kry. Hierna in Johannes 15:9–17 gaan dit oor die onderlinge liefde in sy afwesigheid. Hy sluit af met die woorde: So sal die Vader aan julle gee wat julle ook al in my Naam vra ... Julle moet mekaar liefhê! In Johannes 16:16–24 gaan dit weer oor sy weggaan na die Vader toe en weer sluit die gedeelte af met onderrig oor die gebed (vv. 23–24): Wat julle die Vader ook al in my Naam sal bid, sal Hy vir julle gee. Tot nou toe het julle nog nie in my Naam gebid nie. Bid en julle sal ontvang, sodat julle blydskap volkome kan wees. Gerhard Sauter (2015:173) is korrek as hy beweer dat die hemelvaartserediens jaarliks plaasvind omdat die kerk die rede vier waarom Jesus Christus as Here en as God se erbarming aangeroep word. Open Access http://www.hts.org.za Open Access Page 4 of 7 Page 4 of 7 Original Research Original Research Hom en sy geregtigheid word deel van my. ’n Ongelyke ruiltransaksie (inaequalis permutatio) vind plaas – sy geregtigheid vir my ongeregtigheid – en só vergaan my sonde danksy Christus sodat ek waardig voor God se troon staan en gereed is om deur Hom aangehoor te word. Gebed word afgesluit met ‘Amen’7 – die versekering dat dít wat gebid word, wel verhoor sal word. In die laaste verwerking van sy postil oor gebed, het Luther (2018a [1540]:548) iets bygevoeg oor die wyse en die wagperiode van die verhoring van ’n gebed. Hy vermaan dat daar nie beperkings vir God gestel moet word nie – nie ten opsigte van die manier hoe die gebed verhoor word nie, en ook nie ten opsigte van die tydstip van verhoring nie. Daar moet slegs in vreugdevolle uitsig op die Here gewag word, en onder geen omstandighede kan Hy aan opdragte en ultimatums onderwerp word nie. minderwaardigheidsgevoelens bepaal nie die verhoring van ’n gebed nie. Die barmhartige toewending van God is die waarborg van gebedsverhoring. ’n Akkurate opsomming van Luther se gebedsteologie8 word in Lindberg (2016) gevind: He asserted that Christians pray because God has commanded them to pray and promised to hear them. For this reason, Christians pray to God, not to Mary or the saints. Prayer was the proper response to God; it never originated the relationship with God. Luther rejected the idea that prayer was a good work and rejected practices – such as repetition of prayers and the use of prayers as works of satisfaction – that might lend support to the idea that prayer was a good work. Prayer was not based in the Christian’s worthiness to pray. Need drives her to pray, and she brings all her needs to God, trusting God’s promise to hear her. As every Christian is a priest, every Christian should pray. One should not trust in others’ prayers and should not leave prayer to the clergy. Prayer should come from the heart and be simple. (p. 418) Die tweede9 bede: Laat u koninkryk kom10 In sy eie woorde: [Das] erste, Das man von gott eyne vorheyssung odder zu sage habe unnd die selbe zuvor bedencke, gott der selben vormane, und sich dadurch bewege trostlich [zuversichtlich] zu bitten, dan [denn] ßo gott nit hette heyßen pitten und erhorung zugesagt, mochten alle creaturen mit allen pitten nit eyn kornleyn erlangen. Darauß dan folget, das niemant ettwas von gott erlanget seyner oder seyns gepeets wirdickeit halben, sondern alleyn auß abgrund gottlicher gutickeit, der [...] durch seyn gnedig zusagen unnd heyßen [Gebieten] unß bewegt zu pitten und begeren, auff das wir [...] kuhn werden, trostlich zu bitten’. Hierdie gemoderniseerde weergawe word by Schwarz (2016 [2015]:383) gevind. 8.Vir ’n kort sistematiese oorsig, kan na Hiller (2015) gekyk word. 9.Luther self sou nie tevrede gewees het met hierdie keuse van my nie. Dit was vir hom uiters belangrik dat gebede en nadenke oor gebede met ‘Onse Vader’ sal begin. Die ander bedes moet in die lig van die naam van die Here uitgelê word (vgl. Barth 2009:476 vir tientalle verwysings na hierdie oortuiging). Luther was van mening dat die aanroep van die Vadernaam ’n belangrike aspek is van die vervulling van die eerste bede (‘laat u Naam geheilig word’). Dit dra ook by tot gehoorsaamheid aan die tweede gebod – om die Naam van God te eer (vgl. Schwarz 2016 [2015]: 380–385 vir die samehang van gebed en die eer van God se Naam). 9.Luther self sou nie tevrede gewees het met hierdie keuse van my nie. Dit was vir hom uiters belangrik dat gebede en nadenke oor gebede met ‘Onse Vader’ sal begin. Die ander bedes moet in die lig van die naam van die Here uitgelê word (vgl. Barth 2009:476 vir tientalle verwysings na hierdie oortuiging). Luther was van mening dat die aanroep van die Vadernaam ’n belangrike aspek is van die vervulling van die eerste bede (‘laat u Naam geheilig word’). Dit dra ook by tot gehoorsaamheid aan die tweede gebod – om die Naam van God te eer (vgl. Schwarz 2016 [2015]: 380–385 vir die samehang van gebed en die eer van God se Naam). 6.Dit was vir Luther belangrik om aan te toon dat gebed in die naam van Jesus Christus nie ’n totale breuk met die Ou Testament impliseer nie. Die eenheidsband tussen Ou en Nuwe Testament het hy in die metafoor genadestoel gevind. Die tweede9 bede: Laat u koninkryk kom10 In die Duitse uitleg van die Onse Vader vir gewone lidmate (WA 2, 81–-130; 1991b [1519]) word een van Luther se oudste bewaar is, is deur Luther as die ‘genadestoel van God’ geïnterpreteer. Dáár is gebede verhoor. Volgens Luther bly die ‘genadestoele’ van die Ou Testament in plek, maar Christene het nou ’n nuwe ‘genadestoel’ in Jesus Christus. God is nou ‘liggaamlik’ teenwoordig in Christus, en só is Hy nou vir alle mense toeganklik. Gebed deur Jesus Christus tree in ontmoeting met die genade van die enigste ware God. Hiervan kan gelowiges seker wees! Sy kommentaar (WA 19, 230, 12–20; 1897 [1526]:230) op hierdie vers is van besondere waarde vir die Luther-navorsing: Also musten auch alle die ym lande odder ausser dem lande, wenn sie beten wolten, yhr gebet dahin richten und yhr hertz hefften, an die stet, da Gott leyblich wonete durch sein wort, Auff da sie keynen andern Gott anbetten, denn den, der uber den Cherubin sass auf dem gnaden stuel [vgl. Ex 25:17; Heb 9:5]. Dahin musten alle gebet komen. Gleich wie nu zu unser zeit ym newen testament alle unser gebet mussen zu Christo komen, wilcher ist unser gnaden stuel [Rom 3:25; Heb 4:16], das wyr auch keynen andern Gott widder wissen noch anbeten noch anruffen sollen on [außer] den, der ynn dem menschen Jhesu Christ wonet leyblich. Denn es ist auch sonst keiner mehr. (Schwarz 2016 [2015]:386 se verbeterde teks) 7.In Luther se advies aan Peter Beskendorf (WA 38, 229; 2012c [1535]:360, 18–22), verklaar hy die ‘Amen’ soos volg: Mag God ons help dat wat ons vra, ons werklik wil hê; en laat ons nie daaraan twyfel dat Hy ons gehoor het en ons sal verhoor nie; dat dit ja en nie nee beteken nie en dat dit nie twyfelagtig is nie. Daarom sê ons in vrolikheid ‘Amen. Dit is waar en verseker. Amen. 5.Luther (WA 2, 175, 5–17; 1884 [1519]:175) het die saak van minderwaardigheidsgevoelens, gebedsekerheid en God se barmhartige toewending klassiek in ’n Leer-preek oor gebed en prosessies tydens die kruisweek van 1519 omskryf. God verwag niks van die waardigheid van die bidder en/of die gebed nie, maar slegs vertroue in sy toegesegde barmhartigheid. Die teologie van gebed Sekerheid van geloof en gebedsekerheid is twee kante van dieselfde muntstuk; en albei is in die barmhartigheid van God begrond.5 Die tweede kenmerk van ware gebed is dat ’n bepaalde behoefte (rei necessariae) geopper word. In gebed kan daar nie ’n warboel van ongestruktureerde wense geuiter word nie. Die Heilige Gees identifiseer die één saak uit ’n versameling van gedagtes (animi collectio) wat voor God gebring word. Gebede wat aan rooskranse en willekeurige lyste van behoeftes gekoppel is, het niks met die identifiseringswerk van die Heilige Gees te make nie. Die derde kenmerk is geloof in die belofte van God (fides opus est). Die geloof van die bidder is egter nie die waarborg vir aanhoring en verhoring nie, maar die betroubaarheid (veritas) van God. Geloof is vertroue (fiducia) in die trouheid en betroubaarheid van God wat aan die mens die belofte van verhoring gee. Weereens: Geloof as sodanig, verseker nie verhoring nie, maar vertroue dat God getrou aan sy belofte bly. Luther kritiseer ’n houding van geloofs- selfversekerdheid met sy argument. Gebedsverhoring berus nie op die selfversekerdheid van die bidder nie, maar alleen op God se genadige toewending tot die onwaardige mens. ’n Retories-voortreflike gebed bied geen waarborg vir verhoring nie – die waarborg lê in die deernis van God met die mens in nood. Die vierde kenmerk is dat gebed in erns gedoen word. Dít waarvoor gebid word, moet ’n saak van dringende verwagting (magnopere desiderante) wees. Daar kan nie ’n houding van kom en kyk maar wat gebeur wees nie. Dit sal oneerbiedigheid teenoor God uitstraal, aangesien daar met Hom in gebed gespeel word. Vanselfsprekend sal dit ook op ’n sinnelose mors van tyd neerkom. Die vyfde kenmerk is dat gebed in die naam van Jesus geskied. Hy beveel (iussu) dat daar in sy Naam gebid moet word, aangesien ’n gebed in sy Naam verhoor sal word (Matt 7:7; Joh 16:23). Daar bestaan geen moontlikheid dat ’n gebed in sy Naam nie verhoor sal word nie6 (non potest non fieri exauditio). Christus bid namens my in die hemel. My nood is sy saak. My sonde neem Hy op ’n Akkurate opsomming van Luther se gebedsteologie8 word in Lindberg (2016) gevind: 5.Luther (WA 2, 175, 5–17; 1884 [1519]:175) het die saak van minderwaardigheidsgevoelens, gebedsekerheid en God se barmhartige toewending klassiek in ’n Leer-preek oor gebed en prosessies tydens die kruisweek van 1519 omskryf. God verwag niks van die waardigheid van die bidder en/of die gebed nie, maar slegs vertroue in sy toegesegde barmhartigheid. In sy eie woorde: [Das] erste, Das man von gott eyne vorheyssung odder zu sage habe unnd die selbe zuvor bedencke, gott der selben vormane, und sich dadurch bewege trostlich [zuversichtlich] zu bitten, dan [denn] ßo gott nit hette heyßen pitten und erhorung zugesagt, mochten alle creaturen mit allen pitten nit eyn kornleyn erlangen. Darauß dan folget, das niemant ettwas von gott erlanget seyner oder seyns gepeets wirdickeit halben, sondern alleyn auß abgrund gottlicher gutickeit, der [...] durch seyn gnedig zusagen unnd heyßen [Gebieten] unß bewegt zu pitten und begeren, auff das wir [...] kuhn werden, trostlich zu bitten’. Hierdie gemoderniseerde weergawe word by Schwarz (2016 [2015]:383) gevind. Die tweede9 bede: Laat u koninkryk kom10 Die gevaar van Turkse besetting is één bewys van hierdie werklikheid. Nie dat God hiervoor blameer kan word nie; dit is ons skuld alleen. Ná die vernedering, verhoog hierdie gebed ons. Dit troos ons dat die toekoms aan die ‘vriendelike Meester’, Jesus Christus, behoort. Om hierdie dubbelslagtigheid te verstaan, moet daar ’n besef van die werklikheid van ‘twee ryke’ ontwikkel. Die een ryk is dié van die Duiwel – die vors van hierdie wêreld, die ryk van sonde, ongehoorsaamheid en die dood. Die ander ryk, die ryk van God, is die ryk van genade en lewe. Hierdie twee ryke voer stryd met mekaar, maar daar is gewis net een wenner. Die ryk van die sonde sal altyd aanloklik wees, maar die ryk van God moet in ons lewe die oorhand kry. Die ryk van God is ’n ‘ryk van geregtigheid en waarheid’ (Matt 6:33). Die ‘geregtigheid van Christus’ wys heen na ’n ryk van sondevergewing en lewe. Die gebed ‘laat u koninkryk kom’ beteken dat daar gebid word om bevryding van die ryk van die Duiwel en die aanbreek van die ryk van barmhartigheid. In sy Klein Kategismus van 1529 het Luther (WA 30(I), 300-302; 2012a [1529]) die tweede bede soos volg uitgelê: Wat is dit? Antwoord: Wat is dit? Antwoord: God se ryk kom vanself, ook sonder ons gebed, maar ons bid met hierdie gebed, dat dit ook na ons mag kom. God se ryk kom vanself, ook sonder ons gebed, maar ons bid met hierdie gebed, dat dit ook na ons mag kom. God se ryk kom vanself, ook sonder ons gebed, maar ons bid met hierdie gebed, dat dit ook na ons mag kom. Hoe gebeur dit? Antwoord: Mense maak die fout om te dink dat die koninkryk kom wanneer kerke ter ere van Petrus en Jakobus gebou word en daar mooi rituele plaasvind. Die koninkryk kom egter wanneer wet en evangelie in ons harte woon. Wanneer die hemelse Vader ons sy Heilige Gees gee, sodat ons sy heilige Woord deur sy genade glo en God-welgevallig lewe, hier tydens ons aardse tyd en dáár in ewigheid13. (bl. 586, 36–588, 2) Wanneer die hemelse Vader ons sy Heilige Gees gee, sodat ons sy heilige Woord deur sy genade glo en God-welgevallig lewe, hier tydens ons aardse tyd en dáár in ewigheid13. (bl. tussen Junie 1516 en die begin van die Lydenstyd in 1517, sowel as kort traktate oor die dekaloog in 1518 en die Onse Vader in 1519. Aan die credo het hy deurentyd gewerk. Hierdie Kort vorms was nie alleen kategese nie, maar ook pastoraat. Dit is daarom die eerste Protestantse stigtingsliteratuur (evangelische Erbauungsliteratur) wat meditasie en praktiese gebruik in die oog gehad het (vgl. inleiding van Korsch tot 2012b [1520]:319). 13.Luther se oorspronklike formulering: Dein Reich kome. Was ist das? Antwort: Gottes reich koempt wol on vnser gebet von yhm selbs / Aber wir bitten ynn diesem gebet / das auch zu vns kome. Wie geschicht das? Antwort: Wenn der hymlische Vater vns seinen heiligen geist gibt / das wir seinem heiligen wort / durch seine gnade / gleuben / vnd Goettlich leben / hie zeitlich vnd dort ewiglich. Die tweede9 bede: Laat u koninkryk kom10 Dit is ’n gebed dat ons nie aan hierdie lewe verknog sal bly nie, maar die dood sonder vrees sal nader en na die toekomstige lewe sal uitsien. betekenis aan die tweede bede gegee: Volgens Luther, bevind hierdie wêreld hom in ’n ellendige toestand. Dit is vol van sonde en boosheid en staan onder beheer van die Duiwel. Daarenteen is God se ryk een vol van genade en deugsaamheid, en staan onder beheer van die Heer Jesus Christus. In die tweede geval word daar gebid om geloof dat Christus se genade en barmhartigheid ons ten goede sal kom. Die gebed om die koms van die koninkryk is ’n bede dat ons van ongeloof en aardse nyd weerhou sal word. Dit is ’n gebed dat ons van onkuisheid, tweedrag en oorlog bewaar sal word. Dit is ’n gebed dat toorn en bitterheid nie in ons ’n koninkryk sal opbou nie, maar dat ons deur Christus se genade redelike liefdevolheid, broederlike getrouheid, vriendskap en sagmoedigheid sal opbou. Dit is die bede dat droefnis en swaarmoedigheid nie in ons die oorhand sal kry nie, maar dat ons ’n vreugde en lus aan die barmhartigheid van die Here sal ontwikkel. Dit is die bede dat ons deur die vergewing van Christus oorweldig sal word, sodat ons in vreugdevolle gehoorsaamheid aan al die gebooie sal lewe. Dit is ’n bede dat ons nie traag sal word in die diens aan die Here nie, sodat ons uiteindelik deel aan die ewige erediens kan kry. Dit is ’n gebed dat ons nie aan hierdie lewe verknog sal bly nie, maar die dood sonder vrees sal nader en na die toekomstige lewe sal uitsien. interpretasies van die Here se gebed gevind. Soos hier onder sal blyk, het Luther verskillende invalshoeke deur die jare gebruik om die onderskeie bedes te verduidelik. Sy uitleg van die tweede bede in hierdie geskrif (WA 2, 95, 15–99, 15; 1991b [1519]:224–229) is daarom uniek en is nie in latere geskrifte net so herhaal nie. Volgens Luther doen hierdie gebed twee dinge aan ’n mens: Dit verneder ons; en dit verhoog ons. Dit verneder ons deur die besef dat die koninkryk nog nie tot ons gekom het nie. Hierdie besef ontstaan wanneer daar met werklike erns oor die koms van die ryk gebid en besin word en daar besef word dat die ryk nog ver van ons verwyderd is. 12.Van die begin van Luther se skryfonderneming, het hy die verpligting aanvaar om die kerkmense oor die Christelike geloof te onderrig. Alle Christene moes die vermoë ontwikkel om krities oor prediking en leer te oordeel. Hierdie onderrigtaak (of kategese) is aan die gemeente opgedra. Om die gemeentes hierin te ondersteun, het Luther vir kerklik-bruikbare verklarings van die credo, die dekaloog en die Onse Vader gesorg. Die Kort vorm van die credo, die dekaloog en die Onse Vader (1520) was Luther se eerste kategetiese geskrif. Dit het as basis vir sy kategismusse van 1529 gedien. Die Kort vorms van 1520 is voorafgegaan deur dekaloogprediking 11.Luther weerhou hom van wat Eberhard Jüngel (1990:306) die ‘tirannie van die etiek en spesifiek die tirannie van die politieke doelstellings en waardes’ noem. Die ‘ontvangskarakter van die evangelie’ bly in bewaring. Die tweede9 bede: Laat u koninkryk kom10 In hierdie verband speel sy Kommentaar op Jona (WA 19, 169–251; 1897 [1526]) ’n belangrike rol. In Jona 2:8 sê Jona: ‘Toe my siel in my verbreek het, het ek aan die Here gedink, en my gebed het tot U gekom in u heilige tempel’ (volgens die Luther-Bybel – Luther 2017:911). In die Ou Testament is gebede in plekke waar God ‘liggaamlik’ teenwoordig is, aangehoor. Die plek waar die dekaloog deur die engele op die ark 10.Volgens Peters (1992:70–71) het Luther tot en met sy kategismusse van 1529 die Middeleeuse tradisie gevolg deur op grond van die Vulgaat ‘Adveniat regnum tuum’ hierdie bede as ‘Zu komme Dein Reich’ te vertaal. ’n Mens sou op grond van sy eie teologie verwag dat hy soos volg sou vertaal: ‘Zu uns komme Dein Reich’ of ‘Zukomme uns Dein Reich’ [‘Mag u ryk na ons kom’ of ‘Mag u ryk ons toekom’]. Die latere vertalings in die kategismusse is waarskynlik deur die Boheemse Broeders en Phillip Melanchthon beïnvloed. http://www.hts.org.za Open Access Page 5 of 7 Page 5 of 7 Original Research Original Research betekenis aan die tweede bede gegee: Volgens Luther, bevind hierdie wêreld hom in ’n ellendige toestand. Dit is vol van sonde en boosheid en staan onder beheer van die Duiwel. Daarenteen is God se ryk een vol van genade en deugsaamheid, en staan onder beheer van die Heer Jesus Christus. In die tweede geval word daar gebid om geloof dat Christus se genade en barmhartigheid ons ten goede sal kom. Die gebed om die koms van die koninkryk is ’n bede dat ons van ongeloof en aardse nyd weerhou sal word. Dit is ’n gebed dat ons van onkuisheid, tweedrag en oorlog bewaar sal word. Dit is ’n gebed dat toorn en bitterheid nie in ons ’n koninkryk sal opbou nie, maar dat ons deur Christus se genade redelike liefdevolheid, broederlike getrouheid, vriendskap en sagmoedigheid sal opbou. Dit is die bede dat droefnis en swaarmoedigheid nie in ons die oorhand sal kry nie, maar dat ons ’n vreugde en lus aan die barmhartigheid van die Here sal ontwikkel. Dit is die bede dat ons deur die vergewing van Christus oorweldig sal word, sodat ons in vreugdevolle gehoorsaamheid aan al die gebooie sal lewe. Dit is ’n bede dat ons nie traag sal word in die diens aan die Here nie, sodat ons uiteindelik deel aan die ewige erediens kan kry. 14.Luther het die gebed van die Here onder die opskrif Vater unser im Himmelreich gestel. Die opskrif van die Afrikaanse weergawe in Gesang 267 (Liedboek van die Kerk 2001) lui ‘Ons Vader op die hemeltroon’. Die motief van die koninkryk is ongelukkig met die motief van mag vervang. Dit is heeltemal teenstrydig met Luther se verstaan van gebed. Die Liedboek van die Kerk (2001) volg die Duitse Evangeliese Gesangeboek (EG 2004: lied 344) deur te stel dat Luther die teks in 1539 geskryf het en dat die melodie, afkomstig van die Boheemse Broeders, uit die jaar 1531 stam. Volgens die onlangse navorsing van Heidrich en Schilling (2017:120–121), bestaan daar geen histories-betroubare bewyse vir hierdie aannames nie. ’n Teks in Luther se handskrif wat in bewaring gebly het, is nie gedateer nie. ’n Kopie van Johann Walters se Gesank Buchleyns (1544) meld wel dat die melodie sedert 1539 in gebruik was. Dít is egter nie waar nie, aangesien die melodie sedert 1519 in Boheme bekend was en reeds in 1531 in Duitsland gebruik is. Page 6 of 7 Page 6 of 7 Die ryk van God kom na ons toe op twee maniere: Eerstens, tydelik deur die Woord en die geloof; en tweedens, deur die openbaring by die wederkoms. Ons bid dat albei gestaltes ook na diegene sal kom wat nog nie daarin glo nie. Die bede kom dus daarop neer dat daar gebid word dat die Here ons sy Woord sal gee sodat die evangelie oral oor in die wêreld getrou gepreek sal word. Verder impliseer hierdie bede dat mense die gepredikte Woord sal aanneem en daarvolgens sal lewe sodat die ryk van die Duiwel in die proses vernietig sal word. uitlegtradisie wat deur Tertullianus en Augustinus begin is en wat stel dat die koninkryk in al sy majesteit eers by die eindoordeel aan die lig sal kom. Hierdie benadering is egter die bedding vir die werkgeregtigheid. Om met ’n goeie gewete die eindoordeel tegemoet te tree, moet die koninkryk in die harte aankom. Verder moet daar sigbaar volhard word tot en met die wederkoms en eindoordeel. Hierdie futuristies- eskatologiese aanslag, dompel gelowiges in die onsekerheid van werkgeregtigheid en verdienste-vroomheid. Luther het besef dat dit beter is om by Origenes en Gregorius van Nissa aan te sluit wat oortuig was dat die ryk van genade in die hart van die gelowige, danksy geloof, aanbreek. Die heerskappy van God is sy saak alleen – dit breek nie met ons toedoen daaraan nie. Die almag van sy genade breek nou in die kerk aan en sal in die toekoms by die wederkoms weer aanbreek. God se ryk is daarom alreeds deel van die aardse bedeling, alhoewel dit eers in die toekoms ten volle sal aanbreek. Die genaderyk van die Vader is hier op aarde egter slegs te vinde waar die evangelie van die gekruisigde Heer verkondig word. Luther se insig kom dus op die volgende neer: Die kerk is ‘creatura verbi’ en God se koninkryk is ’n heerskappy deur die woord van die kruis. Aangesien die koninkryk nie net genade is nie, maar ook reg, stel Luther dat die koninkryk ook alreeds aangebreek het toe God sy barmhartige reg in Jesus se kruisiging en opstanding aan die wêreld kom toon het. Ook dít moet vandag verkondig en geglo word. ’n Koninkryksverlange sonder ’n liefde vir die kerk waar die genade en barmhartigheid reeds teenwoordig is danksy die prediking, sou vir Luther ’n totaal vreemde aangeleentheid gewees het. Page 6 of 7 ’n Koninkryk sonder die verkondiging van die heil in Christus, verplaas die Christendom terug in die Middeleeue. Kerk en koninkryk moet saam bedink word, aangesien albei ’n heerskapsgebied van die barmhartige Vader verteenwoordig (vgl. Peters 1992:78–83 met tientalle verwysings na die kategismusse). Vir Luther is die ‘koninkryk van God’ geen kleinigheid nie. Dit is nie iets wat uit die menslike verbeelding of hartsverlange voortspruit nie, maar dit is God se geskenk aan ons. Hierdie geskenk is véél groter as wat ons verbeelding toelaat, aangesien dit uit God se liefde voortspruit wat groter is as menslike voorstellings van skenkende liefde. Die ‘koninkryk’ kan daarom nie ’n produk van ons morele skeppingsdrang wees nie, aangesien dit méér is as wat ons uit ons eie goedheid kan voortbring. In die tweede strofe van sy gesang Onse Vader in die hemelryk14 (tussen 1539 en 1544) kry ons ’n kernagtige samevatting van sy denke oor die tweede bede. Hy belig die volgende aspekte van die koninkryk: Die praesentiese en futuristiese eskatologie; die Heilige Gees wat met sy talle gawes by ons inwoon; die verbreking van die Satan se toorn en groot geweld; en die bewaring van die kerk. Die oorspronklike digwerk (Heidrich & Schilling 2017) lui: Es kom dein reich zu dieser zeit Vnd dort hernach ynn ewigkeit Der heilig geist vns wone bey Mit seinen gaben Mancherley Des Satans zorn vnd gros gewalt Zerbrich fur yhm dein kirch erhalt. (p. 122) 15.Luther se woorde: ‘Vis inquam, ut homo se peccatorem agnoscat et totam vitam suam aliud nihil esse ducat, quam orationem, desyderium, gemitum, suspirium ad misericordiam tuam.’ Die tweede9 bede: Laat u koninkryk kom10 586, 36–588, 2) Die koninkryk kan nie in uiterlike dinge gesien word nie, maar in die innerlike dinge – die nederige en dankbare lewe wat uit die geloof van die hart voortspruit. Hierdie gebed is nie ’n gebed dat ons toegelaat word om na die ryk van God te beweeg nie. Dit is ’n gebed dat die ryk na ons toe moet kom. Dit is egter ook ’n gebed dat ons volgens die kenmerke van die koninkryk sal lewe, want die koninkryk kom ook deur ons liefde vir die medemens. Die koninkryk kom egter nie op grond van ons medemenslike liefde11 nie, maar naasteliefde is ’n bewys dat die koninkryk na ons gekom het en nou in ons harte lewe. In sy uitleg van die tweede bede in Die Groot Kategismus, beklemtoon Luther (WA 30(I), 199–201; 1991a [1529]:102–104) aspekte wat nie elders aandag geniet nie. Volgens Luther kan die ryk van God niks anders beteken nie as dat God sy Seun na die wêreld gestuur het om ons te verlos van die geweld van die Duiwel, en om Hom toe te laat om in geregtigheid te regeer sodat die lewe sonder sonde, dood en boosheid gekenmerk kan word. Hiervan lees ons in die Woord, en die Heilige Gees skep geloof in hierdie belofte. Die bede impliseer ook dat diegene wat die evangelie glo, se lewenshouding daarvan sal getuig, sodat die ryk van genade ook by ander mense byval sal vind. Ook húlle sal deel aan die verlossing kry, sodat almal saam aan die één ryk deel sal kry en daarin bewaar sal word. In 1520 het Luther in sy Kort vorm van die Onse Vader (WA 7, 222–223; 2012b [1520]:351, 1–43),12 die volgende Open Access http://www.hts.org.za Original Research Koninkryk en kerk Luther (WA 5, 127, 35–37; 1892 [1519–1521]:127) het vroeg in sy lewe in sy Voorlesing oor Psalm 5 entoesiasties gesê: ‘U wil, sê ek, dat die mens erken dat hy ’n sondaar is en om daardie rede toesien dat sy ganse lewe niks anders sal wees as ’n gebed, met ’n verlange en begeerte na u barmhartigheid nie.’15 Hy het trouens ’n bietjie later (WA 8, 360, 29; 1889 [1521]:360) beweer ‘dat geloof pure gebed is’. Geloof sonder gebed, en gebed sonder geloof is dus nie denkbaar nie. ‘Ware geloof maak gretig en dors na regte gebed’ (WA 8, 356, 1; 1889 [1521]:356). Luther (WA 51, 455, 10–12; 1991c [1540]:203) het egter laat in sy lewe erken dat ‘... regte gebed die hoogste [maar ook] die swaarste werk op aarde is; [maar dat] dit tegelyk ook die belangrikste erediens en oefening van die geloof is’. Hierdie dialektiek van vreugde aan gebed en onvermoë om te bid, is ’n algemene probleem in die lewe van gelowiges. Trouens, dit is die grondstruktuur van egte teologie! Volgens Luther (WA 50, 659; 2012d [1539]:662) is oratio, meditatio en tentatio [gebed, meditasie en aanvegting] die hoekstene van outentieke teologie. Hy (WA 38, 363; 2012c [1535]:611) het aan Volgens albei kategismusse kan die tweede bede nie van die derde artikel van die Credo ontkoppel word nie. Koninkryk en kerk moet saam bedink word. Die ‘eine heilige Christenheit, die Gemeine der Heiligen’ vorm ’n tematiese eenheid met ‘Gottes König- und Gnadenreich’. Luther se uitleg van kerk en koninkryk het egter met die Middeleeuse uitlegtradisie gebreek. Volgens Luther het die koninkryk reeds in Christus se kruis en opwekking deur die werking van die Gees aangebreek. Die eskatologiese openbaring van dít wat nou nog verborge is, sal niks nuuts aan die lig bring nie. Die oorlog wat reeds gewen is, sal slegs as segetog waargeneem kan word. Deurdat Luther die praesentiese dimensie van die heerskappy van God beklemtoon, breek hy met die http://www.hts.org.za Open Access Page 7 of 7 Page 7 of 7 Original Research Heidrich, J. & Schilling, J. (Hrsg), 2017, Martin Luther: Die Lieder, Carus-Verlag, Stuttgart. sy vriend, Peter Beskendorf, gesê dat ‘hyself dikwels méér uit gebed geleer het [wat die evangelie behels] as uit die lees van boeke’, aangesien gebed (in samehang met meditasie oor die bybelse tekste en worsteling met God) die leerskool vir goeie teologie is (vgl. Koninkryk en kerk Barth 2009:476–479 vir talle aanhalings oor die band tussen gebed, geloof, meditasie, aanvegting en teologie). In die lig hiervan sal ons goed doen om op voetspoor van Luther meer dikwels oor gebed te publiseer. Hiller, D., 2015, s.v. ‘Gebet’, in V. Leppin & G. Schneider-Ludorff (Hrsg.), Das Luther- Lexikon, pp. 236–237, Bückle & Böhm, Regensburg. Jüngel, E., 1990, ‘Der evangelisch verstandene Gottesdienst’, in E. Jüngel (Hrsg.), Wertlose Wahrheit: Zur Identität und Relevanz des christlichen Glaubens., pp. 283–310, Kaiser Verlag, München. (Theologische Erörterungen III). Liedboek van die Kerk, 2001, NG Kerk-Uitgewers, Wellington. Lindberg, C., 2016, ‘Piety, prayer, and worship in Luther’s view of daily life’, in R. Kolb, I. Dingel & L. Batka (eds.), The Oxford handbook of Martin Luther’s Theology, pp. 414–426, Oxford University Press, Oxford. Luther, M., 1884 [1519], ‘Eyn sermon von dem gepeet und procession in der Kreuz wochen’, in J.K.F. Knaake (Hrsg.), D. Martin Luthers Werke, 2. Band, pp. 172–179, Hermann Böhlaus Nachfolger, Weimar (WA 2, 172–179). (Kritische Gesamtausgabe). Befondsing Luther, M., 1991a [1529], ‘Der Groβe Katechismus’, in K. Alant (Hrsg.), Luther Deutsch: Die Werke Luthers in Auswahl, 4. Band 3, erweiterte Aufl, pp. 11–150, Vandenhoeck, Göttingen (WA 30(I), 123–238, 17). (Der neue Glaube). Hierdie navorsing het geen spesifieke toekenning ontvang van enige befondsingsagentskap in die openbare, kommersiële of nie-winsgewende sektore. Luther, M., 1991b [1519], ‘Deutsche Auslegung des Vaterunsers für die einfältigen Laien’, in K. Alant (Hrsg.), Luther Deutsch: Die Werke Luthers in Auswahl, Die Schriftauslegung, 5. Band 5, 4. Aufl., pp. 204–273, Vandenhoeck & Ruprecht, Göttingen (WA 2, 81–129). Vrywaring Luther, M., 2012b [1520], ‘Eine kurze Form der Zehn Gebote, eine kurze Form des Glaubens, eine kurze Form des Vaterunser’, in D. Korsch (Hrsg.), Martin Luther: Deutsch-Deutsche Studienausgabe, Glaube und Leben, Band 1, pp. 317–361, Evangelische Verlagsanstalt, Leipzig (WA 7, 204–229). Die sienings en menings wat in hierdie artikel uitgedruk word, is dié van die outeur (s) en weerspieël nie noodwendig die amptelike beleid of posisie van enige geaffilieerde agentskap van die outeurs nie. Luther, M., 2012c [1535], ‘Eine schlichte Weise zu beten, für einen guten Freund’, in D. Korsch, (Hrsg.), Martin Luther: Deutsch-Deutsche StudienausgabeGlaube und Leben, Band 1, pp. 599–631, Evangelische Verlagsanstalt, Leipzig (WA 38, 358–375). Luther, M., 2012d [1539], ‘Vorrede zum ersten Band der Wittenberger Ausgabe der deutschen Schriften’, in D. Korsch, (Hrsg.), Martin Luther: Deutsch-Deutsche Studienausgabe, Glaube und Leben, Band 1, pp. 657–669, Evangelische Verlagsanstalt, Leipzig. (WA 50, 657–661). Mededingende belange Die outeur verklaar dat hy geen finansiële of persoonlike verbintenis het met enige party wat hom nadelig kon beïnvloed in die skryf van hierdie artikel nie. Luther, M., 1889 [1521], ‘Evangelium von den zehn Aussätzigen vordeutscht und ausgelegtt’, in J.K.F. Knaake et al. (Hrsg.), D. Martin Luthers Werke, 8. Band, pp. 340–397, Hermann Böhlaus Nachfolger, Weimar (WA 8, 340–397). (Kritische Gesamtausgabe). Luther, M., 1892 [1519–1521], ‘Operationes in Psalmos: Psalmus V’, in J.K.F. Knaake et al. (Hrsg.), D. Martin Luthers Werke, 5. Band, pp. 125–199, Hermann Böhlaus Nachfolger, Weimar (WA 5, 125–199). (Kritische Gesamtausgabe). Outeursbydrae Luther, M., 1897 [1526], ‘Der Prophet Jona ausgelegt’, in J.K.F. Knaake et al. (Hrsg.), D. Martin Luthers Werke, 19. Band, pp. 169–251, Hermann Böhlaus Nachfolger, Weimar (WA 19, 169–251). (Kritische Gesamtausgabe). I.W.C.v.W. was die enigeste outeur betrokke by die skryf van die artikel. Luther, M., 1899 [1524], ‘Predigt am Sonntag Dominica Vocem Iocunditatis (Sonntag Rogate) Ioh. XVI am 1. Mai 1524. Nr 28’ in J.K.F. Knaake (Hrsg.), D. Martin Luthers Werke, 15. Band, pp. 546–550, Hermann Böhlaus Nachfolger, Weimar (WA 15, 546–550). (Kritische Gesamtausgabe). Data beskikbaarheidsverklaring Luther, M., 1991c [1540], ‘Ein kurzer Trostzettel für die Christen, dass sie sich im Gebet nicht beirren lassen’, in K. Alant (Hrsg.), Luther Deutsch: Die Werke Luthers in Auswahl, Kirche und Gemeinde, 6. Band 6, 3. Aufl., pp. 203–204, Vandenhoeck & Ruprecht, Göttingen (WA 51, 455–456). Data-deling is nie van toepassing op hierdie artikel nie, aangesien geen nuwe data in hierdie studie geskep of ontleed is nie. Luther, M., 2012a [1529], ‘Der kleine Katechismus für die gemeine Pfarherr und Prediger’, in D. Korsch (Hrsg.), Martin Luther: Deutsch-Deutsche Studienausgabe, Glaube und Leben, Band 1, pp. 574–597, Evangelische Verlagsanstalt, Leipzig (WA 30(I), 264–326). Etiese oorwegings Luther, M., 1907 [1522], ‘Betbüchlein’, in J.K.F. Knaake (Hrsg.), D. Martin Luthers Werke, 10. Band, Zweite Abteilung, pp. 331–406, Hermann Böhlaus Nachfolger, Weimar (WA 10(II), 331–406). (Kritische Gesamtausgabe). Hierdie artikel volg alle etiese standaarde vir navorsing. Luther, M., 1912 [1538], ‘Das XVI Kapitel S. Johannis gepredigte und ausgelegt’ in J.K.F. Knaake (Hrsg.), D. Martin Luthers Werke, 46. Band, pp. 1–113, Hermann Böhlaus Nachfolger, Weimar (WA 46, 1–113). (Kritische Gesamtausgabe). Erkenning Luther, M., 1886 [1520], ‘In secunda adventus dominica. Orationis verae conditiones’, in J.K.F. Knaake (Hrsg.), D. Martin Luthers Werke, 4. Band, pp. 622–624, Hermann Böhlaus Nachfolger, Weimar (WA 4, 622–624). (Kritische Gesamtausgabe). Literatuurverwysings Barth, H-M., 2009, Die Theologie Martin Luthers: Eine kritische Würdigung, Gütersloher Verlagshaus, Gütersloh. Luther, M., 2016 [2014], Aus der Tiefe rufe ich, HeRR, zu Dir: Andachten über das Gebet, zusammengestellt C. van der Woerden, 2. Aufl., Christliche Literatur- Verbreitung, Bielefeld. Bieritz, K-H., 2014 [1986], Das Kirchenjahr: Feste, Gedenk- und Feiertage in Geschichte und Gegenwart, Neu bearbeitet und erweitert von C. Albrecht, CH Beck, München. Luther, M., 2017, Die Bibel nach Martin Luthers Übersetzung: Lutherbibel. Revidiert 2017. Jubiläumsausgabe: 500 Jahre Reformation, mit Sonderseiten zu Martin Luthers Wirken als Reformator und Bibelübersetzer, Deutsche Bibelgesellschaft, Stuttgart. Cobb, P., 2004 [1992], ‘The history of the Christian year’, in C. Jones, G. Wainwright, E. Yarnold & P. Bradshaw (eds.), The study of Liturgy, rev. edn., 6th impression, pp. 455–472, Society for Promoting Christian Knowledge (SPCK), London. Luther, M., 2018a [1540], ‘Another sermon, on prayer’, in B. Mayes (ed.), A year in the gospels with Martin Luther: Sermons from Luther’s Church Postil, transl. J. Langebartels, pp. 546–548, Concordia Publishing House, Saint Louis, MO. Connell, M., 2006, Eternity today: On the liturgical year: Sunday, lent, the three days, the easter season, ordinary time, vol. 2, Continuum, New York. Luther, M., 2018b [1540] ‘Gospel for the fifth Sunday after Easter’, in B. Mayes (ed.), A year in the gospels with Martin Luther: Sermons from Luther’s Church Postil, transl. J. Langebartels, pp. 538–545, Concordia Publishing House, Saint Louis, MO. Ebeling, G., 1975 [1973], ‘Das Gebet’, in G. Ebeling, Wort und Glaube, Band 3. Beiträge zur Fundamentaltheologie, Soteriologie und Ekklesiologie, pp. 405–427, Mohr (Siebeck), Tübingen. Peters, A., 1992, Kommentar zu Luthers Katechismen: Das Vaterunser, Band 3, Hrsg. G. Seebaß, Vandenhoeck & Ruprecht, Göttingen. EG 2004 kyk Evangelische Kirche im Rheinland, Westfalen und Lippische Landeskirche Engelsberger, G., 2013, Gemeinde auf dem Weg durch das Kirchenjahr: Andachten, Meditationen und Gottesdienste für die Zeit von Ostern bis Ewigkeitssontag, Gütersloher Verlagshaus, Gütersloh. Sauter, G., 2015, Schrittfolgen der Hoffnung: Theologie des Kirchenjahres, Gütersloher Verlagshaus, Gütersloh. Senftleben, M., 2019, ‘Informationen zu jedem Sonn- und Festtag im Kirchenjahr’, viewed 8 April 2019, from https://www.daskirchenjahr.de. Evangelische Kirche im Rheinland, Westfalen und Lippische Landeskirche, 2004. Evangelisches Gesangbuch, Gütersloher Verlagshaus, Gütersloh Goldschmidt, S., 2018, Denn du bist unser Gott: Gebete, Texte und Impulse für die Gottesdienste des Kirchenjahres, Neukirchener Verlag, Neukirchen-Vluyn. Schwarz, R., 2016 [2015], Martin Luther – Lehrer der christlichen Religion, 2. Aufl., Mohr Siebeck, Tübingen. Open Access http://www.hts.org.za
https://openalex.org/W4378714982	https://zenodo.org/record/7982582/files/42-29-2023.pdf	English	null	The Expanding Scope of Emulgels: Formulation, Evaluation and Medical Uses	International Journal of Current Science Research and Review	2,023	cc-by	9,138	International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsr www.ijcsrr.org Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 A. Definition Emulgels are semisolid emulsions that contain both emulsion and gel components. They exhibit characteristics of both emulsions and gels, combining the benefits of these delivery systems [3]. Emulgels are hydrophilic lipid structures that provide sustained and controlled release of incorporated active pharmaceutical ingredients. INTRODUCTION Emulgels are novel colloidal delivery systems that combine the properties of emulsions and gels. They exhibit both fluid and semi- solid properties, providing interesting possibilities for drug delivery and other applications. Emulgels can encapsulate both hydrophilic and lipophilic actives and provide controlled release [1]. They also demonstrate enhanced stability compared to emulsions alone. Emulgels represent an exciting new class of delivery vehicles, and this review explores their potential and future prospects [2]. Despite their promise, emulgels have not been extensively reviewed in the literature. This article aims to provide a comprehensive overview of emulgels, focusing on their formulation, characterization, and applications. 3030 Corresponding Author: Prakash Nathaniel Kumar Sarella The Expanding Scope of Emulgels: Formulation, Evaluation and Medical Uses Prakash Nathaniel Kumar Sarella1, Lakshmi Rama Krishna Pravallika Godavari2 1,2 Department of Pharmaceutics, Aditya College of Pharmacy, Surampalem, Andhra Pradesh-533437, India. Prakash Nathaniel Kumar Sarella1, Lakshmi Rama Krishna Pravallika Godavari2 1,2 Department of Pharmaceutics, Aditya College of Pharmacy, Surampalem, Andhra Pradesh-533437, Ind ABSTRACT: Emulgels are semi-solid emulsions that combine the benefits of both emulsions and gels. They provide enhanced stability, sustained release and improved cosmetic properties. Emulgels can be fabricated using a variety of emulsification techniques and gelling agents like carbopol, hydroxypropyl cellulose. Characterization of emulgels includes evaluation of particle size, viscosity, pH, spreadability and drug release. Emulgels have promising applications in topical delivery of drugs and cosmetics, parenteral delivery of drugs and as emulsion-based oral drug delivery systems. Topical emulgels are used in skin care, hair care and cosmetics to provide moisturization, hydration, etc. Parenteral emulgels can deliver drugs in a sustained manner. Oral emulgels improve the absorption of some drugs. Several advantages of emulgels include sustained and controlled release of actives, improved solubility of both hydrophilic and lipophilic drugs, protection from degradation, and enhanced cosmetic elegance. However, emulgel formulation requires specialized emulsification equipment, and they have lower drug loading capacity compared to other semi-solid systems. Emulgels combine the benefits of emulsions and gels, providing a versatile drug and cosmetic delivery platform with unique advantages. Improvements in emulsification techniques, identifying newer gelling agents and permeation enhancers can further enhance the potential of emulgel systems. With growing research on emulgels, these systems are poised to make a significant impact on topical, parenteral and oral delivery in the coming years. Emulgels thus present an exciting prospect for developing innovative and improved formulations. KEYWORDS: Cosmeceuticals, Emulgels, Emulsifying agents, Microemulsions, Rheology, Topical drug delivery B. Characteristics Emugels are unique in sense that they combine the properties of emulsions and gels. Fine tuning their internal and external phases make them more adaptable for loading any kind of drug. The key characteristics and techniques used for the preparation of emulgels are shown in Figure 1. 3030 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 3030 Corresponding Author: Prakash Nathaniel Kumar Sarella International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org Figure 1. Characteristics and preparation techniques for emulgels y V6-i5-42, Impact Factor: 6.789 www.ijcs Figure 1. Characteristics and preparation techniques for emulgels Figure 1. Characteristics and preparation techniques for emulgels Figure 1. Characteristics and preparation techniques for emulgels Some key characteristics [5] of emulgels include: Some key characteristics [5] of emulgels include: Hydrophilic-lipophilic balance (HLB): The HLB of emulsifiers used determines the type of emulsion formed (oil-in water or water-in-oil). Viscosity: Imparts emulgel-like consistency and prevents separation of phases. This is provided by thickeners in the aqueous phase. p pH: The pH of emulgels depends on the compounds used and impacts skin irritation, drug stability and permeation. Most emulgels have a pH in the range of 5 to 8. pH: The pH of emulgels depends on the compounds used and impacts skin irritation, drug stability and permeation. Most emulgels have a pH in the range of 5 to 8. p y g p y y pp A. Components The key components [4] used in emulgels are listed in Table 1. Table 1. Key components of emulgels Sl. No. Components Examples 01 Oil phase Lipophilic solvents Ex. mineral oil, olive oil Lipids Ex. waxes, triglycerides Oils Ex. castor oil, coconut oil 02 Aqueous phase Hydrophilic solvents Ex. water, glycols, Humectants Ex. glycerin, propylene glycol Polymers Ex. carbomer, hydroxypropyl cellulose 03 Emulsifiers Non-ionic Ex. cetostearyl alcohol, cetearyl macromonium chloride, Anionic Ex. sodium lauryl sulfate, Cationic Ex. cetrimonium bromide 04 Surfactants Fluorosurfactants, Polysorbates, Span 80, Tween 80 05 Gelling agents Natural Ex. gelatin, starch, carbomer Synthetic Ex. carbomer, acrylates/C10-30 alkyl acrylate crosspolymer 06 Penetration enhancers Ex. urea, propylene glycol 07 Preservatives Parabens, Formaldehyde, Phenoxyethanol 08 pH modifiers Citric acid, sodium hydroxide FORMULATION OF EMULGELS Emulgels contain several excipients that aid in their formulation and improve their properties. The key excipients [6] used in emulgels include: Emulsifiers: Help in emulsifying the immiscible oil and water phases. Common emulsifiers include sorbitan esters (Span), glyceryl esters (Glyceryl Monooleate), cetostearyl alcohol, etc. The HLB value determines whether an emulsifier will produce O/W or W/O emulsion. Co-emulsifiers: Used along with emulsifiers to improve emulsion formation and stability. Examples include polyethylene glycol, propylene glycol, ethanol, etc. They help in solubilizing ingredients and decreasing interfacial tension. Thickeners: Provide viscosity and gel-like consistency to emulgels. Common thickeners are carbomer, hydroxypropyl cellulose, xyanthan gum, etc. The thickener type and concentration determines whether a fluid, soft solid or firm solid emulgel is obtained. Polymers: Synthetic ( carbomer, acrylates) or natural (xanthan gum, guar gum) polymers are used as thickeners, emulsifiers and to improve release control in emulgels. They absorb water and form hydrogels. Thickeners: Provide viscosity and gel-like consistency to emulgels. Common thickeners are carbomer, hydroxypropyl cellulose, xyanthan gum, etc. The thickener type and concentration determines whether a fluid, soft solid or firm solid emulgel is obtained. Polymers: Synthetic ( carbomer, acrylates) or natural (xanthan gum, guar gum) polymers are used as thickeners, emulsifiers and to improve release control in emulgels. They absorb water and form hydrogels. Surfactants: Used in addition to emulsifiers to improve wetting, solubilization and stability. Examples include cetyl alcohol, cetostearyl alcohol, etc. Anionic, cationic or nonionic surfactants are used depending on the emulsion type. Surfactants: Used in addition to emulsifiers to improve wetting, solubilization and stability. Examples include cetyl alcohol, cetostearyl alcohol, etc. Anionic, cationic or nonionic surfactants are used depending on the emulsion type. Solvents: Used to dissolve oils, increase solubility and modify release. Common solvents for emulgels include water, alcohol, propylene glycol, glycerin, etc. They modify properties like spreadability, drying time, etc. Solvents: Used to dissolve oils, increase solubility and modify release. Common solvents for emulgels include water, alcohol, propylene glycol, glycerin, etc. They modify properties like spreadability, drying time, etc. Penetration Enhancers: Substances that increase the permeability of skin or other tissues. Examples for emulgels include ethanol, propylene glycol, oleic acid, etc. They improve the release of incorporated actives. Penetration Enhancers: Substances that increase the permeability of skin or other tissues. Examples for emulgels include ethanol, propylene glycol, oleic acid, etc. They improve the release of incorporated actives. Preservatives: Prevent the growth of microbes in emulgel formulations. International Journal of Current Science Research and Review Volume 06 Issue 05 May 2023 FORMULATION OF EMULGELS Common preservatives are methyl paraben, propyl paraben, benzyl alcohol, etc. They maintain sterility, stability and prevent irritation when applied to skin or mucous membranes. pH Modifiers: Acids (citric acid or phytic acid) or bases (triethanolamine) are added to adjust the pH of emulgels. The pH impacts properties like viscosity, emulsification, stability, Skin irritation etc. Most emulgels have a pH in the range 5 to 8. PREPARATION OF EMULGELS B. Preparation techniques There are several methods for preparing emulgel formulations. The method selected depends on the type of emulgel, ingredients used and objectives [7]. Some of the main techniques and their characteristic features are discussed below: Low energy emulsification:  Also known as solubilization- dispersion technique.  Requires weak emulsifiers that can solubilize ingredients with mild agitation.  Commonly used for microemulsions.  Provides ultrafine droplets. A. Components 3031 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 ch and Review ww ve their properties. The key excip ommon emulsifiers include sorbit determines whether an emulsifier w and stability. Examples include pol sing interfacial tension. n thickeners are carbomer, hydroxy fluid, soft solid or firm solid emul m) polymers are used as thickener International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org CHARACTERIZATION OF EMULGELS Several parameters [10,11] are evaluated to characterize emulgel formulations are discussed below. A. Droplet Size Analysis: The droplet size of dispersed phase in emulgels is determined by photon correlation spectroscopy, laser diffraction, or scanning electron microscopy. Smaller droplet sizes provide larger surface area and better release. Droplet size ranges from 10-1000 nm for microemulsions to 0.1-100 μm for macroemulsions. A. Droplet Size Analysis: The droplet size of dispersed phase in emulgels is determined by photon correlation spectroscopy, laser diffraction, or scanning electron microscopy. Smaller droplet sizes provide larger surface area and better release. Droplet size ranges from 10-1000 nm for microemulsions to 0.1-100 μm for macroemulsions. B. Viscosity: The viscosity of emulgels is measured using viscometers like Brookfield viscometer, cone and plate viscometer, etc. High viscosity provides emulgel-like consistency while low viscosity yields liquid-like formulations. Viscosity depends on the type and concentration of thickener used. It ranges from 100-1000000 cP for emulgels [12]. B. Viscosity: The viscosity of emulgels is measured using viscometers like Brookfield viscometer, cone and plate viscometer, etc. High viscosity provides emulgel-like consistency while low viscosity yields liquid-like formulations. Viscosity depends on the type and concentration of thickener used. It ranges from 100-1000000 cP for emulgels [12]. C. pH: The pH of emulgels influences properties like stability, irritancy, release and permeation. It is measured using a pH meter or pH strips. Most emulgels have a pH in the range of 5 to 8. Acids or bases can be used to adjust the pH [13]. C. pH: The pH of emulgels influences properties like stability, irritancy, release and permeation. It is measured using a pH meter or pH strips. Most emulgels have a pH in the range of 5 to 8. Acids or bases can be used to adjust the pH [13]. D. Spreadability: Spreadability is assessed by determining the time taken for emulgel to spread over a fixed area on a non- adhesive substrate. It depends on the viscosity, oil content and emulsifier type. Emulgels with good spreadability have a prolonged and uniform spread with less mess [14]. D. Spreadability: Spreadability is assessed by determining the time taken for emulgel to spread over a fixed area on a non- adhesive substrate. It depends on the viscosity, oil content and emulsifier type. Emulgels with good spreadability have a prolonged and uniform spread with less mess [14]. E. C. Factors affecting emulgel formulation and stability Various factors [8,9] affecting the formation and stability of emulgels are: Various factors [8,9] affecting the formation and stability of emulgels are: HLB Value: Appropriate HLB of emulsifiers is critical for emulsion formation and stability. HLB 12-18 produces O/W emulsions while HLB 4-6 gives W/O emulsions. HLB Value: Appropriate HLB of emulsifiers is critical for emulsion formation and stability. HLB 12-18 produces O/W emulsions while HLB 4-6 gives W/O emulsions. y p y Emulsifier Concentration: Optimal amount needed to coat the droplets and prevent coalescence. Too high or too less can destabilize the emulsion. Emulsifier Concentration: Optimal amount needed to coat the droplets and prevent coalescence. Too high or too less can destabilize the emulsion. Temperature: Low temperatures favor W/O emulsions while O/W emulsions are favored at higher temperatures. Temperature affects solubility, viscosity and kinetic energy of molecules. Temperature: Low temperatures favor W/O emulsions while O/W emulsions are favored at higher temperatures. Temperature affects solubility, viscosity and kinetic energy of molecules. Agitation: sufficient agitation is required to overcome the interfacial tension between oil and water phases. High agitation produces smaller droplets. Additives: Surfactants, co-emulsifiers, electrolytes, gels and polymers can improve emulsion stability by increasing charge, decreasing surface curvature, and providing viscosity. International Journal of Current Science Research and Review  Simple but time consuming process. 3033 Corresponding Author: Prakash Nathaniel Kumar Sarella High Pressure Homogenization: High Pressure Homogenization:  Uses high pressure valve homogenizers or microfluidizers to subject the formulation to intense shearing forces. Produces very fine droplets, around 0.2 μm.  Uses high pressure valve homogenizers or microfluidizers to subject the formulation to intense shearing forces. Produces very fine droplets, around 0.2 μm.  Requires use of high HLB emulsifiers.  Limited by energy input and heat generation. Microemulsion technique:  Limited by energy input and heat generation.  Requires appropriate HLB of emulsifiers and oils.  Formulation of oil-in-water (O/W) or water-in-oil (W/O) microemulsions that are thermodynamically stable, iso-tropic and have a droplet size below 100 nm.  Formulation of oil-in-water (O/W) or water-in-oil (W/O) microemulsions that are thermodynamically stable, iso-tropic and have a droplet size below 100 nm.  Provides high stability and encapsulation.  Limited by types of emulsifiers and APIs that can be incorporated. ot and Cold method:  Limited by types of emulsifiers and APIs that can be incorporated. Hot and Cold method:  Heating the oil phase separately and then cooling and mixing with the aqueous phase under high speed stirring.  Heating the oil phase separately and then cooling and mixing with the aqueous phase under high speed stirring.  Followed by increasing homogenization at low temperature.  Produces medium sized droplets. Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 3032 Corresponding Author: Prakash Nathaniel Kumar Sarella International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org CHARACTERIZATION OF EMULGELS Drug Release: The release of incorporated actives from emulgels is evaluated using Franz diffusion cells, dialysis membrane method, etc. Factors affecting release include droplet size, viscosity, pH, solubility, etc. Sustained and controlled release over prolonged periods is desirable for most emulgel applications [15]. Release kinetics can be zero-order, first-order, Higuchi, Korsmeyer-Peppas, etc. Release compounds with different solubility, molecular weight and dose are commonly used. Release assessment of emulgels provides valuable insights into optimizing formulation and application. The details of complete evaluation tests, their significance and techniques used for characterization of emulgels are listed out in Table 2. The details of complete evaluation tests, their significance and techniques used for characterization of emulgels are listed out in Table 2. 3033 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 202 Available at: www.ijcsrr.org Page No. 3030-3041 Table 2. Significance and methods of different emulgel characterization parameters Sl. No. Parameter Significance Technique 01 Particle size Indicates stability and dispersion. Smaller sizes have higher stability Measured using photon correlation spectroscopy, laser diffraction, scanning electron microscopy. 02 Viscosity Relates to spreadability and gel strength. Higher viscosity signifies stronger gel with lower spreadability Evaluated using rotational viscometers, Brookfield viscometer Table 2. Significance and methods of different emulgel characterization parameters Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr. 03 pH Important for stability, drug compatibility and skin irritation. Range of 4-8 is usually acceptable for topical use. Determined using pH meters, pH strips or indicators 04 Spreadability Indicates flow behavior and patients' acceptance. Emulgels should spread easily and retain form Assessed using slips and slides method or cone penetration method. 05 Drug release Reflects sustained release ability and effectiveness. Slower release over longer period is desirable Studied using diffusion cells, dialysis bags, Franz diffusion cells. 06 Spreading coefficient Provides insight into spreadability and adhesion to skin. Higher positive values indicate better spreadability and adhesion. Calculated using interfacial tension measurements 07 Zeta potential Gives information about surface charge and stability. Values around ±30 mV denote acceptable stability. Measured using laser doppler electrophoresis, electrophoretic light scattering. 08 Rheological behavior Provides viscosity profiles under steady and dynamic conditions. Pseudoplastic, thixotropic or viscoelastic behavior is expected. CHARACTERIZATION OF EMULGELS Evaluated using concentric cylinders, plate-plate or tube viscometers under increasing shear rates. 09 Drug permeation Determines penetration of actives into skin and effectiveness. Enhanced permeation leads to improved activity. Assessed using ex-vivo or in-vivo experiments on skin or using synthetic membrane. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr International Journal of Current Science Research and Review ISSN 2581 8341 www.ijcsrr.org Measured using laser doppler electrophoresis, electrophoretic light scattering. Measured using laser doppler electrophoresis, electrophoretic light scattering. B. Parenteral applicaitons Emulgels provide sustained release of drugs through injection. They release drugs over prolonged periods, reducing dosing frequency and maintaining adequate drug levels. Water soluble corticosteroids and antibiotics are commonly incorporated [18]. 3034 Corresponding Author: Prakash Nathaniel Kumar Sarella A. Topical Applications A. Topical Applications p pp Skin Care: Emulgels are used as moisturizers, emollients and for treating conditions like roughness, dullness, irritation, etc. Ingredients like glycerin, cetyl alcohol, chamomile extract, aloe vera, etc. are incorporated [16]. Hair Care: Emulgels containing proteins, oils, keratin, etc. are used as conditioners, styling agents, treatment for dandruff, etc. Coconut oil and glycerin are commonly used [17]. Hair Care: Emulgels containing proteins, oils, keratin, etc. are used as conditioners, styling agents, treatment for dandruff, etc. Coconut oil and glycerin are commonly used [17]. Cosmetics: Emulgels are used as foundations, lip balms, sunscreens, etc. Ingredientsinclude pigments, waxes, silicones, emulsifiers, etc. for desired effects. Sun protection factor depends on the emulgent and sunscreens used [16]. Cosmetics: Emulgels are used as foundations, lip balms, sunscreens, etc. Ingredientsinclude pigments, waxes, silicones, emulsifiers, etc. for desired effects. Sun protection factor depends on the emulgent and sunscreens used [16]. C. Oral Applications The applications of emulgels in parenteral controlled drug delivery are listed out in Table 4 while their applications in oral drug delivery is shown in Table 5 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 Benefits of oral emulgel systems include improved solubility, absorption, sustained release, reduced dosing frequency and improved patient compliance. However, formulating challenges exist for emulsion-based systems. More research is needed to realize their full potential in drug delivery. Various applications of emulgels in skin care, hair care, and other cosmetic products are listed out in Table 3. The applications of emulgels in parenteral controlled drug delivery are listed out in Table 4 while their applications in oral drug delivery is shown in Table 5 Benefits of oral emulgel systems include improved solubility, absorption, sustained release, reduced dosing frequency and improved patient compliance. However, formulating challenges exist for emulsion-based systems. More research is needed to realize their full potential in drug delivery. Various applications of emulgels in skin care, hair care, and other cosmetic products are listed out in Table 3. The applications of emulgels in parenteral controlled drug delivery are listed out in Table 4 while their applications in oral drug delivery is shown in Table 5 Table 3. Applications of topical emulgels (skin care, hair care, cosmetics) Skin care Hair care Cosmetics Moisturizers: Hydration, soothing and softening dry skin. Emulgels contain humectants, emollients and hydrating polymers. Emollients: Reducing roughness, softening skin and relief from scaling/itching. Oil and wax emulgels are effective emollients. Protectants: Forming barrier against environmental aggressors like sun, wind, pollution etc. Emulgels contain UV absorbers, antioxidant emulgels. Calming agents: Reducing inflammation, soothing irritation and relieving pain. Emulgels contain corticosteroids, lidocaine, aloe vera etc. Scrubs: Gentle exfoliation and purification of skin. Emulgels contain abrasives (ground nut shells, oatmeal), surfactants and detergents in proper proportions. Conditioners: Improving manageability, reducing static and detangling hair. Emulgels contain hydrating polymers, silicones, dimethicone etc. Styling agents: Providing hold, shape and volume without stiffening hair. Emulgels contain polymers, wax emulsions, plant extracts (carnauba wax). Treatments: Penetrating deeply to strengthen hair, stimulate hair follicles and promote growth. Emulgels contain vitamins A, E, amino acids, caffeine, peptides etc. Foundations: Covering skin imperfections and providing even tone and texture. C. Oral Applications Emulgels provide oil control, non-greasy feel with emollients, silicones, dimethicone. Primers: Filling fine lines and pores, providing smooth base for makeup application. Emulgels contain silicones, dimethicone, cyclomethicone for mattifying and smoothing effect. Highlighters: Enhancing natural glow and highlighting features. Emulgels contain micronized pigments, pearlescent agents, diethylhexyl 2,6-naphthalate etc for highlighting effect. Lip products: Plumping lips, protecting and hydrating. Emulgel lipsticks and balms contain emollients, humectants, wax emulsions, botanical extracts etc. Nail paints: Coloring nails with chip-resistant and long-lasting polish. Emulgel nail polishes provide smooth and even application with quick drying. Table 3. Applications of topical emulgels (skin care, hair care, cosmetics) Foundations: Covering skin imperfections and providing even tone and texture. Emulgels provide oil control, non-greasy feel with emollients, silicones, dimethicone. Primers: Filling fine lines and pores, providing smooth base for makeup application. Emulgels contain silicones, dimethicone, cyclomethicone for mattifying and smoothing effect. Protectants: Forming barrier against environmental aggressors like sun, wind, pollution etc. Emulgels contain UV absorbers, antioxidant emulgels. Treatments: Penetrating deeply to strengthen hair, stimulate hair follicles and promote growth. Emulgels contain vitamins A, E, amino acids, caffeine, peptides etc. Highlighters: Enhancing natural glow and highlighting features. Emulgels contain micronized pigments, pearlescent agents, diethylhexyl 2,6-naphthalate etc for highlighting effect. Highlighters: Enhancing natural glow and highlighting features. Emulgels contain micronized pigments, pearlescent agents, diethylhexyl 2,6-naphthalate etc for highlighting effect. Calming agents: Reducing inflammation, soothing irritation and relieving pain. Emulgels contain corticosteroids, lidocaine, aloe vera etc. Lip products: Plumping lips, protecting and hydrating. Emulgel lipsticks and balms contain emollients, humectants, wax emulsions, botanical extracts etc. Nail paints: Coloring nails with chip-resistant and long-lasting polish. Emulgel nail polishes provide smooth and even application with quick drying. Lip products: Plumping lips, protecting and hydrating. Emulgel lipsticks and balms contain emollients, humectants, wax emulsions, botanical extracts etc. Scrubs: Gentle exfoliation and purification of skin. Emulgels contain abrasives (ground nut shells, oatmeal), surfactants and detergents in proper proportions. 3035 C di A h P k h N h i l K S ll V l 06 I 05 M 2 Table 4. Applications of parenteral emulgels (controlled drug delivery) Sl.No. Parenteral emulgels Applications 01 Sustained release injectables Providing prolonged therapeutic effect with single administration. Emulgels contain water insoluble drugs, polymers for sustained release. Examples include norethindrone emulsions, testosterone emulsions etc. 02 Prolonged anesthetics Emulgels containing local anesthetics like lidocaine provide pain relief for extended duration with single injection. C. Oral Applications Emulsion-based oral drug delivery systems include: Emulsion-based oral drug delivery systems include: Emulgels: Emulgels contain both emulsions and gels for controlled release of drugs. Oil-in-water or water-in-oil emulsions are used based on solubility of drug [12]. Emulgels: Emulgels contain both emulsions and gels for controlled release of drugs. Oil-in-water or water-in-oil emulsions are used based on solubility of drug [12]. Liquid filled gelatin capsules (LFGCs): Emulsions contained within gelatin capsules. LFGCs provide floating, sink-ing or remained buoyant to release drug at specific sites. Used for site-specific release [10]. Liquid filled gelatin capsules (LFGCs): Emulsions contained within gelatin capsules. LFGCs provide floating, sink-ing or remained buoyant to release drug at specific sites. Used for site-specific release [10]. Self-emulsifying drug delivery systems (SEDDS): Contain emulsifiers and solvents to produce fine O/W or W/O emulsions upon dispersion in aqueous media with low energy. Improve solubility, absorption and bioavailability of drugs [19]. Self-emulsifying drug delivery systems (SEDDS): Contain emulsifiers and solvents to produce fine O/W or W/O emulsions upon dispersion in aqueous media with low energy. Improve solubility, absorption and bioavailability of drugs [19]. Microemulsions: Thermodynamically stable, isotropic and have a droplet size below 100 nm. Microemulsions pro-vide maximum surface area for absorption and enhance solubility. They are suitable for lipophilic, amphiphilic and hydrophilic drugs [20]. Microemulsions: Thermodynamically stable, isotropic and have a droplet size below 100 nm. Microemulsions pro-vide maximum surface area for absorption and enhance solubility. They are suitable for lipophilic, amphiphilic and hydrophilic drugs [20]. 3034 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org Benefits of oral emulgel systems include improved solubility, absorption, sustained release, reduced dosing frequency and improved patient compliance. However, formulating challenges exist for emulsion-based systems. More research is needed to realize their full potential in drug delivery. Various applications of emulgels in skin care, hair care, and other cosmetic products are listed out in Table 3. 3035 Corresponding Author: Prakash Nathaniel Kumar Sarella International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcs ew www.ijcsrr.org 04 Emulgels containing Neurological drugs Drugs for disorders like Parkinson's disease (levodopa), epilepsy (valproic acid) have been developed as emulgel injectables for controlled release 05 Peptide/protein delivery Emulgels provide stable environment and sustained release of therapeutic peptides, hormones, enzymes etc. preventing enzymatic degradation. Examples include insulin emulsions, growth hormone emulsions etc. 06 Vitamin emulgels Parenteral emulsions of fat soluble vitamins A, D, E and K have been developed to provide adequate dosing of these vitamins which would otherwise be difficult to deliver through other routes. 07 Emulgels containing antibiotics Antibiotics, antifungal and antiviral drugs have been formulated as emulgel injectables for site-specific delivery, sustained release and reduced toxicity. Examples include fluconazole emulsions, acyclovir emulsions etc. 08 Cosmeceuticals Injectable emulgels containing hyaluronic acid, retinol, vitamin C, peptides etc. are used for anti-aging, skin rejuvenation and wrinkle reduction with prolonged and anti-aging effects. ing Drugs for disorders like Parkinson's disease (levodopa), epilepsy (valproic acid) have been developed as emulgel injectables for controlled release Emulgels provide stable environment and sustained release of therapeutic peptides, hormones, enzymes etc. preventing enzymatic degradation. Examples include insulin emulsions, growth hormone emulsions etc. Table 5. Applications of oral emulgels Sl. No. Category Applications 01 Lipid insoluble drugs Emulgels improve absorption of drugs having low lipid solubility and high hydrophobicity which cannot be absorbed easily through GIT. Examples include carbamazepine, nimodipine, coenzyme Q10 etc. 02 Peptides and proteins Emulgels protect peptides and proteins from enzymatic degradation and improve their absorption in intact form. Examples include insulin, calcitonin, enzyme replacements etc. 03 Photosensitive drugs Light sensitive drugs get protection from degradation in emulgels. Examples include carotenoids, chlorophyll, flavonoids etc. 04 Poor membrane permeability Emulgels aid in absorption of drugs that cannot permeate through intestinal membranes easily. Examples include poorly absorbed NSAIDs, antihistamines etc. 05 Improved oral bioavailability Emulgels enhance absorption of many drugs leading to better therapeutic effect with same dose. This results in decreased dosage and side effects. Examples of drugs with improved bioavailability through emulgels include paclitaxel, atorvastatin, fenofibrate etc. 06 Treatment of gallstones Oral emulgels containing bile salts help in dissolving gallstones slowly and preventing their recurrence. They maintain supersaturation of bile salts for prolonged period 07 Treatment of infections Antibacterial, antifungal and antiviral drugs have been formulated in emulgels to improve absorption, reduce toxicity and achieve better patient compliance through decreased dosage frequency. Examples include fluconazole, terbinafine, acyclovir etc. C. Oral Applications 03 Anticancer emulges Anti-cancer drugs like vincristine, methotrexate, hydroxycamptothecin etc. have been formulated in emulgel injectables for sustained, targeted and improved effectiveness Table 4. Applications of parenteral emulgels (controlled drug delivery) Sl.No. Parenteral emulgels Applications Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.o 04 Emulgels containing Neurological drugs Drugs for disorders like Parkinson's disease (levodopa), epilepsy (valproic acid) have been developed as emulgel injectables for controlled release 05 Peptide/protein delivery Emulgels provide stable environment and sustained release of therapeutic peptides, hormones, enzymes etc. preventing enzymatic degradation. Examples include insulin emulsions, growth hormone emulsions etc. 06 Vitamin emulgels Parenteral emulsions of fat soluble vitamins A, D, E and K have been developed to provide adequate dosing of these vitamins which would otherwise be difficult to deliver through other routes. 07 Emulgels containing antibiotics Antibiotics, antifungal and antiviral drugs have been formulated as emulgel injectables for site-specific delivery, sustained release and reduced toxicity. Examples include fluconazole emulsions, acyclovir emulsions etc. 08 Cosmeceuticals Injectable emulgels containing hyaluronic acid, retinol, vitamin C, peptides etc. are used for anti-aging, skin rejuvenation and wrinkle reduction with prolonged and anti-aging effects. Table 5. Applications of oral emulgels Sl. No. Category Applications 01 Lipid insoluble drugs Emulgels improve absorption of drugs having low lipid solubility and high hydrophobicity which cannot be absorbed easily through GIT. Examples include carbamazepine, nimodipine, coenzyme Q10 etc. 02 Peptides and proteins Emulgels protect peptides and proteins from enzymatic degradation and improve their absorption in intact form. Examples include insulin, calcitonin, enzyme replacements etc. 03 Photosensitive drugs Light sensitive drugs get protection from degradation in emulgels. Examples include carotenoids, chlorophyll, flavonoids etc. 04 Poor membrane permeability Emulgels aid in absorption of drugs that cannot permeate through intestinal membranes easily. Examples include poorly absorbed NSAIDs, antihistamines etc. 05 Improved oral bioavailability Emulgels enhance absorption of many drugs leading to better therapeutic effect with same dose. This results in decreased dosage and side effects. Examples of drugs with improved bioavailability through emulgels include paclitaxel, atorvastatin, fenofibrate etc. 06 Treatment of gallstones Oral emulgels containing bile salts help in dissolving gallstones slowly and preventing their recurrence. C. Oral Applications They maintain supersaturation of bile salts for prolonged period 07 Treatment of infections Antibacterial, antifungal and antiviral drugs have been formulated in emulgels to improve absorption, reduce toxicity and achieve better patient compliance through decreased dosage frequency. Examples include fluconazole, terbinafine, acyclovir etc. 08 Improved nutrition Fat soluble vitamins A, D, E and K as well as certain minerals have been delivered through emulgels to counter their deficiencies and improve nutritional status. 09 Anti-cancer activity Certain anti-cancer emulgel formulations help in achieving higher drug concentrations at the site of tumor for improved therapeutic effect. Examples include coenzyme Q10, tea polyphenols etc. D. Advantages Emulgel systems offer several advantages over conventional delivery systems [4,10]. However, they also pose some challenges in formulation and application.Some of the advantages of emulgel Systems are discussed below: Sustained and Controlled Release: Emulgel droplets and high viscosity provide sustained release of incorporated actives over prolonged periods. This reduces dosing frequency and maintains therapeutic drug levels. Improved Solubility: Hydrophilic drugs can be solubilized in the aqueous phase while lipophilic drugs dissolve in the oil phase. This enhances the solubility of poorly soluble drugs. Enhanced Stability: Emulgel structure protects light sensitive, oxygen sensitive or microbe sensitive drugs from degradation. The aqueous and oil phases also provide separate environments for compatibility. Improved Cosmetic Elegance: Emulgel semisolid consistency provides a pleasant feel and non-greasy texture suitable for cosmetic and topical applications. Improved Cosmetic Elegance: Emulgel semisolid consistency provides a pleasant feel and non-greasy texture suitable for cosmetic and topical applications. Flexibility: Various grades of viscosity and spreading can be obtained by modifying the thickener concentration and type. This flexibility allows developing emulsions, creams, gels or pastes. The key advantages and challenges of emulgel systems are shown in Table 7 Flexibility: Various grades of viscosity and spreading can be obtained by modifying the thickener concentration and type. This flexibility allows developing emulsions, creams, gels or pastes. The key advantages and challenges of emulgel systems are shown in Table 7 Table 7. Key advantages of emulgel systems Sl.No. Characteristic Advantages 01 Controlled and sustained release Emulgel matrix releases the incorporated drugs in a controlled manner over prolonged period. This results in sustained therapeutic effect, reduced dosing frequency and improved patient compliance. 02 Solubilization of poorly soluble drugs Hydrophilic-lipophilic balance in emulgels helps in solubilizing drugs with poor aqueous solubility. This enhances absorption and bioavailability of such drugs. 03 Protection from degradation Emulgel formulation provides a stable environment protecting labile drugs, peptides and proteins from degradation. This helps improving their effectiveness and stability. 04 Improved cosmetic appeal Emulgel bases provide a smooth, cushioned and emollient feel. This leads to better cosmetic elegance, spreadability and non-greasy feel which enhances patients' acceptance 05 Site-specific delivery Emulgels can be designed to release drugs at specific sites like transdermal, intra-articular, intra-nasal etc. This achieves high local concentrations with minimal systemic absorption and side effects. 06 Improved penetration Some emulgels facilitate penetration of drugs into skin, hair follicles and other layers. This allows enhanced therapeutic effects for conditions affecting these layers. International Journal of Current Science Research and Review Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcs 08 Improved nutrition Fat soluble vitamins A, D, E and K as well as certain minerals have been delivered through emulgels to counter their deficiencies and improve nutritional status. 09 Anti-cancer activity Certain anti-cancer emulgel formulations help in achieving higher drug concentrations at the site of tumor for improved therapeutic effect. Examples include coenzyme Q10, tea polyphenols etc. Table 5. Applications of oral emulgels Certain anti-cancer emulgel formulations help in achieving higher drug concentrations at the site of tumor for improved therapeutic effect. Examples include coenzyme Q10, tea polyphenols etc. 3036 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 3036 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 E. Challenges E. Challenges Challenges [2] involved in the formulation and development of emulgel systems are E. Challenges Challenges [2] involved in the formulation and development of emulgel systems are: nges [2] involved in the formulation and development of emulgel systems are: g Challenges [2] involved in the formulation and development of emulgel systems are: Difficult emulsification: Emulsification requires high-energy inputs and specialized equipment. It is time consuming and can lead to over-processing damaging heat sensitive compounds. Limited drug loading: The space occupied by oil droplets and thickener phases reduces the amount of drug that can be loaded compared to liquid or semisolid systems. Irritation: Some emulsifiers and penetration enhancers used in emulgels can irritate the skin in high concentrations. Proper selection and testing can minimize but not completely eliminate irritation. Phase Separation: In spite of emulsification, limited stability may allow separation of phases over longer storage periods, especially for W/O emulsions. Continuous agitation or using stabilizing agents helps prevent separation. Altered drug release: While sustained release is an advantage, the release profile of drugs can get modified from that of conventional dosage forms. This may not be desirable for all drugs and therapeutic objectives. Altered drug release: While sustained release is an advantage, the release profile of drugs can get modified from that of conventional dosage forms. This may not be desirable for all drugs and therapeutic objectives. The challenges faced in the development of emulgels are shown in Table 8 Table 8. Limitations and challenges with emulgel preparation and use Sl. No. Parameter Limitations/Challenges 01 High equipment cost Specialized equipment like high shear homogenizers, microfluidizers, ultrasonicators etc. are required for preparing emulgel formulations which adds to the total cost. This can limit large scale production of emulgel formulations. 02 Lower drug loading Only a fraction of the total formulation composition can be allocated for the drug due to gel matrix formation and increased aqueous content in emulgels. This results in lower drug loading compared to other semi-solid or liquid formulations. Higher doses may be needed to achieve desired therapeutic effect. 03 Stability issues The emulsified system and gel matrix can make emulgels more prone to instability issues like creaming, sedimentation, phase separation, flocculation etc. Proper selection and optimization of emulsifiers and gelling agents are required to overcome stability challenges which can be difficult to achieve. 04 Difficulty in sterilization The susceptibility of emulgel components like emulsifiers, gelling agents, drugs etc. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 D. Advantages 07 Anti-irritation Emulgel formulations help in masking bitter taste, irritation and unpleasant smell of certain drugs. They provide soothing and cooling sensation. 08 Flexible dosing Emulgels can be designed as solid, semi-solid or liquid formulations based on required consistency and dose. This permits flexible choices of dosage forms for various applications. 09 Combination of drugs Emulgel matrix allows incorporation of drugs with synergistic effects or different mechanisms of action. This provides combined therapeutic benefits in single formulation. 10 Improved patient compliance Advantages of emulgels like sustained release, reduced dosing frequency, improved taste masking, flexible dosing etc. enhance patients' acceptance and compliance. This leads to better therapy outcomes. Table 7. Key advantages of emulgel systems Emulgel formulations help in masking bitter taste, irritation and unpleasant smell of certain drugs. They provide soothing and cooling sensation. Emulgels can be designed as solid, semi-solid or liquid formulations based on required consistency and dose. This permits flexible choices of dosage forms for various applications. Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org E. Challenges to high temperatures or radiation makes sterilization a challenging task. Sterilization is critical for parenteral applications but can deteriorate product quality. Alternative sterilization techniques may need to be explored. 05 Skin irritation Although emulgels minimize irritation in comparison to liquid systems, they may still cause irritation or sensitization to certain components like surfactants, preservatives etc. especially with prolonged and repeated use. Irritation liability needs to be properly assessed during formulation and evaluation. 06 Drug incompatibility Although emulgel matrix provides some protection, it may not prevent all types of interactions between co-administered drugs or drugs and other emulgel components. Drug compatibility needs to be confirmed to avoid any physicochemical or biological incompatibility. 07 Long term effects The effects of prolonged use or exposure to certain emulgel components are not fully known, especially for components used at high concentrations. This can be an important concern, especially for formulations applied topically or injected parenterally. Long term safety needs to be ensured before marketing. Table 8. Limitations and challenges with emulgel preparation and use 3038 Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 3038 Corresponding Author: Prakash Nathaniel Kumar Sarella International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org 3039 Corresponding Author: Prakash Nathaniel Kumar Sarella 3040 Corresponding Author: Prakash Nathaniel Kumar Sarella CONCLUSION Emulgels are innovative semi-solid delivery systems that combine the benefits of emulsions and gels. They offer sus-tained release, improved stability, enhanced cosmetic properties, and versatile applications in drug and cosmetic deli-very. Emulgels can be tailored for optimal performance using different techniques and agents, but require specialized equipment and have lower drug loading. Continued research can help realize their true potential, including combining with nanotechnology, microemulsions, and hydrogels. Emulgels are poised to transform topical, parenteral, and oral delivery in the coming years, representing an exciting prospect for the healthcare and cosmetics industries. In sum-mary, emulgels are a remarkable delivery system that deserves further exploration for optimized therapeutic and cos-metic solutions. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org This enhances safety, patient compliance and acceptability 07 Flexible and multifunctional formulations Emerging areas like Janus particles, functional nanoparticles, temperature/pH responsive systems etc. can enable emulgels for flexible and combined functionalities like controlled release as well as targeting, imaging, therapy, testing etc. These "intelligent" formulations can revolutionize applications of emulgels. e for Exploring longer acting gelling agents, wax emulsions and polymers can result in emulgels providing sustained release over weeks or months from a single administration. This can improve management of chronic conditions. ons Emulgels can potentially be explored for applications beyond current topical, parenteral and oral use like buccal, nasal, rectal, intra-articular delivery etc. They can also be investigated for applications like tissue engineering, gene delivery etc. There is immense scope for improving emulgel formulations by developing better emulsification techniques, identify-ing newer gelling agents, permeation enhancers and solubilizers. Combining emulgels with nanotechnology, micro-emulsions and hydrogels can generate synergistic formulations with enhanced effectiveness [17]. With growing research on preparation, characterization, applications and optimization of emulgel systems, these plat-forms are poised to make significant improvements in topical, parenteral and oral delivery of drugs and cosmetics. Emulgels thus present promising opportunities for developing innovative formulations with improved therapeutic and cosmetic benefits. ACKNOWLEDGEMENT The authors would like to thank Dr. K. Ravishankar, Aditya College of Pharmacy, Surampalem for supporting this work. The authors would like to thank Dr. K. Ravishankar, Aditya College of Pharmacy, Surampalem for sup International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org International Journal of Current Science Research and Review International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.or 03 Sustained release for longer periods Exploring longer acting gelling agents, wax emulsions and polymers can result in emulgels providing sustained release over weeks or months from a single administration. This can improve management of chronic conditions. 04 Wider applications Emulgels can potentially be explored for applications beyond current topical, parenteral and oral use like buccal, nasal, rectal, intra-articular delivery etc. They can also be investigated for applications like tissue engineering, gene delivery etc. 05 Improved stability Use of newer surfactants, polymers, ions etc. with improved emulsifying and gelling abilities can lead to emulgels with higher stability against creaming, sedimentation,phase separation etc. This can reduce wasted material and improve ease of use. 06 Reduced irritation Exploring surfactants, polymers and other emulgel components with non-irritant and hypoallergenic properties can minimize potential for skin irritation, sensitization and other allergic reactions especially for topical use. This enhances safety, patient compliance and acceptability 07 Flexible and multifunctional formulations Emerging areas like Janus particles, functional nanoparticles, temperature/pH responsive systems etc. can enable emulgels for flexible and combined functionalities like controlled release as well as targeting, imaging, therapy, testing etc. These "intelligent" formulations can revolutionize applications of emulgels. www.ijcsrr.org 03 Sustained release for longer periods Exploring longer acting gelling agents, wax emulsions and polymers can result in emulgels providing sustained release over weeks or months from a single administration. This can improve management of chronic conditions. 04 Wider applications Emulgels can potentially be explored for applications beyond current topical, parenteral and oral use like buccal, nasal, rectal, intra-articular delivery etc. They can also be investigated for applications like tissue engineering, gene delivery etc. 05 Improved stability Use of newer surfactants, polymers, ions etc. with improved emulsifying and gelling abilities can lead to emulgels with higher stability against creaming, sedimentation,phase separation etc. This can reduce wasted material and improve ease of use. 06 Reduced irritation Exploring surfactants, polymers and other emulgel components with non-irritant and hypoallergenic properties can minimize potential for skin irritation, sensitization and other allergic reactions especially for topical use. FUTURE PERSPECTIVES Emulgels are promising semi-solid systems that combine the advantages of emulsions and gels. They offer enhanced stability, sustained release, improved cosmetic properties and versatile applications in topical, parenteral and oral drug delivery [21]. The promising areas of research and development for emulgels are illustrated in Figure 2 Figure 2. Promising areas of research and development for emulgels Figure 2. Promising areas of research and development for emulgels Emulgels can be formulated using different emulsification techniques like high shear homogenization, ultrasonication, microemulsification etc. along with various gelling agents such as carbopol, hydroxypropyl cellulose, gelatin etc. Proper selection of emulsifiers, surfactants and gelling polymers can help achieve the desired physicochemical proper-ties and release kinetics for different applications [22]. Besides characterization of particle size, viscosity, pH, spreadability and drug release, evaluating factors like drug loading, drug permeation and skin hydration can provide useful insights into the performance of emulgel formulations. Topical emulgels are promising for skin care, hair care and cosmetics. Parenteral emulgels can deliver drugs in a controlled manner for systemic effects. Oral emulgels can improve absorption of both hydrophilic and lipophilic drugs [23]. Key advantages of emulgels include sustained release, solubilization of both hydrophilic and lipophilic drugs, protection from degradation, improved cosmetic appeal and flexible dosing. However, emulgel preparation requires specialized equipment and they have lower drug loading compared to other semisolid systems. The potential benefits for exploring future perspectives on emulgels are listed out in Table 9. Table 9. Potential benefits of exploring future perspectives on emulgels Sl. No. Potential benefits Future perspectives 01 Enhanced effectiveness Exploring areas like newer gelling agents, emulsifiers, penetration enhancers etc. can lead to formulations with improved drug absorption, bioavailability and therapeutic effect. Use of nanotechnology and microemulsions can also enhance effectiveness. 02 Optimized and controlled release Combining emulgels with intelligent polymers, stimuli responsive systems and touch activated nanoparticles can enable optimized release in response to external stimuli like pH, temperature, pressure etc. This allows activated release at specific sites or times. Table 9. Potential benefits of exploring future perspectives on emulgels Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 International Journal of Current Science Research and Review 4. D.N. Tanaji, Emulgel: A comprehensive review for topical delivery of hydrophobic drugs, Asian Journal of Pharmaceutics (AJP). 12 (2018). 5. A. Shakeel, U. Farooq, T. Iqbal, S. Yasin, F.R. Lupi, D. Gabriele, Key characteristics and modelling of bigels systems: A review, Materials Science and Engineering: C. 97 (2019) 932–953. https://doi.org/10.1016/j.msec.2018.12.075. 5. A. Shakeel, U. Farooq, T. Iqbal, S. Yasin, F.R. Lupi, D. Gabriele, Key characteristics and modelling of bigels systems: A review, Materials Science and Engineering: C. 97 (2019) 932–953. https://doi.org/10.1016/j.msec.2018.12.075. 5. A. Shakeel, U. Farooq, T. Iqbal, S. Yasin, F.R. Lupi, D. Gabriele, Key characteristics and modelling of bigels systems: A review, Materials Science and Engineering: C. 97 (2019) 932–953. https://doi.org/10.1016/j.msec.2018.12.075. 6. N.Ü. Okur, E.Ö. Bülbül, A.P. Yağcılar, P.I. Siafaka, Current Status of Mucoadhesive Gel Systems for Buccal Drug Delivery, Curr Pharm Des. 27 (2021) 2015–2025. https://doi.org/10.2174/1381612824666210316101528. 6. N.Ü. Okur, E.Ö. Bülbül, A.P. Yağcılar, P.I. Siafaka, Current Status of Mucoadhesive Gel Systems for Buccal Drug Delivery, Curr Pharm Des. 27 (2021) 2015–2025. https://doi.org/10.2174/1381612824666210316101528. 7. M. Talat, M. Zaman, R. Khan, M. Jamshaid, M. Akhtar, A.Z. Mirza, Emulgel: an effective drug delivery system, Drug Dev Ind Pharm. 47 (2021) 1193–1199. https://doi.org/10.1080/03639045.2021.1993889. 7. M. Talat, M. Zaman, R. Khan, M. Jamshaid, M. Akhtar, A.Z. Mirza, Emulgel: an effective drug delivery system, Drug Dev Ind Pharm. 47 (2021) 1193–1199. https://doi.org/10.1080/03639045.2021.1993889. 8. V. Singla, S. Saini, B. Joshi, A.C. Rana, Emulgel: A new platform for topical drug delivery, International Journal of Pharma and Bio Sciences. 3 (2012) 485–498. 8. V. Singla, S. Saini, B. Joshi, A.C. Rana, Emulgel: A new platform for topical drug delivery, International Journal of Pharma and Bio Sciences. 3 (2012) 485–498. 9. F. Vanpariya, M. Shiroya, M. Malaviya, Emulgel: A Review, International Journal of Science and Research (IJSR). 10 (2021) 847. 9. F. Vanpariya, M. Shiroya, M. Malaviya, Emulgel: A Review, International Journal of Science and Research (IJSR). 10 (2021) 847. 10. S.K. Yadav, M.K. Mishra, A. Tiwari, A. Shukla, Emulgel: a new approach for enhanced topical drug delivery, Int J Curr Pharm Res. 9 (2016) 15–19. https://doi.org/10.22159/ijcpr.2017v9i1.16628. 10. S.K. Yadav, M.K. Mishra, A. Tiwari, A. Shukla, Emulgel: a new approach for enhanced topical drug delivery, Int J Curr Pharm Res. 9 (2016) 15–19. https://doi.org/10.22159/ijcpr.2017v9i1.16628. 11. K.M. Haneefa, S. Easo, P.V. Hafsa, G.P. Mohanta, C. Nayar, Emulgel: An advanced review, Journal of Pharmaceutical Sciences and Research. 5 (2013) 254. 11. K.M. Haneefa, S. Easo, P.V. Hafsa, G.P. 3041 Corresponding Author: Prakash Nathaniel Kumar Sarella REFERENCES V. Joshi, K. Kumar, D. Khairya, D. Teotia, Emulgel as a Novel Drug Delivery System: A Compre International Journal of Indigenous Herbs and Drugs. (2022) 94–98. 1. V. Joshi, K. Kumar, D. Khairya, D. Teotia, Emulgel as a Novel Drug Delivery System: A Comprehensive Review, International Journal of Indigenous Herbs and Drugs. (2022) 94–98. 2. M. Kankane, V. Nigam, S. Modi, S. Jain, P. Adhikari, Emulgel: A dual release system for hydrop (2022). 3. A. Alexander, A. Khichariya, S. Gupta, R.J. Patel, T.K. Giri, D.K. Tripathi, Recent expansions in an emergent novel drug delivery technology: Emulgel, Journal of Controlled Release. 171 (2013) 122–132. https://doi.org/10.1016/j.jconrel.2013.06.030. Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 06 Issue 05 May 2023 DOI: 10.47191/ijcsrr/V6-i5-42, Impact Factor: 6.789 IJCSRR @ 2023 www.ijcsrr.org International Journal of Current Science Research and Review Mohanta, C. Nayar, Emulgel: An advanced review, Journal of Pharmaceutical Sciences and Research. 5 (2013) 254. 12. V. Sharma, S.K. Nayak, S.R. Paul, B. Choudhary, S.S. Ray, K. Pal, Emulgels, in: Polymeric Gels, Elsevier, 2018: pp. 251–264. 12. V. Sharma, S.K. Nayak, S.R. Paul, B. Choudhary, S.S. Ray, K. Pal, Emulgels, in: Polymeric Gels, Elsevier, 2018: pp. 251–264. 13. K. Light, S. Karboune, Emulsion, hydrogel and emulgel systems and novel applications in cannabinoid delivery: A review, Critical Reviews in Food Science and Nutrition. 62 (2022) 8199–8229. https://doi.org/10.1080/10408398.2021.1926903. 13. K. Light, S. Karboune, Emulsion, hydrogel and emulgel systems and novel applications in cannabinoid delivery: A review, Critical Reviews in Food Science and Nutrition. 62 (2022) 8199–8229. https://doi.org/10.1080/10408398.2021.1926903. ( ) p g 14. S. Sabalingam, M.A. Siriwardhene, A review on emerging applications of emulgel as topical drug delivery system, World Journal of Advanced Research and Reviews. 13 (2022) 452–463. https://doi.org/10.30574/wjarr.2022.13.1.0048. 14. S. Sabalingam, M.A. Siriwardhene, A review on emerging applications of emulgel as topical drug delivery system, World Journal of Advanced Research and Reviews. 13 (2022) 452–463. https://doi.org/10.30574/wjarr.2022.13.1.0048. 15. K. Anand, S. Ray, M. Rahman, A. Shaharyar, R. Bhowmik, R. Bera, S. Karmakar, Nano-emulgel: emerging as a smarter topical lipidic emulsion-based nanocarrier for skin healthcare applications, Recent Patents on Anti-Infective Drug Discovery. 14 (2019) 16–35. https://doi.org/10.2174/1574891X14666190717111531. 16. Y. de Lafuente, A. Ochoa-Andrade, M.E. Parente, M.C. Palena, A.F. Jimenez-Kairuz, Preparation and evaluation of caffeine bioadhesive emulgels for cosmetic applications based on formulation design using QbD tools, International Journal of Cosmetic Science. 42 (2020) 548–556. https://doi.org/10.1111/ics.12638. 17. M. Umekar, K. Wadher, S. Bute, A. Chandewar, N. Kochar, Y. Amgaonkar, Overview of Emulgel as Emergent Topical Delivery: Recent Applications and Advancement, Journal of Pharmaceutical Research International. (2021) 258–268. 17. M. Umekar, K. Wadher, S. Bute, A. Chandewar, N. Kochar, Y. Amgaonkar, Overview of Emulgel as Emergent Topical Delivery: Recent Applications and Advancement, Journal of Pharmaceutical Research International. (2021) 258–268. 18. G. Marti-Mestres, F. Nielloud, Emulsions in health care applications—an overview, Journal of Dispersion Science and Technology. 23 (2002) 419–439. 17. M. Umekar, K. Wadher, S. Bute, A. Chandewar, N. Kochar, Y. Amgaonkar, Overview of Emulgel as Emergent Topical Delivery: Recent Applications and Advancement, Journal of Pharmaceutical Research International. (2021) 258–268. 18. G. Marti-Mestres, F. Nielloud, Emulsions in health care applications—an overview, Journal of Dispersion Science and Technology. 23 (2002) 419–439. Delivery: Recent Applications and Advancement, Journal of Pharmaceutical Research International. (2021) 258 268. 18. G. . B. Panchal, S. Rathi, Topical Emulgel: A Review on state of art., Pharma Science Monitor. 9 (2018). International Journal of Current Science Research and Review Marti-Mestres, F. Nielloud, Emulsions in health care applications—an overview, Journal of Dispersion Science and Technology. 23 (2002) 419–439. 18. G. Marti-Mestres, F. Nielloud, Emulsions in health care applications—an overview, Journal of Dispersion Science and Technology. 23 (2002) 419–439. 19. R.N. Gürsoy, S. Benita, Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs., Biomedicine & Pharmacotherapy. 58 (2004) 173–182. https://doi.org/10.1016/j.biopha.2004.02.001. 19. R.N. Gürsoy, S. Benita, Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs., Biomedicine & Pharmacotherapy. 58 (2004) 173–182. https://doi.org/10.1016/j.biopha.2004.02.001. 20. G.M. Eccleston, Emulsions and microemulsions, in: J. Swarbrick (Ed.), Encyclopedia of pharmaceutical technology, Informa Healthcare, New York, 2007. 20. G.M. Eccleston, Emulsions and microemulsions, in: J. Swarbrick (Ed.), Encyclopedia of pharmaceutical technology, Informa Healthcare, New York, 2007. 21. S. Malavi, P. Kumbhar, A. Manjappa, S. Chopade, O. Patil, U. Kataria, J. Dwivedi, J. Disouza, Topical Emulgel: Basic Considerations in Development and Advanced Research, Indian Journal of Pharmaceutical Sciences. 84 (2022) 1105– 1115. https://doi.org/10.36468/pharmaceutical-sciences.1005. 21. S. Malavi, P. Kumbhar, A. Manjappa, S. Chopade, O. Patil, U. Kataria, J. Dwivedi, J. Disouza, Topical Emulgel: Basic Considerations in Development and Advanced Research, Indian Journal of Pharmaceutical Sciences. 84 (2022) 1105– 1115. https://doi.org/10.36468/pharmaceutical-sciences.1005. 22. B. Panchal, S. Rathi, Topical Emulgel: A Review on state of art., Pharma Science Monitor. 9 (2018 22. B. Panchal, S. Rathi, Topical Emulgel: A Review on state of ar 23. S. Huan, B.D. Mattos, R. Ajdary, W. Xiang, L. Bai, O.J. Rojas, Two-phase emulgels for direct ink writing of skin-bearing architectures, Advanced Functional Materials. 29 (2019) 1902990. https://doi.org/10.1002/adfm.201902990. 23. S. Huan, B.D. Mattos, R. Ajdary, W. Xiang, L. Bai, O.J. Rojas, Two-phase emulgels for direct ink writing of skin-bearing architectures, Advanced Functional Materials. 29 (2019) 1902990. https://doi.org/10.1002/adfm.201902990. Cite this Article: Prakash Nathaniel Kumar Sarella, Lakshmi Rama Krishna Pravallika Godavari (2023). The Expanding Scope of Emulgels: Formulation, Evaluation and Medical Uses. International Journal of Current Science Research and Review, 6(5), 3030-3041 3041 *Corresponding Author: Prakash Nathaniel Kumar Sarella Volume 06 Issue 05 May 2023 Available at: www.ijcsrr.org Page No. 3030-3041
https://openalex.org/W4293724629	https://www.mdpi.com/2076-393X/10/9/1418/pdf?version=1661753789	English	null	Immune Evaluation of Avian Influenza Virus HAr Protein Expressed in Dunaliella salina in the Mucosa of Chicken	Vaccines	2,022	cc-by	8,376	7 Viren SA de CV, Presidente Benito Juárez 110B, José María Arteaga 76135, Queretaro, Mexico 8 Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, Boulevard Marcelino Garcia Barragan #1421, Guadalajara 44430, Jalisco, Mexico * Correspondence: vmpetrone@hotmail.com Citation: Castellanos-Huerta, I.; Gómez-Verduzco, G.; Tellez-Isaias, G.; Ayora-Talavera, G.; Bañuelos- Hernández, B.; Petrone-García, V.M.; Fernández-Siurob, I.; Velázquez- Juárez, G. Immune Evaluation of Avian Inﬂuenza Virus HAr Protein Expressed in Dunaliella salina in the Mucosa of Chicken. Vaccines 2022, 10, 1418. https://doi.org/10.3390/ vaccines10091418 Abstract: Avian inﬂuenza (AI) is a serious threat to the poultry industry worldwide. Currently, vaccination efforts are based on inactivated, live attenuated, and recombinant vaccines, where the principal focus is on the type of virus hemagglutinin (HA), and the proposed use of recombinant proteins of AI virus (AIV). The use of antigens produced in microalgae is a novel strategy for the induction of an immune response in the mucosal tissue. The capacity of the immune system in poultry, particularly in mucosa, plays an important role in the defense against pathogens. This system depends on a complex relationship between specialized cells and soluble factors, which confer protection against pathogens. Primary lymphoid organs (PLO), as well as lymphocytic aggregates (LA) such as the Harderian gland (HG) and mucosa-associated lymphoid tissue (MALT), actively participate in a local immune response which is mainly secretory IgA (S-IgA). This study demonstrates the usefulness of subunit antigens for the induction of a local and systemic immune response in poultry via ocular application. These ﬁndings suggest that a complex protein such as HAr from AIV (H5N2) can successfully induce increased local production of S-IgA and a speciﬁc systemic immune response in chickens. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: avian inﬂuenza; S-IgA; immunoglobulin; mucosal; hemagglutinin; recombinant protein Immune Evaluation of Avian Inﬂuenza Virus HAr Protein Expressed in Dunaliella salina in the Mucosa of Chicken Inkar Castellanos-Huerta 1, Gabriela Gómez-Verduzco 2, Guillermo Tellez-Isaias 3 , Inkar Castellanos-Huerta 1, Gabriela Gómez-Verduzco 2, Guillermo Tellez-Isaias 3 , Guadalupe Ayora-Talavera 4 , Bernardo Bañuelos-Hernández 5 , Víctor Manuel Petrone-García 6,* , Isidro Fernández-Siurob 7 and Gilberto Velázquez-Juárez 8 , , , Guadalupe Ayora-Talavera 4 , Bernardo Bañuelos-Hernández 5 , Víctor Manuel Petrone- Isidro Fernández-Siurob 7 and Gilberto Velázquez-Juárez 8 Guadalupe Ayora-Talavera 4 , Bernardo Bañuelos-Hernández 5 , Víctor Manuel Petrone-Ga Isidro Fernández Siurob 7 and Gilberto Velázquez Juárez 8 p y Isidro Fernández-Siurob 7 and Gilberto Velázquez-Juárez 8 1 Programa de Maestría y Doctorado en Ciencias de la Producción y de la Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de Mexico 04510, Mexico 2 Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Ciudad de Mexico 04510, Mexico 3 2 Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Ciudad de Mexico 04510, Mexico 3 Department of Poultry Science, University of Arkansas, Fayetteville, AK 72701, USA 3 Department of Poultry Science, University of Arkansas, Fayetteville, AK 72701, USA 4 Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autonoma de Yucatan (UADY), Merida 97000, Yucatan, Mexico 5 Escuela de Veterinaria, Universidad De La Salle Bajío, Avenida Universidad 602, Lomas del Campestre, Leon 37150, Guanajuato, Mexico 6 Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores Cuautitlán UNAM, Cuautitlan 54714, Mexico Communication Communication 1. Introduction Avian inﬂuenza (AI) is classiﬁed from a respiratory to a systemic disease, with a high economic and health impact on the poultry industry worldwide [1]. Clinical presentations of AI range from undetected to serious illness with high mortality depending on the virus subtype and factors such as age, sex, and the commercial purpose of the poultry [2]. The AI virus (AIV), a member of the Orthomyxoviridae family [3], presents the following two glyco- proteins on its surface: hemagglutinin (HA) and neuraminidase (NA) [4]. HA, the principal surface antigen, and the most abundant protein on the surface of viruses [5], is a trimer (≈225 kDa) made up of identical monomers (≈75 kDa) that forms a trimeric, elongated, Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/vaccines Vaccines 2022, 10, 1418. https://doi.org/10.3390/vaccines10091418 Vaccines 2022, 10, 1418 2 of 9 rod-shaped protein organized into two polypeptides—HA1 and HA2 polypeptides—and intertwined in a core-helical coiled-coil (stem-like domain) with N-linked oligosaccharide side chains and three globular heads in the monomeric structure of HA0 [6]. The globular head of HA is the viral receptor, which interacts with a cellular receptor (sialic acid) [6,7]. The vaccination strategy against AI is largely based on the use of the HA subtype circulating in the susceptible population [8] through the use of inactivated virus vaccines and recom- binant virus vaccines [9,10]. Another strategy proposed is the expression of recombinant antigenic proteins from AI [10]. In particular, the expression of HA in heterologous systems demonstrates the ability of these systems to produce recombinant proteins as possible antigens against AI with different approaches [6,8,11–14] including a heterologous protein expression system with high potential in various applications such as microalgae [15]. Tra- ditional systems present particularly undesirable characteristics including high production costs, undesirable post-translational modiﬁcations, prolonged cultivation times, and the lack of glycosylation in proteins in some cases [16]; however, microalgae present several advantages for the expression of proteins with particular characteristics, such as the HA protein [17]. p The immune system of poultry, especially in the mucosal tissue, plays a very important role in the defense against pathogens, whose entry or presence in the mucosa is a constant problem in animals [18]. 1. Introduction In general, the immune system depends on a complex relationship between specialized cells and soluble factors, working together to confer protection against pathogens [1,19,20]. In particular, the mucosal immune system of birds has similarities with mammals, such as the presence of mucosa-associated lymphoid tissue (MALT), with some exceptions [19]. Among these, we can highlight the presence of primary lymphoid organs, such as the bursa of Fabricius (FB) and lymphocytic aggregates such as the Harderian gland (HG). The HG is observed in all vertebrates with the exception of ﬁshes, some amphibians and primates, whereas in poultry HG is the major contributor to ocular Ig production [20]. HG is a dominant orbital gland in birds, with a strap-like structure, located anatomically ventral and posteromedial to the eyeball, and connected to the medial angle of the nictitating membrane [21]. HG is responsible for the production of secretory IgA (S-IgA) [22,23], mainly as a result of local stimulation [24]. Therefore, HG participates in the immune response in mucous membranes, mostly in the eyes and upper respiratory tract [24]. S-IgA, a polypeptide of two monomers IgA, is the predominant Ig isotype on most mucosal surfaces; due to its molecular stability and strong anti-inﬂammatory properties, S-IgA is an ideal protective immunity component of mucosal surfaces [25]. S-IgA and immunoglobulin M (IgM) represent an important part of the defenses at the mucosal level due to their function of preventing the adhesion of pathogens such as bacteria and viruses, limiting their pathogenic effect, and neutralizing and facilitating their excretion [26]. However, the parenteral administration of antigens in many cases is inefﬁcient for its induction, hence the mucosal immune system is considered as separate from the systemic immune system [27]. The administration of antigens via mucosa led to the stimulation of a systemic response [28]; however, the main focus is the local stimulation of an immune response due to the need for protection in these tissues. In the case of recombinant antigens from microalgae, due to all advantages over other antigen-production systems [29], it is possible to explore their use as mucosal vaccines, as a complementary stimulation pathway for parenteral immunization [30]. This study aimed to examine the local and systemic immune response of the recombinant HAr protein of AIV expressed in microalgae (Dunaliella salina) in chickens via mucosal administration. 2.2.2. Preparation of HAr Antigen Produced in Dunaliella salina The production of microalgae biomass was carried out in 15 L of PKS (phosphate– potassium–sodium) modiﬁed medium in ﬂasks with magnetic agitation (Corning PYREX glass Proculture® Spinner Flasks 15,000 mL. (New York City, NY, USA). The biomass from the culture was harvested by centrifugation at 13,000 rpm for 15 min at 4 ◦C and stored at −80 ◦C until ready for use. For antigen HAr’s preparation protocol, biomass was ground using a mortar and pestle in liquid nitrogen, suspended in lysis buffer (1% SDS, 10 mM Tris-MOPS, 2 mM MgCl2, 10 mM KCl pH 7.5, and 2 mM PMSF, added before use), and treated to obtain total protein soluble (TPS) according to the methods of a previous report [15]. According to the quantitative densitometry of proteins stained with Coomassie blue, approximately 1.277 mg of recombinant protein was recovered from 10 g wet weight (WW) of D. salina. TPS from D. salina culture extracts was ﬁltered through a PVDF membrane pore diameter of 0.22 µm and veriﬁed by centrifugation. Recombinant protein HAr was concentrated by performing ultra-ﬁltering; upon the 10-fold increase in the sample a sufﬁcient concentration was achieved. The samples were analyzed using a Quick Start™Bradford Protein assay (Bio-Rad, Hercules, CA, USA) mixed with the designated immunomodulator A1, and homogenized to a ﬁnal concentration of ~25 µg of recombinant protein HAr, each in 50 µL. The antigen and immunomodulator A1 were combined at a ratio of 98:2 (v/v, recombinant protein: A1), mixed for 10 min, and maintained at 4◦C until use. One dose consisted of 100 µL of the antigen preparation (HAr), with 50 µL in each eye. A total of 50 µg of recombinant protein HAr was considered as a dose per animal, as previously observed [11]. 2.2.1. Preparation of Virus-Inactivated Antigens Virus-inactivated antigens for mucosa application were prepared as follows: allantoic ﬂuid from 13-day-old embryos was inoculated with AIV H5N2 A/chicken/Hidalgo/28159- 232/1994 (H5N2), and low pathogenicity AIV (LPAIV) with a minimum viral titer of 109.1 50% chicken embryo infectious dose (CEID)/1 mL was harvested and inactivated with 0.2% formaldehyde for 1 h under constant stirring at room temperature and stored for 24 h at 4 ◦C, with a minimum titer of hemagglutination test of 512 hemagglutination units (HAU)/mL, then it was mixed with the immunomodulator A1 at a ratio of 98:2 (v/v, allantoic ﬂuid: A-1), according to the method of Fernandez-Siurob et al. [31]. Immunomodulator A1 was prepared from the lipopolysaccharide mixture as described by Westphal et al. [32], which was extracted from Escherichia coli strains characterized on an outbreak ﬁeld [33]. 2.2. Preparation of Antigens for Mucosa Application 2.2. Preparation of Antigens for Mucosa Application 2.1. Expression of HAr Recombinant Protein 2.1. Expression of HAr Recombinant Protein 2.1. Expression of HAr Recombinant Protein HAr expression was performed in a previously reported study. Brieﬂy, a sequence of the HA gene of the reference strain A/chicken/Hidalgo/28159-232/1994 (H5N2) Genbank # CY006040.1) was used for the agroinﬁltration protocol for expression in D. salina according to the method of Castellanos-Huerta et al. [15]. Vaccines 2022, 10, 1418 3 of 9 2.4. ELISA Test of S-IgA from Lachrymal Fluid Quantiﬁcation of total S-IgA in lachrymal ﬂuid samples was carried out using a commercial ELISA kit (Catalog #: E33-103, Bethyl Laboratories, Inc., Montgomery, TX, USA) following the manufacturer’s guidelines. Samples were diluted at 1:1000 (v/v lachrymal ﬂuid: dilution buffer), prior to their use in the ELISA test. Concentrations of S-IgA were expressed as nanograms of total S-IgA per ml (ng/mL). 2.3. Animal Experiment The trial was performed to determine the antigenicity of the recombinant antigen HAr compared with a virus-inactivated antigen on mucosal application, as previously reported [31]. The animal experimentation consisted of 60 laying hen chickens divided into groups of 20 for treatments A, B, and C. In all of the experiments, animals were housed in poultry coops and divided according to the applied treatments. Treatment A consisted of the positive control via the ocular route with 100 µL (50 µL in each eye) of virus-inactivated antigen/adjuvant prepared as described above. Treatment B consisted of the application of 100 µL (total of 50 µg of recombinant protein HAr) protein/adjuvant (50 µL in each eye) via the ocular route. Treatment C consisted of the negative control including a treatment of PBS/A1 combined at a ratio of 98:2 (v/v, PBS/A1), mixed for 10 min, and maintained at 4 ◦C until its use via the ocular route (total of 100 µL of PBS, 50 µL in each eye). For this experiment, a calibrated dropper was used to deposit a 50 µL drop in each eye according to the manufacturer’s instructions. In all of the experiments, environmental and animal management conditions were upheld according to the standards of laying chicken breeding. Feed and water were supplied ad libitum throughout the entire experiment. For the trial, treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) were applied at 14 and 21 days of age. For each treatment, 10 birds were sampled for total S-IgA Vaccines 2022, 10, 1418 4 of 9 quantitation in lachrymal ﬂuid at 7 and 14 days post-treatment (DPT) (28 and 36 days of age); the samples were analyzed by performing an ELISA test according to the method of Merino-Guzman et al. [34]. A total of 12 birds were bled for serum at 14 and 21 DPT (36 and 42 days of age) for the inhibition of the hemagglutination (HI) assay to determine the serum immunoglobulin levels against AIV H5N2 [35]. All samples were stored at −80 ◦C until use. During the experiment, the experimental animals were handled according to ethical protocols for animal welfare [36]. 2.5. Inhibition of Hemagglutination (HI) Assay The stimulation of the systemic immune response of treatments A, B, and C was evaluated using the HI assay expressed as the geometric mean titer (GMT). All mea- surements of serum AI-speciﬁc antibody levels were analyzed with 4 HAU of the AIV A/chicken/Hidalgo/28159-232/1994 (H5N2) strain, according to the WHO Manual on Avian Inﬂuenza Diagnosis and Surveillance [37]. 2.6. Statistical Analyses Absorbance results in the ELISA test (for total S-IgA in the lachrymal ﬂuid) were extrapolated onto a polynomial calibration chart according to the supplier’s instructions. The data conﬁrmed normal distribution (Shapiro–Wilk test) and homoscedasticity (Levene test). Consequently, the data were subjected to a parametric test and one-way ANOVA, followed by Tukey’s multiple comparison test with the level of statistical signiﬁcance set at p < 0.05. 3.1. ELISA Test of S-IgA from Lachrymal Fluid The quantiﬁcation of total S-IgA determined by performing the ELISA test at 7 DPT in the lachrymal ﬂuid of animals on treatments A (48,334 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL), showed a statistical difference (p > 0.05) compared with treatment C (24,224 ng/mL ± 3496 ng/mL) (Figure 1A). Samples obtained at 14 DPT showed an increase in the local immune response for the concentration of total S-IgA in treat- ment A (57,486 ng/mL ± 7192 ng/mL), with a statistical difference (p > 0.05) compared with treatment B (43,043 ng/mL ± 5733 ng/mL) and treatment C (35,633 ng/mL± 6195 ng/mL) (Figure 1B). No statistical difference was observed between treatments B and C. mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in conc on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0 HI Assay from Serum According to the results, for treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 2.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) at 14 DPT no statistical difference was observed (p > 0.05) (Figure 2A). The systemic immune response observed at 21 DPT pre- ents a statistical difference (p > 0.05) by comparing treatment A (GMT = 38.05 ± 1.54), reatment B (GMT = 33.90 ± 1.86), and treatment C (GMT = 6.72 ± 1.54) (Figure 2B). HI Assay from Serum According to the results, for treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = ± 1.98), and treatment C (GMT = 10.67 ± 1.59) at 14 DPT no statistical difference was rved (p > 0.05) (Figure 2A). The systemic immune response observed at 21 DPT pre- a statistical difference (p > 0.05) by comparing treatment A (GMT = 38.05 ± 1.54), ment B (GMT = 33.90 ± 1.86), and treatment C (GMT = 6.72 ± 1.54) (Figure 2B). Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic immune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). Immune response at 21 DPT was observed in treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference (p-value < 0.05). Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic immune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). 3.2. HI Assay from Serum ELISA test results at 14 DPT showed an increase in concentration of S-IgA on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0.05) compared with treatment B (43,043 ng/mL ± 5733 ng/mL) and treatment C (35,633 ng/mL ± 6195 ng/mL). No statistical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a statistical difference (p < 0.05). Figure 1. ELISA test of total S-IgA from lachrymal fluid of treatments A, B, and C. The concentration of total S-IgA from lachrymal fluid samples was determined using a commercial ELISA kit. (A). ELISA test results of total S-IgA in the lacrimal fluid were observed at 7 days post-treatment (DPT) on treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). The re- sults at 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment A (48,334 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL) compared to treatment C (24,224 ng/mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in concentration of S- IgA on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0.05) compared with treatment B (43,043 ng/mL± 5733 ng/mL) and treatment C (35,633 ng/mL± 6195 ng/mL). No statistical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a statistical difference (p < 0.05). 3.2. HI Assay from Serum According to the results, for treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) at 14 DPT no statistical difference was observed (p > 0.05) (Figure 2A). The systemic immune response observed at 21 DPT pre- sents a statistical difference (p > 0.05) by comparing treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86), and treatment C (GMT = 6.72 ± 1.54) (Figure 2B). Figure 1. ELISA test of total S-IgA from lachrymal fluid of treatments A, B, and C. The concentratio of total S-IgA from lachrymal fluid samples was determined using a commercial ELISA kit. (A ELISA test results of total S-IgA in the lacrimal fluid were observed at 7 days post-treatment (DP on treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in conc on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0 1. ELISA test of total S-IgA from lachrymal fluid of treatments A, B, and C. The concentration l S-IgA from lachrymal fluid samples was determined using a commercial ELISA kit. (A). test results of total S-IgA in the lacrimal fluid were observed at 7 days post-treatment (DPT) atment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). The re- t 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment A 4 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL) compared to treatment C (24,224 ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in concentration of S- n treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0.05) compared reatment B (43,043 ng/mL± 5733 ng/mL) and treatment C (35,633 ng/mL± 6195 ng/mL). No ical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a ical difference (p < 0.05). Figure 1. ELISA test of total S-IgA from lachrymal ﬂuid of treatments A, B, and C. The concentra- tion of total S-IgA from lachrymal ﬂuid samples was determined using a commercial ELISA kit. (A). ELISA test results of total S-IgA in the lacrimal ﬂuid were observed at 7 days post-treatment (DPT) on treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). The results at 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment A (48,334 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL) compared to treatment C (24,224 ng/mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in concentration of S-IgA on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0.05) compared with treatment B (43,043 ng/mL ± 5733 ng/mL) and treatment C (35,633 ng/mL ± 6195 ng/mL). No statistical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a statistical difference (p < 0.05). with treatment B (43,043 ng/mL± 5733 ng/mL) and treatment C (35,633 ng/mL± 6195 ng/mL). No tatistical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a tatistical difference (p < 0.05). .2. mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in conc on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0 Immune response at 21 DPT was observed in treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference (p-value < 0.05). Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic immune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). Immune response at 21 DPT was observed in treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference (p-value < 0 05) Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic immune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). Immune response at 21 DPT was observed in treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference (p-value < 0.05). mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in conc on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0 Immune response at 21 DPT was observed in treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference (p-value < 0.05). I Assay from Serum According to the results, for treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = ± 1.98), and treatment C (GMT = 10.67 ± 1.59) at 14 DPT no statistical difference was ved (p > 0.05) (Figure 2A). The systemic immune response observed at 21 DPT pre- a statistical difference (p > 0.05) by comparing treatment A (GMT = 38.05 ± 1.54), ment B (GMT = 33.90 ± 1.86), and treatment C (GMT = 6.72 ± 1.54) (Figure 2B). Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic mmune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). Immune response at 21 DPT was observed in treatment A GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a statistical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference p-value < 0 05) Figure 2. Inhibition of hemagglutination assay (HI) from serum of treatments A, B, and C. Systemic immune response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI assay expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference was observed (p > 0.05) at 14 DPT. (B). 3.2. HI Assay from Serum y f According to the results, for treatment A (GMT = 8.97 ± 1.30), treatment B (GMT = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) at 14 DPT no statisti- cal difference was observed (p > 0.05) (Figure 2A). The systemic immune response ob- served at 21 DPT presents a statistical difference (p > 0.05) by comparing treatment A (GMT = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86), and treatment C (GMT = 6.72 ± 1.54) (Figure 2B). 5 of 9 Vaccines 2022, 10, 1418 s 2022, 10, x Figure 1. ELISA test of total S-IgA from lachrymal fluid of treatments A, B, and C. The concentration of total S-IgA from lachrymal fluid samples was determined using a commercial ELISA kit. (A). ELISA test results of total S-IgA in the lacrimal fluid were observed at 7 days post-treatment (DPT) on treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). The re- sults at 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment A (48,334 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL) compared to treatment C (24,224 ng/mL ± 3496 ng/mL). (B). ELISA test results at 14 DPT showed an increase in concentration of S- IgA on treatment A (57,486 ng/mL ± 7192 ng/mL) with a statistical difference (p < 0.05) compared with treatment B (43,043 ng/mL± 5733 ng/mL) and treatment C (35,633 ng/mL± 6195 ng/mL). No statistical difference was observed in the cases of treatments B and C at 14 DPT. Literals indicate a statistical difference (p < 0.05). Figure 1. ELISA test of total S-IgA from lachrymal ﬂuid of treatments A, B, and C. The concentra- tion of total S-IgA from lachrymal ﬂuid samples was determined using a commercial ELISA kit. (A). ELISA test results of total S-IgA in the lacrimal ﬂuid were observed at 7 days post-treatment (DPT) on treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C (PBS/A1). The results at 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment A (48,334 ng/mL ± 4196 ng/mL) and B (44,759 ng/mL ± 8002 ng/mL) compared to treatment C (24,224 ng/mL ± 3496 ng/mL). (B). 3.2. HI Assay from Serum The r sults at 7 DPT showed a statistical difference (p < 0.05) in total S-IgA concentration in treatment ( / / ) d ( / / ) d C ( 2. Inhibition of he f t p 4. Discussion The recombinant protein (HAr) was previously produced in a protein-recombinant expression model [15] with advantages in terms of production costs, expression levels, as well as its possible use in the oral vaccination of animals [29]. Therefore, mucosal vaccination was demonstrated in an animal model (poultry) due to the importance of local mucosal immunity against diseases caused by respiratory viruses such as AI [44]. p y The total S-IgA ELISA kit was utilized to determine the reactivity of the ocular applica- tion of antigens by stimulating eye-associated lymphoid tissue [24]. Despite the non-speciﬁc detection of S-IgA against AI, it was possible to determine different levels of S-IgA stimula- tion during the application of both viral antigens and the recombinant protein. At 7 DPT, an increase in the levels of total S-IgA in the lachrymal ﬂuid samples was detected following the application of the viral protein and recombinant protein, compared to the negative control group, thereby demonstrating HG stimulation in the presence of the administered antigens [45]. For the second sampling at 14 DPT, an antigen of viral origin showed a signiﬁcant increase compared to the recombinant antigen and the negative control group in the total S-IgA levels, due to antigenic components of AIV (proteins NA, M) as well as internal viral proteins [46]. The total S-IgA values induced at 14 DPT by the recombinant HAr protein maintained the same level compared to 7 DPT sampling; however, bacterial components in the immunomodulator used called A1 were able to stimulate an adaptive immune response against these bacterial components [47], detectable by the total S-IgA ELISA kit in the negative control group. The results of total S-IgA at 7 DPT showed the ability of viral proteins and the HAr protein to stimulate local immunity. Nevertheless, at 14 DPT, due to the use of an adjuvant based on bacterial components and the absence of viral components, the results observed reﬂect a local immune response with less activity from the HAr protein compared to the complete viral antigen [46], thus reﬂecting the necessity to improve HAr subunit antigens, including the determination of SIgA-speciﬁc antigens against AIV antigens for an accurate evaluation. The HI assay was proposed to determine the ability to stimulate a systemic immune re- sponse through ocular administration of viral proteins and a recombinant protein HAr. [11]. 2. Inhibition of he f t p 4. Discussion ne response of treatment A (virus-inactivated antigen), treatment B (HAr), and treatment C A1) via ocular application at 14 and 21 days post-treatment (DPT) were evaluated by the HI expressed in geometric mean titer (GMT). (A). Treatment A (GMT = 8.97 ± 1.30), treatment B = 12.70 ± 1.98), and treatment C (GMT = 10.67 ± 1.59) indicated that no statistical difference bserved (p > 0.05) at 14 DPT. (B). Immune response at 21 DPT was observed in treatment A = 38.05 ± 1.54), treatment B (GMT = 33.90 ± 1.86) and treatment C (GMT = 6.72 ± 1.54) with a ical difference (p > 0.05) in comparison to each other. Literals indicate a statistical difference ue < 0.05). A description of the response of the mucosal immune system is necessary for the un- derstanding of several pathologies of infectious origin, particularly in the case of AI [38,39]. As is known, the main route of entry for pathogens into an organism is via the mucosa tissue [40]. Therefore, this tissue represents the ﬁrst barrier that needs to be overcome in order to achieve infection and the generation of infectious diseases. For this reason, the mucosa requires a highly specialized immune system for the correct response and protection against various pathogens [38,39]. In the case of birds, these lymphoid organs present some variations with respect to other species [38,41]; however, the specialized Vaccines 2022, 10, 1418 6 of 9 tissue plays a vital role in homeostasis, as in other species. The ocular route for vaccination is widely used in the poultry industry as an option for diseases such as Newcastle disease and avian bronchitis [23,24] due to its practicality, as well as the relevance and participation of lymphocytic aggregates, e.g., HG [20,22,23] The active participation of HG in the produc- tion of an adaptive immune response after ocular administration represents a tool for the local application of antigens [11,42] due to the relevance in the participation of S-IgA in the protection against infections, as well as the induction of immunity after vaccination [43]. Nevertheless, the measurement of this Ig subtype in lachrymal ﬂuid samples [34] is not a regular practice. g p The main objective of this study was to determine the antigenicity of a recombinant protein compared with native viral antigens of AIV. 2. Inhibition of he f t p 4. Discussion The results observed at immunization at 14 days DPT with viral proteins, recombinant protein and the application of PBS/A1, showed no signiﬁcant difference. However, at 21 days DPT, an increase in HI titers was observed in animals subjected to treatments with the virus protein and recombinant HAr protein, compared to the negative control group, demonstrating that immune stimulation via the ocular route is relevant for the local immune response [48]. This was due to the presence of a lymphocyte aggregate HG, [38] as well as its participation in the presentation of antigens to the immune system for the gener- ation of the humoral response at the systemic level [49]. As observed in the measurement of total S-IgA in lachrymal ﬂuid, the stimulus-induced generation of a systemic immune response was also inﬂuenced by the participation of other viral antigens present in the sample of AI [50]; therefore, the presence of a single subunit antigen presents an antigenic capacity not similar to a complete viral native antigen. The increase in GMT titers in the HI assay upon the application of recombinant anti- gens demonstrates AI-speciﬁc systemic immune stimulation by the ocular administration of a recombinant protein [49] since the HI assay measures the reactivity of stimulated Vaccines 2022, 10, 1418 7 of 9 antibodies to AI-speciﬁc immunization [37]. Due to the immunological performance of HAr compared to viral proteins administered under the same adjuvant and application route, it is necessary to continue its study as a possible recombinant antigen for AI before considering viral challenge experiments in animals. The titers obtained from the HI assay, induced by the application of viral antigens and recombinant protein HAr, are suggestive of the minimum protection titers (GMT ≥32) [51]. However, various experiments are required to determine the minimum protection level. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: Inkar Castellanos-Huerta is employed by Viren SA de CV. The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 5. Conclusions The present study described the antigenic capacity of the recombinant protein HAr, applied by the ocular route in experimental animals, in comparison with a complete viral antigen. The results indicate the capacity of protein HAr for stimulation of the local and systemic immune system in an ocular application, for its recognition as a viral protein by HG, as well as its processing and induction of an antibody-speciﬁc immune response. The antibodies generated at the serum level proved to be reactive with a wild-type viral antigen, used in the HI assay, thus demonstrating that protein HAr presents characteristics of an antigen of AI. Nevertheless, viral antigens present more antigenic activity according to the ELISA test S-IgA results and HI assay; therefore, further studies are required to develop a recombinant antigen capable of inducing an immune response similar to that observed in antigens of viral origin. The results of total measured S-IgA in this experiment demonstrate the efﬁciency of a recombinant protein to achieve antigenic stimulation in experimental animals; however, these results require a more speciﬁc evaluation, because total quantiﬁcation is not able to determine the speciﬁc stimulation against protein HAr and its relationship with viral antigens, describing no speciﬁc local immune response. Therefore, its evaluation requires further study. y In this experiment, the AIV H5N2 A/chicken/Hidalgo/28159-232/1994 (H5N2) strain complied with the stipulations of the Mexican authority for use in the production of vaccines against AI and experimentation in vitro. Nevertheless, the absence of evidence of viral challenges in experimental animals leaves the capacity of these types of antigens unevaluated in the presence of pathogenic viruses, so their efﬁcacy as vaccine antigens requires permission from the local authority to carry out their evaluation in viral challenges in order to understand its full scope as a possible vaccine antigen. Author Contributions: Conceptualization, I.C.-H., G.G.-V., G.A.-T. and G.T.-I.; methodology, I.C.-H. and B.B.-H.; software, G.V.-J.; investigation, V.M.P.-G.; resources, G.G.-V.; writing—original draft preparation, I.C.-H.; writing—review and editing, G.T.-I.; funding acquisition, I.F.-S. All authors have read and agreed to the published version of the manuscript. Funding: This research was ﬁnanced with private economic resources by the company VIREN SA DE CV. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. f y Pantin-Jackwood, M. Pathogenicity of Avian Inﬂuenza Viruses in Poultry. Dev. Biol. 2006, 124, 61–67. 1. Saif, Y. Disease of Poultry, 11th ed.; Lowa State Press: Ames, IA, USA, 2003. y p g p 4. Lamb, R.A. Genes and Proteins of the Inﬂuenza Viruses. In The Inﬂuenza Viruses; Springer: Berlin/Heidelberg, Germany, 1989; pp. 1–87. Swayne, D.; Pantin-Jackwood, M. Pathogenicity of Avian Inﬂuenza Viruses in Poultry. Dev. Biol. 2006, 124 S d A B W i D S A A i I ﬂ I St tP l [I t t] S P l P bli hi T y g y y 3. Sendor, A.B.; Weerasuriya, D.; Sapra, A. Avian Inﬂuenza. In StatPearls [Internet]; StatPearls Publishing: Tampa, FL, USA, 2021. References EMBO J. 1986, 5, 1359–1365. [CrossRef] [PubMed] 15. Castellanos-Huerta, I.; Gómez-Verduzco, G.; Tellez-Isaias, G.; Ayora-Talavera, G.; Bañuelos-Hernández, B.; Petrone-García, V.M.; Velázquez-Juárez, G.; Fernández-Siurob, I. Transformation of Dunaliella Salina by Agrobacterium Tumefaciens for the Expression of the Hemagglutinin of Avian Inﬂuenza Virus H5. Microorganisms 2022, 10, 361. [CrossRef] [PubMed] gg g 16. Potvin, G.; Zhang, Z. Strategies for High-Level Recombinant Protein Expression in Transgenic Microalgae: A Review. Biotechnol. Adv. 2010, 28, 910–918. [CrossRef] [PubMed] 17. Bayne, A.-C.V.; Boltz, D.; Owen, C.; Betz, Y.; Maia, G.; Azadi, P.; Archer-Hartmann, S.; Zirkle, R.; Lippmeier, J.C. Vaccination against Inﬂuenza with Recombinant Hemagglutinin Expressed by Schizochytrium sp. Confers Protective Immunity. PLoS ONE 2013, 8, e61790. 18. Schijns, V.E.J.C.; van de Zande, S.; Lupiani, B.; Reddy, S.M. Chapter 20—Practical Aspects of Poultry Vaccination. In Avian Immunology, 2nd ed.; Schat, K.A., Kaspers, B., Kaiser, P., Eds.; Academic Press: Boston, MA, USA, 2014; pp. 345–362, ISBN 978-0-12-396965-1. 19. Ratcliffe, M.J.H.; Härtle, S. Chapter 4—B Cells, the Bursa of Fabricius and the Generation of Antibody Repertoires. In Avian Immunology, 2nd ed.; Schat, K.A., Kaspers, B., Kaiser, P., Eds.; Academic Press: Boston, MA, USA, 2014; pp. 65–89, ISBN 978-0-12-396965-1. 20. Burns, R. The Harderian Gland in Birds: Histology and Immunology. In Harderian Glands; Springer: Berlin/Heidelberg, Germany, 1992; pp. 155–163. 21. Burns, R. Possible Route of Antigen Uptake by the Harderian Gland of the Domestic Fowl. Br. Poult. Sci. 1977, 18, 407–409. [CrossRef] 22. Baba, T.; Kawata, T.; Masumoto, K.; Kajikawa, T. Role of the Harderian Gland in Immunoglobulin A Production in Chicken Lacrimal Fluid. Res. Vet. Sci. 1990, 49, 20–24. [CrossRef] 23. Baba, T.; Masumoto, K.; Nishida, S.; Kajikawa, T.; Mitsui, M. Harderian Gland Dependency of Immunoglobulin A Production in the Lacrimal Fluid of Chicken. Immunology 1988, 65, 67. 24. Gallego, M.; Del Cacho, E.; Felices, C.; Bascuas, J. Immunoglobulin Classes Synthesised by the Chicken Harderian Gland after Local Immunisation. Res. Vet. Sci. 1992, 52, 44–47. [CrossRef] 25. Snoeck, V.; Peters, I.R.; Cox, E. The IgA System: A Comparison of Structure and Function in Different S 455–467. [CrossRef] R.; Cox, E. The IgA System: A Comparison of Structure and Function in Different Species. Vet. Res. 2006, 37 26. Brandtzaeg, P.; Baekkevold, E.S.; Farstad, I.N.; Jahnsen, F.L.; Johansen, F.-E.; Nilsen, E.M.; Yamanaka, T. Regional Specialization in the Mucosal Immune System: What Happens in the Microcompartments? Immunol. Today 1999, 20, 141–151. [CrossRef] y pp p y 27. Mestecky, J.; McGHEE, J.R. References 1. Saif, Y. Disease of Poultry, 11th ed.; Lowa State Press: Ames, IA, USA, 2003. 8 of 9 Vaccines 2022, 10, 1418 5. Wright, P.; Neumann, G.; Kawaoka, Y. Orthomyxoviruses. In Fields Virology; Lippincott-Williams Wilkins: Philadelphia, PA, USA, 2007; pp. 1691–1740. pp 6. Bosch, F.; Orlich, M.; Klenk, H.-D.; Rott, R. The Structure of the Hemagglutinin, a Determinant for the Pathogenicity of Inﬂuenza Viruses. Virology 1979, 95, 197–207. [CrossRef] 7. Chen, J.; Lee, K.H.; Steinhauer, D.A.; Stevens, D.J.; Skehel, J.J.; Wiley, D.C. Structure of the Hemagglutinin Precursor Cleavage Site, a Determinant of Inﬂuenza Pathogenicity and the Origin of the Labile Conformation. Cell 1998, 95, 409–417. [CrossRef] 8. DuBois, R.M.; Aguilar-Yañez, J.M.; Mendoza-Ochoa, G.I.; Oropeza-Almazán, Y.; Schultz-Cherry, S.; Alvarez, M.M.; White, S.W.; Russell, C.J. The Receptor-Binding Domain of Inﬂuenza Virus Hemagglutinin Produced in Escherichia Coli Folds into Its Native, Immunogenic Structure. J. Virol. 2011, 85, 865–872. [CrossRef] [PubMed] J p g gg Immunogenic Structure. J. Virol. 2011, 85, 865–872. [CrossRef] [PubMed] g 9. Vuong, C.N.; Kuttappan, V.A.; Faulkner, O.B.; Berghman, L.R.; Wolfenden, A.D.; Tellez-Isaias, G.; Jonas, M.; Kapczynski, D.R.; Hargis, B.M.; Bielke, L.R. Comparison of Oil Emulsion, Mannosylated Chitosan, and Bacillus Vector Adjuvants for Vaccination against Inﬂuenza in Chickens. J. Appl. Poult. Res. 2020, 29, 653–664. [CrossRef] g J pp [ ] 10. van den Berg, T.; Lambrecht, B.; Marché, S.; Steensels, M.; Van Borm, S.; Bublot, M. Inﬂuenza Vaccines and Vaccination Strategies in Birds. Comp. Immunol. Microbiol. Infect. Dis. 2008, 31, 121–165. [CrossRef] 11. Castellanos-Huerta, I.; Bañuelos-Hernández, B.; Téllez, G.; Rosales-Mendoza, S.; Brieba, L.G.; Esquivel-Ramos, E.; Beltrán-López, J.I.; Velazquez, G.; Fernandez-Siurob, I. Recombinant Hemagglutinin of Avian Inﬂuenza Virus H5 Expressed in the Chloroplast of Chlamydomonas Reinhardtii and Evaluation of Its Immunogenicity in Chickens. Avian Dis. 2016, 60, 784–791. [CrossRef] 12. Bertran, K.; Thomas, C.; Guo, X.; Bublot, M.; Pritchard, N.; Regan, J.T.; Cox, K.M.; Gasdaska, J.R.; Dickey, L.F.; Kapczynski, D.R.; et al. Expression of H5 Hemagglutinin Vaccine Antigen in Common Duckweed (Lemna Minor) Pro- tects against H5N1 High Pathogenicity Avian Inﬂuenza Virus Challenge in Immunized Chickens. Vaccine 2015, 33, 3456–3462. [CrossRef] 13. Landry, N.; Ward, B.J.; Trépanier, S.; Montomoli, E.; Dargis, M.; Lapini, G.; Vézina, L.-P. Preclinical and Clinical Development of Plant-Made Virus-like Particle Vaccine against Avian H5N1 Inﬂuenza. PLoS ONE 2010, 5, e15559. [CrossRef] 14. Kuroda, K.; Hauser, C.; Rott, R.; Klenk, H.; Doerﬂer, W. Expression of the Inﬂuenza Virus Haemagglutinin in Insect Cells by a Baculovirus Vector. 5. Wright, P.; Neumann, G.; Kawaoka, Y. Orthomyxoviruses. In Fields Virology; Lippincott-Williams Wilkins: Philadelphia, PA, USA, 2007; pp. 1691–1740. References The Well-Developed Mucosal Immune Systems of Birds and Mammals Allow for Similar Approaches of Mucosal Vaccination in Both Types of Animals. Front. Nutr. 2018, 5, 60. [CrossRef] 39. Nochi, T.; Jansen, C.A.; Toyomizu, M.; van Eden, W. The Well-Developed Mucosal Immune Systems of Birds and Mammals All f Si il A h f M l V i ti i B th T f A i l F t N t 2018 5 60 [C R f] ; Jansen, C.A.; Toyomizu, M.; van Eden, W. The Well-Developed Mucosal Immune Systems of Birds and 39. Nochi, T.; Jansen, C.A.; Toyomizu, M.; van Eden, W. The Well-Developed Mucosal Immune System Allow for Similar Approaches of Mucosal Vaccination in Both Types of Animals. Front. Nutr. 2018, 5, 6 r Similar Approaches of Mucosal Vaccination in Both Types of Animals. Front. Nutr. 2018, 5, 60. [CrossRef] ky, C.; Anjueie, F.; McGhee, J.R.; Geoige-Chundy, A.; Holmgren, J.; Kieny, M.-P.; Fujiyashi, K.; Mestecky, J.F. 40. Czerkinsky, C.; Anjueie, F.; McGhee, J.R.; Geoige-Chundy, A.; Holmgren, J.; Kieny, M.-P.; Fujiyashi, K.; Mestecky, J.F.; Pierreﬁte- Carle, V.; Rusk, C.; et al. Mucosal Immunity and Tolerance: Relevance to Vaccine Development. Immunol. Rev. 1999, 170, 197–222. [CrossRef] [PubMed] 41. ´Smialek, M.; Tykalowski, B.; Stenzel, T.; Koncicki, A. Local Immunity of the Respiratory Mucosal System in Chickens and Turkeys. Pol. J. Vet. Sci. 2011, 14, 291–297. [CrossRef] [PubMed] 42. van Ginkel, F.W.; De-chu, C.T.; Gulley, S.L.; Toro, H. Induction of Mucosal Immunity in the Avian Harderian Gland with a Replication-Deﬁcient Ad5 Vector Expressing Avian Inﬂuenza H5 Hemagglutinin. Dev. Comp. Immunol. 2009, 33, 28–34. [CrossRef] [PubMed] 43. Gianchecchi, E.; Manenti, A.; Kistner, O.; Trombetta, C.; Manini, I.; Montomoli, E. How to Assess the Effectiveness of Nasal Inﬂuenza Vaccines? Role and Measurement of SIgA in Mucosal Secretions. Inﬂuenza Other Respir. Viruses 2019, 13, 429–437. [CrossRef] 44. Brandtzaeg, P. Role of Mucosal Immunity in Inﬂuenza. Dev. Biol. 2003, 115, 39–48. 45. Zegpi, R.; Breedlove, C.; Gulley, S.; Toro, H. Infectious Bronchitis Virus Immune Responses in the Harderian Gland upon Initial Vaccination. Avian Dis. 2020, 64, 92–95. [CrossRef] 46. Kreijtz, J.; Fouchier, R.; Rimmelzwaan, G. Immune Responses to Inﬂuenza Virus Infection. Virus Res. 2011, 162, 19–30. [CrossRef] 47. Rice, B.E.; Rollins, D.M.; Mallinson, E.T.; Carr, L.; Joseph, S.W. Campylobacter Jejuni in Broiler Chickens: Colonization and Humoral Immunity Following Oral Vaccination and Experimental Infection. Vaccine 1997, 15, 1922–1932. [CrossRef] 48. Tomley, F.F. Recombinant Vaccines for Poultry. References Immunoglobulin A (IgA): Molecular and Cellular Interactions Involved in IgA Biosynthesis and Immune Response. Adv. Immunol. 1987, 40, 153–245. [PubMed] 28. Zhang, X.; Zhang, X.; Yang, Q. Effect of Compound Mucosal Immune Adjuvant on Mucosal and Systemic Immune Responses in Chicken Orally Vaccinated with Attenuated Newcastle-Disease Vaccine. Vaccine 2007, 25, 3254–3262. [CrossRef] [PubMed] y 29. Hempel, F.; Maier, U.G. Microalgae as Solar-Powered Protein Factories. Adv. Technol. Protein Complex Prod. Charact. 2016, 241–262. 29. Hempel, F.; Maier, U.G. Microalgae as Solar-Powered Protein Factories. Adv. Technol. Protein Complex Pro 9 of 9 Vaccines 2022, 10, 1418 30. Specht, E.; Miyake-Stoner, S.; Mayﬁeld, S. Micro-Algae Come of Age as a Platform for Recombinant Protein Production. Biotechnol. Lett. 2010, 32, 1373–1383. [CrossRef] [PubMed] 31. Fernandez-Siurob, I.; Retana, M.; Tellez, G.; Arroyo-Navarro, L.; Bañuelos-Hernandez, B.; Castellanos-Huerta, I. Assessment of Viral Interference Using a Chemical Receptor Blocker against Avian Inﬂuenza and Establishment of Protection Levels in Field Outbreaks. Vaccine 2014, 32, 1318–1322. [CrossRef] 32. Westphal, O. Bacterial Lipopolysaccharides Extraction with Phenol-Water and Further Applications of the Procedure. Methods Carbohydr. Chem 1965, 5, 83–91. y 33. Swayne, D.E. Laboratory Manual for the Isolation and Identiﬁcation of Avian Pathogens; American Association of Avian Pathologists, University of Pennsylvania: Philadelphia, PA, USA, 1998. 34. Merino-Guzmán, R.; Latorre, J.D.; Delgado, R.; Hernandez-Velasco, X.; Wolfenden, A.D.; Teague, K.D.; Graham, L.E.; Mahaffey, B.D.; Baxter, M.F.A.; Hargis, B.M.; et al. Comparison of Total Immunoglobulin A Levels in Different Samples in Leghorn and Broiler Chickens. Asian Pac. J. Trop. Biomed. 2017, 7, 116–120. [CrossRef] J p llegas, P. Titration of Biological Suspensions. A Lab. Man. Isol. Identif. Avian Pathog. 1998, 248–254. 35. Villegas, P. Titration of Biological Suspensions. A Lab. Man. Isol. Identif. Avian Pathog. 1998, 248–254. 36. Olfert, E.D.; Cross, B.M.; McWilliam, A.A. Gu Ottawa, ON, Canada, 1993; Volume 1. 36. Olfert, E.D.; Cross, B.M.; McWilliam, A.A. Guide to the Care and Use of Experimental Animals; Canadian Council on Animal Care: Ottawa, ON, Canada, 1993; Volume 1. Ottawa, ON, Canada, 1993; Volume 1. 37. World Health Organization. WHO Manual on Animal Inﬂuenza Diagnosis and Surveillance; World Healt Switzerland, 2002. rganization. WHO Manual on Animal Inﬂuenza Diagnosis and Surveillance; World Health Organization: Gen 2. 38. Swayne, D.E.; Kapczynski, D. Strategies and Challenges for Eliciting Immunity against Avian Inﬂuenza Virus in Birds. Immunol. Rev. 2008, 225, 314–331. [CrossRef] 39. Nochi, T.; Jansen, C.A.; Toyomizu, M.; van Eden, W. References Vaccine 1991, 9, 4–5. [CrossRef] Kreijtz, J.; Fouchier, R.; Rimmelzwaan, G. Immune R j , J ; , ; , p , , [ ] 47. Rice, B.E.; Rollins, D.M.; Mallinson, E.T.; Carr, L.; Joseph, S.W. Campylobacter Jejuni in Broiler Chickens: Colonization and Humoral Immunity Following Oral Vaccination and Experimental Infection. Vaccine 1997, 15, 1922–1932. [CrossRef] 48. Tomley, F.F. Recombinant Vaccines for Poultry. Vaccine 1991, 9, 4–5. [CrossRef] 49. Mueller, A.; Sato, K.; Glick, B. The Chicken Lacrimal Gland, Gland of Harder, Caecal Tonsil, and Accessory Spleens as Sources of Antibody-Producing Cells. Cell. Immunol. 1971, 2, 140–152. [CrossRef] y g 50. Suarez, D.L.; Schultz-Cherry, S. Immunology of Avian Inﬂuenza Virus: A Review. Dev. Comp. Immunol. 2000, 24, 269–283. [CrossRef] 51. Swayne, D.E.; Suarez, D.L.; Spackman, E.; Jadhao, S.; Dauphin, G.; Kim-Torchetti, M.; McGrane, J.; Weaver, J.; Daniels, P.; Wong, F.; et al. Antibody Titer Has Positive Predictive Value for Vaccine Protection against Challenge with Natural Antigenic- Drift Variants of H5N1 High-Pathogenicity Avian Inﬂuenza Viruses from Indonesia. J. Virol. 2015, 89, 3746–3762. [CrossRef] [PubMed]
https://openalex.org/W3019678432	https://europepmc.org/articles/pmc7188863?pdf=render	English	null	A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling	Signal transduction and targeted therapy	2,020	cc-by	10,044	INTRODUCTION to increases of oxidative stress and inﬂammatory cell inﬁltration in the kidney, recapitulating the phenotypes of renal ﬁbrosis.8 These studies demonstrate renal tubules as the major action site of TGF- β/Smad signaling in renal ﬁbrosis. Renal ﬁbrosis is a very complex and irreversible pathological process, which involves the activation and crosstalk of a variety of proﬁbrotic signaling pathways,1 characterizing the late stages of virtually all types of chronic kidney diseases (CKD). Currently, there are very limited therapeutics to treat renal ﬁbrosis effectively. Aberrant activation of Wnt/β-catenin signaling has been shown to play a pathogenic role in renal ﬁbrosis in obstructive nephro- pathy2 and diabetic nephropathy.3 However, the role of low- density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of Wnt/β-catenin signaling,4 has not been well characterized in these diseases. A recent study reported that high LRP5 levels in patients were associated with faster progression of idiopathic pulmonary ﬁbrosis (IPF) through activation of Wnt/β-catenin signaling,5 implicating an important role of LRP5 in Wnt/β-catenin signaling-related pulmonary ﬁbrotic diseases. β g g Renal tubules are the primary insult targets in a variety of acute kidney injuries and CKD, and they also play a role in the initiation and progression of renal diseases,9 as injured tubule epithelial cells are capable of producing reactive oxygen species,10 growth factors,1,11,12 chemokines,1,12 adhesion molecules,1,12 ﬁbrotic cytokines,1,11,12 and extracellular matrix.1,12 Renal tubule epithelial cells arrested in the prolonged G2/M phase manifest proﬁbrotic effects by expressing ﬁbroblast/myoﬁbroblast markers such as vimentin and alpha-smooth muscle actin (α-SMA).11 In addition, tubule epithelial cells interact with multiple cell types to trigger and promote the inﬂammatory and ﬁbrotic processes.1 Notably, proﬁbrotic phenotypes of epithelial cells are positively correlated with the ﬁbrotic severity in human renal allografts.13 In type 2 diabetic nephropathy, tubulointerstitial lesions also correlate better with renal dysfunctions than glomerulopathy.14 Taking together, these observations suggest a vital role of tubules in contributing to kidney diseases. y The TGF-β/Smad signaling pathway is a potent ﬁbrogenic pathway. Received: 6 December 2019 Revised: 10 February 2020 Accepted: 19 February 2020 1Department of Endocrinology & Metabolism, The Third Afﬁliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510630, China; 2Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; 3Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA and 4NHC Key Laboratory of Hormones and Development (Tianjin Medical University), Tianjin Key Laboratory of Metabolic Diseases, Tianjin Medical University Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin 300134, China Correspondence: Jian-xing Ma (jian-xing-ma@ouhsc.edu) Signal Transduction and Targeted Therapy Signal Transduction and Targeted Therapy Signal Transduction and Targeted Therapy www.nature.com/sigtrans Received: 6 December 2019 Revised: 10 February 2020 Accepted: 19 February 2020 ARTICLE OPEN A novel role of LRP5 in tubulointerstitial ﬁbrosis through activating TGF-β/Smad signaling Xuemin He1,2, Rui Cheng2, Chao Huang2, Yusuke Takahashi2,3, Yanhui Yang2,4, Siribhinya Benyajati2, Yanming Chen1, Xin A. Zhang2 and Jian-xing Ma2,3 Previous studies by us and others demonstrated that activation of Wnt/β-catenin signaling plays a pathogenic role in chronic kidney diseases (CKD). Wnt co-receptor LRP5 variants are reported to associate with autosomal dominant polycystic kidney disease; but their exact roles in this disease and renal ﬁbrosis have not been explored. Here, we observed the upregulation of LRP5 in the renal tubules of both type 1 and type 2 diabetic models and of an obstructive nephropathy model. In the obstructed kidneys, Lrp5 knockout signiﬁcantly ameliorated tubulointerstitial ﬁbrosis and tubular injury without changing Wnt/β-catenin signaling. Instead, decreased levels of TGF-β1 and TGF-β receptors (TβRs) were detected in Lrp5 knockout kidneys, followed by attenuated activation and nuclear translocation of Smad2/3 in the renal tubules, suggesting a regulatory effect of LRP5 on TGF-β/Smad signaling. In consistent with this hypothesis, LRP5 overexpression resulted in enhanced TGF-β/Smad signaling activation in renal tubule epithelial cells. Furthermore, LRP5 was co-immunoprecipitated with TβRI and TβRII, and its extracellular domain was essential for interacting with TβRs and for its pro-ﬁbrotic activity. In addition to stabilizing TβRs, LRP5 increased the basal membrane presentation and TGF-β1-induced internalization of these receptors. Notably, TGF-β1 also induced LRP5 internalization. These ﬁndings indicate that LRP5 promotes tubulointerstitial ﬁbrosis, at least partially, via direct modulation of TGF-β/Smad signaling, a novel, Wnt-independent function. ; https://doi.org/10.1038/s41392-020-0142-x ; https://doi.org/10.1038/s41392-020-0142-x Signal Transduction and Targeted Therapy (2020) 5:45 Signal Transduction and Targeted Therapy (2020) 5:45 Signal Transduction and Targeted Therapy (2020) 5:45 RESULTS Knockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules 4 Knockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules 4 LRP5 is signiﬁcantly upregulated in the renal tubules of diabetic and obstructive nephropathies LRP5 is signiﬁcantly upregulated in the renal tubules of diabetic and obstructive nephropathies LRP5 is known as a co-receptor of Wnt/β-catenin signaling;4 thus, we investigated whether knockout of Lrp5 affected Wnt/β-catenin signaling in the kidney. Under normal conditions, protein levels of another Wnt co-receptor LRP6 and the effector β-catenin in Lrp5−/−kidneys were not different from those in WT kidneys (Supplementary Fig. S4a, b). At day 10 post-UUO, although Wnt/ β-catenin signaling was activated by UUO, levels of non- phosphorylated β-catenin (non-p-β-catenin) and total β-catenin, key indicators of Wnt/β-catenin signaling activation, were not signiﬁcantly altered by Lrp5 knockout (Supplementary Fig. S4c, d). The mRNA levels of Wnt target genes, Cyclin D120 and PPARδ21, as well as nine canonical Wnt ligands including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt9a, Wnt10b, and Wnt16, were not changed by Lrp5 knockout (Supplementary Fig. S4e, f). These results suggest that the alleviation of UUO-induced renal ﬁbrosis in Lrp5−/−mice, at least at day 10 post-UUO, is surprisingly not mediated through the Wnt/β-catenin signaling pathway. The expression and distribution of LRP5 in CKD have not been previously characterized. Firstly, we measured renal LRP5 levels in diabetic models. Western blot analysis showed that renal LRP5 levels were signiﬁcantly increased in 3-month-old Akita mice (type 1 diabetes) and 6-month-old db/db mice (type 2 diabetes) relative to their genetic background- and age-matched wild-type (WT) non-diabetic controls, accompanied by increased renal levels of collagen I and collagen III (Fig. 1a–d). To determine the cellular localization of LRP5 in the kidney, we used an Lrp5 knockout (Lrp5−/−) mouse strain, in which the LacZ reporter gene is knocked in the locus of Lrp5. As shown by X-gal staining, Lrp5 was abundantly expressed in the renal tubules and moderately in the glomeruli (Supplementary Fig. S1a). Staining of LRP5 conﬁrmed the abundant distribution of LRP5 in the renal tubules of non- diabetic mice, and revealed that the diabetes-induced increases of LRP5 predominantly occurred in the renal tubules of Akita, db/db, and OVE26 (type 1 diabetes) mice (Fig. 1e, Supplementary Fig. S2a). RESULTS g β g g p y Previously, Lrp5 deﬁciency was reported to result in decreased TGF-β1 production in a pulmonary ﬁbrosis model.5 Therefore, we examined TGF-β1 levels at day 10 post-UUO. Consistently, total TGF- β1 levels were signiﬁcantly lower in the Lrp5−/−UUO kidneys, compared to those in the WT UUO kidneys (Fig. 3a–c). As TGF-β1 is a potent stimulator of TGF-β/Smad signaling,22 we measured this signaling pathway. UUO increased the levels of phosphorylated Smad2 and Smad3 (p-Smad2/3), Smad2/3, TβRI, and TβRII; mean- while, Lrp5 knockout down-regulated renal levels of these factors in UUO (Fig. 3d, e). Furthermore, we examined TβRI cellular localization at day 5 post-UUO, when the tubular structure was still mostly intact, and the tubulointerstitial ﬁbrosis was prominent. As shown in Fig. 3f, TβRI was predominantly upregulated in the tubules of the UUO kidneys, and not co-localized with α-SMA in the interstitial areas. Lrp5 knockout attenuated the upregulation of TβRI in the UUO kidneys. The nuclear translocation of Smad2/3 was also substantially decreased in Lrp5−/−kidneys compared to WT at day 10 post-UUO (Fig. 3f, g). Furthermore, by crossbreeding Lrp5−/−mice with SBE-Luc mice, a reporter strain for TGF-β/Smad signaling,23 we generated an Lrp5−/−/SBE-Luc strain. Consistently, Lrp5 knockout attenuated UUO- induced Smad2/3 activities at day 10 post-UUO (Fig. 3h). Interest- ingly, the depletion of Lrp5 in the kidney did not affect basal TGF-β/ Smad signaling under normal conditions (Supplementary Fig. S3h, i). Taken together, these results suggest that inhibition of TGF-β/Smad signaling contributes to the ameliorated tubulointerstitial ﬁbrosis in Lrp5−/−UUO kidneys. We further measured renal levels of LRP5 in the UUO model, which is also characterized by signiﬁcant renal ﬁbrosis.2 The LRP5 expression in UUO kidneys was evaluated at day 5 and day 10 post-UUO surgery. LRP5 was signiﬁcantly and persistently upregulated compared to that in the control kidneys, which correlated with the increases of α-SMA and connective tissue growth factor (CTGF) (Fig. 1f, g). Notably, the increase of LRP5 was predominantly in the dilated tubules (Fig. 1h), the major site for the tubulointerstitial ﬁbrosis. Knockout of Lrp5 alleviates the renal tubulointerstitial ﬁbrosis in CKD CKD As LRP5 levels were positively correlated with those of ﬁbrotic factors in diabetic and obstructed kidneys (Fig. 1), we further evaluated the role of LRP5 in renal ﬁbrosis using Lrp5−/−mice. Firstly, Lrp5−/−mice were examined under normal conditions. INTRODUCTION Activation of TGF-β/Smad signaling leads to extracellular matrix synthesis and deposition, podocyte depletion, mesangial expansion, tubule epithelial proﬁbrotic transformation, and myoﬁbroblast activation.6 In the kidneys with unilateral ureteral obstruction (UUO), levels of TGF-β receptor I and II (TβRI and TβRII) are predominantly elevated in renal tubules.7 Overexpression of a constitutively active mutant of TβRI in mouse renal tubules leads g y Although the expression of LRP5 is detected in normal human and mouse kidneys,15 its cellular distribution in the kidney has not been well characterized. Moreover, LRP5 variants are reported to associate with autosomal dominant polycystic kidney disease,16 © The Author(s) 2020 LRP5 interacts with TGF-β/Smad signaling He et al. LRP5 interacts with TGF-β/Smad signaling 2 but the role of LRP5 in renal ﬁbrosis and CKD has not been documented. The present study investigated pro-ﬁbrotic effect of LRP5 in CKD models such as diabetes and UUO, and identiﬁed a new role of LRP5 in regulating the TGF-β/Smad signaling pathway to promote renal ﬁbrosis. tubules by measuring the epithelial marker E-cadherin. Notably, UUO not only downregulates E-cadherin expression,18 but also induces E-cadherin re-distribution to the apical membrane.19 At day 5 post-UUO, Lrp5 knockout attenuated E-cadherin loss (Fig. 2c, d) and prevented E-cadherin re-distribution (Fig. 2e). Taken together, these results demonstrated that Lrp5 ablation alleviates renal tubulointerstitial ﬁbrosis. Signal Transduction and Targeted Therapy (2020) 5:45 RESULTS Lrp5−/−kidneys displayed similar levels of CTGF, ﬁbronectin, collagen I, and collagen III relative to those of controls (Supplementary Fig. S3a, b). In addition, the ratio of kidney weight to body weight, 24-h urine volume, urinary albumin excretion rate (UAE), and urinary albumin to creatinine ratio (ACR) of Lrp5−/−mice were not different from those of WT mice with sham surgery (Supplementary Fig. S2c–f). Periodic acid staining (PAS) also showed no obvious difference in glomerular and tubular structures in Lrp5−/−kidneys (Supplementary Fig. S3g). These ﬁndings suggest that Lrp5 knockout per se does not alter the renal structure and functions. LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells We utilized UUO to examine the effect of Lrp5 knockout on renal ﬁbrosis. At day 10 post-UUO surgery, Lrp5−/−kidneys displayed signiﬁcantly lower levels of CTGF, ﬁbronectin, collagen I, collagen III, and α-SMA, as shown by Western blot analysis (Fig. 2a, b) and by histochemical staining (Fig. 2e), compared to WT kidneys. In addition, we examined Lrp5’s role in renal ﬁbrosis of OVE26 mice, a type 1 diabetic model which displays progressive albuminuria, GFR decline and renal ﬁbrosis,17 recapitulating the phenotypes in diabetic patients. In consistent with the observa- tions in the UUO model, Lrp5 deﬁciency also resulted in signiﬁcant reductions of collagen I, collagen III, and α-SMA in the kidneys of OVE26 mice (Supplementary Fig. S2b–d). Due to LRP5’s polarized distribution in renal tubules, we next investigated LRP5’s effect on Our results demonstrated renal tubules as the major action site of UUO-induced LRP5 and TGF-β/Smad signaling, and renal tubule epithelial cells were thus utilized as a cell model in this study. Primary proximal tubule epithelial cells (PTECs) were isolated and conﬁrmed by immunostaining of sodium-glucose linked transpor- ter 1 (SGLT-1) (Supplementary Fig. S5). In Lrp5−/−PTECs, TGF-β1- elicited Smad2/3 phosphorylation was attenuated compared to that in WT PTECs (Fig. 4a). On the other hand, overexpression of LRP5 in HKC-8 cells, a human proximal tubule epithelial cell line, potentiated TGF-β1-induced Smad2/3 phosphorylation (Fig. 4b) and nuclear translocation (Fig. 4c). Measurement of nuclear fractions further demonstrated higher nuclear p-Smad2/3 levels Signal Transduction and Targeted Therapy (2020) 5:45 expressing cells (Fig. 4d, e). As shown by 3TP-Lux say a reporter system of Smad2/3 transcriptional and α-SMA.25–27 Consistently, ﬁbronectin, CTGF, expression was enhanced by LRP5 overexpression an LRP5 interacts with TGF-β/Smad signaling He et al. Signal Transduction and Targeted Therapy (2020) 5:45 RESULTS 1 Levels of LRP5 are upregulated in the renal tubules of CKD. a, c Western blot analyses and (b, d) densitometry quantiﬁcation of LRP5, collagen III, and collagen I in the kidney homogenates from 3-month-old Akita and 6-month-old db/db mice, as well as their respective age- and genetic background-matched, non-diabetic controls (n = 8–10). e Immunohistochemical staining of LRP5 (brown color) in the kidneys of Akita and db/db mice and their respective non-diabetic controls (n = 5–6; scale bar = 100 μm). The lower panels are enlarged images of the boxed areas in the upper panels. f Western blot analyses and (g) densitometry quantiﬁcation of LRP5, α-SMA, and CTGF in the kidney homogenates from WT mice with sham surgery or UUO at day 5 and day 10 post-surgery (n = 5–6). h Immunohistochemical staining of LRP5 (brown color) in the kidneys from WT mice with sham surgery or UUO at day 5 and day 10 (n = 5; scale bar = 75 μm). The lower panels are enlarged images of the boxed areas in the upper panels. Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.05; p < 0.01, by unpaired Student’s t-test LRP5 interacts with TβRs and promotes the formation of TβRI/TβRII heterodimers and TβRII levels were persistently decreased in the LRP5- overexpressing group (Fig. 6a, b). Interestingly, TGF-β1 also decreased the membrane levels of LRP5, correlating with the changes of membrane TβRs (Fig. 6a, b). We also examined the knockdown effect of LRP5 using a short hairpin RNA targeting LRP5 (shLRP5). Contrary to the effect of LRP5-overexpression, basal membrane levels of TβRI and TβRII were signiﬁcantly decreased by LRP5 knockdown alone, compared to those in the control group. Furthermore, the TGF-β1-induced membrane presentation of its receptors was attenuated by LRP5 knockdown at 7.5–15 min (Fig. 6c, d). We showed that UUO-induced prominent over-expression of LRP5 and TβRI in renal tubules. To understand whether UUO-induced LRP5 was co-localized with TβRI, we examined the UUO kidneys at day 5. Indeed, LRP5 and TβRI were co-localized in the dilated tubules of UUO kidneys (Fig. 5a). Using co-immunoprecipitation (Co-IP) in normal condition, endogenous LRP5 was found to co- precipitate with both endogenous TβRI and TβRII in renal tubule epithelial cells (Fig. 5b). By forced overexpression of LRP5 and TβRI in 293A cells, LRP5 also interacted with TβRI, which was independent of TGF-β1 (Fig. RESULTS 5c, d). Similar to TβRI, TβRII was co- precipitated with LRP5, independent of TGF-β1 (Fig. 5e, f). To identify the structural domain of LRP5 responsible for interacting with TβRs, we generated plasmids expressing the full-length LRP5 (LRP5FL), an LRP5 truncation mutant lacking the extracellular domain (LRP5ΔN), or an LRP5 truncation mutant lacking the intracellular domain (LRP5ΔC). LRP5FL and LRP5ΔC, but not LRP5ΔN, were associated with TβRI (Fig. 5g). 3TP-luciferase activity assay further conﬁrmed that only LRP5FL and LRP5ΔC potentiated TGF-β/Smad signaling in HKC-8 cells (Fig. 5h), suggesting that the extracellular domain of LRP5 confers the potentiating effect on TGF-β/Smad signaling. In addition, we examined the dynamics of internalized TβRs by evaluating the cytosolic fractions. In the control group, cytosolic TβRI and TβRII levels were transiently reduced at 7.5–15 min of TGF-β1 treatment, compared to those at 0 min, due to the trafﬁcking of these receptors to the plasma membrane. After 7.5–15 min, cytosolic TβRI and TβRII were gradually increased. With LRP5 overexpression, cytosolic TβRs were continuously increased after TGF-β1 treatment (Fig. 6f, g). Immunostaining of internalized TβRs at 7.5 min also conﬁrmed enhanced internaliza- tion of these receptors by LRP5 overexpression (Fig. 6e). Interestingly, we noticed that LRP5 was also internalized by upon TGF-β1 stimulation, correlating with the changes of TβRs. The increases of cytosolic TβRs were unlikely to result from de novo synthesis, as treatment of TGF-β1 for 30 min did not increase the total levels of these receptors (Fig. S7). On the contrary, knockdown of LRP5 induced decreases of cytosolic levels of TβRs (Fig. 6h, i). Taken together, these results suggest that LRP5 potentiates the basal level membrane presentation of TβRs and their internalization elicited by TGF-β1. Next, we evaluated the effect of LRP5 on the formation of TβRI/ TβRII heterodimers, an essential step for signaling activation.28 In the absence of TGF-β1, TβRI-His failed to co-precipitate with TβRII in the low-density lipoprotein receptor (LDLR) control group, consistent with the report that TβRI is separated from TβRII in quiescent state.29 In the LRP5-expressing cells, a signiﬁcant amount of TβRII co-precipitated with TβRI was detected (Fig. 5i), suggesting that LRP5 alone promotes the formation of TβRI/TβRII heterodimers. Notably, LRP5 was also co-precipitated with TβRI/ TβRII heterodimers. Upon TGF-β1 stimulation, TβRI quickly binds to TβRII, forming TβRI/TβRII heterodimers which subsequently activates Smad2/328. RESULTS LRP5 interacts with TGF-β/Smad signaling He et al. 3 expressing cells (Fig. 4d, e). As shown by 3TP-Lux say, a reporter system of Smad2/3 transcriptional RP5 overexpression enhanced the transcriptional mad2/3 induced by TGF-β1 (Fig. 4f). We also measured on of its downstream target genes ﬁbronectin, CTGF, and α-SMA.25–27 Consistently, ﬁbronectin, CTGF, and α-SMA expression was enhanced by LRP5 overexpression and attenuated by Lrp5 knockout (Fig. 4g, h). Thus, we have demonstrated that LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. and α-SMA.25–27 Consistently, ﬁbronectin, CTGF, and α-SMA expression was enhanced by LRP5 overexpression and attenuated by Lrp5 knockout (Fig. 4g, h). Thus, we have demonstrated that LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. in LRP5-overexpressing cells (Fig. 4d, e). As shown by 3TP-Lux luciferase assay, a reporter system of Smad2/3 transcriptional activity,24 LRP5 overexpression enhanced the transcriptional activity of Smad2/3 induced by TGF-β1 (Fig. 4f). We also measured the expression of its downstream target genes ﬁbronectin, CTGF, Signal Transduction and Targeted Therapy (2020) 5:45 Fig. 1 Levels of LRP5 are upregulated in the renal tubules of CKD. a, c Western blot analyses and (b, d) densitometry quantiﬁcation of LRP5, collagen III, and collagen I in the kidney homogenates from 3-month-old Akita and 6-month-old db/db mice, as well as their respective age- and genetic background-matched, non-diabetic controls (n = 8–10). e Immunohistochemical staining of LRP5 (brown color) in the kidneys of Akita and db/db mice and their respective non-diabetic controls (n = 5–6; scale bar = 100 μm). The lower panels are enlarged images of the boxed areas in the upper panels. f Western blot analyses and (g) densitometry quantiﬁcation of LRP5, α-SMA, and CTGF in the kidney homogenates from WT mice with sham surgery or UUO at day 5 and day 10 post-surgery (n = 5–6). h Immunohistochemical staining of LRP5 (brown color) in the kidneys from WT mice with sham surgery or UUO at day 5 and day 10 (n = 5; scale bar = 75 μm). The lower panels are enlarged images of the boxed areas in the upper panels. Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.05; p < 0.01, by unpaired Student’s t-test LRP5 interacts with TGF-β/Smad signaling He et al. LRP5 interacts with TGF-β/Smad signaling He et al. LRP5 interacts with TGF-β/Smad signaling H l 4 Fig. RESULTS Indeed, after TGF-β1 treatment for 15 min, a signiﬁcant amount of co-precipitated TβRII was detected in the LDLR control group. In the LRP5 group, co-precipitation of TβRII was decreased (Fig. 5i), which is ascribed to the fact that TβRI dissociates from TβRII after Smad2/3 becomes phosphorylated.30 Furthermore, we noticed that LRP5 overexpression signiﬁcantly increased the protein levels but not mRNA levels of TβRs (Supplementary Fig. S6a–c). As shown by protein stability analysis, LRP5 prolonged the half-lives of TβRs (Supplementary Fig. S6d, e). Signal Transduction and Targeted Therapy (2020) 5:45 DISCUSSION Th The present study identiﬁed a previously undocumented pro- ﬁbrotic function of LRP5 through direct interactions with TβRs in renal ﬁbrosis. Notably, LRP5 was upregulated in the renal tubules of CKD models. Moreover, knockout of Lrp5 ameliorated the tubulointerstitial ﬁbrosis in UUO kidneys, independent of Wnt/ β-catenin signaling. The results of Smad2/3 nuclear translocation and luciferase activity in SBE-Luc reporter mice provided solid in vivo evidence that Lrp5 knockout signiﬁcantly attenuated UUO- induced TGF-β/Smad signaling activation. On the other hand, overexpression of LRP5 potentiated TGF-β/Smad signaling in renal tubule epithelial cells. As to the mechanism underlying this proﬁbrotic activity of LRP5, the present study showed for the ﬁrst time that LRP5 interacted directly with TβRI and TβRII, prolonged their half-lives, promoted the formation of TβRI/TβRII hetero- dimers, and enhanced membrane presentation and TGF-β1- elicited internalization of these receptors. Our studies presented evidence suggesting that the pro-ﬁbrotic activity of LRP5 in the kidney is through a mechanism independent of Wnt/β-catenin signaling, which may represent a new pathogenic mechanism for the progression of renal ﬁbrosis in CKD. LRP5 promotes the basal level membrane presentation and ligand-induced internalization of TβRs The availability of TβRs on the plasma membrane is another important factor affecting TGF-β signaling.28 Firstly, LRP5 was detected in the membrane, and its basal membrane levels were ~60% of total LRP5 in endogenous and forced overexpressed conditions (Supplementary Fig. S1b, d, e). Cells were fractionated to examine the dynamics of membrane TβRs in response to TGF- β1 stimulation. Notably, LRP5 overexpression alone increased the basal membrane levels of TβRI and TβRII. Upon TGF-β1 treatment, membrane TβRI and TβRII levels were transiently increased at 7.5–15 min of TGF-β1 treatment in the control pcDNA3 vector group, and then gradually decreased. However, membrane TβRI p g This study showed that LRP5 was upregulated in the renal tubules of both type 1 and type 2 diabetic models (Fig. 1e) and of the UUO model (Fig. 1h), consistent with previous studies.31,32 In alignment, we found that both high glucose and TGF-β1 potently LRP5 interacts with TGF-β/Smad signaling He et al. ckout of Lrp5 alleviates the renal tubulointerstitial ﬁbrosis in CKD. DISCUSSION Th a, c Western blot analyses and (b, d) densitometry quantiﬁc nectin, collagen I, and collagen III in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–7), and (c, d) E-ca kid f WT d L / i d ( 6) S i i f ll III (b l l b 200 5 g. 2 Knockout of Lrp5 alleviates the renal tubulointerstitial ﬁbrosis in CKD. a, c Western blot analyses and (b, d) densitometry quantiﬁc TGF, ﬁbronectin, collagen I, and collagen III in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–7), and (c, d) E-cad MA in the kidneys from WT and Lrp5−/−mice at day 5 post-surgery (n = 5–6). e Staining of collagen III (brown color; scale bar = 200 μ rius red (red color; scale bar = 200 μm) in the kidneys from WT and Lrp5−/−mice with sham surgery or UUO at day 10 post-surgery, an -SMA (green color; scale bar = 75 μm) and E-cadherin (green color; scale bar = 100 μm) in the kidneys from WT and Lrp5−/−mice with r UUO at day 5 post-surgery. The lower panels are enlarged images of the boxed areas in the upper panels. White arrows indic istribution of E-cadherin in the basolateral membrane of tubules; red arrows indicate re-distribution of E-cadherin to the apical m ubules. n = 5–6. Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.01, by two-way ANOVA w multiple comparisons t of Lrp5 alleviates the renal tubulointerstitial ﬁbrosis in CKD. a, c Western blot analyses and (b, d) densitometry quant n, collagen I, and collagen III in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–7), and (c, d) E- eys from WT and Lrp5−/−mice at day 5 post-surgery (n = 5–6). e Staining of collagen III (brown color; scale bar = 200 olor; scale bar = 200 μm) in the kidneys from WT and Lrp5−/−mice with sham surgery or UUO at day 10 post-surgery, olor; scale bar = 75 μm) and E-cadherin (green color; scale bar = 100 μm) in the kidneys from WT and Lrp5−/−mice wit 5 post-surgery. The lower panels are enlarged images of the boxed areas in the upper panels. White arrows ind E-cadherin in the basolateral membrane of tubules; red arrows indicate re-distribution of E-cadherin to the apica . Signal Transduction and Targeted Therapy (2020) 5:45 DISCUSSION Th d Western blot analys etry quantiﬁcation of p-Smad2/3, total Smad2/3, TβRI and TβRII in the kidneys from WT and Lrp5−/−mice at day 10 post-sur munostaining of TβRI (red color; scale bar = 100 μm) and α-SMA (green color; scale bar = 100 μm) in the kidney sections fro mice at day 5 post-surgery (n = 5–7), and of Smad2/3 (red color; scale bar = 25 μm), and (g) quantiﬁcation of nuclear Smad ections from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–10). h Luciferase activity measurement in the kidney hom /SBE-Luc and Lrp5−/−/SBE-Luc mice at day 10 post-surgery (n = 5–6). Each lane represents an individual mouse. All v d as mean ± SEM. p < 0.01; n.s., not signiﬁcant, by two-way ANOVA with pair-wise multiple comparisons Signal Transduction and Targeted Therapy (2020) 5:45 ockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules. a Western blot analyses, (b) densitometry qua LISA of total TGF-β1 in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 6–8). d Western blot analys etry quantiﬁcation of p-Smad2/3, total Smad2/3, TβRI and TβRII in the kidneys from WT and Lrp5−/−mice at day 10 post-su munostaining of TβRI (red color; scale bar = 100 μm) and α-SMA (green color; scale bar = 100 μm) in the kidney sections fro ice at day 5 post-surgery (n = 5–7), and of Smad2/3 (red color; scale bar = 25 μm), and (g) quantiﬁcation of nuclear Smad ctions from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–10). h Luciferase activity measurement in the kidney hom SBE-Luc and Lrp5−/−/SBE-Luc mice at day 10 post-surgery (n = 5–6). Each lane represents an individual mouse. All as mean ± SEM. p < 0.01; n.s., not signiﬁcant, by two-way ANOVA with pair-wise multiple comparisons Fig. 3 Knockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules. a Western blot analyses, (b) densitometry quantiﬁcation and (c) ELISA of total TGF-β1 in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 6–8). d Western blot analyses and (e) densitometry quantiﬁcation of p-Smad2/3, total Smad2/3, TβRI and TβRII in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–7). DISCUSSION Th Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.01, by two-way ANOVA risons ion and Targeted Therapy (2020)5:45 Fig. 2 Knockout of Lrp5 alleviates the renal tubulointerstitial ﬁbrosis in CKD. a, c Western blot analyses and (b, d) densitometry quantiﬁcation of (a, b) CTGF, ﬁbronectin, collagen I, and collagen III in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–7), and (c, d) E-cadherin and α- SMA in the kidneys from WT and Lrp5−/−mice at day 5 post-surgery (n = 5–6). e Staining of collagen III (brown color; scale bar = 200 μm) and picro- sirius red (red color; scale bar = 200 μm) in the kidneys from WT and Lrp5−/−mice with sham surgery or UUO at day 10 post-surgery, and staining of α-SMA (green color; scale bar = 75 μm) and E-cadherin (green color; scale bar = 100 μm) in the kidneys from WT and Lrp5−/−mice with sham surgery or UUO at day 5 post-surgery. The lower panels are enlarged images of the boxed areas in the upper panels. White arrows indicate polarized distribution of E-cadherin in the basolateral membrane of tubules; red arrows indicate re-distribution of E-cadherin to the apical membrane of tubules. n = 5–6. Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.01, by two-way ANOVA with pair-wise multiple comparisons Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. 3 Knockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules. a Western blot analyses, (b) densitometry quan (c) ELISA of total TGF-β1 in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 6–8). d Western blot analyse i iﬁ i f S d2/3 l S d2/3 TβRI d TβRII i h kid f WT d L 5 / i d 10 He et al. 6 ockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules. a Western blot analyses, (b) densitometry qua LISA of total TGF-β1 in the kidneys from WT and Lrp5−/−mice at day 10 post-surgery (n = 6–8). DISCUSSION Th g, h Western blot analyses of ﬁbronectin, CTGF, and α-SMA (g) in primary PTECs from WT and Lrp5−/−mice exposed to ndicated concentrations of TGF-β1 for 24 h, and (h) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for followed by the treatment with/without 2 ng/ml TGF-β1 for 24 h. Representative images are shown from at least 3 independent riments. All values are expressed as mean ± SEM. p < 0.01, by two-way ANOVA with pair-wise multiple comparisons β g g He et al. 7 Fig. 4 LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. a, b Western blot analyses of p-Smad2/3 and total Smad2/3 (a) in WT PTECs and Lrp5−/−PTECs treated with 2 ng/ml TGF-β1 for the indicated times, and (b) in HKC-8 cells after transfection of a control plasmid (pcDNA3) or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for the indicated times. c Immunostaining of Smad2/3 (red color; scale bar = 25 μm) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min. d Western blot analyses and (e) densitometry quantiﬁcation of nuclear levels of p-Smad2/3 and Smad2/3 in the fractions extracted from HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min (n = 6). f 3TP-Lux luciferase activity assay in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 hr, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control (n = 3). g, h Western blot analyses of ﬁbronectin, CTGF, and α-SMA (g) in primary PTECs from WT and Lrp5−/−mice exposed to the indicated concentrations of TGF-β1 for 24 h, and (h) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 24 h. Representative images are shown from at least 3 independent experiments. All values are expressed as mean ± SEM. DISCUSSION Th f Immunostaining of TβRI (red color; scale bar = 100 μm) and α-SMA (green color; scale bar = 100 μm) in the kidney sections from WT and Lrp5−/−mice at day 5 post-surgery (n = 5–7), and of Smad2/3 (red color; scale bar = 25 μm), and (g) quantiﬁcation of nuclear Smad2/3 in the kidney sections from WT and Lrp5−/−mice at day 10 post-surgery (n = 5–10). h Luciferase activity measurement in the kidney homogenates from WT/SBE-Luc and Lrp5−/−/SBE-Luc mice at day 10 post-surgery (n = 5–6). Each lane represents an individual mouse. All values are expressed as mean ± SEM. p < 0.01; n.s., not signiﬁcant, by two-way ANOVA with pair-wise multiple comparisons Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. eased LRP5 levels in renal tubule epithelial cells in vitro microvilli of renal tubules, although no obvious polarized LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. a, b Western blot analyses of p-Smad2/3 and total Smad2/3 (a) in PTECs and Lrp5−/−PTECs treated with 2 ng/ml TGF-β1 for the indicated times, and (b) in HKC-8 cells after transfection of a control plasmid NA3) or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for the indicated times. c unostaining of Smad2/3 (red color; scale bar = 25 μm) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 8 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min. d Western blot analyses and (e) densitometry quantiﬁcation of ear levels of p-Smad2/3 and Smad2/3 in the fractions extracted from HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid essing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min (n = 6). f 3TP-Lux luciferase activity assay in HKC-8 after transfection of the 3TP-Lux plasmid, a renilla plasmid, and a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 hr, followed by the ment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of NA3 control (n = 3). Signal Transduction and Targeted Therapy (2020) 5:45 DISCUSSION Th p < 0.01, by two-way ANOVA with pair-wise multiple comparisons microvilli of renal tubules, although no obvious polarized distribution of LRP5 was detected in those of non-diabetic kidneys (Supplementary Fig. S1c). We assume that this polarized distribu- tion of LRP5 may play a role in reabsorbing glucose by renal tubules in response to diabetes, a property of LRP5 which has been demonstrated in mammary epithelial cells.33 increased LRP5 levels in renal tubule epithelial cells in vitro (Supplementary Fig. S8), which recapitulated the changes of LRP5 in diabetic and UUO kidneys, respectively. However, TGF-β1 did not signiﬁcantly prolong the half-life of LRP5 protein (Fig. S9), suggesting that the increase of LRP5 may be regulated at the transcriptional level. Thus, it remains to be elucidated how TGF-β1 regulates the transcription of LRP5. Notably, diabetes-induced LRP5 was predominantly located on the apical membrane and increased LRP5 levels in renal tubule epithelial cells in vitro (Supplementary Fig. S8), which recapitulated the changes of LRP5 in diabetic and UUO kidneys, respectively. However, TGF-β1 did not signiﬁcantly prolong the half-life of LRP5 protein (Fig. S9), suggesting that the increase of LRP5 may be regulated at the transcriptional level. Thus, it remains to be elucidated how TGF-β1 regulates the transcription of LRP5. Notably, diabetes-induced LRP5 was predominantly located on the apical membrane and On the other hand, LRP5 affected TGF-β1 levels, as our results showed that renal levels of total TGF-β1 were reduced in Lrp5−/− Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. 8 UUO kidneys, which was consistent with decreased total TGF-β1 production in IPF.5 It is not well understood how LRP5 affects total TGF-β1 levels in these ﬁbrotic diseases. The regulation of TGF-β1 levels by LRP5 is unlikely through Wnt/β-catenin signaling,12 as the Wnt/β-catenin signaling activity was not signiﬁcantly altered by Lrp5 knockout at day 10 post-UUO (Supplementary Fig. S4c–f). It has been reported that TGF-β/Smad signaling upregulates TGF- β1 expression,34 which may explain the regulation of total TGF-β1 by LRP5. Interestingly, Lrp5 deﬁciency did not reduce the response to TGF-β1 in lung ﬁbroblasts and alveolar epithelial cells,5 which is contrast to our observation in renal tubule epithelial cells (Fig. 4). It is noteworthy that LRP5 expression was not measured in lung ﬁbroblasts or alveolar epithelial cells in the previous study, while LRP5 was abundant in renal tubule epithelial cells. Signal Transduction and Targeted Therapy (2020) 5:45 MATERIALS AND METHODS property which is also possessed by neuropilin-1,35 another co- receptor of TGF-β/Smad signaling. It is not known why over- expression of this Wnt co-receptor alone could trigger the formation of TβRI/TβRII heterodimers, which theoretically only occurs when TGF-β1 is present. Notably, when TGF-β1 was applied, LRP5 internalization was detected, sharing the same trend as TβRs, which also indirectly indicates that LRP5 binds to TβRI/ TβRII heterodimers to undergo internalization. Animal models Akita, db/db, Lrp5−/−(B6.129P2-Lrp5tm1Dgen/J), and SBE-Luc mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Lrp5−/−mice were cross-bred with SBE-Luc mice to generate Lrp5−/−/SBE-Luc mice, cross-bred with OVE26 mice to generate Lrp5+/−/OVE26 mice. Eight-week-old mice were randomly assigned to sham surgery or UUO surgery according to an established protocol.2 Brieﬂy, the left ureter was double ligated using 4-0 silk. Ureters of the sham control were manipulated without ligations. Left kidneys were collected for analyses at day 5 or day 10 post-surgery. All the procedures on mice were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center (protocol number: 14-032-SSHT). β g It is well-known that the crosstalk between Wnt/β-catenin signaling and TGF-β/Smad signaling occurs at multiple subcellular levels including the extracellular, cytoplasmic, and nuclear levels.36 TGF-β/Smad increases the levels of Wnt ligands and enhances Wnt ligand-elicited β-catenin stabilization in vascular smooth muscle cells.37 In addition, TGF-β1 suppresses DKK-1 to promote Wnt/ β-catenin signaling activation in human ﬁbroblasts.38 On the other hand, suppression of Wnt/β-catenin signaling results in attenua- tion of TGF-β/Smad-mediated ﬁbrosis.38 Binding of transcription factors Smad4 and β-catenin/Lef1 has also been reported.39 Our study focused on analyzing changes at day 10 post-UUO, when we did not detect any signiﬁcant changes in canonical Wnt ligands, the effector, or downstream target genes (Supplementary Fig. S4c–f). However, it does not exclude the possibility that Lrp5 knockout affects Wnt/β-catenin signaling activation at other time points after UUO. We are aware and acknowledge that it is very challenging to exclude the involvement of Wnt/β-catenin signal- ing when measuring TGF-β/Smad signaling in ﬁbrotic disease conditions, due to their reciprocal regulations and interactions. MATERIALS AND METHODS Notably, in the study of Lrp5 deﬁciency in pulmonary ﬁbrosis, the authors also acknowledged that LRP5 may play a β-catenin- independent role to regulate ﬁbrosis, supporting our ﬁnding in the kidney.5 LRP6, another LDLR family member, mediates the inhibitory effect of DKK-1 through the TGF-β-JNK signaling axis in renal pericytes,40 suggesting a role of LRP6 independent of β-catenin. This study thus attests LRP5’s effect on promoting the ﬁbrotic process independent of β-catenin. Measurements of renal functions Measurements of renal functions Twenty-four-hour urine samples were collected. Urinary creatinine was measured by HPLC as previously described.3 Urinary albumin was measured using an ELISA kit for mouse albumin (Exocell, Philadelphia, PA). culture, transfection, fractionation, and Co-IP assays Cell culture, transfection, fractionation, and Co-IP assays PTECs were isolated according to an established protocol.44 Brieﬂy, the cortex of the kidney was dissected out carefully and chopped into small pieces. Then 1 mg/ml of collagenase solution was applied and incubated at 37 °C for 30 min, with gentle agitation. The digestion was terminated by FBS and then sequentially ﬁltered. Fragmented proximal tubules were collected, and maintained in renal epithelial cell basal medium with a growth kit. The purity of PTECs was conﬁrmed by immunostaining of SGLT1 (07-1417, EMD Millipore, Temecula, CA). Cells of passage 2–5 were used for experiments. p HKC-8, a human renal proximal tubule epithelial cell line (a kind gift from Dr. L. Racusen, the Johns Hopkins University, Baltimore, MD), and HEK293A cells were transfected and then cultured for 48 h before the treatments. Plasmids expressing LRP5FL, LRP5ΔN, and LRP5ΔC were generated. pcDNA3 plasmid was purchased from the Thermo Fisher Scientiﬁc (Waltham, MA), and LDLR-Flag plasmid was obtained from the Addgene (Cambridge, MA). p p β LRP5 levels are upregulated in CKD, correlating with renal ﬁbrosis. Thus, it will be very interesting to study if human patients with loss-of-function mutations of LRP5 are less likely to develop renal ﬁbrosis in CKD. In addition, we observed increased LRP5 levels in the diabetic glomeruli, but whether LRP5 regulates the survival and foot effacement of podocytes remains to be explored under diabetic conditions. Furthermore, LRP5 expression is detected in macrophages41 and monocytes,42 cells that play critical roles in the renal ﬁbrogenesis.43 It is undetermined if LRP5 plays a role in these inﬂammatory cells and what signaling pathways mediate its effect. DISCUSSION Th e, f IP of (e) LRP5 or (f) TβRII in HKC-8 cells co- transfected with TβRII and LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRII and LRP5 were precipitated using their speciﬁc antibodies. The precipitated proteins were immunoblotted for TβRII and LRP5. g IP of TβRI in HEK293A cells co-transfected with TβRI-His and LRP5FL-Flag, LRP5ΔN-Flag or LRP5ΔC-Flag for 48 h. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His and Flag. h Measurement of 3TP-Lux luciferase activity in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid and a control plasmid or plasmid expressing LRP5FL, LRP5ΔN, or LRP5ΔC for 48 h, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control (n = 3). i IP of TβRI in HKC-8 cells co-transfected with TβRI-His and LRP5-Flag or LDLR-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His, Flag, and TβRII. Arrowheads indicate non-speciﬁc bands; arrows indicate target proteins. All values are expressed as mean ± SEM. p < 0.01; n.s., not signiﬁcant, by two-way ANOVA with pair-wise multiple comparisons DISCUSSION Th Therefore, it is unclear whether the divergent responses to TGF-β1 are attributed to different expression levels of LRP5 in these cell types. In the absence of TGF-β1, increased formation of TβRI/TβRII heterodimers was observed after LRP5 overexpression (Fig. 5i), a Signal Transduction and Targeted Therapy (2020) 5:45 UUO kidneys, which was consistent with decreased total TGF-β1 production in IPF.5 It is not well understood how LRP5 affects total TGF-β1 levels in these ﬁbrotic diseases. The regulation of TGF-β1 levels by LRP5 is unlikely through Wnt/β-catenin signaling,12 as the Wnt/β-catenin signaling activity was not signiﬁcantly altered by Lrp5 knockout at day 10 post-UUO (Supplementary Fig. S4c–f). It has been reported that TGF-β/Smad signaling upregulates TGF- β1 expression,34 which may explain the regulation of total TGF-β1 by LRP5. Interestingly, Lrp5 deﬁciency did not reduce the response to TGF-β1 in lung ﬁbroblasts and alveolar epithelial cells,5 which is contrast to our observation in renal tubule epithelial cells (Fig. 4). It is noteworthy that LRP5 expression was not measured in lung ﬁbroblasts or alveolar epithelial cells in the previous study, while LRP5 was abundant in renal tubule epithelial cells. Therefore, it is unclear whether the divergent responses to TGF-β1 are attributed to different expression levels of LRP5 in these cell types. In the absence of TGF-β1, increased formation of TβRI/TβRII heterodimers was observed after LRP5 overexpression (Fig. 5i), a LRP5 interacts with TGF-β/Smad signaling He et al. LRP5 interacts with TGF-β/Smad signaling 9 Fig. 5 LRP5 interacts with TβRs and stimulates the formation of TβRI/TβRII heterodimers. a Immunostaining of LRP5 (green color; scale bar = 25 μm) and TβRI (red color; scale bar = 25 μm) in the kidney sections from WT mice at day 5 post-surgery showing co-localization of LRP5 and TβRI in the injured tubules of UUO kidneys (n = 5–7). b IP of endogenous LRP5 in HKC-8 cells. LRP5 was precipitated with its speciﬁc antibody. The precipitated proteins were immunoblotted for TβRI, TβRII, and LRP5. c, d IP of (c) TβRI and (d) LRP5 in HEK293A cells co-transfected with TβRI-His and LRP5-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 15 min. TβRI-His was precipitated by Ni-NTA resin and LRP5-Flag by anti-Flag resin. The precipitated proteins were immunoblotted for His and Flag. Signal Transduction and Targeted Therapy (2020) 5:45 MATERIALS AND METHODS In summary, we have identiﬁed a new proﬁbrotic role of LRP5 in driving renal ﬁbrosis via direct interaction with TGF-β/Smad signaling, suggesting LRP5 expression as a prognostic marker for the progression of CKD. Cycloheximide (Sigma-Aldrich, Saint Louis, MO) treatment at 50 μM was used to measure the protein stability, with DMSO (Sigma- Aldrich, Saint Louis, MO) as control. Fractionation of cell lysates was performed according to the manufacturer’s instructions (BioVision Technology, Chester Spring, PA). For Co-IP assays, cells were washed with PBS, lysed in the IP buffer,45 and then proteins were pulled down using anti-Flag resin (Thermo Fisher Scientiﬁc, Waltham, MA), Ni-NTA resin (Thermo Fisher Scientiﬁc, Waltham, MA), and TβRII antibody (sc-17792, Santa Cruz, Dallas, TX). Precipitated proteins were then subjected to Western blot analysis. Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. 10 5 promotes the basal membrane presentation and ligand-induced internalization of TβRs. a, c, f, h Western blot analyses ometry quantiﬁcation of TβRI, TβRII, and LRP5 in the cell factions from HKC-8 cells after transfection with (a, b, f, g) a contr r a plasmid expressing LRP5, and (c, d, h, i) a control plasmid (sh-scramble) or a plasmid expressing shRNA for LRP5 (s wed by the treatment of 2 ng/ml TGF-β1 for the indicated times. Cells were fractionated and immunoblotted for TβRI, pan-cadherin and α-tubulin as loading controls (n = 3). e Immunostaining of internalized TβRI, TβRII, and LRP5 in HKC-8 n of pcDNA3 or a plasmid expressing LRP5 together with either a plasmid expressing TβRI or TβRII for 48 hr, follow of 2 ng/ml TGF-β1 for 7.5 min (green color; scale bar = 10 μm). Representative images are shown from at least three ind s. All values are expressed as mean ± SEM. p < 0.05; p < 0.01, by unpaired Student’s t-test Fig. 6 LRP5 promotes the basal membrane presentation and ligand-induced internalization of TβRs. a, c, f, h Western blot analyses and (b, d, g, i) densitometry quantiﬁcation of TβRI, TβRII, and LRP5 in the cell factions from HKC-8 cells after transfection with (a, b, f, g) a control plasmid (pcDNA3) or a plasmid expressing LRP5, and (c, d, h, i) a control plasmid (sh-scramble) or a plasmid expressing shRNA for LRP5 (shLRP5) for 48 h, followed by the treatment of 2 ng/ml TGF-β1 for the indicated times. REFERENCES et al. Wnt coreceptor Lrp5 is a driver of idiopathic pulmonary ﬁbrosis. Am. J. Respir. Crit. Care Med. 190, 185–195 (2014). 6. Bottinger, E. P. & Bitzer, M. TGF-beta signaling in renal disease. J. Am. Soc. Nephrol. 13, 2600–2610 (2002). 7. Sutaria, P. M. et al. Transforming growth factor-beta receptor types I and II are expressed in renal tubules and are increased after chronic unilateral ureteral obstruction. Life Sci. 62, 1965–1972 (1998). 8. Gentle, M. E. et al. Epithelial cell TGFbeta signaling induces acute tubular injury and interstitial inﬂammation. J. Am. Soc. Nephrol. 24, 787–799 (2013). 9. Chevalier, R. L. The proximal tubule is the primary target of injury and progression of kidney disease: role of the glomerulotubular junction. Am. J. Physiol. Ren. Physiol. 311, F145–F161 (2016). 10. Bagby, S. P. Diabetic nephropathy and proximal tubule ROS: challenging our glomerulocentricity. Kidney Int. 71, 1199–1202 (2007). 11. Yang, L. et al. Epithelial cell cycle arrest in G2/M mediates kidney ﬁbrosis after injury. Nat. Med. 16, 535–543 (2010). Internalization of membrane receptors HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc- 390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS. Cells were then incubated in pre-warmed medium containing 2 ng/ml TGF-β1 at 37 °C for 7.5 min to stimulate the internalization. Non- internalized primary antibodies were removed with 0.2 M glycine buffer (pH 2.5). Then the cells were ﬁxed with 4% paraformaldehyde and permeabilized with 0.1% Brij98 in PBS, and blocked with 20% goat serum in PBS. Internalized primary antibodies were detected with Alexa-488 conjugated goat anti- rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientiﬁc, Waltham, MA). 12. He, X. et al. Pigment epithelium-derived factor, a noninhibitory serine protease inhibitor, is renoprotective by inhibiting the Wnt pathway. Kidney Int. 91, 642–657 (2017). 13. Xu-Dubois, Y. C. et al. Epithelial phenotypic changes are associated with a tubular active ﬁbrogenic process in human renal grafts. Hum. Pathol. 44, 1251–1261 (2013). 14. Brocco, E. et al. Renal structure and function in non-insulin dependent diabetic patients with microalbuminuria. Kidney Int. Suppl. 63, S40–S44 (1997). 15. Figueroa, D. J. et al. Competing interests The authors declare no competing interests. ACKNOWLEDGEMENTS Real-time PCR Real-time PCR was performed as described previously.3 Sequences of primer pairs for genes were listed in Table S1 or referred to a documented study.12 We thank the Diabetes COBRE Imaging Facility, and the Diabetic Animal Core at the University of Oklahoma Health Sciences Center for the technical supports. This study was supported by a grant from the Oklahoma Center for the Advancement of Science and Technology (HR16-041) and NIH grants EY012231, EY018659, EY028949, EY019309, and GM122744, a grant from National Nature Science Foundation of China NO. 81700631 and Science & Technology Development Fund of Tianjin Education Commission for Higher Education 2016YD05. Western blot analysis and ELISA y Western blot analysis was performed as previously described.46 Antibodies used for Western blot analysis were listed in the Table S2. GAPDH, β-actin, α-tubulin, and pan-cadherin were used as loading controls. Individual band in Western blots was semi- quantiﬁed by densitometry using the AlphaView software (Alpha Innotech, Santa Clara, CA). The densitometry of target proteins was normalized to that of individual housekeeping protein. Then the normalized ratio values were averaged in the control group. The averages of other groups were divided by that of the control group, to obtain the “fold change over the control” (with control group as “1”). TGF-β1 ELISA was performed according to the manufacturer’s instruction (R&D Systems, Minneapolis, MN). AUTHOR CONTRIBUTIONS X.H., R.C., and J.M. designed experiments. X.H. wrote the draft of the manuscript, R.C., Y.T., S.B., Y.C., and J.M. edited it. X.H., R.C., C.H., Y.Y., and X.Z. performed experiments, X.H., R.C., and J.M. designed experiments. X.H. wrote the draft of the manuscript, R.C., Y.T., S.B., Y.C., and J.M. edited it. X.H., R.C., C.H., Y.Y., and X.Z. performed experiments, d X H l d th d t Y T t t d th LRP5 d LRP5 t t l id X.H., R.C., and J.M. designed experiments. X.H. wrote the draft of the manuscript, R.C., Y.T., S.B., Y.C., and J.M. edited it. X.H., R.C., C.H., Y.Y., and X.Z. performed experiments, and X.H. analyzed the data. Y.T. constructed the LRP5 and LRP5 mutant plasmids. MATERIALS AND METHODS Cells were fractionated and immunoblotted for TβRI, TβRII, and LRP5, with pan-cadherin and α-tubulin as loading controls (n = 3). e Immunostaining of internalized TβRI, TβRII, and LRP5 in HKC-8 cells after transfection of pcDNA3 or a plasmid expressing LRP5 together with either a plasmid expressing TβRI or TβRII for 48 hr, followed by the treatment of 2 ng/ml TGF-β1 for 7.5 min (green color; scale bar = 10 μm). Representative images are shown from at least three independent experiments. All values are expressed as mean ± SEM. p < 0.05; **p < 0.01, by unpaired Student’s t-test Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. He et al. 11 REFERENCES Luciferase activities measurement Luciferase activities measurement Measurement of luciferase activity in cell lysates or kidney Luciferase activities measurement Measurement of luciferase activity in cell lysates or kidney homogenates was performed according to the manufacturer’s 1. Liu, Y. Cellular and molecular mechanisms of renal ﬁbrosis. Nat. Rev. Nephrol. 7, 684–696 (2011). Measurement of luciferase activity in cell lysates or kidney homogenates was performed according to the manufacturer’s instructions (Promega, Madison, WI). 2. He, W. et al. Wnt/beta-catenin signaling promotes renal interstitial ﬁbrosis. J. Am. Soc. Nephrol. 20, 765–776 (2009). 3. Zhou, T. et al. Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy. Diabetologia 55, 255–266 (2012). Immunohistochemistry, immunoﬂuorescence, picro-sirius red, X- gal staining, and PAS Immunohistochemistry, immunoﬂuorescence, picro-sirius red, X- gal staining, and PAS d d f l ld h 4. Mao, J. et al. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7, 801–809 (2001). g g Kidney cryosections were used for X-gal staining (Sigma-Aldrich, Saint Louis, MO) as previously described,45 or immunostaining of Smad2/3 (sc-8332, Santa Cruz, Dallas, TX). Parafﬁn-embedded kidney sections were used for picro-sirius red staining (Roche Life Science, Indianapolis, IN), PAS (Promega, Madison, WI), immuno- histochemical staining of LRP5 (ab38311, Abcam, Cambridge, MA) and collagen III (NBP2-15946, BD Pharmingen, San Jose, CA), and immunoﬂuorescence staining of α-SMA (sc-32251, Santa Cruz, Dallas, TX), E-cadherin (610182, BD Biosciences, San Jose, CA), TβRI (NBP1-01037, Novus, Littleton, CO), SGLT-2 (ab37296, Abcam, Cambridge, MA) and LRP5 (ab38311, Abcam, Cambridge, MA). HKC-8 cells were ﬁxed, penetrated or non-penetrated, and used for LRP5 staining (sc-390267, Santa Cruz, Dallas, TX) and Smad2/3 (sc-8332, Santa Cruz, Dallas, TX). g g Kidney cryosections were used for X-gal staining (Sigma-Aldrich, Saint Louis, MO) as previously described,45 or immunostaining of Smad2/3 (sc-8332, Santa Cruz, Dallas, TX). Parafﬁn-embedded kidney sections were used for picro-sirius red staining (Roche Life Science, Indianapolis, IN), PAS (Promega, Madison, WI), immuno- histochemical staining of LRP5 (ab38311, Abcam, Cambridge, MA) and collagen III (NBP2-15946, BD Pharmingen, San Jose, CA), and immunoﬂuorescence staining of α-SMA (sc-32251, Santa Cruz, Dallas, TX), E-cadherin (610182, BD Biosciences, San Jose, CA), TβRI (NBP1-01037, Novus, Littleton, CO), SGLT-2 (ab37296, Abcam, Cambridge, MA) and LRP5 (ab38311, Abcam, Cambridge, MA). HKC-8 cells were ﬁxed, penetrated or non-penetrated, and used for LRP5 staining (sc-390267, Santa Cruz, Dallas, TX) and Smad2/3 (sc-8332, Santa Cruz, Dallas, TX). 5. Lam, A. P. REFERENCES 8, 171–179 (1997 26. Hu, B., Wu, Z. & Phan, S. H. Smad3 mediates transforming growth factor-beta- induced alpha-smooth muscle actin expression. Am. J. Respir. Cell Mol. Biol. 29, 397–404 (2003). 41. Borrell-Pages, M., Romero, J. C., Juan-Babot, O. & Badimon, L. Wnt pathway activation, cell migration, and lipid uptake is regulated by low-density lipoprotein receptor-related protein 5 in human macrophages. Eur. Heart J. 32, 2841–2850 (2011). 27. Ignotz, R. A. & Massague, J. Transforming growth factor-beta stimulates the expression of ﬁbronectin and collagen and their incorporation into the extra- cellular matrix. J. Biol. Chem. 261, 4337–4345 (1986). 42. Borrell-Pages, M., Carolina Romero, J. & Badimon, L. LRP5 and plasma cholesterol levels modulate the canonical Wnt pathway in peripheral blood leukocytes. Immunol. Cell Biol. 93, 653–661 (2015). 28. Shi, Y. & Massague, J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685–700 (2003). 29. Ozdamar, B. et al. Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science 307, 1603–1609 (2005). 43. Dufﬁeld, J. S. Macrophages and immunologic inﬂammation of the kidney. Semin. Nephrol. 30, 234–254 (2010). 30. Chen, Y. G. Endocytic regulation of TGF-beta signaling. Cell Res. 19, 58–70 (2009). 44. Terryn, S. et al. A primary culture of mouse proximal tubular cells, established on collagen-coated membranes. Am. J. Physiol. Ren. Physiol. 293, F476–F485 (2007). 31. Jiang, M. Q. et al. HuangQi decoction improves renal tubulointerstitial ﬁbrosis in mice by inhibiting the up-regulation of Wnt/beta-catenin signaling pathway. Cell Physiol. Biochem 36, 655–669 (2015). 45. Lee, K. et al. Receptor heterodimerization as a novel mechanism for the regula- tion of Wnt/beta-catenin signaling. J. Cell Sci. 127, 4857–4869 (2014). 32. Wang, L. et al. Astragaloside IV inhibits the up-regulation of Wnt/beta-catenin signaling in rats with unilateral ureteral obstruction. Cell Physiol. Biochem. 33, 1316–1328 (2014). 46. Park, K. et al. Identiﬁcation of a novel inhibitor of the canonical Wnt pathway. Mol. Cell Biol. 31, 3038–3051 (2011). 33. Chin, E. N. et al. Lrp5 has a Wnt-independent role in glucose uptake and growth for mammary epithelial cells. Mol. Cell Biol. 36, 871–885 (2015). REFERENCES Expression of the type I diabetes-associated gene LRP5 in macrophages, vitamin A system cells, and the Islets of Langerhans suggests multiple potential roles in diabetes. J. Histochem Cytochem 48, 1357–1368 (2000). 16. Cnossen, W. R. et al. LRP5 variants may contribute to ADPKD. Eur. J. Hum. Genet 24, 237–242 (2016). 17. Zheng, S. et al. Development of late-stage diabetic nephropathy in OVE26 dia- betic mice. Diabetes 53, 3248–3257 (2004). 18. Lian, Y. G., Zhou, Q. G., Zhang, Y. J. & Zheng, F. L. VEGF ameliorates tubu- lointerstitial ﬁbrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition. Acta Pharm. Sin. 32, 1513–1521 (2011). 19. Vidyasagar, A. et al. Tubular expression of heat-shock protein 27 inhibits ﬁbro- genesis in obstructive nephropathy. Kidney Int. 83, 84–92 (2013). 20. Shtutman, M. et al. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl Acad. Sci. USA 96, 5522–5527 (1999). Statistical analysis Experiments were performed at least three times, and repre- sentative data was presented. All values were expressed as mean ± SEM. Statistical comparisons among groups were performed using two-way ANOVA with pair-wise multiple comparisons or unpaired Student’s t-test. Statistical signiﬁcance was set at p < 0.05. 21. He, T. C., Chan, T. A., Vogelstein, B. & Kinzler, K. W. PPARdelta is an APC-regulated target of nonsteroidal anti-inﬂammatory drugs. Cell 99, 335–345 (1999). 22. Meng, X. M., Nikolic-Paterson, D. J. & Lan, H. Y. TGF-beta: the master regulator of ﬁbrosis. Nat. Rev. Nephrol. 12, 325–338 (2016). 23. Lin, A. H. et al. Global analysis of Smad2/3-dependent TGF-beta signaling in living mice reveals prominent tissue-speciﬁc responses to injury. J. Immunol. 175, 547–554 (2005). Signal Transduction and Targeted Therapy (2020) 5:45 LRP5 interacts with TGF-β/Smad signaling He et al. LRP5 interacts with TGF-β/Smad signaling He et al. 12 39. Nishita, M. et al. Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann’s organizer. Nature 403, 781–785 (2000). 24. Wrana, J. L. et al. TGF beta signals through a heteromeric protein kinase receptor complex. Cell 71, 1003–1014 (1992). 25. Grotendorst, G. R. Connective tissue growth factor: a mediator of TGF-beta action on ﬁbroblasts. Cytokine Growth Factor Rev. 8, 171–179 (1997). 40. Ren, S. et al. LRP-6 is a coreceptor for multiple ﬁbrogenic signaling pathways in pericytes and myoﬁbroblasts that are inhibited by DKK-1. Proc. Natl Acad. Sci. USA 110, 1440–1445 (2013). on ﬁbroblasts. Cytokine Growth Factor Rev. Signal Transduction and Targeted Therapy (2020) 5:45 REFERENCES Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 34. Bierie, B. & Moses, H. L. Transforming growth factor beta (TGF-beta) and inﬂammation in cancer. Cytokine Growth Factor Rev. 21, 49–59 (2010). 35. Glinka, Y., Stoilova, S., Mohammed, N. & Prud’homme, G. J. Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta. Carcinogenesis 32, 613–621 (2011). 36. van den Bosch, M. H. et al. Wnts talking with the TGF-beta superfamily: WISPers about modulation of osteoarthritis. Rheumatology 55, 1536–1547 (2016). 37. DiRenzo, D. M. et al. A crosstalk between TGF-beta/Smad3 and Wnt/beta-catenin pathways promotes vascular smooth muscle cell proliferation. Cell Signal. 28, 498–505 (2016). , pathways promotes vascular smooth muscle cell proliferation. Cell Signal. 28, 498–505 (2016). 38. Akhmetshina, A. et al. Activation of canonical Wnt signalling is required for TGF- beta-mediated ﬁbrosis. Nat. Commun. 3, 735 (2012). © The Author(s) 2020
https://openalex.org/W4361852514	https://ejournal.uinsaizu.ac.id/index.php/komunika/article/download/6810/3134	English	null	Nahdlatul Ulama Da'wah Dynamics: Socio-Cultural Change and Disruption	Komunika	2,022	cc-by-sa	8,738	Abstract The dynamics of da'wah and social and cultural change/disruption, especially since the middle of the last century, have challenged major civic organizations such as Nahdlatul Ulama (NU). The Nahdlatul Ulama is often considered a conservative organization, unprogressive, and even often stigmatized, but this organization continues to fly its wings and capture the code of change. This study examines the dynamics of Nahdlatul Ulama's da'wah method, especially in the deal with changes and socio-cultural disruptions. This paper uses qualitative methods with a transdisciplinary approach. It consists of a sociological, historical, and cultural approach, mainly emphasizing integrating da'wah and communication science. The subjects of this study are the Chairmen at PBNU for the 2015-2020 period—data analysis in this study by data reduction, display, conclusion, and verification. The results of this study expose: (1) The era of disruption has a tremendous impact on the da'wah method (2) Nahdlatul Ulama utilizes digital da'wah Media, such as social media (Twitter, Facebook, Instagram, Youtube, and Google Plusme), NU Online, 146 Chanel, Aswaja TV, Nutizen, Islamic.com, and Film. (3) Nahdlatul Ulama's da'wah materials contain: countering fundamentalism, radicalism, terrorism, and Wahabism by strengthening aswaja content, ukhuwah trilogy (ukhuwwah bashariyyah, ukhuwwah wathaniyyah, ukhuwwah Islamiyyah). (4) Nahdlatul Ulama continues to revive slametan, praise, tahlilan, dhikr after prayer, maulid nabi, berzanji, halal bi halal, sarong, skullcap, turban, blangkon, istighasah, recitation, pilgrimage, haul, and bahth al-masa'il. Keywords: : Nahdlatul Ulama, Da'wah dinamics, Disruption, Socio-cultural change. Keywords: : Nahdlatul Ulama, Da'wah dinamics, Disruption, Socio-cultural change. Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption Siti Uswatun Khasanah1 1Universitas Islam Djakarta (UID), Jakarta, Indonesia Article Information Submitted August 8, 2022 Revision September 3, 2022 Accepted September 14, 2022 Published October 30, 2022 Komunika: Jurnal Dakwah dan Komunikasi Vol. 16, No. 2, Oktober 2022, 183-199 ISSN 1978-1261 (print); 2548-9496 (online) Komunika: Jurnal Dakwah dan Komunikasi Vol. 16, No. 2, Oktober 2022, 183-199 ISSN 1978-1261 (print); 2548-9496 (online) KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 183 Author Correspondence: Siti Uswatun Khasanah, Email: sitiuswatunkhasanah@uid.ac.id Copyright © 2022 Dortje L. Y. Lopulalan, Selvianus Salakay, Yustina Sopacua, Ronald Alfredo Introduction era by accelerating effective and productive approaches, methods, materials, and techniques (Khasanah, 2021). The rapid advance of information technology in the contemporary era spurred acceleration in culture, ideology, and the development of science. Da'wah should provide changes (disruptions) and rearrange their da'wah system, structuring new formulating materials, methods, targets, and how to deal with problems. Da'wa should answer the directions and challenges of the times. In objective reality, da'wah is required to respond to the trends of the contemporary Basit (2013) states four da'wah methods in the contemporary era: (1) Da'wah can be developed as an object of science and developed according to the requirements and necessities of society, (2) Changing the paradigm of da'wah science to Islamic communication science by synthesizing theories of communication science with da'wah theories derived from Islamic KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 183 *Author Correspondence: Siti Uswatun Khasanah, Email: sitiuswatunkhasanah@uid.ac.id Copyright © 2022 Dortje L. Y. Lopulalan, Selvianus Salakay, Yustina Sopacua, Ronald Alfredo 183 Siti Uswatun Khasanah teachings, (3) The preacher should increase their ability following the development of science and technology, (4) Utilize community's communication media. or community interactions, including changes in cultural matters. Thus, social change transforms toward a state distinct from the previous form. Disruption continues to spur advances in information technology, culture, ideology, economics, and the development of science. For this reason, da'wah must respond to trends by accelerating new approaches to be more effective and productive, such as acceleration in materials, methods, and techniques. The intended social change in da'wah activities is planned change. An example of da'wah of social movement in reforming society is exemplified by the Prophet Muhammad. The apostle's da'wah includes various aspects, including strengthening the socio-religious element by supporting the creed of the people starting with the construction of mosques and maintaining socio-political and socio- economic by applying for alms orders and prohibiting usury and encouraging work ethic (Syamsuddin, 2016). The disruption era is associated with rapid communication and information technology development, which initiated the digital industry revolution 4.0. Advancements in information technology, computing, automation, and robotization characterize disruptive technology. This change followed a massive domino effect, including in religious behavior. The Internet has also changed patterns of religious behavior (Kasali, 2017). Bayu Setiaji (Kompas, 2020) stated that disruption is an innovation that replaces all old systems in new ways or techniques. Introduction It replaces old physical technology with more efficient and effective digital technology. For example, in print media that turned into online media, traditional motorcycle taxis are less competitive than online motorcycle taxis, and traditional stores are inferior to online stores and others. According to Fukuyama (1999), disruption in society is inevitable. The change is conditioned by the power of information technology that upholds democratic values, such as freedom and equality. The changes in the "information society" are the strengths and advantages of disruption. The purpose of disruption is welfare, democracy, awareness of human rights, and concern for the environment. Nahdlatul Ulama is an independent socio- religious organization with the independence to determine its attitudes and activities. The NU upholds democracy, the constitution, and the law. NU consists of values of openness, cooperativeness, ideology, moderation, and pluralism (Hefner, 2020). The NU counters discrimination, radicalism, anarchism, and terrorism (Aziz, 2016). In addition to being Indonesia's largest reckoned civic organization, NU dares to stand out amid socio-political constellations—for example, the term Islam Nusantara Social change is a transformation from the social's symptoms starting from the individual to the more complex. The sign of social change is a continuity disruption in social unity, although the situation is relatively small. The social change includes structures, functions, values, norms, institutions, and all aspects resulting from human, organizational, According to Abdurahman Wahid, NU's practice of "intermarriage" between religion and the social sciences of the humanities, the pros and cons of the NU thoughts of the NU pattern have continually been interesting. KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 184 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption The NU is often considered a conservative organization, unprogressive, and even often stigmatized, but in reality, it continues to fly its wings and capture the code of change. Therefore, researchers are interested in seeing the dynamics of NU da'wah, especially toward socio-cultural changes and disruptions. 2015-2020 period; observation results; works/books published by NU and written by NU figures and PBNU documents. Secondary data includes literature studies of theories and relevant information, such as books, journals, magazines, and printed or electronic media. Data collection techniques use semi- structured interviews, participatory observation (participatory observation), and documentation. Data analysis in this study utilizes data reduction (reduction), testing or presenting data (display), drawing conclusions (conclusion drawing), and verification (Suyanto: 2007; Milles, 1986). Method The research uses qualitative methods with a case study approach to study/ explore and understand the meaning of social problems. A qualitative approach means discovering and understanding the hidden value/meaning behind people's lives, history, behavior, concepts or phenomena, and social issues. A case study approach allows for an in-depth investigation of a case, accumulating complete information using various data collection procedures based on a predetermined time. The data includes an event, activity, process, or program (Creswell, 2016). Result In the era of social disruption and change, the role of da'wah is correspondingly a means of educating, criticizing, social supervision, protecting the environment, scientific development, building the welfare of the people, and empowering the community. The results of this study reveal that: First: The era of disruption has had an impact on social and cultural changes, including in da'wah. In this case, NU has utilized digital da'wah media, new media, and conventional media. The distinctions in traditional and contemporary media characteristics are presented in Table 1 below. The subject of this study is the management of Nahdlatul Ulama organization (chairmen of PBNU) for the 2015-2020 management period. Primary data were obtained from field data interviews with the general chairman of PBNU, chairman of the NU Da'wah Institute (LDNU), and representatives of the management for the KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 185 Table .1. Characteristics of Conventional Media vs. New Media Characteristics Conventional Media New Media 1 Content Limited by space/tend to be determined, local factors, and the presence of sensors Limited by space/tend to be determined, local factors, and the presence of sensors 2 Orientation of Fuction Elite class, ignoring global publications (universality) All social types, global access Table .1. Characteristics of Conventional Media vs. New Media Characteristics Conventional Media New Media 1 Content Limited by space/tend to be determined, local factors, and the presence of sensors Limited by space/tend to be determined, local factors, and the presence of sensors 2 Orientation of Fuction Elite class, ignoring global publications (universality) All social types, global access Table .1. Characteristics of Conventional Media vs. New Media KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 185 Siti Uswatun Khasanah 3 Institution Elite class, ignoring global publications Centralized flexible, anonymity, user as owner, only for capital access 4 Public Acess Low, one-way Significantly broad, multi-directional, interactive, freedom, equality (Processed from various sources: Sparks, Colin. (2001), Bennett, W. Lance & Entman, Robert M. (eds), Pavlik, John V. (1996.) 3 Institution Elite class, ignoring global publications Centralized flexible, anonymity, user as owner, only for capital access 4 Public Acess Low, one-way Significantly broad, multi-directional, interactive, freedom, equality (Processed from various sources: Sparks, Colin. (2001), Bennett, W. Lance & Entman, Robert M. (eds), Pavlik, John V. (1996.) "The Islamic typology in Indonesia is a typical Islamic typology. Result Khasa'is has an extensive scope, initiating from history, thoughts, traditions, ijtihad, and how to preach to social interaction. Among the main Khasais of Islam Nusantara are as follows: i'tidal, tawassut (moderate), tasamuh (tolerant), tawazun (Balanced), tasyawur (Happy to deliberate), ta'awun wa tadhamun (Mutual Cooperation). This value adapts to the community's culture and customs that align with Shari'a, prioritizing (Da'wah kindly, not with anger, violence, and hatred). In addition, social interaction is realized by being happy and staying in touch. Furthermore, NU aims to achieve harmony between religion and the state, not a secular nor religious state. It includes the concept that the love of the motherland is part of the faith. NU implements 3 ukhuwah (fraternity), Islamiyah, wathaniyah, and basyariyah. In addition, NU is open to new thoughts following Islam's basic principles, providing equality in religious, social, and state life. There is no strict separation of space between men and women that blends Islam with culture, Islam with nationalism, and blends Islam with a wealth of traditions." (Interview with Said Aqil Siradj). Second: NU utilizes various media for da'wah, both verbal, essay, electronic media, and digital media, as a result of interviews with Nahdlatul Ulama administrators. "NU utilizes digital da'wah media, such as social media (Twitter, Facebook, Instagram, Youtube, Google Plusme), NU Online, 146 Chanel, Aswaja TV, Nutizen, Islamic.com, and Film." (Interview with Khairul Anam) "NU utilizes digital da'wah media, such as social media (Twitter, Facebook, Instagram, Youtube, Google Plusme), NU Online, 146 Chanel, Aswaja TV, Nutizen, Islamic.com, and Film." (Interview with Khairul Anam) Third, NU's da'wah materials contain countering fundamentalism, radicalism, terrorism, and Wahabism by strengthening aswaja content and the ukhwah trilogy (ukhuwwah bashariyyah, ukhuwwah wathaniyyah, ukhuwwah Islamiyyah). Fourth, the development of da'wah is based on local culture. NU continues to revive the tradition, namely tasyakuran, in welcoming the birth of children, circumcision, reading shalawat before praying/waiting for other jama'ah, tahlilan, dhikr after prayer, commemorating the Prophet's maulid, recitation of al-berzanji (biography of the Prophet), halal bi halal, wearing a sarong, wearing skullcap, turban, blangkon, istighasah, grand recitation, walisongo pilgrimage, nyekar/pilgrimage of ancestral tombs, haul, bahth al-masa'il, defending the Republic of Indonesia and Pancasila as the ideology of the Indonesian nation. The obligations of all societal levels and young figures of NU are expected to continue to inherit the local culture and lead the strength and existence of the Republic of Indonesia. 3. Nahdlatoel Oelama News 3. Nahdlatoel Oelama News Nahdlatul Ulama Da'wah Media Some of the dynamics of NU's da'wah in terms of media are: Result NU's da'wah in a multicultural society aims to develop patterns and models to convey a da'wah message that targets a combination of cultures, ethnicities, languages, and religions. Therefore, how NU's exertions in implementing multicultural da'wah based on local wisdom during a pluralist society and also how to develop the Nahdlatul Ulama (NU) da'wah model, which has been committed to prioritizing the principles of fraternity, courtesy, and awareness, needs to be highlighted to the surface. Finally, the KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 186 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption community has new choices in diversity that continue to grow (Said Aqil Siradj: 2022) "Islamic society must be integrated with the culture as long as the culture is suitable for Islam. "We" must not oppose it except for cultures contrary to sharia, such as infidelity, gambling, drunkenness, and others (Said Aqil Siradj: 2019, interview). monthly magazine based in PWNU East Java. It was established in 1978. H. Anas Thohir drove the magazine, then continued by the vice chairman of PWNU. Among the NU figures who have been journalists for this magazine is Dr. KH. A.Hasyim Muzadi, Drs. H. Choirul Anam, H. Solihin Hidayat, Drs. H. Fuad Anwar, Drs. H. Masduqi Baidlowi, H. Sholeh Hayat, etc. (Fadeli and Subhan,2010). This magazine is the singular NU media that has survived; although a regional branch publishes the magazine, its market segment is NU figures and pilgrims nationally. community has new choices in diversity that continue to grow (Said Aqil Siradj: 2022) "Islamic society must be integrated with the culture as long as the culture is suitable for Islam. "We" must not oppose it except for cultures contrary to sharia, such as infidelity, gambling, drunkenness, and others (Said Aqil Siradj: 2019, interview). 2. LINO News Oral proselytizing is one of NU's conventional da'wah, using da'wah media in spoken words (spoken and addressed through the sense of hearing). Preaching delivers over the podium or pulpit, conferences, radio, telephone, and others. Da'wah with conventional media remains the PBNU Da'wah Institute routinely conducts preacher cadre training. PBNU publishes the LINO bulletin, which stands for Lailatul Ijtima Nahdlatoel Oelama, in the 1937s. The LINO bulletin contains explicit news about the routine activities of the Nahdlatul Ulama organization called Lailatul Ijtima'. Lailatul Ijtima is the custom of the Kyai gathering to discuss various important issues between them, both regarding religious issues and various social and community issues. Apart from discussing news about Lailatul Ijtima's activities, the LINO bulletin also contains obituaries regarding NU members who have died. The LINO news includes religious issues, organizational communication, politics, and society. This bulletin was also published in Semarang in the 1st970s with Prof. Dr. Saifuddin Zuhri as the supervisor. Every month the NU institution holds a public recitation on the PBNU (Executive Board of Nahdlatul Ulama) page, which begins with tahlil, istighosah, shalawat, and public lectures. In addition to being carried out by the NU da'wah institute, NU preachers also convey their da'wah orally to forums for scientific studies, religious studies, mosques, majlis taklim, and others (Interview with Agus Salim). B. Written Da'wah Nahdlatoel Oelama News is one of the NU media directed by K.H. Mahfuz Siddiq in the 1934s and headquartered on Sasak Street Number 23 Surabaya. Nahdlatul Oelama News continues Surabaya Swara Nahdlatoel Oelama, pioneered by K.H. Wahab Hasbullah. Nahdlatul Oelama news is a simple magazine Some of the da'wah media carried out by PBNU (Executive Board of Nahdlatul Ulama) in papers such as: 1. Aula 5. Obor Revolusi with 18 pages, published every middle of the month. In addition, there is also a Bintang Sembilan magazine which belongs to PP Anshor. The magazine is, abbreviated as Be- Es, founded in 1951 and was the mandate of the congress in Surabaya. The Obor Revolusi was a daily newspaper in Surabaya during the critical period after the 1965 G-30 S/PKI tragedy. The Obor Revolusi was published at the end of 1964, prepared to strengthen the opinion of the NU Party. Although the operation was small, this newspaper was highly respected to compensate for the PKI's Suara Rakyat newspaper. The two are immortal enemies, claiming and striking each other out. Whenever the PKI takes unilateral action, the Obor Revolusi will contain it massively as a headline. After the PKI was disbanded, the government or the Obor Revolusi declined for a long time until it finally stopped publishing. 7. Risalah Islamiyah Risalah Islamiyah is a NU magazine published by missi Islam in 1969, received a Publication Permit (SIT) from the Ministry of Information dated August 22, 1969. The magazine was published and existed until 1980. However, since 1982 the magazine has ceased publication because its leading institution (missi Islam) is also in a vacuum. After the reformation era, PBNU revived the newspaper as a public daily in collaboration with Jawa Pos and based in Graha Pena Surabaya. Long after, Duta Masyarakat separated from Jawa Pos and had its own office on Kapuas Street Surabaya. However, then the newspaper collapsed again and discontinued to published. In 2001 Duta Masyarakat was revived and based in Kutisari Indah Barat VI/I under the P.T. Duta Aksara, one of the businesses owned by the Bisma Foundation. 4. Duta Masyarakat Duta Masyarakat is Nu's official newspaper and has been running since 1954. The newspaper is located in the Menteng area, the former office of PP Muslimat NU (Nahdlatul Oelama Central Administrator). The newspaper used a loud and bold language style due to the newspaper's aim as a propaganda tool. The courage in conveying the news led to this magazine being banned twice by the New Order government. 6. Oetoesan Nahdlatoel Oelama Oetoesan Nahdlatoel Oelama is an NU magazine published in the 1st930s, besides Swara Nahdlatoel Oelama was established first and managed by KH. Wahab Hasbullah and KH. Abdullah Ubaid. This magazine is based in Surabaya. This magazine was quite helpful to the development of NU at that period, but this magazine only lasted a moment. In 1974 the Duta Masyarakat newspaper ceased publication due to the New Order government's anti-critical media political stance. In addition, it is also caused by internal conflicts among NU members. The sponsorship for the newspaper from the PBNU fund was stopped, and at the same time, many branch administrators wanted to avoid paying the bills because they considered the newspaper their own, so it did not need to be paid. 1. Aula PWNU (Nahdlatul Ulama Regional Administrator) East Java branch publishes a KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 187 Siti Uswatun Khasanah 10. Santri The Santri is communication media belonging to Rabithah Ma'ahid Islamiyah (RMI). They were published in Surabaya in 1996 with an office on Raya Darmo Street No.96 Surabaya. In 2004 the magazine briefly stopped and republished. However, after the result of Solo muktamar in 2004 and after the transition of management, Santri Magazine is no longer published. 9. Risalah Nahdlatul Ulama (www.risalahnu. com) Risalah Nahdlatul Ulama is PBNU's official magazine and was founded in 1978. The magazine pioneers were Slamet Efendy Yusuf, Anwar Nuris, and Saiful Masykur. It was revived in May 2007 by the initiators and managers of this second magazine period (Drs. H. Mustafa Helmy and H. Zis Muzahid, M.Si.). Risalah NU published monthly with 114 pages. Based on the 7th floor of PBNU Office, Jl. Kramat Raya 164 Central Jakarta. 8. Warta NU Warta NU Tabloid belongs to the Executive Board of Nahdlatul Oelama (PBNU), managed under the coordination of the Lajnah Ta'lif wan Nasyr (LTN) Centre. It has been KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 188 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption published since 1985 with Ichwan Sam as the general leader and is based at the PBNU office, Keramat Raya Street No.164 Central Jakarta. The founders of this tabloid are Ichwan Sam, Slamet Efendi Yusuf, and Arifin Junaidi. official publications, such as: a) Organizing a books challenge to form a publishing business system and process in internal PBNU. b) trial publication of 5 book titles within three months (successful) ( Report of the Ta'lif Wa Nasyr PBNU institution, 2019). Through the Warta NU tabloid, PBNU socializes organizational decisions regarding muktamar and munas (National Conference) results and further important decisions. This tabloid contains an exclamation to restore the major struggle of Nahdlatul Ulama which is politically independent. This tabloid reached 48,000 copies in national circulation in its heyday but was eventually discontinued. Since 1995, this tabloid has been reissued with an office at PWNU East Java branch, Raya Darmo Street No.96 Surabaya. However, since 2005 this tabloid has yet to be published again. 11. Suluh Nahdlatul Ulama The magazine was published in Surabaya in 1957. It is headed by Umar Burhan and handled by H. Mahfudz Syamsul Hadi (Nahdlatul Ulama regional administrator chairman) and H. Sholeh Mukmin (Nahdlatul Ulama regional administrator secretary), based at Nahdlatul Ulama regional administrator East Java branch, Raya Darmo Street No.96 Surabaya. This magazine is tongue-in-cheek between NU party administrators and the community. 9. Risalah Nahdlatul Ulama (www.risalahnu. com) 12. Suara ANO The Ansoru Nahdlatil Oelama Executive Board (PB ANO) published the magazine in 1937. Almost all branch managers subscribe to ANO, even in the southern Central Java Regional Commissioner (Banyumas, Purbalingga, Banjarnegara, Wonosobo, Temanggung, Magelang, Puworejo, Kebumen, Karanganyar, Cilacap, and Purwokerto). Based on the annual report of the Ta'lif Wa Nasyr institution, the 2019 Nahdlatul Ulama Executive Board in Surakarta, it is stated that the management of the Risalah NU magazine is published monthly. Risalah NU bulletin (www. risalahnu.com) is published every Friday, and the publication of NU Treatise book with a thematic every three months. Division to publish the book NU Treatise aims to provide PBNU's ability to collect manuscripts, and 13. Suara Ansor Suara Ansor is a media belonging to Centre Administrator (PP) of Gerakan Pemuda KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 189 Siti Uswatun Khasanah C. Da'wah with Radio Ansor(GP Ansor) published in the 1965s. The magazine was published in a heated national political atmosphere due to the G 30 S/ PKI tragedy. As a medium of struggle, Suara Ansor highlighted many national political developments. NU has streaming radios that can be accessed via Google Play or downloaded through the App Store. The radio has programs including Burdah by Gus Mus, Kajian Tafsir, Cahaya Sufi, and general recitation. The live radio broadcasts can be accessed on radio. nu.or.id. One reason NU keeps radio as a means of da'wah is that radio has several advantages. These advantages are as detailed by Onong Uchjana Effendy (1995) below: 16. Jurnal ilmiah Islam Nusantara 3) Radio broadcasting has a strong appeal. The appeal comes from the combination of music, narration, and sound effects realized through announcers' voices, singing, radio talk shows, radio dramas, quizzes, and much more. With the evolution of communication and telecommunications technologies, the differentiation and specifications of mass communication media are getting sharper. 3) Radio broadcasting has a strong appeal. The appeal comes from the combination of music, narration, and sound effects realized through announcers' voices, singing, radio talk shows, radio dramas, quizzes, and much more. With the evolution of communication and telecommunications technologies, the differentiation and specifications of mass communication media are getting sharper. Jurnal Islam Nusantara is published by the Ta'lif wan Nasyir Institute (LTN) PBNU Jakarta, Indonesia, with the primary objective of disseminating critical and original analysis from researchers and academic practitioners. Aim and Scope this journal is about various issues of contemporary Islamic thought across perspectives based on the Nusantara Islamic approach. This journal emphasizes multiple issues of modern Islamic thought across arguments based on the Nusantara Islamic approach. Its scope consists of; (1) Qur'an and Hadith, (2) Theology, (3) Islamic Mysticism, (4) Islamic Education and Management, (5) Islamic Law, (6) Islamic Economics, (7) Islamic Art and History, and (8) philosophy. 14. Swara Nadhlatoel Oelama Swara Nahdlatoel Oelama is an NU magazine published in the 1930s. It rises monthly in the middle of the month. During its seven-year journey, the magazine was directly directed by KH. Wahab Hasbullah. The headquarter of Swara Nadhlatoel Oelama is located on Sasak Street No. 23 Surabaya. 1) Radio sends a direct message. It is through a simple process to achieve that goal, diverse with newspapers, which have to go through the printing process and distribution network of retailers before they get into the hands of consumers. 15. Taswirul Afkar Taswirul Afkar is a Journal published by the Nahdlatul Ulama Institute for the Study and Development of Human Resources (Lakpesdam NU). Based on Haji Ramli Street No. 22 A Menteng in Tebet, South Jakarta with Homepage www.lakpesdam.or.id. The journal is published per three months with about 150 pages. 2) Radio broadcasting has no distance or obstacles. No matter how far away the target group is, while wave frequencies still cover the location, information can be transmitted simultaneously and instantly wherever radio frequencies are available. The actuality of radio is the highest among other mass communication media. F. Da'wah Through Social Media Da'wah using social media is a rising style and the most appropriate solution for many problems in the era of social media disruption. In addition to covering an extensive area, social media provides various conveniences for both the preachers and the pilgrims (madhu) in amar ma'ruf nahi munkar (upholding the truth and forbidding wickedness) activities. Aswaja TV can be consumed by anyone worldwide, not exclusively through satellite TV. This TV station has also launched a live- streaming service. The NU UPDATE program is one of the programs from the NU Channel that is particular for the latest news updates or responses and opinions related to Nahdlatul Ulama both in Politics, Economy, and Religion. Aswaja TV aims to provide positive information for Indonesia people through the program Ngaji Online-Tafsir al-Jailani, Ngaji kitab Qasidah Burdah, Ngaji Online kitab Minjhajul Abidin, and various other scripture recitations. According to Dedik, one of the editors of @islam.co stated that NU was belatedly in creating content in online media, but the media was alive and growing. NU has several online media and platforms: NU Online, 146 Chanel, Aswaja TV, Nutizen, and Islamic.com (Interview with Dedik). Some social media platforms, such as Twitter, Facebook, and blogs/websites, da'wah media as the warmest new media, offer possibilities for interactive spaces. Discussions highlighting interactive elements can be found on media websites, blogs, Facebook, Twitter, and Youtube. Based on the annual report of the Ta'lif Wa Nasyr institution, the 2019 Nahdlatul Ulama Executive Board in Surakarta has reached 160 thousand followers. The annual report of the Ta'lif Wa Nasyr institution of the Nahdlatul Ulama Executive Board in 2019 stated that NU's social media performance, in this case, D. Da'wah with Television Analog and previously existing digital television channels are now evolving into internet television (TVLive streaming). Aswaja TV is one of the Islamic TV channels KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 190 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption Examples of technologies involving new media include: in Indonesia that carries out the concept of ahlussunnah wal jama'ah (experts in practicing the sunnah, adherents of the sunnah, or followers of the sunnah). This Islamic television was launched on July 25, 2013/17 Ramadan 1434H, coinciding with the night of Nuzulul Qur'an 1434H. Aswaja TV uses the jargon Ahl al-sunnah wa al-jama'ah Nahdliyin Network, The Real Indonesian Moslem Channel. Aswaja TV is an original Indonesian Islamic channel accessed through Parabola TV. Using the Palapa satellite, Aswaja TV's broadcasts cover the entire archipelago and Southeast Asia, Australia, Hong Kong, and Taiwan. in Indonesia that carries out the concept of ahlussunnah wal jama'ah (experts in practicing the sunnah, adherents of the sunnah, or followers of the sunnah). This Islamic television was launched on July 25, 2013/17 Ramadan 1434H, coinciding with the night of Nuzulul Qur'an 1434H. Aswaja TV uses the jargon Ahl al-sunnah wa al-jama'ah Nahdliyin Network, The Real Indonesian Moslem Channel. Aswaja TV is an original Indonesian Islamic channel accessed through Parabola TV. Using the Palapa satellite, Aswaja TV's broadcasts cover the entire archipelago and Southeast Asia, Australia, Hong Kong, and Taiwan. a) Internet and Websites, b) Digital TV/ Plasma TV, c) Digital Cinema/3D Cinema, d) Supercomputer/Laptop, e) DVD/CD/Blu-ray, f) MP3 Player, g) mobile phone/PDA, h) video game, i) RSS feed, j) video streaming, etc. Most new media technologies are digital, inclusive, interactive, manipulative, connected, dense, incompressible, and unbiased. E. Da'wah Through New Media New Media is a term that describes the convergence of computerized digital communication technologies and networks. New media is a medium that conveys information from a source to the recipient of information (intermediary). The main elements of new media are digitization and convergence. The Internet is proof of convergence because it incorporates several other media capabilities such as audio, video, and text (Quail, 2006) KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 191 Siti Uswatun Khasanah the Facebook fan page reached 16 million likes, and the version of Instagram social media reached 2.1 million followers. The Youtube platform can be accessed on the NU Channel or by clicking the website: http:// nucultural, Instagram id: @nu.channels. situation and being remembered more than the spoken word). In essence, da'wah bil-hal is an implementation of da'wah bil qudwah (exemplary) and da'wah bil amal (action). In other words, bil-hal da'wah is carried out through personal qualities and activities that directly touch the needs of society. The official website belongs to PBNU. It was established in 2002 with the office at PBNU Office Kramat Raya Street No.164 Central Jakarta, website address: www. nu.or.id. In 2005 this site received the best Indonesian Site award from Komputer Aktif magazine in the social and community category. Since 2006 NU Online has been writing in Indonesian, Arabic, and English. This site is the most effective way to disseminate information on NU development news worldwide. In addition to being fast, it is also easy and cheap. NU Online pioneered the application of information technology at NU organization. The NU Online division in the second semester of 2019 averaged 4.5 outstanding readers in the first semester (Report by Ta'lif Wa Nasyr PBNU institution, 2019). Da'wah bil-hal is essential for Muslims to touch their hearts and improve the community. Some examples of da'wah bil-hal that PBNU has carried out are educational scholarships, social benefits, social services, mass medicine, the establishment of boarding schools, the establishment of schools, and the establishment of universities (Aceng, 2016). Da'wah bil-hal PBNU is realized by carrying out da'wah activities as follows: Religious aspects include PBNU recitation, both routine and tentative (once every three months)/conditional, such as commemorating religious holidays, istighasah, joint prayer, etc. Compensation for poor orphans. Creating majis taklim and others. E. Da'wah Through New Media This spiritual aspect under the Nahdlatul Ulama Da'wah Institution (LDNU) is to implement the Nahdlatul Ulama policy in Islamic spiritual development yang adhere to the ahlussunnah wal Jama'ah understanding. Other NU autonomous bodies, such as Fatayat, Muslimat, Ansor, PMII, IPPNU, and IPNU, also apply the religious aspect. NU also has the Nahdlatul Ulama Mosque Ta'mir Institute (LTMNU), which implements the Nahdlatul Ulama policy in mosque development and administration. The Bahth al-Masa'il Nahdlatul Ulama (LBMNU) institution is tasked with discussing the issues of maudlu'iyyah (thematic) and waqi'iyyah (actual). In addition, the Ta'lif wan Nasyr Nahdlatul In addition to the Youtube channel, NU has a video platform under the Ta'lif wan Nasyr Institution (LTN) or a Communication and Information institution under the auspices of the Nahdlatul Ulama Executive Board (PBNU) called 164 Channel. Da'wah (Da'wah by Charity) Da'wah bil-hal is a da'wah that focuses more on charitable enterprises or real works that can be enjoyed and raise the community's dignity, respect, and welfare. Da'wah bil-hal prioritizes authentic (actual) activity and refers to the oral expression of al-hal afsah min oral al-maqal (talking about the reality of the KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 192 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption Ulama (LTNNU) Institute is responsible for developing the writing, translation, and publication of books and information media according to the understanding of Ahl al-sunnah wa al-jama'ah. implementing the Nahdlatul Ulama policy in Islamic boarding school development and religious education. PBNU also takes a role in institutional advocacy of boarding schools and oversees discussing the pesantren needs. 2. Economic Aspects. Da'wah bil-hal, in the financial aspect, is manifested in the activities of cooperative formations. In constructing this collaborative, almost all autonomous bodies of NU have cooperatives, such as Yasmin (Fatayat NU) and Ansor Mart/Ansor retail. NU zakat management/Amil Zakat Institution, Infaq, and Shadaqah Nahdlatul Ulama (LAZISNU) are responsible for collecting, managing, and distributing zakat and shadaqah to their receiver (mustahiq). NU-Care LAZIZNU is spread across 388 branches (in 29 countries) and 26 provinces in Indonesia (Banom Report and PBNU Institution, 2019). The Nahdlatul Ulama Economic Institute (LPNU) implements the Nahdlatul Ulama policy in the economic development of Nahdlatul Ulama members. Meanwhile, the Nahdlatul Ulama Waqf and Land (LWPNU) Institutions manage and develop land, buildings, and other waqf property owned by Nahdlatul Ulama. 4. Organizational Aspects. The administrative aspect of the KH Said Sirodj era (2015-2021) was realized by providing organizational training at all NU levels from the center to the branch. There are five types and models of NU cadre development, as stated in the 33rd NU Muktamar Mandate held in Jombang in 2015. The coaching model carried out among others is. 4. a. Structural Regeneration. This structuralization cadre is called the Nahdatul Ulama Cadre Madrasah (MKNU). This regeneration is obligatory and is followed by all levels of NU management, from executive boards to branch administrators, institutional administrators, and autonomous organization administrators. This regeneration aims to increase administrators' capacity to lead, guide and mobilize citizens and manage organizations. MKNU in the fourth week of July 2019 has been carried out 140 times (www.nu.or.id) 3. Da'wah (Da'wah by Charity) The renewal of NU mobilizers aims to prepare cadres with the extraordinary task of strengthening, securing, maintaining, and transforming the values of N.U.'s struggle to drive civil, religious, social, and national life and fight for the establishment of Islam with ahlussunnah wal Jama'ah value. a. Programmatic activities. Programmatic, social activities include providing scholarships for orphans and compensation for the poor. Programmatic health programs are health education, especially providing health within the family and community. b. Activities of an unprogrammed charity. Unprogrammed social activities in the social aspect of health include actions of a conditional character. The activity is carried out when there are certain circumstances. For example, NU cares about Konawe floods, NU shares blessings, NU sacrifices, renovations of mosques, and NU cares about healthy children of the archipelago (Report of autonomous and PBNU Management Institutions 2019). b. Activities of an unprogrammed charity. Unprogrammed social activities in the social aspect of health include actions of a conditional character. The activity is carried out when there are certain circumstances. For example, NU cares about Konawe floods, NU shares blessings, NU sacrifices, renovations of mosques, and NU cares about healthy children of the archipelago (Report of autonomous and PBNU Management Institutions 2019). d. Functional regeneration; Functional regeneration aims to prepare executives with roles, duties, and responsibilities in five primary parts: As trainers, facilitators, and instructors in various educational, training, and regeneration activities, a researcher who can carry out essential research activities in NU environment; as a team leader for Rukyatul Hilal's actions (determination of the beginning of fasting and Eid al-Fitr), as a partner in various fields, community expander or community organizer in multiple sectors. d. d. Functional regeneration; Functional regeneration aims to prepare executives with roles, duties, and responsibilities in five primary parts: As trainers, facilitators, and instructors in various educational, training, and regeneration activities, a researcher who can carry out essential research activities in NU environment; as a team leader for Rukyatul Hilal's actions (determination of the beginning of fasting and Eid al-Fitr), as a partner in various fields, community expander or community organizer in multiple sectors. On the other hand, NU also continues the da'wah on global issues to develop and discusses Human Rights, Gender, and Democracy. 1. Human Rights (HAM). 1. Human Rights (HAM). Humanitarian issues are global problems that transcend ethnic, racial, and ideological boundaries. Da'wah (Da'wah by Charity) Aspects of Education, PBNU has concerns in education by establishing the Maarif Nahdlatul Ulama Education Institute (LP Maarif NU), tasked with implementing Nahdlatul Ulama policies in formal education and teaching, such as establishing Ma'arif schools. The Nahdlatul Ulama Higher Education Institute (LPTNU) is responsible for developing Nahdlatul Ulama higher education. The Rabithah Ma'ahid Islamiyah Nahdlatul Ulama (RMINU) institution is at the cost of 3. Aspects of Education, PBNU has concerns in education by establishing the Maarif Nahdlatul Ulama Education Institute (LP Maarif NU), tasked with implementing Nahdlatul Ulama policies in formal education and teaching, such as establishing Ma'arif schools. The Nahdlatul Ulama Higher Education Institute (LPTNU) is responsible for developing Nahdlatul Ulama higher education. The Rabithah Ma'ahid Islamiyah Nahdlatul Ulama (RMINU) institution is at the cost of b. The regeneration of youth is called Education and Development of Religious Insights (PPWK). The renewal of youth aims to prepare candidates for the top leadership of Nahdlatul Ulama (NU syuriah) ranks at all levels of leadership KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 193 Siti Uswatun Khasanah based on regeneration at the level of autonomous institutions. based on regeneration at the level of autonomous institutions. (management). It is hoped that young successor cadres will emerge prepared as NU Syuriah. 5. Social and Health Aspects. Da'wah bil-hal in social and health aspects is divided into two groups of activities, specifically: c. Regeneration of NU Mobilizers. The resurrection of NU mobilizers is called NU Mobilizing Cadre Education (PKPNU). The renewal of NU mobilizers aims to prepare cadres with the extraordinary task of strengthening, securing, maintaining, and transforming the values of N.U.'s struggle to drive civil, religious, social, and national life and fight for the establishment of Islam with ahlussunnah wal Jama'ah value. c. Regeneration of NU Mobilizers. The resurrection of NU mobilizers is called NU Mobilizing Cadre Education (PKPNU). The renewal of NU mobilizers aims to prepare cadres with the extraordinary task of strengthening, securing, maintaining, and transforming the values of N.U.'s struggle to drive civil, religious, social, and national life and fight for the establishment of Islam with ahlussunnah wal Jama'ah value. c. Regeneration of NU Mobilizers. The resurrection of NU mobilizers is called NU Mobilizing Cadre Education (PKPNU). Da'wah (Da'wah by Charity) Rejection of all forms of discrimination, injustice, coercion, restraint, and intimidation of the will is inherent in every country, tribe, religion, and class. Efforts to protect and enforce human rights are a global and humanitarian issue. All groups, nations, and ideologies in every corner of e. Professional cadres; Professional regeneration aims to prepare competent and credible NU cadres to occupy certain positions in the executive, legislative, judicial, and tertiary institutions and state companies at the national and regional levels. It prepares functional and professional regeneration KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 194 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption the world reflect the struggle for human rights, a common ideal of realizing a just, prosperous, and prosperous society far from oppression, bloodshed, violence, and injustice. idea of equality between women and men (within boundaries that do not conflict with their nature). Some decisions within NU scholars reflect this argument, such as: As an example of the case committed by the Myanmar military against Rohingya Muslims, PBNU expressed the following attitudes: (1) Condemning all forms of violence that violate human values; It is unacceptable to any form of violence in any name. (2) Islam condemns violence. There is no religion or ideology in the world that justifies violence. (3) Call on all heads around the world to take a proactive stance against all forms of violence; (4) Call on all nations of the world to continue to mobilize humanitarian solidarity to bring peace to all nations; (5) NU appeals and urges relevant parties, particularly the international community and the United Nations, to take concrete steps immediately regarding incidents of violence against Rohingya Muslims in Myanmar. (6) Call on ASEAN, particularly the Government of Myanmar, to take immediate concrete steps to recognize citizenship status; (7) Call on the Indonesian government to take diplomatic measures to ensure respect for human rights in Myanmar. 1) Decree of the Nu Syuriah Council dated 17 Sya'ban 1376 H / March 19, 1957 AD in Surabaya, which allowed women to become legislative members (People's Representative Council); 2) A 1961 NU Muktamar decree in Salatiga allowed a woman to be the head of a village and; 3) The 1997 decision of Munas Alim Ulama in NTB, gave the permit to women for public roles to become president and vice president. Da'wah (Da'wah by Charity) Therefore, it is not entirely reasonable for Islam to ideologically reject women's leadership or subordinate women. 3. Democracy Democracy is now recognized as the best system for governing any country. The relationship between Islam and democracy regarding the potential of Islamic democracy as a religion, culture, and civilization remains controversial. Democracy is a historical phenomenon in human life. The concept of Shura is equated with democracy. Ali Abdur Raziq wrote the book al-Islam wa Usul al-Hukm in 1926. The central argument is that a nation is acceptable to Islam if it meets the three pillars of the nation's pattern. (1) justice (al'is), (2) equality of degrees (al-musawah), (3) democracy (ash- shura). Khilafah is not the singular form of an Islamic state (Shirazi:1999). Law enforcement (al-qist), safeguarding the 3. Democracy Democracy is now recognized as the best system for governing any country. The relationship between Islam and democracy regarding the potential of Islamic democracy as a religion, culture, and civilization remains controversial. Democracy is a historical phenomenon in human life. The concept of Shura is equated with democracy. Ali Abdur Raziq wrote the book al-Islam wa Usul al-Hukm in 1926. The central argument is that a nation is acceptable to Islam if it meets the three pillars of the nation's pattern. (1) justice (al'is), (2) equality of degrees (al-musawah), (3) democracy (ash- shura). Khilafah is not the singular form of an Islamic state (Shirazi:1999). Law enforcement (al-qist), safeguarding the Gender 2. The empowerment of women is NU's primary concern for community development. This ideal underlies women's organizations such as IPPNU, Muslimat NU, and Fatayat NU. NU rejected the traditional argument in pre- independence times, where women were only seen as "konco wingking" (unequal to men). Conceptually, NU developed an The empowerment of women is NU's primary concern for community development. This ideal underlies women's organizations such as IPPNU, Muslimat NU, and Fatayat NU. NU rejected the traditional argument in pre- independence times, where women were only seen as "konco wingking" (unequal to men). Conceptually, NU developed an KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 195 Siti Uswatun Khasanah ukhuwwah, and Islam has been considered universal human values accepted, upheld, and practiced by all groups. NU expresses this understanding by accepting Pancasila, not Islamic law, as the basis of the state. Pancasila is considered not to be contrary to the principles of Islamic law because its rules do not include aspects contrary to Islamic law's regulations or objectives (Maqassidu al Syariah). 6) Utilization of information and communication technology to develop the Nusantara Islamic movement. 7) It Prioritizes the principle of fighting for independence as a national identity to face global challenges. In terms of cultural proselytizing, some of the things that NU accomplishes include the following: 1. There is a tradition in NU: Halal bi halal every year, haul, silaturrahim every holiday (Eid al-Fitr), ketupat holiday (festival ketupat), reading sholawat accompanied by flying, almsgiving, which is termed a rescue of 7 days, 40 days, 100 days, and 1000 days, tingkepan, baby shower, bridal ceremony, a bridal parade which includes the wedding ceremony and held Walimatul 'Urs both by women's families and men's families, and other traditions. NU's da'wah strategy in encountering the currents of globalization for other Nusantara communities is Saptawikrama (Seven cultural strategies) of Islam Nusantara. The concept of this movement was born during the PBNU Lesbumi National Meeting on January 26, 2016 (Siraj, 2016). The seven steps of the cultural strategy (Al Qawa'id Al-Sab'ah) are as follows. The seven steps of the cultural strategy (Al Qawa'id Al-Sab'ah) are as follows. 1) It collects and blends movements based on the archipelago's customs, traditions, and culture. 2. Gender In terms of dressing, some wear sarongs, skullcaps, traditional clothes of Betawi, Javanese, Papuan, Balinese, Madura, and many other traditional clothes models, especially seen in wedding packages where the bride and groom are arranged and displayed in the wedding ceremony, and so on. 2) Develop a model of Sufi education (tarbiyah wa ta'lim) that is closely related to the reality of each educational unit, mainly formal educational institutions (ma'arif), which is managed by Rabitah Ma'ahid Islamiyyah (RMI) 3) It builds an independent discourse to interpret local wisdom and Islamic culture in an ontologically and epistemologically scientific manner. 3. Regarding tolerance for Islamic teachings, there is Id praying twice in the field, in mosques, and even on feast days. Some tarawih pray with twenty rakaat, and some are eight rakaat. In the event of aqiqah, some are filled with shalawatan, and some are filled with tahlilan 4) It gathered strength as a child of the nation formed by Bhinneka Tunggal Ika to rebuild the maritime civilization of the archipelago. 4. Regarding tolerance with cultures that contain history or teachings, some areas are prohibited from slaughtering cows, such as in Kudus Central Java. It is a form of tolerance of Sunan Kudus to Hindu 5) The revitalization of various cultural arts in the Bhineka Tungal Ika area is based on harmony, peace, tolerance, empathy, cooperation, and excellence in art, culture, and science. KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 196 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption teachings that purify it, the custom of brides using Janur Kuning, kembang mayang, and others. a religious practice based on cultural attitudes and locality during the globalization era. The outcome is the growth of nationalism and Pancasila ideology (Report of Autonomous and the PBNU Institute: 2019, 188). Intolerance with other religions, there are national holidays due to Islamic holidays, Christian holidays, Hinduism, Buddhism, Confucianism, and other holidays (Interview with Said Aqil Siradj). Aswaja NU Islam and Islam Nusantara develop internationality through dai shipments abroad (such as Japan, the Netherlands, Cairo, and Egypt). The Islamic da'wah movement rahmatan li al-'alamin developed Islam Nusantara in other countries such as Arabia, Pakistan, Russia, and others, creating Islam well, the establishment of PCI (such as Malaysia, Australia, Singapore, Brunei, Thailand, Vietnam, Cambodia, Japan, China, Hongkong, New Zealand and others), ICIS, Reconciling Afghanistan, providing UNUSIA education scholarships. Gender In addition to da'wah in various areas of life, NU also conducts internal and external da'wah and understands and strengthens the value of Aswaja (Islam Nusantara). The program is n NU's university is socialization and training of the Aswaja curriculum at NU's university, schools, and Islamic boarding schools and strengthening Islamic justice (washatiyah) in schools to counteract radicalism. MKNU and PKPNU training results in value strengthening NU values activities (fikrah, amaliyah, and harokah), forming aswaja teacher militancy and caring for, maintaining, and indicting the ideology of aswaja an-nahdhiyah and Islam rahmatan lil'alamin and publishing Aswaja books for teachers and students. Development of da'wah bi al-halal, bi al-maal, bi al-qalam, and bi al-lisaan through majlis taklim (Interview with Agus Salim, 2019). Conclusion Da'wah in the disruption era can be interpreted as implementing Islamic treatises into real life. This da'wah aims to encourage people to realize the values of Islamic teachings and cultural values that guide all aspects of their lives. Furthermore, implementing da'wah activity is understood as a process of social interaction in a global society. The da'wah message cannot be separated from other subsystems such as religion, culture, economy, education, and community. The understanding of aswaja and the socialization of aswaja's stream is now more than just in schools and pesantren but through social media. It is strengthening the Islamic Nusantara cultural strategy and escort, protection, and development of Nusantara cultural assets, such as the collection and inventory of the regional languages and scripts of the archipelago, conversion, and translation of ancient books and inscriptions of the archipelago, a leather puppet show on 1 Muharram, preservation and learning of writing Carakan Cirebon and others. Output, the participants knew and understood the nature of Nusantara Islam as In the era of disruption, NU remains anchored in the ideology of Aswaja An- Nahdiyah, by utilizing conventional da'wah media and new media/digital media simultaneously. With da'wah material remains by strengthening Aswaja content, ukhwah trilogy (ukhuwwah bashariyyah, ukhuwwah washatiyyah, ukhuwwah Islamiyyah), Hubb KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 197 Siti Uswatun Khasanah Dedik. (2019). Wawancara. Jum’at, 08 -09 Maret 2019, Hotel Bintang Wisata Mandiri, Jakarta Pusat. al-Wathan Min al-Iman, peace, and human rights. NU's Islamic style and orientation remain with the tradition of Nusantara cultural piety ranging from discussions of high-class religious-social issues to "religious visits." The steps aim to defend the Republic of Indonesia and strengthen Pancasila as the Indonesian nation's ideology. Fukuyama, The Great Disruption, h. 137-139; Human Nature and Social Order, dan bab 11 tentang Self-Organization. https://nasional.republika.co.id/berita/ nasional/hukum/18/12/05/pj9jgy354- pbnu-pembunuhan-pekerja-di-papua- kejahatan-kemanusiaan. References https://www.nu.or.id/post/read/110351/ ini-lima-jenis-pendidikan-kader-dalam- nu. ………. Andi Faisal, “The Contribution of Dakwah to Communication Studies: Risale-I Nur Collection Perspective." Kasali, Rhenald (1998). Focusing: Strategi Beriklan di Media Massa. Jakarta, Gramedia. Abdul Aziz, Aceng, M. Harfin Zuhdi, dkk. (2016). Islam Ahl al-sunnah wa al-Jama’ah: Sejarah, Pemikiran, dan Dinamika NU di Indonesia. Jakarta: Pengurus Lembaga Pendidikan Ma’arif NU Pusat. Keputusan Bahtsul Masail Maudhu’iyah PWNU Jawa Timur tentang Islam Nusantara, hal. 3. Di Universitas Negeri Malang. Anggaran Dasar NU. (2016). Hasil-Hasil Muktamar KE-33 NU. Jakarta: Lembaga Ta’lif wan Nasyr PBNU. Khasanah, Siti Uswatun. (2022). Dakwah Kontemporer Nahdlatul Ulama; Perubahan dan Disrupsi Sosial Budaya, Keagamaan dan Politik. Depok: Yayasan Karimatul Hasanah Al-Mubarok. Bakti, Andi Faisal. (2004). “Communication and Family Planning in Islam Indonesia: South Sulawesi Muslim Perceptions of a Global Development Program." Jakarta: INIS. Kompasiana. 2010. “Memahami Istilah- Istilah Baru New Media”. http://new media.kompasiana.com/2010/02/05/ memahami-istilah-media-baru-new- media. Basith, Abdul. (2013). Berdakwah Dengan Cerdas. Jurnal Komunikasi Islam UIN Surabaya, Nomor 01, Juni. Laporan Banom dan Lembaga Pengurus Besar Nahdlatul Ulama Tahun 2019. Bungin, M. Burhan (2007) Sosiologi Komunikasi: Teori, Paradigma, Dan Diskursus Teknologi Komunikasi Di Masyarakat. Jakarta: Kencana Prenadamedia Group. Laporan lembaga Ta’lif Wa Nasyr Pengurus Besar Nahdlatul Ulama Tahun 2019. Mahmuddin. (2016). “Dakwah Kontemporer dan Radikalisme Agama di Bulukumba”, Jurnal al Ulum, Volume 16 No.02 Desember. Creswell, John W. (2016). Research Design : Pendekatan Metode Kualitatif, Kuantitatif dan Campuran. Edisi Keempat (Cetakan Kesatu). Yogyakarta : Pustaka Pelajar KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 198 Nahdlatul Ulama Da’wah Dynamics: Socio-Cultural Change and Disruption Martono. Nanang. (2014). Sosiologi Perubahan Sosial: Perspektif Klasik, Modern, Posmodern, dan Psikolonial. Depok: PT. Raja Grafindo Persada. Salvatore Simarmat. (2014).“Media Baru, Ruang Publik Baru, dan Transformasi Komunikasi Politik Di Indonesia”, Inter Setiaji, Bayu. (2020) Direktur dari PT Lutan Edukasi. https://biz.kompas.com/ read/2020/03/11/230918328/tips- tips-agar-siap-menghadapi-era-disrupsi. Mc Quail, Denis. (2006). Teori Komunikasi Massa Suatu Pengantar. Alih bahasa oleh Agus Dharma dan Aminudin Ram. Jakarta: Erlangga. Siradj, Said Aqil (2019). Wawancara. Tanggal 25 Mei 2019, Jam 19.00-20.30 WIB di Kantor PBNU. Milles. Matthew B. dan Micheal Huberman. (1986). Qualitative Data Analysis: A Sourcebook of New Methods (Beverly Hills: Sage Publication. Siradj, Said Aqil. (1999). Islam Kebangsaan: Fiqih Demokrasi Kaum Santri. Jakarta: Pustaka Ciganjur Pavlik, John V. (1996). New Media Technology, Cultural and Commercial Perspectives. USA: Allyn and Bacon. Sparks, Colin. (2001), Bennett, W. Lance & Entman, Robert M. (eds), Pavlik, John V. References (1996.) Pedoman, Aqidah dan Asas NU, Hasil-Hasil Muktamar KE-33 NU, (Jakarta: Lembaga Ta’lif wan Nasyr PBNU, 2016). Sparks, Colin. (2001). “The Internet and the Global Public Sphere.” Dalam Bennett, W. Lance & Entman, Robert M. (eds). Mediated Politics: Communication in the Future of Democracy. United Kingdom: Cambridge University Press. Robert W. Hefner. (2000). Civil Islam; Muslim and Democration in Indonesia. Princeton, New Jersey: Princeton University Press. Salim, Agus. (2019). Wawancara; Ketua Lembaga Dakwah/ LD PBNU. Tempat: Kantor PBNU, Jakarta Pusat. Hari: Rabu, Tanggal, 10 April 2019, Jam 15.30- 17.30 WIB. Dan Khairul Anam, Minggu: 07 April. Suyanto. Bagong dan Sutinah (edt.). (2007). Metode Penelitian Sosial: Berbagai Alternatif Pendekatan. Jakarta: Kencana. Syamsuddin, AB. (2016). Pengantar Sosiologi Dakwah. Jakarta: Kencana Prenadamedia Group. KOMUNIKA: Jurnal Dakwah dan Komunikasi −Vol 16, No. 2 (2022) 199
https://openalex.org/W2111810559	https://europepmc.org/articles/pmc3384616?pdf=render	English	null	Geographic Inequalities in All-Cause Mortality in Japan: Compositional or Contextual?	PloS one	2,012	cc-by	10,164	Abstract doi:10.1371/journal.pone.0039876 Editor: Noel Christopher Barengo, Fundacio´n para la Prevencio´n y el Control de las Enfermedades Cro´nicas No Transmisibles en A Argentina Editor: Noel Christopher Barengo, Fundacio´n para la Prevencio´n y el Control de las Enfermedades Cro´nicas No Transmisibles en Ame´rica Latina (FunPRECAL), Argentina Editor: Noel Christopher Barengo, Fundacio´n para la Prevencio´n y el Control de las Enfermedades Cro´nicas No Transmisibles en Ame´rica Latina (FunPRECAL), Argentina Received March 22, 2012; Accepted May 28, 2012; Published June 27, 2012 Copyright: 2012 Suzuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Suzuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: SVS is supported by a Robert Wood Johnson Investigator Award in Health Policy Research and by a career development award from the National Institutes of Health (NHLBI K25 HL081275). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests ex * E-mail: etsuji-s@cc.okayama-u.ac.jp Etsuji Suzuki1*, Saori Kashima1,2, Ichiro Kawachi3, S. V. Subramanian3 1 Department of Epidemiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 2 Department of Public Health and Health Policy, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan, 3 Department of Society, Human Development, and Health, Harvard School of Public Health, Boston, Massachusetts, United States of America Abstract Background: A recent study from Japan suggested that geographic inequalities in all-cause premature adult mortality have increased since 1995 in both sexes even after adjusting for individual age and occupation in 47 prefectures. Such variations can arise from compositional effects as well as contextual effects. In this study, we sought to further examine the emerging geographic inequalities in all-cause mortality, by exploring the relative contribution of composition and context in each prefecture. Methods: We used the 2005 vital statistics and census data among those aged 25 or older. The total number of decedents was 524,785 men and 455,863 women. We estimated gender-specific two-level logistic regression to model mortality risk as a function of age, occupation, and residence in 47 prefectures. Prefecture-level variance was used as an estimate of geographic inequalities in mortality, and prefectures were ranked by odds ratios (ORs), with the reference being the grand mean of all prefectures (value = 1). Results: Overall, the degree of geographic inequalities was more pronounced when we did not account for the composition (i.e., age and occupation) in each prefecture. Even after adjusting for the composition, however, substantial differences remained in mortality risk across prefectures with ORs ranging from 0.870 (Okinawa) to 1.190 (Aomori) for men and from 0.864 (Shimane) to 1.132 (Aichi) for women. In some prefectures (e.g., Aomori), adjustment for composition showed little change in ORs, while we observed substantial attenuation in ORs in other prefectures (e.g., Akita). We also observed qualitative changes in some prefectures (e.g., Tokyo). No clear associations were observed between prefecture-level socioeconomic status variables and the risk of mortality in either sex. Conclusions: Geographic disparities in mortality across prefectures are quite substantial and cannot be fully explained by differences in population composition. The relative contribution of composition and context to health inequalities considerably vary across prefectures. Citation: Suzuki E, Kashima S, Kawachi I, Subramanian SV (2012) Geographic Inequalities in All-Cause Mortality in Japan: Compositional or Contextual? PLoS ONE 7(6): e39876. June 2012 \| Volume 7 \| Issue 6 \| e39876 PLoS ONE \| www.plosone.org Vital statistics and census data Data on deaths was obtained from the Report of Vital Statistics: Occupational and Industrial Aspects [13], which is compiled by the Ministry of Health, Labour and Welfare every five fiscal years since 1970, coinciding with the Population Census. The latest year for which data are available is 2005. In the death notifications, respondents are asked to fill in the decedent’s occupation at the time of death, and one of the following persons is obliged to submit the notification: (1) relatives who lived with the decedent, (2) other housemates, (3) landlord, estate owner, land/house agent, or (4) relatives who do not live with the decedent [14]. In 2005 fiscal year (i.e., from April 1, 2005 to March 31, 2006), occupation at the time of death was recorded for each decedent following the fourth revision of the Japan Standard Occupational Classification [15], which includes the following 11 groups: (1) specialist and technical workers, (2) administrative and managerial workers, (3) clerical workers, (4) sales workers, (5) service workers, (6) security workers, (7) agriculture, forestry and fishery workers, (8) transport and communication workers, (9) production process and related workers, (10) workers not classifiable by occupation, and (11) non-employed (a full description of each occupational group is available on-line in English [15]). Note that the group ‘‘non- employed’’ includes the unemployed as well as the non-labor force (e.g., home-makers, students, and the retired). Although the Census distinguishes the unemployed from home-makers, the vital records combine these categories as ‘‘non-employed.’’ The response variable, proportion of deaths in each cell, was modeled with allowances made for the varying denominator in each cell. We estimated a multilevel binomial logit link model, which consisted of a fixed part and a random part. Based on the results of the fixed part, we can estimate the relations between occupation and mortality, conditional on individual age variation, while the results of the random part allow estimation of prefecture- level variations in the risk of mortality. The prefecture-level variance was used as an estimate of geographic inequalities in mortality. The importance of measures of between-area variation has been emphasized for a better understanding of the socio- spatial patterning of health [12,20–22]. To fit the models, we used Bayesian estimation procedures as implemented via Markov chain Monte Carlo (MCMC) methods by using MLwiN 2.25 [23,24]. Vital statistics and census data We used default diffuse priors for all the parameters, meaning that we did not favor a priori any particular values of the estimates [24]. We obtained maximum- likelihood estimates for starting values of the distribution, then 500 simulations as discarded burn-in, then 50,000 further simulations to get the distribution of interest. Based on the mean as well as the 2.5th and 97.5th percentiles of the posterior distributions, odds ratios (ORs) and 95% credible intervals (CIs) for all-cause mortality were obtained for each variable. We used the Deviance Information Criterion (DIC) to compare the goodness-of-fit of each model [24]. The DIC statistic is a combination of the fit to the data and complexity, with larger DIC values suggesting worse performance. To present the results of geographic inequalities in mortality, we created maps showing prefecture-level residuals by using ArcGIS (ESRI Japan Inc., version 10.0). Denominator data for the calculation of mortality rates was obtained from the Population Census which has been conducted by the Ministry of Internal Affairs and Communications every five years since 1920 [16]. The 2005 Population Census was taken as of October 1, 2005. In the Census questionnaire, occupation was assessed by the following question [16]: ‘‘Description of work – Describe in detail the duties you are assigned to perform.’’ The questionnaires are delivered to every household, and one person in each household completes it on behalf of the household members. We used ‘‘production process and related workers’’ as the referent category because they were the largest and the second largest occupational category in men and women, respectively, excluding non-employed. First, we examined the prefecture-level variance in mortality without including any explanatory variables as follows: We restricted the analysis to those who are aged 25 or older to exclude students. Further, deaths records missing information on age or residence were excluded from the analysis, along with records with populations of 0 as well as cells with proportions being exceeding 1. As a result, the total number of decedents was 524,785 men and 455,863 women, in 47 prefectures (Table S1 and Figure 1). logit pij ~b0zu0j, ½null model where pij is a proportion of deaths in cell i in prefecture j. Prefecture-level random effect of the intercept (u0j) was assumed to be normally distributed with a mean of 0 and variance, s2 u0. Geographic Inequalities in Mortality in Japan nications every five years since 1959. We obtained the following three variables for each prefecture from the 2004 Survey and divided them into tertiles; Gini coefficient for yearly income, average yearly income, and average savings [17]. These variables were calculated among two-or-more-person households. Although household income and savings may follow skewed distributions, median income or savings were not available. context in each prefecture could substantially vary across areas, and thus the findings of the present study are expected to be very useful in providing clearer implications to mitigate the emerging geographic inequalities across prefectures. In line with most literature on area effects on health [12], we used sex, age, and occupation as a measure of composition whereas we used prefecture-level socioeconomic status as a measure of context. To provide a comprehensive perspective, the data of this study are census based and cover the whole of Japan. Statistical analysis The data had a two-level structure of 5,687 cells for men and 5,617 cells for women at level 1, nested within 47 prefectures at level 2. Each prefecture had a maximum 121 cells (11 age groups times 11 occupational groups), and the maximum number of cells in the present data set was 5,687 (121 cells times 47) for each sex (Tables S1 and S2). We thus conducted gender-specific two-level logistic regression analysis to model mortality risk as a function of age, occupation, and residence in 47 prefectures. We used multilevel statistical procedures because of their ability to model complex variance structures at multiple levels [18]. The lowest unit of analysis was ‘‘cells,’’ and our models are structurally identical to models with individuals at level 1 [19]. Vital statistics and census data Based on the prefecture-level variance, prefectures were ranked by ORs, with the reference being the grand mean of all prefectures (value = 1), and uncertainty was estimated by 95% CIs. Note that an estimate of the parameter b0 in null model represents an estimate of logarithm of the grand-mean odds for mortality among all the where pij is a proportion of deaths in cell i in prefecture j. Prefecture-level random effect of the intercept (u0j) was assumed to be normally distributed with a mean of 0 and variance, s2 u0. Based on the prefecture-level variance, prefectures were ranked by ORs, with the reference being the grand mean of all prefectures (value = 1), and uncertainty was estimated by 95% CIs. Note that an estimate of the parameter b0 in null model represents an estimate of logarithm of the grand-mean odds for mortality among all the Introduction prefectures have increased since 1995 even after adjusting for individual age and occupation in each prefecture, providing suggestive evidence of common ecologic effects of place where people live [10]. Previous studies have demonstrated the presence of geographic health inequalities between regions, between countries, and within countries [1,2]. The bulk of studies on social and geographic inequalities in health have derived primarily from the United States and western European countries [3–8]. Meanwhile, although Japan has the lowest mortality in developed world, the magnitude and patterning of health inequalities within the nation remains less understood. Recently, Suzuki et al [9] examined the time-trends in social and geographic inequalities in all-cause premature adult mortality in Japan, which suggested that spatial health disparities have widened in both sexes during the decades following the collapse of the asset bubble in the early 1990s. According to this study, geographic inequalities across 47 In the present study, we further examine the emerging geographic inequalities in all-cause adult mortality across prefec- tures in both sexes, in terms of compositional effects (i.e., effects due to the different characteristics of individuals residing in different areas) and contextual effects (i.e., effects due to features and characteristics of the area over and above the characteristics of residents) [11]. In so doing, we sought to establish whether or not the pattern of geographic inequalities in the nation is largely reflective of the variation in the composition of the areas. We hypothesized that the relative contribution of composition and PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 1 Geographic Inequalities in Mortality in Japan June 2012 \| Volume 7 \| Issue 6 \| e39876 Measures of prefecture-level socioeconomic status We derived prefecture-level socioeconomic status variables from the National Survey of Family Income and Expenditure [17], which has been conducted by the Ministry of Internal Affairs and Commu- PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 2 Geographic Inequalities in Mortality in Japan Figure 1. A blank map of Japan. We show the locations of 47 prefectures in Japan. doi:10.1371/journal.pone.0039876.g001 Geographic Inequalities in Mortality in J Figure 1. A blank map of Japan. We show the locations of 47 prefectures in Japan. doi:10.1371/journal.pone.0039876.g001 cell types across 47 prefectures. Then, we entered age and 11 occupations as level-1 variables as follows: cell types across 47 prefectures. Then, we entered age and 11 occupations as level-1 variables as follows: income/savings, we also entered Gini coefficient and average yearly income/savings into the model simultaneously. We repeated these analyses by stratifying the subjects into those aged less than 65 and those aged 65 or older. logit pij ~b0z P 10 k~1 bkxkijz P 10 l~1 clwlijzu0j, ½model 1 PLoS ONE \| www.plosone.org Geographic Inequalities in Mortality in Japan Prefectures with a lower estimate of odds for all-cause mortality are ranked higher. Note that CI and OR stand for credible interval and odds ratio, respectively. doi:10.1371/journal.pone.0039876.g002 Finally, to calculate the mean predicted probabilities of mortality for the 6 occupational groups, we removed 10 dummy variables of occupations in model 2, and entered 5 dummy variables for the 6 occupational groups as level-1 variables as follows: logit pij ~b0z P 10 k~1 bkxkijz P 6 m~2 dmWmijzP 6 m~1 umjWmij: ½model 3 We calculated the predicted probabilities of mortality among those aged 55 to 59 because they constitute the largest population in both sexes (excluding those aged 75 or older in women). Note that, in models 2 and 3, we did not allow the intercept to vary across prefectures; rather we employed separate coding for each prefecture-level random effect term [19]. Geographic Inequalities in Mortality in Japan Geographic Inequalities in Mortality in Japan Geographic Inequalities in Mortality in Japan Figure 2 Unadjusted and adjusted prefecture level residua fixed occupational differential on mortality to vary randomly across prefectures as follows: logit pij ~b0z P 10 k~1 bkxkijz P 10 l~1 clwlijz P 6 m~1 umjWmij, ½model 2 Figure 2. Unadjusted and adjusted prefecture-level residuals for all-cause mortality among men in 47 prefectures, Japan, 2005. Prefecture-level residuals are described in odds ratios with the reference being the grand mean of all prefectures. Red diamond and blue square represent point estimates of residuals from null model and model 1, respectively. Horizontal bars represent their 95% credible intervals. Prefectures with a lower estimate of odds for all-cause mortality are ranked higher. Note that CI and OR stand for credible interval and odds ratio, respectively. doi:10.1371/journal.pone.0039876.g002 where W1ij, W2ij, W3ij, W4ij, W5ij, and W6ij denote coding variables for clerical, technical and managerial occupations, sales and service occupations, agriculture, forestry and fishery occupa- tions, production and transport occupations, unclassifiable occu- pations, and non-employed, respectively, of cell i in prefecture j. Thus, u1j, u2j, u3j, u4j, u5j, and u6j represent prefecture-level random effects among the corresponding occupations. They were assumed to be normally distributed with a mean of 0 and variances of s2 u1, s2 u2, s2 u3, s2 u4, s2 u5, and s2 u6, respectively. We ranked prefectures by the 6 aggregated occupational groups based on the prefecture-level occupation-specific variances. where W1ij, W2ij, W3ij, W4ij, W5ij, and W6ij denote coding variables for clerical, technical and managerial occupations, sales and service occupations, agriculture, forestry and fishery occupa- tions, production and transport occupations, unclassifiable occu- pations, and non-employed, respectively, of cell i in prefecture j. Thus, u1j, u2j, u3j, u4j, u5j, and u6j represent prefecture-level random effects among the corresponding occupations. They were assumed to be normally distributed with a mean of 0 and variances of s2 u1, s2 u2, s2 u3, s2 u4, s2 u5, and s2 u6, respectively. We ranked prefectures by the 6 aggregated occupational groups based on the prefecture-level occupation-specific variances. Figure 2. Unadjusted and adjusted prefecture-level residuals for all-cause mortality among men in 47 prefectures, Japan, 2005. Prefecture-level residuals are described in odds ratios with the reference being the grand mean of all prefectures. Red diamond and blue square represent point estimates of residuals from null model and model 1, respectively. Horizontal bars represent their 95% credible intervals. PLoS ONE \| www.plosone.org Supplementary analyses As a supplementary analysis, we examined occupation-specific geographic inequalities in mortality. In this analysis, following the previous report of the Population Census [25], we summarized the 11 occupations into 6 groups to increase the statistical power as follows: I. clerical, technical and managerial occupations (i.e., (1) specialist and technical workers, (2) administrative and managerial workers, and (3) clerical workers), II. sales and service occupations (i.e., (4) sales workers, (5) service workers, and (6) security workers), III. agriculture, forestry and fishery occupations (i.e., (7) agricul- ture, forestry and fishery workers), IV. production and transport occupations (i.e., (8) transport and communication workers and (9) production process and related workers), V. unclassifiable occu- pations (i.e., (10) workers not classifiable by occupation), and VI. non-employed (i.e., (11) non-employed). Then, we entered 6 prefecture-level random effect terms corresponding to the 6 aggregated occupational groups into model 1 in order to allow the where xkij and wlij denote 10 dummy variables of age and occupation, respectively, of cell i in prefecture j. Like null model, based on the ‘‘adjusted’’ prefecture-level variance, prefectures were ranked by ORs, with the reference being the grand mean of all prefectures (value = 1), and uncertainty was estimated by 95% CIs. Note that an estimate of the parameter b0 in model 1 represents an estimate of logarithm of the grand-mean odds for mortality among production process and related workers (i.e., the reference category for occupation) aged 25 to 29 years (i.e., the reference category for age) across 47 prefectures. Subsequently, to explore the possible contextual effects by area- level deprivation, the prefecture-level socioeconomic status vari- able was entered into model 1 separately. Furthermore, to examine the joint effects of income inequality and average June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 3 Geographic Inequalities in Mortality in Japan = 1). We should note that the degree of change varied substantially across 47 prefectures. In some prefectures, adjustment for age and occupation yielded little change in ORs, while other prefectures exhibited striking changes. For example, as noted above, Saitama ranked at the top in null model with more than 30% lower odds for mortality in both sexes, whereas Kochi ranked at the bottom with more than 20% higher odds for mortality in both sexes. However, once we adjusted for their composition in model 1, the point estimates of ORs became close to 1, and none of them were statistically significant. In other words, Saitama and Kochi were seemingly the best and the worse prefectures, respectively, in terms of the risk for all-cause mortality, which is likely due to their composition, not context. Figure 3. Unadjusted and adjusted prefecture-level residuals for all-cause mortality among women in 47 prefectures, Japan, 2005. Prefecture-level residuals are described in odds ratios with the reference being the grand mean of all prefectures. Red diamond and blue square represent point estimates of residuals from null model and model 1, respectively. Horizontal bars represent their 95% credible intervals. Prefectures with a lower estimate of odds for all-cause mortality are ranked higher. Note that CI and OR stand for credible interval and odds ratio, respectively. doi:10.1371/journal.pone.0039876.g003 Notably, we observed qualitative changes of ORs in some prefectures – from significantly higher ORs to significantly lower ORs, and vice versa. For example, among men, the ORs in Shimane (No. 32), Kumamoto (No. 43), and Kagoshima (No. 46) were significantly high when we did not adjust for the composition in each prefecture (null model) while they became significantly low adjusting for their composition (model 1). By contrast, in Tochigi (No. 9), Chiba (No. 12), Tokyo (No. 13), Shizuoka (No. 22), Aichi (No. 23), and Kyoto (No. 26), the pattern was reversed. The results of geographic inequalities among men and women are also shown by using maps in Figures 4 and 5, respectively. Figures S1 and S2 show the patterns of age-stratified geographic inequalities among men and women, respectively. Overall, the patterns were relatively similar between the age groups when we adjusted for compositions in each prefecture (model 1) although we observed qualitative changes between age groups in some prefectures; for example, we observed significantly low odds for mortality among those aged less than 65 in both sexes in Chiba (No. Contextual effects by prefecture-level socioeconomic status Overall, we found little evidence of the association between prefecture-level socioeconomic status and the risk of mortality in both sexes, conditional on individual age and occupation (Table 2). When we stratified the subjects by age, however, there was a suggestion of an inverse association between average savings and mortality among men aged less than 65 (Table 3). No clear patterns were observed for other indicators of prefecture-level socioeconomic status. When we examined the joint effects of income inequalities and average income/savings, no substantial changes was observed (data not shown). Geographic Inequalities in Mortality in Japan 12) whereas we observed significantly high odds for mortality among those aged 65 or older in both sexes. Overall geographic inequalities in all-cause mortality doi:10.1371/journal.pone.0039876.g002 When adjusting for age and occupations in model 1, almost all of the prefecture-level residuals moved toward the null (i.e., OR PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 4 Geographic Inequalities in Mortality in Japan Overall geographic inequalities in all-cause mortality Overall geographic inequalities in all-cause mortality In Figures 2 and 3, we show the results of geographic inequalities in all-cause mortality across 47 prefectures among men and women, respectively. Note that these Figures show both unadjusted and adjusted prefecture-level residuals for mortality based on the results of the random part in null model and model 1, respectively. (See Table 1 for the results of the fixed part of model 1.) Overall, the degree of geographic inequalities was more pronounced in null model (see red diamonds in Figures 2 and 3). In null model, estimates of variances of the intercepts for men and women were 0.025 (standard error (SE): 0.005) and 0.023 (SE: 0.005), respectively, and unadjusted prefecture-specific ORs for mortality ranged from 0.681 (95% CI: 0.652, 0.712) in Saitama (No. 11) to 1.277 (95% CI: 1.214, 1.343) in Kochi (No. 39) for men and from 0.676 (95% CI: 0.647, 0.706) in Saitama (No. 11) to 1.231 (95% CI: 1.170, 1.295) in Kochi (No. 39) for women. By contrast, when we adjusted for the composition (i.e., age and occupations) of each prefecture in model 1, estimates of variances of the intercepts were substantially reduced in both sexes; 0.005 (SE: 0.001) and 0.004 (SE: 0.001) among men and women, respectively. Adjusted prefecture-specific ORs ranged from 0.870 (95% CI: 0.839, 0.901) in Okinawa (No. 47) to 1.190 (95% CI: 1.155, 1.226) in Aomori (No. 2) for men and from 0.864 (95% CI: 0.833, 0.897) in Shimane (No. 32) to 1.132 (95% CI: 1.107, 1.158) in Aichi (No. 23) for women (see blue squares in Figures 2 and 3). When adjusting for age and occupations in model 1, almost all Figure 2. Unadjusted and adjusted prefecture-level residuals for all-cause mortality among men in 47 prefectures, Japan, 2005. Prefecture-level residuals are described in odds ratios with the reference being the grand mean of all prefectures. Red diamond and blue square represent point estimates of residuals from null model and model 1, respectively. Horizontal bars represent their 95% credible intervals. Prefectures with a lower estimate of odds for all-cause mortality are ranked higher. Note that CI and OR stand for credible interval and odds ratio, respectively. June 2012 \| Volume 7 \| Issue 6 \| e39876 Geographic inequalities in all-cause mortality by occupational groups Based on the results of the random part in model 2, Table 4 shows variations in all-cause mortality across 47 prefectures by the 6 aggregated occupational groups. (See Table 1 for the results of the fixed part of model 2. The DIC values of model 2 were smaller than those of model 1 in both sexes, suggesting better fit to the data.) In both sexes, unclassifiable occupations had the highest variation, and the variations among non-employed were close to 0. Among men, the variation was higher in non-manual workers (i.e., I. clerical, technical and managerial occupations and II. sales and service occupations) than manual workers (i.e., III. agriculture, forestry and fishery occupations and IV. production and transport occupations), whereas the pattern was reversed among women. Overall chi-squared values of the random parts in model 2 for men Figure 3. Unadjusted and adjusted prefecture-level residuals for all-cause mortality among women in 47 prefectures, Japan, 2005. Prefecture-level residuals are described in odds ratios with the reference being the grand mean of all prefectures. Red diamond and blue square represent point estimates of residuals from null model and model 1, respectively. Horizontal bars represent their 95% credible intervals. Prefectures with a lower estimate of odds for all-cause mortality are ranked higher. Note that CI and OR stand for credible interval and odds ratio, respectively. doi:10.1371/journal.pone.0039876.g003 June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 5 PLoS ONE \| www.plosone.org Geographic Inequalities in Mortality in Japan Table 1. Odds ratios for all-cause mortality associated with fixed parameters, along with the Deviance Information Criterion, Japan, 2005. Geographic inequalities in all-cause mortality by occupational groups Men Women Model 1a Model 2b Model 1a Model 2b Characteristics OR 95% CI OR 95% CI OR 95% CI OR 95% CI Age (y) 25–29 1.00 Reference 1.00 Reference 1.00 Reference 1.00 Reference 30–34 1.44 1.37, 1.51 1.42 1.35, 1.50 1.45 1.35, 1.55 1.43 1.32, 1.55 35–39 2.11 2.02, 2.21 2.10 2.00, 2.20 2.01 1.88, 2.15 1.98 1.84, 2.14 40–44 3.08 2.95, 3.22 3.04 2.91, 3.18 3.28 3.08, 3.50 3.24 3.00, 3.49 45–49 4.68 4.49, 4.88 4.59 4.40, 4.79 4.78 4.50, 5.08 4.72 4.39, 5.07 50–54 7.36 7.08, 7.64 7.20 6.91, 7.50 6.99 6.60, 7.39 6.90 6.44, 7.40 55–59 11.60 11.18, 12.04 11.42 10.98, 11.88 11.24 10.64, 11.88 11.11 10.38, 11.90 60–64 10.15 9.78, 10.53 10.03 9.65, 10.43 12.73 12.06, 13.45 12.60 11.77, 13.48 65–69 11.80 11.37, 12.25 11.68 11.23, 12.14 17.16 16.26, 18.12 16.98 15.87, 18.17 70–74 18.36 17.70, 19.05 18.18 17.50, 18.90 30.24 28.67, 31.90 29.93 28.00, 32.00 $75 57.39 55.35, 59.51 56.91 54.79, 59.11 153.32 145.45, 161.62 151.79 142.07, 162.18 Occupation Specialist and technical workers 3.16 3.09, 3.23 3.26 3.14, 3.40 3.28 3.13, 3.44 3.37 3.02, 3.77 Administrative and managerial workers 3.20 3.11, 3.28 3.27 3.13, 3.41 7.96 7.53, 8.43 8.21 7.30, 9.23 Clerical workers 0.96 0.93, 0.99 0.99 0.95, 1.04 0.97 0.92, 1.02 1.00 0.89, 1.12 Sales workers 1.69 1.65, 1.74 1.77 1.71, 1.84 2.05 1.96, 2.15 2.08 1.90, 2.28 Service workers 4.05 3.95, 4.16 4.24 4.09, 4.40 2.43 2.32, 2.54 2.47 2.26, 2.70 Security workers 1.78 1.70, 1.86 1.84 1.74, 1.94 16.27 14.18, 18.66 16.57 14.15, 19.40 Agriculture, forestry and fishery workers 3.33 3.26, 3.41 3.05 2.88, 3.24 2.31 2.21, 2.41 2.34 2.08, 2.63 Transport and communication workers 1.80 1.75, 1.86 1.81 1.75, 1.87 12.63 11.34, 14.08 12.57 11.26, 14.03 Production process and related workers 1.00 Reference 1.00 Reference 1.00 Reference 1.00 Reference Workers not classifiable by occupation 7.75 7.53, 7.98 8.34 6.60, 10.53 10.49 9.96, 11.04 11.35 8.93, 14.42 Non-employed c 9.98 9.81, 10.16 9.08 8.56, 9.63 6.86 6.62, 7.12 7.03 6.24, 7.91 Deviance Information Criterion 78,803.48 74,117.36 50,873.53 49,658.28 Table 1. Odds ratios for all-cause mortality associated with fixed parameters, along with the Deviance Information Criterion, Japan, 2005. and women were 82.375 (21 degrees of freedom, P,0.01) and 70.504 (21 degrees of freedom, P,0.01), respectively. correlation coefficients between II. sales and service occupations and IV. production and transport occupations were also high (0.911 and 0.777 in men and women, respectively). PLoS ONE \| www.plosone.org Discussion they cannot directly determine whether differences across areas are due to characteristics of the areas themselves or to differences between the characteristics of individuals residing in different areas [34]. We should also note that, due to ecologic fallacy [35], their findings cannot be necessarily extrapolated to the association between socioeconomic status and individual health. To examine geographic inequalities in all-cause mortality in Japan, we used the 2005 vital statistics and census data. The present findings demonstrate the presence of substantial geo- graphic variations in both sexes across 47 prefectures, even after adjusting for the composition (i.e., age and occupation) of each prefecture. Adjusting for age and occupation, ORs for all-cause mortality ranged from 0.870 in Okinawa to 1.190 in Aomori in men, while they ranged from 0.864 in Shimane to 1.132 in Aichi in women. In other words, even when taking into account the differentials of compositions in each prefecture, the risk for all- cause mortality varied by as much as 30% across prefectures. Subsequently, we used three different, but related prefecture-level socioeconomic status variables to examine their possible contex- tual effects – Gini coefficients for yearly income, average yearly income, and average savings. Although there was an indication of an inverse association between average savings and mortality among men aged less than 65 years, no clear patterns were observed for other prefecture-level variables. The patterns of geographic inequalities were relatively similar between non- manual occupations and production and transport occupations, primarily among men. In this study, we employed a novel multilevel approach and used the results of the random part of multilevel models to examine the geographic inequalities in all-cause mortality, by simultaneously adjusting for composition and context [18]. Indeed, rather than seeing the random part of multilevel models as a nuisance in an attempt to identify the fixed effects, estimating variance would add substantive information into the boundaries of the collectives to which individuals belong [20–22]. In particular, the present study would be of great use to assess the relative contribution of composition and context to the geographic inequalities across 47 prefectures. In some prefectures, adjustment for age and occupation showed little change in ORs for mortality, which implies that their composition played only a minor role. For example, adjustment for age and occupation showed little change in ORs in Aomori (No. 2) in both sexes, and they remained significantly high. Geographic inequalities in all-cause mortality by occupational groups Although we observed a strong correlation between I. clerical, technical and managerial occupations and IV. production and transport occupations among men (i.e., 0.807), we did not observe this pattern among women (i.e., 0.538). In Tables S3 and S4, we show the rankings of 47 prefectures by the occupational groups among men and women, respectively. The corresponding patterns of geographic inequalities are also illustrated using maps in Figures S3 and S4, respectively. Table S5 shows the results of prefecture-level variance and covariance among the 6 occupational groups. Overall, men and women revealed a similar pattern (see signs of the covariances). In both sexes, the correlation coefficients between I. clerical, technical and managerial occupations and II. sales and service occupations were high (0.944 and 0.856 in men and women, respectively), and the See Table S6 for the predicted number of all-cause mortality for each occupational group among those aged 55 to 59, which was calculated from the results of model 3. PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 6 Geographic Inequalities in Mortality in Japan Figure 4. Unadjusted and adjusted geographic inequalities in all-cause mortality among men, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among men. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. doi:10.1371/journal.pone.0039876.g004 Figure 4. Unadjusted and adjusted geographic inequalities in all-cause mortality among men, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among men. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. doi:10.1371/journal.pone.0039876.g004 PLoS ONE \| www.plosone.org Discussion This result suggests that composition matters much less than context, implying a possibility of contextual detrimental determinant(s) of health in the prefecture, e.g., economic, environmental, or social. Obviously, a possibility that this pattern emerges due to an omitted composition of the prefecture cannot be ruled out since the information about other indicators of composition (e.g., income, education, etc.) was not available. It is notable, however, that we observed substantial attenuation in ORs when adjusting for age and occupation in Previous studies from Japan have analyzed geographic inequal- ities in health by examining the relationship between area-level socioeconomic status and health outcomes in the corresponding areas, such as life expectancy and age-adjusted mortality rates [26–33]. These ecologic studies would be useful to document and monitor inequalities in health, showing the possible relationship between area-level deprivation and health. We should note, however, that the relevance of these studies is often limited since PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 7 Geographic Inequalities in Mortality in Japan Figure 5. Unadjusted and adjusted geographic inequalities in all-cause mortality among women, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among women. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. doi:10.1371/journal.pone.0039876.g005 Figure 5. Unadjusted and adjusted geographic inequalities in all-cause mortality among women, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among women. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. doi:10.1371/journal.pone.0039876.g005 other prefectures. For example, in Akita (No. 5), unadjusted ORs were remarkably high by approximately 20% in both sexes, whereas they moved toward the null after adjusting for age and occupation, and OR was no longer statistically significant in women. Discussion (Note that Akita is a neighboring prefecture to Aomori as shown in Figure 1.) This finding indicates that composition in Akita played a significant role in lowering its health status in term of the risk for all-cause mortality. At the same time, the findings suggest that, once adjusting for its composition, the (unspecified) contextual effect(s) in Akita is approximately equivalent to the grand mean of all prefectures, in terms of inequalities in all-cause mortality. To summarize, based on the present findings, we can weigh the impact of composition against the impact of context on the apparent pattern of geographic inequalities, which would provide a useful clue as to direct our attention toward more effective interventions. Table 2. Odds ratios for all-cause mortality associated with prefecture-level socioeconomic status variables, Japan, 2005a. Men Women OR 95% CI OR 95% CI Gini coefficients for yearly incomeb Low 1.00 Reference 1.00 Reference Middle 0.98 0.94, 1.02 0.96 0.92, 1.00 High 0.98 0.94, 1.03 0.98 0.94, 1.02 Average yearly incomeb High 1.00 Reference 1.00 Reference Middle 0.97 0.92, 1.02 0.96 0.92, 1.00 Low 0.99 0.94, 1.04 0.97 0.93, 1.01 Average savingsb High 1.00 Reference 1.00 Reference Middle 1.00 0.95, 1.06 0.99 0.95, 1.03 Low 0.99 0.94, 1.04 0.98 0.94, 1.02 CI; credible interval, OR; odds ratio. aThese odds ratios were adjusted for age and occupations. Prefecture-level variables were adjusted for separately. bThese variables were calculated among two-or-more-person households. doi:10.1371/journal.pone.0039876.t002 Table 2. Odds ratios for all-cause mortality associated with prefecture-level socioeconomic status variables, Japan, 2005a. Notably, we observed qualitative changes before and after adjusting for age and occupation in some prefectures. In particular, in Chiba (No. 12), Tokyo (No. 13), and Aichi (No. 23), although the adjusted prefecture-level ORs for mortality were significantly high in both sexes, they were apparently ‘‘masked’’ by their composition in unadjusted analyses – their unadjusted ORs were remarkably low by approximately 20%. This phenomenon would be explained as a result of skewed distributions of composition(s) in these prefectures; the distribution is skewed to those who have a lower risk for mortality, which outweighs the ‘‘negatives’’ of the context in these prefectures (see Table 1). June 2012 \| Volume 7 \| Issue 6 \| e39876 Discussion Notably, compared with manual workers, the risk for mortality was higher among upper non-manual workers (i.e., specialist and technical workers and administrative and managerial workers), June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 PLoS ONE \| www.plosone.org 8 Geographic Inequalities in Mortality in Japan Table 3. Odds ratios for all-cause mortality associated with prefecture-level socioeconomic status variables when stratified by age, Japan, 2005a. Men Women Less than 65 65 or older Less than 65 65 or older OR 95% CI OR 95% CI OR 95% CI OR 95% CI Gini coefficients for yearly incomeb Low 1.00 Reference 1.00 Reference 1.00 Reference 1.00 Reference Middle 0.99 0.91, 1.07 0.97 0.92, 1.02 0.99 0.91, 1.07 0.96 0.92, 1.01 High 0.98 0.91, 1.06 0.98 0.93, 1.03 1.01 0.92, 1.10 0.98 0.94, 1.02 Average yearly incomeb High 1.00 Reference 1.00 Reference 1.00 Reference 1.00 Reference Middle 0.98 0.90, 1.07 0.96 0.91, 1.01 0.96 0.88, 1.05 0.96 0.92, 1.00 Low 1.03 0.95, 1.11 0.97 0.93, 1.01 1.04 0.96, 1.13 0.97 0.93, 1.01 Average savingsb High 1.00 Reference 1.00 Reference 1.00 Reference 1.00 Reference Middle 1.03 0.95, 1.11 1.00 0.95, 1.05 1.00 0.92, 1.09 1.00 0.96, 1.05 Low 1.09 1.01, 1.17 0.98 0.93, 1.03 1.05 0.96, 1.14 0.98 0.94, 1.02 CI; credible interval, OR; odds ratio. aThese odds ratios were adjusted for age and occupations. Prefecture-level variables were adjusted for separately. bThese variables were calculated among two-or-more-person households. doi:10.1371/journal.pone.0039876.t003 cause mortality associated with prefecture-level socioeconomic status variables when stratified by age, Table 3. Odds ratios for all-cause mortality associated with prefecture-level socioeconomic status variables when stratified by age, Japan, 2005a. CI; credible interval, OR; odds ratio. aThese odds ratios were adjusted for age and occupations. Prefecture-level variables were adjusted for separately. bThese variables were calculated among two-or-more-person households. doi:10.1371/journal.pone.0039876.t003 We explored the possible contextual effects of prefecture-level socioeconomic status by using three variables. Apparently, each indicator of area socioeconomic status may be tapping into different aspects of the social environment and may be differently associated with specific health outcomes [12]. Note that we examined them after adjusting for individual age and occupation, in contrast with previous ecologic studies [26–33]. A previous review suggested that the studies in income inequality are more which is different from the typical hierarchical pattern in industrialized western European and North American countries [3,4,6]. Discussion A recent study from Japan suggested that this remarkable pattern emerged among men following the collapse of the asset bubble in the early 1990s [9]. These discussions highlight the significance of examining the pattern of geographic inequalities in terms of composition and context, so that researchers can present its true picture. Table 4. Variation in all-cause mortality between 47 prefectures by occupation groups, Japan, 2005a. Men Women Residuals on logit scale Range of OR Residuals on logit scale Range of OR Occupation Estimate 95% CI Estimate 95% CI Clerical, technical and managerial occupations 0.038 0.021, 0.055 0.666 to 1.357 0.016 0.007, 0.024 0.796 to 1.303 Sales and service occupations 0.044 0.024, 0.063 0.576 to 1.387 0.027 0.013, 0.041 0.722 to 1.326 Agriculture, forestry and fishery occupations 0.023 0.012, 0.034 0.751 to 1.408 0.055 0.028, 0.082 0.591 to 1.403 Production and transport occupations 0.031 0.017, 0.044 0.638 to 1.350 0.055 0.023, 0.086 0.681 to 1.609 Unclassifiable occupations 0.550 0.306, 0.795 0.201 to 4.454 0.515 0.279, 0.751 0.270 to 5.398 Non-employedb 0.005 0.003, 0.008 0.850 to 1.174 0.004 0.002, 0.006 0.862 to 1.126 CI; credible interval, OR; odds ratio. aThese variations were calculated from model 2. All the differential tests of the variations were statistically significant, except for clerical, technical and managerial occupations vs. sales and service occupations among men (P = 0.318), clerical, technical and managerial occupations vs. agriculture, forestry and fishery occupations among men (P = 0.126), clerical, technical and managerial occupations vs. production and transport occupations among men (P = 0.278), sales and service occupations vs. agriculture, forestry and fishery occupations among men (P = 0.058), agriculture, forestry and fishery occupations vs. production and transport occupations among men (P = 0.377), sales and service occupations vs. agriculture, forestry and fishery occupations among women (P = 0.067), sales and service occupations vs. production and transport occupations among women (P = 0.050), and agriculture, forestry and fishery occupations vs. production and transport occupations among women (P = 0.996). bNon-employed includes the unemployed as well as the non-labor force. doi:10.1371/journal.pone.0039876.t004 Table 4. Variation in all-cause mortality between 47 prefectures by occupation groups, Japan, 2005 Variation in all-cause mortality between 47 prefectures by occupation groups, Japan, 2005a. C ; c ed b e te a , O ; odds at o. aThese variations were calculated from model 2. Supporting Information Figure S1 Unadjusted and adjusted geographic inequal- ities in all-cause mortality among men, stratified by age groups, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among men. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. (PDF) Figure S1 Unadjusted and adjusted geographic inequal- ities in all-cause mortality among men, stratified by age groups, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among men. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. (PDF) Figure S2 Unadjusted and adjusted geographic inequal- ities in all-cause mortality among women, stratified by age groups, Japan, 2005. We show the overall geographic inequalities in all-cause mortality across 47 prefectures among women. Unadjusted and adjusted inequalities were estimated from null model and model 1, respectively. Prefecture-level residuals are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. (PDF) There are some limitations of this study. First, as a composition of each prefecture, only the information about sex, age, and occupation at the time of death were available. Occupations have been used as a dominant measure of socioeconomic position or occupational hazard, and researchers have been increasingly recognizing that occupation-based socio- economic position may also reflect social networks [39]. Recent studies from Japan have indicated the significance of workplace social networks and social capital to health status among Japanese workers [40–42]. We should, however, note that occupations reflect only certain aspects of socioeconomic position, and in particular, the most appropriate way of defining socioeconomic position among women might not be occupation. To overcome this, we used the finest occupational classification available in the present data set, which could adjust for other omitted compositional variables (e.g., education). Supporting Information However, we should carefully interpret the findings among the group ‘‘non- employed’’ because this group included the unemployed as well as the non-labor force. Second, the smallest geographic unit available was the prefecture, and we could not explore geographic inequalities in finer detail. Although the prefecture may be a useful and valid unit of analysis since it is the unit that has direct administrative authority in the economic, education, and health sectors [43], we should note that the choice of spatial unit can lead to different conclusions regarding the pattern of geographic inequalities [12,44,45]. Third, a possibility of numerator/denominator bias between the two sources of information (i.e., vital statistics and census, respectively) cannot be ruled out. Although this type of measurement error may occur homogeneously across prefectures, it could exhibit varying degrees of adjustment if the person recording the notification of deaths tends to misclassify some specific occupations. Figure S3 Geographic inequalities in all-cause mortal- ity by occupational groups among men, Japan, 2005. We show the geographic inequalities in all-cause mortality across 47 prefectures for the six aggregated occupational groups, conditional on individual age and occupation. Prefecture-level residuals from model 2 are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. (PDF) Figure S4 Geographic inequalities in all-cause mortal- ity by occupational groups among women, Japan, 2005. We show the geographic inequalities in all-cause mortality across 47 prefectures for the six aggregated occupational groups, conditional on individual age and occupation. Prefecture-level residuals from model 2 are described by odds ratios, with the reference being the grand mean of all prefectures. Prefectures with lower odds for mortality are blue, and those with higher odds are red. The prefectures with non-significant residuals are gray. (PDF) Discussion All the differential tests of the variations were statistically significant, except for clerical, technical and managerial occupations vs. sales and service occupations among men (P = 0.318), clerical, technical and managerial occupations vs. agriculture, forestry and fishery occupations among men (P = 0.126), clerical, technical and managerial occupations vs. production and transport occupations among men (P = 0.278), sales and service occupations vs. agriculture, forestry and fishery occupations among men (P = 0.058), agriculture, forestry and fishery occupations vs. production and transport occupations among men (P = 0.377), sales and service occupations vs. agriculture, forestry and fishery occupations among women (P = 0.067), sales and service occupations vs. production and transport occupations among women (P = 0.050), and agriculture, forestry and fishery occupations vs. production and transport occupations among women (P = 0.996). bNon-employed includes the unemployed as well as the non-labor force. June 2012 \| Volume 7 \| Issue 6 \| e39876 PLoS ONE \| www.plosone.org 9 Geographic Inequalities in Mortality in Japan supportive in large areas, e.g., states, regions, and metropolitan areas, because in that context income inequality serves as a measure of the scale of social stratification [36]. As has been noted previously [37], a prefecture is similar to a state in the United States in terms of its population size and variations in income inequality. Although we thoroughly investigated their possible effects, no clear patterns were observed except for an inverse association between average savings and mortality among men aged less than 65 years. We should note, however, that the measures of area socioeconomic status in this study provide only truncated information about the context of areas [38]. More importantly, we lacked information at the individual level on the socioeconomic variables measured at the prefecture level, i.e., household income and household savings, which precludes a rigorous examination of true causal operation at the prefecture level. Further studies are warranted to explore contextual effects in more detail by including a sufficient number of variables measured at the individual level. PLoS ONE \| www.plosone.org References 7. Davey Smith G, Krieger N (2008) Tackling health inequities. BMJ 337: a1526. 8. Kawachi I, Kennedy BP (2002) The Health of Nations: Why Inequality is Harmful to Your Health. New York, NY: The New Press. 33. Fukuda Y, Nakamura K, Takano T (2004) Wide range of socioeconomic factors associated with mortality among cities in Japan. Health Promot Int 19: 177–187. 9. Suzuki E, Kashima S, Kawachi I, Subramanian SV (2012) Social and geographic inequalities in premature adult mortality in Japan: a multilevel observational study from 1970 to 2005. BMJ Open 2: e000425. Available: http://bmjopen.bmj.com/content/2/2/e000425.full. Accessed 23 May 2012. 34. Diez-Roux AV (2001) Investigating neighborhood and area effects on health. Am J Public Health 91: 1783–1789. 35. Wakefield J (2008) Ecologic studies revisited. Annu Rev Public Health 29: 75– 90. http://bmjopen.bmj.com/content/2/2/e000425.full. Accessed 23 10. Subramanian SV, Glymour MM, Kawachi I (2007) Identifying causal ecologic effects on health: a methodological assessment. In: Galea S, editor. Macrosocial Determinants of Population Health. New York, NY: Springer. 301–331. 36. Wilkinson RG, Pickett KE (2006) Income inequality and population health: a review and explanation of the evidence. Soc Sci Med 62: 1768–1784. 37. Shibuya K, Hashimoto H, Yano E (2002) Individual income, income distribution, and self rated health in Japan: cross sectional analysis of nationally representative sample. BMJ 324: 16–19. 11. Diez-Roux AV (2002) A glossary for multilevel analysis. J Epidemiol Community Health 56: 588–594. 12. Riva M, Gauvin L, Barnett TA (2007) Toward the next generation of research into small area effects on health: a synthesis of multilevel investigations published since July 1998. J Epidemiol Community Health 61: 853–861. 38. Cummins S, Macintyre S, Davidson S, Ellaway A (2005) Measuring neighbourhood social and material context: generation and interpretation of ecological data from routine and non-routine sources. Health Place 11: 249– 260. since July 1998. J Epidemiol Community Health 61: 853–861. 13. Ministry of Health, Labour and Welfare. Overview Report of Vital Statistics in FY 2005: Occupational and Industrial Aspects Available: http://www.mhlw.go. jp/english/database/db-hw/orvf/index.html. Accessed 23 May 2012. 39. Galobardes B, Shaw M, Lawlor DA, Davey Smith G, Lynch J (2006) Indicators of socioeconomic position. In: Oakes JM, Kaufman JS, editors. Methods in Social Epidemiology. San Francisco, CA: Jossey-Bass. 47–85. 14. Ministry of Health, Labour and Welfare. Outline of Vital Statistics in Japan. Available: http://www.mhlw.go.jp/english/database/db-hw/outline/index. html. Accessed 23 May 2012. 40. Author Contributions Conceived and designed the experiments: ES SVS. Performed the experiments: ES. Analyzed the data: ES SVS. Contributed reagents/ materials/analysis tools: ES. Wrote the paper: ES. Data collection: ES. Created the maps: SK. Revised the manuscript for intellectual content: SK IK SVS. Supervised the study: SVS. Conceived and designed the experiments: ES SVS. Performed the experiments: ES. Analyzed the data: ES SVS. Contributed reagents/ materials/analysis tools: ES. Wrote the paper: ES. Data collection: ES. Created the maps: SK. Revised the manuscript for intellectual content: SK IK SVS. Supervised the study: SVS. Geographic Inequalities in Mortality in Japan Geographic Inequalities in Mortality in Japan Table S6 Predicted number of all-cause mortality (per 100,000) by each occupation group, Japan, 2005. (PDF) References Suzuki E, Takao S, Subramanian SV, Doi H, Kawachi I (2009) Work-based social networks and health status among Japanese employees. J Epidemiol Community Health 63: 692–696. 15. Ministry of Internal Affairs and Communications. Japan Standard Occupational Classification. Available: http://www.stat.go.jp/english/index/seido/ shokgyou/index-co.htm. Accessed 23 May 2012. 41. Suzuki E, Takao S, Subramanian SV, Komatsu H, Doi H, et al. (2010) Does low workplace social capital have detrimental effect on workers’ health? Soc Sci Med 70: 1367–1372. 16. Ministry of Internal Affairs and Communications. Population Census. Available: http://www.stat.go.jp/english/data/kokusei/index.htm. Accessed 23 May 2012. 42. Suzuki E, Fujiwara T, Takao S, Subramanian SV, Yamamoto E, et al. (2010) Multi-level, cross-sectional study of workplace social capital and smoking among Japanese employees. BMC Public Health 10: 489. Available: www. biomedcentral.com/1471-2458/10/489. Accessed 23 May 2012. 17. Ministry of Internal Affairs and Communications. National Survey of Family Income and Expenditure. Available: http://www.stat.go.jp/english/data/ zensho/index.htm. Accessed 23 May 2012. y 18. Raudenbush SW, Bryk AS (2002) Hierarchical Linear Models: Applications and Data Analysis Methods. Thousand Oaks, CA: Sage Publications. y 18. Raudenbush SW, Bryk AS (2002) Hierarchical Linear Models: Ap 43. Hasegawa T (2001) Japan: historical and current dimensions of health and health equity. In: Evans T, Whitehead M, Diderichsen F, Bhuiya A, Wirth M, editors. Challenging Inequities in Health: From Ethics to Action. New York, NY: Oxford University Press. 90–103. 19. Subramanian SV, Duncan C, Jones K (2001) Multilevel perspectives on modeling census data. Environment and Planning A 33: 399–417. 44. Krieger N, Chen JT, Waterman PD, Soobader MJ, Subramanian SV, et al. (2002) Geocoding and monitoring of US socioeconomic inequalities in mortality and cancer incidence: does the choice of area-based measure and geographic level matter?: The Public Health Disparities Geocoding Project. Am J Epidemiol 156: 471–482. 20. Merlo J, Yang M, Chaix B, Lynch J, Ra˚stam L (2005) A brief conceptual tutorial on multilevel analysis in social epidemiology: investigating contextual phenom- ena in different groups of people. J Epidemiol Community Health 59: 729–736. 21. Merlo J, Ohlsson H, Lynch KF, Chaix B, Subramanian SV (2009) Individual and collective bodies: using measures of variance and association in contextual epidemiology. J Epidemiol Community Health 63: 1043–1048. 45. Reijneveld SA, Verheij RA, de Bakker DH (2000) The impact of area deprivation on differences in health: does the choice of the geographical classification matter? J Epidemiol Community Health 54: 306–313. p gy J p y 22. The authors thank Kelvyn Jones for his advice on statistical analysis. The authors thank Kelvyn Jones for his advice on statistical analysis. Table S1 Description of data used for multilevel models analyzing all-cause mortality in 47 prefectures, Japan, 2005. Table S2 Detailed description of data used for multi- level models analyzing all-cause mortality in 47 prefec- tures, Japan, 2005. (PDF) In conclusion, the results of the present study demonstrate that geographic inequalities in all-cause mortality are not simply a passive reflection of composition in each prefecture. Indeed, the present findings suggest that the relative contribution of compo- sition and context to health inequalities substantially vary across 47 prefectures, even between neighboring prefectures. Although we should note that compositional and contextual explanations are not mutually exclusive [46–49], the significance of context to human health cannot be over-emphasized [34,50–53], and further attention should be given to evaluating their relative contribution to the pattern of geographic inequalities in other countries. Based on the present findings, future research is needed to understand the specific determinants of emerging geographic inequalities in Japan – either compositional or contextual. Table S3 Prefecture-level residuals for all-cause mor- tality by occupations among men, Japan, 2005. (PDF) Table S4 Prefecture-level residuals for all-cause mor- tality by occupations among women, Japan, 2005. (PDF) Table S5 Variance and covariance matrices of prefec- ture-level variances of each occupation group, Japan, 2005. (PDF) PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 PLoS ONE \| www.plosone.org 10 References 28. Nakaya T, Dorling D (2005) Geographical inequalities of mortality by income in two developed island countries: a cross-national comparison of Britain and Japan. Soc Sci Med 60: 2865–2875. 1. Friel S, Marmot MG (2011) Action on the social determinants of health and health inequities goes global. Annu Rev Public Health 32: 225–236. , ( ) health inequities goes global. Annu Rev Public Health 32: 225–236. 2. Evans T, Whitehead M, Diderichsen F, Bhuiya A, Wirth M, editors (2001) Challenging Inequities in Health: From Ethics to Action. New York, NY: Oxford University Press. 2. Evans T, Whitehead M, Diderichsen F, Bhuiya A, Wirth M, editors (2001) Challenging Inequities in Health: From Ethics to Action. New York, NY: Oxford University Press. 29. Fukuda Y, Nakamura K, Takano T (2005) Cause-specific mortality differences across socioeconomic position of municipalities in Japan, 1973–1977 and 1993– 1998: increased importance of injury and suicide in inequality for ages under 75. Int J Epidemiol 34: 100–109. 3. Krieger N, Rehkopf DH, Chen JT, Waterman PD, Marcelli E, et al. (2008) The fall and rise of US inequities in premature mortality: 1960–2002. PLoS Med 5: e46. Available: http://www.plosmedicine.org/article/info%3Adoi%2F10. 1371%2Fjournal.pmed.0050046. Accessed 23 May 2012. 30. Fukuda Y, Umezaki M, Nakamura K, Takano T (2005) Variations in societal characteristics of spatial disease clusters: examples of colon, lung and breast cancer in Japan. Int J Health Geogr 4: 16. Available: http://www.ij- healthgeographics.com/content/4/1/16. Accessed 23 May 2012. 1371%2Fjournal.pmed.0050046. Accessed 23 May 2012. j p y 4. Braveman P, Egerter S, Williams DR (2011) The social determinants of health: coming of age. Annu Rev Public Health 32: 381–398. 4. Braveman P, Egerter S, Williams DR (2011) The social d coming of age. Annu Rev Public Health 32: 381–398. 31. Fukuda Y, Nakamura K, Takano T (2005) Municipal health expectancy in Japan: decreased healthy longevity of older people in socioeconomically disadvantaged areas. BMC Public Health 5: 65. Available: http://www. biomedcentral.com/1471-2458/5/65. Accessed 23 May 2012. 5. Thomas B, Dorling D, Davey Smith G (2010) Inequalities in premature mortality in Britain: observational study from 1921 to 2007. BMJ 341: c3639. 6. Mackenbach JP, Stirbu I, Roskam AJ, Schaap MM, Menvielle G, et al. (2008) Socioeconomic inequalities in health in 22 European countries. N Engl J Med 358: 2468–2481. 32. Fukuda Y, Nakamura K, Takano T (2004) Municipal socioeconomic status and mortality in Japan: sex and age differences, and trends in 1973–1998. Soc Sci Med 59: 2435–2445. 51. Diez-Roux AV (2004) The study of group-level factors in epidemiology: rethinking variables, study designs, and analytical approaches. Epidemiol Rev 26: 104–111. 52. Krieger N (1994) Epidemiology and the web of causation: Has anyone seen the spider? Soc Sci Med 39: 887–903. 53 S ki E (2012) Ti h d l S S i M d 75 452 456 References Næss Ø, Leyland AH (2010) Analysing the effect of area of residence over the life course in multilevel epidemiology. Scand J Public Health 38: 119–126. 46. Macintyre S, Ellaway A, Cummins S (2002) Place effects on health: how can we conceptualise, operationalise and measure them? Soc Sci Med 55: 125–139. p gy 23. Rasbash J, Browne WJ, Healy M, Cameron B, Charlton C (2012) MLwiN Version 2.25. Centre for Multilevel Modelling, University of Bristol. Rasbash J, Browne WJ, Healy M, Cameron B, Charlton C (2012 Version 2.25. Centre for Multilevel Modelling, University of Bristo 47. Cummins S, Curtis S, Diez-Roux AV, Macintyre S (2007) Understanding and representing ‘place’ in health research: a relational approach. Soc Sci Med 65: 1825–1838. 24. Browne WJ (2012) MCMC Estimation in MLwiN, v2.25: Centre for Multilevel Modelling, University of Bristol. 25. Ministry of Internal Affairs and Communications. Population Census: Explanation of Terms. Available: http://www.stat.go.jp/english/data/ kokusei/2000/terms.htm#Occupation. Accessed 23 May 2012. 48. Diez-Roux AV (2011) Complex systems thinking and current impasses in health disparities research. Am J Public Health 101: 1627–1634. p y 26. Fukuda Y, Nakao H, Yahata Y, Imai H (2007) Are health inequalities increasing in Japan? The trends of 1955 to 2000. Biosci Trends 1: 38–42. 49. Stafford M, Bartley M, Mitchell R, Marmot M (2001) Characteristics of individuals and characteristics of areas: investigating their influence on health in the Whitehall II study. Health Place 7: 117–129. 26. Fukuda Y, Nakao H, Yahata Y, Imai H (2007) Are health inequal in Japan? The trends of 1955 to 2000. Biosci Trends 1: 38–42. 27. Fukuda Y, Nakamura K, Takano T (2007) Higher mortality in areas of lower socioeconomic position measured by a single index of deprivation in Japan. Public Health 121: 163–173. 50. Diez-Roux AV (1998) Bringing context back into epidemiology: variables and fallacies in multilevel analysis. Am J Public Health 88: 216–222. PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 June 2012 \| Volume 7 \| Issue 6 \| e39876 11 52. Krieger N (1994) Epidemiology and the web of causation: Has anyone seen the spider? Soc Sci Med 39: 887–903. 53. Suzuki E (2012) Time changes, so do people. Soc Sci Med 75: 452–456. p 53. Suzuki E (2012) Time changes, so do people. Soc Sci Med 75: 452–456. spider? Soc Sci Med 39: 887–903. 53. Suzuki E (2012) Time changes, so do people. Soc Sci Med 75: 452–456. Geographic Inequalities in Mortality in Japan PLoS ONE \| www.plosone.org June 2012 \| Volume 7 \| Issue 6 \| e39876 12
https://openalex.org/W2884678251	https://www.frontiersin.org/articles/10.3389/fpsyg.2018.01106/pdf	English	null	Neurocognitive Development of the Resolution of Selective Visuo-Spatial Attention: Functional MRI Evidence From Object Tracking	Frontiers in psychology	2,018	cc-by	15,795	Neurocognitive Development of the Resolution of Selective Visuo-Spatial Attention: Functional MRI Evidence From Object Tracking Kerstin Wolf1,2,3, Elena Galeano Weber1,3, Jasper J. F. van den Bosch4†, Steffen Volz5, Ulrike Nöth5, Ralf Deichmann5, Marcus J. Naumer4, Till Pfeiffer2 and Christian J. Fiebach1,3,5* 1 Department of Psychology, Goethe University Frankfurt, Frankfurt am Main, Germany, 2 Institute of Psychology, University of Education Karlsruhe, Karlsruhe, Germany, 3 IDeA Center for Individual Development and Adaptive Education, Frankfurt am Main, Germany, 4 Institute of Medical Psychology, Goethe University Frankfurt, Frankfurt am Main, Germany, 5 Brain Imaging Center, Goethe University Frankfurt, Frankfurt am Main, Germany Citation: Wolf K, Galeano Weber E, van den Bosch JJF, Volz S, Nöth U, Deichmann R, Naumer MJ, Pfeiffer T and Fiebach CJ (2018) Neurocognitive Development of the Resolution of Selective Visuo-Spatial Attention: Functional MRI Evidence From Object Tracking. Front. Psychol. 9:1106. doi: 10.3389/fpsyg.2018.01106 Wolf K, Galeano Weber E, van den Bosch JJF, Volz S, Nöth U, Deichmann R, Naumer MJ, Pfeiffer T and Fiebach CJ (2018) Neurocognitive Development of the Resolution of Selective Visuo-Spatial Attention: Functional MRI Evidence From Object Tracking. Front. Psychol. 9:1106. doi: 10.3389/fpsyg.2018.01106 Keywords: selective attention, visuo-spatial attention, object tracking, development, functional magnetic resonance imaging (fMRI) ORIGINAL RESEARCH published: 30 July 2018 doi: 10.3389/fpsyg.2018.01106 Edited by: Peter Klaver, University of Surrey, United Kingdom Our ability to select relevant information from the environment is limited by the resolution of attention – i.e., the minimum size of the region that can be selected. Neural mechanisms that underlie this limit and its development are not yet understood. Functional magnetic resonance imaging (fMRI) was performed during an object tracking task in 7- and 11-year-old children, and in young adults. Object tracking activated canonical fronto-parietal attention systems and motion-sensitive area MT in children as young as 7 years. Object tracking performance improved with age, together with stronger recruitment of parietal attention areas and a shift from low-level to higher- level visual areas. Increasing the required resolution of spatial attention – which was implemented by varying the distance between target and distractors in the object tracking task – led to activation increases in fronto-insular cortex, medial frontal cortex including anterior cingulate cortex (ACC) and supplementary motor area, superior colliculi, and thalamus. This core circuitry for attentional precision was recruited by all age groups, but ACC showed an age-related activation reduction. Our results suggest that age-related improvements in selective visual attention and in the resolution of attention are characterized by an increased use of more functionally specialized brain regions during the course of development. Reviewed by: Chris Lange-Küttner, London Metropolitan University, United Kingdom Valerio Santangelo, University of Perugia, Italy *Correspondence: Christian J. Fiebach ﬁebach@psych.uni-frankfurt.de †Present address: Jasper J. F. van den Bosch, MRC Cognition and Brain Sciences Unit, University of Cambridge, Cambridge, United Kingdom Specialty section: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology Received: 11 March 2018 Accepted: 11 June 2018 Published: 30 July 2018 Wolf K, Galeano Weber E, van den Bosch JJF, Volz S, Nöth U, Deichmann R, Naumer MJ, Pfeiffer T and Fiebach CJ (2018) Neurocognitive Development of the Resolution of Selective Visuo-Spatial Attention: Functional MRI Evidence From Object Tracking. Front. Psychol. 9:1106. doi: 10.3389/fpsyg.2018.01106 INTRODUCTION Visual selective attention is the ability to modulate perception in order to optimize processing according to our current task goals (Carrasco, 2011). We can apply visual selective attention rather coarsely or we can scrutinize our environment with a ﬁne grain – limited by what has been termed the resolution of attention (He and Cavanagh, 1996). The resolution of attention is the minimum size of the focus of attention, i.e., of the region that can be selected by attention, and is as small as 3–5 min of arc in foveal vision in adults (Toet and Levi, 1992; Intriligator and Cavanagh, 2001). July 2018 \| Volume 9 \| Article 1106 1 Frontiers in Psychology \| www.frontiersin.org Neurocognitive Development of Attentional Resolution Wolf et al. Developmental research suggests that several aspects of children’s selective attention undergo substantial changes in early childhood (e.g., Ridderinkhof and van der Stelt, 2000; Scerif, 2010), including the resolution of attention (Wolf and Pfeiﬀer, 2014). The ability to constrict the focus of attention to increasingly smaller sizes changes markedly between 7 years of age and adulthood; for 9- to 13-year-old children and adolescents, it is about twice the size as for adults and for 7-year-olds three times the size as for adults (Wolf and Pfeiﬀer, 2014). Similar ﬁndings have been reported by studies using ﬂanker (Enns and Girgus, 1985; Pastò and Burack, 1997) and crowding paradigms (Bondarko and Semenov, 2005; Jeon et al., 2010; Huurneman et al., 2014; Norgett and Siderov, 2014). However, the neural mechanisms underlying the development of attentional resolution are at present unknown. This problem touches upon two intimately related aspects, namely the development of the neurocognitive mechanisms underlying visuo-spatial selective attention per se and the speciﬁc development of the precision of visuo-spatial selective attention. Developmental research on both topics is so far scarce. is considered to be a parameter requiring higher attentional precision; Alvarez and Franconeri, 2007) results in increased activation in FEF and primary visual areas. Similarly, Drew et al. (2013) used event-related brain potentials to study the eﬀect of increasing item speed during object tracking, and observed an increase in the contralateral delay activity – which supposedly originates from a source in the lateral intraparietal sulcus (Todd and Marois, 2004; Xu and Chun, 2006). INTRODUCTION In summary, there is relatively limited research on the neuro- cognitive development of visuo-spatial selective attention and a considerable lack of empirical work concerning the neural mechanisms underlying the resolution of the focus of attention and its development. The aim of the present study is to ﬁll this gap by examining brain activation patterns during an object tracking task that systematically varies the required spatial precision of selective attention (Figure 1). To achieve this, the minimum distance between the to-be-tracked object and distractors was varied in four steps, i.e., 2.8, 5.6, 8.4, and 11.2 arcmin. Using functional magnetic resonance imaging (fMRI), BOLD activation changes were determined in children aged 7 years, 11 years, and in young adults, to identify brain mechanisms underlying the developmental changes in the resolution of spatial attention previously documented in behavioral work using the same paradigm (Wolf and Pfeiﬀer, 2014). Regarding the neural basis of visual selective attention, adult and primate research has converged on the view that selective attention can be conceived as a modulation of perceptual processes, with modulatory signals originating from a fronto- parietal network comprising frontal eye ﬁelds (FEF), posterior parietal cortex (PPC, most speciﬁcally the superior parietal lobule/SPL and intraparietal sulcus/IPS), and the superior colliculi, and with sensory areas as targets of these modulatory signals (for reviews see Baluch and Itti, 2011; Moore and Zirnsak, 2017). Based upon previous developmental studies on object tracking and voluntary attention (Booth et al., 2003; Beaton et al., 2010; Wendelken et al., 2011), it was hypothesized that all age groups would recruit the canonical network activated during object tracking (i.e., the fronto-parietal attention network – including the FEF and posterior parietal areas SPL and IPS – as well as motion area MT). At the same time, however, it was predicted that increasing age would be associated with activation increases within the fronto-parietal regions of this network during tracking. Increased demands with regard to spatial precision (i.e., reducing the minimum distance between the to-be-tracked object and distractors during the tracking phase) were expected to be associated with increased activity within the FEF and posterior parietal areas across all age groups (Shim et al., 2009; Drew et al., 2013). This increase in activity due to higher precision demands was expected to be the more pronounced the older the participants were. INTRODUCTION ) Despite obvious developmental changes in the ability to voluntarily deploy attention (Scerif, 2010; Amso and Scerif, 2015), only few studies have so far explored the neural mechanisms underlying age-related changes in this ability (Booth et al., 2003; Beaton et al., 2010; Wendelken et al., 2011; Vuontela et al., 2013; Hwang et al., 2014; Farrant and Uddin, 2015). These studies suggest that children as young as 8 years of age recruit fronto-parietal networks largely similar to adults during tasks requiring top-down control of attention. At the same time, however, both quantitative (Wendelken et al., 2011; Vuontela et al., 2013; Hwang et al., 2014) as well as qualitative (Konrad et al., 2005; Rubia et al., 2010) diﬀerences and diﬀerences with regard to functional connectivity (Hwang et al., 2014) in the recruitment of attention networks can be observed. MATERIALS AND METHODS While empirical evidence regarding the neural basis of the development of selective attention is scarce, it is non-existent with regard to the development of attentional resolution. Also, research in adults has so far not produced a clear empirical basis regarding the neural mechanisms underlying the control of spatially precise attentional selection. Intriligator and Cavanagh (2001) suggested that the spatial extent of the selection region is controlled by the fronto-parietal attention network. Although this proposal has so far not been investigated directly, initial evidence for fronto-parietal involvement in spatiotemporal attentional precision comes from a study by Shim et al. (2009). This study demonstrates that increasing the speed of the items to be tracked (which – besides spatial attentional precision – Frontiers in Psychology \| www.frontiersin.org Participants 1500 ms 330 ms 2040 ms 300 ms max. 8385 ms Black screen Start Correct! Response Static display Marking Movement Target? Feedback Warning Start HF pulse Static display Loading 1 2 3 4 5 6 7 8 2.6 arcmin 5.6 arcmin 8.4 arcmin 11.2 arcmin A B FIGURE 1 \| Object tracking task. (A) Trial procedure. At the beginning of the trial, a black screen was presented for 8.2 s, followed by a warning (2.34 s). Subsequently, a static display of 8 disks was presented for 1.105 s. Then, one of the disks was marked as target by highlighting it six times with a red circle (1.98 s). After another presentation of the static display for 330 ms, the disks moved in a pseudorandom manner for 7 s. The panel above the trial procedure illustrates the closest-possible encounters between target and a distractor during the tracking phase in the different distance conditions. After the tracking phase, the disks were replaced by digits (1–8) and the participant overtly named the disk that they thought was the target. The response was followed by a feedback display of variable duration, so that the time between loading of the tracking sequence and removal of the feedback was constant 20.6 s. (B) Timing of events. Each trial was a separate fMRI acquisition with a duration of 10 × TR = 25.6 s. Acquisition started during the feedback period of the preceding trial (compare Trial n vs. Trial n+1). Loading of the stimulus sequence was synchronized with the 6th TR. and visual acuity of at least 20/20 was ensured by a visual acuity test with a standardized Landolt C eye chart (Schairer Ophthal-Technik, Stuttgart, Germany). Participants were screened for psychiatric conditions by using the Strengths and Diﬃculties Questionnaire (SDQ; Goodman, 1997) for the children samples and the Symptom Checklist-90-Revised (SCL- 90-R; Franke, 2002) for the adult sample. Children were additionally rated for symptoms of ADHD by their parents on the ADHD subscale of a test for diagnosis of mental disorders in children and adolescents (DISYPS-KJ; Döpfner and Lehmkuhl, 2003). All participants had scores within ± 1 SD of the mean of a normative sample in the respective tests. IQ scores were estimated by using Raven’s Standard Progressive Matrices (SPM; Horn, 2009) for children and Standard Progressive Matrices- Plus (SPM-Plus; Raven et al., 2000) for adults. Participants Mean IQ scores were 108 for 7-year-olds (SD: 11.05), 101 for 11-year-olds (SD: 14.22), and 108 for adults [SD: 11.65; F(2,37) = 1.67, p = 0.20]. failed to complete the study (3 subjects) or because of excessive movement (8 subjects; see section on “fMRI Data Analysis” below for exclusion criteria). Two children of the older group were removed because of incomplete fMRI data. The ﬁnal sample consisted of 35 participants: 12 7-year-old children (6 females; mean age: 7.4, range: 6.5–8.0), 11 11-year-old children (5 females; mean age: 11.5, range: 11.1–12.0), and 12 adults (6 females; mean age: 24.7, range: 21.4–34.1). This sample size was determined based on several considerations, including the moderate to strong eﬀect sizes obtained in our previous behavioral study (Wolf and Pfeiﬀer, 2014), the fact that the object tracking paradigm generally elicits robust BOLD activation in attentional networks (e.g., Culham et al., 1998; Howe et al., 2009), but also the potential strain of the fMRI session for the youngest age group. Adults received monetary compensation or course credit for participation. Children received a gift token and a certiﬁcate of participation, and parents received monetary compensation for travel costs. The study was conducted in compliance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of the University Hospital Frankfurt am Main. All participants or their parents gave written informed consent prior to testing. Frontiers in Psychology \| www.frontiersin.org Participants Forty-nine healthy, right-handed participants from three age groups (7-year-olds, 11-year-olds, and adults) participated in the study. All participants were without history of neurologic/psychiatric disorders, developmental disorders of scholastic skills, or Attention Deﬁcit Hyperactivity Disorder (ADHD). Letters were distributed to the parents of children in the ﬁrst and ﬁfth grades of several primary and secondary schools in Frankfurt (Germany) in order to recruit 7- and 11-year-olds. Participants of the adult sample were students of Goethe University Frankfurt. 11 children of the youngest group were removed from the sample because they either July 2018 \| Volume 9 \| Article 1106 2 Wolf et al. Neurocognitive Development of Attentional Resolution A B 20600 ms Break 8200 ms Time 1105 ms 1980 ms 7000 ms min. 1500 ms 330 ms 2040 ms 300 ms max. 8385 ms Trial n Trial n+1 Time 5 TR Black screen Start Correct! Response Static display Marking Movement Target? Feedback Warning Start HF pulse Static display Loading 1 2 3 4 5 6 7 8 5 TR 2.6 arcmin 5.6 arcmin 8.4 arcmin 11.2 arcmin FIGURE 1 \| Object tracking task. (A) Trial procedure. At the beginning of the trial, a black screen was presented for 8.2 s, followed by a warning (2.34 s). Subsequently, a static display of 8 disks was presented for 1.105 s. Then, one of the disks was marked as target by highlighting it six times with a red circle (1.98 s). After another presentation of the static display for 330 ms, the disks moved in a pseudorandom manner for 7 s. The panel above the trial procedure illustrates the closest-possible encounters between target and a distractor during the tracking phase in the different distance conditions. After the tracking phase, the disks were replaced by digits (1–8) and the participant overtly named the disk that they thought was the target. The response was followed by a feedback display of variable duration, so that the time between loading of the tracking sequence and removal of the feedback was constant 20.6 s. (B) Timing of events. Each trial was a separate fMRI acquisition with a duration of 10 × TR = 25.6 s. Acquisition started during the feedback period of the preceding trial (compare Trial n vs. Trial n+1). Loading of the stimulus sequence was synchronized with the 6th TR. A B 20600 ms 8200 ms Time 1105 ms 1980 ms 7000 ms min. Experimental Procedures On two occasions before the fMRI session, children were acquainted with the environment and sounds of an MRI scanner using a mock scanner set-up. During fMRI scanning, participants performed an object tracking task in which they had to track one target with focal attention in the presence of seven identically looking moving distractors for a time interval of 7 s (see Children and adults participated in a separate behavioral testing session before scanning. Right-handedness was assessed by the Edinburgh Handedness Inventory (Oldﬁeld, 1971) July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 3 Neurocognitive Development of Attentional Resolution Wolf et al. Figure 1). Stimuli were presented with E-Prime 2.0 Professional (Psychology Software Tools, Inc., Sharpsburg, PA, United States) running on a PC (Shuttle Barebone SG31G2S, Intel Core 2 Duo, Windows XP) at a frame rate of 60 Hz. Images were projected onto a screen at the rear end of the MR scanner bore with an LCD projector (Sony, VPL-PX20) equipped with a custom- made lens. The screen could be seen by the participant on a mirror fastened to the head coil of the MR scanner. Each trial started with the presentation of a black screen (8.4 s) followed by a display indicating the immediate start of the task (2.34 s). Subsequently, a small quadratic black display (0.85◦) containing eight static, near white disks (each 1.42 arcmin in diameter, RGB 200/200/200) was presented at the center of a dark gray background (RGB 30/30/30) for 1.105 s. During the next 1.980 s, one of the eight disks was marked as target by highlighting it six times for 165 ms with a red circle. After another 330 ms of the static display, the disks started to move in a pseudorandom manner. 16 of those quadrants (repeated visits possible). Movement sequences were generated ahead of the experiment using Java (Sun Microsystems, Santa Clara, CA, United States) and only sequences satisfying the criteria mentioned above were included in the experiment. The same movement sequences were presented to all participants. After 7 s of movement, the disks stopped and were replaced by digits (1–8; 0.11◦). Subjects were asked to verbally communicate the number that corresponded to the target’s position, during the silent period between two trials/fMRI runs (see below). Experimental Procedures In case of a collision between two disks, the disks repulsed each other at the speciﬁed distance and this repulsion also took place according to the law of reﬂection. Minimum center-to-center distance between two distractors (i.e., non-target stimuli) was a constant 4 arcmin throughout the whole experiment. In contrast, minimum center-to-center distance between the target and distractors was manipulated in four steps between trials (but kept constant within one trial). Speciﬁcally, target and distractor never came closer (center-to-center) than 2.8, 5.6, 8.4, or 11.2 arcmin, respectively. Each trial was acquired as a separate functional MRI run to reduce head motion inﬂuences on fMRI data and to facilitate the acquisition of spoken responses without inducing additional motion artifacts. Data acquisition for each run started during the feedback phase of the previous run – to achieve steady state tissue magnetization before the start of the stimulus presentation of each trial – and stopped approximately 2 s after the presentation of the response display. Each functional run lasted for 25.6 s and was followed by a 5.44 s break during which participants gave their verbal response. Each trial thus had a ﬁxed total duration of 31.04 s. Participants were explicitly instructed to withhold their response until the end of data acquisition of the current run to make their response audible. Temporal coordination between data acquisition and stimulus presentation during each trial/run was accomplished by time-locking the loading of the tracking display to the 6th TR. Figure 1 illustrates the coordination of stimulus presentation and measurement. Within each trial, movement of the target and the distractors had to meet a number of criteria apart from the minimum distances between targets and distractors. Those criteria were deﬁned to ensure comparable movement of the disks in the diﬀerent conditions. During one trial, there had to be exactly six target-distractor encounters and ﬁve collisions between the target and the edge of the tracking ﬁeld. In addition, the tracking ﬁeld was divided into six sections along both the horizontal and vertical axes, resulting in 36 quadrants. During each trial, the target had to visit exactly Adults and 11-year-old children completed 60 experimental trials, i.e., 12 trials per distance condition (2.8, 5.6, 8.4, or 11.2 arcmin) and 12 additional trials of a baseline condition. Experimental Procedures The answer was transferred to the control room outside the scanner room by a MR-compatible microphone that was fastened to the headphones used for noise protection. Verbal responses were recorded digitally and monitored by the investigator who pressed the appropriate number on a keyboard connected to the PC that controlled stimulus presentation. The answers logged into the experimental software were later compared with the recorded response to determine the correctness of the participants’ responses. The investigator’s response terminated the presentation of the number display and a display containing visual feedback was presented. For the two older samples, the words “correct” and “incorrect” were displayed in German on the screen and the younger sample was additionally presented a green rectangle for a correct response and a red rectangle for an incorrect response. The maximum response interval was 8.385 s and the minimum time interval for feedback presentation was 1.500 s. In case of an earlier response, the duration of the feedback was prolonged so that total duration of the period between the loading of the tracking display and removal of the feedback display was 20.6 s. The feedback display was replaced by the black screen of the next trial. During the movement sequences, the disks moved independently from each other. The rate of movement of each disk from one frame to the next (33 ms) was determined by a 2-component-vector V = (x,y) coding the horizontal and vertical movement of the disk. Both values of V ranged between −2 and +2 pixels, with positive values corresponding to rightward and upward movements and negative values corresponding to movements in the opposite direction. Each disk was assigned a random vector Vt = (x,y) at the start of the movement phase. This vector was conserved for at least ﬁve frames and then changed with a probability of 10% per frame. In case of a change of movement, both components of the vector were randomly and independently increased or decreased by the value ‘1’ [i.e., Vt+n = (x+/−1, y+/−1)]. If the value of one of the components exceeded ‘2,’ this component was reset to ‘2’ and if a disk was assigned V = (0,0), a new vector was calculated in order to prevent a halt of the disk. The disks bounced oﬀthe edges of the tracking ﬁeld according to the law of reﬂection. Frontiers in Psychology \| www.frontiersin.org fMRI Data Analysis For a detailed characterization of the patterns of developmental changes in signiﬁcant brain regions, spheres (diameter: 12 mm) were centered on peak voxels of clusters showing age eﬀects; subsequently, mean beta values across all voxels included in the sphere were extracted and used to visualize developmental trends and to conduct direct mean comparisons between the three age groups. Data were preprocessed and analyzed using Statistical Parametric Mapping (SPM; Wellcome Trust Center for Neuroimaging1). EPI images were motion corrected via realignment to the ﬁrst image of the ﬁrst run. To reduce residual errors after realignment, ArtRepair toolbox for high motion samples (Stanford Psychiatric Neuroimaging Laboratory2) was applied to the EPI data sets that had been prepared by a ﬁrst step of spatial smoothing (4 mm FWHM). This algorithm removes interpolation errors from the image time series on a voxel-by-voxel basis (Grootoonk et al., 2000) and has been recommended in preference to adding motion regressors to the design matrix in high motion samples (Mazaika, 2009; Wilke, 2012; Fassbender et al., 2017). In a second step, ArtRepair was used to identify noisy volumes on the basis of frame-to-frame movement (threshold: 1.5 mm, calculated using Pythagoras’ theorem based on the 6 translation and rotation parameters derived from SPM’s image realignment process; Mazaika, 2009; Wilke, 2012, 2014; Power et al., 2015) and signal ﬂuctuations, which were then corrected via interpolation between the nearest non-repaired scans (“scrubbing”; Power et al., 2012, 2015; Siegel et al., 2014). Trials containing more than two interpolated volumes and participants with more than 15% corrected trials across the whole experiment were excluded from further analysis (see Turner and Miller, 2013; Fassbender et al., 2017 for comparable thresholds). Subsequently, the functional data were normalized (MNI152) and further smoothed (7 mm FWHM), resulting in a ﬁnal spatial smoothness of 8 mm FWHM. To ensure children’s motivation, the task was embedded into a medieval scenario. According to this scenario, each target represented a thief that the participants had to track before they could ﬁnally convict him. For each successful conviction, participants were rewarded a virtual bag of gold coins. Before each break, they were reported their intermediate results and at the end of the experiment they were informed about their total result. All age groups underwent the same scenario. fMRI Data Analysis The introductory story was presented on a laptop computer before participants entered the scanner room, as well as ﬁve practice trials that could be repeated for 7-year-olds. Inside the scanner, subjects completed four further practice trials. 1http://www.ﬁl.ion.ucl.ac.uk/spm 2http://cibsr.stanford.edu/tools/human-brain-project/artrepair-software.html 3https://afni.nimh.nih.gov/pub/dist/doc/program_help/3dClustSim.html fMRI Data Acquisition q During data acquisition, participants were instructed to lie as still as possible and their head movement was restricted by cushions. Earplugs and headphones dampened scanner noise. Functional images were acquired on a dedicated 3 Tesla head MRI scanner (Magnetom Allegra, Siemens, Erlangen, Germany) equipped with a Tx/Rx head coil, using a T2∗-weighted BOLD- sensitive gradient-echo echo planar imaging (EPI) sequence [repetition time (TR) = 2,560 ms, echo time (TE) = 30 ms] with 32 slices (thickness = 3 mm, gap = 1 mm, ﬁeld of view = 192 mm × 192 mm, matrix size 64 × 64, in-plane resolution = 3 mm × 3mm, ﬂip angle = 90◦). Slices were tilted by 30◦from axial toward coronal orientation to reduce susceptibility induced signal losses (Deichmann et al., 2003). The parameters governing the EPI readout were deliberately chosen to reduce acoustic scanner noise (band width 1,860 Hz/pixel, echo spacing 740 µs). To allow for exact timing, the fMRI sequence was modiﬁed in a way that after each acquisition of N consecutive volumes, a delay T was inserted, where N and T could be chosen freely by the researcher. For the parameters chosen in this study, each functional run (corresponding to one trial) consisted of N = 10 volumes (total acquisition time of 25.6 s; see above), followed by T = 5.44s (see above). The ﬁrst two volumes of each run were discarded, restricting the analysis to fMRI data acquired under steady state conditions. After acquisition of the functional runs, a whole brain structural data set with 1 mm isotropic resolution was acquired, using a T1-weighted magnetization prepared-rapid gradient echo (MP- RAGE) sequence (TR = 1,700 ms, TE = 1.44 ms, TI = 800 ms, ), g p At the ﬁrst level of statistical analysis, a general linear model (GLM) was calculated in which each run (i.e., trial) was modeled by a separate regressor with a square wave representing the tracking period (high-pass ﬁlter 128 s cut-oﬀ). Contrast estimates were computed for each subject by contrasting parameter estimates (beta images) of all runs belonging to one condition with parameter estimates of all runs of another condition or with zero, respectively. Resulting contrast images were submitted to random-eﬀects group analyses by performing t-tests (to determine the signiﬁcance of a certain contrast across all participants) or full-factorial ANOVA (to compare age groups). Experimental Procedures However, the latter was not included into the present analysis of the experiment because it turned out not to be successful in suppressing tracking-related brain activations. 7-year-old children completed only three distance conditions, as a previous behavioral study had shown that 7-year-olds performed the task at chance level at 2.8 arcmin (Wolf and Pfeiﬀer, 2014). The July 2018 \| Volume 9 \| Article 1106 4 Neurocognitive Development of Attentional Resolution Wolf et al. ﬁeld of view = 256 mm × 256 mm × 160 mm, matrix size 256 × 256 × 160, ﬂip angle = 7◦). trials for the two older samples were presented in four blocks of 15 trials (7 min 45 s each) and the trials of the youngest sample were presented in six blocks of eight trials (4 min 8 s each). Each block contained an equal number of trials of every distance condition. The sequence of trials within blocks was pseudorandomly determined such that the same condition was never repeated immediately, and ﬁxed across subjects. The sequence of blocks was randomized across subjects. After each block, participants were oﬀered to take a break of individual length. 7-year-old children were taken out of the scanner after half of the experiment to allow them to move. The entire measurement (excluding breaks and including the anatomical scan) lasted approximately 36 min for the two older samples and 30min for 7-year-old children. fMRI Data Analysis fMRI Data Analysis Data were preprocessed and analyzed using Statistical Parametric Mapping (SPM; Wellcome Trust Center for Neuroimaging1). EPI images were motion corrected via realignment to the ﬁrst image of the ﬁrst run. To reduce residual errors after realignment, ArtRepair toolbox for high motion samples (Stanford Psychiatric Neuroimaging Laboratory2) was applied to the EPI data sets that had been prepared by a ﬁrst step of spatial smoothing (4 mm FWHM). This algorithm removes interpolation errors from the image time series on a voxel-by-voxel basis (Grootoonk et al., 2000) and has been recommended in preference to adding motion regressors to the design matrix in high motion samples (Mazaika, 2009; Wilke, 2012; Fassbender et al., 2017). In a second step, ArtRepair was used to identify noisy volumes on the basis of frame-to-frame movement (threshold: 1.5 mm, calculated using Pythagoras’ theorem based on the 6 translation and rotation parameters derived from SPM’s image realignment process; Mazaika, 2009; Wilke, 2012, 2014; Power et al., 2015) and signal ﬂuctuations, which were then corrected via interpolation between the nearest non-repaired scans (“scrubbing”; Power et al., 2012, 2015; Siegel et al., 2014). Trials containing more than two interpolated volumes and participants with more than 15% corrected trials across the whole experiment were excluded from further analysis (see Turner and Miller, 2013; Fassbender et al., 2017 for comparable thresholds). Subsequently, the functional data were normalized (MNI152) and further smoothed (7 mm FWHM), resulting in a ﬁnal spatial smoothness of 8 mm FWHM. At the ﬁrst level of statistical analysis, a general linear model (GLM) was calculated in which each run (i.e., trial) was modeled by a separate regressor with a square wave representing the tracking period (high-pass ﬁlter 128 s cut-oﬀ). Contrast estimates were computed for each subject by contrasting parameter estimates (beta images) of all runs belonging to one condition with parameter estimates of all runs of another condition or with zero, respectively. Resulting contrast images were submitted to random-eﬀects group analyses by performing t-tests (to determine the signiﬁcance of a certain contrast across all participants) or full-factorial ANOVA (to compare age groups). Activations are reported at a family-wise corrected signiﬁcance level of 0.05, by combining a voxel-level p-value of 0.001 (uncorrected) with a cluster-extent threshold (k = 62) determined by a Monte-Carlo simulation (10,000 iterations) using the AFNI routine 3dClustSim3 (voxel size 2 mm × 2 mm × 2 mm; 2nd nearest neighbor clustering). Frontiers in Psychology \| www.frontiersin.org Behavioral Results Accuracy (i.e., the probability of picking the correct item after the 7 s tracking period; chance level = 12.5%) increased with increasing age and increasing target-distractor-distance (Figure 2). Two separate repeated measures ANOVAs were calculated to statistically support this observation, which was necessary because the most diﬃcult condition (2.8 arcmin) was not acquired in the youngest participant group due to its diﬃculty (see section “Materials and Methods”). The ﬁrst analysis compared all three age groups in those conditions completed by all groups, i.e., 5.6, 8.4, and 11.2 arcmin. Accuracy increased with age [F(2,32) = 95.85, p < 0.0001], with signiﬁcant increases between all age groups [both t(21) > 2.6, p < 0.025; Bonferroni corrected signiﬁcance threshold 0.05/2 = 0.025]. Reduced performance for 7-year-olds compared to 11-year-olds and adults was visible even in the easiest condition (11.2 arcmin; both t > 4, p < 0.0001). In addition, there was a signiﬁcant main eﬀect of distance [F(2,64) = 49.44, p < 0.0001] reﬂecting that accuracy increased with distance [both t(34) > 2.5, p < 0.025]. A robust age group × distance interaction [F(4,64) = 18.21, p < 0.0001] reﬂects that the distance eﬀect is driven by the 7- year-olds [5.6 vs. 8.4 arcmin: t(11) = 8.75, p < 0.0001; 8.4 vs. 11.2 arcmin: t(11) = 2.57; p = 0.026] while 11-year-olds and adults showed no distance-dependent improvements between these distance conditions (all t < 2.52). RESULTS 2.8 and 5.6 arcmin distance [t(22) = 13.37, p < 0.0001] and between 5.6 and 8.4 arcmin [t(22) = 2.89, p < 0.01], but not between 8.4 and 11.2 arcmin [t(22) = 1.79, p = 0.09; see also previous paragraph]. Optimal performance was thus reached by both older groups already at a distance of 8.4 arcmin. An age group x distance interaction [F(3,63) = 6.66, p < 0.01] was due to the fact that 11-year-olds and adults only diﬀered in performance in the closest distance condition [2.8 arcmin; t(21) = 3.40, p < 0.0125; all other p > 0.0125; Bonferroni corrected signiﬁcance threshold 0.05/4 = 0.0125]. fMRI Data Acquisition Activations are reported at a family-wise corrected signiﬁcance level of 0.05, by combining a voxel-level p-value of 0.001 (uncorrected) with a cluster-extent threshold (k = 62) determined by a Monte-Carlo simulation (10,000 iterations) using the AFNI routine 3dClustSim3 (voxel size 2 mm × 2 mm × 2 mm; 2nd nearest neighbor clustering). For a detailed characterization of the patterns of developmental changes in signiﬁcant brain regions, spheres (diameter: 12 mm) were centered on peak voxels of clusters showing age eﬀects; subsequently, mean beta values across all voxels included in the sphere were extracted and used to visualize developmental trends and to conduct direct mean comparisons between the three age groups. 1http://www.ﬁl.ion.ucl.ac.uk/spm 2http://cibsr.stanford.edu/tools/human-brain-project/artrepair-software.html 3https://afni.nimh.nih.gov/pub/dist/doc/program_help/3dClustSim.html July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 5 Neurocognitive Development of Attentional Resolution Wolf et al. Age-Independent Tracking-Related Brain Activations To determine regions showing age-independent activation during the tracking of moving objects, we assessed activation in the largest-distance tracking condition (11.2 arcmin) relative to zero and independent of age group. In developmental neurocognitive research, diﬀerences in activation between age groups are often accompanied by diﬀerences in task performance, which leaves open the possibility of attributing diﬀerences in neural activation to either group membership or level of performance or both (Church et al., 2010). Accounting for performance diﬀerences is thus an important prerequisite for isolating the unique eﬀects of age, which was accomplished here by including performance as a covariate of no interest. Across age groups, tracking activated an extended bilateral fronto-parietal network (Figure 3A and Table 1; for age group-speciﬁc brain activations, see Figure 3B) consisting of the SPL, aIPS and pIPS in parietal cortex, FEF, and MT. In addition, right anterior insula and subcortical areas including putamen and claustrum, and the cerebellum were activated during tracking of moving objects. In a second ANOVA, performance across all four distance conditions was compared, but only for the two older samples. Again, better performance was observed in adults than in 11-year-olds [F(1,21) = 12.91, p < 0.005] as well as a performance increase with increasing distance [F(3,63) = 173.18, p < 0.0001]. Increases in accuracy were observed between 7-year-olds 11-year-olds Adults 20 30 40 50 60 70 80 90 100 2.8 5.6 8.4 11.2 Distance (arcmin) Accuracy (%) FIGURE 2 \| Mean accuracy of each age group as a function of target-distractor-distance in the object tracking task. Note that the most difﬁcult condition (i.e., 2.8 arcmin) was not acquired in the group of 7-year-old children, as it was too difﬁcult for children of that age. Error bars represent 95% conﬁdence intervals. FIGURE 2 \| Mean accuracy of each age group as a function of target-distractor-distance in the object tracking task. Note that the most difﬁcult condition (i.e., 2.8 arcmin) was not acquired in the group of 7-year-old children, as it was too difﬁcult for children of that age. Error bars represent 95% conﬁdence intervals. July 2018 \| Volume 9 \| Article 1106 6 Neurocognitive Development of Attentional Resolution Wolf et al. 11 year of age [all t(21) < 1, p > 0.34]. Developmental eﬀects deviated from this pattern in the mid-cingulate gyrus, which showed a signiﬁcant monotonic age-related increase between 7-year-olds and adults [7 years vs. Age-Independent Tracking-Related Brain Activations adults: t(22) = 3.80, p < 0.005; 7 years vs. 11 years: t(21) = 1.69, p = 0.11; 11 years vs. adults: t(21) = 2.33, p = 0.03; Bonferroni corrected signiﬁcance threshold 0.05/3 = 0.017]. Activation in primary visual cortex around the calcarine sulcus showed the reverse pattern and decreased between 7 and 11 years of age [t(21) = 4.04, p < 0.005] and, at trend level, between age 11 and adults [t(21) = 2.05, p = 0.053]. all age groups FEF MT SPL AIPS PIPS 0 11.7 T adults 11-year-olds 7-year-olds 0 14.9 T A B Left hemisphere Right hemisphere FIGURE 3 \| Brain activation during object tracking. (A) Simple effect of tracking across all age groups, i.e., largest distance condition (11.2 arcmin) tested against zero. See Table 1 for further details. (B) Age group speciﬁc brain activations during tracking. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). AIPS, anterior intraparietal sulcus; FEF, frontal eye ﬁelds; MT, motion area MT/V5; PIPS, posterior intraparietal sulcus; SPL, superior parietal lobe. B B Age-Independent Mechanisms Underlying the Resolution of Attention y g Investigating the neural mechanisms underlying increasing spatial precision of tracking, i.e., the resolution of the focus of attention, required contrasting two conditions with diﬀerences in performance that are as large as possible (thus assuring an actual diﬀerence in task diﬃculty) while avoiding ﬂoor eﬀects in performance to assure considerable eﬀort even in the easier distance condition. Critically, the diﬀerence in performance of the diﬀerent age groups needed to be comparable. These criteria were met for diﬀerent pairs of conditions for the diﬀerent age groups, i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-old children. A 2 (distance: large vs. small) × 3 (age group) ANOVA showed that performance diﬀerences (7-year- olds 43.75%, 11-year-olds 51.52%, adults 39.58%) between these distance conditions did not diﬀer statistically between age groups [F(2,32) = 1.50, p = 0.24]. Absolute performance levels (i.e., average of easy and diﬃcult conditions), however, diﬀered between age groups [F(2,32) = 12.12, p < 0.001]. This diﬀerence was accounted for by including the average performance level as a covariate of no interest into fMRI analyses. 14.9 Increasing the demands on the spatial precision of selective attention yielded increased activation bilaterally fronto-insular cortex (FIC) and basal ganglia. In addition, a large bilateral cluster containing anterior cingulate cortex (ACC), medial and lateral superior frontal gyrus and supplementary motor area, as well as a cluster containing thalamus and the superior colliculi were increasingly activated with increasing demands on spatial precision (Figure 5A and Table 3; for age group-speciﬁc brain activations, see Figure 5B). FIGURE 3 \| Brain activation during object tracking. (A) Simple effect of tracking across all age groups, i.e., largest distance condition (11.2 arcmin) tested against zero. See Table 1 for further details. (B) Age group speciﬁc brain activations during tracking. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). AIPS, anterior intraparietal sulcus; FEF, frontal eye ﬁelds; MT, motion area MT/V5; PIPS, posterior intraparietal sulcus; SPL, superior parietal lobe. Age Effects on Brain Systems Underlying Attentional Resolution While all age groups thus recruit a similar network during tracking, we also expected age-related changes within this network. Age-related diﬀerences (again controlling for performance diﬀerences) were observed in bilateral MT extending into occipital areas, right SPL extending into IPS and precuneus, left inferior parietal lobule (IPL)/IPS, the mid-portion of the right cingulate gyrus, and in right primary visual cortex (Figure 4 and Table 2). Parietal areas and visual area MT showed a common developmental increase of activation magnitude between 11 years of age and adulthood [all t(21) > 4, p < 0.0001], whereas there were no activation diﬀerences between 7 and In a ﬁnal step, age diﬀerences in the neural substrates underlying increased spatial precision were examined, again taking into account diﬀerences in absolute performance levels. One area was found within the described network for increased spatial precision, this was the ACC. Here, precision-related brain activation dropped between 7 and 11 years of age [t(21) = 4.92, p < 0.0001], while ACC activity in 11-year-olds and adults diﬀered only at trend level [t(21) = 2.34; p = 0.029, Bonferroni corrected threshold 0.05/2 = 0.025; Figure 6 and Table 4]. In addition, four areas that belonged neither to the precision-related July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 7 Neurocognitive Development of Attentional Resolution Wolf et al. TABLE 1 \| Simple effect of object tracking (greatest distance condition (11.8 arcmin) > zero) across all age groups. TABLE 1 \| Simple effect of object tracking (greatest distance condition (11.8 arcmin) > zero) across all age groups. DISCUSSION In this study, we investigated both the neural mechanisms underlying the resolution of attention and their developmental change from childhood to adulthood. For this purpose, we examined brain activation (i.e., BOLD signals) during a visual object-tracking task that requires increasingly ﬁne-grained spatial precision cross-sectionally in children aged 7 years, children aged 11 years, and young adults. Across all age groups, performance dropped with increasing proximity between target and distractors, and thus with the requirement of increasingly narrowing down the focus of attention. Adults and 11-year-old children could sustain high performance up to a higher level of required precision. Given the similar performance patterns across diﬃculty levels of 11-year-olds and adults, the behavioral data of this study indicate that the major development in the ability to constrict the attentional focus is achieved between 7 and 11 years of age (which is consistent with our previous behavioral work; Wolf and Pfeiﬀer, 2014). Anatomical region Cerebellum (not depicted in Figure 3A) BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Tmax, maximum t statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). activation. Increasing the resolution of spatial attention – i.e., narrowing the focus of attention – involved a distinct set of brain areas, particularly the FIC and ACC, the superior colliculi, thalamus, and striatum. Developmental changes in the neural mechanisms underlying an increased resolution of the attentional focus were seen in only one of these areas, i.e., in the ACC, where an age-related reduction was observed. In the following, we discuss several implications of these results for understanding the development of visuo-spatial attention and of the ability to control the size of the focus of attention. network nor to the fronto-parietal tracking network (i.e., superior frontal gyrus, posterior cingulate gyrus, precuneus, and postero- lateral occipital cortex) showed a comparable activation decrease between 7 and 11 years of age [all t(21) > 4.14, p < 0.0001], but an increase between 11 years of age and adulthood [all t(21) > 2.97; p < 0.009]. Age Effects on Brain Systems Underlying Attentional Resolution MNI coordinates Anatomical region Hem BA k Tmax x y z Anterior insula, inferior frontal gyrus, putamen, claustrum R 13 314 7.05 38 16 8 Precentral gyrus, superior frontal gyrus, medial superior frontal gyrus, middle frontal gyrus R 6/24/32 1347 10.47 24 −10 50 Precentral gyrus, superior frontal gyrus, medial superior frontal gyrus, supplementary motor area, middle frontal gyrus L 24 1152 10.66 −28 −12 48 Precentral gyrus, inferior frontal gyrus R 6/9 127 5.00 52 2 32 Superior parietal lobe, anterior intraparietal sulcus, inferior parietal lobe, postcentral gyrus, precuneus, posterior intraparietal sulcus, middle temporal gyrus, middle occipital gyrus L 5/7/19/37/39/40 3447 13.80 −20 −56 56 Middle temporal gyrus, inferior temporal gyrus, middle occipital gyrus, posterior intraparietal sulcus, precuneus, inferior parietal lobe, superior parietal lobe, anterior intraparietal sulcus, paracentral lobe, postcentral gyrus R 5/7/19/37/39/40 4716 11.82 46 −60 6 Cerebellum (not depicted in Figure 3A) L 106 5.78 −24 −40 −42 L/R 75 4.11 −2 −74 −32 BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Tmax, maximum t statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). Frontiers in Psychology \| www.frontiersin.org Neurocognitive Development of Object Tracking Networks Within the attentional tracking network, parietal regions and motion area MT feature a protracted development that showed the most pronounced age-related increase in tracking- related activation between 11 years of age and adulthood. It can be excluded that this developmental pattern reﬂects performance changes rather than maturation of neural systems, as (i) the most pronounced performance improvements occurred between 7 and 11 years of age, which is distinct from the developmental pattern in brain activations, and (ii) performance diﬀerences were statistically controlled for in the fMRI analyses. The protracted neurocognitive development of attention-related temporal and parietal systems clearly dissociates from the developmental pattern seen in primary visual cortex, which shows age-related decrease of tracking-related activity, particularly between 7 and 11 years of age. 11 Object tracking activated a canonical tracking network (Culham et al., 1998; Jovicich et al., 2001; Howe et al., 2009; Alnaes et al., 2015) that largely coincides with established attention systems (Moore and Zirnsak, 2017), already for 7-year-old children. A subset of these areas – i.e., right SPL, left IPL/IPS, and bilateral area MT – were recruited during tracking to a higher degree with increasing age, while a region around the calcarine sulcus showed a developmental decrease in Based on this dissociation of developmental changes in primary visual vs. attention-related brain systems, three conclusions can be derived. First, our ﬁndings conﬁrm and extend earlier work showing activation of the canonical fronto- parietal attention network in 11-year-old children during tracking (Beaton et al., 2010) and generally a large degree of accordance between children’s and adults’ activations during the July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 8 Neurocognitive Development of Attentional Resolution Wolf et al. -20 0 20 -20 0 20 -20 0 20 -20 0 20 0 F mean beta-value MT (left) MT (right) Calcarine s. MCG IPL/IPS MT SPL Calcarine s. Neurocognitive Development of Object Tracking Networks 0 25.5 F -20 0 20 mean age -20 0 20 adults 11 ys 7 ys SPL IPL/IPS MCG A B MCG adults 11 ys 7 ys adults 11 ys adults 11 ys 7 ys 7 ys adults 11 ys 7 ys FIGURE 4 \| (A) ANOVA (F-statistic) results for age differences in tracking-related brain activation: middle cingulate gyrus (14, −18, 44; Fmax = 16.6), left inferior parietal lobe/intraparietal sulcus (−42, −38, 46; Fmax = 14.14), right superior and inferior parietal lobe (34, −50, 58; Fmax = 17.5), middle and inferior occipito-temporal cortex (right: 46, −70, 0; Fmax = 23.34; left: −44, −78, 12; Fmax = 25.51), calcarine sulcus (10, −90, 12; Fmax = 17.01). (B) Mean beta-values for the different age groups for the regions showing age-related differences in tracking. Error bars represent the 95% conﬁdence intervals. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). AIPS, anterior intraparietal sulcus; FEF, frontal eye ﬁelds; IPL/IPS, inferior parietal lobe and intraparietal sulcus; MCG, mid-portion of cingulate gyrus; MT, motion area MT/V5; PIPS, posterior intraparietal sulcus; SPL, superior parietal lobe. White labels indicate activation increases; red label indicates activation decrease. See Table 2 for further details. (see also Olesen et al., 2006; Velanova et al., 2008; Wendelken et al., 2011) suggests an increasing reliance on parietal sites and the functions they provide, as the attention system matures. Jointly with the FEF, the PPC is assumed to form a priority map of the visual ﬁeld (Baluch and Itti, 2011), i.e., a topologically organized map of the external environment encoding the relative importance of objects or events occurring at the corresponding location of the visual scene (Gottlieb et al., 1998; Thompson and Bichot, 2005; Gottlieb and Balan, 2010). Activity in this map is supposed to be driven both by salient (i.e., capturing attention in a bottom-up manner) external stimuli (Santangelo, 2015) and by the demands of the task at hand, and is assumed to guide attention or other goal-driven behavior toward the locations/objects of high relevance (Gottlieb and Balan, 2010; Nardo et al., 2011). Within the fronto-parietal priority network, the unique function of PPC seems to be to bind visuo-spatial, motor, and cognitive information to eventually deliver a spatial priority representation that can be easily translated into action (Gottlieb, 2007). As they become older, children may make increasing use of this complex representation to optimize their performance in attention-demanding tasks. Neurocognitive Development of Object Tracking Networks -20 0 20 -20 0 20 -20 0 20 -20 0 20 0 F mean beta-value MT (left) MT (right) Calcarine s. MCG IPL/IPS MT SPL Calcarine s. 0 25.5 F -20 0 20 mean age -20 0 20 adults 11 ys 7 ys SPL IPL/IPS MCG A B MCG adults 11 ys 7 ys adults 11 ys adults 11 ys 7 ys 7 ys adults 11 ys 7 ys Third, our data indicate a qualitative developmental change among visual systems recruited to support attentionally guided visual processing from a reliance on primary visual areas by the youngest age group toward more task-optimized processing in higher-order visual area MT by the older age groups. The developmental decrease in activation of primary visual areas – that mature earlier during development (Conner et al., 2004) – suggests that the children (from both age groups) relied more on lower-level visual representations coded in this region to accomplish the task at hand, whereas adults seemed to rely more on the higher-level, motion-sensitive area MT where we observed an age-dependent increase in activation. Lower activation of MT by younger participants suggests that it may not be fully matured at the age of our samples of children – which has indeed been demonstrated both functionally (Klaver et al., 2008) and structurally (Sowell et al., 2003). The ﬁndings of this study thus extend the proposal of Klaver et al. (2008), who suggested that with development, higher-level visual processes increasingly substitute FIGURE 4 \| (A) ANOVA (F-statistic) results for age differences in tracking-related brain activation: middle cingulate gyrus (14, −18, 44; Fmax = 16.6), left inferior parietal lobe/intraparietal sulcus (−42, −38, 46; Fmax = 14.14), right superior and inferior parietal lobe (34, −50, 58; Fmax = 17.5), middle and inferior occipito-temporal cortex (right: 46, −70, 0; Fmax = 23.34; left: −44, −78, 12; Fmax = 25.51), calcarine sulcus (10, −90, 12; Fmax = 17.01). (B) Mean beta-values for the different age groups for the regions showing age-related differences in tracking. Error bars represent the 95% conﬁdence intervals. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). AIPS, anterior intraparietal sulcus; FEF, frontal eye ﬁelds; IPL/IPS, inferior parietal lobe and intraparietal sulcus; MCG, mid-portion of cingulate gyrus; MT, motion area MT/V5; PIPS, posterior intraparietal sulcus; SPL, superior parietal lobe. White labels indicate activation increases; red label indicates activation decrease. See Table 2 for further details. Neurocognitive Development of Object Tracking Networks deployment of voluntary, i.e., goal-driven, attention (Booth et al., 2003; Wendelken et al., 2011; Vuontela et al., 2013). Second, the observation of increasing involvement of PPC with age deployment of voluntary, i.e., goal-driven, attention (Booth et al., 2003; Wendelken et al., 2011; Vuontela et al., 2013). Second, the observation of increasing involvement of PPC with age TABLE 2 \| Age differences in activation elicited by object tracking. MNI coordinates Anatomical region Hem BA k Fmax x y z Middle cingulate gyrus R 24/31 175 16.60 14 −18 44 Inferior parietal lobe, intraparietal sulcus L 40 113 14.14 −42 −38 46 Superior parietal lobe, anterior intraparietal sulcus, inferior parietal lobule, precuneus, superior occipital gyrus R 7/39/40 570 17.50 34 −50 58 Middle temporal gyrus, inferior temporal gyrus, middle occipital gyrus, superior occipital gyrus, inferior occipital gyrus, lingual gyrus R 18/19/37/39 1296 23.34 46 −70 0 Middle temporal gyrus, middle occipital gyrus, inferior occipital gyrus, lingual gyrus L 18/19/39 725 25.51 −44 −78 12 Calcarine sulcus R 17/18 78 17.01 10 −90 12 BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Fmax, maximum F statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). TABLE 2 \| Age differences in activation elicited by object tracking. TABLE 2 \| Age differences in activation elicited by object tracking. Anatomical region BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Fmax, maximum F statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 9 Wolf et al. Neurocognitive Development of Attentional Resolution MTG FIC ACC/ medSFG/ SFG Thalamus SC 0 7.1 T e r e h p s i m e h t h g i R e r e h p s i m e h tf e L s dlo -r a e y - 7 stlu d a 11-year-olds all age groups 0 9.8 T Lateral Lateral l a i d e M l a i d e M A B FIGURE 5 \| Brain activation related to attentional precision. (A) Activation related to increasing the spatial precision of the focus of attention, independent of age group. For the different age groups, contrasts of different difﬁculty conditions (i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-old children) were fed into the analysis. For the rationale behind the choice of the respective difﬁculty conditions, see section “Age-Independent Mechanisms Underlying the Resolution of Attention”. See Table 3 for further details. (B) Age group speciﬁc brain activations related to increasing spatial precision (based on the same contrasts of different difﬁculty conditions for the different age groups as depicted in A). Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). ACC, anterior cingulate cortex; FIC, fronto-insular cortex; MTG, middle temporal gyrus; SC, superior colliculus; SFG, superior frontal gyrus. MTG FIC ACC/ medSFG/ SFG Thalamus SC 0 7.1 T e r e h p s i m e h t h g i R e r e h p s i m e h tf e L all age groups A B stlu d a 0 9.8 T Lateral M l a i d e M Lateral Lateral Lateral FIGURE 5 \| Brain activation related to attentional precision. (A) Activation related to increasing the spatial precision of the focus of attention, independent of age group. Frontiers in Psychology \| www.frontiersin.org Anatomical region For the different age groups, contrasts of different difﬁculty conditions (i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-old children) were fed into the analysis. For the rationale behind the choice of the respective difﬁculty conditions, see section “Age-Independent Mechanisms Underlying the Resolution of Attention”. See Table 3 for further details. (B) Age group speciﬁc brain activations related to increasing spatial precision (based on the same contrasts of different difﬁculty conditions for the different age groups as depicted in A). Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). ACC, anterior cingulate cortex; FIC, fronto-insular cortex; MTG, middle temporal gyrus; SC, superior colliculus; SFG, superior frontal gyrus. lower-order visual-perceptual processes, to the domain of attention. space at 7 years of age to a metric representation where objects are grouped into areas at 11 years of age. If this were the case, the age-related changes observed in neuronal activation patterns may (at least partly) be related to this transition. While there is, to our knowledge, yet no developmental evidence regarding the neuronal correlates of diﬀerent spatial representations, there is some neurocognitive research on the development of a spatial ability that is conceptually closely related to the idea of processing objects as object-in-place units or as objects grouped into areas, i.e., the concept of local vs. holistic processing (Navon, 1977). This research proposes that in hierarchical conﬁgurations of stimuli, adults have a strong (attentional) bias to process conﬁgurations on a global level (i.e., the whole conﬁguration), whereas children younger than 10 years predominantly process these conﬁgurations on a local level (i.e., on a single-unit The neuronal changes observed during tracking of objects may also partly be accounted for by age-dependent diﬀerences in spatial processing abilities (Jahn et al., 2012; Brockhoﬀ et al., 2016). It has been shown that these abilities develop markedly during the age range examined here (Stiles et al., 2013). More speciﬁcally, younger children (4–6 years of age) represent objects within topological (i.e., non-metric) space and bind single objects to places, whereas older children (8–10 years of age) represent objects within Euclidean (i.e., metric) space and group objects into areas determined by vectors of a spatial axis system (Lange-Küttner, 2006; Lange-Küttner and Küttner, 2015). Anatomical region Thalamus, superior colliculi, midbrain Cerebellum BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Tmax, maximum t statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Tmax, maximum t statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). visual areas (Poirel et al., 2011, 2014). This observation leaves open the possibility that some of the changes observed in the present study, both behaviorally and at the level of brain activation, may be interpreted in terms of changes in the computations underlying diﬀerent types of spatial processing. Under this perspective, younger participants who display a bias toward local processing might predominantly process the objects in the display in a single-unit manner – which might be mediated by stronger involvement of primary visual areas. Older participants, in contrast, might rather process the whole spatial conﬁguration relying on an increasingly mature fronto-parietal network. Further research is yet required to investigate this idea. -0.6 0 0.6 SFG ACC -0.6 0 0.6 lat. occ. cortex -0.6 0 0.6 PCG -0.6 0 0.6 Precuneus SFG lat. occ. cortex ACC Precuneus PCG 0 24 F mean contrast-value mean age -0.6 0 0.6 adults 11 ys 7 ys adults 11 ys 7 ys adults 11 ys 7 ys A B FIGURE 6 \| (A) ANOVA (F-statistic) results of age differences for increased demands in attentional precision (again based on the contrasts 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-old children): Left superior and middle frontal gyrus (−24, 40, 36; Fmax = 15.86), anterior cingulate cortex (14, 50, 12; Fmax = 19.5), left posterior cingulate gyrus (−26, −36, 36; Fmax = 23.97), precuneus (4, −56, 36; Fmax = 15.4), left superior/middle occipital gyrus (−20, −88, 22; Fmax = 11.69). Anatomical region (B) Mean contrast-values (i.e., difference in beta-values between the respective easy and difﬁcult tracking condition) for the regions showing age-related differences with increasing spatial precision. Error bars represent the 95% conﬁdence intervals. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). ACC, anterior cingulate cortex; PCG, posterior cingulate gyrus; SFG, superior frontal gyrus. See Table 4 for further details. -0.6 0 0.6 SFG ACC -0.6 0 0.6 lat. occ. cortex -0.6 0 0.6 PCG -0.6 0 0.6 Precuneus SFG lat. occ. cortex ACC Precuneus PCG 0 24 F mean contrast-value mean age -0.6 0 0.6 adults 11 ys 7 ys adults 11 ys 7 ys adults 11 ys 7 ys A B A Anatomical region It can be assumed that children in our study switched from a predominantly topological, one object-in-place representation of July 2018 \| Volume 9 \| Article 1106 10 Neurocognitive Development of Attentional Resolution Wolf et al. TABLE 3 \| Activation related to increased demands in precision (i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-olds) across all age groups. TABLE 3 \| Activation related to increased demands in precision (i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-olds) across all age groups. TABLE 3 \| Activation related to increased demands in precision (i.e., 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-olds) across all age groups. MNI coordinates Anatomical region Hem BA Tmax k x y z Anterior insula, claustrum, putamen, inferior frontal gyrus, precentral gyrus L 6/9/13/44/45/47 7.09 901 −34 22 8 Anterior insula, claustrum, putamen, inferior frontal gyrus, precentral gyrus, middle frontal gyrus R 9/13/44/45/46/47 6.69 1008 30 20 −6 Anterior cingulate cortex, medial superior frontal gyrus, superior frontal gyrus, supplementary motor area, middle cingulate cortex R/L 6/8/9/10/24/32/33 6.98 4347 −8 38 26 Middle temporal gyrus R 21 4.17 82 56 −30 −8 Middle temporal gyrus, superior temporal gyrus L 22/39 4.05 108 −54 −56 10 Thalamus, superior colliculi, midbrain L/R 5.41 1294 −8 −14 8 Striatum R 3.89 70 14 12 4 Cerebellum L 4.17 128 −18 −50 −34 BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Tmax, maximum t statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). Neural Mechanisms Underlying the Resolution of the Focus of Attention While tracking-related activity was mainly seen in the fronto- parietal attention network, the set of brain regions that were evoked to achieve increased spatial precision (i.e., FIC, medial and lateral superior frontal cortex, and thalamus and superior colliculi) largely conforms to a diﬀerent functional network that has been identiﬁed in the investigation of resting-state fMRI connectivity, i.e., the so-called salience network (Seeley et al., 2007). This network is thought to highlight the (neural) salience of external stimuli by increasing the amount of cognitive resources available for their processing (Menon and Uddin, 2010; Uddin, 2015). This is supposedly achieved by control signals from the salience network to the fronto-parietal attention network, which in turn eﬀects the stimulus highlighted by the salience network to have preferential access to the brain’s attentional and memory resources (Sridharan et al., 2008; Menon and Uddin, 2010; Chen et al., 2013, 2016; Goulden et al., 2014). Given that increased attentional precision in the present study lead to increased activity in the upstream salience network independent of age group, an important conclusion from our data may thus be that willingly increasing the spatial precision of attention is a function of the insula-cingulate salience network rather than the fronto-parietal attention network. FIGURE 6 \| (A) ANOVA (F-statistic) results of age differences for increased demands in attentional precision (again based on the contrasts 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-old children): Left superior and middle frontal gyrus (−24, 40, 36; Fmax = 15.86), anterior cingulate cortex (14, 50, 12; Fmax = 19.5), left posterior cingulate gyrus (−26, −36, 36; Fmax = 23.97), precuneus (4, −56, 36; Fmax = 15.4), left superior/middle occipital gyrus (−20, −88, 22; Fmax = 11.69). (B) Mean contrast-values (i.e., difference in beta-values between the respective easy and difﬁcult tracking condition) for the regions showing age-related differences with increasing spatial precision. Error bars represent the 95% conﬁdence intervals. Statistical threshold: p < 0.05 (FEW corrected; cf. section “Materials and Methods”). ACC, anterior cingulate cortex; PCG, posterior cingulate gyrus; SFG, superior frontal gyrus. See Table 4 for further details. level; Dukette and Stiles, 2001; Harrison and Stiles, 2009). Anatomical region BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Fmax, maximum F statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). Within this network, the diﬀerent components are ascribed distinct and ﬁnely coordinated functions (Menon and Uddin, 2010). The FIC is assumed to be responsible for the detection of behaviorally relevant or perceptually salient stimuli (Seeley et al., 2007; Uddin, 2015), that is stimuli that are either of high relevance for current behavioral goals or that exhibit a high degree of sensory prominence relative to their background (Santangelo, 2015). The FIC is supposed to be the ultimate source of the control signals (Crottaz-Herbette and Menon, 2006; Chand and Dhamala, 2016b) which are then passed on to ACC (Menon and Uddin, 2010). The ACC is thought to sustain the control signals initiated by the FIC and to promote those signals ﬁrstly to the components of the fronto-parietal network and secondly to cortical areas related to motion and action (Crottaz-Herbette and Menon, 2006; Menon and Uddin, 2010; Chand and Dhamala, 2016a). The fact that in the data presented here activation around ACC spreads into motor areas like SMA appears compatible with this assumption. Besides FIC and ACC, the superior colliculi – that are not considered part of the salience network – also showed increased activity during increased attentional precision. Despite their subcortical location, the superior colliculi are in fact generally considered to be an integral part of the fronto-parietal attention network (Lovejoy and Krauzlis, 2010; Baluch and Itti, 2011). Recent evidence further suggests that the superior colliculi work in tight coupling with the thalamus – a supposed subcortical component of the salience network (Seeley et al., 2007) – to convey attentional control (Wurtz et al., 2011). We thus suggest that the modulation of visual processing required during increased spatial precision is not accomplished by the cortical components of the dorsal fronto-parietal attention network but by a routing of control signals generated by the salience network via the thalamus and superior colliculi. Anatomical region salience network is similar to adults by the age of seven (Fair et al., 2012b; Power et al., 2012; Supekar and Menon, 2012; Grayson and Fair, 2017). The results of this study deviate from earlier studies that observed either a decrease (Supekar and Menon, 2012) or an increase in FIC function with age in tasks requiring cognitive control (for a review see Houdé et al., 2010). Instead an astonishing stability of FIC activation across age was observed, which suggests mature FIC function already from an early age. In contrast, under increased spatial precision demands, increased activation in the most anterior part of ACC was observed in the 7-year-old sample compared to the two older samples. y p p p Findings from research on the development of functional networks may help to elucidate this developmental change. Several studies have demonstrated that while the salience network’s basic structure in children is similar to that of adults, reﬁnements take place as to the salience network’s within- and between-network connectivity in several regards (Fair et al., 2012a,b; Gu et al., 2015; Marek et al., 2015). Firstly, studies have demonstrated that brain development between childhood and adulthood is characterized by a trend from local interconnectivity in children to long-range functional coupling in adults (Fair et al., 2009). More speciﬁcally, these studies show that connectivity between anatomically close regions of interest slowly decreases until adulthood whereas long-range connectivity, especially anterior-posterior links, gradually increases. Secondly, the interplay between FIC and ACC (with the FIC being a major source of control signals for ACC in adults; Menon and Uddin, 2010) seems to change between childhood and adulthood in that the directed causal inﬂuence of FIC on ACC has been found to increase between childhood and adulthood (Supekar and Menon, 2012). Thirdly, the salience network as a whole as well as its individual components show age-related changes in integration with other networks, especially the somatomotor network presumably resulting in a more rapid access from the salience network to the motor system to control goal-directed behavior (Grayson et al., 2014; Marek et al., 2015). In sum, these results indicate that a ‘calibration’ of ACC function both with regard to within-network as well as with regard to between- network interaction takes place during childhood (and into adulthood). We speculate that these reﬁnements come along with altered ACC activation in younger children. The diﬀerent Neural Mechanisms Underlying the Resolution of the Focus of Attention Interestingly, the shift from (predominantly) local to holistic processing seems to be related to morphometric gray matter changes in regions similar to the ones identiﬁed in the present study, i.e., the fronto-parietal attention network and primary July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 11 Neurocognitive Development of Attentional Resolution Wolf et al. TABLE 4 \| Age differences in activation elicited by increased demands in precision (based on contrasts for difﬁculty conditions 2.8 > 5.6 arcmin for adults and 11-year-olds, and 5.6 > 11.2 arcmin for 7-year-olds; cf. section “Age-Independent Mechanisms Underlying the Resolution of Attention”). MNI coordinates Anatomical region Hem BA k Fmax x y z Anterior cingulate cortex, medial superior frontal gyrus R 10/32 193 19.50 14 50 12 Superior frontal gyrus, middle frontal gyrus L 9 64 15.86 −24 40 36 Posterior cingulate gyrus, postcentral gyrus, precentral gyrus, middle frontal gyrus L 31 469 23.97 −26 −36 36 Precuneus, posterior cingulate gyrus, middle cingulate gyrus R/L 7/31 535 15.40 4 −56 36 Superior occipital gyrus, middle occipital gyrus, middle temporal gyrus L 18/19 122 11.69 −20 −88 22 BA, approximate Brodmann’s area; Hem, hemisphere; L, left; R, right; MNI, coordinates referring to the Montreal Neurological Institute template brain included in the SPM software package; Fmax, maximum F statistic in the cluster; k, cluster size in voxels. Activation is reported for a threshold of p < 0.05 (corrected), constituted by a voxel-level threshold of p < 0.001 and a cluster-level threshold of k > 62 (determined using 3dClustSim). Neurocognitive Development of the Resolution of the Focus of Attention The networks recruited to handle increasing attentional precision demands are remarkably similar in children and adults: already participants from the 7-year-old sample activated the salience regions discussed above as well as the superior colliculi and thalamus. This result is consistent with developmental functional connectivity studies suggesting that the basic organization of the July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 12 Neurocognitive Development of Attentional Resolution Wolf et al. empirical ﬁndings oﬀer diﬀerent possible explanations. Increased ACC activation in this age group may be a result of stronger local frontal connectivity entailing stronger frontal activations to accomplish the computations that adults conduct by relying more on long-distance networks. Alternatively it may be a consequence of weak control signals from FIC that necessitate stronger involvement of the ACC to compensate for weaker input from upstream FIC or it may be related to stronger eﬀort to forward information to downstream networks like the somatomotor network. The appropriateness of these proposals will have to be determined by further research. paradigm elicited very strong behavioral eﬀects of age group and distance (eﬀect sizes of η2 = 0.86 and η2 = 0.61, respectively) and for their interaction (η2 = 0.53). Also, the activation eﬀects obtained in the present study were strong, particularly the age eﬀects on attentional resolution, which are at the focus of our interest (cf. Table 4). Our study, thus, does not seem to suﬀer from a general problem in identifying attention-related brain activations or developmental age eﬀects. Nevertheless, we cannot exclude that power may be insuﬃcient for observing more subtle eﬀects. Future studies with larger sample sizes will have to resolve this. Relating the changes in ACC activation to behavioral development, the aforementioned spatial processing framework might provide yet another account for age eﬀects. According to this framework, the youngest age group – in contrast to the two older two age groups – might represent the to-be- tracked dot in a topological, one-object-unit mode. However, representation of the complete conﬁguration might indeed be helpful when tracking objects (as this might improve the ability to, e.g., calculate trajectories, remember object locations etc.; Allen et al., 2004; Oksama and Hyönä, 2004; Scholl, 2009). Presumably, making use of these abilities is especially helpful when the objects within the display get closer (Dukette and Stiles, 2001). FUNDING This research was funded by the Initiative for the Development of Scientiﬁc and Economic Excellence of the state of Hessen (LOEWE; IDeA-Center) and by an Emmy Noether grant from the German Research Foundation (DFG FI848/3-1 to CF). CF was further supported by a Consolidator grant from the European Research Council (ERC Grant Agreement No. 617891). ACKNOWLEDGMENTS We thank Ulrike Basten for her comments on an earlier version of this manuscript and Alexandra Tinnermann for assistance during data collection. We also thank the headmasters and teachers of several Frankfurt elementary and secondary schools for giving us the opportunity to recruit our participants among their students. Neurocognitive Development of the Resolution of the Focus of Attention Given the limitations of the spatial representation system available to young children, increased ACC involvement in this group might represent a compensatory mechanism, e.g., by increasing the salience of the single to-be-tracked object which is still represented in a less elaborated spatial mode in order to fulﬁll the task in a successful manner. AUTHOR CONTRIBUTIONS KW, RD, TP, and CF conceived and planned the experiments. KW, EGW, JvdB, SV, and UN performed the experiments. KW, EGW, and CF analyzed the data. KW and CF interpreted the data and wrote the manuscript. EGW, JvdB, and RD critically revised the manuscript. All authors approved the version to be published. CONCLUSION This study demonstrates that general maturation principles apply to central aspects of selective attention, i.e., the ability to track a target with attention and the ability to select targets with increasingly ﬁne-grained attentional precision. Firstly, the core networks supporting both abilities are principally on-line from an early age on. However, secondly, reﬁnements are both observed in quantitative and qualitative terms and can be interpreted as an optimized recruitment of structures needed to perform the task at hand. More generally, the results of this study support an emerging picture of increased use of more widely distributed and functionally specialized networks with development. This neurofunctional change enables the developing observer to perform visuo-attentional tasks with increasing spatial precision. Apart from activation in the insula-cingulate salience network, a number of further areas showed increased precision-related activity especially in the youngest sample. Such activations in additional, i.e., not typically task-related areas in children are common in developmental fMRI studies (e.g., Konrad et al., 2005; Velanova et al., 2008; Rubia et al., 2010) and interpreted either in terms of ineﬃcient or compensatory recruitment forced by the fact that mature networks cannot yet be activated by younger children (e.g., Geier et al., 2009) or as a result of still-changing communication between networks during childhood and adolescence (Gu et al., 2015; Marek et al., 2015). A region that frequently shows such properties is the precuneus (e.g., Scherf et al., 2006; Velanova et al., 2009). It was proposed that additional precuneus recruitment may reﬂect children’s limited access to other parietal regions responsible for more sophisticated processing in adults (Scherf et al., 2006). Interestingly, in the present study precuneus as well as posterior cingulate gyrus and lateral occipital cortex showed a common developmental pattern characterized by greatest recruitment in 7- year-olds, signiﬁcantly reduced activation during late childhood, and an intermediate level of activation in adults. Older children thus seem to rely less on these areas than the other two age groups. REFERENCES Drew, T., Horowitz, T. S., and Vogel, E. K. (2013). Swapping or dropping? Electrophysiological measures of diﬃculty during multiple object tracking. Cognition 126, 213–223. doi: 10.1016/j.cognition.2012.10.003 Allen, R., McGeorge, P., Pearson, D., and Milne, A. B. (2004). Attention and expertise in multiple target tracking. Appl. Cogn. Psychol. 18, 337–347. doi: 10.1002/acp.975 Dukette, D., and Stiles, J. (2001). The eﬀects of stimulus density on children’s analysis of hierarchichal patterns. Dev. Sci. 4, 233–251. doi: 10.1111/1467-7687. 00168 Alnaes, D., Sneve, M. H., Richard, G., Skatun, K. C., Kaufmann, T., Nordvik, J., et al. (2015). Functional connectivity indicates diﬀerential roles for the intraparietal sulcus and the superior parietal lobule in multiple object tracking. Neuroimage 123, 129–137. doi: 10.1016/j.neuroimage.2015.08.029 Enns, J. T., and Girgus, J. S. (1985). Developmental changes in selective and integrative visual attention. J. Exp. Child Psychol. 40, 319–337. doi: 10.1016/ 0022-0965(85)90093-1 123, 129–137. doi: 10.1016/j.neuroimage.2015.08.029 Alvarez, G. A., and Franconeri, S. L. (2007). How many objects can you track? Evidence for a resource-limited attentive tracking mechanism. J. Vis. 7, 1–10. doi: 10.1167/7.13.14 Fair, D. A., Bathula, D., Nikolas, M. A., and Nigg, J. T. (2012a). Distinct neuropsychological subgroups in typically developing youth inform heterogeneity in children with ADHD. Proc. Natl. Acad. Sci. U. S. A. 109, 6769–6774. doi: 10.1073/pnas.1115365109 Amso, D., and Scerif, G. (2015). The attentive brain: insights from developmental cognitive neuroscience. Nat. Rev. Neurosci. 16, 606–619. doi: 10.1038/nrn4025 Fair, D. A., Nigg, J. T., Iyer, S., Bathula, D., Mills, K. L., Dosenbach, N., et al. (2012b). Distinct neural signatures detected for ADHD subtypes after controlling for micro-movements in resting state functional connectivity MRI data. Front. Syst. Neurosci. 6:80. doi: 10.3389/fnsys.2012.00080 Baluch, F., and Itti, L. (2011). Mechanisms of top-down attention. Trends Neurosci. 34, 210–224. doi: 10.1016/j.tins.2011.02.003 Beaton, E. A., Stoddard, J., Lai, S., Lackey, J., Shi, J., Ross, J. L., et al. (2010). Atypical functional brain activation during a multiple object tracking task in girls with Turner syndrome: neurocorrelates of reduced spatiotemporal resolution. Am. J. Intellect. Dev. Disabil. 115, 140–156. doi: 10.1352/1944-7558-115. 2.140 Fair, D. A., Cohen, A. L., Power, J. D., Dosenbach, N. U. F., Church, J. A., Miezin, F., et al. (2009). Functional brain networks develop from a “local to distributed” organization. PLoS Comput. Biol. 5:e1000381. doi: 10.1371/journal. pcbi.1000381 Farrant, K., and Uddin, L. Q. (2015). Asymmetric development of dorsal and ventral attention networks in the human brain. Dev. Cogn. Neurosci. 12, 165–174. doi: 10.1016/j.dcn.2015.02.001 Bondarko, V. REFERENCES M., and Semenov, L. A. (2005). Visual acuity and the crowding eﬀect in 8- to 17-year-old schoolchildren. Hum. Physiol. 31, 532–538. doi: /10.1007/ s10747-005-0093-1 Booth, J. R., Burman, D. D., Meyer, J. R., Lei, Z., Trommer, B. L., Davenport, N., et al. (2003). Neural development of selective attention and response inhibition. Neuroimage 20, 737–751. doi: 10.1016/S1053-8119(03) 00404-X Fassbender, C., Mukherjee, P., and Schweitzer, J. B. (2017). Minimizing noise in pediatric task-based functional MRI; Adolescents with developmental disabilities and typical development. Neuroimage 149, 338–347. doi: 10.1016/ j.neuroimage.2017.01.021 Brockhoﬀ, A., Papenmeier, F., Wolf, K., Pfeiﬀer, T., Jahn, G., and Huﬀ, M. (2016). Viewpoint matters: exploring the involvement of reference frames in multiple object tracking from a developmental perspective. Cogn. Dev. 37, 1–8. doi: 10.1016/j.cogdev.2015.10.004 Franke, G. H. (2002). SCL-90-R. Symptom-Checkliste von L.R. Derogatis. Göttingen: Beltz Test GmbH. Geier, C. F., Garver, K., Terwilliger, R., and Luna, B. (2009). Development of working memory maintenance. J. Neurophysiol. 101, 84–99. doi: 10.1152/jn. 90562.2008 Carrasco, M. (2011). Visual attention: The past 25 years. Vis. Res. 51, 1484–1525. doi: 10.1016/j.visres.2011.04.012 Goodman, R. (1997). The strengths and diﬃculties questionnaire: a research note. J. Child Psychol. Psychiatry 38, 581–586. doi: 10.1111/j.1469-7610.1997.tb 01545.x Chand, G. B., and Dhamala, M. (2016a). Interactions among the brain default- mode, salience, and central-executive networks during perceptual decision- making of moving dots. Brain Connect. 6, 249–254. doi: 10.1089/brain.2015. 0379 Gottlieb, J. (2007). From thought to action: the parietal cortex as a bridge between perception, action and cognition. Neuron 53, 9–16. doi: 10.1016/j.neuron.2006. 12.009 Chand, G. B., and Dhamala, M. (2016b). The salience network dynamics in perceptual decision-making. Neuroimage 134, 85–93. doi: 10.1016/j.neuro image.2016.04.018 Gottlieb, J., and Balan, P. F. (2010). Attention as a decision in information space. Trends Cogn. Sci. 14, 240–248. doi: 10.1016/j.tics.2010.03.001 Chen, A. C., Oathes, D. J., Chang, C., Bradley, T., Zhou, Z. W., Williams, L., et al. (2013). Causal interactions between fronto-parietal central executive and default-mode networks in humans. Proc. Natl. Acad. Sci. U. S. A. 110, 19944–19949. doi: 10.1073/pnas.1311772110 Gottlieb, J., Kusunoki, M., and Goldberg, M. E. (1998). The representation of visual salience in monkey parietal cortex. Nature 391, 481–484. doi: 10.1038/35135 Goulden, N., Khusnulina, A., Davis, N. J., Bracewell, R. M., Bokde, A. L., McNulty, J. P., et al. (2014). The salience network is responsible for switching between the default mode network and the central executive network: replication from DCM. Neuroimage 99, 180–190. doi: 10.1016/j.neuroimage.2014. Limitations The relatively small sample size of the present study may constitute a potential limitation. However, the object tracking July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 13 Neurocognitive Development of Attentional Resolution Wolf et al. REFERENCES T., Hamid, J., Maurer, D., and Lewis, T. L. (2010). Developmental changes during childhood in single-letter acuity and its crowding by surrounding contours. J. Exp. Child Psychol. 107, 423–437. doi: 10.1016/j.jecp.2010.05.009 Ridderinkhof, K. R., and van der Stelt, O. (2000). Attention and selection in the growing child: views derived from developmental psychophysiology. Biol. Psychol. 54, 55–106. doi: 10.1016/S0301-0511(00)00053-3 Jovicich, J., Peters, R. J., Koch, C., Braun, J., Chang, L., and Ernst, T. (2001). Brain areas speciﬁc for attentional load in a motion-tracking task. J. Cogn. Neurosci. 13, 1048–1058. doi: 10.1162/089892901753294347 Rubia, K., Hyde, Z., Halari, R., Giampietro, V., and Smith, A. (2010). Eﬀects of age and sex on developmental neural networks of visual-spatial attention allocation. Neuroimage 51, 817–827. doi: 10.1016/j.neuroimage.2010.02.058 Klaver, P., Lichtensteiger, J., Bucher, K., Dietrich, T., Loenneker, T., and Martin, E. (2008). Dorsal stream development in motion and structure-from-motion perception. Neuroimage 39, 1815–1823. doi: 10.1016/j.neuroimage.2007.11.009 Santangelo, V. (2015). Forced to remember: when memory is biased by salient information. Behav. Brain Res. 283, 1–10. doi: 10.1016/j.bbr.2015. 01.013 Konrad, K., Neufang, S., Thiel, C. M., Specht, K., Hanisch, C., Fan, J., et al. (2005). Development of attentional networks: An fMRI study with children and adults. Neuroimage 28, 429–439. doi: 10.1016/j.neuroimage.2005.06.065 Scerif, G. (2010). Attention trajectories, mechanisms and outcomes: at the interface between developing cognition and environment. Dev. Sci. 13, 805–812. doi: 10.1111/j.1467-7687.2010.01013.x Lange-Küttner, C. (2006). Drawing boundaries: from individual to common region - the development of spatial region attribution in children. Br. J. Dev. Psychol. 24, 419–427. doi: 10.1348/026151005X50753 j Scherf, K. S., Sweeney, J. A., and Luna, B. (2006). Brain basis of developmental change in visuospatial working memory. J. Cogn. Neurosci. 18, 1045–1058. doi: 10.1162/jocn.2006.18.7.1045 Lange-Küttner, C., and Küttner, E. (2015). How to learn places without spatial concepts: does the what-and-where reaction time system in children regulate learning during stimulus repetition? Brain Cogn. 97, 59–73. doi: 10.1016/j. bandc.2015.04.008 Scholl, B. J. (2009). “What have we learned about attention from multiple-object tracking (and vice versa)?,” in Computation, Cognition, and Pylyshyn, eds D. Dedrick and L. M. Trick (Cambridge, MA: MIT Press), 49–77. Lovejoy, L. P., and Krauzlis, R. J. (2010). Inactivation of primate superior colliculus impairs covert selection of signals for perceptual judgments. Nat. Neurosci. 13, 261–266. doi: 10.1038/nn.2470 Seeley, W. W., Menon, V., Schatzberg, A. F., Keller, J., Glover, G. H., Kenna, H., et al. (2007). Dissociable intrinsic connectivity networks for salience processing and executive control. J. Neurosci. REFERENCES July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 14 Neurocognitive Development of Attentional Resolution Wolf et al. Houdé, O., Rossi, S., Lubin, A., and Joliot, M. (2010). Mapping numerical processing, reading, and executive functions in the developing brain: an fMRI meta-analysis of 52 studies including 842 children. Dev. Sci. 13, 876–885. doi: 10.1111/j.1467-7687.2009.00938.x Pastò, L., and Burack, J. A. (1997). A developmental study of visual attention: issues of ﬁltering eﬃciency and focus. Cogn. Dev. 12, 523–535. doi: 10.1016/S0885- 2014(97)90021-6 Poirel, N., Leroux, E., Pineau, A., Houde, O., and Simon, G. (2014). Changes in cortical thickness in 6-year-old children open their mind to a global vision of the world. Biomed Res. Int. 2014:362349. doi: 10.1155/2014/ 362349 Howe, P. D., Horowitz, T. S., Akos Morocz, I., Wolfe, J. M., and Livingstone, M. S. (2009). Using fMRI to distinguish components of the multiple object tracking task. J. Vis. 9, 10–10. doi: 10.1167/9.4.10 Poirel, N., Simon, G., Cassotti, M., Leroux, G., Perchey, G., Lanoe, C., et al. (2011). The shift from local to global visual processing in 6-year-old children is associated with grey matter loss. PLoS One 6:e20879. doi: 10.1371/journal. pone.0020879 Huurneman, B., Cox, R. F., Vlaskamp, B. N., and Boonstra, F. N. (2014). Crowded visual search in children with normal vision and children with visual impairment. Vis. Res. 96, 65–74. doi: 10.1016/j.visres.2014.01.004 Hwang, S., White, S. F., Nolan, Z. T., Sinclair, S., and Blair, R. J. (2014). Neurodevelopmental changes in the responsiveness of systems involved in top down attention and emotional responding. Neuropsychologia 62, 277–285. doi: 10.1016/j.neuropsychologia.2014.08.003 Power, J. D., Barnes, K. A., Snyder, A. Z., Schlaggar, B. L., and Petersen, S. E. (2012). Spurious but systematic correlations in functional connectivity MRI networks arise from subject motion. Neuroimage 59, 2142–2154. doi: 10.1016/j. neuroimage.2011.10.018 Intriligator, J., and Cavanagh, P. (2001). The spatial resolution of visual attention. Cogn. Psychol. 43, 171–216. doi: 10.1006/cogp.2001.0755 Power, J. D., Schlaggar, B. L., and Petersen, S. E. (2015). Recent progress and outstanding issues in motion correction in resting state fMRI. Neuroimage 105, 536–551. doi: 10.1016/j.neuroimage.2014.10.044 Jahn, G., Papenmeier, F., Meyerhoﬀ, H. S., and Huﬀ, M. (2012). Spatial reference in multiple object tracking. Exp. Psychol. 59, 163–173. doi: 10.1027/1618-3169/ a000139 Raven, J., Raven, J. C., and Court, J. H. (2000). Manual for Raven’s Progressive Matrices and Vocabulary Scales: Section 3. Standard progressive matrices (Including the Parallel and Plus Versions). Oxford: Oxford Psychologists Press. Jeon, S. REFERENCES 27, 2349–2356. doi: 10.1523/JNEUROSCI. 5587-06.2007 Marek, S., Hwang, K., Foran, W., Hallquist, M. N., and Luna, B. (2015). The contribution of network organization and integration to the development of cognitive control. PLoS Biol. 13:e1002328. doi: 10.1371/journal.pbio.1002328 Shim, W. M., Alvarez, G. A., Vickery, T. J., and Jiang, Y. V. (2009). The number of attentional foci and their precision are dissociated in the posterior parietal cortex. Cereb. Cortex 20, 1341–1349. doi: 10.1093/cercor/bhp197 Mazaika, P. (2009). Motion Correction and Despike Functions. Available at: http://cibsr.stanford.edu/content/dam/sm/cibsr/documents/tools/methods/ artrepair-software/MotionandDespike.pdf [accessed July 16, 2017]. Siegel, J. S., Power, J. D., Dubis, J. W., Vogel, A. C., Church, J. A., Schlaggar, B. L., et al. (2014). Statistical improvements in functional magnetic resonance imaging analyses produced by censoring high-motion data points. Hum. Brain Mapp. 35, 1981–1996. doi: 10.1002/hbm.22307 Menon, V., and Uddin, L. Q. (2010). Saliency, switching, attention and control: a network model of insula function. Brain Struct. Funct. 214, 655–667. doi: 10.1007/s00429-010-0262-0 Moore, T., and Zirnsak, M. (2017). Neural mechanisms of selective visual attention. Annu. Rev. Psychol. 68, 47–72. doi: 10.1146/annurev-psych-122414-033400 Sowell, E. R., Peterson, B. S., Thompson, P. M., Welcome, S. E., Henkenius, A. L., and Toga, A. W. (2003). Mapping cortical change across the human life span. Nat. Neurosci. 6, 309–315. doi: 10.1038/nn1008 Nardo, D., Santangelo, V., and Macaluso, E. (2011). Stimulus-driven orienting of visuo-spatial attention in complex dynamic environments. Neuron 69, 1015– 1028. doi: 10.1016/j.neuron.2011.02.020 Sridharan, D., Levitin, D. J., and Menon, V. (2008). A critical role for the right fronto-insular cortex in switching between central-executive and default-mode networks. Proc. Natl. Acad. Sci. U.S.A. 105, 12569–12574. doi: 10.1073/pnas. 0800005105 Navon, D. (1977). Forest before trees: the precedence of global features in visual perception. Cogn. Psychol. 9, 353–383. doi: 10.1016/0010-0285(77)90012-3 g y Norgett, Y., and Siderov, J. (2014). Foveal crowding diﬀers in children and adults. J. Vis. 14. doi: 10.1167/14.12.23 Stiles, J., Akshoomoﬀ, N., and Haist, F. (2013). “The development of visuospatial processing,” in Comprehensive Developmental Neuroscience: Neural Circuit Development and Function in the Healthy and Diseased Brain, eds J. L. R. Rubinstein and P. Rakic (Amsterdam: Elsevier), 271–296. Oksama, L., and Hyönä, J. (2004). Is multiple object tracking carried out automatically by an early vision mechanism independent of higher-order cognition? An individual diﬀerence approach. Vis. Cogn. 11, 631–671. doi: 10.1080/13506280344000473 Rubinstein and P. Rakic (Amsterdam: Elsevier), 271–296. Supekar, K., and Menon, V. (2012). REFERENCES 05.052 Chen, T., Cai, W., Ryali, S., Supekar, K., and Menon, V. (2016). Distinct global brain dynamics and spatiotemporal organization of the salience network. PLoS Biol. 14:e1002469. doi: 10.1371/journal.pbio.1002469 Grayson, D. S., and Fair, D. A. (2017). Development of large-scale functional networks from birth to adulthood: a guide to the neuroimaging literature. Neuroimage 160, 15–31. doi: 10.1016/j.neuroimage.2017.01.079 Church, J. A., Petersen, S. E., and Schlaggar, B. L. (2010). The “Task B problem” and other considerations in developmental functional neuroimaging. Hum. Brain Mapp. 31, 852–862. doi: 10.1002/hbm.21036 Conner, I. P., Sharma, S., Lemieux, S. K., and Mendola, J. D. (2004). Retinotopic organization in children measured with fMRI. J. Vis. 4, 509–523. doi: 10.1167/ 4.6.10 Grayson, D. S., Ray, S., Carpenter, S., Iyer, S., Dias, T. G., Stevens, C., et al. (2014). Structural and functional rich club organization of the brain in children and adults. PLoS One 9:e88297. doi: 10.1371/journal.pone.0088297 Grootoonk, S., Hutton, C., Ashburner, J., Howseman, A. M., Josephs, O., Rees, G., et al. (2000). Characterization and correction of interpolation eﬀects in the realignment of fMRI time series. Neuroimage 11, 49–57. doi: 10.1006/nimg. 1999.0515 Crottaz-Herbette, S., and Menon, V. (2006). Where and when the anterior cingulate cortex modulates attentional response: combined fMRI and ERP evidence. J. Cogn. Neurosci. 18, 766–780. doi: 10.1162/jocn.2006.18. 5.766 Culham, J. C., Brandt, S. A., Cavanagh, P., Kanwisher, N. G., Dale, A. M., and Tootell, R. B. (1998). Cortical fMRI activation produced by attentive tracking of moving targets. J. Neurophysiol. 80, 2657–2670. doi: 10.1152/jn.1998.80. 5.2657 Gu, S., Satterthwaite, T. D., Medaglia, J. D., Yang, M., Gur, R. E., Gur, R. C., et al. (2015). Emergence of system roles in normative neurodevelopment. Proc. Natl. Acad. Sci. U.S.A. 112, 13681–13686. doi: 10.1073/pnas.150282 9112 Deichmann, R., Gottfried, J. A., Hutton, C., and Turner, R. (2003). Optimized EPI for fMRI studies of the orbitofrontal cortex. Neuroimage 19, 430–441. doi: 10.1016/S1053-8119(03)00073-9 Harrison, T. B., and Stiles, J. (2009). Hierarchichal forms processing in adults and children. J. Exp. Child Psychol. 103, 222–240. doi: 10.1016/j.jecp.2008.09.004 He, S., and Cavanagh, P. (1996). Attentional resolution and the locus of visual awareness. Nature 383, 334–338. doi: 10.1038/383334a0 Döpfner, M., and Lehmkuhl, G. (2003). Diagnostik-System für Psychische Störungen im Kindes- und Jugendalter nach ICD-10 und DSM-IV (DISYPS-KJ). Bern: Huber. Horn, R. (2009). Standard Progressive Matrices (SPM). Frankfurt: Pearson Assessment. REFERENCES Developmental maturation of dynamic causal control signals in higher-order cognition: a neurocognitive network model. PLoS Comput. Biol. 8:e1002374. doi: 10.1371/journal.pcbi.10 02374 Oldﬁeld, R. C. (1971). The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9, 97–113. doi: 10.1016/0028-3932(71)90067-4 Thompson, K. G., and Bichot, N. P. (2005). A visual salience map in the primate frontal eye ﬁeld. Prog. Brain Res. 147, 251–262. doi: 10.1016/S0079-6123(04) 47019-8 Olesen, P. J., Macoveanu, J., Tegner, J., and Klingberg, T. (2006). Brain activity related to working memory and distraction in children and adults. Cereb. Cortex 17, 1047–1054. doi: 10.1093/cercor/bhl014 July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 15 Neurocognitive Development of Attentional Resolution Wolf et al. Todd, J. J., and Marois, R. (2004). Capacity limit of visual short-term memory in human posterior parietal cortex. Nature 428, 751–754. doi: 10.1038/ nature02466 Wilke, M. (2012). An alternative approach towards assessing and accounting for individual motion in fMRI timeseries. Neuroimage 59, 2062–2072. doi: 10.1016/ j.neuroimage.2011.10.043 Toet, A., and Levi, D. M. (1992). The two-dimensional shape of spatial interaction zones in the parafovea. Vis. Res. 32, 1349–1357. doi: 10.1016/0042-6989(92) 90227-A Wilke, M. (2014). Isolated assessment of translation or rotation severely underestimates the eﬀects of subject motion in fMRI data. PLoS One 9:e106498. doi: 10.1371/journal.pone.0106498 Turner, B. O., and Miller, M. B. (2013). Number of events and reliability in fMRI. Cogn. Aﬀect. Behav. Neurosci. 13, 615–626. doi: 10.3758/s13415-013- 0178-2 Wolf, K., and Pfeiﬀer, T. (2014). The development of attentional resolution. Cogn. Dev. 29, 62–80. doi: 10.1016/j.cogdev.2013.09.004 Wurtz, R. H., McAlonan, K., Cavanaugh, J., and Berman, R. A. (2011). Thalamic pathways for active vision. Trends Cogn. Sci. 15, 177–184. doi: 10.1016/j.tics. 2011.02.004 Uddin, L. Q. (2015). Salience processing and insular cortical function and dysfunction. Nat. Rev. Neurosci. 16, 55–61. doi: 10.1038/ nrn3857 Xu, Y., and Chun, M. M. (2006). Dissociable neural mechanisms supporting visual short-term memory for objects. Nature 440, 91–95. doi: 10.1038/nature 04262 Velanova, K., Wheeler, M. E., and Luna, B. (2008). Maturational changes in anterior cingulate and frontoparietal recruitment support the development of error processing and inhibitory control. Cereb. Cortex 18, 2505–2522. doi: 10.1093/ cercor/bhn012 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Velanova, K., Wheeler, M. E., and Luna, B. (2009). July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org REFERENCES The maturation of task set- related activation supports late developmental improvements in inhibitory control. J. Neurosci. 29, 12558–12567. doi: 10.1523/JNEUROSCI.1579-09.2009 Copyright © 2018 Wolf, Galeano Weber, van den Bosch, Volz, Nöth, Deichmann, Naumer, Pfeiﬀer and Fiebach. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Vuontela, V., Jiang, P., Tokariev, M., Savolainen, P., Ma, Y., Aronen, E., et al. (2013). Regulation of brain activity in the fusiform face and parahippocampal place areas in 7-11-year-old children. Brain Cogn. 81, 203–214. doi: 10.1016/j. bandc.2012.11.003 Wendelken, C., Baym, C. L., Gazzaley, A., and Bunge, S. A. (2011). Neural indices of improved attentional modulation over middle childhood. Dev. Cogn. Neurosci. 1, 175–186. doi: 10.1016/j.dcn.2010.11.001 July 2018 \| Volume 9 \| Article 1106 Frontiers in Psychology \| www.frontiersin.org 16
https://openalex.org/W4233787419	https://www.researchsquare.com/article/rs-35908/latest.pdf	English	null	Education in A New Place and Social Support Are Needed to Improve The Health of North Korean Defectors: A Cross-Sectional Study	Research Square (Research Square)	2,020	cc-by	5,580	Education in a new place and social support are needed to improve the health of North Korean defectors: A cross-sectional study Jihyun Lee Seoul National University School of Dentistry Yui Park Seoul National University School of Dentistry DONGHUN HAN (  dhhan73@snu.ac.kr ) Seoul National University School of Dentistry https://orcid.org/0000-0001-6427-3137 Ji Won Yoo University of Nevada Las Vegas Wenlian Zhou University of Nevada Las Vegas Pearl Kim University of Nevada Las Vegas Jay J. Shen (  jay.shen@unlv.edu ) University of Nevada Las Vegas Research Keywords: Education, North Korean defectors, Self-rated health, Social support Posted Date: June 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-35908/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License Read Full License Abstract Background The number of North Korean defectors (NKDs) escaping to South Korea has increased. The health status of NKDs is an essential factor for a successful settlement into South Korean society. However, no studies have been conducted on the health status of NKDs in terms of education and social support. The aim of this study was to determine the effects of education and social support on the self- rated health status among NKDs. Methods This study utilized data gained from face-to-face interviews with 126 NKDs. A multivariable logistic regression and path analysis were performed to assess the effects of education in South Korea and social support with their self-rated health status and to explore the complex relationships between direct and indirect effects of the variables. Results NKDs who did not experience regular education in South Korea responded that they were in poor health compared to their counterpart (OR=5.78). Although a direct association between education in South Korea and self-rated health was not shown, there was an indirect path from education in South Korea to self-rated health through social support. Conclusions Participation in regular education is important for the health status of NKDs. Social policies and NKD assistance programs should consider and reflect the combination of education and social support interventions relevant to the health status of NKDs. Research Keywords: Education, North Korean defectors, Self-rated health, Social support Posted Date: June 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-35908/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Background North Korea’s economic difficulties after the fall and dissolution of communism in Eastern Europe and the Soviet Union and a great flood in 1995 that caused the Great Famine led to the deaths of millions of North Koreans due to starvation and hunger-related illnesses during the late 1990s [1]. Since then, many North Korean defectors (NKDs) have escaped to South Korea. The highest concentration of military power in the world is on the border between North and South Korea, so NKDs often pass through third countries such as China, Thailand, and Laos to enter South Korea. Despite the huge barriers and great danger, the number of NKDs entering South Korea has been steady at 1000 ~ 2000 annually since 2002, and the cumulative total by September 2019 was 33,247 [2]. Despite the financial and medical support by Korean Government, NKDs have experienced various difficulties in settling into South Korean society and managing their health conditions [3]. According to a survey of NKDs in 2018, 62.2% of NKDs suffered from stress in overall daily life, while only 54.4% of South Koreans suffered from stress. Furthermore, 14.6% of NKDs had suicidal thoughts compared to 5.1% of South Koreans. Moreover, a particularly notable part was that the "physical/mental illness and disability" was the second highest cause of suicidal ideation (23.3%) [4]. Education can be considered as one of the most critical factors that potentially affect the adjustment of NKDs to South Korean society and their health. The health effects of education attainment against risk of morbidity and mortality are well established [5–7]. High education is shown to protect against a decline in self-rated health (SRH) [8, 9], chronic disease [10], and mortality [11]. As another essential component of human life, social support has been known to be associated with health and well-being. Social Page 2/15 Page 2/15 isolation entails a mortality risk comparable in magnitude to that of smoking, a sedentary lifestyle, obesity, and alcohol abuse [12]. Previous evidence suggests that education and social relationships interact with each other to affect health. Some research in this area has found that the protective effects of social support on health are stronger among individuals with high education levels [13, 14]. These studies have demonstrated that those with high levels of education benefit most from the protective qualities of social support, and the social support-health connection is almost nonexistent among those with lower levels of education. Methods This was a cross-sectional study to assess the association of education and social support with self- rated health. It was approved by the Institutional Review Board of the Seoul National University School of Dentistry (No.S-D20170041). Background Conversely, one study found that the protective qualities of social support are stronger among individuals who are less educated [15]. Yet, additional empirical evidence has demonstrated that social support is predictive of worse, rather than better health outcomes among individuals with a low level of education [13, 14]. Education is not only a determinant of health but also an opportunity to form social relationships. Especially for NKDs, being a part of a social community in South Korean society, such as a regular education in South Korea, provides them with good opportunities to receive social support and may have an important health-promoting impact. Previous studies [16, 17] showed that facilitating the visit of educational institutions for refugees is an essential factor for successful social inclusion. However, literature is scarce on the exact nature of the links among education in South Korea, social support, and self-rated health status among NKDs. We hypothesized that education in South Korea, social support, and self-rated health status would be directly associated with each other. We also hypothesized that education in South Korea would be directly and indirectly associated with self-rated health status through social support. Therefore, the aims of this study was to identify the individual effects of education and social support on the self-rated health status, respectively, and then to further assess the path from education in South Korea to self-rated health status through social support among NKDs. Variables The dependent variable was self-rated health status. It was measured with a 5-point scale (very good, good, moderate, poor, and very poor) and reclassified into two groups (good/moderate and poor). Independent variables were education in South Korea and social support. Education in South Korea was reclassified into 1) college or more, 2) middle/high school, and 3) no education experience in South Korea. Social support score was measured by the Korean version of the Duke-UNC social support questionnaire which was developed by Suh et al. [18] The tool consists of 13 questions with a 5-point scale. After summing up, the score ranges from 13 to 65 points, and a higher score indicates more social support. The reliability of the Korean version of the Duke-UNC social support questionnaire was a Cronbach's ⍺=0.89 at the time of its development. Social support was categorized as a lower/higher group based on the median score (34 point). Confounding variables were age (continuous), gender (male/female), living with family (yes/no), residence period in South Korea (continuous), smoking (yes/no), and drinking (yes/no). Residence period in South Korea was calculated by months from entrance date into South Korea to October 2017 when the data collection was completed. Participants The study participants were recruited with cooperation from the Hana Center, alternative schools for NKDs and the Catholic Bishop's Conference of Korea National Reconciliation Committee in Seoul and Gyeonggi Province. A total of 137 participants were recruited, and the purpose and method of this study were explained to them. All the participants voluntarily agreed to join this study and gave their written consent. The criteria for selection and exclusion were as follows. Page 3/15 Page 3/15 - Selection criteria · those who completed education at the Hana center · those who completed education at the Hana center · those who can read and answer the questionnaire by themselves or who can listen to the explanation of the questionnaire and answers · those who can read and answer the questionnaire by themselves or who can listen to the explanation of the questionnaire and answers · those willing to participate in this study - Exclusion Criteria · those with a severe mental illness condition · those who want to quit Statistical analysis After excluding missing values, 126 participants were included in the final sample for analysis. The characteristics of the participants were analyzed using frequency, percent, means, and standard deviations. Chi-square tests for categorical variables and independent t-tests for continuous variables were used to assess the associations of education in South Korea and confounders with subjective poor health (Table 1). The association between social support and self-rated health status (Supplementary Table 1 and Fig. 1) and the association between social support and education in South Korea (Supplementary Table 2 and Fig. 2) were evaluated by analysis of variance (ANOVA) and analysis of covariance (ANCOVA) controlling for confounders, respectively. The association between education in South Korea and subjective poor health was assessed using logistic regression models adjusting for the Page 4/15 above mentioned confounders (Model 1 in Table 2). Social support was regarded as a mediator, and logistic regression analysis was done controlling for confounders in model 1 and social support (Model 2 in Table 2). The role of the social support was evaluated by the percent (%) excess odd explained, which was calculated as [(ORadj confounders – ORadj confounders + mediator) / (ORadj confounders -1) X 100 (%)] in this above mentioned confounders (Model 1 in Table 2). Social support was regarded as a mediator, and logistic regression analysis was done controlling for confounders in model 1 and social support (Model 2 in Table 2). The role of the social support was evaluated by the percent (%) excess odd explained, which was calculated as [(ORadj confounders – ORadj confounders + mediator) / (ORadj confounders -1) X 100 (%)] in this study [19]. The % excess odd explained represents the degree to which a mediator explains the relationship between education in South Korea and subjective poor health. Finally, to assess the direct and indirect effects of the education of NKDs in South Korea on their health, we set up a model that hypothesizes that education in South Korea affects their SRH directly and indirectly by their perceived social support. A path analysis was conducted using AMOS 23.0 [20]. Path coefficients were estimated by maximum likelihood estimation. Maximum Likelihood Bootstrapping was conducted to test the significance of the indirect effect with bootstrap samples of 2,000 and 95% bias-corrected confidence intervals. Model fit was evaluated with proper indices. Statistical analysis Table 1 Related factors of self-rated health status among North Korean defectors (n = 126) Self-rated health status Independent Variables good/moderate poor (n = 98) (n = 28) p-Value Age (years) 29.88 [14.71] 48.89 [16.98] < 0.001 Gender Male 29 [29.6] 2 [7.1] 0.029 Female 69 [70.4] 26 [92.9] Living with family No 25 [25.5] 10 [35.7] 0.288 Yes 73 [74.5] 18 [64.3] Residence period in South Korea (months) 67.35 [53.50] 97.93 [52.20] 0.009 Smoking Yes 19 [19.4] 3 [10.7] 0.286 No 79 [80.6] 25 [89.3] Drinking Yes 66 [67.3] 14 [50.0] 0.093 No 32 [32.7] 14 [50.0] Education in South Korea No 16 [16.3] 16 [57.1] < 0.001 Middle/high school 46 [46.9] 7 [25.0] College or more 36 [36.7] 5 [17.9] Notes. Means for continuous variables are reported with standard deviation in brackets. Frequencies for categorical variables are reported with column percent in brackets. p-values were obtained by independent t-test in continuous variables and chi-square test in categorical variables. Table 1 Notes. Means for continuous variables are reported with standard deviation in brackets. Frequencies for categorical variables are reported with column percent in brackets. p-values were obtained by independent t-test in continuous variables and chi-square test in categorical variables. Page 5/15 Page 5/15 Table 2 Table 2 Unadjusted and adjusted social support score according to self-rated health status Social support score Self-rated health status p-Value Very good (n = 10) Good (n = 39) Moderate (n = 49) Poor (n = 18) Very poor (n = 10) Unadjusted Mean 27.60a 32.00ab 34.27abc 36.67bc 41.30c 0.001 [SD] [9.66] [8.15] [6.76] [8.27] [7.51] Adjusted Mean 28.47a 32.06ab 34.13bc 37.20 cd 39.86d 0.007 [SE] [2.37] [1.18] [1.07] [1.84] [2.47] Unadjusted means are reported with standard deviation in brackets. Adjusted means are reported with standard error in brackets. p-values were obtained by ANOVA and ANCOVA controlling for age, gender, living with family, residence period in South Korea, smoking, and drinking. SD = standard deviation; SE = standard error. Unadjusted means are reported with standard deviation in brackets. Adjusted means are reported with standard error in brackets. p-values were obtained by ANOVA and ANCOVA controlling for age, gender, living with family, residence period in South Korea, smoking, and drinking. SD = standard deviation; SE = standard error. Results Descriptive results are listed in Table 1. Age ranges from 13 to 74 years old (mean age: 34.1 years old) and 41.3% were in their twenties. The NKDs were dominantly females (75.4%) and most of them lived with family (72.2%). The range of the residence period in South Korea was 1 to 213 months (mean length of residence in South Korea: 74.14 months), and two thirds of the participants lived in South Korea for 1 ~ 5 years (37.3% for 12 ~ 59 months and 31.7% for 60 ~ 119 months). Smokers and drinkers were 17.5% and 63.5%, respectively. Additionally, 78.6% of the participants completed a regular educational curriculum in South Korea. The average score of social support was 33.9 with a minimum score of 13 and a maximum score of 51 points. Subjective health conditions showed that "good" and "moderate" were 38.9%, respectively, which surpassed “poor” (22.2%). ANOVA and ANCOVA results are presented in Tables 2 and 3. The association between social support and self-rated health status is shown in Table 2. Social support scores increased as self-rated health status deteriorated. Individuals with at least a college degree reported a lower social support score (that is, a high social support status) than those who had less than a college degree (Table 3). Page 6/15 Table 3 Table 3 d social support score according to education in South Korea d and adjusted social support score according to education in South Korea Unadjusted and adjusted social support score according to education in South Korea Social support score Education in South Korea p-Value College or more (n = 41) Middle/high school (n = 53) No (n = 32) Unadjusted Mean 31.29a 33.77ab 37.59b 0.004 [SD] [8.45] [7.77] [7.50] Adjusted Mean 31.90 33.05 38.01 0.020 [SE] [1.27] [1.14] [1.63] Unadjusted means are reported with standard deviation in brackets. Adjusted means are reported with standard error in brackets. p-values were obtained by ANOVA and ANCOVA controlling for age, gender, living with family, residence period in South Korea, smoking, and drinking. SD = standard deviation; SE = standard error. Unadjusted means are reported with standard deviation in brackets. Adjusted means are reported with standard error in brackets. p-values were obtained by ANOVA and ANCOVA controlling for age, gender, living with family, residence period in South Korea, smoking, and drinking. SD = standard deviation; SE = standard error. Unadjusted means are reported with standard deviation in brackets. Adjusted means are reported with standard error in brackets. p-values were obtained by ANOVA and ANCOVA controlling for age, gender, living with family, residence period in South Korea, smoking, and drinking. SD = standard deviation; SE = standard error. In an adjusted logistic regression analysis controlling for age, gender, living with family, residence period in South Korea, and smoking and drinking, those who did not experience a regular educational curriculum in South Korea showed 5.78 odds of self-reported poor health (Model 1 in Table 4, p-value = 0.024). This association was attenuated to 3.95 in model 2 (p-value = 0.096). The % excess odd explained for no educational experience in South Korea was 38.23% when social support was included in model 2. Table 3 Page 7/15 Page 7/15 Table 4 Table 4 Association between education in SK and subjective poor health Independent Variables Model 1 Model 2 Odds Ratio 95% CI Odds Ratio 95% CI Education in SK College or more 1.00 [Reference] 1.00 [Reference] Middle/high school 2.05 [0.47–8.95] 2.11 [0.46–9.67] No 5.78 [1.26–26.57] 3.95 [0.78–19.93] Age (years, continuous) 1.05 [1.00-1.09] 1.04 [1.00-1.09] Gender (reference = male) 5.53 [0.56–54.47] 8.20 [0.70-96.59] Living with family (reference = yes) 2.48 [0.84–8.51] 1.58 [0.46–5.39] Residence period in SK (months, continuous) 1.00 [0.99–1.02] 1.01 [0.99–1.02] Smoking (reference = no) 3.23 [0.40-26.03] 5.85 [0.57–60.34] Drinking (reference = no) 1.25 [0.36–4.35] 1.05 [0.28–3.90] Social support (score, continuous) 1.10 [1.02–1.18] Model1 was adjusted for age, gender, living with family, residence period in SK, smoking, and drinking. Model2 was adjusted for age, gender, living with family, residence period in SK, smoking, drinking, and social support. CI = confidence interval; SK = South Korea. We evaluated the path model of direct and indirect effects between the three variables of interest, namely education in South Korea, social support, and self-rated health status along with a control variable age. As shown in Fig. 1 and Table 5, the path from Education in South Korea to SRH was not significant, in Association between education in SK and subjective poor health We evaluated the path model of direct and indirect effects between the three variables of interest, namely education in South Korea, social support, and self-rated health status along with a control variable age. As shown in Fig. 1 and Table 5, the path from Education in South Korea to SRH was not significant, in other words, the direct effect of education was not supported (p = 0.064). Paths from education to social support (β = 0.30, p = 0.001) and from social support to health (β = 0.30, p = 0.001) were all statistically significant, and the strength was big as the effect of age on health (β =-0.31, p = 0.001). Thus, education Page 8/15 in South Korea did not directly affect SRH but did indirectly affect it through social Support. The fit of the path model was assessed using the indices: χ2/df = 2.64; Normed Fit Index (NFI) = 0.945; Comparative Fit Index (CFI) = 0.961; and Standardized Root Mean Square Residual (SRMR) = 0.0107. All the indices were above the recommended criteria for good or fit [21]. Table 3 Table 5 Direct, indirect and total effects of Education in South Korea on Self-rated Health Dependent Variable Independent Variable Direct Effect Indirect Effect Total Effect R2 Self-rated Health Education in South Korea 0.16 0.089 0.25 0.29 Social Support 0.30* 0.30 Age -0.31* -0.31 Social Support Education in South Korea 0.30* 0.30 0.09 p < 0.05, p < 0.01, *p < 0.001 Discussion In other words, education affects health indirectly through social support. This result is not consistent with other studies [5–9] that argued a direct education effect on health. However, the significant path from education to social support and the one from social support to health were in agreement with previous studies. Previous evidence has supported that greater social support is associated with more education [30, 31]. Formal education may promote interpersonal skills and friendships and provide more resources (e.g., income and leisure time) to develop and maintain relationships [32]. Social support may also promote healthier lifestyles by enhancing individuals’ motivation, normative environment, and access to information [33–35]. The significant indirect effect of education on health through social support is in the same line with studies showing that education and social relationships interact with each other to affect health [13, 14]. It is noteworthy that the strength of this indirect effect of education (0.25) and direct effect of social support (0.30) on health is comparable to the strength of the age effect (-0.31) on health. There could be many other mediating factors between education and health. In this study, we proposed social support as one of the factors. Further study is recommended to discover other factors that link education and health. Therefore, based on our findings, there is a need for a policy enforcing education and social integration activities in the NKD group for their health. This study has limitations. First, the selection of study participants was restricted. There are 33247 NKDs throughout South Korea. However, the participants in this study were selected by a snowball sampling method mainly from the residents in the Seoul and Gyeonggi metropolitan areas. Therefore, the study participants may not be considered as a representative sample of the NKD population. This led to a wide range for the confidence interval in the results of the logistic regression analysis in this study. Therefore, the interpretation of the study results should be performed carefully. However, considering the limited opportunities to contact NKDs and restrictions on information collection from NKDs, these limitations are inevitable. Second, because this study was a cross-sectional survey research, it is difficult to infer causality among the variables. A well-designed longitudinal follow-up study will be needed to find underlying factors affecting the health status of NKDs on which proper health promotion and intervention strategies and programs can be developed. Discussion This study analyzed the association of receiving education in South Korea and social support with self- reported health status among NKDs and the link among those three variables. This is the first study that observed the independent main effect of receiving education in South Korea on health among NKDs. Our findings indicate that receiving education in South Korea is positively associated with self-rated health status, even when potential confounding factors are taken into consideration. Like other populations, educational attainment is a significant predictor of morbidity, mortality, and other indices of physical health among NKDs [22–24]. Receiving education in South Korea among NKDs is also significantly associated with social support. Those with college education in South Korea, relative to those without, had a higher level of social support. It is evident that education in South Korea is associated with psychological aspects of health that may be considered as an important part of social relationships. Our findings also indicate that social support is positively associated with better health. The buffering hypothesis suggests that social support affects health by attenuating the physiological effects of psychosocial stress [25]. Social support has been shown to have both buffering effects and direct beneficial effects on general morbidity [26, 27]. Evidence suggests that socioeconomic status (SES) and social relationships can interact with each other to affect health. Some studies have found that the protective qualities of social support are stronger among individuals with a low SES [15, 28]. Social support is particularly important for individuals with a lower SES. However, some research reports that the protective effects of social support on health are stronger among individuals with a high SES [13, 14, 29]. Page 9/15 This study’s finding further supports that not only receiving education in South Korea is associated with the health status of NKDs—independence of socio-demographic factors and health behaviors—but that also social support has an important role in the association between education in South Korea and self- rated health among NKDs. However, we are not clear on the exact pathway from education to health which remains to be elucidated. The hypothesis that is the most interesting to us is the potential effects of receiving education in South Korea on self-rated health either through social support or not. Our finding showed that education in South Korea did not directly affect their health, but education leads to social support, which affects their health. Abbreviations ANCOVA analysis of covariance ANOVA analysis of variance CFI Comparative Fit Index NFI Normed Fit Index NKDs North Korean defectors SES socioeconomic status SRH self-rated health SRMR standardized root mean square residual Discussion Third, although the questionnaires used in this study had proven validity and reliability, it was still challenging for some NKDs with different social and cultural backgrounds to fully understand and answer the questionnaires. It is necessary to develop tools to better measure the health status of this vulnerable group. Page 10/15 Ethics approval and consent to participate This study was approved by the Institutional Review Board of the Seoul National University School of Dentistry (No.S-D20170041). Informed consent was obtained from all participants. Conclusions Despite the aforementioned limitations, this study has some vital findings. The NKDs encounter challenges in adapting to a new political and social system in South Korea. To assist NKDs to overcome these challenges and improve their health condition, social integration policies and interventions focusing on enrollment in regular education curriculums/programs and participating in social interaction activities are essential. Further research should develop related intervention programs for NKDs and assess their outcomes. Consent for publication Informed consent was obtained from all participants. Authors' contributions All authors have made substantial contributions to conception and design of the study. Han DH and Shen JJ contributed equally to conception, design, and interpretation of study. Lee J drafted this manuscript. Park Y carried out the survey and made a dataset. Yoo JW, Kim P, and Zhou W revised it critically for important intellectual content. All authors have given final approval of the version to be published. Availability of data and materials Availability of data and materials Page 11/15 The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements Not applicable. Funding This was a self-funded study. Dental Research Institute supported the cost of English editing. was a self-funded study. Dental Research Institute supported the cost of Eng Competing interests The authors declare that they have no competing interests. References 1. Noland M, Robinson S, Wang T. Famine in North Korea: Causes and Cures. Econ Dev Cult Change. 2001;49:741–67. 2. Ministry of Unification. Settlement Support for North Korean Defectors. 2020. https://www.unikorea.go.kr/eng_unikorea/whatwedo/support/ Accessed 06 May 2020. 3. Park SM. Primary health care for vulnerable population. Korean J Fam M 4. Korea Hana Foundation. 2018 Social Integration Survey of North Korean Refugees in South Korea. Seoul: Korea Hana Foundation; 2019. p. 85–87. 5. Baker DP, Leon J, Smith Greenaway EG, Collins J, Movit M. The education effect on population health: A reassessment. Popul Dev Rev. 2011;37:307–32. 5. Baker DP, Leon J, Smith Greenaway EG, Collins J, Movit M. The education effect on population health: A reassessment. Popul Dev Rev. 2011;37:307–32. 6. Herd P, Goesling B, House JS. Socioeconomic position and health: The differential effects of education versus income on the onset versus progression of health problems. J Health Soc Behav. 2007;48:223–38. 6. Herd P, Goesling B, House JS. Socioeconomic position and health: The differential effects of education versus income on the onset versus progression of health problems. J Health Soc Behav. 2007;48:223–38. 7. Marmot MG, Shipley MJ. Do socioeconomic differences in mortality persist after retirement? 25 year follow up of civil servants from the first Whitehall study. BMJ. 1996;313:1170–80. 7. Marmot MG, Shipley MJ. Do socioeconomic differences in mortality persist after retirement? 25 year follow up of civil servants from the first Whitehall study. BMJ. 1996;313:1170–80. 8. Meyer OL, Castro-Schilo L, Aguilar-Gaxiola S. Determinants of mental health and self-rated health: A model of socioeconomic status, neighborhood safety, and physical activity. Am J Public Health. 2014;104:1734–41. Page 12/15 Page 12/15 9. Assari S, Lankarani MM. Demographic and Socioeconomic Determinants of Physical and Mental Self-rated Health across 10 Ethnic Groups in the United States. Int J Epidemiol Res. 2017;3:185–93. 10. Choi AI, Weekley CC, Chen SC, et al. Association of educational attainment with chronic disease and mortality: The Kidney Early Evaluation Program (KEEP). Am J Kidney Dis. 2011;58:228–34. 10. Choi AI, Weekley CC, Chen SC, et al. Association of educational attainment with chronic disease and mortality: The Kidney Early Evaluation Program (KEEP). Am J Kidney Dis. 2011;58:228–34. 11. Gakidou E, Cowling K, Lozano R, Murray CJ. Increased educational attainment and its effect on child mortality in 175 countries between 1970 and 2009: A systematic analysis. Lancet. 2010;376:959–74. 12. Holt-Lunstad J, Smith TB, Layton JB. Social relationships and mortality risk: A meta-analytic review. PLoS Med. References 2010;7:e1000316. 13. Gorman BK, Sivaganesan A. The role of social support and integration for understanding socioeconomic disparities in self-rated health and hypertension. Soc Sci Med. 2007;65:958–75. 14. Fagundes CP, Bennett JM, Alfano CM, et al. Social support and socioeconomic status interact to predict Epstein-Barr virus latency in women awaiting diagnosis or newly diagnosed with breast cancer. Health Psychol. 2012;31:11–9. 15. Brummett BH, Barefoot JC, Vitaliano PP, Siegler IC. Associations among social support, income, and symptoms of depression in an educated sample: the UNC Alumni Heart Study. Int J Behav Med. 2003;10:239–50. 16. Thomas R. The Right to Quality Education for Refugee Children Through Social Inclusion. J Hum Rights Soc Work. 2016;1:193–201. 17. Gušić S, Malešević A, Cardeña E, Bengtsson H, Søndergaard H. “I Feel Like I do Not Exist:” A Study of Dissociative Experiences Among War-Traumatized Refugee Youth. Psychol Trauma. 2018;10:542– 50. 18. Suh SY, Im YS, Lee SH, Park MS, Yoo TW. A study for the development of Korean version of the Duke- UNC functional social support questionnaire. J Korean Acad Fam Med. 1997;18:250–60. 19. Szklo M, Nieto J. Epidemiology. Beyond the basics. 3rd ed. London: Jones & Bartlett Publishers; 2014. pp. 161–2. 20. Amos [computer program]. Version 23.0. Chicago: IBM SPSS Inc; 2014. 21. Hu L, Bentler PM. Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct Equ Model. 1999;6:1–55. 21. Hu L, Bentler PM. Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct Equ Model. 1999;6:1–55. 22. Strand BH, Grøholt EK, Steingrímsdóttir ÓA, Blakely T, Graff-Iversen S, Naess Ø. Educational inequalities in mortality over four decades in Norway: Prospective study of middle aged men and women followed for cause specific mortality, 1960–2000. BMJ. 2010;340:c654. 23. Case A, Deaton A. Rising morbidity and mortality in midlife among white non-Hispanic Americans in the 21st century. Proc Natl Acad Sci USA. 2015;112:15078–83. 23. Case A, Deaton A. Rising morbidity and mortality in midlife among white non-Hispanic Americans in the 21st century. Proc Natl Acad Sci USA. 2015;112:15078–83. 24. Viner RM, Hargreaves DS, Ward J, Bonell C, Mokdad AH, Patton G. The health benefits of secondary education in adolescents and young adults: An international analysis in 186 low-, middle-and high- income countries from 1990 to 2013. SSM Popul Health. 2017;3:162–71. Page 13/15 Page 13/15 25. Cohen S, Wills TA. Stress, social support, and the buffering hypothesis. Psychol Bull. References 1985;98:310– 57. 26. McKay DA, Blake RL Jr, Colwill JM, et al. Social supports and stress as predictors of illness. J Fam Pract. 1985;20:575–81. 27. Blake RL Jr, McKay DA. A single-item measure of social supports as a predictor of morbidity. J Fam Pract. 1986;22:82–4. 28. North RJ, Holahan CJ, Moos RH, Cronkite RC. Family support, family income, and happiness: a 10- year perspective. J Fam Psychol. 2008;22:475–83. 29. Krause N. Anticipated support, received support, and economic stress among older adults. J Gerontol B Psychol Sci Soc Sci. 1997;52:P284–93. 30. Mirowsky J, Ross CE. Education, social status, and health. Hawthorne: A 30. Mirowsky J, Ross CE. Education, social status, and health. Hawthorne: Aldine de Gruyter; 2003. 31. Cutler DM, Lleras-Muney A. Understanding differences in health behaviors by education. J Health Econ. 2010;29:1–28. 32. Cutler DM, Lleras-Muney A. Education and health: Evaluating theories and evidence. In: Schoeni RF, House JS, Kaplan GA, Pollack H, editors. Making Americans healthier: Social and economic policy as health policy. New York: Russell Sage Foundation; 2008. pp. 29–60. 33. Uchino BN. Social support and health: a review of physiological processes potentially underlying links to disease outcomes. J Behav Med. 2006;29:377–87. 33. Uchino BN. Social support and health: a review of physiological processes potentially underlying links to disease outcomes. J Behav Med. 2006;29:377–87. 34. Chatzisarantis NLD, Hagger MS, Brickell T. Using the construct of perceived autonomy support to understand social influence within the theory of planned behavior. Psychol Sport Exerc. 2008;9:27– 44. 34. Chatzisarantis NLD, Hagger MS, Brickell T. Using the construct of perceived autonomy support to understand social influence within the theory of planned behavior. Psychol Sport Exerc. 2008;9:27– 44. 35. Gillison FB, Standage M, Cumming SP, Zakrzewski-Fruer J, Rouse PC, Katzmarzyk PT. Does parental support moderate the effect of children's motivation and self-efficacy on physical activity and sedentary behaviour? Psychol Sport Exerc. 2017;32:153–61. 35. Gillison FB, Standage M, Cumming SP, Zakrzewski-Fruer J, Rouse PC, Katzmarzyk PT. Does parental support moderate the effect of children's motivation and self-efficacy on physical activity and sedentary behaviour? Psychol Sport Exerc. 2017;32:153–61. 35. Gillison FB, Standage M, Cumming SP, Zakrzewski-Fruer J, Rouse PC, Katzmarzyk PT. Does parental support moderate the effect of children's motivation and self-efficacy on physical activity and sedentary behaviour? Psychol Sport Exerc. 2017;32:153–61. Figures Page 14/15 Figure 1 Path diagram of direct and indirect effects of Education in South Korea on Self-rated Health. p < 0.05, p < 0.01, p < 0.001 Figure 1 Path diagram of direct and indirect effects of Education in South Korea on Self-rated Health. p < 0.05, p < 0.01, p < 0.001 Path diagram of direct and indirect effects of Education in South Korea on Self-rated Health. p < 0.05, p < 0.01, *p < 0.001 Page 15/15 Page 15/15
https://openalex.org/W2098443684	https://europepmc.org/articles/pmc3528661?pdf=render	English	null	Endothelial cells influence the sodium nitroprusside mediated inhibition of platelet aggregation by an as yet unkown pathway	Thrombosis journal	2,012	cc-by	4,468	Abstract In addition, Suzuki and coworkers suggest a synergistic effect of SNP and fi- brinolytic agents [10]. In vitro studies have shown that NO inhibits platelet adhesion to endothelial cells [11] as well as platelet aggregation [12,13], platelet activation [5-7,14,15] and clot formation [16,17] and thrombus growth under shear stress [18]. However, these results could not always be confirmed by in vivo studies. Several studies have been performed investigating the mechan- ism of SNP action on platelet aggregation, but the exact means of interaction still is not understood [19,20]. Most authors confirm the NO dependent inhibition of platelet activation by SNP. Woods and coworkers clearly demon- strated that the platelet response to SNP in hyperten- sives is lower due to reduced sensitivity of the platelets to NO [21]. Sogo and coworkers showed that in the ab- sence of endothelial cells the effect of SNP is exclusively cGMP dependent, which is in accordance with our results [22]. In addition this direct interference with Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Endothelial cells influence the sodium nitroprusside mediated inhibition of platelet aggregation by an as yet unkown pathway Hans-Georg Topf, Manfred Rauh, Wolfgang Rascher, Jörg Dötsch and Jens M Klinge Hans-Georg Topf, Manfred Rauh, Wolfgang Rascher, Jörg Dötsch and Jens M Klinge © 2012 Topf et al.; licensee BioMed Central Ltd .This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The clinical use of Sodium nitroprusside (SNP) may be associated with an alteration of platelet function. The main focus of this study was the effect of SNP on platelet aggregation in the absence or presence of endothelial cells. Methods: Platelets were incubated with different concentrations of SNP with and without endothelial cells. Platelet aggregation was induced by ADP. Results: Platelet aggregation was significantly inhibited by all concentrations of SNP. Endothelial cells significantly increased this inhibitory effect of SNP. Time course studies showed an inverse correlation of incubation time to platelet aggregation inhibition in the absence of endothelial cells, and a direct correlation in the presence of endothelial cells. Blocking platelet and endothelial cell guanylate cyclase with 1 H-(1,2,4)-oxadiazolo(4,3-a) quinoxalin-1-one (ODQ), or pretreatment of the endothelial cells with cyclooxygenase – inhibitors, had no influence on the increased inhibitory effect of the endothelial cells. Cyanide reversed the inhibitory effect of SNP completely. Conclusion: Endothelial cells play an important role in the SNP mediated inhibition of platelet aggregation. The effect is reversible only by cyanide, not by blocking classical NO signal transduction. Keywords: Nitric oxide (NO), Platelet aggregation, Sodium-nitro-prusside (SNP), Cyanide, Endoth Similar results confirming the synergistic effect of NO donors and other antiplatelet drugs, could be demon- strated by Negrescu et al. [9]. In addition, Suzuki and coworkers suggest a synergistic effect of SNP and fi- brinolytic agents [10]. In vitro studies have shown that NO inhibits platelet adhesion to endothelial cells [11] as well as platelet aggregation [12,13], platelet activation [5-7,14,15] and clot formation [16,17] and thrombus growth under shear stress [18]. However, these results could not always be confirmed by in vivo studies. Several studies have been performed investigating the mechan- ism of SNP action on platelet aggregation, but the exact means of interaction still is not understood [19,20]. Most authors confirm the NO dependent inhibition of platelet activation by SNP. Woods and coworkers clearly demon- strated that the platelet response to SNP in hyperten- sives is lower due to reduced sensitivity of the platelets to NO [21]. Sogo and coworkers showed that in the ab- sence of endothelial cells the effect of SNP is exclusively cGMP dependent, which is in accordance with our results [22]. In addition this direct interference with Similar results confirming the synergistic effect of NO donors and other antiplatelet drugs, could be demon- strated by Negrescu et al. [9]. * Correspondence: hans-georg.topf@uk-erlangen.de Klinik für Kinder und Jugendliche, University of Erlangen-Nuremberg, Loschgestr 1591054, Erlangen, Germany Introduction The administration of Sodium nitroprusside (SNP) is a highly effective strategy in pulmonary and systemic hypertension crisis, both in adults and in neonates [1,2]. The most common SNP application is intravenous, but inhalation and endotracheal application is used as well [3,4]. The main mechanism of action may be explained by the release of NO which results in a dilation of small vessels both in the systemic and the pulmonary circula- tion. A well known side effect of NO is the inhibition of platelet function [5,6]. This interference of platelet acti- vation is due to the stimulation of the soluble guanylate cyclase which increases the concentration of cGMP and inhibits the calcium-dependant platelet activation [7]. Just recently Jensen et al. published a study on the po- tential synergistic effect of Dipyridamole and NO donors, such as sodiumnitroprusside, to prolong the in- hibition of thrombin-induced platelet shape change. [8] * Correspondence: hans-georg.topf@uk-erlangen.de Klinik für Kinder und Jugendliche, University of Erlangen-Nuremberg, Loschgestr 1591054, Erlangen, Germany Page 2 of 5 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 controls. To study the effect of endothelial cells on ag- gregation, the incubation was performed on culture plates with, and without endothelial cells. platelet activity, cGMP stimulates cyclooxygenase activ- ity in endothelial cells, resulting in increased concentra- tions of prostacyclins and the subsequent inhibition of platelet activation [23-25]. SNP concentrations were 100 μM, 500 μM and 1000 μM. Time course studies were performed with 100 μM SNP, and incubation times of the whole blood samples were 0, 1, 2 and 4 hours. As well as this NO dependent effect other mechanisms seem to have an important impact on the inhibitory ac- tion of SNP in the presence of endothelial cells. Levin et al. already mentioned that the supernatant taken from SNP-incubated endothelial cells, had a surprising inhibi- tory effect on platelet aggregation not due to NO alone [26]. As total centrifugation time was 20 minutes (s. below) and SNP was added to the whole blood sample before centrifugation contact time between SNP and platelets was at least 20 minutes. Samples were kept at 37°C dur- ing the whole experiment. In the present study we wanted to evaluate the role of endothelial cells on the antiaggregatory effect of SNP. Material and methods Reagents Sodium nitroprusside (SNP), Cyanide and ADP were pur- chased from Sigma laboratories and 1 H-(1,2,4)oxadia- zolo(4,3-a)quinoxalin-1-one (ODQ) was purchased from Alexis Biochemicals. PBS was obtained from Boehringer, Germany and Protaminhydrochloride (Protamin ICN 1000) was from ICN, Frankfurt, Germany. Acetyl sali- cylic acid (ASS) was obtained from Bayer, Leverkusen, Germany and indomethacin (Liomethacin) was purchased from Chiesi Pharmaceutica, Parma, Italy. To study whether SNP has an effect on the endothelial production of prostaglandins by activating cyclooxy- genases (COX I and II), two cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin were used. Endo- thelial cells were incubated for 5 min with 3 ml of either 1 mM acetylsalicylic acid or 10 μM indomethacin. Suffi- cient suppression of the cyclooxygenases in HUVEC by ASA or indomethacin has been previously described [28]. After thorough washing with PBS the experiments were repeated as described above. Platelet aggregation f For preparation of platelet-rich plasma the whole blood samples were centrifuged for 10 minutes at 800 r.p.m. Platelet-poor plasma was obtained by further centrifuga- tion of PRP at 3800 r.p.m for 10 minutes. Platelet count was then adjusted with PPP to 250,000/μL. Platelet re- sponse to ADP (20 μM) was recorded with a Lumiag- gregometer (Aggregometer 840, Elvi). Introduction An additional aim was to investigate wether the time span between the incubation of platelets with SNP and measurement of the antiaggregatory effect provides an explanation for the sometimes contradictory results of in vitro and in vivo studies. The third goal was to test the reversibility of the antiaggregatory effects of SNP by cyanide and classical NO pathway inhibition. Endothelial cell culture For the endothelial cell experiments we used a well established configuration using human umbilical endo- thelial cells (HUVEC). HUVEC were isolated from fresh human umbilical cord vein by enzymatic disaggregation with collagenase, and were used between passages 2 and 5. HUVEC were cultured as previously described [27], the plates were washed with PBS before the blood sample was added and Heparin was neutralized with 75 I.U. of Protamin. Blood samples Venous blood was drawn from 4 healthy volunteers and coagulation was prevented immediately using 3.8% sodium citrate 1:10. The volunteers took no medication two weeks prior to blood sampling. To study the effect of cyanide on the effect of SNP, we added cyanide to whole blood in one set-up. In another experiment we added cyanide to platelet rich plasma (PRP) after the addition of ADP. A control group proved that cyanide itself has no effect on platelet aggregation. Incubation with SNP The effect of different concentrations of SNP and differ- ent incubation times was tested. After adding SNP to 5 mL of whole blood, samples were placed on culture plates. A new plate was used for each concentration and incubation time. Samples without SNP were used as Data analysis Data represent the mean +/−SD of at least 4 experiments. Mechanism of SNP – mediated inhibition of platelet function We wished to study the mechanism by which SNP inhibits platelet aggregation. NO signal transduction by cGMP was inhibited using the guanylate cyclase inhibitor 1 H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). ODQ (100 μM) was added to the sample 2 min- utes before adding 100 μM SNP to allow the reaction of ODQ with the guanylate cylcase. Platelet rich plasma was then obtained from the blood sample. The platelet function was tested using the Born method. Aggregation was induced with ADP (20 μM) after the platelet count had been adjusted to 250,000/μL. Discussion d In our study we could clearly demonstrate that endothe- lial cells play an important role in the antiaggregatory ef- fect of SNP. Incubation with endothelial cells increased both the inhibitory effect on platelet aggregation and the duration of this effect. Levin et al. already mentioned that the supernates taken from SNP-incubated endothe- lial cells had an inhibiting effect on platelet aggregation [26]. They speculated that their data could be explained by the synergy of NO and PGI2 [26]. The Levin study used supernatants of SNP-incubated endothelial cells and did not preincubate the endothelial cells with cyclooxygenase inhibitors, which may have led to differ- ent results. In contrast to their experimental setting, the platelets in our study were in direct contact with the endothelial cells during SNP incubation, which suggests possible additional interactions between platelets and endothelial cells. It has been well established that PGs of the E series and PGI2 inhibit platelet aggregation by counteracting the proaggregatory effect of thromboxane A2 [29]. This could explain the additional inhibitory affect of the endothelial cells in the presence of NO. However, this explanation seems insufficient, as the pre- incubation of endothelial cells with cyclooxygenase inhi- bitors, suppressing the PGI2 production, did not change the SNP effect as expected. In an earlier study we could show that the effect of other NO-donors, such as SNAP, is increased by the PG pathway. Induction of PGI2 via NO, following reduced platelet aggregation, could be blocked by preincubation of the endothelial cells with cyclooxygenases [28]. The effect of SNP was not attenu- ated by the preincubation of the endothelial cells with a cyclooxygenase inhibitor. This is in contrast to the con- clusions of Levin and coworkers [26]. Sogo et al. could clearly demonstrate that, in the absence of endothelial cells, the effect of SNP is exclusively cGMP-dependent [22], which should be the case if the effect is due to NO Effect of SNP on platelet aggregation, effect of endothelial cells After 1 hour of incubation SNP inhibited platelet aggre- gation in a dose-dependent manner. 100 μM, 500 μM and 1000 μM of SNP were tested (data not shown). This was statistically significant for all concentrations of SNP. These experiments were not repeated with endothelial cells. Time course studies were performed with 100 μM of SNP, as the 100 μmol curve at one hour was at the middle of normal aggregation and total inhibition, so changes in both directions would be detectable. A time dependent reduction of platelet aggregation could be detected with an inverse correlation to incubation time. Endothelial cells significantly increased the SNP – mediated inhibition of platelet function after 2 and 4 hours of incubation (Additional file 1 2nd and 4th column). Effect of ODQ on SNP mediated inhibition of platelet aggregation Incubation with ODQ decreased the effect on platelet ag- gregation at 0 h of incubation time. ODQ did not inhibit the SNP mediated-effect on platelet aggregation later in the presence of endothelial cells. (Additional file 1 6th column) Results The difference in platelet aggregation of controls with, and without, endothelial cells was not significant differ- ent. (Additional file 1 1st and 3rd column). We could show that ADP induced thrombocyte aggregation was intact even 4 h after blood sampling, with and without endothelial cells. Effect of cyanide on SNP mediated inhibition of platelet aggregation Cyanide (2000 μM) alone did not influence platelet aggregation. Cyanide added to whole blood prior to addition of SNP repressed the effect of SNP in all cases. The addition of cyanide after the preparation of platelet- rich plasma, prior to the addition of ADP only inhibited the SNP effect of the endothelial cell experiments (Additional file 1 5th column). Data analysis D t Data analysis Data represent the mean +/−SD of at least 4 experiments. Data represent the mean +/−SD of at least 4 experiments. Page 3 of 5 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 The levels of aggregation given in percent were detected every 20 seconds for 6 minutes. The following parameters were used for statistical analysis: For each experiment, all values of the aggregation curves were compared. Data were analyzed with Prism 3.0 using the Kruskal-Wallis-test and p ≤0.05 was accepted as being significant. In case of multiple comparisons p – values were corrected according to Dunn. Results were grouped according to incubation time: Group 1 = no incubation time, group 2 = incubation time of 1 hour, group 3 = in- cubation time of 2 hours, group 4 = incubation time of 4 hours. salicylic acid and indomethacin (in the graph called COX I) did not influence the effect of SNP on platelet aggregation. The results were therefore combined. The pretreatment of endothelial cells with COX inhibitors did not interfere with the effect of SNP incubation (Additional file 1 7th column). 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. 2. Patel HP, Mitsnefes M: Advances in the pathogenesis and management of hypertensive crisis. Curr Opin Pediatr 2005, 17(2):210–214. 2. Patel HP, Mitsnefes M: Advances in the pathogenesis and management of hypertensive crisis. Curr Opin Pediatr 2005, 17(2):210–214. 3. Palhares DB, Figueiredo CS, Moura AJ: Endotracheal inhalatory sodium nitroprusside in severely hypoxic newborns. J Perinat Med 1998, 26(3):219–224. Our results without endothelial cells show an effect of SNP on platelets which is in accordance with other in vitro studies [7,26,30]. 4. Schreiber MD, et al: Direct comparison of the effects of nebulized nitroprusside versus inhaled nitric oxide on pulmonary and systemic hemodynamics during hypoxia-induced pulmonary hypertension in piglets. Crit Care Med 2002, 30(11):2560–2565. 4. Schreiber MD, et al: Direct comparison of the effects of nebulized nitroprusside versus inhaled nitric oxide on pulmonary and systemic hemodynamics during hypoxia-induced pulmonary hypertension in piglets. Crit Care Med 2002, 30(11):2560–2565. 5. Brune B, Hanstein K: Rapid reversibility of nitric oxide induced platelet inhibition. Thromb Res 1998, 90(2):83–91. 6. Mellgren K, et al: Effect of nitric oxide gas on platelets during open hea operations. Ann Thorac Surg 1998, 65(5):1335–1341. Another interesting fact is that the addition of cyanide is able to reverse the effect, not only after immediate ad- ministration, as shown by Brune et al. [5], but even after 4 h of incubation with SNP. This effect has not been described before in the literature. Although we do not have an explanation for this observation, we speculate that a cGMP independent pathway may be responsible for this effect. p g 5. Brune B, Hanstein K: Rapid reversibility of nitric oxide induced platelet inhibition. Thromb Res 1998, 90(2):83–91. 6. Mellgren K, et al: Effect of nitric oxide gas on platelets during open heart operations. Ann Thorac Surg 1998, 65(5):1335–1341. p g 7. Moro MA, et al: cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci U S A 1996, 93(4):1480–1485. 8. 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. Jensen BO, et al: Dipyridamole synergizes with nitric oxide to prolong inhibition of thrombin- induced platelet shape change. Platelets 2011, 22(1):7–18. In summary, our results show that endothelial cells play an important role in the SNP- mediated inhibition of platelet activation, which can be reversed only by cyanide. We could demonstrate that this effect is not mediated by activation of endothelial cyclooxygenases, as the inhibition of cyclooxygenases had no influence on the results. It is not dependent on cGMP, as it is not influenced by the guanylate cyclase inhibitor ODQ. Thus we suggest a cGMP independent, possibly not yet known pathway for platelet inhibition by SNP/endothelial cells. 9. Negrescu EV, et al: Interaction of antiplatelet drugs in vitro: aspirin, iloprost, and the nitric oxide donors SIN-1 and sodium nitroprusside. Cardiovasc Drugs Ther 1995, 9(4):619–629. 10. Suzuki Y, et al: Synergistic disaggregation of platelets by the products of endothelial cells or their analogs. Haematologia (Budap) 2000, 30(2):81–90 10. Suzuki Y, et al: Synergistic disaggregation of platelets by the products of endothelial cells or their analogs. Haematologia (Budap) 2000, 30(2):81–90. 11. Radomski MW, et al: Platelet adhesion to human vascular endothelium is modulated by constitutive and cytokine induced nitric oxide. Cardiovasc Res 1993, 27(7):1380–1382. 11. Radomski MW, et al: Platelet adhesion to human vascular endothelium is modulated by constitutive and cytokine induced nitric oxide. Cardiovasc Res 1993, 27(7):1380–1382. 12. Kurata M, Tanaka K, Usami M: Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood. Gen Pharmacol 1997, 28(5):671–673. 12. Kurata M, Tanaka K, Usami M: Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood. Gen Pharmacol 1997, 28(5):671–673. 13. Cheung PY, et al: Inhaled nitric oxide and inhibition of platelet aggregation in critically ill neonates. Lancet 1998, 351(9110):1181–1182. 13. Cheung PY, et al: Inhaled nitric oxide and inhibition of platelet aggregation in critically ill neonates. Lancet 1998, 351(9110):1181–1182. The identification of this unknown antiaggregatory metabolite could be of great interest, since its effect on ADP-dependent platelet aggregation is long-lasting, but reversible by cyanide. We speculate that other less toxic ion metabolites could also reverse this effect, thus offer- ing the opportunity for antidot treatment. 14. Radomski MW, Palmer RM, Moncada S: The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 1987, 92(3):639–646. 15. 1. Benitz WE, et al: Use of sodium nitroprusside in neonates: efficacy and safety. J Pediatr 1985, 106(1):102–110. Radomski MW, Palmer RM, Moncada S: Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol 1987, 92(1):181–187. 15. Radomski MW, Palmer RM, Moncada S: Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol 1987, 92(1):181–187. p 16. Nielsen VG, Geary BT, Baird MS: Effects of DETANONOate, a nitric oxide donor, on hemostasis in rabbits: an in vitro and in vivo thrombelastographic analysis. J Crit Care 2000, 15(1):30–35. p 16. Nielsen VG, Geary BT, Baird MS: Effects of DETANONOate, a nitric oxide donor, on hemostasis in rabbits: an in vitro and in vivo thrombelastographic analysis. J Crit Care 2000, 15(1):30–35. Competing interests 19. Gold ME: Pharmacology of the nitrovasodilators. Antianginal, antihypertensive, and antiplatelet actions. Nurs Clin North Am 1991, 26(2):437–450. p g All authors have read the manuscript and conflict of interest statement and have approved their submission for publication; all authors declare that they do not have competing interests. 20. Erne P, et al: Vasodilating agents and platelet function: intracellular free calcium concentration, cyclic nucleotides, and shape-change response. J Cardiovasc Pharmacol 1986, 8(Suppl 8):S102–S106. Effect of COX-inhibition on SNP mediated reduction of platelet aggregation Since endothelial cells play an important role in the SNP-induced inhibition of platelet aggregation, the effect of COX inhibitors and of ODQ was tested only after in- cubation with endothelial cells. The cox inhibitors acetyl Page 4 of 5 Page 4 of 5 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 release. In our setting the effect of SNP is not attenuated by the guanylate cyclase inhibitor ODQ, which is sup- posed to block the cGMP-dependent NO effect. This opposes the hypothesis that the effect of SNP is exclu- sively cGMP dependent in the presence of endothelial cells. These results highlight the importance of the endothelial cell in SNP metabolism and platelet inter- action. Our data do not allow us to decide which path- way is responsible for the results measured. Because the effect of endothelial cells on platelet aggregation could not be reversed by incubation with a COX-inhibitor, we speculate that activation of endothelial cyclooxygenases by SNP may not be too important for the antiaggregatory ef- fect of SNP. release. In our setting the effect of SNP is not attenuated by the guanylate cyclase inhibitor ODQ, which is sup- posed to block the cGMP-dependent NO effect. This opposes the hypothesis that the effect of SNP is exclu- sively cGMP dependent in the presence of endothelial cells. These results highlight the importance of the endothelial cell in SNP metabolism and platelet inter- action. Our data do not allow us to decide which path- way is responsible for the results measured. Because the effect of endothelial cells on platelet aggregation could not be reversed by incubation with a COX-inhibitor, we speculate that activation of endothelial cyclooxygenases by SNP may not be too important for the antiaggregatory ef- fect of SNP. JD established the endothelial cultures in the lab and gave relevant input on the test up during incubation. All authors have read and approved the final manuscript. Received: 25 May 2011 Accepted: 13 March 2012 Published: 7 May 2012 Received: 25 May 2011 Accepted: 13 March 2012 Published: 7 May 2012 Additional file 17. Dambisya YM, Lee TL: A thromboelastography study on the in vitro effects of L-arginine and L- NG-nitro arginine methyl ester on human whole blood coagulation and fibrinolysis. Blood Coagul Fibrinolysis 1996, 7(7):678–683. Additional file 1: Comparison of aggregation at timepoints 0, 1, 2 and 4 hours. 18. Williams RH, Nollert MU: Platelet-derived NO slows thrombus growth on a collagen type III surface. Thromb J 2004, 2(1):11. Acknowledgments The authors thank Ida Allabauer and Ulla Jacobs for excellent technical assistance, Dr. B Brüne (University of Kaiserslautern) for discussion of results and Patricia Schmid for language corrections. Additional file 1: Comparison of aggregation at timepoints 0, 1, 2 and 4 hours. Authors’ contributions H-GT was performing the experiments and wrote the manuscript. MR and JK established the born method for measuring platelet aggregation and gave helpful input on pre-analytical pitfalls when working with platelets. WR and pp 21. Woods JD, Edwards JS, Ritter JM: Inhibition by nitroprusside of platelet calcium mobilization: evidence for reduced sensitivity to nitric oxide in essential hypertension. J Hypertens 1993, 11(12):1369–1373. 21. Woods JD, Edwards JS, Ritter JM: Inhibition by nitroprusside of platelet calcium mobilization: evidence for reduced sensitivity to nitric oxide in essential hypertension. J Hypertens 1993, 11(12):1369–1373. Page 5 of 5 Page 5 of 5 Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 22. Sogo N, et al: Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms. Biochem Biophys Res Commun 2000, 279(2):412–419. 23. Salvemini D, et al: Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A 1993, 90(15):7240–7244. Proc Natl Acad Sci U S A 1993, 90(15):7240–7244. 24. Salvemini D, Masferrer JL: Interactions of nitric oxide with cyclooxygenase: in vitro, ex vivo, and in vivo studies. Methods Enzymol 1996, 269:12–25. 25. Vane JR, Anggard EE, Botting RM: Regulatory functions of the vascular endothelium. N Engl J Med 1990, 323(1):27–36. 25. Vane JR, Anggard EE, Botting RM: Regulatory functions of the vascular endothelium. N Engl J Med 1990, 323(1):27–36. g 26. Levin RI, Weksler BB, Jaffe EA: The interaction of sodium nitroprusside with human endothelial cells and platelets: nitroprusside and prostacyclin synergistically inhibit platelet function. Circulation 1982, 66(6):1299–1307. g 26. Levin RI, Weksler BB, Jaffe EA: The interaction of sodium nitroprusside with human endothelial cells and platelets: nitroprusside and prostacyclin synergistically inhibit platelet function. Circulation 1982, 66(6):1299–1307. 27. Dotsch J, et al: Recovery from withdrawal of inhaled nitric oxide and kinetics of nitric oxide-induced inhibition of nitric oxide synthase activity in vitro. Intensive Care Med 2000, 26(3):330–335. 27. Dotsch J, et al: Recovery from withdrawal of inhaled nitric oxide and kinetics of nitric oxide-induced inhibition of nitric oxide synthase activity in vitro. Intensive Care Med 2000, 26(3):330–335. 28. Klinge JM, et al: Endothelial cells play an important role in the antiaggregatory effect of nitric oxide. Crit Care Med 2003, 31(7):2010–2014. 29. Konturek SJ, Pawlik W: Physiology and pharmacology of prostaglandins. Dig Dis Sci 1986, 31(2 Suppl):6S–19S. 30. Brune B, Zhou J, von Knethen A: Nitric oxide, oxidative stress, and apoptosis. Kidney Int Suppl 2003, 84:S22–S24. Topf et al. Thrombosis Journal 2012, 10:6 http://www.thrombosisjournal.com//10/1/6 Authors’ contributions doi:10.1186/1477-9560-10-6 Cite this article as: Topf et al.: Endothelial cells influence the sodium nitroprusside mediated inhibition of platelet aggregation by an as yet unkown pathway. Thrombosis Journal 2012 10:6. 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https://openalex.org/W2966000359	https://europepmc.org/articles/pmc6916180?pdf=render	English	null	A randomised controlled trial of treatments of childhood anxiety disorder in the context of maternal anxiety disorder: clinical and cost‐effectiveness outcomes	Journal of child psychology and psychiatry and allied disciplines	2,019	cc-by	15,944	A randomised controlled trial of treatments of childhood anxiety disorder in the context of maternal anxiety disorder: clinical and cost-effectiveness outcomes Cathy Creswell,1,2,3* Mara Violato,4* Susan Cruddace,3 Stephen Gerry,5 Lynne Murray,3 Roz Shafran,6 Alan Stein,1,7 Lucy Willetts,8 Emma McIntosh,9 and Peter J. Cooper3 1Department of Psychiatry, University of Oxford, Oxford, UK; 2Department of Experimental Psychology, University of Oxford, Oxford, UK; 3School of Psychology and Clinical Language Sciences, University of Reading, Reading, UK; 4Nufﬁeld Department of Population Health, University of Oxford, Oxford, UK; 5Nufﬁeld Department of Orthopaedics, Rheumatology & Musculoskeletal Science, University of Oxford, Oxford, UK; 6Population, Policy and Practice, UCL Great Ormond Street Institute of Child Health, London, UK; 7MRC/Wits Rural Public Health and Health Transitions Research Unit (Agincourt), University of the Witwatersrand, Johannesburg, South Africa; 8Berkshire Healthcare NHS Foundation Trust, Berkshire, UK; 9Institute of Health and Wellbeing, University of Glasgow, Glasgow, UK Background: This study evaluated whether clinical and economic outcomes from CBT for child anxiety disorders in the context of maternal anxiety disorders are improved by adding treatment focused on (a) maternal anxiety disorders or (b) mother–child interactions. Methods: Two hundred and eleven children (7–12 years, 85% White British, 52% female) with a primary anxiety disorder, whose mothers also had a current anxiety disorder, were randomised to receive (a) child-focused CBT with nonspeciﬁc control interventions (CCBT+Con), (b) CCBT with CBT for the maternal anxiety disorder (CCBT+MCBT), or (c) CCBT with an intervention targeting the mother–child interaction (CCBT+MCI). A cost-utility analysis from a societal perspective was conducted using mother/child combined quality-adjusted life years (QALYs). [Trial registration: https://doi.org/10.1186/isrctn19762288]. Results: MCBT was associated with immediate reductions in maternal anxiety compared to the nonspeciﬁc control; however, after children had also received CCBT, maternal outcomes in the CCBT+MCI and CCBT+Con arms improved and CCBT+MCBT was no longer superior. Neither CCBT+MCBT nor CCBT+MCI conferred a beneﬁt over CCBT+Con in terms of child anxiety disorder diagnoses post-treatment [primary outcome] (adj RR: 1.22 (95% CI: 0.88, 1.67), p = .23; adj RR: 1.21 (95% CI: 0.88, 1.65), p = .24, respectively) or global improvement ratings (adj RR: 1.25 (95% CI: 0.99, 1.57), p = .06; adj RR: 1.18 (95% CI: 0.93, 1.50), p = .17) or six and 12 months later. No signiﬁcant differences between the groups were found on the main economic outcome measures (child/mother combined QALY mean difference: CCBT+MCBT vs. CCBT+Con: 0.04 (95% CI: 0.12, 0.04), p = .29; CCBT+MCI vs. CCBT+Con: 0.02 (95% CI: 0.05, 0.09), p = .54). doi:10 1111/jcpp 13089 doi:10 1111/jcpp 13089 Journal of Child Psychology and Psychiatry 61:1 (2020), pp 62–7 Journal of Child Psychology and Psychiatry 61:1 (2020), pp 62–76 Journal of Child Psychology and Psychiatry 61:1 (2020), pp 62–76 doi:10.1111/jcpp.13089 A randomised controlled trial of treatments of childhood anxiety disorder in the context of maternal anxiety disorder: clinical and cost-effectiveness outcomes CCBT+MCI was associated with nonsigniﬁcantly higher costs than CCBT (mean difference: £154 (95% CI: £1,239, £1,547), p = .83) but, when taking into account sampling uncertainty, it may be cost-effective compared with CCBT alone. Conclusions: Good outcomes were achieved for children and their mothers across treatment arms. There was no evidence of signiﬁcant clinical beneﬁt from supplementing CCBT with either CBT for the maternal anxiety disorder or treatment focussed on mother–child interactions, but the addition of MCI (and not MCBT) may be cost-effective. Keywords: Child; anxiety; mother; parent–child interaction; cognitive behaviour therapy. Soler, & Choke, 2013). However, where a parent has an anxiety disorder, rates of child recovery have been found to be as little as half compared to when there is no parental anxiety disorder (Bodden, B€ogels et al., 2008; Hudson et al., 2014). Two studies to date have examined whether speciﬁcally targeting elevated parental anxiety might beneﬁt child treatment out- come (with treatments delivered in group formats). One of these studies reported a positive impact on anxious children of parents with high trait anxiety (Cobham, Dadds, & Spence, 1998), at least in the short term, while the other reported no beneﬁt for anxious children of parents with a current anxiety disorder (Hudson et al., 2014). Notably, both of these studies administered relatively brief treatments for parental anxiety (4 and 3.75 hr, respectively) and neither brought about signiﬁcant reductions in © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Conﬂict of interest statement: No conﬂicts declared. Joint ﬁrst authors. Participants Participants comprised 211 children, aged 7–12 years (mean age = 10.22, SD = 1.58), with a current anxiety disorder, together with their mothers, who also had a current anxiety disorder. The study was powered to provide 90% power at the 5% (two-sided) signiﬁcance level to detect a 30% difference in the primary outcome – that is the proportion of children who recovered from their primary anxiety disorder at the post- treatment assessment in the CCBT+MCI or CCBT+MCBT arms compared to the CCBT+Con arm. The estimated remission rate for the CCBT+Con group was 40% (based on Cobham et al., 1998). A difference of 30% in the proportion of anxiety-free children following completion of the treatment was considered to be the minimum that would be clinically worthwhile taking into account the increased resources required and change to service delivery that would be required if either of these interventions was found to be effective and implemented in practice. The sample size was deﬁned on the basis of two independent trials as recommended by Machin, Campbell, Fayers, and Pinol (1997). The required sample size of 56 children per group was increased to allow for an estimated 20% loss to follow-up. In view of the roles of parental anxiety as well as parenting cognitions and behaviour in maintaining child anxiety, the current study tested the hypoth- esis that clinical outcomes for child anxiety disor- ders presenting in the context of parental anxiety disorders will be improved by supplementing indi- vidual child CBT with (a) treatment of parental anxiety disorders or (b) treatment focused on par- ent–child interactions. This study also evaluated whether supplementing CBT with these adjunct treatments represents good value for money. The few published cost-effective- ness analyses of the treatment of child anxiety mainly concern the value of child CBT compared with a wider family approach (Bodden, B€ogels et al., 2008; Bodden, Dirksen, & Bogels, 2008) or with a parent-directed treatment for child anxiety (Simon, Dirksen, B€ogels, & Bodden, 2012). There is, how- ever, a paucity of evidence on the short-term and long-term cost-effectiveness of alternative conﬁgura- tions of treatment types involving maternal and child treatment combinations, which are likely to be of particular relevance when maternal anxiety disorder is present. Furthermore, no economic evaluations of the treatment of child anxiety have taken into account both the direct effects to the child and ‘spillover’ effects, for example, to the parents and wider society. Participants For outcomes, this is achieved by using quality-adjusted life years (QALY), a generic measure of outcome that can be aggregated across individuals (Weinstein, Torrance, & McGuire, 2009) and allows for examination of both direct treatment and spillover effects (Tilford et al., 2015). For costs, this is achieved by measuring broader impacts on education and employment. Aggregating direct and spillover effects may alter cost-effectiveness proﬁles, a particularly appropriate strategy when the eco- nomic evaluation adopts a societal perspective. Indeed, recommendations from a recent review of family-based therapies suggest that future research The study focussed on mothers as (a) intergenerational associations for anxiety disorders are most commonly found between mothers and their children (Cooper, Fearn, Willetts, Seabrook, & Parkinson, 2006), (b) mothers are most commonly the primary caregivers in the study region, and (c) maternal and paternal behaviours may differ in how they are associated with childhood anxiety (B€ogels & Perotti, 2011). Participants were recruited between June 2008 and May 2011, with the last follow-up assessment in February 2013. Participating children were all referred to a university research clinic for treatment by a health or educational professional. The inclusion criteria were (a) the child must (i) be aged 7–12 years; (ii) have a current primary diagnosis of a DSM-IV anxiety disorder (if speciﬁc phobia, this must be comorbid with another anxiety disorder); and (b) the child’s mother must (i) be the primary carer; and (ii) meet criteria for a current DSM-IV anxiety disorder. The exclusion criteria were (i) signiﬁcant physical or intellectual impairment (including autistic spectrum disorders) in the child or mother; (ii) current prescription of psychotropic medication for the child or mother that had not been at a stable dose for at least one month and without agreement to maintain that dose throughout the study; and (iii) the child had previously received six or more sessions of CCBT for an anxiety disorder. Participating children and mothers were given a complete description of the study orally and in writing before giving written informed assent/consent. A total of 676 children were assessed for eligibility; 435 families did not meet the inclusion criteria and 30 declined to participate (see Figure 1). Introduction d d Anxiety disorders are among the most common psychological disorders in childhood affecting 2.6%– 5.2% of children under the age of 12 years (e.g. Costello, Egger, & Angold, 2004). They negatively affect children’s functioning (Essau, Conradt, & Petermann, 2000), raise the risk for disorders in adolescence and adulthood (Woodward & Fergusson, 2001), and carry a substantial health and social cost (Bodden, Dirksen, & Bogels, 2008). There is consid- erable evidence for the efﬁcacy of cognitive behaviour therapy (CBT) for these disorders, with 59% of anx- ious children no longer meeting criteria for an anxiety disorder following CBT (James, James, Cowdrey, doi:10.1111/jcpp.13089 63 Child and maternal anxiety disorder parental anxiety. The question therefore remains open whether successful treatment of parental anx- iety might beneﬁt child outcome. in this ﬁeld should include such spillover effects to the rest of the family as well as consider cost and effectiveness over a longer time horizon (Tubeuf & Guthmuller, 2017). Further, few evaluations to date have formally aggregated QALYs as a means of capturing spillover effects. The current study there- fore tested the value for money of the two supple- mentary interventions, considering spillover effects, societal impacts and including a one-year follow-up. It is possible that targeting parent–child interac- tions that are likely to interfere with the child’s treatment – that is reducing parent cognitions and behaviours that limit the child’s autonomy and promote the child’s focus on threat (Creswell, Mur- ray, Stacey, & Cooper, 2011; Hudson & Rapee, 2004) – may be of beneﬁt for anxious children in the context of parental anxiety disorder (Settipani, O’Neil, Podell, Beidas, & Kendall, 2013). Indeed, parental cognitions characterised by expectations that the child will be frightened and feel out of control in the face of a challenge and behaviours characterised by intrusiveness and modelling of fear have been found to occur more commonly in anxious compared to nonanxious parents of children with anxiety disorders (Creswell, Apetroaia, Murray, & Cooper, 2013; Moore, Whaley, & Sigman, 2004). © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. J Child Psychol Psychiatr 2020; 61(1): 62–76 J Child Psychol Psychiatr 2020; 61(1): 62–76 Cathy Creswell et al. J Child Psychol Psychiatr 2020; 61(1): 62–76 Assessed for eligibility (n = 676) Randomised (n = 211) CCBT+Con (n = 71) CCBT+MCBT (n = 69) CCBT+MCI (n = 71) Assessment 1B (n = 64) Assessment 1B (n = 64 ) Assessment 1B (n = 70) Not assessed (n = 7) Reasons: • Mother in other therapy (n = 1) • Mother –did not attend (n = 5) • Withdrew- Child improved (n = 1) Not assessed (n = 5) Reasons: • Mother did not attend (n = 5) Not assessed (n = 1) Reasons: • Withdrew -Child improved (n = 1) Not assessed (n = 8) Reasons: • Did not attend (n = 5) • Treatment complete but declined assessment (n = 3) Not assessed (n = 5) Reasons: • Did not attend (n = 1) • Treatment complete but declined assessment (n = 4) Not assessed (n = 8) Reasons: • Did not attend (n = 5) • Treatment complete but declined assessment (n = 2)* • Exclude – Mother serious mental health concerns (n = 1) Total Excluded (n = 465) Not meeting inclusion criteria (n = 435) Reasons: • Mother not anxious (n = 311) • Mother other diagnosis (n = 7) • Mother not primary care giver (n = 2) • Child outside age range (n = 3) • Child Autistic Spectrum Disorder (n = 8) • Child Attention/Behavior Disorder Primary (n = 38) • Child Mood Disorder Primary (n = 6) • Child PTSD primary (n = 6) • Child OCD Primary (n = 13) • Child no Anxiety Disorder (n = 24) • Child-other (n = 17) • No consent to participate (n = 30) Assessment 2 (n = 56, 78.9% of 71) Assessment 2 (n = 59, 85.5% 0f 69) Assessment 2 (n = 62, 87% of 71) PRIMARY OUTCOME Assessment 3 (n = 49) 69% Assessment 3 (n = 52) 75% Assessment 3 (n = 51) 72% Assessment 4 (n = 43) 61% NB includes (n = 2) who did not complete A3 Assessment 4 (n = 48) 70% NB includes (n = 3) who did not complete A3 Assessment 4 (n = 46) 65% NB includes (n = 4) who did not complete A3 Not assessed (n = 11) Reasons: • Did not attend (n = 5) • Withdrew- left country (n = 2) • Declined A3 but completed A4 (n = 4) Not assessed (n =7) Reasons: • Did not attend (n = 4) • Declined A3 but completed A4 (n = 3) Not assessed (n =7) Reasons: • Did not attend (n = 5) • Declined A3 but completed A4 (n = 2) Not assessed (n = 8) Reasons: • Did not attend (n = 7) • Lost contact (n = 1) Not assessed (n = 7) Reasons: • Did not attend (n = 6) • Left country (n = 1) Not assessed (n = 9) Reasons: • Did not attend (n = 6) • Withdrew- lost contact (n = 3) CCBT+Con CCBT+MCBT CCBT+MCI Figure 1 Consort diagram. Procedure The study was approved by the Berkshire Research Ethics Committee (07/H0505/156) and the University of Reading © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child d Ad l t M t l H lth © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. 64 Measures Structured diagnostic interviews with children and parents. Children were assigned diagnoses on the basis of the Anxiety Disorders Interview Schedule for DSM-IV for Chil- dren – Child and Parent Versions (ADIS-C/P; Silverman & Albano, 1996). The presence or absence of a current maternal anxiety disorder was assigned on the basis of the Anxiety Disorders Interview Schedule for DSM-IV (ADIS-IV; DiNardo, Barlow, & Brown, 1994). For the ADIS-C/P overall diagnoses and clinical severity ratings (CSRs)were assigned if the child met diagnostic criteria on the basis of either child or parent report. Those with a CSR of 4 or more (at least moderate psychopathol- ogy) were considered to meet diagnostic criteria. Assessors were thoroughly trained to ensure high levels of reliability (presence/ absence of child diagnosis j = .98 (child report), .98 (mother report); CSR intraclass correlation = .99 (child report), .99 (mother report); presence/absence of maternal diagnosis j = .97; CSR intraclass correlation = .99). The primary outcome derived was absence of the child’s primary (most impairing) anxiety disorder; secondary outcomes derived from the ADIS-c/ p were absence of all anxiety disorder diagnoses and CSR of the primary anxiety disorder, as prespeciﬁed in the trial protocol and statistical analysis plan (see Appendices S1 and S2). Randomisation was performed externally at the Centre for Statistics in Medicine (University of Oxford) on receipt of anonymised participant information by fax. Patients were randomised with a 1:1:1 ratio, with minimisation for child age and gender, type of child anxiety disorder, and baseline severity of both child and maternal primary anxiety disorder. These minimisation variables were selected as they have been most frequently (albeit not consistently) identiﬁed as predictors of response to treatment for child anxiety disorders (Ginsburg et al., 2011; Hudson et al., 2015). The minimisation algorithm included a 20% random element. Assessments of maternal anxiety disorder and parenting were made before and immediately following the interven- tions. Assessments of child anxiety disorder status and severity were conducted before and following treatment, as well as at six and 12 months following treatment. Different research staff assessed the mother and the child. Assessments of maternal and child health-related quality of life were made on ﬁve occasions: before treatment (Assess- ment 1), mid-way through treatment (1B), immediately post-treatment (2), and at the six (3)- and 12-month (4) follow-up. Data on the use of other health and social care resources, as well as non-NHS cost-generating (e.g. J Child Psychol Psychiatr 2020; 61(1): 62–76 CCBT+Con, child-focused cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + CBT to target maternal anxiety disorder; CCBT+MCI, CCBT + intervention to target the mother–child interaction [Colour ﬁgure can be viewed at wileyonlinelibrary.com] 64 Cathy Creswell et al. J Child Psychol Psychiatr 2020; 61(1): 62–76 Assessed for eligibility (n = 676) CCBT+Con (n = 71) CCBT+MCBT (n = 69) Assessment 1B (n = 64) Assessment 1B (n = 70) Assessment 1B (n = 64 ) Assessment 2 (n = 56, 78.9% of 71) Assessment 2 (n = 62, 87% of 71) Assessment 2 (n = 59, 85.5% 0f 69) PRIMARY OUTCOME Assessment 3 (n = 51) 72% Assessment 3 (n = 49) 69% Assessment 3 (n = 52) 75% Assessment 4 (n = 43) 61% NB includes (n = 2) who did not complete A3 Assessment 4 (n = 46) 65% NB includes (n = 4) who did not complete A3 Assessment 4 (n = 48) 70% NB includes (n = 3) who did not complete A3 ( ) did not complete A3 did not complete A3 Figure 1 Consort diagram. CCBT+Con, child-focused cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + CBT to target maternal anxiety disorder; CCBT+MCI, CCBT + intervention to target the mother–child interaction [Colour ﬁgure can be viewed at wileyonlinelibrary.com] © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. 65 Child and maternal anxiety disorder doi:10.1111/jcpp.13089 Research Ethics Committee (07/48). Participants were ran- domised to one of three treatment arms: (a) child cognitive behaviour therapy plus control interventions to balance for therapist time (CCBT+Con) (see below); (b) CCBT plus cognitive behaviour therapy for maternal anxiety disorder (CCBT+MCBT); or (c) CCBT plus treatment focused on the mother–child interaction (CCBT+MCI). Each of the three arms included nonspeciﬁc therapeutic interventions to ensure treat- ment arms were balanced for therapist contact with both children and mothers (see Table 1). Measures Given that mothers presented with a range of anxiety disorders and given evidence for the efﬁcacy of transdiagnostic CBT treatments for anxiety disorders (Newby, McKinnon, Kuyken, Gilbody, & Dalgleish, 2015), we followed a manu- alised transdiagnostic treatment for adult disorders which aimed to reverse the putative cognitive behavioural maintain- ing mechanisms identiﬁed through individual formulation This manual has not previously been systematically evaluated; however, promising results were obtained in a case series evaluation (McManus, Clark, Muse, & Shafran, 2015). Observers who were blind to treatment arm coded parental behaviours on scales developed by Murray et al. (2012) and adapted by Creswell et al. (2013) to be suitable for children aged 7–12 years. Ratings were given for each minute of the interaction on 5-point scales (1 = none, 5 = pervasive/strong). Since interactions varied in duration, total scores across tasks reﬂected the sum of the mean scores for each task. The following behaviours were considered: maternal expressed anxiety, overprotection, intrusiveness and positivity (warmth and encouragement). For each coder, in each task, a second coder independently scored a random sample of 25 videotapes. Intraclass correlations showed acceptable agreement across all indices (range .60–1.00; mean: .86). In the treatment arms that did not involve MCBT, mothers received nondirective counselling (NDC), a supportive individ- ual intervention that was not focussed speciﬁcally on reducing symptoms of anxiety. NDC was provided by one of four qualiﬁed counsellors (supervised by a highly experienced counsellor/psychotherapist) following the manual of Borkovec and Costello (1993). As shown in Appendix S3, the MCBT and NDC were clearly distinct. MCBT and NDC were delivered ﬁrst, before the delivery of CCBT (see Table 1). Maternal expectations were assessed before initiating the challenge tasks (Creswell et al., 2013). Immediately after receiving the instructions for each task, mothers were taken to a separate room and asked to provide ratings regarding (a) how their child would feel about doing the task (0 = not scared at all, 10 = extremely scared); and (b) how much their child could do about how the task went (0 = nothing at all, 10 = a lot). Ratings were combined across the three tasks to represent their expectations across a range of challenge contexts. Mother–Child Interaction treatment (MCI). Measures The MCI intervention consisted of 10 sessions delivered over 8 weeks by one of ﬁve qualiﬁed clinical psychologists or cognitive behaviour therapists (supervised by an experienced clinical psychologist): eight sessions were with the mother alone and two were with the mother and child together. This was a novel intervention designed to target potentially anxiogenic features of the mother–child relationship. Speciﬁcally, it aimed to enhance maternalautonomy promoting cognitions(suchasconﬁdencein the child’s ability to face challenge) and behaviours, and reduce potentially anxiogenic behaviours. This was achieved through a combination of speciﬁc strategies from existing family interven- tions for childhood anxiety (Lyneham et al., 2003) with the addition of video-feedback techniques developed and piloted by the trial investigators. The two joint mother and child sessions involved the mother and child completing structured tasks that were video-recorded for reference in later sessions. Health-related quality of life. Mother and child health- related quality of life (HRQoL) was measured using responses to the EuroQol EQ-5D-3L (Dolan, Gudex, Kind, & Williams, 1995) and EQ-5D-Youth version (Wille et al., 2010) instruments, respectively. Responses for mother and child at each time point were converted into utility weights using the UK population tariff (Dolan et al., 1995). Total quality-adjusted life years (QALYs) were calculated for each mother and child, using the area under the curve approach after linear interpolation between the time points. Mother–child dyad QALYs were obtained by additively combining individual mother and child QALYs (Weinstein et al., 2009). A broad, societal perspective (including parental productivity and school impacts) was adopted in assessing resource use and costs. In addition to the intervention costs (captured through therapists’ logs completed at each therapy session), values were attached for measured school absence, time off work, and lost leisure time (for mothers), use of non-NHS services (e.g. educa- tional services) and personal costs of medications, all of which was captured through parent diaries. An NHS perspective was also adopted as per NICE guidance (NICE, 2013). To balance therapist contact, sessions that focused on the promotion of a healthy lifestyle (Healthy Living Control, HLC) were delivered in the treatment arms that did not receive the MCI intervention. This intervention was principally concerned with family diet and exercise based on existing interventions applied within school settings (BDA, 2003) and was delivered by one of eight therapists (under supervision of an experienced clinical psychologist). MCI/HLC was delivered in parallel with CCBT. Measures Cathy Creswell et al. 66 J Child Psychol Psychiatr 2020; 61(1): 62–76 CBT. Treatment was delivered by one of seven qualiﬁed clinical psychologists or cognitive behaviour therapists, following a manual based on the ‘Cool Kids’ (Lyneham, Abbott, Wignall, & Rapee, 2003) and ‘Coping Cat’ (Kendall & Hedtke, 2006) programmes (both of which have established efﬁcacy; Hudson et al., 2009; Walkup et al., 2008). While these manuals include more than eight treatment sessions, there is evidence of good outcomes from similar CBT content for child anxiety disorders delivered in eight hours or less (e.g. Gallagher, Rabian, & McCloskey, 2004; Ginsburg & Drake, 2002). CCBT was supervised by a highly experienced, clinical psychologist and accredited cognitive behaviour therapist. Treatment focused on helping children to identify and challenge negative thinking styles, gradually increasing exposure to feared stimuli and developing problem-solving skills. Mothers were included at the beginning and end of each session to share progress and feedback. As described in Appendix S3, CCBT treatment adherence was equivalent across treatment arms. subscale of the Strengths and Difﬁculties Questionnaire (child, parent, teacher report) and a teacher report measure of Child Adjustment to School. Descriptions of measures and outcomes are provided in Table S1. Assessment of parenting and parental expecta- tions. In order to establish whether the intervention that targeted mother–child interactions did successfully alter mater- nal responses, we conducted laboratory observations under conditions of mild social, performance and physical challenge (Creswell et al., 2013). The social threat task involved the child preparing anddelivering a speech, withtheir mother’s support, to a research assistant with a hand-held video camera. The perfor- mance task involved the child attempting difﬁcult tangram puzzles (following the procedure of Hudson & Rapee, 2001). The physicalthreat taskrequiredchildrentoinvestigatethecontentof four chambers within a mysterious ‘black box’. To account for prior experience, the assessment was modiﬁed at the post- treatment assessment point; in the social stress task, the child was required to present to a panel rather than a single research assistant, the tangram puzzles were more difﬁcult, and the black box was accompanied by sound effects (i.e. rustling/scratching). CBT for maternal anxiety disorder (MCBT). MCBT consisted of eight one-hour weekly sessions delivered by one of ﬁve qualiﬁed clinical psychologists or cognitive behaviour therapists (all supervised by a highly experienced, clinical psychologist and accredited cognitive behaviour therapist) over 8 weeks. Measures As shown in Appendix S3, MCI and HLC were clearly distinct. Measures educa- tional) services and days off school or work, were obtained from parent report. All assessors were blind to treatment allocation. The trial was preregistered as follows https://doi. org/10.1186/isrctn19762288. The protocol is provided in Appendix S1. Clinical Global Impressions: Improvement (CGI- I). Overall improvement in child anxiety was assessed using the Clinical Global Impression – Improvement scale (CGI-I), a seven-point scale from 1 = very much improved to 7 = very much worse; scores of 1 and 2 are accepted to represent treatment success (Guy, 1976). Overall mean inter-rater reli- ability for the assessment team was high (ICC = .96). Symptoms of anxiety and comorbid difﬁculties were also assessed as secondary outcomes using the Spence Child Anxiety Scale (child, parent, teacher report), Child Anxiety Impact Scale (child, parent report), Short Mood and Feelings Questionnaire (child, parent report), conduct problems Table 1 Overview of design Table 1 Overview of design CCBT+MCBT CCBT+MCI CCBT+Con Assessment 1 Pretreatment Diagnostic assessment (mother and child) + laboratory observation of mother–child interaction Treatment 1 (number of sessions) MCBT (8) NDC (2) NDC (8) Assessment 1B Mid-treatment Diagnostic assessment (mother and child) Treatment 2 (number of sessions) CCBT (8) + HLC (Mother:2; Child + mother: 2) CCBT (8) + MCI (Mother:8; Child + Mother: 2) CCBT (8) + HLC (Mother:2; Child + mother: 2) Assessment 2 Post-treatment Diagnostic assessment (mother and child) + laboratory observation of mother–child interaction Assessment 3 6 months post-treatment Diagnostic assessment (child) Assessment 4 12 months post-treatment Diagnostic assessment (child) Total therapy sessions Mother: 10 Child: 2 Child + Mother: 2 Mother: 10 Child: 2 Child + Mother: 2 Mother: 10 Child: 2 Child + Mother: 2 CCBT, child-focused cognitive behaviour therapy; CCBT-Con, child-focused cognitive behaviour therapy plus nonspeciﬁc control interventions; MCBT, cognitive behaviour therapy targeting maternal anxiety diagnoses; NDC, nondirective counselling for mothers; HLC, Healthy Living Control; MCI, mother–child interaction focussed treatment. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. CCBT, child-focused cognitive behaviour therapy; CCBT-Con, child-focused cognitive behaviour therapy plus nonspeciﬁc control interventions; MCBT, cognitive behaviour therapy targeting maternal anxiety diagnoses; NDC, nondirective counselling for mothers; HLC, Healthy Living Control; MCI, mother–child interaction focussed treatment. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health Manipulation checks We ﬁrst examined the extent to which delivery of the adjunct treatments was associated with change in the putative maintenance mechanisms that were targeted; maternal anxiety disorder status and change in maternal cognitions and behaviours. MCBT was successful in changing maternal anxiety disorder status. At Assessment 1B (i.e. after com- pleting MCBT), 58.5% (n = 38) of mothers in the MCBT arm had recovered from their primary diag- nosis compared to 36.5% (n = 23) in the CCBT+Con arm (who received NDC) (adj RR: 1.63 (95% CI: 1.13, 2.36), p = .009). The recovery rate in the MCI arm was not different from that in the CCBT+Con arm (who both received NDC) (adj RR: 1.22 (95% CI: 0.83, 1.81), p = .31). Current best-practice methods were adhered to for conduct- ing and reporting economic evaluations alongside trials (Drummond, Sculpher, Claxton, Stoddart, & Torrance, 2015; Husereau et al., 2013; NICE, 2013; Petrou & Gray, 2011). A broad societal perspective cost-utility analysis framework was adopted for the base case to assess the cost-effectiveness of (a) CCBT+MCBT compared to CCBT; and (b) CCBT+MCI compared with CCBT. Costs of the nonspeciﬁc treatments (NDC/HLC) were excluded in the base-case analyses. This was in recogni- tion of the fact that the main aim of any economic evaluation is to estimate the likely cost-effectiveness of a change in clinical practice in real-world settings, rather than in ideal controlled conditions (as in efﬁcacy trials). Those costs were instead included in some of the sensitivity analyses. Costs were expressed in pounds sterling (£) at 2011/2012 prices. Given the short time-frame of the trial and follow-up, discounting was not applied to costs or effects. The base-case analyses were performed on an ITT basis. Mean imputation methods were used for missing resource use and health outcomes deemed highly deterministic (e.g. face-to-face therapists con- tact), and multiple imputation for other resources (e.g. use of medications), under the assumption of missing at random (Faria, Gomes, Epstein, & White, 2014). For each mother and child participant, all components of treatment costs, stratiﬁed by category of resource use and other broader societal costs (educational services, travel costs, time off school/work) were calculated by multiplying units of resource use by their unit costs (see Table S2). These values were then summed to obtain a total cost for each child/mother. Treatment Child Cognitive Behaviour Therapy (CCBT). All children received eight-weekly one-hour sessions of individual Child Cognitive Behaviour Therapy (CCBT). All children received eight-weekly one-hour sessions of individual © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child d Ad l t M t l H lth doi:10.1111/jcpp.13089 67 Child and maternal anxiety disorder CCBT) and are detailed in Table S3. All analyses were conducted using Stata Software: Release 12, StataCorp 2011. Results Baseline characteristics were well balanced across treatment arms (see Table 2). Numbers of available participants for each treatment arm are shown in Figure 1. No important harms due to any of the interventions or trial procedures were reported dur- ing the trial. Analysis A comprehensive statistical analysis plan was prepared before embarking on the analysis (Appendix S4). ( ) In line with our hypotheses and power analysis, we set out to make two sets of pairwise comparisons (MCBT+CCBT vs. CCBT+Con; and MCI+CCBT vs. CCBT+Con). Dichotomous outcome variables (e.g. diagnostic outcomes) were analysed using a modiﬁed Poisson regression approach with robust error variance adjusting for the minimisation factors [child age, child gender, type of child anxiety disorder (GAD, social phobia, SAD, other), baseline severity of the child’s and the mother’s primary anxiety disorder (ADIS Clinician Severity Rating)]. This approach allows for adjusted relative risks to be presented (Zou, 2004). All analyses are presented based on the intention-to-treat (ITT) population. Sensitivity analyses of the primary endpoints included: (a) no adjustment for minimisa- tion criteria, (b) per-protocol population (i.e. those participants who had received at least half of the treatment sessions and had data for the post-treatment assessments) and (c) multiple imputation analysis (see Appendix S2). Primary outcome results did not change based on the prespeciﬁed sensitivity analyses, nor when best- and worst-case scenarios were used in the context of the multiple imputation analysis. Continuous outcomes were modelled using linear regression, adjusted for baseline scores and minimisation factors. No adjustments were made to the conﬁdence intervals or p-values to account for multiplicity. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Manipulation checks Effects were identiﬁed and measured using child/mother QALYs, derived from the EQ- 5D-Y and EQ-5D-3L child and mother report, respectively. Incremental mean costs and effects, and the associated 95% CIs, were estimated comparing the two intervention groups for each of the two comparisons (CCBT+MCBT vs. CCBT; and CCBT+MCI vs. CCBT). Incremental cost-effectiveness ratios (ICERs) were estimated and reported where relevant. Uncer- tainty in the cost-effectiveness results was analysed using cost-effectiveness acceptability curves (CEACs) over a range of potential threshold values that the health system and the wider society might be willing to pay for an additional QALY gained (Fenwick, Marshall, Levy, & Nichol, 2006). Extensive sensitivity analyses were performed for each of the two base- case comparisons (CCBT+MCBT vs. CCBT; and CCBT+MCI vs. At assessment 2 (i.e. after the children had received CCBT), many of the mothers in the CCBT+Con arm had recovered from their primary disorder and there was, therefore, no longer a superiority in this respect for the CCBT+MCBT arm (CCBT+MCBT- CCBT+Con adj RR: 1.23 (95% CI: 0.90–1.68, p = .21; CCBT+MCI- CCBT+Con adj RR: 1.27 (95% CI: 0.93–1.74, p = .13). Change in observed overprotection (across all three mother–child tasks) at the end of all treatment (Assessment 2) was greater in the CCBT+MCI arm compared to CCBT+Con (adjusted mean difference in change from baseline, (0.03 (0.06, 0.004), p = .03). There was no difference in change in observed overprotection between the CCBT+MCBT and CCBT+Con arms (0.02 (0.05, 0.009), p = .17). There were no signiﬁcant differences between CCBT+Con and either CCBT+MCBT or CCBT+MCI in terms of change in observed intrusive- ness, positive behaviours or maternal expressed anxiety (See Table S4 and S5). Differences between the CCBT+Con and CCBT+MCI arms were also found on change in measures of maternal expectations of how scared the child would be (0.67 (1.26, 0.07); p = .03) and how in control the child would feel (0.53 (0.01, 1.05); p = .05), with mothers in the CCBT+MCI arm predicting that their child would be less scared and more in control. While a similar pattern was found, the corresponding differences between the CCBT+Con and CCBT+MCBT arms did not quite J Child Psychol Psychiatr 2020; 61(1): 62–76 68 Cathy Creswell et al. Child outcomes Clinical Global Impressions of Improvement (CGI- I). As shown in Table 4, at the post-treatment assessment (Assessment 2), there were no signiﬁ- cant differences between the groups in terms of rates of ‘much’ or ‘very much’ improvement (CCBT+MCBT vs. CCBT+Con adj RR: 1.25 (95% CI: 0.99, 1.57), p = .06; CCBT+MCI vs. CCBT+Con adj RR: 1.18 (95% CI: 0.93, 1.50), p = .17). Differences were even smaller at the six month (Assessment 3) (CCBT+MCBT vs. CCBT+Con adj RR: 0.97 (95% CI: 0.79, 1.19), p = .77; CCBT+MCI vs. CCBT+Con adj RR: 1.16 (95% CI: 0.94, 1.33), p = .22) and 12 months post-treatment assessment (Assessment 4; CCBT+MCBT vs. CCBT+Con adj RR: 1.02 (95% CI: 0.82, 1.27), p = .83; CCBT+Con vs. CCBT+MCI adj RR: 1.05 (95% CI: 0.85, 1.30), p = .63). Recovery from anxiety diagnoses. Rates of recov- ery from the primary diagnosis and all anxiety diagnoses are shown in Table 3, and statistical analyses are summarised in Table 4. There were no signiﬁcant differences between either the CCBT+Con and the CCBT+MCBT (adj RR: 1.22 (95% CI: 0.88, 1.67), p = .23) or the CCBT+MCI arm (adj RR: 1.21 (95% CI: 0.88, 1.65), p = .24) on the primary out- come, that is recovery from primary anxiety disorder at the post-treatment assessment (Assessment 2). Similarly, there were no signiﬁcant post-treatment differences on recovery from all anxiety disorders between the CCBT+Con and the CCBT+MCBT (1.06 (95% CI: 0.63, 1.78), p = .82) or CCBT+MCI arms (adj RR: 1.48 (95% CI: 0.92, 2.37). At six months post-treatment (Assessment 3), again there was no difference between the CCBT+Con arm and either the CCBT+MCBT (Free of primary adj RR 1.09 (95% CI: 0.81, 1.46), p = .57; Free of all adj RR 1.04 (95% CI: 0.70, 1.53), p = .86) or the CCBT+MCI arm [Free of primary adj RR: 1.26 (95% CI: 0.97, 1.64), p = .08; Free of all adj RR: 1.04 (95% CI: 0.71, 1.55)]. Similarly, at 12 months post-treatment, there was Manipulation checks aChildren with speciﬁc phobia and selective mutism were only included if also met diagnostic criteria for another comorbid anxiety disorder. disorder. bThese mothers had a current anxiety disorder but MDD had the highest severity. ese mothers had a current anxiety disorder but MDD h no difference between the CCBT+Con arm and either the CCBT+MCBT (Free of primary adj RR: 0.85 (95% CI: 0.65, 1.12), p = .26; Free of all adj RR: 0.89 (95% CI: 0.61, 1.31), p = .57) or the CCBT+MCI arm (Free of primary adj RR: 1.04 (95% CI: 0.82, 1.30), p = .77; Free of all adj RR: 1.01 (95% CI: 0.70, 1.45), p = .97). reach the conventional level of statistical signiﬁ- cance (Scared: 0.53 (1.12, 0.07), p = .08; Con- trol: 0.50 (0.03, 1.02), p = .06). A summary of the results of statistical analyses for maternal anxiety, behaviours and cognitions is given in Table S5. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health Manipulation checks Table 2 Baseline characteristics by treatment arm Table 2 Baseline characteristics by treatment arm Table 2 Baseline characteristics by treatment arm CCBT+Con N = 71 CCBT+MCBT N = 69 CCBT+MCI N = 71 Child age (mean, SD) 10.28 (1.49) 10.29 (1.56) 10.10 (1.68) Ethnicity n (%) White British 67 (94.37) 58 (84.06) 55 (77.46) Sex n (%) Male 34 (47.89) 35 (50.72) 32 (45.07) Parent marital status n (%) married/living with partner 39 (54.93) 50 (72.46) 46 (64.79) Family socioeconomic statusn (%) ‘higher’/‘professional’ 29 (40.85) 39 (56.52) 38 (53.52) Child: ADIS-c/p primary anxiety disorder Separation anxiety disorder n (%) 19 (26.76) 16 (23.19) 21 (29.58) Social anxiety disorder n (%) 16 (22.54) 18 (26.09) 14 (19.72) Generalised anxiety disorder n (%) 22 (31.00) 20 (28.96) 24 (33.80) Speciﬁc phobiaa n (%) 8 (11.27) 11 (15.94) 5 (7.04) Panic disorder Agoraphobia n (%) 4 (5.63) 2 (2.90) 4 (5.63) Selective mutisma n (%) 0 0 1 (1.41) Anxiety disorder not otherwise speciﬁed n (%) 2 (2.82) 2 (2.90) 2 (2.82) ADIS-c/p primary diagnosis severity (CSR)Mean (SD) 5.65 (0.80) 5.71 (0.79) 5.69 (0.79) Mother: ADIS primary disorder Social anxiety disorder n (%) 9 (12.70) 14 (20.3) 11 (15.49) Generalised anxiety disorder n (%) 37 (52.10) 35 (50.7) 40 (56.34) Speciﬁc phobiaa n (%) 12 (16.90) 17 (24.64) 9 (12.68) Panic disorder Agoraphobia n (%) 3 (4.2) 1 (1.4) 3 (4.23) Other (ADNOS, hypochondriasis, PTSD, OCD) 5 (7.04) 2 (2.9) 8 (11.27) MDDb n (%) 5 (7) 0 0 ADIS primary diagnosis severity (CSR)Mean (SD) 5.24 (1.01) 5.17 (0.89) 5.31 (0.98) ADNOS, anxiety disorder not otherwise speciﬁed; CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CSR, clinical severity rating; MCBT, CCBT + maternal cognitive behaviour therapy; MCI, CCBT + mother–child interaction treatment; MDD, major depressive disorder; OCD, obsessive-compulsive disorder; PTSD, post-traumatic stress disorder. aChildren with speciﬁc phobia and selective mutism were only included if also met diagnostic criteria for another comorbid anxiety disorder. bThese mothers had a current anxiety disorder but MDD had the highest severity. CCBT+Con N = 71 CCBT+MCBT N = 69 CCBT+MCI N = 71 ADNOS, anxiety disorder not otherwise speciﬁed; CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CSR, clinical severity rating; MCBT, CCBT + maternal cognitive behaviour therapy; MCI, CCBT + mother–child interaction treatment; MDD, major depressive disorder; OCD, obsessive-compulsive disorder; PTSD, post-traumatic stress disorder. Economic evaluation Missing resource use data were prevalent (Table S6). Mother and child combined HRQoL results are presented in Table S7 (unadjusted differences) and Tables S8 and S9 (differences adjusted for child’s and mother’s baseline utilities). At 12 months, in the © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. doi:10.1111/jcpp.13089 Child and maternal anxiety disorder 69 Table 3 Primary and secondary categorical (child) outcomes Initial assessment (1) Post-treatment assessment (2) 6-month post-treatment assessment (3) 12-month post-treatment assessment (4) n (%) free of primary diagnosis CCBT+ Con 0 27 (48.21) 29 (59.18) 31 (72.09) CCBT+MCBT 0 35 (58.33) 34 (64.15) 30 (60.00) CCBT+MCI 0 37 (59.68) 38 (74.51) 34 (73.91) n (%) free of all anxiety diagnoses CCBT_+Con 0 16 (28.57) 23 (46.94) 23 (53.49) CCBT+MCBT 0 18 (30.00) 25 (47.17) 23 (46.00) CCBT+MCI 0 25 (40.32) 24 (47.06) 24 (52.17) n (%) CGI-I ‘much’/’very much’ improved CCBT +Con 0 36 (64.29) 39 (79.59) 33 (76.74) CCBT+MCBT 0 48 (80.00) 41 (77.36) 39 (78.00) CCBT+MCI 0 47 (75.81) 45 (88.24) 37 (80.43) See Table S1 for descriptive statistics for secondary continuous outcomes. CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cognitive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; CGI-I Clinical Global Impression – Improvement. Table 3 Primary and secondary categorical (child) outcomes p y CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cognitive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; CGI-I Clinical Global Impression – Improvement. base-case analysis, the CCBT+Con arm conferred slightly higher QALYs than the CCBT+MCBT arm (CCBT+MCBT vs. CCBT+Con: 0.04 (95% CI: 0.12, 0.04), p = .29), whilst the CCBT+MCI arm conferred slightly higher QALYs than the CCBT+Con arm (CCBT+MCI vs. CCBT+Con: 0.02 (95% CI: 0.05, 0.09), p = .54). Neither of these differences was statistically signiﬁcant. Mean NHS and societal cost differences for the two comparisons are reported in Tables S10–S12. When a societal perspective was adopted (base-case analysis; Table S11), CCBT+MCBT was £482 more costly than CCBT (95% CI: £827, £1,791); this was not signiﬁcant (p = .47). However, from an NHS perspective, costs for CCBT+MCBT were £797 signiﬁcantly greater than CCBT (95% CI: £603, £991; p < .001). © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Economic evaluation For CCBT+MCI, when a societal perspective was adopted [base-case analysis; Table S10), CCBT+MCI was £154 more costly than CCBT (95% CI: £1,239, £1,547)], again an insigniﬁcant difference (p = .83). The NHS perspective costs for CCBT+MCI, on the other hand, were £808 greater than for CCBT (95% CI: £610, £1,006), a statistically signiﬁcant differ- ence (p < .001). probability that the CCBT+MCI arm was cost-effec- tive compared to CCBT (Figure 2, Panel 2, and Table S9 base-case line) was between 60% and 64% based on UK National Institute for Health and Care Excellence thresholds for accepted levels of willingness to pay for an additional QALY (usually between £20,000 and £30,000). Sensitivity analyses reinforced the ﬁndings of the base-case analyses, with the CCBT+MCBT arm not being cost-effective compared with the CCBT arm across the majority of sensitivity analyses (Table S8) [other than the sce- nario where the CCBT+MCBT arm was less costly but also less effective (SA 4 and SA 10)]. For the comparison between the CCBT+MCI arm and CCBT (Table S9), the CCBT+MCI arm may be a cost- effective alternative to CCBT from a societal perspec- tive (insigniﬁcantly higher costs or less costly, insigniﬁcantly higher QALYs), as reﬂected by the probability of cost-effectiveness being greater than 60% overall. Discussion CCBT+MCBT 0.85 0.65–1.12 .257 CCBT+MCI 1.04 0.82–1.30 .766 Free from all anxiety diagnoses at post-treatment assessment (2) Treatment CCBT+Con Ref. CCBT+MCBT 1.06 0.63–1.78 .816 CCBT+MCI 1.48 0.92–2.37 .102 Free from all anxiety diagnoses at 6-month post-treatment assessment (3) Treatment CCBT+Con Ref. CCBT+MCBT 1.04 0.70–1.53 .860 CCBT+MCI 1.04 0.71–1.55 .814 Free from all anxiety diagnoses at 12-month post-treatment assessment (4) Treatment CCBT+Con Ref. CCBT+MCBT 0.89 0.61–1.31 .569 CCBT+MCI 1.01 0.70–1.45 .972 CGI-I ‘much’/’very much’ improved at post-treatment assessment (2) Treatment CCBT+Con Ref. CCBT+MCBT 1.26 1.00–1.59 .054 CCBT+MCI 1.20 0.95–1.53 .133 CGI-I ‘much’/’very much’ improved at 6-month post-treatment assessment (3) Treatment CCBT+Con Ref. CCBT+MCBT 0.97 0.79–1.19 .771 CCBT+MCI 1.16 0.94–1.33 .216 CGI-I ‘much’/’very much’ improved at 12-month post- treatment assessment (4) Treatment CCBT+Con Ref. CCBT+MCBT 1.02 0.82–1.27 .834 CCBT+MCI 1.05 0.85–1.30 .628 Adjusted for child age, child gender, type of child anxiety disorder (GAD, social phobia, SAD, other), baseline severity (ADIS-C/P CSR) of the child’s primary anxiety disorder and baseline severity (ADIS-IV mother self-report) of the mother’s primary anxiety disorder. CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cogni- tive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; CGI-I, Clinical Global Impression – Improvement; Ref., reference category. Parameter Adjusted RR 95% CI p- value Free from primary diagnosis at post-treatment assessment (2) Treatment CCBT+Con Ref. CCBT+MCBT 1.18 0.83–1.62 .285 CCBT+MCI 1.22 0.90–1.67 .203 Despite these potential broader economic beneﬁts, it is important to consider the lack of signiﬁcant clinical effects of the two active adjunct interventions (MCI/MCBT). The study was powered to detect a 30% difference in the proportion of recovered children, based on a projected recovery rate from child CBT of 40% (Cobham et al., 1998). While a 30% difference in recovery between CBT plus the adjunct treatments and CBT plus the control interventions might be considered conservative (as smaller differences may still be clinically meaningful), an anticipated success rate of 40% from individual CBT could have been considered optimistic, given ﬁndings from a more recent trial – the ﬁrst to examine treatment of child anxiety disorder in the context of diagnosed parental anxiety disorder – where a recovery rate of only 33% was found (Hudson et al., 2014). © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Discussion Good outcomes were achieved for both children and their mothers across all treatment arms but neither adding treatment of maternal anxiety (MCBT) nor treatment of the mother–child relationship (MCI) to individual child-focused CBT conferred a signiﬁcant beneﬁt to children with anxiety disorders whose mothers also had a current anxiety disorder. Although adding both adjunct treatments achieved higher child recovery rates post-treatment than the CCBT plus control treatment arm (where neither maternal anxiety nor potentially anxiogenic parent- ing received speciﬁc therapeutic attention), the advantages were not statistically signiﬁcant. How- ever, from a broad societal perspective combining mother/child QALYs and costs, including productiv- ity and school impacts, the economic evaluation indicated that CCBT+MCI arm (but not the CCBT+MCBT arm) may be cost-effective compared Key cost drivers in both of these base-case com- parisons were the differential treatment delivery costs in both the CCBT+MBCT and CCBT+MCI arms compared with CCBT only (Tables S11 and S12). While the CCBT+Con arm had higher absences from school and work costs than the other two arms, few differences were statistically signiﬁcant although, notably, the lower costs of mother’s lost days of work in the CCBT+MCI arm than in the CCBT+Con arm were signiﬁcant (Table S12). Taking sampling uncertainty into consideration, the cost-effectiveness acceptability curves (CEACs) for the two base-case analyses showed that, in view of the joint distribution of incremental mean costs and effects, the CCBT+MBCT arm was not likely to be a cost-effective alternative to CCBT (Figure 2, Panel 1, and Table S8, base-case line). The J Child Psychol Psychiatr 2020; 61(1): 62–76 70 Cathy Creswell et al. Table 4 Statistical analyses for child primary and secondary categorical outcomes Table 4 Statistical analyses for child primary and secondary categorical outcomes afﬁrms the importance of including wider family/ society born costs and outcomes in mental health economic evaluations (Creswell et al., 2017; Tilford et al., 2015; Tubeuf & Guthmuller, 2017). Table 4 Statistical analyses for child primary and secondary categorical outcomes Parameter Adjusted RR 95% CI p- value Free from primary diagnosis at post-treatment assessment (2) Treatment CCBT+Con Ref. CCBT+MCBT 1.18 0.83–1.62 .285 CCBT+MCI 1.22 0.90–1.67 .203 Free from primary diagnosis at 6-month post-treatment assessment (3) Treatment CCBT+Con Ref. CCBT+MCBT 1.09 0.81–1.46 .566 CCBT+MCI 1.26 0.97–1.64 .077 Free from primary diagnosis at 12-month post-treatment assessment (4) Treatment CCBT+Con Ref. Discussion However, the CCBT plus control treatment arm in the current study performed considerably better than expected, with 48% of children being free of their primary anxiety disorder and 64% ‘much’ or ‘very much’ improved immediately post-treatment, and over 70% free of their primary diagnosis and ‘much/very much improved’ a year after treatment. Indeed, these suc- cess rates are similar to those found from more intensive (14 session) CCBT for children with anxiety disorders, regardless of parental anxiety disorder status, where the proportions ‘much’/’very much’ improved were 60% at post-treatment (Walkup et al., 2008) and 72% at six-month follow-up (Piacentini et al., 2014), respectively. The unexpectedly high rate of recovery within the CCBT plus control arm in the current study is unlikely to be a function of particular features of our sample, as our study population was comparable to other clinic samples in the literature. There could, however, have been an added value of the nonspeciﬁc interventions, all of which provided some level of parental support. This raises the possibility that the modest outcomes typically found for anxious children in the context of parental anxiety disorder are in fact a result of other associated factors – for example, other stressors experienced by the family (see, e.g. Schleider et al., 2015) – which might have been addressed to some extent in this trial by the generic support received by all mothers. Free from primary diagnosis at 6-month post-treatment assessment (3) Free from primary diagnosis at 12-month post-treatment assessment (4) t CCBT+Con Ref. CCBT+MCBT 0.85 0.65–1.12 .257 CCBT+MCI 1.04 0.82–1.30 .766 Free from all anxiety diagnoses at post-treatment assessment (2) Treatment CCBT+Con Ref. CCBT+MCBT 1.06 0.63–1.78 .816 CCBT+MCI 1.48 0.92–2.37 .102 Free from all anxiety diagnoses at 6-month post-treatment assessment (3) Treatment CCBT+Con Ref. CCBT+MCBT 1.04 0.70–1.53 .860 CCBT+MCI 1.04 0.71–1.55 .814 Free from all anxiety diagnoses at 12-month post-treatment assessment (4) CGI-I ‘much’/’very much’ improved at post-treatment (2) / y p p assessment (2) ent CCBT+Con Ref. CCBT+MCBT 1.26 1.00–1.59 .054 CCBT+MCI 1.20 0.95–1.53 .133 CGI-I ‘much’/’very much’ improved at 6-month post-treatment assessment (3) Treatment CCBT+Con Ref. CCBT+MCBT 0.97 0.79–1.19 .771 CCBT+MCI 1.16 0.94–1.33 .216 CGI-I ‘much’/’very much’ improved at 12-month post- treatment assessment (4) treatment assessment (4) Treatment CCBT+Con Ref. CCBT+MCBT 1.02 0.82–1.27 .834 CCBT+MCI 1.05 0.85–1.30 .628 There could, however, have been an added value of the nonspeciﬁc interventions, all of which provided some level of parental support. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Discussion This raises the possibility that the modest outcomes typically found for anxious children in the context of parental anxiety disorder are in fact a result of other associated factors – for example, other stressors experienced by the family (see, e.g. Schleider et al., 2015) – which might have been addressed to some extent in this trial by the generic support received by all mothers. Adjusted for child age, child gender, type of child anxiety disorder (GAD, social phobia, SAD, other), baseline severity (ADIS-C/P CSR) of the child’s primary anxiety disorder and baseline severity (ADIS-IV mother self-report) of the mother’s primary anxiety disorder. CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cogni- tive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; CGI-I, Clinical Global Impression – Improvement; Ref., reference category. Given the novelty of the adjunctive treatments, the degree to which they were successful in altering their respective targets also needs to be considered. In the one previous study that assessed the impact of adding CBT for parental anxiety disorders to CBT for child anxiety disorders (Hudson et al., 2014), the failure to ﬁnd signiﬁcant differences in child out- comes may have been attributable to the parental CBT not reducing parental anxiety. In the current study, however, compared to nondirective coun- selling, MCBT was associated with a signiﬁcant to CCBT. Notably, the probability of the CCBT+MCI arm being cost-effective lessened when a more restricted NHS perspective was adopted and the gains from reduced school absenteeism and moth- ers’ productivity/employment excluded. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child d Ad l t M t l H lth © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Discussion 72 Table 5 (continued) Adjusteda Mean difference (95% CI) p value Ref 0.12 (2.71, 2.48) .928 1.99 (0.48, 4.46) .113 Ref 1.85 (4.24, 0.55) .130 0.69 (1.61, 3.00) .552 Ref 1.06 (3.07, 0.96) .301 1.04 (3.08, 1.00) .314 Ref 1.61 (3.83, 0.61) .154 0.65 (2.91, 1.61) .570 Ref 0.61 (1.82, 3.04) .619 0.83 (3.11, 1.46) .474 Ref 0.30 (0.26, 0.86) .289 0.22 (0.33, 0.77) .427 Ref 0.09 (0.59, 0.76) .803 0.10 (0.54, 0.75) .748 Ref 0.03 (0.68, 0.62) .925 0.07 (0.60, 0.74) .832 Ref 0.27 (0.65, 1.18) .562 0.21 (0.68, 1.10) .637 Ref 0.04 (0.60, 0.52) .897 0.06 (0.49, 0.61) .836 Ref 0.09 (0.66, 0.49) .763 0.19 (0.77, 0.39) .515 Ref 0.51 (1.05, 0.02) .060 0.64 (1.19, 0.09) .022 Ref 0.16 (0.57, 0.89) .668 0.20 (0.50, 0.89) .575 Ref 0.52 (1.30, 0.27) .190 0.46 (1.24, 0.31) .236 Ref 0.78 (1.98, 0.42) .196 0.26 (1.42, 0.90) .654 Ref 0.18 (1.71, 1.34) .805 0.50 (0.99, 2.00) .490 Ref 1.10 (3.11, 0.90) .275 0.40 (2.37, 1.57) .684 Ref 1.50 (4.65, 1.66) .343 0.03 (2.92, 2.98) .985 Ref 2.52 (0.62, 5.66) .110 2.16 (0.77, 5.09) .139 Treatment arm N Adjusteda Mean change (95% CI) Adjusteda Mean difference (95% CI) p value Depression symptoms (SMFQ-c) – 12- month post-treatment assessment (4) CCBT+Con 28 4.08 (5.95, 2.21) Ref CCBT+MCBT 35 4.20 (5.91, 2.49) 0.12 (2.71, 2.48) .928 CCBT+MCI 37 2.09 (3.72, 0.47) 1.99 (0.48, 4.46) .113 Depression symptoms (SMFQ-p) – mid- treatment assessment (1B) CCBT+Con 40 1.32 (2.97, 0.32) Ref CCBT+MCBT 38 3.17 (4.86, 1.47) 1.85 (4.24, 0.55) .130 CCBT+MCI 44 0.63 (2.20, 0.94) 0.69 (1.61, 3.00) .552 Depression symptoms (SMFQ-p) – post- treatment assessment (2) CCBT+Con 34 4.60 (6.03, 3.18) Ref CCBT+MCBT 38 5.66 (7.03, 4.29) 1.06 (3.07, 0.96) .301 CCBT+MCI 35 5.64 (7.07, 4.22) 1.04 (3.08, 1.00) .314 Depression symptoms (SMFQ-p) – 6- month post-treatment assessment (3) CCBT+Con 36 4.25 (5.82, 2.67) Ref CCBT+MCBT 38 5.86 (7.40, 4.32) 1.61 (3.83, 0.61) .154 CCBT+MCI 36 4.90 (6.49, 3.30) 0.65 (2.91, 1.61) .570 Depression symptoms (SMFQ-p) – 12- month post-treatment assessment (4) CCBT+Con 30 5.14 (6.81, 3.48) Ref CCBT+MCBT 29 4.54 (6.25, 2.82) 0.61 (1.82, 3.04) .619 CCBT+MCI 33 5.97 (7.55, 4.39) 0.83 (3.11, 1.46) .474 Conduct problems (SDQ-c) – mid- treatment assessment (1B) CCBT+Con 54 0.48 (0.88, 0.08) Ref CCBT+MCBT 58 0.18 (0.56, 0.21) 0.30 (0.26, 0.86) .289 CCBT+MCI 65 0.26 (0.62, 0.11) 0.22 (0.33, 0.77) .427 Conduct problems (SDQ-c) – post- treatment assessment (2) CCBT+Con 47 0.61 (1.08, 0.14) Ref CCBT+MCBT 47 0.52 (1.00, 0.05) 0.09 (0.59, 0.76) .803 CCBT+MCI 55 0.50 (0.94, 0.07) 0.10 (0.54, 0.75) .748 Conduct problems (SDQ-c) – 6-month post-treatment assessment (3) CCBT+Con 42 0.88 (1.35, 0.42) Ref CCBT+MCBT 44 0.91 (1.37, 0.46) 0.03 (0.68, 0.62) .925 CCBT+MCI 42 0.81 (1.28, 0.34) 0.07 (0.60, 0.74) .832 Conduct problems (SDQ-c) – 12-month post-treatment assessment (4) CCBT+Con 31 1.21 (1.87, 0.55) Ref CCBT+MCBT 34 0.94 (1.58, 0.30) 0.27 (0.65, 1.18) .562 CCBT+MCI 38 1.00 (1.59, 0.40) 0.21 (0.68, 1.10) .637 Conduct problems (SDQ-p) – mid- treatment assessment (1B) CCBT+Con 42 0.14 (0.54, 0.26) Ref CCBT+MCBT 46 0.18 (0.56, 0.21) 0.04 (0.60, 0.52) .897 CCBT+MCI 50 0.08 (0.45, 0.29) 0.06 (0.49, 0.61) .836 Conduct problems (SDQ-p) – post- treatment assessment (2) CCBT+Con 37 0.65 (1.06, 0.24) Ref CCBT+MCBT 41 0.74 (1.12, 0.35) 0.09 (0.66, 0.49) .763 CCBT+MCI 40 0.84 (1.23, 0.45) 0.19 (0.77, 0.39) .515 Conduct problems (SDQ-p) – 6-month post-treatment assessment (3) CCBT+Con 39 0.47 (0.86, 0.09) Ref CCBT+MCBT 42 0.99 (1.36, 0.62) 0.51 (1.05, 0.02) .060 CCBT+MCI 41 1.11 (1.49, 0.73) 0.64 (1.19, 0.09) .022 Conduct problems (SDQ-p) – 12-month post-treatment assessment (4) CCBT+Con 32 1.04 (1.56, 0.53) Ref CCBT+MCBT 32 0.89 (1.40, 0.38) 0.16 (0.57, 0.89) .668 CCBT+MCI 38 0.85 (1.31, 0.38) 0.20 (0.50, 0.89) .575 Conduct problems (SDQ-t) – post- treatment assessment (2) CCBT+Con 18 0.35 (0.21, 0.91) Ref CCBT+MCBT 22 0.17 (0.68, 0.35) 0.52 (1.30, 0.27) .190 CCBT+MCI 23 0.11 (0.62, 0.39) 0.46 (1.24, 0.31) .236 Conduct problems (SDQ-t) – 6-month post-treatment assessment (3) CCBT+Con 12 0.57 (0.36, 1.50) Ref CCBT+MCBT 18 0.21 (0.98, 0.57) 0.78 (1.98, 0.42) .196 CCBT+MCI 22 0.31 (0.37, 1.00) 0.26 (1.42, 0.90) .654 Conduct problems (SDQ-t) – 12-month post-treatment assessment (4) CCBT+Con 9 0.09 (1.21, 1.03) Ref CCBT+MCBT 11 0.27 (1.30, 0.75) 0.18 (1.71, 1.34) .805 CCBT+MCI 12 0.41 (0.54, 1.36) 0.50 (0.99, 2.00) .490 Child Adjustment to School (CAS-t) – post-treatment assessment (2) CCBT+Con 18 0.86 (2.32, 0.60) Ref CCBT+MCBT 24 1.96 (3.22, 0.70) 1.10 (3.11, 0.90) .275 CCBT+MCI 25 1.26 (2.48, 0.04) 0.40 (2.37, 1.57) .684 Child Adjustment to School (CAS-t) – 6- month post-treatment assessment (3) CCBT+Con 11 1.43 (3.85, 0.99) Ref CCBT+MCBT 17 2.92 (4.91, 0.94) 1.50 (4.65, 1.66) .343 CCBT+MCI 23 1.40 (3.02, 0.23) 0.03 (2.92, 2.98) .985 Child Adjustment to School (CAS-t) – 12-month post-treatment assessment (4) CCBT+Con 9 2.56 (4.76, 0.37) Ref CCBT+MCBT 10 0.04 (2.23, 2.14) 2.52 (0.62, 5.66) .110 CCBT+MCI 12 0.40 (2.29, 1.48) 2.16 (0.77, 5.09) .139 CAIS-c/p, Child Anxiety Impact Scale child/parent report; CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control Treatment arm N Adjusteda Mean chan (95% CI) Depression symptoms (SMFQ-c) – 12- month post-treatment assessment (4) CCBT+Con 28 4.08 (5.95, 2.21) CCBT+MCBT 35 4.20 (5.91, 2.49) CCBT+MCI 37 2.09 (3.72, 0.47) Depression symptoms (SMFQ-p) – mid- treatment assessment (1B) CCBT+Con 40 1.32 (2.97, 0.32) CCBT+MCBT 38 3.17 (4.86, 1.47) CCBT+MCI 44 0.63 (2.20, 0.94) Depression symptoms (SMFQ-p) – post- treatment assessment (2) CCBT+Con 34 4.60 (6.03, 3.18) CCBT+MCBT 38 5.66 (7.03, 4.29) CCBT+MCI 35 5.64 (7.07, 4.22) Depression symptoms (SMFQ-p) – 6- month post-treatment assessment (3) CCBT+Con 36 4.25 (5.82, 2.67) CCBT+MCBT 38 5.86 (7.40, 4.32) CCBT+MCI 36 4.90 (6.49, 3.30) Depression symptoms (SMFQ-p) – 12- month post-treatment assessment (4) CCBT+Con 30 5.14 (6.81, 3.48) CCBT+MCBT 29 4.54 (6.25, 2.82) CCBT+MCI 33 5.97 (7.55, 4.39) Conduct problems (SDQ-c) – mid- treatment assessment (1B) CCBT+Con 54 0.48 (0.88, 0.08) CCBT+MCBT 58 0.18 (0.56, 0.21) CCBT+MCI 65 0.26 (0.62, 0.11) Conduct problems (SDQ-c) – post- treatment assessment (2) CCBT+Con 47 0.61 (1.08, 0.14) CCBT+MCBT 47 0.52 (1.00, 0.05) CCBT+MCI 55 0.50 (0.94, 0.07) Conduct problems (SDQ-c) – 6-month post-treatment assessment (3) CCBT+Con 42 0.88 (1.35, 0.42) CCBT+MCBT 44 0.91 (1.37, 0.46) CCBT+MCI 42 0.81 (1.28, 0.34) Conduct problems (SDQ-c) – 12-month post-treatment assessment (4) CCBT+Con 31 1.21 (1.87, 0.55) CCBT+MCBT 34 0.94 (1.58, 0.30) CCBT+MCI 38 1.00 (1.59, 0.40) Conduct problems (SDQ-p) – mid- treatment assessment (1B) CCBT+Con 42 0.14 (0.54, 0.26) CCBT+MCBT 46 0.18 (0.56, 0.21) CCBT+MCI 50 0.08 (0.45, 0.29) Conduct problems (SDQ-p) – post- treatment assessment (2) CCBT+Con 37 0.65 (1.06, 0.24) CCBT+MCBT 41 0.74 (1.12, 0.35) CCBT+MCI 40 0.84 (1.23, 0.45) Conduct problems (SDQ-p) – 6-month post-treatment assessment (3) CCBT+Con 39 0.47 (0.86, 0.09) CCBT+MCBT 42 0.99 (1.36, 0.62) CCBT+MCI 41 1.11 (1.49, 0.73) Conduct problems (SDQ-p) – 12-month post-treatment assessment (4) CCBT+Con 32 1.04 (1.56, 0.53) CCBT+MCBT 32 0.89 (1.40, 0.38) CCBT+MCI 38 0.85 (1.31, 0.38) Conduct problems (SDQ-t) – post- treatment assessment (2) CCBT+Con 18 0.35 (0.21, 0.91) CCBT+MCBT 22 0.17 (0.68, 0.35) CCBT+MCI 23 0.11 (0.62, 0.39) Conduct problems (SDQ-t) – 6-month post-treatment assessment (3) CCBT+Con 12 0.57 (0.36, 1.50) CCBT+MCBT 18 0.21 (0.98, 0.57) CCBT+MCI 22 0.31 (0.37, 1.00) Conduct problems (SDQ-t) – 12-month post-treatment assessment (4) CCBT+Con 9 0.09 (1.21, 1.03) CCBT+MCBT 11 0.27 (1.30, 0.75) CCBT+MCI 12 0.41 (0.54, 1.36) Child Adjustment to School (CAS-t) – post-treatment assessment (2) CCBT+Con 18 0.86 (2.32, 0.60) CCBT+MCBT 24 1.96 (3.22, 0.70) CCBT+MCI 25 1.26 (2.48, 0.04) Child Adjustment to School (CAS-t) – 6- month post-treatment assessment (3) CCBT+Con 11 1.43 (3.85, 0.99) CCBT+MCBT 17 2.92 (4.91, 0.94) CCBT+MCI 23 1.40 (3.02, 0.23) Child Adjustment to School (CAS-t) – 12-month post-treatment assessment (4) CCBT+Con 9 2.56 (4.76, 0.37) CCBT+MCBT 10 0.04 (2.23, 2.14) CCBT+MCI 12 0.40 (2.29, 1.48) CAIS-c/p, Child Anxiety Impact Scale child/parent report; CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cognitive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; SCAS-c/p, Spence Children’s Anxiety Scale – child/parent report; SDQ-p, Strengths and Difﬁculties Questionnaire – parent report; SMFQ-c/p, Short Mood and Feelings Questionnaire – child/parent report. Discussion This indi- cates that costs associated with childhood anxiety disorders are likely to be underestimated if a purely healthcare provider perspective is adopted, and it doi:10.1111/jcpp.13089 Child and maternal anxiety disorder Child and maternal anxiety disorder 71 Table 5 Statistical analyses for child secondary continuous outcomes Treatment arm N Adjusteda Mean change (95% CI) Anxiety symptoms (SCAS-c) – mid- treatment assessment (1B) CCBT+Con 53 7.71 (10.87, 4.56) CCBT+MCBT 60 6.15 (9.13, 3.17) CCBT+MCI 64 6.02 (8.89, 3.15) Anxiety symptoms (SCAS-c) – post- treatment assessment (2) CCBT+Con 45 19.68 (23.48, 15.89) CCBT+MCBT 46 13.71 (17.49, 9.92) CCBT+MCI 52 15.73 (19.27, 12.19) Anxiety symptoms (SCAS-c) – 6-month post-treatment assessment (3) CCBT+Con 41 17.85 (22.73, 12.98) CCBT+MCBT 43 16.59 (21.35, 11.83) CCBT+MCI 41 17.60 (22.52, 12.67) Anxiety symptoms (SCAS-c) – 12-month post-treatment assessment (4) CCBT+Con 31 18.11 (23.18, 13.03) CCBT+MCBT 34 18.92 (23.82, 14.01) CCBT+MCI 37 17.98 (22.59, 13.38) Anxiety symptoms (SCAS-p) – mid- treatment assessment (1B) CCBT+Con 42 8.30 (11.68, 4.93) CCBT+MCBT 44 8.14 (11.45, 4.83) CCBT+MCI 48 9.38 (12.54, 6.21) Anxiety symptoms (SCAS-p) – post- treatment assessment (2) CCBT+Con 36 18.00 (21.09, 14.88) CCBT+MCBT 39 16.77 (19.75, 13.78) CCBT+MCI 38 18.30 (21.35, 15.25) Anxiety symptoms (SCAS-p) – 6-month post-treatment assessment (3) CCBT+Con 36 17.44 (21.03, 13.84) CCBT+MCBT 41 16.62 (20.00, 13.25) CCBT+MCI 38 19.17 (22.72, 15.61) Anxiety symptoms (SCAS-p) – 12-month post-treatment assessment (4) CCBT+Con 30 22.37 (26.62, 18.12) CCBT+MCBT 31 16.36 (20.58, 12.15) CCBT+MCI 33 20.74 (24.78, 16.71) Anxiety symptoms (SCAS-t) – post- treatment assessment (2) CCBT+Con 7 4.02 (11.27, 3.23) CCBT+MCBT 14 4.94 (9.90, 0.03) CCBT+MCI 12 5.31 (10.53, 0.10) Anxiety symptoms (SCAS-t) – 6-month post-treatment assessment (3) CCBT+Con 4 1.88 (15.75, 12.00) CCBT+MCBT 9 15.07 (22.85, 7.29) CCBT+MCI 15 10.26 (15.89, 4.64) Anxiety symptoms (SCAS-t) – 12-month post-treatment assessment (4) CCBT+Con 4 7.90 (17.17, 1.37) CCBT+MCBT 4 0.04 (18.30, 18.22) CCBT+MCI 5 11.90 (23.14, 0.66) Child Anxiety Impact Scale (CAIS-c) – mid-treatment assessment (1B) CCBT+Con 52 4.30 (7.36, 1.24) CCBT+MCBT 58 6.48 (9.41, 3.55) CCBT+MCI 62 7.19 (9.97, 4.41) Child Anxiety Impact Scale (CAIS-c) – post-treatment assessment (2) CCBT+Con 44 8.19 (12.10, 4.28) CCBT+MCBT 45 6.28 (10.20, 2.37) CCBT+MCI 53 6.49 (10.05, 2.93) Child Anxiety Impact Scale (CAIS-c) – 6- month post-treatment assessment (3) CCBT+Con 38 12.45 (15.06, 9.84) CCBT+MCBT 43 11.02 (13.50, 8.54) CCBT+MCI 41 10.75 (13.29, 8.21) Child Anxiety Impact Scale (CAIS-c) – 12-month post-treatment assessment (4) CCBT+Con 29 12.83 (17.14, 8.51) CCBT+MCBT 33 9.25 (13.41, 5.09) CCBT+MCI 37 11.71 (15.48, 7.93) Child Anxiety Impact Scale (CAIS-p) – mid-treatment assessment (1B) CCBT+Con 39 2.39 (4.69, 0.08) CCBT+MCBT 39 5.57 (7.89, 3.25) CCBT+MCI 40 4.77 (7.05, 2.48) Child Anxiety Impact Scale (CAIS-p) – post-treatment assessment (2) CCBT+Con 33 10.19 (12.37, 8.01) CCBT+MCBT 35 12.95 (15.09, 10.80) CCBT+MCI 31 10.15 (12.45, 7.84) Child Anxiety Impact Scale (CAIS-p) – 6- month post-treatment assessment (3) CCBT+Con 35 9.25 (12.12, 6.37) CCBT+MCBT 37 12.13 (14.95, 9.31) CCBT+MCI 34 9.69 (12.68, 6.70) Child Anxiety Impact Scale (CAIS-p) – 12-month post-treatment assessment (4) CCBT+Con 28 9.50 (12.44, 6.56) CCBT+MCBT 27 12.11 (15.19, 9.04) CCBT+MCI 31 12.15 (14.99, 9.32) Depression symptoms (SMFQ-c) – mid- treatment assessment (1B) CCBT+Con 52 2.83 (3.96, 1.70) CCBT+MCBT 60 2.09 (3.15, 1.03) CCBT+MCI 64 2.48 (3.50, 1.45) Depression symptoms (SMFQ-c) – post- treatment assessment (2) CCBT+Con 46 5.03 (6.35, 3.71) CCBT+MCBT 47 2.25 (3.57, 0.93) CCBT+MCI 54 2.70 (3.92, 1.48) Depression symptoms (SMFQ-c) – 6- month post-treatment assessment (3) CCBT+Con 40 3.81 (5.33, 2.29) CCBT+MCBT 44 3.52 (4.97, 2.06) CCBT+MCI 39 3.97 (5.52, 2.41) Table 5 Statistical analyses for child secondary continuous outcomes Table 5 Statistical analyses for child secondary continuous outcomes Treatment arm N Adjusteda Mean change (95% CI) Adjusteda Mean difference (95% CI) p value Anxiety symptoms (SCAS-c) – mid- treatment assessment (1B) CCBT+Con 53 7.71 (10.87, 4.56) Ref CCBT+MCBT 60 6.15 (9.13, 3.17) 1.57 (2.82, 5.96) .482 CCBT+MCI 64 6.02 (8.89, 3.15) 1.69 (2.61, 5.99) .438 Anxiety symptoms (SCAS-c) – post- treatment assessment (2) CCBT+Con 45 19.68 (23.48, 15.89) Ref CCBT+MCBT 46 13.71 (17.49, 9.92) 5.97 (0.54, 11.41) .031 CCBT+MCI 52 15.73 (19.27, 12.19) 3.96 (1.28, 9.19) .137 Anxiety symptoms (SCAS-c) – 6-month post-treatment assessment (3) CCBT+Con 41 17.85 (22.73, 12.98) Ref CCBT+MCBT 43 16.59 (21.35, 11.83) 1.26 (5.56, 8.08) .715 CCBT+MCI 41 17.60 (22.52, 12.67) 0.26 (6.77, 7.28) .943 Anxiety symptoms (SCAS-c) – 12-month post-treatment assessment (4) CCBT+Con 31 18.11 (23.18, 13.03) Ref CCBT+MCBT 34 18.92 (23.82, 14.01) 0.81 (7.96, 6.34) .823 CCBT+MCI 37 17.98 (22.59, 13.38) 0.12 (6.73, 6.97) .972 Anxiety symptoms (SCAS-p) – mid- treatment assessment (1B) CCBT+Con 42 8.30 (11.68, 4.93) Ref CCBT+MCBT 44 8.14 (11.45, 4.83) 0.17 (4.62, 4.96) .944 CCBT+MCI 48 9.38 (12.54, 6.21) 1.07 (5.75, 3.62) .653 Anxiety symptoms (SCAS-p) – post- treatment assessment (2) CCBT+Con 36 18.00 (21.09, 14.88) Ref CCBT+MCBT 39 16.77 (19.75, 13.78) 1.22 (3.15, 5.59) .581 CCBT+MCI 38 18.30 (21.35, 15.25) 0.32 (4.77, 4.14) .888 Anxiety symptoms (SCAS-p) – 6-month post-treatment assessment (3) CCBT+Con 36 17.44 (21.03, 13.84) Ref CCBT+MCBT 41 16.62 (20.00, 13.25) 0.82 (4.11, 5.74) .743 CCBT+MCI 38 19.17 (22.72, 15.61) 1.73 (6.86, 3.40) .505 Anxiety symptoms (SCAS-p) – 12-month post-treatment assessment (4) CCBT+Con 30 22.37 (26.62, 18.12) Ref CCBT+MCBT 31 16.36 (20.58, 12.15) 6.01 (0.01, 12.02) .050 CCBT+MCI 33 20.74 (24.78, 16.71) 1.62 (4.27, 7.51) .585 Anxiety symptoms (SCAS-t) – post- treatment assessment (2) CCBT+Con 7 4.02 (11.27, 3.23) Ref CCBT+MCBT 14 4.94 (9.90, 0.03) 0.92 (10.69, 8.85) .847 CCBT+MCI 12 5.31 (10.53, 0.10) 1.29 (10.86, 8.27) .782 Anxiety symptoms (SCAS-t) – 6-month post-treatment assessment (3) CCBT+Con 4 1.88 (15.75, 12.00) Ref CCBT+MCBT 9 15.07 (22.85, 7.29) 13.19 (30.33, 3.94) .123 CCBT+MCI 15 10.26 (15.89, 4.64) 8.39 (24.79, 8.01) .296 Anxiety symptoms (SCAS-t) – 12-month post-treatment assessment (4) CCBT+Con 4 7.90 (17.17, 1.37) Ref CCBT+MCBT 4 0.04 (18.30, 18.22) 7.86 (12.83, 28.55) .244 CCBT+MCI 5 11.90 (23.14, 0.66) 4.00 (18.25, 10.25) .351 Child Anxiety Impact Scale (CAIS-c) – mid-treatment assessment (1B) CCBT+Con 52 4.30 (7.36, 1.24) Ref CCBT+MCBT 58 6.48 (9.41, 3.55) 2.18 (6.51, 2.15) .322 CCBT+MCI 62 7.19 (9.97, 4.41) 2.89 (7.03, 1.26) .171 Child Anxiety Impact Scale (CAIS-c) – post-treatment assessment (2) CCBT+Con 44 8.19 (12.10, 4.28) Ref CCBT+MCBT 45 6.28 (10.20, 2.37) 1.91 (3.73, 7.55) .505 CCBT+MCI 53 6.49 (10.05, 2.93) 1.70 (3.63, 7.03) .530 Child Anxiety Impact Scale (CAIS-c) – 6- month post-treatment assessment (3) CCBT+Con 38 12.45 (15.06, 9.84) Ref CCBT+MCBT 43 11.02 (13.50, 8.54) 1.43 (2.21, 5.07) .437 CCBT+MCI 41 10.75 (13.29, 8.21) 1.70 (1.97, 5.37) .361 Child Anxiety Impact Scale (CAIS-c) – 12-month post-treatment assessment (4) CCBT+Con 29 12.83 (17.14, 8.51) Ref CCBT+MCBT 33 9.25 (13.41, 5.09) 3.57 (2.59, 9.74) .253 CCBT+MCI 37 11.71 (15.48, 7.93) 1.12 (4.59, 6.83) .698 Child Anxiety Impact Scale (CAIS-p) – mid-treatment assessment (1B) CCBT+Con 39 2.39 (4.69, 0.08) Ref CCBT+MCBT 39 5.57 (7.89, 3.25) 3.18 (6.51, 0.15) .061 CCBT+MCI 40 4.77 (7.05, 2.48) 2.38 (5.67, 0.91) .154 Child Anxiety Impact Scale (CAIS-p) – post-treatment assessment (2) CCBT+Con 33 10.19 (12.37, 8.01) Ref CCBT+MCBT 35 12.95 (15.09, 10.80) 2.76 (5.86, 0.34) .080 CCBT+MCI 31 10.15 (12.45, 7.84) 0.04 (3.20, 3.28) .980 Child Anxiety Impact Scale (CAIS-p) – 6- month post-treatment assessment (3) CCBT+Con 35 9.25 (12.12, 6.37) Ref CCBT+MCBT 37 12.13 (14.95, 9.31) 2.88 (6.92, 1.16) .160 CCBT+MCI 34 9.69 (12.68, 6.70) 0.44 (4.61, 3.73) .835 Child Anxiety Impact Scale (CAIS-p) – 12-month post-treatment assessment (4) CCBT+Con 28 9.50 (12.44, 6.56) Ref CCBT+MCBT 27 12.11 (15.19, 9.04) 2.61 (6.88, 1.66) .227 CCBT+MCI 31 12.15 (14.99, 9.32) 2.65 (6.72, 1.42) .199 Depression symptoms (SMFQ-c) – mid- treatment assessment (1B) CCBT+Con 52 2.83 (3.96, 1.70) Ref CCBT+MCBT 60 2.09 (3.15, 1.03) 0.74 (0.82, 2.30) .350 CCBT+MCI 64 2.48 (3.50, 1.45) 0.36 (1.18, 1.90) .649 Depression symptoms (SMFQ-c) – post- treatment assessment (2) CCBT+Con 46 5.03 (6.35, 3.71) Ref CCBT+MCBT 47 2.25 (3.57, 0.93) 2.78 (0.88, 4.68) .004 CCBT+MCI 54 2.70 (3.92, 1.48) 2.33 (0.52, 4.14) .012 Depression symptoms (SMFQ-c) – 6- month post treatment assessment (3) CCBT+Con 40 3.81 (5.33, 2.29) Ref CCBT+MCBT 44 3 52 ( 4 97 2 06) 0 30 ( 1 82 2 42) 783 Adjusteda Mean difference (95% CI) p value Ref 1.57 (2.82, 5.96) .482 1.69 (2.61, 5.99) .438 Ref 5.97 (0.54, 11.41) .031 3.96 (1.28, 9.19) .137 Ref 1.26 (5.56, 8.08) .715 0.26 (6.77, 7.28) .943 Ref 0.81 (7.96, 6.34) .823 0.12 (6.73, 6.97) .972 Ref 0.17 (4.62, 4.96) .944 1.07 (5.75, 3.62) .653 Ref 1.22 (3.15, 5.59) .581 0.32 (4.77, 4.14) .888 Ref 0.82 (4.11, 5.74) .743 1.73 (6.86, 3.40) .505 Ref 6.01 (0.01, 12.02) .050 1.62 (4.27, 7.51) .585 Ref 0.92 (10.69, 8.85) .847 1.29 (10.86, 8.27) .782 Ref 13.19 (30.33, 3.94) .123 8.39 (24.79, 8.01) .296 Ref 7.86 (12.83, 28.55) .244 4.00 (18.25, 10.25) .351 Ref 2.18 (6.51, 2.15) .322 2.89 (7.03, 1.26) .171 Ref 1.91 (3.73, 7.55) .505 1.70 (3.63, 7.03) .530 Ref 1.43 (2.21, 5.07) .437 1.70 (1.97, 5.37) .361 Ref 3.57 (2.59, 9.74) .253 1.12 (4.59, 6.83) .698 Ref 3.18 (6.51, 0.15) .061 2.38 (5.67, 0.91) .154 Ref 2.76 (5.86, 0.34) .080 0.04 (3.20, 3.28) .980 Ref 2.88 (6.92, 1.16) .160 0.44 (4.61, 3.73) .835 Ref 2.61 (6.88, 1.66) .227 2.65 (6.72, 1.42) .199 Ref 0.74 (0.82, 2.30) .350 0.36 (1.18, 1.90) .649 Ref 2.78 (0.88, 4.68) .004 2.33 (0.52, 4.14) .012 Ref 0.30 (1.82, 2.42) .783 0.16 (2.36, 2.04) .887 (continued) J Child Psychol Psychiatr 2020; 61(1): 62–76 Cathy Creswell et al. CAIS-c/p, Child Anxiety Impact Scale child/parent report; CCBT+Con, child cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+MCBT, CCBT + maternal cognitive behaviour therapy; CCBT+MCI, CCBT + mother–child interaction treatment; SCAS-c/p, Spence Children’s Anxiety Scale – child/parent report; SDQ-p, Strengths and Difﬁculties Questionnaire – parent report; SMFQ-c/p, Short Mood and Feelings Questionnaire – child/parent report. aAdjusted for child age, child gender, type of child anxiety disorder (GAD, social phobia, SAD, other), baseline severity (ADIS Clinician Severity Rating) of the child’s primary anxiety disorder baseline severity (ADIS Mother self report) of the mother’s primary Discussion p p ; Q /p, g Q /p p aAdjusted for child age, child gender, type of child anxiety disorder (GAD, social phobia, SAD, other), baseline severity (ADIS Clinician Severity Rating) of the child’s primary anxiety disorder, baseline severity (ADIS Mother self-report) of the mother’s primary anxiety disorder and baseline questionnaire score. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. 73 Child and maternal anxiety disorder doi:10.1111/jcpp.13089 Panel 1 – CCBT+MCBT vs CCBT+Con Panel 2 – CCBT+MCI vs CCBT+Con Figure 2 Cost-effectiveness acceptability curves for the base-case analyses. CCBT+Con, child-focused cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+ MCBT, CCBT + CBT to target maternal anxiety disorder; CCBT+MCI, CCBT + intervention to target the mother–child interaction [Colour ﬁgure can be viewed at wileyonlinelibrary.com] Panel 2 – CCBT+MCI vs CCBT+Con Panel 2 – CCBT+MCI vs CCBT+Con Panel 1 – CCBT+MCBT vs CCBT+Con Figure 2 Cost-effectiveness acceptability curves for the base-case analyses. CCBT+Con, child-focused cognitive behaviour therapy + nonspeciﬁc control interventions; CCBT+ MCBT, CCBT + CBT to target maternal anxiety disorder; CCBT+MCI, CCBT + intervention to target the mother–child interaction [Colour ﬁgure can be viewed at wileyonlinelibrary.com] inclusion of nonspeciﬁc interventions to balance therapist contact, a design which allowed for isolat- ing the effects of speciﬁcally targeting maternal anxiety and parenting responses, and the inclusion of a broad societal perspective, prospectively designed economic evaluation measuring and valu- ing both mother and child costs and outcomes. These strengths need to be considered in the light of various limitations. Although we allowed for 20% loss to follow-up, by the one year post-treatment, assessment retention was down to 61% in the CCBT+Con arm. Although there were no clear base- line differences between completers and those who dropped out, it is of concern that the greatest drop- out occurred during the eight-session maternal counselling phase which may not have been a sufﬁciently acceptable treatment approach for some families. Whether dropouts overrepresented those with good or bad treatment outcomes cannot be determined, although the sensitivity analyses that were conducted gave a consistent pattern of results which suggest that this was not the case. Other limitations include the relatively restricted demo- graphic characteristics of the participants, who were predominantly of nonminority ethnicity and rela- tively high socioeconomic status. Discussion We elected to focus on middle childhood (7–12 years) as anxiety disor- ders can be reliably diagnosed at this age, particular parental behaviours have been observed in the context of parent anxiety disorder at this age (Creswell et al., 2013), and it is likely that the nature of parental inﬂuences on child anxiety varies with child age (Connell & Goodman, 2002; Maccoby, 1992). We also focused on intervening with mothers as parental inﬂuences have also been shown to differ according to parent gender (B€ogels & Perotti, 2011). As a result, however, the ﬁndings cannot be gener- alised to younger children, to adolescents or to interventions with fathers or other caregivers. The study also included children and mothers with a reduction in the frequency of maternal anxiety disorder. However, after the children had received treatment, there was no longer a signiﬁcant differ- ence in maternal disorder status, with all treatment arms showing relatively high rates of recovery from maternal disorders. This is consistent with previous ﬁndings indicating that reductions in child anxiety are associated with reductions in parental anxiety (Settipani et al., 2013). We conducted observational assessments of par- ental responses before and after treatment for child anxiety disorders which provided evidence that, in terms of a relative change in overprotective beha- viours, the mother–child interaction treatment was successful. The MCI intervention was also associ- ated with change in maternal cognitions associated with conﬁdence in child coping (i.e. reduced predic- tions regarding child fear and increased predictions regarding child control). Despite these positive ben- eﬁts, no signiﬁcant beneﬁt to child outcome was conferred. Possible reasons for this may be that the changes were not of sufﬁcient magnitude to be of beneﬁt, or, as noted above, that change in these factors is not in fact critical to changing child anxiety. Notably, there was no speciﬁc beneﬁt for the MCI intervention on measures of maternal expressed anxiety, intrusiveness or positive beha- viours. While it is possible that the intervention was ineffective with respect to these dimensions, it may well be that these parental behaviours changed equally across groups in response to improvements in child anxiety (Silverman, Kurtines, Jaccard, & Pina, 2009). However, it may also reﬂect limitations in the extent to which these laboratory-based obser- vational tasks assess parenting dimensions as they are expressed in the home. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. Correspondence Cathy Creswell, Department of Experimental Psychol- ogy, University of Oxford, Anna Watts Building, Rad- cliffe Observatory Quarter, Woodstock Road, Oxford, OX2 6HG, UK; Email: cathy.creswell@psych.ox.ac.uk Cathy Creswell, Department of Experimental Psychol- ogy, University of Oxford, Anna Watts Building, Rad- cliffe Observatory Quarter, Woodstock Road, Oxford, OX2 6HG, UK; Email: cathy.creswell@psych.ox.ac.uk Discussion Cost utility analysis – CCBT+MCBT versus CCBT(+Con). Table S8. Cost utility analysis – CCBT+MCBT versus CCBT(+Con). Table S9. Cost utility analysis – CCBT+MCI versus CCBT(+Con). Table S9. Cost utility analysis – CCBT+MCI versus CCBT(+Con). Table S10. Societal cost mean differences – CCBT+MCBT versus CCBT; CCBT+ MCI versus CCBT (+Con). Table S10. Societal cost mean differences – CCBT+MCBT versus CCBT; CCBT+ MCI versus CCBT (+Con). Table S11. Treatment resource use mean differences – CCBT+MCBT versus CCBT+Con. Table S11. Treatment resource use mean differences – CCBT+MCBT versus CCBT+Con. Table S12. Treatment resource use mean differences – CCBT+MCI versus CCBT+Con. Table S12. Treatment resource use mean differences – CCBT+MCI versus CCBT+Con. Acknowledgements This trial was supported by grants from the Medical Research Council/National Institute of Health Research Efﬁcacy and Mechanism Evaluation program (09/800/ 17), Berkshire Healthcare Foundation Trust and the Thames Valley Comprehensive Local Research Network; C.C. was supported by MRC Clinician Scientist Fellowship (G0601874).Trialregistrationnumber:ISRCTN19762288 (doi.10.1186/ISRCTN19762288). The full trial report can be found here: doi: 10.3310/hta19380 In sum, the current study showed that good clinical outcomes can be achieved for children with anxiety disorders in the context of maternal anxiety disorder by providing high-quality individual child CBT together with some parental support. However, although offering CBT to mothers for their own anxiety disorders gave an early boost to the timing of maternal recovery, it did not lead to a signiﬁcant clinical beneﬁt in terms of child anxiety treatment outcome and (given the higher costs) was not cost- effective. An adjunctive treatment to target parenting responses had a nonsigniﬁcant positive effect on child outcomes, which, despite the higher costs, may be cost-effective. The ﬁndings also suggest that reductions in child anxiety may have a positive effect on maternal mental health. The authors thank the participating families, the managers, administrators, therapists, assessors, coders, clinical supervisors and the Trial Steering and Data Management and Ethics Committee members (Chairs: Professor Jonathan Hill & John Geddes). The authors also thank Liz White for her support in preparing the manuscript. This report presents inde- pendent research managed by the National Institute for Health Research (NIHR). The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reﬂect those of the NHS, the NIHR, MRC, CCF, NETSCC or the Department of Health. The authors have declared that they have no competing or potential conﬂicts of interest. Discussion The study had several notable strengths, including the use of reliable, blind raters to make assessments of child and maternal anxiety and maternal beha- viours and cognitions before and after treatment, the Cathy Creswell et al. J Child Psychol Psychiatr 2020; 61(1): 62–76 74 broad range of anxiety disorders, and further work needs to be done which takes account of the precise form of parental and child anxiety. Appendix S4. Multiple imputation approach. pp p p pp Table S1. Scores at each time point on continuous secondary outcome measures. p y For the health economics analyses, data on resource use and costs of additional health and personal social services beyond the NHS treatment costs were characterised by a high percentage of missing data. However, the economic results were conﬁrmed by multiple sensitivity analyses, including a variation that best approximated a complete-case scenario. The base-case economic evaluations excluded the costs of the two nonspeciﬁc interven- tions (i.e. family healthy living and nondirective counselling for mothers), which were introduced to match for therapist time/contact across the trial arms, and are not commonly delivered in ‘real-world’ settings. While the sensitivity analyses that were conducted including those costs conﬁrmed and rein- forced the base-case results, it should be acknowl- edged that the nonspeciﬁc interventions may not have been completely neutral in their effect. However, in the absence of a valid counterfactual, we are unable to quantify that potential impact. Furthermore, this study should be regarded as providing an indication of the short-term likely cost-effectiveness of CCBT+MCBT or CCBT+MCI compared to CCBT, with further research required to determine cost-effective- ness in the longer term. Finally, generalising these economic ﬁndings to other populations and settings (e.g. different healthcare systems) is challenging and, with the added complexity associated with capturing the broader societal impact as well as the combined mother/child impacts, the cost-effectiveness results are best interpreted with caution. Table S2. Unit costs. Table S3. Sensitivity analyses for base-case compar- isons. Table S4. Descriptive data: Maternal anxiety disorders, behaviours and cognitions. Table S5. Manipulation checks: Statistical analyses for maternal anxiety disorders, behaviours and cognitions. Table S6. Health Economic data completeness (per- centage of missing data reported). Table S7. Child and mother combined Quality Adjusted Life Years (QALYs) gained – CCBT+MCBT versus CCBT+Con; CCBT+MCI versus CCBT+Con. Q y j Life Years (QALYs) gained – CCBT+MCBT versus CCBT+Con; CCBT+MCI versus CCBT+Con. Table S8. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. References BDA (2003). Family health. Birmingham, UK: British Dietetic Association. Bodden, D.H., B€ogels, S.M., Nauta, M.H., De Haan, E., Ringrose, J., Appelboom, C., . . . & Appelboom-Geerts, K.C. (2008). Child versus family cognitive-behavioral therapy in clinically anxious youth: An efﬁcacy and partial effectiveness study. Journal of the American Academy of Child and Adolescent Psychiatry, 47, 1384–1394. DiNardo, P.A., Barlow, D.H., & Brown, T.A. (1994). Anxiety disorders interview schedule for DSM-IV.: Client interview schedule. Lifetime Version. Oxford, UK: Oxford University Press. Bodden, D.H., Dirksen, C.D., & Bogels, S.M. (2008). Societal burden of clinically anxious youth referred for treatment: A cost-of-illness study. Journal of Abnormal Child Psychology, 36, 487–497. Dolan, P., Gudex, C., Kind, P., & Williams, A. (1995). A social tariff for EuroQol: Results from a UK general population survey. York, UK: Centre for Health Economics University of York. B€ogels, S., & Perotti, E. (2011). Does father know best? A formal model of the paternal inﬂuence on childhood social anxiety. Journal of Child and Family Studies, 20, 171–181. Drummond, M.F., Sculpher, M.J., Claxton, K., Stoddart, G.L., & Torrance, G.W. (2015). Methods for the economic evalua- tion of health care programmes. Oxford, UK: Oxford Univer- sity Press. Borkovec, T., & Costello, E. (1993). Efﬁcacy of applied relax- ation and cognitive-behavioral therapy in the treatment of generalized anxiety disorder. Journal of Consulting and Clinical Psychology, 61, 611. Essau, C.A., Conradt, J., & Petermann, F. (2000). Frequency, comorbidity and psychosocial impairment of anxiety disor- ders in German adolescents. Journal of Anxiety Disorders, 14, 263–279. Cobham, V.E., Dadds, M.R., & Spence, S.H. (1998). The role of parental anxiety in the treatment of childhood anxiety. Journal of Consulting and Clinical Psychology, 66, 893–905. Faria, R., Gomes, M., Epstein, D., & White, I.R. (2014). A guide to handling missing data in cost-effectiveness analysis conducted within randomised controlled trials. Pharma- coeconomics, 32, 1157–1170. Connell, A.M., & Goodman, S.H. (2002). The association between psychopathology in fathers versus mothers and children’s internalizing and externalizing behavior prob- lems: A meta-analysis. Psychological Bulletin, 128, 746–773. Fenwick, E., Marshall, D.A., Levy, A.R., & Nichol, G. (2006). Using and interpreting cost-effectiveness acceptability curves: An example using data from a trial of management strategies for atrial ﬁbrillation. BMC Health Services Research, 6, 1. Cooper, P.J., Fearn, V., Willetts, L., Seabrook, H., & Parkinson, M. (2006). Affective disorder in the parents of a clinic sample of children with anxiety disorders. Journal of Affective Disorders, 93, 205–212. Key points Treatment outcomes for children with anxiety disorders are impaired in the context of concurrent parental anxiety disorder but it is unclear how to improve outcomes. Treatment outcomes for children with anxiety disorders are impaired in the context of concurrent parental anxiety disorder but it is unclear how to improve outcomes. In this RCT for children with anxiety disorders whose mothers also had a current anxiety disorder, individual child-focused CBT was supplemented with treatments to (a) improve maternal anxiety (MCBT) or (b) alter maternal responses to her child when faced with challenge (MCI). In this RCT for children with anxiety disorders whose mothers also had a current anxiety disorder, individual child-focused CBT was supplemented with treatments to (a) improve maternal anxiety (MCBT) or (b) alter maternal responses to her child when faced with challenge (MCI). p g The adjunct treatments were successful in improving maternal anxiety and responses; however, this did not lead to a signiﬁcant beneﬁt in terms of child anxiety treatment outcome. g The adjunct treatments were successful in improving maternal anxiety and responses; however, this did not lead to a signiﬁcant beneﬁt in terms of child anxiety treatment outcome. g y Good child outcomes were achieved for children with anxiety disorders in the context of maternal anxiety disorder by providing high-quality individual child CBT together with some parental support. Reductions in child anxiety may also have a positive effect on maternal mental health Good child outcomes were achieved for children with anxiety disorders in the context of maternal anxiety disorder by providing high-quality individual child CBT together with some parental support. Reductions in child anxiety may also have a positive effect on maternal mental health. y y p MCI (but not MCBT) may be a cost-effective psychological approach for the treatment of child anxiety problems in the context of maternal anxiety disorders. y y p MCI (but not MCBT) may be a cost-effective psychological approach for the treatment of child anxiety problems in the context of maternal anxiety disorders. Creswell, C., Violato, M., Fairbanks, H., White, E., Parkinson, M., Abitabile, G., . . . & Cooper, P.J. (2017). Clinical outcomes and cost-effectiveness of brief guided parent-delivered cog- nitive behavioural therapy and solution-focused brief ther- apy for treatment of childhood anxiety disorders: A randomised controlled trial. The Lancet Psychiatry, 4, 529– 539. Supporting information pp g Additional supporting information may be found online in the Supporting Information section at the end of the article: Peter J. Cooper, School of Psychology and Clinical Language Sciences, University of Reading, Reading, Berkshire, RG6 6AL, UK; Email: p.j.cooper@reading. ac.uk Peter J. Cooper, School of Psychology and Clinical Language Sciences, University of Reading, Reading, Berkshire, RG6 6AL, UK; Email: p.j.cooper@reading. ac.uk Appendix S1. Study protocol. Appendix S2. Statistical analysis plan. Appendix S3. Adherence and ﬁdelity. Appendix S1. Study protocol. Appendix S1. Study protocol. Appendix S2. Statistical analysis plan. Appendix S3. Adherence and ﬁdelity. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child d Ad l t M t l H lth doi:10.1111/jcpp.13089 75 Child and maternal anxiety disorder © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. References In R.G. Heimberg, C.L. Turk & D.S. Mennin (Eds.), Generalized anxiety disor- der: Advances in research and practice (pp. 51–74). New York: Guilford Press. Piacentini, J., Bennett, S., Compton, S.N., Kendall, P.C., Birmaher, B., Albano, A.M., . . . & Ginsburg, G. (2014). 24- and 36-week outcomes for the Child/Adolescent Anxiety Multimodal Study (CAMS). Journal of the American Academy of Child and Adolescent Psychiatry, 53, 297–310. Hudson, J.L., Rapee, R.M., Deveney, C., Schniering, C.A., Lyneham, H.J., & Bovopoulos, N. (2009). Cognitive-behav- ioral treatment versus an active control for children and adolescents with anxiety disorders: A randomized trial. Journal of the American Academy of Child and Adolescent Psychiatry, 48, 533–544. Schleider, J.L., Ginsburg, G.S., Keeton, C.P., Weisz, J.R., Birmaher, B., Kendall, P.C., . . . & Walkup, J.T. (2015). Parental psychopathology and treatment outcome for anx- ious youth: Roles of family functioning and caregiver strain. Journal of Consulting and Clinical Psychology, 83, 213–224. Settipani, A., O’Neil, A., Podell, L., Beidas, S., & Kendall, P.C. (2013). Youth anxiety and parent factors over time: Direc- tionality of change among youth treated for anxiety. Journal of Clinical Child and Adolescent Psychology, 42, 9–21. Hudson, J.L., Keers, R., Roberts, S., Coleman, J.R., Breen, G., Arendt, K., . . . Heiervang, E.R. (2015). Clinical predictors of response to cognitive-behavioral therapy in pediatric anxiety disorders: the Genes for Treatment (GxT) study. Journal of the American Academy of Child & Adolescent Psychiatry, 54, 454–463. Silverman, W.K., & Albano, A.M. (1996). Anxiety disorders interview schedule for DSM-IV.: Parent interview schedule, Vol. (1). UK: Oxford University Press. Husereau, D., Drummond, M., Petrou, S., Carswell, C., Moher, D., Greenberg, D., . . . & Loder, E. (2013). Consol- idated health economic evaluation reporting standards (CHEERS) statement. Cost Effectiveness and Resource Allocation, 11, 1. Silverman, W.K., Kurtines, W.M., Jaccard, J., & Pina, A.A. (2009). Directionality of change in youth anxiety treatment involving parents: An initial examination. Journal of Con- sulting and Clinical Psychology, 77, 474. Simon, E., Dirksen, C., B€ogels, S., & Bodden, D. (2012). Cost- effectiveness of child-focused and parent-focused interven- tions in a child anxiety prevention program. Journal of Anxiety Disorders, 26, 287–296. James, A.C., James, G., Cowdrey, F.A., Soler, A., & Choke, A. (2013). Cognitive behavioural therapy for anxiety disorders in children and adolescents. Cochrane Database Syst, Rev, 6. Tilford, J.M., Payakachat, N., Kuhlthau, K.A., Pyne, J.M., Kovacs, E., Bellando, J., . . . & Frye, R.E. (2015). References Costello, E.J., Egger, H.L., & Angold, A. (2004). Developmental Epidemiology of Anxiety Disorders. In T.H. Ollendick & J.S. March (Eds.), Phobic and anxiety disorders in children and adolescents: A clinician’s guide to effective psychosocial and pharmacological interventions (pp. 61–91). New York: Oxford University Press. Gallagher, H.M., Rabian, B.A., & McCloskey, M.S. (2004). A brief group cognitive-behavioral intervention for social pho- bia in childhood. Journal of Anxiety Disorders, 18, 459–479. Ginsburg, G.S., & Drake, K.L. (2002). School-based treatment for anxious African-American adolescents: A controlled pilot study. Journal of the American Academy of Child and Adolescent Psychiatry, 41, 768–775. Creswell, C., Apetroaia, A., Murray, L., & Cooper, P. (2013). Cognitive, affective, and behavioral characteristics of moth- ers with anxiety disorders in the context of child anxiety disorder. Journal of Abnormal Psychology, 122, 26. Ginsburg, G.S., Kendall, P.C., Sakolsky, D., Compton, S.N., Piacentini, J., Albano, A.M., . . . & Rynn, M.A. (2011). Remission after acute treatment in children and adolescents with anxiety disorders: Findings from the CAMS. Journal of Consulting and Clinical Psychology, 79, 806–813. Creswell, C., Murray, L., Stacey, J., & Cooper, P.J. (2011). Parenting and child anxiety. In W.K. Silverman & A.P. Field (Eds.), Anxiety disorders in children and adolescents (2nd edn, pp. 299–322). Cambridge, UK: Cambridge University Press. Guy, W. (Ed.). (1976). Clinical global impressions. In ECDEU assessment manual for psychopharmacology (pp. 217–221). Rockville, MD: National Institute of Mental Health. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. 76 Cathy Creswell et al. J Child Psychol Psychiatr 2020; 61(1): 62–76 NICE. (2013). National Institute for Health and Care Excel- lence. Guide to the methods of technology appraisal. Avail- able from: https://www.nice.org.uk/process/pmg9/ resources/guide-to-the-methods-of-technology-appraisal- 2013-pdf-2007975843781 [last accessed 17 June 2019]. Hudson, J.L., Newall, C., Rapee, R.M., Lyneham, H.J., Sch- niering, C.C., Wuthrich, V.M., . . . & Gar, N.S. (2014). The impact of brief parental anxiety management on child anxiety treatment outcomes: A controlled trial. Journal of Clinical Child and Adolescent Psychology, 43, 370–380. Hudson, J.L., & Rapee, R.M. (2001). Parent-child interactions and anxiety disorders: An observational study. Behaviour Research and Therapy, 39, 1411–1427. Petrou, S., & Gray, A. (2011). Economic evaluation alongside randomised controlled trials: Design, conduct, analysis, and reporting. British Medical Journal, 342, d1548. Hudson, J.L., & Rapee, R.M. (2004). From Anxious Tempera- ment to Disorder: An Etiological Model. © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health. References Treatment for sleep problems in children with autism and caregiver spillover effects. Journal of Autism and Developmental Disorders, 45, 3613–3623. Kendall, P.C., & Hedtke, K.A. (2006). Cognitive-behavioral therapy for anxious children: Therapist manual. Ardmore, PA: Workbook. Lyneham, H., Abbott, M., Wignall, A., & Rapee, R.M. (2003). The cool kids family program - therapist manual. Sydney, Australia: Macquarie University. Tubeuf, S., & Guthmuller, S. (2017). Economic evaluation of family-based therapies for children and adolescents: What do we know?. Global and Regional Health Technology Assessment, 4, grhta. 5000272. Maccoby, E.E. (1992). The role of parents in the socialization of children: An historical overview. Developmental Psychology, 28, 1006. Walkup, J.T., Albano, A.M., Piacentini, J., Birmaher, B., Compton, S.N., Sherrill, J.T., . . . & Waslick, B. (2008). Cognitive behavioral therapy, sertraline, or a combination in childhood anxiety. New England Journal of Medicine, 359, 2753–2766. Machin, D., Campbell, M.J., Fayers, P.M., & Pinol, A.P.Y. (1997). Sample size tables for clinical studies (2nd edn). Oxford, UK: Blackwell Science Ltd. McManus, F., Clark, G., Muse, K., & Shafran, R. (2015). Case- series evaluating a transdiagnostic cognitive-behavioural treatment for co-occurring anxiety disorders. Behavioural and cognitive psychotherapy, 43, 744–758. Weinstein, M.C., Torrance, G., & McGuire, A. (2009). QALYs: The basics. Value in Health, 12, S5–S9. Wille, N., Badia, X., Bonsel, G., Burstr€om, K., Cavrini, G., Devlin, N., . . . & Herdman, M. (2010). Development of the EQ-5D-Y: A child-friendly version of the EQ-5D. Quality of Life Research, 19, 875–886. Moore, P.S., Whaley, S.E., & Sigman, M. (2004). Interactions between mothers and children: impacts of maternal and child anxiety. Journal of Abnormal Psychology, 113, 471. Murray, L., Lau, P.Y., Arteche, A., Creswell, C., Russ, S., Della Zoppa, L., . . . & Cooper, P.J. (2012). Parenting by anxious mothers: Effects of disorder subtype, context, and child characteristics. Journal of Child Psychology and Psychiatry, 53, 188–196. Woodward, L.J., & Fergusson, D.M. (2001). Life course outcomes of young people with anxiety disorders in adoles- cence. Journal of the American Academy of Child and Adolescent Psychiatry, 40, 1086–1093. Zou, G. (2004). A modiﬁed poisson regression approach to prospective studies with binary data. American Journal of Epidemiology, 159, 702–706. Newby, J.M., McKinnon, A., Kuyken, W., Gilbody, S., & Dalgleish, T. (2015). Systematic review and meta-analysis of transdiagnostic psychological treatments for anxiety and depressive disorders in adulthood. Clinical Psychology Review, 40, 91–110. References Accepted for publication: 22 May 2019 First published online: 31 July 2019 © 2019 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health.
https://openalex.org/W4251681345	https://zenodo.org/record/1818928/files/article.pdf	English	null	President Wilson's Scientific Appointments	Science	1,916	public-domain	2,295	SCIENCE BEREACOLLEGE announces a gift of $10,000 from the late James TalcotO of New York City, received shortly before his death. This gift was part of a total pledge for $40,000 for the erection of a girls' dormitory, which will be ready for occupancy on January 1. point at issue. In fairness to all concerned it is necessary to call attention to a few scien- tific appointments made by the Wilson admin- istration about which the writers failed to en- lighten the readers of SCIENCE and The Scien- tific Nonthly. In the first place, it has been generally understood (and even claimed by some of the parties interested) that the original adminis- tration slate contemplated the appointment of E. Lester Jones to the position of commis- sioner of fisheries. That this slate was broken is much to the credit of the Americah Society of Naturalists and the American Society of Zoologists. But what followed 2 The presi- dent immediately appointed Mr. Jones deputy commissioner of fisheries. That position, in many respects, even more important to science than that of the commissionership itself, and which should have been filled only upon the recommendation of the commissioner, was at once filled by the appointment of Mr. Jones. The commissioner of fisheries was not even consulted. He was completely ignored by the president and the secretary of commerce not only in this case but in other important ap- pointments in the bureau of fisheries, a few of which may be mentioned. One of the first was the appointment, without even consulting the commissioner of fisheries, of a young man as private secretary to the commissioner. It would seem that the chief of an important bureau should be permitted to select his own private secretary, the position being so distinc- tively personal and confidential. The young man appointed was, it is understood, from the home town of John H. Rothermel, at that time a congressman from Pennsylvania and chairman of a committee of the House that had been for some years conducting certain fur-seal hearings. The young man was neither a stenographer nor a typewriter (it was said he was a plumber). It was said at the time (and there is every reason to believe it was true) that he was appointed as a spy to keep Rothermel and Henry W. Elliott informed as to the commissioner's relations to fur-seal matters, in which Rothermel at that time was very active--so active, indeed, that at the next THE New York School of Dental Hy,' wene has become allied with the new Columbia Uni- versity School of Dentistry and the College of Physici~ans and Surgeons. The school will open on September 21, classes being held in the Vanderbilt Clinic. IRVING (Brown, '12), in- H. BLAKE, A.M. structor in the Oregon Agricultural College, has been appointed instructor in the depart- ment of zoology, Syracuse University. Mk. CHARLES recently of the Peabody COLBY, College for Teachers, Nashville, Tenn., has become instructor in geography at the Uni- versity of Chicago. Mk. CHARLES recently of the Peabody COLBY, College for Teachers, Nashville, Tenn., has become instructor in geography at the Uni- versity of Chicago. AT the University of Chicago, Anton Julius Carlson, of the department of physiology, and Charles Manning Child, of the department of zoology, have been promoted to professorships. Lee Irving Knight, of the department of botany, has been promoted to an assistant pro- fessorship. New appointments are : Ernest Watson Burgess, of Ohio State University, to be assistant professor in the department of sociology and anthropology; Professor Jean Piccard, of the University of Lausanne, Switzerland, to be assistant professor in t.he department of chemistry, and Dr. W. B. Sharpe and William E. Cary, to be instructors' in the department of hygiene 'and bacteriology. PRESIDENT WILSON'S SCIENTIFIC APPOINT- MENTS THE two articles in SCIENCE of August 25, 1916, on " Scientific Appointments under the Government " and " President Wilson's Scien- tific Appointments " are interesting and sug- gestive, but not entirely convincing. They do not fully cover the question; the writers were apparently not familiar with a number of facts which have a very important bearing upon the SCIENCE election, Ize was unable to explain certain charged irregularities and his constituents de- clined to return him to congress. might very properly decline to reverse his opinioll until he had secured further infornla- tion. Tlic administration thought this infor- mation could be secured by sending a special commission to the islands. To assist in select- ing the members of the commissio~l the predi- dent aslied the National Academy of Sciences, the secretary of the Smithsonian Institution and the secretary of agriculture each to nomi- nate one meniber of the commission. This was done. The Natioual Academy of Scicrlces nomiliated a very able and distinguihed zool- ogist, Dr. George 11. Parker, of Harvard Uni- versity. The commission went to the seal is- lands in the summer of 1914, made a stutly of the seal herd and, upon their return to TITash- ington, submitted a very comprehensive re- port, in which, evidently to the wrprist, o l the semetary of commerce, every importa~lt thiug for which Clark, Jordan, Evermaan, Stej- neger, Luczas, Osborn, Townse~ld, Merrianl, Zemb1it.y arid others familiar with fur-seal matters had contended, was sustained. Another flagrant violation of the principles of the civil sercice and a total disregard of fit- ness mas the appointment of one El. 0. Smith, of l'alestine, Illinois, as chief Alaska salmon agent. This appointment was made without consulting the commissioiier of fisheries or the chief of the Alaslia fisheries service, and after the secretary of commerce had assured the commissioner of fisheries that he would pro- mote to the positiorl the assistant Alaska sal- mon agent, Mr. Ward T. Bower, a thoroughly competent and experienced man. H. 0. Smith openly claimed that his appointment was made at tllc illstaizce of Senator James IInrnilton 'ewis, of Illinois. The duties of the Alaslia salmon agent, like those of a deputy commissioner of fisheries, are highly technical, and require special linowledge ant1 experience of the fisheries. Neither Mr. Jones nor Mr. FIREFLIES FLASHING IN UNISON INSCIENCE of February 4, 1916, page 169, I recorded for the first time an observation made fifty years ago of a large number of fireflies flashing in perfect unison. I have been on the lookout ever since that time for a confirma- tion of my observations, consulting every book on entomology and watching the fireflies ever since for the recurrence of this phenom- enon without success. I n Na.ture for December 9, 1915, is recorded a paper by W. G. Blair, Esq., entitled ''Luminous Insects " in which reference is made to the remarkable synchron- ism of the flashes in certain species of Euro- pean fireflies. A somewhat extended extract is given from Mr. Blair's address. A copy of this paper was sent to my friend Professor E. B. Poulton, of Oxford, and in return he has sent me a proof sheet from a book he is editing entitled " A Naturalist in Borneo" by R. Shelford, who died a few years ago, a former assistant of Professor Poulton. I am taking the liberty of presenting an extract from this advanced page : The statement in Tlze Scientific Monthly article that E,. Lester Jones "has proved to be an efficient executive" was probably made without intimate knowledge of the facts. It is well known in the bureau of fisheries that just the reverse was true, as was clearly shown by the very extravagant and unbusiness-like administration of Alaska fishery matters of which Mr. Jones took entire charge. Two or three illustrations may be given. It is under- stood that the sending of supplies to the seal islands under Mr. Jones's management cost the government several thousand dollars more than it had cost before, and yet the natives suffered severely for want of food. On the opposite bank was a small tree growing close to the water's edge, which was covered with thousands of fire+flies, small beetles of the family Lampyrida?; and I observed that the light emitted by these little creatures pulsated in a regular synchronous rhythm, so that at one mo- ment the tree would be one blaze of light, whilst at another the light would be dim and uncertain. This concerted action of thousands of insects is very remarkable and not easy of explanation. BARTONWARRENEVERMAN BARTONWARRENEVERMAN PRESIDENT WILSON'S SCIENTIFIC APPOINT- MENTS Smith possessed even elementary knowledge of fii;hes or fisher- ies; it was apparent that neither could tell a salmon from a sucker. Each of them inade at least oue tour of inspection of the Alaslia fisheries, bringing discredit upon the bureau wherever they went, so lacliing mere they in linowledge or apprecPdtion of the problems of the fisheries. The voluminous and profusely illustrated report by the deputy commissioner will probably nevc'r be cxcelled in the number of inaccuracies, absurd statements, fairy stor- ies arid erroneous conclusions it contains. The report contained n number of rcxcom- me~ldatione, the most important of which was the immediate repeal of the law whic11 pro- hibits commercial killing of seals, and for which Mr. ltedfield had voted and which he had said, as late as October 13, 1913, ara3" :I sou~id and wise one." Dr. Parlccr and his associates submitted their report to the commissioner of fisherieq 011 January 23, 1915, by whom iL was proml~tly transmitted to Secretary Redfield on Jan ualy 25. Although the report contained reconl- mendations of ~ ~ i t a l importance to the fur-seal herd, which if actccl upon promptly would saTc hundreds of thousands of dollars to the government as well as save the seal herd From irreparable injury, Mr. Redfield pigeor~lloled the report for more than three weeks and did not transmit it to congress until February 17, only a few days before congress adjourned. And, very strangely, and to the great disap- pointmcrrt of the commission, Mr. Redfic4d studiously refrained from calling attention to any of tlze reconlmendations of the commis- sioil; nor did lie make any recommenclatio~ hiniself that congress sllould takc any action One other case may be mentioned, one with which the National Acadr:m;y of Sciences is c.oncerned. In the spring of 1914 the admin- istration decided to sencl a special comniissioli of zoologists to the seal islands of Alaska. The secretary of commerce, when a meniber of congress, had voted for a hill which pro- hibits all commercial Irillil~g of fur srals for five years in spite of the fact that every zool- ogist in America, England, Russia and Japan who had studied our fur-seal herd adriscd against sucl? a conrse. Having taken ii position favoring the sus- pension of commercial Billiilg the secretary SCIENCE SEPTEMBER 15, 19161 387 on the recommendations of the commission. PRESIDENT WILSON'S SCIENTIFIC APPOINT- MENTS In fact, it is understood that it was Mr. Red- field's desire that congress should not take any action. He wholly ignored, and wished con- gress to ignore, the recommendations of the commission named by the National Academy of Sciences, the secretary of the Smithsonian Institution and the secretary of agriculture. I t would be proper for the National Academy of Sciences, the official adviser of the govern- ment on scientific matters, to ask the president what action, if any, has been taken on the recommendations of the board which it as- sisted in naming; and if called upon again for expert advice, the academy would do well to inquire whether any attention would be paid to its advice when given. on the recommendations of the commission. In fact, it is understood that it was Mr. Red- field's desire that congress should not take any action. He wholly ignored, and wished con- gress to ignore, the recommendations of the commission named by the National Academy of Sciences, the secretary of the Smithsonian Institution and the secretary of agriculture. I t would be proper for the National Academy of Sciences, the official adviser of the govern- ment on scientific matters, to ask the president what action, if any, has been taken on the recommendations of the board which it as- sisted in naming; and if called upon again for expert advice, the academy would do well to inquire whether any attention would be paid to its advice when given. fisheries a thoroughly business-like adminis- tration, during which &ore real productive scientific work was done than ever before by the bureau. FIREFLIES FLASHING IN UNISON Another instance of it was mentioned by COX certain ants that are found very frequently pro- ceeding in columns' along the floor of the jungle, when alarmed, knock their heads against the leaves or dead sticks which they happn to be traversing; every member of a community makes the necessary movement at the same time, and as the movements are rapid a distinct loud rattling sound is heard. In this case the action is prob- ably a danger-signal, and we can understand- theoretically at any rate-how it was brought A certain important scientific investigation of the Alaska salmon, begun in 1910 and which required at least six years to reach conclusive results, was stopped in 1914, thus breaking the continuity of the investigation, with the result that the whole thing must be done over again if the results are to be of any vtxlue. If these illustrations of inefficiency are not enough, inquiry might be made regarding the boat Roosevelt purchased by Mr. Jones for the Alaska service. But if the appointment of a politician to the head of a scientific bureau is justified because the appointee proves to be a good executive,. then President McKinley's appointment of Mr. Bowers as Commissioner of Fisheries is fully justified, as Mr. Bowers proved to be an excellent executive, who gave the bureau of
https://openalex.org/W4210525985	https://refubium.fu-berlin.de/bitstream/fub188/34179/2/Materials%20Corrosion%20-%202022%20-%20Kingsbery%20-%20Effect%20of%20KCl%20deposits%20in%20high-temperature%20corrosion%20on%20chromium-rich%20steels%20in.pdf	English	null	Effect of KCl deposits in high‐temperature corrosion on chromium‐rich steels in SO<sub>2</sub>‐containing atmosphere	Materials and corrosion	2,022	cc-by	8,145	Funding information Deutsche Forschungsgemeinschaft, Grant/Award Number: DFG‐416318834 Deutsche Forschungsgemeinschaft, Deutsche Forschungsgemeinschaft, Grant/Award Number: DFG‐416318834 K E Y W O R D S high‐temperature corrosion, KCl, microstructure, SO2, steel alloy Phillip Kingsbery1,2 \| Christiane Stephan‐Scherb1,2 1Department of Earth Science, Freie Universität Berlin, Berlin, Germany 2Division of Materialography, Fractography and Ageing of Engineered Materials, Bundesanstalt für Materialforschung und ‐prüfung, Berlin, Germany This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Materials and Corrosion published by Wiley‐VCH GmbH. Abstract High‐temperature corrosion was studied under multiple chemical loads on ferritic‐austenitic model alloys (Fe–13Cr, Fe–18Cr–12Ni, and Fe‐25Cr–20Ni) with KCl deposit under 0.5% SO2/99.5% Ar gas atmosphere at 560°C. Post- exposure characterization was done by X‐ray diffraction and scanning electron microscopy. In a pure SO2/Ar environment a protective Cr O 2 3 scale was formed by all samples. The introduction of KCl deposits causes the scale to be nonprotective and multilayered, consisting of CrS, FeS, Cr O , Fe O 2 3 3 4, and Fe O 2 3. The impact of the microstructure and alloying elements is discussed. Correspondence Phillip Kingsbery, Department of Earth Science, Freie Universität Berlin, Malteserstraße 74‐100, 12247 Berlin, Germany. © 2022 The Authors. Materials and Corrosion published by Wiley‐VCH GmbH. Correspondence Email: phillip.kingsbery@fu-berlin.de Received: 2 November 2021 \| Accepted: 9 December 2021 DOI: 10.1002/maco.202112901 1 \| Accepted: 9 December 2021 Received: 2 November 2021 DOI: 10.1002/maco.202112901 Effect of KCl deposits in high‐temperature corrosion on chromium‐rich steels in SO2‐containing atmosphere Phillip Kingsbery1,2 Phillip Kingsbery1,2 \| Christiane Stephan‐Scherb1,2 high‐temperature corrosion, KCl, microstructure, SO2, steel alloy This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2.2 \| Sample pretreatment and exposure The samples were cut into 10 × 20 × 3 mm coupons and ground to Grit 600/P1200. The samples were exposed at 560°C for 330h to a vo- lumetric gas mixture of 0.5% ∕ SO 99.5% 2 Ar (pS = 3. 9 × 10 , pO = 1. 1 × 10 2 −11 2 −16, calculated using FactSage 8.0) in a tubular furnace. Exposure experiments with and without KCl deposits were performed. The at- mosphere and flow rate are given in Table 2. A schematic overview of the experimental setup is shown in Figure 1. The gas intake is regulated by a flow controller to ensure a well‐defined gas mixture inside the furnace with a constant flow rate. The goal of this study is to gain an understanding of the underlying mechanism of high‐temperature corro- sion, especially in the presence of salt deposits, con- sidering active chloride corrosion, oxidation, and sulfidation. Furthermore, the impact of Cr and Ni in such environments on corrosion behavior is investigated. For the experiments including the KCl deposit, the samples were placed in alumina crucibles and partially embedded in KCl before being placed into the furnace (see Figure 1b). The crucibles and KCl were both dried in an oven for 1 day at 250°C before each experiment to purify the KCl from any adsorbed water according to DIN ISO 17224.[9] KCl + H O 2 atmosphere. The lower corrosion rate was linked to the formation of K SO 2 4, which does not attack the protective Cr O 2 3 scale to form K CrO 2 4, as is the case for an SO2‐free atmosphere. is chosen as an intermediary alloy between Fe–13Cr and Fe–25Cr–20Ni, to show the effect of increasing Cr and Ni content and is comparable to alloy TP 347H. The studies on KCl induced corrosion, coupled with the humid atmosphere and under the influence of SO2 did not include the consideration of different alloy mi- crostructures (for instance ferritic–ferritic/austenitic). However, the impact of KCl in a dry, SO2 containing atmosphere has not been studied. As SO2 is a product of biomass combustion it is crucial to understand the cor- rosion mechanism of boiler tubes in SO2 bearing atmo- spheres with and without KCl deposits. To gain insight into the corrosion mechanism this study will focus on model alloys derived off of commercially used steels for boiler tubes: X20, TP 347H, and HR3C.[8] 1 \| INTRODUCTION and oxidizing atmospheres. Hernas et al.[3] found that for steels with Cr contents between 8.96 wt% and 18.5 wt% the corrosion resistance increases with increasing Cr content in an N + 9% O + 0.2% HCl + 0.08% SO 2 2 2 atmosphere at 600°C and 700°C. The environmental impact of power generation has become one of the most important topics of the 21st century. One initiative to reduce the use of fossil fuels has been co‐firing of coal power plants using biofuel, which consists of or- ganic matter such as straw and wood. The use of biofuel presents new challenges to the materials used as boiler tubes due to the different chemical environments. The chemistry of biomass, wood, and straw, for example, is quite different from fossil fuels. In Germany, the main quantity of biomass types used for energy fabrication is ligneous fuels. Besides the main constituents carbon, oxy- gen, and hydrogen, a significant amount of KCl (e.g., straw: up to 1 wt%) can be present, which is known to cause se- vere deposit‐induced corrosion.[1,2] The effect of KCl deposits on the corrosion mechan- ism in a humid atmosphere has been studied by Lehmusto et al.[4] on Sanicro 28 at 550°C (20% O2; 10% H O 2 volumetric mixture), Pettersson[5] on Fe‐18Cr‐10Ni at 600°C (5% O2; 40% H O 2 volumetric mixture) and Jonsson et al.[6] on 304L at 600°C (5% O2; 40% H O 2 vo- lumetric mixture). In the presence of oxygen and water, the KCl attacks Cr O 2 3 to form K CrO 2 4, destroying the protective chromia scale. Karlsson et al.[7] studied the influence of SO2 and KCl on the initial stages of corrosion of stainless steel 304L at 600°C in a volumetric mixture of 0.5% O2 and 40% H O 2 .[7] The study by Karlsson et al.[7] showed, that the addition of SO2 in a wet atmosphere leads to a decreased corrosion rate, in comparison to a The corrosion resistance of high‐temperature alloys used in energy applications depends on the Cr‐content. Cr affects the corrosion resistance of steels in sulfidising 758 \| www.matcorr.com Materials and Corrosion. 2022;73:758–770. 758 \| www.matcorr.com KINGSBERY AND STEPHAN‐SCHERB 759 2.3 \| Sample post‐characterization (a) shows the oxidation simulation facility and (b) the way the sample is mounted within the crucible FIGURE 1 Schematic drawing of experimental setups. (a) shows the oxidation simulation facility and (b) the way the sample is mounted within the crucible FIGURE 1 Schematic drawing of experimental setups. (a) shows the oxidation simulation facility and (b) the way the sample is mounted within the crucible ic drawing of experimental setups. (a) shows the oxidation simulation facility and (b) the way the sample i ibl FIGURE 2 Scanning electron microscopy‐backscattered electron images showing the surface of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni FIGURE 2 Scanning electron microscopy‐backscattered electron images showing the surface of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni finished with a 1μm diamond suspension. For the KCl exposed samples, the entire preparation was done water‐ free, to preserve possible soluble phases. These samples were finished using P4000 SiC paper instead of the dia- mond suspension. through the scale indicating a thicker scale compared to Fe–13Cr. Individual spots show spallation of the oxide scale. Fe–25Cr–20Ni shows a continuous and smooth sur- face scale. However, the sample shows multiple areas with spallation. The cross‐sections were carbon‐coated to improve conductivity before analysis by SEM and energy dis- persive X‐ray analysis (EDX) using a Zeiss Sigma 300 VP and the equipped Quantax Xflash 60mm2 detectors. Line scans were produced utilizing the EDX map data using Bruker's Esprit program. 3 \| RESULTS Fe–18Cr–12Ni (Figure 3b) and Fe–25Cr–20Ni (Figure 3c show a thin, continuous layer (<1μm) as well as some islands. Compared to Fe–13Cr, increasing the Cr content transforms the corrosion islands into a continuous layer, increasing the scale thickness (see Figure 3c). 3.1.2 \| Cross‐section analysis The SEM‐BSE cross‐section images of samples exposed to SO2 are displayed in Figure 3. Fe–13Cr (Figure 3a) shows a very thin (<1μm) oxide layer and comparatively larger islands (~2μm). 2.3 \| Sample post‐characterization This study focuses on three model alloys purchased from HMW Hauner GmbH. All samples were vacuum‐melted, mixed, hot, and cold rolled with minimum Fe purity of 99.95% and Cr, Ni purities of 99.9%. The chemical com- position and phase of the microstructure are given in Table 1. α denotes the body‐centered cubic phase of iron (ferrite), γ the face‐centered cubic phase (austenite). The microstructure was determined through light microscopy of the etched samples. After gas exposure, the surface of samples with KCl de- posit was characterized by X‐ray diffraction (XRD) using a Panalytical Empyrean diffractometer utilizing Cu K‐α X‐rays. The as‐reacted KCl deposit was scraped off the alloys, together with the scale, and turned into a powder. This mixture was then analyzed in theta–2theta geometry. The utilized custom alloys are similar in composition to commercially used ones, namely, Fe–13Cr to X20 and Fe–25Cr–20Ni to HR3C.[8] The custom alloy Fe–18Cr–12Ni Additionally, the surface was analyzed using scan- ning electron microscopy (SEM). Images of samples ex- posed to 0.5% ∕ SO 99.5% 2 Ar were obtained using a Tescan Vega 3 (Fe–13Cr) and Zeiss Sigma 300 VP (Fe–18Cr–12Ni, Fe–25Cr–20Ni). Samples exposed to KCl deposits were analyzed using a Zeiss Sigma 300 VP. The acceleration voltage was 20 kV for all samples. TABLE 1 Elemental composition of studied samples obtained by electron microprobe analysis on a JEOL JXA 8200 Superprobe utilizing ZAF correction, values given in wt% TABLE 1 Elemental composition of studied samples obtained by electron microprobe analysis on a JEOL JXA 8200 Superprobe utilizing ZAF correction, values given in wt% Metallographic cross‐sections were prepared by em- bedding the samples into an epoxy resin, ground, and Sample Fe Cr Ni Phase Fe–13Cr Bal. 13.5 ± 0.4 0.0 ± 0.0 α Fe–18Cr–12Ni Bal. 18.5 ± 0.5 11.8 ± 0.2 α + γ Fe–25Cr–20Ni Bal. 24.5 ± 0.7 19.1 ± 0.4 α + γ TABLE 2 Corrosive atmosphere used in this study, values given in vol% Ar SO2 Flow rate (m/s) Corrosive atmosphere 99.5 0.5 1.4–1.5 × 10−2 TABLE 2 Corrosive atmosphere used in this study, values given in vol% Note: ZAF is the abbreviation for the three effects impacting the characteristic X‐ray intensity when performing quantitative analysis: atomic number(Z), absorbtion(A) and fluorescence(F). KINGSBERY AND STEPHAN‐SCHERB 760 FIGURE 1 Schematic drawing of experimental setups. 3.2 \| 0.5% ∕ SO 99.5% 2 Ar + KCl Fe–18Cr–12Ni shows a homogeneous Fe, Cr, and Ni distribution in the base alloy. Just below the alloy/scale interface a larger Cr depletion zone (~1μm) is observed, while the scale consists of Cr O 2 3. 3.1.1 \| Surface structure and morphology postexposure (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 5 Backscattered electron surface images of samples (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C FIGURE 5 Backscattered electron surface images of samples (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C 3.1.1 \| Surface structure and morphology postexposure Initially, the effect of chromium content on oxidation/sul- fidation (without KCl deposit) was investigated. The SEM backscattered electron (BSE) surface images of samples after exposure to 0.5% ∕ SO 99.5% 2 Ar are shown in Figure 2. Initially, the effect of chromium content on oxidation/sul- fidation (without KCl deposit) was investigated. The SEM backscattered electron (BSE) surface images of samples after exposure to 0.5% ∕ SO 99.5% 2 Ar are shown in Figure 2. The line scans in Figure 4 show the elemental variation from the base alloy (left side) to the surface (right side). Fe–13Cr shows constant Fe and Cr levels in the bulk alloy. Towards the surface, a small area (<1μm) of Cr depletion below the alloy/scale interface is detected. The Cr depletion zone is the distance from the point where Cr levels fall below the nominal Cr amount to the surface of the alloy. The scale consists of Fe, Cr, and O. Sulfur was not detected by SEM‐EDX. p ∕ g Fe–13Cr shows only slight surface damage by corro- sion. The corrosion islands are localized close to scrat- ches, which are artifacts of sample pretreatment. Fe–18Cr–12Ni developed a large area showing oxide islands on the surface. Scratch marks cannot be seen KINGSBERY AND STEPHAN‐SCHERB 761 FIGURE 3 Scanning electron microscopy‐backscattered electron images of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni FIGURE 3 Scanning electron microscopy‐backscattered electron images of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni FIGURE 4 Line scans obtained by energy dispersive X‐ray analysis of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 4 Line scans obtained by energy dispersive X‐ray analysis of samples exposed to 0.5% ∕ SO 99.5% 2 Ar for 330 h at 560°C. 3.2.1 \| Surface structure and morphology— post‐exposure Fe–25Cr–20Ni shows constant amounts of Fe, Cr, and Ni. However, 6μm below the surface a local enrich- ment of Cr at the cost of Fe and Ni is observed. This might be an artifact of insufficient homogenization during alloy preparation. Fe–25Cr–20Ni shows the deepest Cr depletion zone of all samples. The scale consists of Cr O 2 3. The SEM‐BSE surface images of samples exposed to KCl deposits and 0.5% SO2 are depicted in Figure 5. Fe–13Cr shows crystals of KCl and a densely packed, fiber‐like iron oxide. Cracks can be observed along the surface. KINGSBERY AND STEPHAN‐SCHERB 762 were not identified in the scale of Fe–13Cr by XRD analysis. Fe–18Cr–12Ni shows KCl crystals overgrown with individual needles of iron oxide. The iron oxide seems to grow in oriented plates. The thin side is oriented towards the surface with large vacancies in between iron oxide plates. Fe–18Cr–12Ni and Fe–25Cr–20Ni show a very similar diffraction pattern. Both show reflections corresponding Fe O , Cr O , Fe O 2 3 2 3 3 4, KCl, and K SO 2 4. The surface of Fe–25Cr–20Ni after SO2 exposure shows similar morphology to Fe–18Cr–12Ni. KCl crystals are observed with iron oxide needles overgrowing them. The KCl on top of the samples shows several cracks. The Fe–Cr–oxide (marked in Figure 5c) is denser than the iron oxide next to it. The iron oxide shows the same oriented plates as seen in Fe–18Cr–12Ni, the Fe–Cr‐oxide grows randomly and does not show a clear orientation. 4 \| DISCUSSION The sample shows Cr depletion along grain bound- aries in the base alloy (see Figure 9d). At the alloy/scale interface S, Fe, and Cr are enriched (Figure 9f,b,d). Above the S layer, a high O concentration was observed (Figure 9e). At the former gas side interface, KCl is covering the scale (Figure 9c,g). 3.2.2 \| Cross‐section analysis The SEM‐BSE cross‐section images of KCl‐exposed samples are shown in Figure 8 for comparison. All g y Figure 6 shows the surface EDX of Fe–13Cr after exposure to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. The surface shows two distinct structures: large crystals and a very porous phase below. FIGURE 7 X‐ray diffraction of 0.5% ∕ SO 99.5% 2 Ar + KCl‐ exposed samples Figure 6 shows the surface EDX of Fe–13Cr after exposure to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. The surface shows two distinct structures: large crystals and a very porous phase below. Figure 6a shows that the crystals are partially over- grown by a darker phase. Cross‐referencing that with the elemental maps in Figure 6 shows that the darker phase consists of K, O, and S. The crystals underneath KCl. The porous phase below and next to the crystals seem to consist of mainly iron oxides with some K and Cl being detected (see Figures 6b,c,d,f). The model alloys Fe–18Cr–12Ni and Fe–25Cr–20Ni show a similar surface composition, as verified by XRD (see Figure 7). FIGURE 7 X‐ray diffraction of 0.5% ∕ SO 99.5% 2 Ar + KCl‐ exposed samples The scale of Fe–13Cr mainly consists of Fe O 2 3 and KCl, as well as K SO 2 4 (see Figure 7). Fe O 3 4 and Cr O 2 3 FIGURE 6 Surface scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–13Cr exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C, showing KCl overgrown by K SO 2 4. (a) shows the backscattered electron image of the surface and (b–f) the element maps [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 6 Surface scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–13Cr exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C, showing KCl overgrown by K SO 2 4. 3.2.2 \| Cross‐section analysis This is followed by a Ni enrichment towards the alloy/scale interface at the cost of Fe and Cr, reaching Ni contents of up to 50 wt% at the interface (see Figure 11i). The scale at the alloy/scale interface consists of Cr, S, and O (see Figure 11d,f,e) Towards the former gas side Fe concentration increases at the expense of Cr (Figure 11b,d). S is only found close to the alloy/scale interface, as CrS closer to the metal and as FeS in the outer layer. The scale/gas interface consists of Fe and O. Similar to Fe–18Cr–12Ni, Fe–25Cr–20Ni (Figure 11i) shows areas of chloride formation deep within the bulk alloy. This is followed by a Ni enrichment towards the alloy/scale interface at the cost of Fe and Cr, reaching Ni contents of up to 50 wt% at the interface (see Figure 11i). The scale of Fe–25Cr–20Ni (Figure 8c) is layered and shows subsurface damage similar to Fe–18Cr–12Ni. An in- ternal corrosion attack caused a zone of rough, dark gray patches and an extensive corrosion network closer to the alloy/scale interface. The scale seems to consist of several layers though, more closely resembling Fe–13Cr's layered scale. It shows a dark layer close to the alloy/scale interface and a porous surface towards the scale/atmosphere interface. The scale at the alloy/scale interface consists of Cr, S, and O (see Figure 11d,f,e) Towards the former gas side Fe concentration increases at the expense of Cr (Figure 11b,d). S is only found close to the alloy/scale interface, as CrS closer to the metal and as FeS in the outer layer. The scale/gas interface consists of Fe and O. Figure 9 shows the cross‐section SEM‐EDX of elemental distribution maps of Fe–13Cr after exposure to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. 3.2.2 \| Cross‐section analysis (a) shows the backscattered electron image of the surface and (b–f) the element maps [Color figure can be viewed at wileyonlinelibrary.com] KINGSBERY AND STEPHAN‐SCHERB 763 FIGURE 8 Scanning electron microscopy‐backscattered electron images of samples (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni exposed to 0.5% SO2/99.5% Ar + KCl for 330 h at 560°C FIGURE 8 Scanning electron microscopy‐backscattered electron images of samples (a) Fe–13Cr, (b) Fe–18Cr–12Ni, and (c) Fe–25Cr–20Ni exposed to 0.5% SO2/99.5% Ar + KCl for 330 h at 560°C samples show a complex, multicomponent scale, that exceeds 100μm. The linescan in Figure 10i identifies the dark spots at around 300μm to be FeCl2. The linescan furthermore shows a Fe and Cr depletion and a Ni enrichment towards the alloy/scale interface. The areas enriched in Ni show higher amounts of O. Fe–13Cr (Figure 8a) shows a rough alloy/scale in- terface and only very little damage to the bulk alloy. The corrosion attack in the alloy seems to be limited to an area close to the alloy/scale interface along grain boundaries. The scale shows several distinct phases. The area closest to the alloy shows a rough morphology with small droplets. Further outward a thin and dark layer can be observed, separating the rough area from the large, light gray area, which ends in a porous, needle‐like area. The scale shows Cr enrichment at the alloy/scale interface along with O or Cl. Notably, this sample shows a thick and porous ∕ Cr O CrCl 2 3 3 layer (d ~ 80μm) close to the alloy/scale interface (seen in Figure 10d,e,g). Fe is predominantly found at the scale/atmosphere interface in conjunction with O. Figure 11 shows the cross‐section SEM‐EDX elemental distribution maps of Fe–25Cr–20Ni after exposure to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. Fe–18Cr–12Ni (Figure 8b) shows an extensive corro- sion attack in the base alloy (henceforth called internal corrosion attack). Large patches of a rough, dark gray area can be seen within the alloy, as well as a large in- ternal corrosion network. The scale consists of multiple components, which do not exhibit an orderly layering but instead are concentrated in different regions. Similar to Fe–18Cr–12Ni, Fe–25Cr–20Ni (Figure 11i) shows areas of chloride formation deep within the bulk alloy. 4.1 \| Main results The experiments show the growth of a thin scale in a 0.5% ∕ SO 99.5% 2 Ar atmosphere for all samples (Figure 12). The scale consists of Cr O 2 3 and Fe O 2 3 in the case of Fe–13Cr. Both higher alloyed samples formed Cr O 2 3 instead and showed spallation of the scale. Figure 10 shows the cross‐section SEM‐EDX ele- mental distribution maps of Fe–18Cr–12Ni after ex- posure to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. KINGSBERY AND STEPHAN‐SCHERB 764 FIGURE 9 Cross section scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–13Cr exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–g) element maps. (h) shows the linescan from the base alloy to the gas [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 9 Cross section scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–13Cr exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–g) element maps. (h) shows the linescan from the base alloy to the gas [Color figure can be viewed at wileyonlinelibrary.com] 4.2 \| 0.5% ∕ SO 99.5% 2 Ar atmosphere 4.2.1 \| Influence of Cr and microstructure • Thin oxide scale on all samples • Spallation of the scale 4.2.1 \| Influence of Cr and microstructure 4.2 \| 0.5% ∕ SO 99.5% 2 Ar atmosphere • Thin oxide scale on all samples 4.2.1 \| Influence of Cr and microstructure Samples exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl show a large corrosion attack with scales exceeding 100μm (see Figure 12). The addition of KCl leads to the formation of K SO 2 4 as well as an extensive scale of iron oxides, Cr O 2 3, FeS, and CrS. The SEM‐EDX linescan on the cross‐section of Fe–13Cr after gas exposure (Figure 4a) shows that Fe–13Cr formed a scale consisting of Fe and Cr oxides. Increas- ing the Cr content to 18 wt% (Fe–18Cr–12Ni) or higher caused Cr O 2 3 formation (see Figure 4b,c). The increasing amount of Cr in the corrosion product is clearly linked to the increasing Cr content in the bulk material. All sam- ples show a Cr depletion zone just under the scale, which increases with increasing Cr content. This is likely due to Cr O 2 3 formation needing more Cr to form compared to a mixed Fe and Cr oxide scale. The microstructure is also noteworthy being different for the three alloys. Fe–13Cr is purely ferritic as opposed to the ferritic–austenitic microstructure of Fe–18Cr–12Ni and Fe–25Cr–20Ni (see Table 1). Calculating the diffusion coefficient at 560°C, in a matrix made purely of Fe, using the formulas (see The alloys Fe–18Cr–12Ni and Fe–25Cr–20Ni show internal corrosion, forming Fe‐ and Cr chlorides and enriching the alloy/scale interface in Ni. • Large multicomponent scale consisting of FeS, CrS, Fe O , Fe O 3 4 2 3, and Cr O 2 3 • Densely packed Fe O 2 3 at a surface of Fe–13Cr (ferri- tic), ferritic–austenitic samples showed a lower density • Ferritic–austenitic samples showed strong internal corrosion • Ferritic–austenitic samples showed Ni enrichment at the alloy/scale interface KINGSBERY AND STEPHAN‐SCHERB 765 FIGURE 10 Cross section scanning electron microscopy–energy dispersive X‐ray analysis elemental distribution maps of Fe–18Cr–12Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–h) element maps. (i) shows the linescan through the metal and (j) the linescan through the scale [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 10 Cross section scanning electron microscopy–energy dispersive X‐ray analysis elemental distribution maps of Fe–18Cr–12Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–h) element maps. 4.2.1 \| Influence of Cr and microstructure (i) shows the linescan through the metal and (j) the linescan through the scale [Color figure can be viewed at wileyonlinelibrary.com] Table 3) given by Buffington et al.,[10] Bowen et al.[11] and Hirano et al.[12] leads to the values displayed in Table 4. The diffusion coefficients show, that every metal present in the model alloys diffuses much quicker in a ferritic matrix as compared to an austenitic one. This explains the thinner Cr depletion zone in the ferritic Fe–13Cr and the larger Cr depletion in the ferritic–austenitic Ni‐ bearing steels. the exposure. This is surprising, considering neither KCl nor K SO 2 4 melt at 560°C.[13] Okoro et al.[13] observed the same phenomenon and explained it through the formation of a eutectic mixture of KCl and Fe or Cr chlorides, where melting temperatures were observed between 200°C and 500°C.[14] 4.3 \| 0.5% ∕ SO 99.5% 2 Ar + KCl Overall Ni‐bearing samples seem to be more susceptible to corrosion attack. Comparing the internal corrosion of Fe–13Cr and Fe–18Cr–12Ni in Figure 8a,b, the internal corrosion affects a much larger zone and leads to dif- ferent corrosion products if Ni is present. A similar result can be observed for Fe–25Cr–20Ni in Figure 8c. 4.3.1 \| Sample surface The salt bearing surface shown in Figure 6 is re- presentative of all studied samples. K SO 2 4 overgrows the KCl crystals when exposed to SO2. K SO 2 4 was also con- firmed in all samples by the XRD experiments, as shown in Figure 7. The shape of the grain boundaries and the smooth texture of K SO 2 4 indicate that it melted during Figure 13a shows the location of detected chlorides within the base alloy. The penetration depth denotes the distance from the alloy/scale interface to the position of the chlorides. KINGSBERY AND STEPHAN‐SCHERB 766 FIGURE 11 Cross section scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–25Cr–20Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–h) element maps. (i) shows the linescan through the metal and (j) the linescan through the scale [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 11 Cross section scanning electron microscopy‐energy dispersive X‐ray analysis elemental distribution maps of Fe–25Cr–20Ni exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl for 330 h at 560°C. (a) shows the backscattered electron image and (b–h) element maps. (i) shows the linescan through the metal and (j) the linescan through the scale [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 12 Comparison of the scale thickness developed in a pure 0.5% SO2 containing atmosphere compared to SO + KCl 2 Fe–13Cr did not form any chlorides within the base alloy. However, alloying with Ni caused chlorides to form within a zone in the alloy. For Fe–18Cr–12Ni, the chloride zone started at 225 ± 69μm and extended until 578 ± 134μm. Fe–25Cr–20Ni showed a much narrower zone from 235 ± 22μm to 313 ± 29μm. Figure 13b shows the average internal corrosion zone and the scale thickness. The internal corrosion zone grows noticeably in the Ni alloyed system. Fe–13Cr shows comparatively low internal corrosion at 61 ± 35μm. The introduction of Ni into the alloy leads to an internal corrosion zone of 1013 ± 85μm for sample Fe–18Cr–12Ni. Further increasing the amount of Ni and Cr reduces the internal corrosion area significantly to 314 ± 19μm in sample Fe–25Cr–20Ni. By comparison, the scale thickness only slightly varies between Fe–13Cr and Fe–18Cr–12Ni, however, Fe–25Cr–20Ni shows a thinner scale. 4.3.1 \| Sample surface Sample Fe–13Cr (at 0 wt% Ni in the graphs) is purely ferritic, the samples Fe–18Cr–12Ni and Fe–25Cr–20Ni are ferritic–austenitic KINGSBERY AND STEPHAN‐SCHERB \| 767 TABLE 3 Diffusion coefficient formulas for D (cm2/s), R = gas constant (J/Kmol)[10–12] Diffusing element Fe Cr Ni In α −Fe 2.0 × eR K −60000 ×833.15 8.52 × eR K −59900 ×833.15 1.4 × eR K −58700 ×833.15 In γ −Fe 0.18 × eR K −64500 ×833.15 10.8 × eR K −69700 ×833.15 0.77 × eR K −67000 ×833.15 TABLE 3 Diffusion coefficient formulas for D (cm2/s), R = gas constant (J/Kmol)[10–12] TABLE 4 Calculated diffusion coefficient D (cm2/s) at 560°C Diffusing element Fe Cr Ni In α −Fe 3.5 × 10−4 1.5 × 10−3 2.9 × 10−4 In γ −Fe 1.6 × 10−5 4.6 × 10−4 4.9 × 10−5 TABLE 4 Calculated diffusion coefficient D (cm2/s) at 560°C The negative effect of SO2 on the corrosion behavior of Ni‐based superalloys has been shown by both Huczkowski et al.[15] and Oleksak et al.[16] at tempera- ture ranges 550–650°C. Oleksak et al. saw mass changes in all studied samples at 600°C with a mass change factor of 2390 comparing 95% ∕ ∕ CO 4% H O 1% O 2 2 2 atmosphere to the same atmosphere with 0.1% SO2 in alloy 617. Huczkowski et al. and Oleksak et al. determined that the poor corrosion behavior is due to the transition from oxides to sulfates. Due to the enrichment of Ni at the alloy/scale interface for samples Fe–18Cr–12Ni and Fe–25Cr–20Ni (Figures 10 and 11i) it is reasonable to assume a similar behavior in these samples, however, sulfates replacing oxides was not observed. FIGURE 13 Effect of alloying elements on corrosion behavior. (a) Cl penetration depth and (b) zone affected by corrosion. Sample Fe–13Cr (at 0 wt% Ni in the graphs) is purely ferritic, the samples Fe–18Cr–12Ni and Fe–25Cr–20Ni are ferritic–austenitic On the basis of the distribution of Cr in Figures 9–11, the likely cause for the different internal oxidation be- havior is the density of the Cr layer. Fe–13Cr shows a thin but dense layer of Cr O 2 3 which provides a diffusion barrier for Cl. Comparing this to the Cr distribution in Fe–18Cr–12Ni (Figure 10d) it is evident that the Cr O 2 3 is not dense enough to protect the metal from further Cl corrosion. 4.3.1 \| Sample surface Fe–25Cr–20Ni shows a denser layer of Cr O 2 3. The dense Cr O 2 3 layer is a diffusion barrier for Cl, which is reflected in the smaller internal corrosion area is shown in Figure 13b. The beneficial effect of the in- creased amount of Cr outweighs the potential downside of increased Ni in the form of Ni‐sulfides or an increasing amount of austenite. Fe–18Cr–12Ni and Fe–25Cr–20Ni the difference in in- ternal corrosion area is evident (see Figure 13a). The impact of the microstructure of a Fe–16Cr–0.2C alloy on the corrosion behavior in an SO2 bearing at- mosphere has recently been studied by Falk et al.[18] The study showed that the microstructure impacts the formed product. A much thinner corrosion layer was formed on ferritic grains compared to bainitic ones which was at- tributed to much faster diffusion paths in bainite. The slower diffusion of Fe and Cr in austenite, as shown in Table 4, is likely a reason for the comparatively poor performance of the ferritic–austenitic alloys com- pared to the ferritic Fe–13Cr. The diffusion coefficient at 560°C is an order of magnitude larger in ferrite compared to austenite. The effect of the lower diffusion velocity, at a constant exposure time of 330 h, can be observed in the linescans. Fe–13Cr (Figure 9h) shows no Cr depletion. Both Fe–18Cr–12Ni (Figure 10i) and Fe–25Cr–20Ni 4.3.1 \| Sample surface FIGURE 12 Comparison of the scale thickness developed in a pure 0.5% SO2 containing atmosphere compared to SO + KCl 2 towards Ni being a detriment to the corrosion resistance of deposit‐induced corrosion. The presence of Ni leads to the formation of both chlorides as well as nickel oxides in the base alloy. The results from Figure 13 in combination with the Ni enrichment observed in Figures 10 and 11i point KINGSBERY AND STEPHAN‐SCHERB 767 \| 767 The negative effect of SO2 on the corrosion behavior of Ni‐based superalloys has been shown by both Huczkowski et al.[15] and Oleksak et al.[16] at tempera- ture ranges 550–650°C. Oleksak et al. saw mass changes in all studied samples at 600°C with a mass change factor of 2390 comparing 95% ∕ ∕ CO 4% H O 1% O 2 2 2 atmosphere to the same atmosphere with 0.1% SO2 in alloy 617. Huczkowski et al. and Oleksak et al. determined that the poor corrosion behavior is due to the transition from oxides to sulfates. Due to the enrichment of Ni at the alloy/scale interface for samples Fe–18Cr–12Ni and Fe–25Cr–20Ni (Figures 10 and 11i) it is reasonable to assume a similar behavior in these samples, however, sulfates replacing oxides was not observed. On the basis of the distribution of Cr in Figures 9–11, the likely cause for the different internal oxidation be- havior is the density of the Cr layer. Fe–13Cr shows a thin but dense layer of Cr O 2 3 which provides a diffusion barrier for Cl. Comparing this to the Cr distribution in TABLE 3 Diffusion coefficient formulas for D (cm2/s), R = gas constant (J/Kmol)[10–12] Diffusing element Fe Cr Ni In α −Fe 2.0 × eR K −60000 ×833.15 8.52 × eR K −59900 ×833.15 1.4 × eR K −58700 ×833.15 In γ −Fe 0.18 × eR K −64500 ×833.15 10.8 × eR K −69700 ×833.15 0.77 × eR K −67000 ×833.15 TABLE 4 Calculated diffusion coefficient D (cm2/s) at 560°C Diffusing element Fe Cr Ni In α −Fe 3.5 × 10−4 1.5 × 10−3 2.9 × 10−4 In γ −Fe 1.6 × 10−5 4.6 × 10−4 4.9 × 10−5 FIGURE 13 Effect of alloying elements on corrosion behavior. (a) Cl penetration depth and (b) zone affected by corrosion. oxidizing environments, the formation of K CrO 2 4 was observed.[4,5,7] (Figure 11i) show a severe Cr depletion towards the al- loy/scale interface. This is likely due to the slower dif- fusion of Fe and Cr in the austenite, which is detrimental in the event of spallation. Experiments were conducted with comparable steel (304L, Fe–18Cr–10Ni) at 600°C by Pettersson[5] in an oxidizing atmosphere (5% ∕ O 95% N + KCl 2 2 ) and Karlsson et al.[7] in a humid atmosphere (5% ∕ O2 ∕ ∕ 40% H O 300 ppm SO N + KCl 2 2 2 ) showed that the in- itial reaction involved the destruction of Cr O 2 3 to form K CrO 2 4. In the present study, no K CrO 2 4 could be de- tected, presumably due to the lower amount of oxygen (0.5% SO2 [pO = 1 × 10 2 −16] vs. Pettersson's 5% O2[SO = 5 × 10 2 ‐2]) available. Instead, K SO 2 4 was ver- ified through EDX and XRD. In Karlsson's humid atmosphere[7] K SO 2 4 was also identified as the reaction product of K CrO 2 4 and SO2. The microstructure also has an impact on the grain orientation and density of the formed corrosion product. The samples exposed to 0.5% ∕ SO 99.5% 2 Ar + KCl show highly oriented grains at the surface, in particular for the partially austenitic samples Fe–18Cr–12Ni and Fe–25Cr–20Ni (see Figure 5b,c). The orientation relation of ferrite, magnetite, and he- matite has been shown previously by Stephan‐Scherb et al.[19] The orientation of hematite is therefore likely due to the orientation of the phase below it. The large gaps between hematite plates could therefore be due to an en- ergetically unfavorable orientation of the phase below, leading to selective hematite growth and a lower density. The impact can be seen in the internal corrosion (Figure 8), which shows a much stronger attack in the austenitic‐ ferritic samples (Fe–18Cr–12Ni and Fe–25Cr–20Ni) when compared to the ferritic Fe–13Cr. The density of the iron oxides in sample Fe–13Cr (see Figure 5a) is much higher than in samples Fe–18Cr–12Ni and Fe–25Cr–20Ni (Figures 5b,c). The higher density hinders the transport of Cl2 through the scale, limiting the corrosion attack. Besides K SO 2 4 no other sulfates were found in our experiments, likely due to the low oxygen partial pres- sure. A Factsage 8.0 calculation shows that at the given sulfur partial pressure, an oxygen partial pressure of 10−13 or higher would be required to form stable metal sulfates. Therefore, only metal sulfides are expected. This means, that in the presence of SO2 the KCl prefers to form K SO 2 4 instead of attacking Cr O 2 3. This supports the beneficial properties found for sulfur‐containing com- pounds in biofuel combustion. This could imply a better corrosion behavior of steels in atmospheres containing SO2. The increase of Ni at the alloy/scale interface is likely due to the formation of FeCl2 and CrCl2. NiCl2 is not formed, since the Gibbs energy is highest for NiCl2 (−174.2 kJ/mol at 600°C) in comparison to FeCl2 and CrCl2 (−232.1 and −286.0 kJ/mol at 600°C respec- tively).[20,21] This causes Ni to enrich at the interface which then partially reacts with available oxygen to form NiO. The Cl attack seems to depend on the micro- structure of the alloy. The ferritic sample shows very little internal corrosion, while the ferritic–austenitic samples showed a much more extensive internal corro- sion zone. Increasing the amount of Cr is beneficial for corrosion resistance, leading to a smaller internal corro- sion zone. This is in line with the results shown by Montero et al.,[22] who observed a Ni enrichment and showed that Ni‐based alloys demonstrated a lower cor- rosion resistance in lignite combustion than austenitic steels and the beneficial effect of Cr. Montero et al. at- tributed the detrimental effect of Ni to breakaway caused by NiS as well as NiSO4, which can promote hot corro- sion. This is counteracted by Cr through the protective properties of Cr O 2 3. Several authors[23–25] have found a beneficial effect through the addition of sulfates and sulfur. Aho et al.[23] showed that introducing Fe SO 2 4 reduces the mass flow of Cl through the formation of SO2, which can prevent Cl deposition or reduce high‐temperature chlorine‐induced corrosion. Davidsson et al.[24] showed, that elemental S and (NH ) SO 4 2 4 reduced KCl in the flue gas, leading to a reduction of KCl deposits. On the basis of the findings in this study and ther- modynamic calculations (using FactSage 8.0), the reac- tion of KCl and SO2 with chromium‐rich steels happens in multiple stages. Only the relevant phases are shown in the reactions below. First, the atmospheric SO2 reacts with the KCl de- posits on the sample surface. This leads to the formation of K SO 2 4, which was shown through EDX and XRD analysis in Figures 6 and 7. → SO + KCl 4 × 10 K SO + 2 × 10 Cl , 2 −10 2 4 −10 2 (1) dG = −1.2 × 10 J. −2 (2) → SO + KCl 4 × 10 K SO + 2 × 10 Cl , 2 −10 2 4 −10 2 ( (1) (1) (2) dG = −1.2 × 10 J. −2 (2) (2) This initial reaction provides Cl2, which can then at- tack the metal. 4.3.3 \| Impact of the microstructure Finally, the microstructure needs to be considered con- cerning its impact on the corrosion behavior, as the ad- dition of Ni changes the microstructure from purely ferritic to ferritic–austenitic.[17] When comparing the purely ferritic Fe–13Cr with the ferritic–austenitic KINGSBERY AND STEPHAN‐SCHERB 768 5 \| CONCLUSION CrCl2 and FeCl2 have a high evaporation pressure.[21] For FeCl2 considerable evaporation is expected above 536°C.[26] This study shows the impact of KCl in a 0.5% ∕ SO 99.5% 2 Ar environment on ferritic and ferritic‐austenitic steel alloys, highlighting the importance of the alloying ele- ments Cr and Ni on corrosion behavior. This study shows the impact of KCl in a 0.5% ∕ SO 99.5% 2 Ar environment on ferritic and ferritic‐austenitic steel alloys, highlighting the importance of the alloying ele- ments Cr and Ni on corrosion behavior. → FeCl (s) FeCl (g), 2 2 (5) → CrCl (s) CrCl (g). 2 2 (6) (5) All model alloys showed very little corrosion in a pure SO2 environment. They developed a thin, protective scale consisting of Cr O 2 3. Fe–13Cr additionally formed Fe O 2 3. All samples showed a small region of Cr depletion un- derneath the scale, which increased with increasing amounts of Cr in the alloy. This is likely due to the spallation that was observed with higher amounts of Cr as well as the Ni‐induced microstructure change[17] from purely ferritic to ferritic–austenitic, causing a slower diffusion rate. (6) CrCl2(g) and FeCl2(g) are then transported towards the surface, where the sulfur and oxygen partial pres- sures are high enough to initiate the reaction: → dG CrCl + SO 0 .7 CrCl (s) + 0 .2 Cr O + 1 .9 × 10 Cl +1 .7 × 10 S (g), = −1.3 × 10 J. 2 2 3 2 3 −6 2 −2 2 5 (7) → dG FeCl + SO FeCl (s) + 2 .4 × 10 Fe O + 1 .3 × 10 Cl + 4 .8 × 10 S (g), = −4 .7 × 10 J. 2 2 2 −5 2 3 −9 2 −6 2 4 (8) → dG CrCl + SO 0 .7 CrCl (s) + 0 .2 Cr O + 1 .9 × 10 Cl +1 .7 × 10 S (g), = −1.3 × 10 J. 2 2 3 2 3 −6 2 −2 2 5 (7) (7) KCl induced corrosion caused extensive damage to the alloy. The samples all showed a thick scale con- sisting of Fe and Cr oxides and sulfides. Ni‐containing samples showed a large internal corrosion zone be- neath the original sample surface. 4.3.4 \| Attack mechanism →x y z Fe Cr Ni + Cl FeCl (s) + CrCl (s) + Ni, x y z 2 2 2 Unlike the KCl corrosion in oxidizing atmospheres, the onset of Cl‐induced corrosion in SO2 is likely different. In (3) KINGSBERY AND STEPHAN‐SCHERB 769 FIGURE 14 Schematic overview of the proposed corrosion mechanism depending on the microstructure. α denotes ferritic steels (i.e., Fe–13Cr), α γ + the ferrite+austenite samples (i.e., Fe–18Cr–12Ni and Fe–25Cr–20Ni) KINGSBERY AND STEPHAN‐SCHERB \| 7 FIGURE 14 Schematic overview of the proposed corrosion mechanism depending on the microstructure. α denotes ferritic steels (i.e., Fe–13Cr), α γ + the ferrite+austenite samples (i.e., Fe–18Cr–12Ni and Fe–25Cr–20Ni) ≈ dG −2.5 × 10 J. 5 (4) (4) 5 \| CONCLUSION This was especially pronounced in the Fe–18Cr–12Ni sample, showing an internal corrosion layer thickness of up to 1 mm. The Ni‐containing samples showed a Ni enrichment towards the alloy/scale interface, which is attributed to extensive Cl attacks and the formation of NiO. Ni enrichment can lead to a poorer corrosion resistance through the formation of Ni‐sulfides and ‐sulfates.[15,16,22] → dG FeCl + SO FeCl (s) + 2 .4 × 10 Fe O + 1 .3 × 10 Cl + 4 .8 × 10 S (g), = −4 .7 × 10 J. 2 2 2 −5 2 3 −9 2 −6 2 4 (8) (8) The sulfur can then react with the alloy to form FeS, CrS and NiS. This reaction is responsible for the scale growth and provides the Cl needed to continue the active Cl corrosion, as described by Grabke,[27] with Cl2 attacking the base alloy and forming Fe and Cr chlorides. These transform into a gas to once again react towards the surface with SO2 to form new Fe and Cr oxides and set free Cl2. ORCID [19] C. Stephan‐Scherb, M. Menneken, K. Weber, L. AgudoJácome, G. Nolze, Corros. Sci. 2020, 174, 108809. Phillip Kingsbery http://orcid.org/0000-0002-0401-2450 Christiane Stephan‐Scherb http://orcid.org/0000-0001- 6775-0023 [20] H. J. Grabke, M. Spiegel, A. Zahs, Mater. Res. 2004, 7, 89. [21] A. Zahs, M. Spiegel, H. J. Grabke, Corros. Sci. 2000, 42, 1093. [22] X. Montero, M. Rudolphi, M. C. Galetz, Mater. Corros. 2019, 70, 2179. ACKNOWLEDGMENTS The authors would like to thank Johannes Vrijmoed for valuable thermodynamic discussions and assistance with the SEM and Moritz Liesegang for assistance with the electron microprobe analysis and XRD measurements. A schematic overview of the corrosion mechanism is depicted in Figure 14. KINGSBERY AND STEPHAN‐SCHERB 770 This study was funded by the German Research Foun- dation (DFG‐416318834). Open Access funding enabled and organized by Projekt DEAL. [8] K. B. Yoo, Y. He, H. S. Lee, S. Y. Bae, D. S. Kim, KEPCO J. Electr. Power Energy 2018, 4, 25. [9] ISO 17224:2015(E). Corrosion of metals and alloys—Test method for high‐temperature corrosion testing of metallive materials by application of a deposit of salt, ash, or other substances, International Organization for Standardization, Geneva, CH, 2015. AUTHOR CONTRIBUTIONS [11] A. W. Bowen, G. M. Leak, Metall. Trans. 1970, 1695. Phillip Kingsbery: analysis, investigation, writing— original draft and visualization. Christiane Stephan‐ Scherb: conceptualization, supervision, writing—review and editing [12] K. Hirano, M. Cohen, B. L. Averbach, Acta Metall. 1961, 440. [13] S. C. Okoro, M. Montgomery, F. J. Frandsen, K. Pantleon, Energy Fuels 2014, 28, 6447. [14] H. P. Michelsen, F. Frandsen, K. Dam‐Johansen, O. H. Larsen, Fuel Process. Technol. 1998, 54, 95. [15] P. Huczkowski, D. J. Young, T. Olszewski, A. Chyrkin, W. J. Quadakkers, Oxid. Metals 2018, 89, 651. DATA AVAILABILITY STATEMENT The raw data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. [16] R. P. Oleksak, J. H. Tylczak, G. R. Holcomb, Ö. N. Doğan, JOM 2020, 72, 1822. [17] W. Bleck (Ed.), Werkstoffkunde Stahl für Studium und Praxis, 5th ed., Verlag Mainz, Aachen, Germany 2014. [18] F. Falk, O. Sobol, C. Stephan‐Scherb, Corros. Sc CONFLICT OF INTERESTS The authors declare that there are no conflicts of interest. [10] F. S. Buffington, K. Hirano, M. Cohen, Acta Metall. 1961, 434. AUTHOR CONTRIBUTIONS Phillip Kingsbery: analysis, investigation, writing— original draft and visualization. Christiane Stephan‐ Scherb: conceptualization, supervision, writing—review and editing REFERENCES [1] Y. Shao, J. Wang, F. Preto, J. Zhu, C. C. Xu, Energies 2012, 5, 5171. [23] M. Aho, K. Paakkinen, R. Taipale, Fuel 2013, 103, 562. [1] Y. Shao, J. Wang, F. Preto, J. Zhu, C. C. Xu, Energies 2012, 5, 5171. [24] K. O. Davidsson, L.‐E. Åmand, B.‐M. Steenari, A.‐L. Elled D. Eskilsson, B. Leckner, Chem. Eng. Sci. 2008, 63, 5314. [2] H. P. Nielsen, F. J. Frandsen, K. Dam‐Johansen, L. L. Baxter, Prog. Energy Combust. Sci. 2000, 26, 283. [25] Y. Zheng, P. A. Jensen, A. D. Jensen, B. Sander, H. Junker, Fuel 2007, 86, 1008. [3] A. Hernas, M. Imosa, B. Formanek, J. Cizner, J. Mater. Process. Technol. 2004, 157–158, 348. [26] H. J. Grabke, Mater. High Temp. 1993, 11, 23. [4] J. Lehmusto, M. Sattari, M. Halvarsson, L. Hupa, Corros. Sci. 2021, 183, 109332. [27] H. J. Grabke, E. Reese, M. Spiegel, Corros. Sci. 1995, 37, 1023. [5] J. Pettersson, H. Asteman, J.‐E. Svensson, L.‐G. Johansson, Oxid. Metals 2005, 64, 23. How to cite this article: P. Kingsbery, C. Stephan‐Scherb, Mater. Corros. 2022;73:758–770. https://doi.org/10.1002/maco.202112901 [6] T. Jonsson, J. Froitzheim, J. Pettersson, J.‐E. Svensson, L.‐G. h l O id l [6] T. Jonsson, J. Froitzheim, J. Pettersson, J.‐E. Svensson, L. Johansson, M. Halvarsson, Oxid. Metals 2009, 72, 213. 6] T. Jonsson, J. Froitzheim, J. Pettersson, J.‐E. Sven Johansson, M. Halvarsson, Oxid. Metals 2009, 72, 213. [7] S. Karlsson, T. Jonsson, J. Hall, J.‐E. Svensson, J. Liske, Energy Fuels 2014, 28, 3102.
W2088033053.txt	https://zenodo.org/records/2052377/files/article.pdf	de		Über atypischen Lichen scrophulosorum	Archives of dermatological research	1,905	public-domain	3,335	&us der iIautkranken~tation de~ st~dti~chen ~rankenhauseq zu Frankfurt a. ~. (Oberarzt Dr. Earl IIerxheimer), Uber atypischen Lichen scrophulosorum. Von Dr. reed. Friedrieh Sehiirmann. Jadassohn konnte 1898 Atypieen im klinischen Bflde des Lichen scrophulosorum nach zwei Riehtungen feststellen: er sah erythematSse Kreise und psoriasiforme linsengrol~e Elfloreszenzen. R 6 n a machte dann 1900 auf eine Abortivform des Lichen scrophulosorum aufmerksam, die er 5fters bei Kranken mit Lupus vulgaris oder Scrophuloderma beobachtete, und die aus unregelma~ig gruppierten, kaum sichtbaren Papeln oder kleinen schuppenden, polygonalen, einfachen erythematSsen Flecken bestand, die in Kreisen oder Kreissegmenten angeordnet waren. Dieses Exanthem war in der Mehrzah] der Fa]le so schwach entwickelt, daft man es kaum bemerkte. In der Umgebung dieser Affektion konnte man in einigen Fallen typischen Lichen scrophulosorum finden. L. P a u t r i e r gruppiert unter~ den typischen Lichen scr,)phnlosornm die drei Arten perifollikularer Tuberkulide B o e c k s : Die Pityriasis simplex des Kindergesiehtes, das Ekzema scrophnlosorum und das papulo-squameuse perifollikulare Tuberkulid: ,,~ notre avis ce ne sent lg que trois formes quelque modifi6es du lichen scrofuleux" und will fiir diese drei Affektionen nicht die Aufstellung neuer Typen gelten lassen. In diesem Archly Bd. LXIX teilt V. K l i n g m i i l l e r drei FKlle mit, die er als ,Ubergangsformen yore Lichen serophulosorum zu den Tuberkuliden" bezeichnet. Der erste dieser 25* 380 Schfirmann. drei Fhlle kam dem Bilde des Eczema scrophulosorum sehr nahe; doch waren die einzelnen papulSsenKnStchen selbst bei grSSerenPlaques deutlich als Einzeleffloreszenzen zu erkennen. Gegen einen typischen Lichen scrophulosorum sprach die GrSf~e and starke Infiltration der gnStchen, ebenso fehlte die strenge Lokalisation an den Follikeln. Bei dem zweiten Fall sprach tier mikroskopische Befund wie auch das klinische Bild ffir die Diagnose Lichen scrophulosorum, hTeben dem typischen Lichen scrophulosorum fanden sich jedoch noch grSBere infiltrierte bl~uliche Herde am Ges~l~ und den unteren Extremit~ten, die nicht als Lichen-Effloreszenzen gedeutet werden konnten, and die auch histologisch erhebliche Unterschiede yon diesem zeigten. Zwischen beiden Formen bemerkte man deutliche Uberg~inge. Da nun auch die Abheilung beider Ausschlagsformen parallel ging, so nimmt K l i n g m f i l l er in diesem Fall eine Ubergangsform yore Lichen scrophulosoram zu einem Tuberkulid oder wenigstens zu einer tuberkulidKhnlichen Dermatose an, wofiir auch das histologische Bild sprach. Im dritten Falle war die ~bergangsform noch deutlicher. Man konnte feststellen, dab sich einfache lichenoide Effloreszenzen, die tells papulSs~ tells pustulSs, tells squamSs waren, in starker infiltrierte, bl~iulich verFKrbte Herde umgewandelt batten, die aufGlasdruck einen gelblichen Pigmentfleck zuriicklie~en und mit einer peripher pigmentierten, kreisrunden, eingelassenen Narbe heilten. Da kein Material zur Verfiigung stand, konnte dieser letzte Fall nicht histologisch untersucht werden. Einen diesen nahe stehenden Krankheitsbefund erhob W. P i c k in diesem Jahre. Er berichtet fiber ein eigenartiges, lichenoides Exanthem, bei dem das histologische Bild vollkommen jenem des Lichen scrophulosorum entsprach. Die GrSl~e des KnStchen jedoch, ihre Gestalt, ihr Glanz und ihr Abheilen mitS/arbenbildung schieden die Affektion klinisch yore typischen Lichen scrophulosorum. Diesen Beobachtungen yon K l i n g m i i l l e r und P i c k schlieBen sich drei kiirztich yon uns beobachtete Krankheitsbilder in mancher Hinsicht an. Uber atypisehen Lichen serophulosorum. 381 F a l l I. Elly P., 2 Jahre, Aufnahme 9./I. 1904. Anamnestisch nichts zu eruieren. Es handelt sich um ein gu~ gen~ihrtes Kind mit starkem pannieulus adiposus. Innere Organe normal. Auf beiden Glat/ien~ sowie am rechten Oberarm fiber dem Deltamuskel finden sieh fluktu~orende Abszesse; die fiber den Glutton sind etwa haselnuBgroB, die am Arm wallnuBgrol~. Die Haut fiber den Abszessea ist vorgew61bt, erscheint aber sonst normal, keine entziindliehe RSte, hSchstens elne leicht bl~uliehe Yerf/~rbung. Die Abszesse seheinen indolent zu sein, das Kind /~uBert bei Druck auf sie keine Sehmerzen. Ferner finder sieh am Rumpfe and zwar in der Bauchgegend, sowie auf den hinteren und seitlichen unteren Teiten des Rfickens ein Kn6tchenaussehlag. Dieser stellt sich dar in Form yon stecknadelkopfgroBen, br~unlichen Kn6tchen~ die grSBtentoils auf ihrer Spitze ein Sehiippohen tragen. Die Anordnung der KnStchen l~Bt an manchen Stellen eine deutliche Gruppierung zu Kreisen nnd Linien erkennen. Alle KnStchea bilden isolierte Effloreszenzen, nirgends finder sich eino Konfluenz. Aus den inzidierten Abszessen entleert sich reichlich rahmiger Eiter, dessert UnSersuchung auf Tbe.-Bazillen negativ ausf~llt. In der rechten GlutAalgegend finden sieh stecknadelkopfgrofie bis linsengrol~e Effloreszenzen tells mehr spitzig und das Hautniveau leicht iiberragend, toils mehr flach im Hau~nlveau selbst liegend. Die ersteren erinnern sehr an die oben beschriebene Affektlon am Stamme. Sie tragen wie diese ein zentrales Schiippchen. Veto Lichen scrophulosorum unterscheiden sie sich dadurch, dab sie weniger br~unlieh als mehr gelblich gef~rbt sind. Nach Entfernung der Schuppe bleibt eiu Trichter zurSck. Im Gegensatz h~erzu zeigea die grSfiten Effioreszenzen eine blaurote Verf~rbung, zu der sich ein ausgesprochener Lichenglatlz gesellt, so dab diese Effloreszenzen, zumal sie auch eine zentrale Delle zeigen, sehr an den Lichen ruber planus erinnern. Diese Effloreszenzen zeigen an den Obersehenkeln oft eine Anordnung in unregelm~l~ige Linien. Die KnStehen sind rund, zwischen den kleinsten and grSl~ten Effloreszenzen finden sich alle m6g]iehen Uberg~inge. Drei Tage uach Aufnahme des Status zeigten sieh neue Effloreszenzen am linken Knie und dem oberen Drittel des Unterschenkels. Fiinf Tage naehher zeigten die Abszesse geringe Abszedierung. Temp. 40"8~ Die Drfisen in der reehten Axilla zeigten sich geschwollen. Einige Tage sp~ter sehien es, als ob die Effloreszenzen am linken Obersehenkel in Rfiekbfldung begriffon w~ren, ihre Farbe war viol blasser geworden, ein Toil derselben war spurlos versehwuuden, dagegen zeigte sich der Lichen scrophulos0rum am Stamme unver~ndert. Die AbszeI~st~tte fiber dem Deltamuskel zeigte eine Fistel, die sieh naeh der AehselhShle zu erstreekte. Auf Druck entleerte sieh reiehlieh Eiter. Spaltung der Fistel. Da die Temperatur anhielt und ebenfalls die Sekretion aus der AehselhShle, wurde das Kind auf die chirurgisehe Station verlegt, we der Lichen scrophulosorum unter Lebertranbehandlung naeh etlicher Zeit spurlos verschwand. 382 Schfirmann. Der histologische Befund an einem aus dem Oberschenkel exzidierten Stiickchen Haut war folgender: Die pathologischen Elemente ]okalisierten sioh um Ha~rbalg und Talgdrtisen. Hier ist ein circumscriptes Infiltrat, bestehend aus massenhaften Lymphocyten, vereinzelten polynuklegren Zellen, grS~eren plasmareichen Zellen und vielen Mastze]len; das Infiltrat ist keilfSrmig um den HaarbMg herum angeordnet. An manchen Stellen bemerkt man spindelfSrmige Bindegewebszellen, die das Infiltrat in schmalen Ztigen durchsetzen. Manche Stellen des Infiltrates finden sich mit Haufen yon neugebildeten Kapillaren durchsetzt. W~hrend nun rings um den Infiltratenkeil das Gewebe des tieferliegenden Coriuras, mit Ausnahme yon Infiltraten l~ings der Blutgefd~e normal ist, sind die Papillen und die subpapilliire Schicht im ganzen Gesichtsfelde yon dem an~egebenen Infiltra~ erfiillt, wobei nament]ich die grol~e Zahl der neugebildeten Kapillaren auff~llt. Die Epidermis ist an der Stelle des Infiltratenkeiles etwas verbreitert. Keine Tuberkelbildung. Keine Riesenzellen. Keine Tuberkelbazillen. F~irbetechnik: Hs in Formolalkohol, Einbetten in Paraffin, F~irbung mit H~matoxylin-Eosin, Cressylechtviolett, Polychrom-Methylenblau, Ziehl-Neelsen.. F a l l 2. Karl D., 15 Jahre. Aufgenommen 15./V. 1901 cntlassen 9&/VL 1904. Dieser Fall betrifft einen 15j~ihrigen Schubmaeherlehrling, der yore 20.ft. his 15./V. 1904 auf der ehirurgischen Abteilung wegen Skrofuloderma behandel~ worden war. Die Drtisen batten seit 6 Jahren bestanden. Seehs Wochen vor seiner Verlegung auf die Hautkrankenabteilung wurde er operiert. Drei Wochen darnach trat eln Ausschlag auf~ der als parasit~res Ekzem diagnostizier~ und mit Teer behandelt wurde. Bei der Ubernahme des grazil gebauten Jungen auf die Hautkrankenstation fanden sich zu beiden Seiten des Halses Operationsnarben. Auf tier Wang% am Kieferwinkel, eine marksttiekgro~e ger6tete Stelle, in deren Mifte eine Fistelmtindung, in der Peripherie KnStehen. Mandibulardr~isen beiderseits bis zu KirschgrS~e geschwollen, ebenso zeigten sieh die Zervikal-, Axillar-, Inguinaldrtisen vergrSBert. Die Perkussion der Lunge ergibt geringen Tiefstand tier rechten Apex. Auskultatorisch niehts Pathologisches nachweisbar. Die iibrigen 0rgane ohne bemerkenswerten Refund. Die Haut zeigte tiberall ziemlich starke Pigmentation, war dtinn, trocken, etwas schuppend und leieht faltbar. An den oberen Extremit~ten kein Lichen pilaris, an den unteren ganz geringer (forme rouge). Auf dem Stamme und zwar sl0eziell an den Sei~enw~nden zwlsehen Uber atypi~ehen Lichen serophulosorum. 383 vorderer und hinterer Axillarlinie, zum Teil aueh vorne bis zur Mittel_ linie reichend und auch auf das Kreuzbein fibergreifend fiudet sich ein Exanthem, des die mittlere Bauchgegend unterhalb des Nabels, sowie den grSBten Tell des Rfickens frei l~Bt. Das Exanthem besteht aus KnStchen, die wie kleine Strophuluspapeln aussehen~ in Gruppen stehen und zum Teile an der Spitze schuppen. Die einzelnen KnStehen sind ]ebhaft rot und steeknadelkopf- bis hirsekorngroB, andere sind deutlich gl~nzend, planes- bis hanfkorngroB und tiefer rot. Die Dermatose ruff keinen Juckreiz hervor. Neben Lichen scrophulosorum kam diagnostiseh Ekzema serofulosorum in Betracht. Nach mehrt~giger Behandlung mit essigsaurer Tonerde, Umsehl~gen, B~dern und Salizylvaseline hatte man beim Darfiberfahren mit der Hand ein reibeisenartiges Gef~hl. Eine zur Sieherung der Diagnose vorgenommene Injektion yon I/10 Milligramm Alt-Tuberkulin ffihrte zu einer geringen Reaktion der Dermatose: Man hatte den Eindruck, als ob die rote Farbe der KnStchen tiefer geworden sei; auBerdem machte sich in den folgenden Tagen eine vermehrte Absehuppung der Epidermis ira Bereiche der Affek~ion bemerkbar, aueh trugen mehr EiTlore~zenzen wie bisher zentrale Seh~ppehen. Am zweiten Tage nach der Injektiou stieg die Temperatur plStzlieh auf 38'5~ dabei zeigte sieh eine RStuag and Schwellung an der Injektionsstelle~ die als Erysipel imponierte. In den folgenden Tagen schwanden Fieber und RSte. An der Stelle, we die RSte bestanden hatte, maehte sleh eine ziemlich starke Absehuppung bemerkbar~ auch traten einige Follikulitiden auf. W~hrend seines Aufenthaltes auf der Hautkrankenabteilung erhielt Patient 3 E~lSffel Leber~ran pro die, die in Verbindung ~mit Umsehl~igen yon Lebertran] die Affektion wesentlieh besserten. Patient war nicht zu bewegen l~nger in Hospitalbehandlung zu bleiben und wurde mit diesem Befunde nach Hause entlassen. Ein am Stature exstirpiertes planes KnStchen wurde in Serienschnitte zerlegt. Es zeigte s~ch die Epidermis bedeutender verbreitert als in Fall I~ namentlich das Stratum Malpighii. Die s~mtlichen Zellen dieses Tells des Ep[thels waren gequollen. Die Papillen waren auch hier mit einem Infiltrate angefiillt, des haupts~ch]ich aus Lymphocyten, Kapillaren und gewucherten Endothelien bestand. An einigen Stellen war das Infiltrat auoh subpapill~ir vorhanden, sonst im Cerium nur entlang einiger Papillen mit Ausnahme der Tiefe des Ceriums, we es auch um eine Haarpapille herum sich befand. Die Untersuchung auf Tuberkelbazfllen fiel bier nach mehreren Methoden und bei einer Anzahl yon je 70 Schnitten negativ aus. Aueh zeigten sich nirgendwo Miliartuberke!. 384 Sehfirmann. Fiirbetechnik: H/irten in F o r m a l a l k o h o l ; E i n b e t t e n in Paraffin; F ~ r b u n g d e r S c h n i t t e n a c h den M e t h o d e n yon Ziehl-Neelsen und Gabber-Ernst. F a l l 3. Frau A. Br. (Dieser Fall wurde uns yon Herrn Kollegen Th. B Rr ambulant demonstriert. Hieffir, wie ffir die l)berlassung zur VerSffentliehung sagen wir dem Herrn Kollegen besten Dank.) Es handelt sich um eine etwa 50 Jahre alte Patientin, Mutter yon 13 Kindern, yon denen noeh drei leben. Abgesehen you 5frets aufgetretenen Drfisenschwellungen will Patientin friiher hie krank gewesen sein. S t a t u s p r a e s e n s Friihjahr 1904: Grol]e, kr~ftig gebaute Frau mit ziemlich starkem Fettpolster. Am Halse zahlreiche unregelm/il~ige I~arben. Auf der ]inken Mamma ein ova]es zirka & c m langes und 2 c~n breites Ulcus mit scharfen RRndern und schmierigem Grunde. An den Armen unregelmM3ig zerstreute, gelbliche, kleinpapulSse Effloreszenzen, zum Tell mit eitrigem Belage. Auf dem Rficken fiber den SchulterblRttern finden sich gelbliehe~ Schfippchen tragende, mehr spitzige KnStchen, die in Gruppen gestellt sind. In der Lumbalgegend deutliohe grSl~ere und flachere lichenoide Efftoreszenzen, d. h. fiber steeknadelkopfgrol~e KnStchen, zum Tell den Follikeln entsprechend, yon sattroter Farbe und wachsartigem Glanze. Eine Dellenbildung l ~ t sieh nicht mit Sicherheit konstatieren. Diagnose: Skrofuloderma der Mammargegend, tuberkulSse Drfisen, Lichen scrofulosorum. Unter roborierender DiRt und Sehwarzsalbenverband heilte alas Ulcus. Auf unsere Bitte stellte sich Patientin am 25. Juli 1904 auf der ttautkrankenstation vor. Das Krankheitsbild hatte sich wie folgt verRndert : Auf dem Rfieken fiber beiden SehulterblRttern finden sieh noch Gruppen spitziger Papelchen, links dunkler gef/irbt als reehts, in der Ausdehnung einer Kinderhand, dicht gedrRngt, kaum fiber die Oberhaut erhaben. An dem Ubergange zu den Oberarmen finden sich nach deren Rfiekseite zu mannshandgro~e Plaques yon gelblich schmutziger Farbe und vielen kleinen Sehiippehen. Bei nRherem Zusehen sind aueh hier massenhafte KnStchen sehr dicht gedrRngt vorhanden und yon der oben angegebenen Farbe. Die Lichen planus Rhnlichen Papeln auf dem Kreuzbein sind verschwunden. Ein histologischer iBefund konnte aus Mangel an Material nicht erhoben werden. Bei d e r B e t r a c h t u n g u n s e r e r d r e i F ~ l l e f~llt uns vor a l l e m das N e b e n e i n a n d e r g e h e n zweier ~ul3erlich d i f f e r e n t e r Affektionen auf. D i e p l a n e n K n S t c h e n k a m e n u n d g i n g e n mit d e n s p i t z e n des t y p i s c h e n L i c h e n s c r o p h u l o s o r u m . Differentiald i a g n o s t i s c h gegen L i c h e n s c r o p h u l o s o r u m in F r a g e k o m m e n d e Uber atypischen Lichen scrophulosorum. 385 Affektionen konnten keine geniigende ErklErung fiir unser Kranl~heitsbild abgeben. Gegen die Diagnose Tuberkulid sprach das Fehlen einer zentralen Nekrose und die ganze Form der Affektion, gegen Ache cachecticorum das Fehlen der Vereiterung, gegen Eczema scrophulosorum das Fehlen der F1Echenerkrankung. Die L e i c h t e n s t e r n s c h e Form der akuten Miliartuberkulose auf der Haut besteht in Einzeleffioreszenzen, die ganz akut auftreten und verschwinden. Am schwierigsten ist die Scheidung vom Lichen planus et acuminatus. Jedoch hilft hier der Farbenunterschied bei den aku, mlnierten Effioreszenzen, der Pr~dilektions-Sitz am Stamme, das Auftreten in Gruppen, der Decursus morbi, das gleichzeitige Bestehen anderweitiger tuberkulSser Erkrankungen sowiedie spurlose Heilung auf Leberthran. Es kann sich demnach nur um einen allerdings yon dem typischen t t e b r a s c h e n Krankheitsbilde abweichenden Lichen scrophulosorum handeln. Nach H eb r a bildet der Lichen scrophulosorum hirsekorngrol]e, blaI~gelbe bis braunrote KnStchen, die im Innern nie ein Fluidum tragen, stets in Gruppen stehen, oft Kreise oder Kreissegmente bilden, in denen bisweilen noch einzelne Stellen stgrker pigmentiert erscheinen, jene Stellen n~mlieh, wo friiher KnStchen sal]en. Einige KnStchen erscheinen stets mit einer geringen Menge Schiippchen bedeckt. Subjektive Erscheinungen fehlen, besenders ist kein Juckreiz vorhanden. Die Affektion zeigt sich besonders an Brust, Bauch und Riicken seltener an den Extremitgten. Der Verlauf ist tr~ge, o h n e B l ~ i s c h e n o d e r Pustelbildung. Verschieden yon diesem Bild des typisehen Lichen scropulosorum ist unsere Affektion eigentlich nur durch die betr~ichtliche GrSl~e, durch die Farbe und dureh den Glanz ciniger Einzeleffloreszenzen. Da die Dermatose jedoch im iibrigen alle Merkmale des Lichen serophulosorum zeigt, und da aul]erdem gewichtige Momente, wie das Vorkommen bei unzweifelhaft tuberkulSsen lndividuen und der parallele Verlauf mit einem typischen Lichen scrophulosorum die Diagnose Lichen scrophulosorum stiitzen, so ist es fiir uns nicht zweifelhaft, dal] wir es in den vor]iegenden F~llen mit der n~mlichen Dermatose zu tun haben. Ist dieses jedoch der Fall, so diirfte es sigh emptehlen, da auch andere gleichartige Beobachtungen vor- 386 Schiirmann. liegen, analog wie beim Lichen ruber eino akuminier~e und eine plane Form zu unterscheiden. Man wird sich selbstversts hiiten miissen, umschriebene liehenifizierte Herde mit unserer Affektion zu verwechseln. Was die histologisehen Befunde bei unseren Fs angeht, so stimmen sie mit den Resul~a~en der Untersuchungen K a p o s i s fiberein. Dieser demonstrierte die KnStehen des Lichen scrophulosorum als das Resultat einer Zellinfiltration und Exsudation um die Haarfollikel und die dazn gehSrigen TMgdrfisen, sowie in die, die Follikelm[indung zuniehst begrenzenden Papillen. 3 a k o b i widerspraeh den Untersuchungsergebnissen K a p osis. Er fund in den Infiltraten die Struktur typischer Miliartuberkel und sah e in Sts das in seinem morphologischen and tinktoriellen Verbalten dem Tuberkelbazilhs entsprach. Dieser Ansicht, dal~ der Lichen scrophulosorum eine eehte Hauttuberkulose sei, wollen sps Untersucher unter ihnen L u k a s i e w i e z , J a d a s s o h n , R i e h l nicht beipflichten. J a d a s s o h n und U n n a sehen vielmehr die Frage fiber die Tbk.-Natur des Lichen serophulosorum als noeh nieht spruchreif an. H a l l o p e a u und B o e c k sowie S c h w e n i n g e r und B u z z i, die nach Tuberkelinjektionen eine verbreitete Eruption yon Lichen scrophul, auftreten sahen und H a l l o p e a u , der die KnStchen in der Umgebung unzweifelhaft tuberkulSser tterde auffand~ wollen die tuberkulSse Grundlage uniter Zweifel gestellt wissen und neigen der Ansicht zu, da~ die KnStehen der Toxinwirkung der Tuberkelbazillen ihre Entstehung verdanken. Beobachtungen yon W o l f f , der fiber Tuberkelbazillenfunde, zwar nicht in den typischen KnStchen selbst, wohl aber in neben diesen zur Vereiterung tendierenden KnStchen, die er yon den ersteren ableitet, schreibt und die positiven Impferfolge, woriiber J a k o b i und H a u s h a 1t e r berichten, erscheinen uns mit Rficksicht auf das AusgangsmateriM und die mangelhafte Kontrolle der Impftiere nieht einwandsfrei. N e i s s e r tr~gt ebenfalls naeh dem positiven Ausfalle der Tuberkelimpfung kein Bedeaken, die Erkrankung den echten Formen der bazill~ren H~uttuberk~lose zazurechnen und schl~gt fiir sie den Namen Tuberculosis miliopapulosa aggregata Uber atypischen Lichen scrophulosorum. 387 vor. Dagegen halten T o u t o n und K l i n g m i i l l e r die Frage fiir nicht absolut spruchreif. Letzterer sieht den Lichen scrophulosorum als eine bei TuberkulSsen auftretende Dermatose an, die durch die Toxine der Tuberkelbazillen entsteht und zwar tritt sie nur be~ solchen ]ndividuen auf, die auch sonst an den chronisehen Formen der Tuberkulose leiden. Unsere F~lle sind nur geeignet, diese Annahme K l i n g m i i l l e r s zu stiitzen, denn unsere Patienten hatten alle andere Manifestationen tier Tuberkulose und wir kSnnen hinzufiigen, daft diese nicht bei einem der ca. 200 Fs yon Lichen seropulosorum der Beobachtungen K. H e r x h e i m e r s fehlten. Aul~erdem kommt hinzu der positive Ansfall der Tuberkulinreaktion und zwar reagierte Fall II nicht nur mit s sondern auch typisch 5rtlieh. 388 S chiirmann. Literatur. B o e c k : Die Exantheme der Tuberkulose. Archiv. Band XLH, 1898. Hallopeau: Sur un cas de lichen scrophul. Annales. 1892. pag. 284 und 451, Haushalter: Deux cas de lichen scroph, chez t'enfanr nature de la tuberculeux de l~affection 1898. J a d a s s o h n : Lichen scrophul. Bericht des III. internationalen Dermatologenkongresses~ London 1896. Jakobi: Pathologic und Pathogenese d. Lichen serophul. Verhandlg, der deutschen dermatologischeu Ges. 1892. K a p o s i : Uber Lichen scrophul. /s 1869. Lehrbuch. K1 i n g m f i l l e r : Beitr~ge zur Tuberkulos e der Haut, Archly 1904. L u k a s i e w i c z: Uber Lichen scrophul. Archiv. Bd. XXVI, 1894. N e i s s e r u. J a d a s s o h n : Hautkrankheiten. P au t r i e r : Les Tubereulose cutan~es atypiques. 1903. P i c k : Archly. 1904. R i c h l: Lichen scrophnl. Archiv. 1892. Schweninger und B n z z i : Zwei Briefe aus Berlin, Monatshefte 1890. U n n a : Folliculitis scrophu]. Histopathologie 1894. Wolff: VI. KongreI~ der dtsch. Derm. Ges. 1899.
https://openalex.org/W2535197851	https://www.epj-conferences.org/10.1051/epjconf/201714008003/pdf	English	null	Clustering and melting in a wet granular monolayer	EPJ web of conferences	2,017	cc-by	3,723	1 Introduction for diﬀerent NESS, and surface melting was found to be associated with the emerging 1/f noise [12]. For partially wet granular materials, liquid mediated particle-particle interactions lead to dramatically diﬀerent collective behavior than noncohesive dry granular mate- rials, even if the mean liquid ﬁlm thickness is only tens of atomic layers thick [1]. In our planet largely cov- ered with water, understanding how wetting liquid plays a role in determining the transitions of granular materi- als between solid-like, liquid-like or gas-like states is im- portant concerning the prediction of natural disasters such as landslide or debris ﬂow, as well as the enhancement of the eﬃciency in industrial processes [2]. In quasi- two-dimensions, driven noncohesive granular monolayers have attracted continuous attentions in the past decade, because the dissipative nature of granular matter marks it as a model system to understand phase transitions far from thermodynamic equilibrium [3–5]. Particularly, a previous investigation on agitated dry granular monolay- ers suggests a two-stage melting scenario [6] in accor- dance to the Kosterlitz, Thouless, Halperin, Nelson and Young (KTHNY) theory for crystals in two-dimensions (2D) [7, 8]. For the partially wet case, the capillary bridges formed between adjacent particles, which give rise to short-ranged attractive interactions, can be considered as ‘molecular bonds’ in the model system. The nonequi- librium stationary states (NESS) depend on the balance between energy injection through, e.g., mechanical agita- tions, and dissipation through, e.g., inelastic collisions or viscous forces from wetting liquid [9, 10]. In a previous investigation, it was shown that a 2D wet granular crys- tal under horizontal swirling motion (i.e., horizontal vibra- tions in two orthogonal directions with a phase shift of π/2 in a plane perpendicular to gravity) melts from its free sur- face [11]. In a following investigation, the ﬂuctuations of the internal structures quantiﬁed by the bond-orientational order parameters (BOOP) show diﬀerent types of noises Here, we focus on the collective behavior of a wet granular monolayer subjected to vertical vibrations along the direction of gravity. From both the internal structure and the mobility of the particles, we identify three distinct regimes: Clustering of mobilized particles, reorganization of small clusters into a hexagonal crystal with defects, and a continuous melting initiated at the defects. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Clustering and melting in a wet granular monolayer Philipp Ramming1 and Kai Huang1,⋆ 1Experimentalphysik V, Universität Bayreuth, 95440 Bayreuth, Germany Abstract. We investigate experimentally the collective behavior of a wet granular monolayer under vertical vibrations. The spherical particles are partially wet such that there are short-ranged attractive interactions between adjacent particles. As the vibration strength increases, clustering, reorganizing and melting regimes are identiﬁed subsequently through a characterization with the bond-orientational order parameters and the mean kinetic energy of the particles. The melting transition is found to be a continuous process starting from the defects inside the crystal. ⋆e-mail: kai.huang@uni-bayreuth.de , 08003 (2017) 140 EPJ Web of Conferences Powders & Grains 2017 , 08003 (2017) 140 EPJ Web of Conferences Powders & Grains 2017 DOI: 10.1051/ 714008003 epjconf/201 2 Experimental Setup and Procedure (c) Reconstructed image of (a) based on the positions and local structures of all par- ticles. (d) Color coding for the local structure (free, line, square, or hexagonal) identiﬁed with bond-orientational order parame- ters and a sketch deﬁning the position of particle i and one of its neighbors j in the laboratory system. ! Figure 1. Schematic of the experimental setup (a) and the con tainer (b). Shaking control, camera triggering, acceleration de tection, and image capturing are carried out automatically with a computer program. In (b), the spacer is a PTFE ring used to ﬁ th ti l i t l Figure 1. Schematic of the experimental setup (a) and the con- tainer (b). Shaking control, camera triggering, acceleration de- tection, and image capturing are carried out automatically with a computer program. In (b), the spacer is a PTFE ring used to conﬁne the particles into a monolayer. Figure 2. (a) A sample raw image from the high speed camera. Eight black spheres are embedded in the PTFE ring symmetri- cally in the azimuth direction for scaling purposes. (b) Negative image of (a) after the background is removed. (c) Reconstructed image of (a) based on the positions and local structures of all par- ticles. (d) Color coding for the local structure (free, line, square, or hexagonal) identiﬁed with bond-orientational order parame- ters and a sketch deﬁning the position of particle i and one of its neighbors j in the laboratory system. acceleration. The collective behavior of the sample is cap- tured with a high speed camera (IDT MotionScope M3) mounted above the container. The camera is triggered by a synchronized multi-pulse generator to capture images at ﬁxed phases of each vibration cycle. More details on the set-up control can be found in Ref. [14]. ter with a sub-pixel resolution [15]. In order to detect all the particles for all frames captured, the threshold used in the Hough transformation is automatically adjusted such that the correct number of particles are detected. 2 Experimental Setup and Procedure Figure 1a shows a sketch of the experiment setup. We use a computer program to adjust vibration parameters, take images, and collect acceleration data automatically in or- der to explore the parameter space in a well-controlled manner. Cleaned white glass beads (SiLibeads P) with a diameter d = 2.0 ± 0.02 mm and a density ρp = 2.47 ± 0.05 g/cm3, after being mixed with puriﬁed wa- ter (LaborStar TWF, surface tension σ = 0.072 N/m), are added into a cylindrical polytetraﬂuoroethylene (PTFE) container. The height H = 2.60 ± 0.05 mm and inner di- ameter D = 13.00 cm of the container is controlled by the embedded spacer (see Figure1b). H is chosen to be only slightly larger than d to ensure a monolayer. The global area fraction is Nd2/D2 ≈50.5% with particle number N = 2134. The liquid content is deﬁned as W ≡Vw/Vg, where Vw and Vg are the volume of the wetting liquid and that of the glass beads correspondingly. It is kept within 3% so that cohesion arises mainly from liquid bridges formed between adjacent particles [13]. The container is agitated vertically against gravity with an electromag- netic shaker (Tira TV50350). The frequency f and ampli- tude z0 of the sinusoidal vibrations are controlled with a function generator (Agilent FG33220) and the dimension- less acceleration Γ = 4π2 f 2z0/g is measured with an ac- celerometer (Dytran 3035B2), where g is the gravitational , 08003 (2017) 140 EPJ Web of Conferences , 08003 (2017) 140 EPJ Web of Conferences Powders & Grains 2017 DOI: 10.1051/ 714008003 epjconf/201 Powders & Grains 2017 ! Figure 1. Schematic of the experimental setup (a) and the con- tainer (b). Shaking control, camera triggering, acceleration de- tection, and image capturing are carried out automatically with a computer program. In (b), the spacer is a PTFE ring used to conﬁne the particles into a monolayer. Figure 2. (a) A sample raw image from the high speed camera. Eight black spheres are embedded in the PTFE ring symmetri- cally in the azimuth direction for scaling purposes. (b) Negative image of (a) after the background is removed. 2 Experimental Setup and Procedure Sub- sequently, the position of particle i at frame number n is rescaled with (xi, yi) = ϵ(k)(xi0, yi0), where phase k is the modulo of n over K, and (xi0, yi0) is the position of particle i in the original image. At the beginning of each experimental run, the sample is driven to the gas-like state with Γ ≈40 for 10 min- utes to have a reproducible initial condition with the wet- ting liquid homogeneously distributed. Subsequently, Γ is quenched to ≈4 and increases stepwise to ≈32 at a ﬁxed f = 50 Hz. In each step, M = 500 images are taken by the high speed camera with a frame rate of 500 frames per second, corresponding to K = 10 frames per vibration cy- cle. The local structure of a particle is identiﬁed with BOOP [16, 17], which are calculated as follows: As shown in Figure 2(d), we ﬁrst identify bonds connecting a parti- cle to all its neighbors, e.g., rij pointing from particle i to j. The neighbors are identiﬁed with the criterion \|rij\| ≤rc, where the critical bond length rc = 1.05d is slightly larger than the particle diameter. From the azimuth (φij) and po- lar angles (θij = 0 for the 2D case here) of all bonds origi- nating from particle i, we calculate Figure 2 shows the image analysis procedure, from raw images captured by the high speed camera (a) to the local structures of individual particles (c) identiﬁed with BOOP. Because shaking is along the viewing direction of the cam- era, the images captured at various phases of each vibra- tion cycle have a slightly diﬀerent scale, which leads to a shift of each particle position up to a few pixels, depending on the particle location and Γ. For particles at the rim of the container, the deviation can reach 0.15d at Γ = 15. In order to correct this error, we group images according to the capturing phase k and use diﬀerent scales for diﬀerent phases. The scales are obtained with ϵ(k) = ⟨ld(k)/D1⟩, where ld(k) is the distance between the centers of two op- positely placed marker particles in pixels (see Figure 2a), D1 = 135.0 mm is the measured length, and ⟨...⟩denotes an average over all pairs of markers and all frames at phase k. 3 Results and discussion Figure 3 presents the collective behavior of the wet spher- ical particles as Γ increases. It is quantiﬁed with two order parameters. The percentage of particles in a locally hexag- onal packing (i.e., appear red in the reconstructed images), ξ6, is used as a measure of the static structure. The rescaled kinetic energy ˜Ek(t) ≡⟨\|vi(t)−¯v(t)\|2⟩/(fd)2 is used to char- acterize particle dynamics, where vi(t) is the velocity of particle i at time t, ¯v(t) = N i=1 vi(t)/N corresponds to the mean velocity of all particles at time t, and ⟨...⟩denotes an average over all particles and frames. The particle velocity is obtained with vi(t) = [ri(t + Δt) −ri(t)]/Δt, where the time step Δt = 1/(K f) is determined by the frame rate of the camera. Based on visual inspections (see the top panel of Figure 3) as well as characterizations with the two order parameters, the following three regimes are identiﬁed. Figure 3. Upper panel shows the local structure of all particles at various Γ and W = 3%. (a) Percentage of particles in a hexag- onal structure, ξ6 as a function of Γ for diﬀerent W. (b) Rescaled kinetic energy, ⟨˜Ek⟩, averaged over all particles and M −1 conse- quent frames as a function of Γ. Both order parameters suggest three regimes separated by the two dash lines (located at Γ = 7.7 and 15.5 respectively). p g g In the clustering regime, the particles are gradually be- ing mobilized, and subsequently form small clusters due to the formation of capillary bridges. At the initial Γ ≈4.0, no movements of the particles are observed from the top view images. As Γ increases to ≈4.7, some particles start to move and form small clusters (see the reconstructed im- age at Γ = 6.8). The critical acceleration Γc can be un- derstood from the force balance Fd = Fb + G, where the magnitude of the capillary force Fb, gravity G and driv- ing force Fd can be estimated with \|Fb\| = πσd cos α with contact angle α ≈0, \|G\| = πρpd3g/6, and \|Fd\| = ΓcG respectively [18]. Consequently, we estimate Γc ≈4.5, which agrees well with the experiments. Above Γc, the mobilized particles start to form chains as well as fractal structures. Correspondingly, the order parameter ξ6 grows with the agglomeration process from ≈0.25 to more than 0.60. onal structure. Initially, ξ6 decays slightly as Γ grows. 2 Experimental Setup and Procedure Qlm(i) = 1 Nb Nb j=1 Ylm(φij, θij), (1) (1) where Nb(i) is the total number of bonds for particle i, and Ylm corresponds to the spherical harmonics of bond rij. Subsequently, we obtain BOOP of weight l with Ql = 4π 2l + 1 l m=−l \|Qlm\|2. (2) (2) After image enhancement (see Figure 2b), we apply a Hough algorithm to locate the positions of all particles in all frames in a Cartesian system centered at the image cen- Here, Q6 is chosen as the order parameter because of its sensitivity to the hexagonal order. Based on a compari- 2 , 08003 (2017) 140 EPJ Web of Conferences Powders & Grains 2017 DOI: 10.1051/ 714008003 epjconf/201 Figure 3. Upper panel shows the local structure of all particles at various Γ and W = 3%. (a) Percentage of particles in a hexag- onal structure, ξ6 as a function of Γ for diﬀerent W. (b) Rescaled kinetic energy, ⟨˜Ek⟩, averaged over all particles and M −1 conse- quent frames as a function of Γ. Both order parameters suggest three regimes separated by the two dash lines (located at Γ = 7.7 and 15.5 respectively). son of Q6 to the values obtained from perfectly hexagonal, square, line structures, as well as for free particles (see Figure 2d), the local structure of each individual particle is identiﬁed (see Figure 2c). In comparison to other local measures such as coordination number or local area frac- tion, the advantage of using BOOP is that the inﬂuence of particles on the edge of a cluster is minimized, which is essential for analyzing the structure of small clusters. Acknowledgements We thank Ingo Rehberg, Simeon Völkel, and Andreas Zip- pelius for helpful discussions. This work is partly sup- ported by the Deutsche Forschungsgemeinschaft through Grant No. HU1939/4-1. 3 Results and discussion re- constructed image at Γ = 17.5), and sometimes the whole cluster swirls collectively. The reason for this collective behavior will be a topic of further investigations. References [1] K. Huang, M. Sohaili, M. Schröter, S. Herminghaus, Phys. Rev. E 79, 010301 (2009) [2] K. Huang, Habilitation thesis, University of Bayreuth (2014) [3] P.M. Reis, R.A. Ingale, M.D. Shattuck, Phys. Rev. Lett. 96, 258001 (2006) Concerning the inﬂuence of liquid content, our results show that the amount of wetting liquid plays a role in de- termining the amount of particles in a liquid-like state, as soon as the particles are eﬀectively mobilized in the reor- ganizing and melting regimes. However, it does not in- ﬂuence the transitions between diﬀerent regimes, which appear to be predominately determined by the agitation strength. In the melting regime, the averaged kinetic en- ergy for both W agrees with each other, indicating that the energy dissipation from the rupture of liquid bridges does not play a dominating role in determining the mobility of the particles. [4] T. Müller, D. de las Heras, I. Rehberg, K. Huang, Phys. Rev. E 91, 062207 (2015) [5] A. Fortini, K. Huang, Phys. Rev. E 91, 032206 (2015) [6] J.S. Olafsen, J.S. Urbach, Phys. Rev. Lett. 95, 098002 (2005) [7] J.M. Kosterlitz, D.J. Thouless, J. Phys. C: Solid State Phys. 6, 1181 (1973) [8] K.J. Strandburg, Rev. Mod. Phys. 60, 161 (1988) [9] F. Gollwitzer, I. Rehberg, C.A. Kruelle, K. Huang, Phys. Rev. E 86, 011303 (2012) [10] T. Müller, K. Huang, Phys. Rev. E 93, 042904 (2016) 3 Results and discussion This feature can be attributed to the enhanced mobility of clusters, because intense interactions between rigid clus- ters can drive more particles into the liquid-like phase and consequently lead to a lower ξ6. Note that the transition into the liquid-like state requires the rupture of capillary bridges, and the rupture energy is ∝ √ W [20], therefore ξ6 is smaller for W = 2% in comparison to 3% for a given ag- itation strength. For the same reason, the mobility of par- ticles for W = 2% is slightly larger than 3%, because less energy is required for rupturing at a given energy injec- tion. As Γ approaches the transition to the melting regime (iii), the interactions between clusters become less impor- tant because of the merging of clusters. The ﬂuctuations of ξ6 suggest that the internal structure of the merged cluster reorganizes frequently. Visual inspections reveal that the frequent generation and diminishing of defects in the crys- talline structure (see the reconstructed image for Γ = 14.7) account for the ﬂuctuations. As Γ increases further, visual inspections reveal that more and more compact structures (reminiscent to those observed in a previous investigation of wet granular mono- layers under shear [19]) are forming along with a more signiﬁcant growth of ξ6 in the range of 6.5 ≤Γ ≤7.7. In- terestingly, the mobility of particles grows rapidly in this range (see Figure 3b), suggesting that the enhanced mobil- ity of particles facilitates the agglomeration process. The rapid growth of ˜Ek in this regime could be attributed to the additional freedom gained during the rupture of cap- illary bridges between the particles and the container bot- tom. Note that overcoming the capillary force at Γc does not necessarily lead to the rupture of capillary bridges, as the latter also requires the relative separation distance to be greater than the rupture distance of the bridge. As melting starts at Γ = 15.5, a sudden drop of ξ6 along with a rapid growth of ˜Ek are observed. Diﬀer- ent from a previous investigation on horizontally driven systems [11], melting does not predominately start from the surface of the crystal. 3 Results and discussion Instead, melting through cluster breaking, preferably along the defects, is more likely to be In the reorganizing regime starting at Γ ≈7.7, the small clusters merge and form a large one with a hexag- 3 , 08003 (2017) 140 EPJ Web of Conferences Powders & Grains 2017 DOI: 10.1051/ 714008003 epjconf/201 vibrations. The defects mediated melting transition ob- served in this nonequilibrium system triggers the question whether this transition falls into the scenario of KTHNY theory or not. observed. For the case of horizontal driving in a plane per- pendicular to gravity, the mobility of the particles are con- ﬁned in a strict monolayer. For the vertical vibrations used here, neighboring particles may move out of phase due to the small gap above the granular layer. It facilitates the formation of topological defects at which melting starts. Further increase of Γ leads to a slow erosion of the crystal by the particles in the liquid-like phase, and consequently a gradual decay of ξ6. At Γ ≈23, melting completes for both liquid contents. Further increase of agitation strength leads to a continuous growth of ˜Ek with Γ, because energy injection increases. Strong density ﬂuctuations are present at high Γ, even though the container is leveled carefully before each experimental run. Occasionally, the particles are accumulated at the side of the container (see, e.g. re- constructed image at Γ = 17.5), and sometimes the whole cluster swirls collectively. The reason for this collective behavior will be a topic of further investigations. observed. For the case of horizontal driving in a plane per- pendicular to gravity, the mobility of the particles are con- ﬁned in a strict monolayer. For the vertical vibrations used here, neighboring particles may move out of phase due to the small gap above the granular layer. It facilitates the formation of topological defects at which melting starts. Further increase of Γ leads to a slow erosion of the crystal by the particles in the liquid-like phase, and consequently a gradual decay of ξ6. At Γ ≈23, melting completes for both liquid contents. Further increase of agitation strength leads to a continuous growth of ˜Ek with Γ, because energy injection increases. Strong density ﬂuctuations are present at high Γ, even though the container is leveled carefully before each experimental run. Occasionally, the particles are accumulated at the side of the container (see, e.g. 4 Conclusions [11] C. May, M. Wild, I. Rehberg, K. Huang, Phys. Rev. E 88, 062201 (2013) To summarize, the collective behavior of a wet granular monolayer under vertical vibrations is investigated experi- mentally. The internal structures of the wet granular as- semblies are obtained with the bond-orientational order parameter Q6, and the dynamics is characterized with the averaged mobility of individual particles. From both inter- nal structures of the particles and the mean kinetic energy, three regimes (clustering, reorganizing and melting) are identiﬁed. If the peak vibration acceleration Γ is greater than a critical value, at which the driving force is suﬃ- cient to overcome the capillary force and gravity, the par- ticles are mobilized and form small compact clusters. In the reorganizing regime, the clusters merge into a hexag- onal crystal with defects. Further increase of Γ leads to a continuous melting predominately from the defects inside the crystal, reminiscent to a second-order phase transition. The diﬀerence to surface melting observed in a horizon- tally driven system [11] can be attributed to the additional degree of freedom of the particles along the direction of [12] K. Huang, New J.Phys. 17, 083055 (2015) [13] M. Scheel, R. Seemann, M. Brinkmann, M. Di Michiel, A. Sheppard, B. Breidenbach, S. Herminghaus, Nat. Mater. 7, 189 (2008) [14] K. Huang, I. Rehberg, Phys. Rev. Lett. 107, 028001 (2011) [15] C. Kimme, D. Ballard, J. Sklansky, Communications of the ACM 18, 120 (1975) [16] P.J. Steinhardt, D.R. Nelson, M. Ronchetti, Phys. Rev. B 28, 784 (1983) [17] Y. Wang, S. Teitel, C. Dellago, J. Chem. Phys. 122, 214722 (2005) [18] L. Butzhammer, S. Völkel, I. Rehberg, K. Huang, Physical Review E 92, 012202 (2015) [19] K. Huang, M. Brinkmann, S. Herminghaus, Soft Matter 8, 11939 (2012) [20] K. Huang, K. Roeller, S. Herminghaus, Eur. Phys. J. Spec. Top. 179, 25 (2009) 4 4
https://openalex.org/W4361882677	https://figshare.com/articles/journal_contribution/Supplementary_Methods_from_JMJD2B_Promotes_Epithelial_Mesenchymal_Transition_by_Cooperating_with_-Catenin_and_Enhances_Gastric_Cancer_Metastasis/22450296/1/files/39901362.pdf	English	null	Supplementary Methods from JMJD2B Promotes Epithelial–Mesenchymal Transition by Cooperating with β-Catenin and Enhances Gastric Cancer Metastasis	null	2,023	cc-by	2,451	Supplementary materials and methods 1 Supplementary materials and methods 1 2 Reagents, small interfering RNA (siRNA) and plasmids 3 Chemical modified siRNAs targeting Snail and scramble siRNA wer 4 from GenePharma (Shanghai GenePharma Co.,Ltd, China). The siRNA sequ 5 as following: siJMJD2B#1, 5′- UCUCCAUCACCUGCCUCAAGCA 6 siJMJD2B #2, 5′-GACCTGCTACGAAGTGAACTT -3′ and 7 5′-CAGGACUCUAAUCCAGAGUTT-3′. E-cadherin promoter 8 pEcad−1008/+49 was kindly provided by Dr. Y.S.Chang (Chang-Gung U 9 Kwei-shan, Taoyuan, Taiwan 333, Republic of China). TCF/LEF-1 10 (TOP-FLASH) and mutation vector (FOP-FLASH) were bought from 11 (Addgene plasmid 12456 and 12457, Cambridge, MA). 12 13 Luciferase Reporter Assays 14 BGC823 cells were plated into 24-well plate, and incubated overnight 15 and then treated with TGF-β1 (5 ng/ml) and/or JMJD2B siRNA or control siR 16 the following day, VimPro-Luc vector, VimProMut-Luc vector, pEcad− 17 vector, pTOP-FLASH or pFOP-FLASH reporter plasmids (0.5 μg eac 18 separately transfected into the treated cells as above. 24 hours later, luciferas 19 was examined by a dual luciferase reporter assay system (Promega, Madi 20 USA), and firefly luciferase activity was normalized to the control pRL-T 21 activity included in the kit. 22 BGC823 cells were plated into 24-well plate, and incubated overnight 23 On the next day, cells were co-transfected with VimPro-Luc vector, VimPro 24 vector, pEcad−1008/+49 vector, pTOP-FLASH or pFOP-FLASH reporter 25 (0.25 μg each) and HA-JMJD2B plasmid (0.25 μg each). 24 hours later, l 26 activity was examined as above. 27 BGC823 cells were plated into 24-well plate, and incubated overnight 28 and then treated with JMJD2B siRNA or control siRNA for 24h. Then th 29 were transfected with flag-β-catenin plasmids (0.5 μg). On the follow 30 pTOP-FLASH or pFOP-FLASH reporter plasmids (0.5 μg each) were s 31 transfected into the treated cells as above. 24 hours later, luciferase acti 32 examined. 33 BGC823 cells were plated into 24-well plate, and incubated overnight 34 and then were co-transfected with flag-β-catenin and HA-JMJD2B plasmid 35 each). 24 hours later, luciferase activity was examined as above. 36 Each experiment was done in triplicate and repeated at least three times. 37 38 Reagents, small interfering RNA (siRNA) and plasmids 3 Chemical modified siRNAs targeting Snail and scramble siRNA were bought 4 from GenePharma (Shanghai GenePharma Co.,Ltd, China). The siRNA sequences are 5 as following: siJMJD2B#1, 5′- UCUCCAUCACCUGCCUCAAGCACAA-3′; 6 siJMJD2B #2, 5′-GACCTGCTACGAAGTGAACTT -3′ and siSnail, 7 5′-CAGGACUCUAAUCCAGAGUTT-3′. E-cadherin promoter vector 8 pEcad−1008/+49 was kindly provided by Dr. Y.S.Chang (Chang-Gung University, 9 Kwei-shan, Taoyuan, Taiwan 333, Republic of China). TCF/LEF-1 reporter 10 (TOP-FLASH) and mutation vector (FOP-FLASH) were bought from Addgene 11 (Addgene plasmid 12456 and 12457, Cambridge, MA). 12 Luciferase Reporter Assays 14 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C, 15 and then treated with TGF-β1 (5 ng/ml) and/or JMJD2B siRNA or control siRNA. On 16 the following day, VimPro-Luc vector, VimProMut-Luc vector, pEcad−1008/+49 17 vector, pTOP-FLASH or pFOP-FLASH reporter plasmids (0.5 μg each) were 18 separately transfected into the treated cells as above. 24 hours later, luciferase activity 19 was examined by a dual luciferase reporter assay system (Promega, Madison, WI, 20 USA), and firefly luciferase activity was normalized to the control pRL-TK renilla 21 activity included in the kit. 22 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C. 23 On the next day, cells were co-transfected with VimPro-Luc vector, VimProMut-Luc 24 vector, pEcad−1008/+49 vector, pTOP-FLASH or pFOP-FLASH reporter plasmids 25 (0.25 μg each) and HA-JMJD2B plasmid (0.25 μg each). 24 hours later, luciferase 26 activity was examined as above. 27 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C, 28 and then treated with JMJD2B siRNA or control siRNA for 24h. Then these cells 29 were transfected with flag-β-catenin plasmids (0.5 μg). On the following day, 30 pTOP-FLASH or pFOP-FLASH reporter plasmids (0.5 μg each) were separately 31 transfected into the treated cells as above. 24 hours later, luciferase activity was 32 examined. 33 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C, 34 and then were co-transfected with flag-β-catenin and HA-JMJD2B plasmid (0.25 μg 35 each). 24 hours later, luciferase activity was examined as above. 36 Each experiment was done in triplicate and repeated at least three times. 37 38 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C, 15 and then treated with TGF-β1 (5 ng/ml) and/or JMJD2B siRNA or control siRNA. On 16 the following day, VimPro-Luc vector, VimProMut-Luc vector, pEcad−1008/+49 17 vector, pTOP-FLASH or pFOP-FLASH reporter plasmids (0.5 μg each) were 18 separately transfected into the treated cells as above. 24 hours later, luciferase activity 19 was examined by a dual luciferase reporter assay system (Promega, Madison, WI, 20 USA), and firefly luciferase activity was normalized to the control pRL-TK renilla 21 activity included in the kit. 22 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C. Western blot 10 2 Western blot analysis was done for detecting expression of EMT markers and 11 cytoplasmic and nuclear β-catenin in JMJD2B-depleted gastric cells. Cellular proteins 12 (80 μg) were separated by SDS-PAGE and transferred to PVDF or nitrocellulose 13 membranes (Millipore, Billerica, MA, USA). It was blocked with 5% nonfat milk for 14 2h at room temperature. Then the membrane was incubated with human speciﬁc 15 antibodies against JMJD2B (A301-478A, Bethyl Laboratories, Montgomery, USA), 16 E-cadherin, Snail, vimentin (Cell Signaling Technology, Billerica, MA, USA) and 17 β-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA). Thereafter it was 18 incubated with anti-mouse or rabbit horseradish peroxidase-conjugated IgG and 19 developed with the enhanced chemiluminescence kit (Millipore). Actin was a loading 20 control. 21 To detect the expression of cytoplasmic and nuclearβ-catenin, cytoplasmic and 22 nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic 23 Extraction Kit (Thermo-Pierce, Rockford, Illinois, USA) according to the 24 manufacturer’s protocol. Human speciﬁc antibodies against JMJD2B (A301-478A, 25 Bethyl Laboratories, Montgomery, USA), E-cadherin, Snail, vimentin, β-catenin (Cell 26 Signaling Technology, Billerica, MA, USA) and β-actin (Santa Cruz Biotechnology, 27 Santa Cruz, California, USA) were used. 28 29 Apoptosis assay 30 Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling 31 (TUNEL; Beyotime Institute of Biotechnology, China) was employed for analysis of 32 apoptosis in mouse lung metastatic samples according to the manufacturer's 33 instructions. 34 35 Immunohistochemistry 36 Immunohistochemical analysis was done in slides of paraffin-embedded human 37 primary gastric cancer and mouse lung metastatic tissue specimens. Briefly, slides of 38 Western blot analysis was done for detecting expression of EMT markers and 11 cytoplasmic and nuclear β-catenin in JMJD2B-depleted gastric cells. Cellular proteins 12 (80 μg) were separated by SDS-PAGE and transferred to PVDF or nitrocellulose 13 membranes (Millipore, Billerica, MA, USA). It was blocked with 5% nonfat milk for 14 2h at room temperature. Then the membrane was incubated with human speciﬁc 15 antibodies against JMJD2B (A301-478A, Bethyl Laboratories, Montgomery, USA), 16 E-cadherin, Snail, vimentin (Cell Signaling Technology, Billerica, MA, USA) and 17 β-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA). Thereafter it was 18 incubated with anti-mouse or rabbit horseradish peroxidase-conjugated IgG and 19 developed with the enhanced chemiluminescence kit (Millipore). Actin was a loading 20 control. Luciferase Reporter Assays 14 23 On the next day, cells were co-transfected with VimPro-Luc vector, VimProMut-Luc 24 vector, pEcad−1008/+49 vector, pTOP-FLASH or pFOP-FLASH reporter plasmids 25 (0.25 μg each) and HA-JMJD2B plasmid (0.25 μg each). 24 hours later, luciferase 26 activity was examined as above. 27 BGC823 cells were plated into 24-well plate, and incubated overnight at 37°C, 34 and then were co-transfected with flag-β-catenin and HA-JMJD2B plasmid (0.25 μg 35 each). 24 hours later, luciferase activity was examined as above. 36 Each experiment was done in triplicate and repeated at least three times. 37 38 1 TGF-β1 and/or JMJD2B siRNA treated gastric cells (BGC823, HGC27 and 1 MGC803) were ﬁxed with 4% paraformaldehyde and treated with 0.3% Triton-X100 2 for cell membranes permeabilization. Cells were incubated with speciﬁc antibodies 3 against vimentin (ab92547, Abcam, Cambridge, UK), β-catenin (ab32572, Abcam), 4 E-cadherin (#3195S, Cell signaling technology) at a dilution of 1:200 overnight at 5 4°C. The cells were then incubated with Alexa 594-conjugated anti-rabbit. We 6 counterstained the nuclei with DAPI (Beyotime, China). Finally, images were 7 acquired under a FluoView 1000 confocal microscope (Olympus). 8 TGF-β1 and/or JMJD2B siRNA treated gastric cells (BGC823, HGC27 and 1 MGC803) were ﬁxed with 4% paraformaldehyde and treated with 0.3% Triton-X100 2 for cell membranes permeabilization. Cells were incubated with speciﬁc antibodies 3 against vimentin (ab92547, Abcam, Cambridge, UK), β-catenin (ab32572, Abcam), 4 E-cadherin (#3195S, Cell signaling technology) at a dilution of 1:200 overnight at 5 4°C. The cells were then incubated with Alexa 594-conjugated anti-rabbit. We 6 counterstained the nuclei with DAPI (Beyotime, China). Finally, images were 7 acquired under a FluoView 1000 confocal microscope (Olympus). 8 9 Western blot 10 21 To detect the expression of cytoplasmic and nuclearβ-catenin, cytoplasmic and 22 nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic 23 Extraction Kit (Thermo-Pierce, Rockford, Illinois, USA) according to the 24 manufacturer’s protocol. Human speciﬁc antibodies against JMJD2B (A301-478A, 25 Bethyl Laboratories, Montgomery, USA), E-cadherin, Snail, vimentin, β-catenin (Cell 26 Signaling Technology, Billerica, MA, USA) and β-actin (Santa Cruz Biotechnology, 27 Santa Cruz, California, USA) were used. 28 Apoptosis assay 30 Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling 31 (TUNEL; Beyotime Institute of Biotechnology, China) was employed for analysis of 32 apoptosis in mouse lung metastatic samples according to the manufacturer's 33 instructions. 34 Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling 31 (TUNEL; Beyotime Institute of Biotechnology, China) was employed for analysis of 32 apoptosis in mouse lung metastatic samples according to the manufacturer's 33 instructions. 34 Immunohistochemical analysis was done in slides of paraffin-embedded human rimary gastric cancer and mouse lung metastatic tissue specimens. Briefly, slides of Immunohistochemical analysis was done in slides of paraffin-embedded human primary gastric cancer and mouse lung metastatic tissue specimens Briefly slides o 2 3 paraffin-embedded tissue specimens were de-paraffinized, and the antigens retrieval 1 was done by boiling in a microwave oven in 0.01 M citrate buffer (pH 6.0). Then the 2 slides were incubated with a primary anti-JMJD2B antibody (1:100, Bethyl 3 Laboratories, USA) at 4°C overnight, and followed by HRP-conjugated secondary 4 antibody (Zhongshan Goldenbridge, China). Immunostaining was visualized with 3.3′ 5 -diaminobenzidine (DAB Zhongshan Goldenbridge, China). Hematoxylin was used 6 for nuclear counterstaining. Speciﬁc antibodies against JMJD2B (1:100, A301-478A, 7 Bethyl Laboratories), P53 (1:100, Zhongshan Goldenbridge, China) and P21(1:100, 8 #3733-1, Epitomics, Burlingame, CA) were used. 9 10 Scratch wound-healing assay 11 HGC27 and MGC803 cells were seeded in 6-well plates and transfected with 12 JMJD2B siRNA and control siRNA. The conﬂuent cell monolayer was scratched with 13 a clean pipette tip and the plates were washed twice with PBS before adding fresh 14 medium. The cell migration was observed and measured after 24h. Each assay was 15 repeated 3 times. 16 17 Matrigel invasion assay 18 The Matrigel invasion was performed at a 24-well Transwell chamber (Costar, 19 New York, NY, USA). The insert was coated with 40 μl Matrigel (BD Biosciences, 20 Franklin Lakes, NJ, USA). HGC27 and MGC803 cells were collected after 21 transfection with JMJD2B siRNA and control siRNA for 48h and transferred to the 22 chamber inserts in serum-free medium (200 μl containing 1×105cells). The bottom 23 chamber contained medium with 20% FBS as a chemoattractant. The cells were 24 incubated in a humidiﬁed incubator at 37°C for 48h. Then the chamber inserts were 25 washed, ﬁxed and stained with 0.1% crystal violet. Each experiment was done in 26 triplicate. Apoptosis assay 30 27 28 Mouse model 29 BGC823 cells transfected with JMJD2B siRNA and control siRNA for 48h were 30 collected and then 1 × 106 cells suspended in 300 μl PBS were injected into the tail 31 veins of 6-week-old male nude mice (Shanghai Slac Laboratory Animal Co. Ltd, 32 Shanghai, China). Thirty days later, the mice were killed and their lungs were 33 obtained for evaluation of metastasis. JMJD2B silencing in the metastatic lung tissues 34 was detected by immunohistochemical analysis. All research involving animals was 35 conducted in accordance with the guidelines established by Ethics Committee of 36 Shandong University (Jinan, PR.China). 37 paraffin-embedded tissue specimens were de-paraffinized, and the antigens retrieval 1 was done by boiling in a microwave oven in 0.01 M citrate buffer (pH 6.0). Then the 2 slides were incubated with a primary anti-JMJD2B antibody (1:100, Bethyl 3 Laboratories, USA) at 4°C overnight, and followed by HRP-conjugated secondary 4 antibody (Zhongshan Goldenbridge, China). Immunostaining was visualized with 3.3′ 5 -diaminobenzidine (DAB Zhongshan Goldenbridge, China). Hematoxylin was used 6 for nuclear counterstaining. Speciﬁc antibodies against JMJD2B (1:100, A301-478A, 7 Bethyl Laboratories), P53 (1:100, Zhongshan Goldenbridge, China) and P21(1:100, 8 #3733-1, Epitomics, Burlingame, CA) were used. 9 3 antibody (Zhongshan Goldenbridge, China). Immunostaining was visualized with 3.3′ 5 -diaminobenzidine (DAB Zhongshan Goldenbridge, China). Hematoxylin was used 6 for nuclear counterstaining. Speciﬁc antibodies against JMJD2B (1:100, A301-478A, 7 Bethyl Laboratories), P53 (1:100, Zhongshan Goldenbridge, China) and P21(1:100, 8 #3733-1, Epitomics, Burlingame, CA) were used. 9 10 Scratch wound-healing assay 11 HGC27 and MGC803 cells were seeded in 6-well plates and transfected with 12 JMJD2B siRNA and control siRNA. The conﬂuent cell monolayer was scratched with 13 a clean pipette tip and the plates were washed twice with PBS before adding fresh 14 medium. The cell migration was observed and measured after 24h. Each assay was 15 repeated 3 times. 16 17 Matrigel invasion assay 18 The Matrigel invasion was performed at a 24-well Transwell chamber (Costar, 19 New York, NY, USA). The insert was coated with 40 μl Matrigel (BD Biosciences, 20 Franklin Lakes, NJ, USA). HGC27 and MGC803 cells were collected after 21 transfection with JMJD2B siRNA and control siRNA for 48h and transferred to the 22 chamber inserts in serum-free medium (200 μl containing 1×105cells). The bottom 23 chamber contained medium with 20% FBS as a chemoattractant. Apoptosis assay 30 The cells were 24 incubated in a humidiﬁed incubator at 37°C for 48h. Then the chamber inserts were 25 washed, ﬁxed and stained with 0.1% crystal violet. Each experiment was done in 26 triplicate. 27 28 Mouse model 29 BGC823 cells transfected with JMJD2B siRNA and control siRNA for 48h were 30 collected and then 1 × 106 cells suspended in 300 μl PBS were injected into the tail 31 veins of 6-week-old male nude mice (Shanghai Slac Laboratory Animal Co. Ltd, 32 Shanghai, China). Thirty days later, the mice were killed and their lungs were 33 obtained for evaluation of metastasis. JMJD2B silencing in the metastatic lung tissues 34 was detected by immunohistochemical analysis. All research involving animals was 35 conducted in accordance with the guidelines established by Ethics Committee of 36 Shandong University (Jinan, PR.China). 37 10 3
https://openalex.org/W4237492477	https://refubium.fu-berlin.de/bitstream/fub188/26344/1/2020_Bahr_etal.pdf	English	null	Ketogenic diet and fasting diet as Nutritional Approaches in Multiple Sclerosis (NAMS): protocol of a randomized controlled study	Research Square (Research Square)	2,019	cc-by	8,488	Ketogenic diet and fasting diet as Nutritional Approaches in Multiple Sclerosis (NAMS): protocol of a randomized controlled study Lina Samira Bahr1†, Markus Bock2,3,4†, Daniela Liebscher5, Judith Bellmann-Strobl1,6, Liane Franz1, Alexandra Prüß5, Dania Schumann5, Sophie K. Piper7,8, Christian S. Kessler5,9, Nico Steckhan5, Andreas Michalsen5,9, Friedemann Paul1,6,7 and Anja Mähler6,7,10* Friedemann Paul1,6,7 and Anja Mähler6,7,10* Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: anja.maehler@charite.de †Lina Samira Bahr and Markus Bock contributed equally to this work. 6Experimental and Clinical Research Center, a cooperation between Charité—Universitätsmedizin Berlin and Max Delbruck Center for Molecular Medicine, Berlin, Germany 7Berlin Institute of Health, Berlin, Germany Full list of author information is available at the end of the article Bahr et al. Trials (2020) 21:3 https://doi.org/10.1186/s13063-019-3928-9 Bahr et al. Trials (2020) 21:3 https://doi.org/10.1186/s13063-019-3928-9 Bahr et al. Trials (2020) 21:3 https://doi.org/10.1186/s13063-019-3928-9 Open Access Abstract Background: Multiple sclerosis (MS) is the most common inflammatory disease of the central nervous system in young adults that may lead to progressive disability. Since pharmacological treatments may have substantial side effects, there is a need for complementary treatment options such as specific dietary approaches. Ketone bodies that are produced during fasting diets (FDs) and ketogenic diets (KDs) are an alternative and presumably more efficient energy source for the brain. Studies on mice with experimental autoimmune encephalomyelitis showed beneficial effects of KDs and FDs on disease progression, disability, cognition and inflammatory markers. However, clinical evidence on these diets is scarce. In the clinical study protocol presented here, we investigate whether a KD and a FD are superior to a standard diet (SD) in terms of therapeutic effects and disease progression. Methods: This study is a single-center, randomized, controlled, parallel-group study. One hundred and eleven patients with relapsing–remitting MS with current disease activity and stable immunomodulatory therapy or no disease-modifying therapy will be randomized to one of three 18-month dietary interventions: a KD with a restricted carbohydrate intake of 20–40 g/day; a FD with a 7-day fast every 6 months and 14-h daily intermittent fasting in between; and a fat-modified SD as recommended by the German Nutrition Society. The primary outcome measure is the number of new T2-weighted MRI lesions after 18 months. Secondary endpoints are safety, changes in relapse rate, disability progression, fatigue, depression, cognition, quality of life, changes of gut microbiome as well as markers of inflammation, oxidative stress and autophagy. Safety and feasibility will also be assessed. Discussion: Preclinical data suggest that a KD and a FD may modulate immunity, reduce disease severity and promote remyelination in the mouse model of MS. However, clinical evidence is lacking. This study is the first clinical study investigating the effects of a KD and a FD on disease progression of MS. Trial registration: ClinicalTrials.gov, NCT03508414. Retrospectively registered on 25 April 2018. Keywords: Multiple sclerosis, Dietary intervention, Ketogenic diet, Intermittent fasting, Anti-inflam © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. * Correspondence: anja.maehler@charite.de Correspondence: anja.maehler@charite.de †Lina Samira Bahr and Markus Bock contributed equally to this work. 6Experimental and Clinical Research Center, a cooperation between Charité—Universitätsmedizin Berlin and Max Delbruck Center for Molecular Medicine, Berlin, Germany 7Berlin Institute of Health, Berlin, Germany Full list of author information is available at the end of the article Introduction Profound reduction of either overall calorie or carbo- hydrate intake decreases glucose and insulin levels, thereby driving the body to produce ketone bodies from internal or external fats. These ketone bodies provide an alternative energy source for the brain. They might even be more efficient (per unit oxygen) than glucose [24]. Furthermore, ketone bodies seem to stimulate mito- chondrial biogenesis and reduce mitochondrial perme- ability [25, 26]. Multiple sclerosis (MS) is the most common chronic in- flammatory autoimmune disease of the central nervous system. It leads to neurodegeneration and is a major cause of progressive disability and early retirement in young adults [1–3]. According to estimates, more than 2 million people worldwide are affected [4]. Relapsing–re- mitting MS (RRMS) is the most common form of MS and is characterized by relapses and periods of remission as well as systemic inflammation of the central nervous system and progressive neurodegeneration from the earliest disease stages [5, 6]. KDs and fasting diets (FDs) both drastically reduce carbohydrate intake and it has been suggested that the resulting ketone bodies facilitate the regeneration of demyelinated axons [20]. It is thus feasible that either of these dietary approaches could have a therapeutic bene- fit in MS patients. Currently, there is no cure for MS, but several immu- nomodulatory therapies are available that may slow dis- ease progression [7]. However, all of them may have substantial side effects and patients respond differently due to the complex nature of the disease. Consequently, there is a need for complementary therapies such as spe- cific diets that may reduce MS symptoms, improve the patient’s quality of life and even delay disease progres- sion. A recent review reported that up to 70% of MS pa- tients already use alternative and complementary medicine [8]. Fasting not only induces ketosis, but might also acti- vate autophagy in the brain, liver and muscle [27]. Macroautophagy is a key quality-control pathway in cells through which nonnuclear parts of the cell and cytoplas- mic macromolecules are renewed and mobilized, re- spectively. It is thought to mediate the anti-aging effects of calorie restriction [28]. Since some of the effects of fasting can be replicated with a KD, autophagy might also be relevant for KDs. Indeed, current evidence from a rat model suggests that a KD can attenuate seizure-induced neuronal injury via autophagy [29]. Introduction It is widely accepted that a combination of genetic susceptibility and environmental factors causes MS [9]. That nutrition might be among these factors is supported by the fact that MS is more frequent in western countries than in less-developed nations [10]. The so-called western diet is high in energy, saturated fats and sugars. Of note, a meal high in fat and refined carbohydrates has been shown to cause a more pronounced postprandial insulin se- cretion and enhanced inflammation compared to a meal high in fiber and fruits in healthy lean subjects [11]. In line with this, a 12-week ketogenic diet (KD) decreased insulin resistance and several serum inflammatory markers in overweight men and women [12]. The KD was originally conceptualized to mimic the biochemical effects associated with fasting [30] and was used as an alternative treatment in pharmaco-resistant childhood epilepsy as early as the 1920s [31]. Since then, KDs have also been shown to improve symptoms in other neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [32, 33]. In the case of MS, there is preclinical evidence for the efficacy of KDs and FDs from experimental autoimmune encephalomyelitis (EAE), the established animal model of MS. A KD slowed disease progression, improved motor disability and hippocampal atrophy, reversed le- sions and suppressed inflammatory cytokines and react- ive oxygen species [34]. A fasting mimicking diet was shown to delay onset and slow disease progression. This was accompanied by increased corticosterone levels, autoreactive lymphocyte apoptosis and oligodendrocyte regeneration during the fasting [35]. These findings are supported by other EAE studies showing beneficial ef- fects of intermittent fasting and chronic calorie restric- tion [36–38]. These are important findings since insulin resistance seems to be more common in MS patients than in healthy controls and is associated with higher Expanded Disability Status Scale (EDSS) scores [13, 14]. Further- more, elevated serum levels of the pro-inflammatory cytokine IL-17 in MS patients [14] may contribute to the impaired glucose and insulin metabolism in MS [15]. The cerebral glucose hypometabolism that occurs in MS patients [16–18] is thought to reflect mitochondrial dysfunction in neuronal cells [19]. Although the mech- anism is not yet defined, it is feasible that neurodegener- ative processes driven partly by oxidative stress contribute to this mitochondrial dysfunction [20]. In- deed, several studies have demonstrated increased markers of oxidative stress and decreased compensatory antioxidative capacity in MS patients [14, 21–23]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bahr et al. Trials (2020) 21:3 Bahr et al. Trials (2020) 21:3 Page 2 of 9 Bahr et al. Trials Introduction Changes in the gut microbiome are associated with many disease states, including autoimmune diseases such as MS, and the gut microbiome is intricately linked to our immune system and inflammatory responses. The typical western diet is associated with gut microbial imbalance (dysbiosis), low-grade inflammation and neuroinflammation [39]. Bahr et al. Trials (2020) 21:3 Bahr et al. Trials (2020) 21:3 Page 3 of 9 Page 3 of 9 Page 3 of 9 Interestingly, recent studies have shown that gut dysbiosis can also occur in MS patients [40–42]. Both KDs and FDs may positively affect the gut microbiota by enhancing gut microbial diversity [42, 43]. Thus, changes of the gut micro- biome could be implicated in the effects of our dietary regimens. restricted to 20–40 g/day; a FD with a 7-day fast every 6 months and 14-h daily intermittent fasting in between; and a SD that is predominantly vegetarian and fat- modified as recommended by the German Nutrition So- ciety. The institutional review board of Charité—Univer- sitätsmedizin Berlin approved the study and written informed consent is obtained from all participants prior to study entry (Additional files 1 and 2). The study is conducted in accordance with the Declaration of Helsinki in its currently applicable version, the guide- lines of the International Conference on Harmonization of Good Clinical Practice (ICH-GCP) and applicable German laws. For further details refer to the SPIRIT checklist (Additional file 3). We have already obtained clinical evidence for the feasibility and safety of KDs and FDs in MS patients and showed that they might improve health-related quality of life [35]. Another more recent pilot study which tested the safety and tolerability of a modified Atkins diet found decreased body mass index, body fat mass, fa- tigue and depression scores in 20 RRMS patients. This was, however, an uncontrolled study with just a 6- month intervention period, which cannot corroborate ef- fects on disease progression [44]. Study design ≥1 relapse or ≥1 new T2 lesion or ≥1 contrast- enhancing lesion on MRI within the last 2 years This is a single-center, randomized, controlled, three- armed, parallel-group study conducted at the NeuroCure Clinical Research Center at Charité—Universitätsmedi- zin Berlin. Recruitment started in April 2017 (Clinical- Trials.gov identifier: NCT03508414). Patients are recruited from all over Germany. Recruitment strategies include specific study calls on the website of the German Multiple Sclerosis Society and the distribution of study flyers in specialized neurological practices as well as in- formation events and lectures for patients. We intend to randomize 111 RRMS patients to one of three 18-month dietary interventions: a KD with a carbohydrate intake Agreement that possible incidental findings will be communicated Body mass index between 19 and 45 kg/m2 Ability to give verbal and written consent Health insurance Participants Patient information and informed consent have been prepared in accordance with the guidelines of the in- stitutional review board of Charité—Universitätsmedi- zin Berlin. Potential participants receive both forms at least 24 h before a consultation with a study physician who personally explains all study procedures. If he or she is willing to participate and had sufficient time to ask questions, written informed consent is given. Afterward, inclusion and exclusion criteria are assessed. The main inclusion criteria are a definite diagnosis of RRMS according to the 2017 McDonald criteria [46], a stable immunomodulatory therapy or no disease-modifying therapy for at least 6 months, an EDSS score below 4.5 [47] and disease activity within the last 2 years before study entry. This is defined as at least one new lesion on brain MRI and/or at least one relapse. Participants are recruited in cohorts of 15–25 patients. Here, we present the protocol of a randomized, con- trolled clinical study investigating the effects of a KD and a FD compared to a standard diet (SD) in RRMS. The SD is a healthy, predominantly vegetarian diet that follows the recommendations of the German Nutrition Society (DGE). MS patients adhering to a similar diet for 12 months showed improved body mass index, metabolic markers and fatigue but no changes in brain MRI out- comes [45]. Thus, we hypothesize that KDs and FDs are superior to the SD with respect to new lesions on cranial T2- weighted MRI after 18 months compared to baseline. We further hypothesize differential effects of our inter- ventions in men and women, because of sex-specific metabolic differences (e.g. fat and carbohydrate metabol- ism). We further expect larger treatment effects in pa- tients without MS medication. Secondary endpoints are safety aspects of the dietary interventions, brain volume, annualized relapse rate, dis- ability progression, changes in gut microbiome, metabo- lomics of stool and blood, immunological effects, cognition, fatigue, depression, muscle strength, walking endurance and quality of life. Methods Stable immunomodulatory therapy or no disease- modifying therapy ≥6 months before study entry Complete list of inclusion criteria Relapsing–remitting MS 18–65 years of age EDSS < 4.5 Relapsing–remitting MS 18–65 years of age y EDSS < 4.5 Dietary interventions Nutritional counseling for all three interventions takes place in small groups within 10 sessions over 18 months (five sessions in months 1–3, five sessions in months 4– 18). The group setting facilitates exchange between pa- tients, management of dietary challenges, monitoring of compliance and recording of adverse events. Between the group sessions, patients have the possibility to con- tact their nutritional counselor via email or telephone at any time. KD. During nutritional counseling, patients are instructed to start by limiting carbohydrate intake to just 20 g/day for 4 weeks in order to establish ketosis. Then, patients increase their carbohydrate intake by 5 g each week until they reach their individual maximum (approximately 40 g) to maintain stable ketosis. All carbohydrates relevant for elevating blood glucose are limited to 40–50 g/ day. In addition, the glycemic index and glycemic load of carbohydrates have to be below 50 and 6, respectively. This ketogenic diet is equivalent to a traditional ketogenic diet, but with a liberalized macronutrient composition of 70–80% fats, 15–20% proteins and 5–10% carbohydrates (compared to a traditional ketogenic diet with 90% fat, 6% proteins and 4% carbohydrates). To aid in the adjustment and determine their individual carbohydrate intake limits, patients receive a hand-held ketone-meter to measure and record blood concentrations of ketone body β-hydroxybutyrate at regular intervals. Values should be between 0.5 and 3.0 mmol/L. Contraindications for MRI such as metallic implants, cardiac pacemakers and claustrophobia Retention of patients is promoted by close and fre- quent contact with their nutritional counselor and study physician via telephone, email and study visits. In case of relevant issues, visits outside the study schedule are offered. Discontinuation criteria are withdrawal of consent, subsequent occurrence of an exclusion criterion (e.g. change of immunomodulatory therapy), lack of compli- ance and medical reasons for stopping the intervention. For all three groups, compliance is defined as attend- ing at least 8 of 10 group sessions and each of six study visits. Food records measure compliance to the pre- scribed intervention. In the KD group, the majority of blood ketone measurements should be ≥0.5 mmol/L. For the FD group, there are regular additional meetings during each of the three fasting periods. These meetings have to be attended, enabling the physician and the counselor to evaluate compliance. FD. Patients in the FD group fast for 7 days every 6 months. Dietary interventions During this intensive fasting episode, patients only consume vegetable juices and vegetable broth, yielding a daily calorie intake between 200 and 350 kcal. Tea and water is available ad libitum to ensure sufficient fluid intake. Two days before and 3 days after fasting, patients eat a low-calorie vegetarian diet for preparation and aftercare. The fasting is initialized by an intestinal clearing with lax- atives (e.g. Glauber’s Salt by FX-Passage). Outside these intensive fasting episodes, patients intermit- tently fast for 14 h daily on 7 days of the week in order to maintain fasting effects. For compliance reasons, participants may take one “cheat day” per week at which they follow the fasting rules less FD. Patients in the FD group fast for 7 days every 6 months. During this intensive fasting episode, patients only consume vegetable juices and vegetable broth, yielding a daily calorie intake between 200 and 350 kcal. Tea and water is available ad libitum to ensure sufficient fluid intake. Two days before and 3 days after fasting, patients eat a low-calorie vegetarian diet for preparation and aftercare. The fasting is initialized by an intestinal clearing with lax- atives (e.g. Glauber’s Salt by FX-Passage). Outside these intensive fasting episodes, patients intermit- tently fast for 14 h daily on 7 days of the week in order to maintain fasting effects. For compliance reasons, participants may take one “cheat day” per week at which they follow the fasting rules less We offer patients who meet a discontinuation criterion to attend the remaining study visits for follow-up outside the study protocol. We assess all outcomes except for exploratory blood parameters (autophagy, oxidative stress) in these patients. Complete list of exclusion criteria Start or change of immunomodulatory therapy < 6 months before or during the study Page 4 of 9 Bahr et al. Trials (2020) 21:3 Page 4 of 9 Bahr et al. Trials Relapse or cortisone treatment within 30 days before study entry Clinically relevant metabolic, progressive or malignant diseases Intake of > 1 g/day omega-3 fatty acid supplements Significant cognitive-cooperative impairment Insulin-dependent diabetes mellitus (type I) Participation in another interventional study Weight loss diet or loss of more than 5 kg within 2 months before study entry Insufficient mental ability for cooperation Eating disorders Kidney stones Therapy with oral anticoagulants Pregnancy and breastfeeding Suspected lack of compliance Smokers (> 5 cigarettes per day) Known alcohol and drug abuse Inability to give informed consent or apply to the study protocol Contraindications for MRI such as metallic implants, cardiac pacemakers and claustrophobia scan. We use a stratified block randomization with variable block length. This will ensure a homogeneous distribution of interventions over these strata. An exter- nal statistician who is not involved in the study carries out the randomization according to predefined randomization lists. Power calculation Based on the results of other MS studies in our research center in comparable patient cohorts, an average of three new T2 lesions after 18 months is expected in the SD group. Sample size was calculated with the Wilcoxon (Mann–Whitney) rank-sum test and the significance level was adjusted (Bonferroni) for the two comparisons (KD vs. SD and FD vs. SD). With a sample size of 33 pa- tients per group and a two-sided significance level of 0.025, there is a power of 80% to detect a probability of 0.72 that an observation from group 1 is smaller than that from group 2. This corresponds to a standardized effect size of 0.83 and an expected mean difference in the number of new T2 lesions in each of the interven- tion groups of 0.5 compared to the SD group after 18 months (Query Advisor 7.0). We expect a dropout rate of approximately 10% and therefore plan to enroll 37 pa- tients per group, 111 patients in total. Outcome parameters Our primary endpoint is the number of new lesions on cranial T2-weighted MRI after 18 months compared to baseline. The secondary MRI endpoint is brain atrophy determined by percentage brain volume change. All MRI scans will be performed in a 3-Tesla MRI scanner (Tim Trio; Siemens, Erlangen, Germany) and will be assessed by an experienced evaluator who is blinded to both clin- ical data and interventional allocation. Randomization Eligibility of patients will be determined at the screening visit by a trained physician. Afterward, randomization is done in three strata to distribute possible confounding factors equally to the dietary interventions. Strata are sex (man or woman), MS medication (yes or no) and lesion load (< 15 or ≥15 lesions) in the baseline brain MRI Bahr et al. Trials (2020) 21:3 Page 5 of 9 Page 5 of 9 kidney and liver parameters in peripheral venous blood. In addition, we want to investigate metabolic, hormonal and immunological effects of the diets. For this, periph- eral blood mononuclear cells are isolated for further analysis. In addition, markers of oxidative stress, espe- cially reaction products of anti-oxidative enzymes, are of interest. The polyamine level and cell metabolites will be measured as secondary markers of nutrient availability and autophagy activation in the cell. Stool samples are collected for 16s rRNA sequencing. For a detailed over- view of assessments and endpoints, see Table 1. strictly. During intermittent fasting, patients are counseled to eat a diet according to the recommen- dations of the DGE (equivalent to the SD group). Patients additionally attend group sessions every other day during fasting. A physician and a dietician with expertise on fasting monitor these sessions. During the first intensive fasting episode, these ses- sions are every other day. In the subsequent fasting episodes, the meetings are less frequent (three or four meetings). g SD. Patients are counseled to adhere to a healthy, non-calorie-restricted diet according to the rec- ommendations of the DGE. This diet is predom- inantly vegetarian with reduced consumption of meat, animal fats, eggs and egg products. Low-fat milk and dairy products are recommended to pro- vide calcium. Dietary recommendations are de- signed to modify the omega-6 to omega-3 fatty acid ratio to 5:1. SD. Patients are counseled to adhere to a healthy, non-calorie-restricted diet according to the rec- ommendations of the DGE. This diet is predom- inantly vegetarian with reduced consumption of meat, animal fats, eggs and egg products. Low-fat milk and dairy products are recommended to pro- vide calcium. Dietary recommendations are de- signed to modify the omega-6 to omega-3 fatty acid ratio to 5:1. We will inform participants about any conspicuous findings and refer them to their general practitioner for further treatment. After completion of the study, partici- pants have the possibility to be referred to appropriate outpatient nutritional counseling. Adverse events There are no major risks for patients participating in this study. Minor adverse events of a ketogenic diet and fast- ing can be headaches, feelings of hunger, fatigue, irrit- ability and dizziness. These side effects are transient and will stop after a few days. Patients will be asked for toler- ability of the interventions and any adverse events will be recorded. We do not expect serious adverse events due to our dietary interventions. Data management h Trials (2020) 21:3 Table 1 SPIRIT flow diagram of the NAMS study Visit –1, screening 0, baseline 1, start intervention 2 3 4 5 Month −4 (max) −1 0 3 9 15 18 Informed consent x Demographics x Inclusion/exclusion criteria x x x x x x x Case history x Medication x x x x x x x Vital signs x x x x x Bioelectrical impedance analysis x x x Anthropometric data x x x x x x Hand grip strength x x x Safety laboratory x x x x x Research laboratory x x x Urine sample x x x x x Stool sample x x x Multiple Sclerosis Quality of Life-54 x x x Questionnaire on activities x x x Beck Depression Inventory II x x x Fatigue Severity Scale x x x Physical examination x x x Multiple Sclerosis Functional Composite x x x Symbol Digit Modalities Test x x x Six-minute walk test x x x Expanded Disability Status Scale x x x Relapse query x x x x x x x AE/SAE query x x x x x x MRI x x x Four-day food record x x x Nutritional counseling x * * * * AE adverse event, MRI magnetic resonance imaging, SAE serious adverse event There are 10 group sessions for nutritional counseling within 18 months. The fasting diet group has additional meetings during their 7-day-fasts at baseline and after 6 and 12 months Table 1 SPIRIT flow diagram of the NAMS study AE adverse event, MRI magnetic resonance imaging, SAE serious adverse event There are 10 group sessions for nutritional counseling within 18 months. The fasting diet group has additional meetings during their 7-day-fasts at baseline and after 6 and 12 months ic resonance imaging, SAE serious adverse event or nutritional counseling within 18 months. The fasting diet group has additional meetings during their 7-day-fasts at baseline and We only obtain consent for usage of data and samples for the research question described in this protocol. Thus, we do not intend to use participant data or bio- logical samples in ancillary studies. intervention adjusted for the baseline value is lower in the KD and FD groups than in the SD group. Data management h Each participant will receive a unique identifier upon study entry. This identifier will be used for all data docu- mentation to ensure the participant’s confidentiality. Data will be gathered in source documents and then transferred onto paper case report forms. Later, all data will be digitized and stored in a central database. To im- prove accuracy of data entry, entries will be verified for proper format and expected range as well as double- checked. Overall data quality will be assured by inde- pendent monitoring throughout the study. However, due to the minimal risks of our dietary interventions, a data monitoring committee is deemed unnecessary. Data will be stored for 10 years after study completion and then deleted. Any modification to the current study protocol will be submitted to the institutional review board, all trial participants and the trial investigators. Additional secondary endpoints are the annualized re- lapse rate, neurological–functional disability progression (EDSS, Multiple Sclerosis Functional Composite), cogni- tion (Symbol Digit Modalities Test), fatigue (Fatigue Severity Scale), depression (Beck Depression Inventory II), muscle strength (handgrip dynamometer), walking endurance (6-min walk test) and quality of life (MSQoL- 54). In a subset of patients, we will evaluate glucose vari- ability from continuous glucose measurements over 14 days (FreeStyle Libre Sensors; Abbott). All endpoints will be assessed at baseline, 9 and 18 months by trained and experienced personnel. For safety monitoring, body weight and composition, dietary intake and vital signs are recorded at regular in- tervals as well as routine laboratory tests of blood count, Bahr et al. Trials Page 6 of 9 Bahr et al. Trials (2020) 21:3 Bahr et al. Data management h Although the sample size calculation was conservatively based on the Mann–Whitney U test, the final analysis of the pri- mary endpoint will be based on non-parametric analysis of covariance (ANCOVA) on ranks with the treatment group as a fixed factor and adjustment for the baseline number of T2 lesions. The resulting coefficients will be reported with 95% confidence intervals and their corre- sponding p-values compared to a two-sided Bonferroni- adjusted significance level of 0.025 [48]. Results will be personally explained to all study partici- pants, presented on national and international conferences, published in peer-reviewed journals, and disseminated to neurologists and the medical laity. We will comply with the official eligibility guidelines for authorship for all publica- tions and do not intend to use professional writers. Discussion Fourth, controlling and ensuring adherence to dietary interventions is rather challenging. This is especially true in an outpatient setting during such a long study period. Some patients might be disappointed with their inter- ventional allocation and surreptitiously include some features of the desired group into their diet. This can only be avoided by close and frequent contact between counselor and patient. Furthermore, changing estab- lished dietary preferences can be rather challenging and might lead to compliance issues. Patients with suspected or known lack of compliance are excluded from the per- protocol analysis. To date, this is the first study that investigates long-term efficacy of KDs and FDs on T2 hyperintense lesion load and clinical measures of disease activity and progression in MS patients. This is also the first study that investi- gates therapeutic effects of repeated prolonged fasting combined with daily intermittent fasting. There are sev- eral reasons for the scarcity of such large-scale, long- term studies. The most prominent reasons are, of course, the high demand of financial and human re- sources. Further, patients often prefer one particular intervention and are therefore prone to withdraw con- sent when randomized to an undesired intervention. Once patients have committed to such a study, it is chal- lenging to convey dietary interventions in a way that suf- ficient motivation is aroused. Only this will ensure dietary adherence for the whole intervention period. One great strength of our study is that patients in the KD group measure their blood ketones at home. Many other studies with low-carbohydrate–high-fat diets did not verify ketosis, which limits the interpretation of re- sults. Although we do not know the actual concentra- tions of ketone bodies in the brain, blood ketone concentrations have been shown to drive the brain’s overall metabolic rate [50]. There are some critical aspects of our study design. First, we include patients with any MS medication or no medication. From a methodological point of view, in- cluding only patients on the same treatment or un- treated patients would have been preferable. However, in light of the variety of available treatment options, we would not be able to recruit a sufficient number of pa- tients with the same medication within an acceptable time span. Furthermore, by including patients with different treatments, putative positive results will be more generalizable. Data analysis We do not plan any interim analyses. In the case of more than 5% missing for the primary endpoint, we plan to replace missing values in the outcome parameter using multiple imputation [49] under the missing at random as- sumption in addition to the complete case analyses. Confirmatory analysis will be conducted based on the intention-to-treat (ITT) population. The aim is to show that KDs and FDs are superior to the SD, meaning that the number of new T2 lesions after 18 months of dietary Bahr et al. Trials (2020) 21:3 Bahr et al. Trials (2020) 21:3 Bahr et al. Trials (2020) 21:3 Page 7 of 9 Page 7 of 9 As a sensitivity analysis, the primary endpoint will be analyzed in the per-protocol population. This population includes all randomized patients who meet the study eli- gibility criteria and fulfill all compliance criteria through- out the study. treatment and no treatment. Having a stratum for every treatment would have resulted in a sample size too un- realistic to recruit and finance. Besides, transferability to clinical routine is only given if the study population, at least in principle, reflects the clinical routine with its di- verse application of different treatment options. We intend to perform two subgroup analyses, men vs. women and MS medication vs. no MS medication, by adding another main effect for sex or medication, respectively, as well as an interaction term. These sub- group analyses will be analyzed in an exploratory man- ner to generate possible hypotheses for follow-up studies without adjustment for multiple comparisons. For these, p-values may not be interpreted as confirmative. Second, a dietary intervention study cannot be blinded completely. Thus, expectations and observer bias cannot be ruled out. However, we try to minimize bias by not communicating any longitudinal data during the study to patients and study personnel in contact with them. Even more important, MRI analysts who evaluate the primary endpoint are blinded for both clinical data and interventional allocation. Safety analysis will include calculation of frequencies and rates of adverse and serious adverse events within 18 months of the interventions. Third, we undertake MRI scans of both the cranium and the spinal cord to evaluate disease activity before study entry in order to prevent selection bias. The study MRI, however, only analyzes the cranium, thereby neglecting spinal cord lesions. This entails the risk of overlooking disease activity. Data analysis However, newly developing spinal cord lesions in MS are often associated with clin- ical symptoms. Thus, they would be recorded as a re- lapse within regular clinical assessments. Furthermore, cerebral lesions are more frequent and, therefore, consti- tute a reliable marker for disease activity. Secondary endpoints will be analyzed in an explorative manner and descriptives will be given using the mean and standard deviation for sufficiently normal distrib- uted metric variables, the median with limits of the interquartile range (IQR: 25th and 75th percentiles) for skewed metric or ordinal data, as well as absolute and relative frequencies for count data. Funding g This study is funded by grants from the Walter and Ilse Rose-foundation and Myelin-Project e.V. FP is supported by Deutsche Forschungsgemeinschaft (DFG Exc 257). 10. Dua TR, P. Atlas multiple sclerosis resources in the world 2008. Accessed 12 November 2019 [Available from: https://www.who.int/mental_health/ neurology/Atlas_MS_WEB.pdf. 10. Dua TR, P. Atlas multiple sclerosis resources in the world 2008. Accessed 12 November 2019 [Available from: https://www.who.int/mental_health/ neurology/Atlas_MS_WEB.pdf. 11. Ghanim H, Abuaysheh S, Sia CL, Korzeniewski K, Chaudhuri A, Fernandez- Real JM, et al. Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance. Diabetes Care. 2009;32(12):2281–7. 11. Ghanim H, Abuaysheh S, Sia CL, Korzeniewski K, Chaudhuri A, Fernandez- Real JM, et al. Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance. Diabetes Care. 2009;32(12):2281–7. Discussion Thus, we decided to include all pa- tients who have been stable on or off treatment for at least 6 months. Stratification only differentiates between Further strengths are the randomized study design, large sample size, long intervention of 18 months and blinded outcome assessment. Moreover, we focus on both MRI-based measures of disease activity and pro- gression and several patient-related endpoints, such as fatigue, depression and quality of life. Considering epidemiological and preclinical data, on the one hand, and the lack of clinical data, on the other, this study has the potential to provide essential data on the Bahr et al. Trials (2020) 21:3 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Consent for publication Not applicable. Consent for publication Not applicable. efficacy of KDs and FDs. By analyzing metabolic markers, oxidative stress and the gut microbiome, we hope to shed some light on the mechanisms that underlie these dietary regimens. A better mechanistic understanding might also be applicable to other neurodegenerative diseases. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests Th h d l The authors declare no competing interests. The final trial dataset will be available to the investigators. The funders of the study are neither involved in the study design nor the interpretation of data or the publishing of the results. In conclusion, KDs and FDs have the potential for a safe and inexpensive complementary treatment option in MS and our study might close the gap between promis- ing preclinical data and the lack of clinical data. Author details 1 1NeuroCure Clinical Research Center and Department of Neurology, Charité—Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt Universität Berlin, Berlin, Germany. 2Institute of Biochemistry, University Medicine Berlin—Charité, Charitéplatz 1, D-10117 Berlin, Germany. 3Department of Medicine B, Ruppin General Hospital, Brandenburg Medical School, 16816 Neuruppin, Germany. 4Department of Hand Surgery, Upper Extremity and Foot Surgery, Center for Orthopedics and Trauma Surgery, Hospital Waldfriede, Argentinische Allee 40, 14163 Berlin, Germany. 5Institute of Social Medicine, Epidemiology & Health Economics, Charité—Universitätsmedizin Berlin, Berlin, Germany. 6Experimental and Clinical Research Center, a cooperation between Charité—Universitätsmedizin Berlin and Max Delbruck Center for Molecular Medicine, Berlin, Germany. 7Berlin Institute of Health, Berlin, Germany. 8Institute of Biometry and Clinical Epidemiology, Abbreviations EDSS: Expanded Disability Status Scale; FD: Fasting diet; ICH- GCP: International Conference on Harmonization of Good Clinical Practice; KD: Ketogenic diet; MRI: Magnetic resonance imaging; MS: Multiple sclerosis; NAMS: Nutritional Approaches in Multiple Sclerosis; QoL: Quality of life; RRMS: Relapsing–remitting multiple sclerosis; SD: Standard diet Received: 19 July 2019 Accepted: 22 November 2019 Received: 19 July 2019 Accepted: 22 November 2019 Received: 19 July 2019 Accepted: 22 November 2019 Trial status This article is based on the study protocol version 1.4 of 23 September 2019. The NAMS started on 24 May 2017. Recruitment will probably continue until March 2020. References fl 1. Pfleger CC, Flachs EM, Koch-Henriksen N. Social consequences of multiple sclerosis (1): early pension and temporary unemployment—a historical prospective cohort study. Mult Scler. 2010;16(1):121–6. 1. Pfleger CC, Flachs EM, Koch-Henriksen N. Social consequences of multiple sclerosis (1): early pension and temporary unemployment—a historical prospective cohort study. Mult Scler. 2010;16(1):121–6. 1. Pfleger CC, Flachs EM, Koch-Henriksen N. Social consequences of multiple sclerosis (1): early pension and temporary unemployment—a historical prospective cohort study. Mult Scler. 2010;16(1):121–6. 2. Flachenecker P, Stuke K, Elias W, Freidel M, Haas J, Pitschnau-Michel D, et al. Multiple sclerosis registry in Germany: results of the extension phase 2005/ 2006. Dtsch Arztebl Int. 2008;105(7):113–9. 2. Flachenecker P, Stuke K, Elias W, Freidel M, Haas J, Pitschnau-Michel D, et al. Multiple sclerosis registry in Germany: results of the extension phase 2005/ 2006. Dtsch Arztebl Int. 2008;105(7):113–9. Acknowledgements h h h k The authors thank Michael Scheel (Charité—Universitätsmedizin Berlin) for developing and overseeing the MRI protocol. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Funds of Charité – Universitätsmedizin Berlin. 3. Krieger SC, Cook K, De Nino S, Fletcher M. The topographical model of multiple sclerosis: a dynamic visualization of disease course. Neurol Neuroimmunol Neuroinflamm. 2016;3(5):e279. 3. Krieger SC, Cook K, De Nino S, Fletcher M. The topographical model of multiple sclerosis: a dynamic visualization of disease course. Neurol Neuroimmunol Neuroinflamm. 2016;3(5):e279. 4. Kingwell E, Marriott JJ, Jette N, Pringsheim T, Makhani N, Morrow SA, et al. Incidence and prevalence of multiple sclerosis in Europe: a systematic review. BMC Neurol. 2013;13:128. Authors’ contributions JB-S, MB, AMi, FP and AMä designed the study and drafted the study protocol. LSB, LF, DL, AP, DS, MB, CSK, NS and AMä developed the interventional concept. LSB, AMi, FP and AMä procured funding. SKP performed statistical planning, sample size calculation and randomization. LSB and AMä drafted the manuscript. All authors read and approved the final version. 7. Dorr J, Paul F. The transition from first-line to second-line therapy in multiple sclerosis. Curr Treat Options Neurol. 2015;17(6):354. 7. Dorr J, Paul F. The transition from first-line to second-line therapy in multiple sclerosis. Curr Treat Options Neurol. 2015;17(6):354. multiple sclerosis. Curr Treat Options Neurol. 2015;17(6):354. 8. Yadav V, Shinto L, Bourdette D. Complementary and alternative medicine for the treatment of multiple sclerosis. Expert Rev Clin Immunol. 2010;6(3): 381–95. 8. Yadav V, Shinto L, Bourdette D. Complementary and alternative medicine for the treatment of multiple sclerosis. Expert Rev Clin Immunol. 2010;6(3): 381–95. 9. Wekerle H. Nature, nurture, and microbes: the development of multiple sclerosis. Acta Neurol Scand. 2017;136(Suppl 201):22–5. 9. Wekerle H. Nature, nurture, and microbes: the development of multiple sclerosis. Acta Neurol Scand. 2017;136(Suppl 201):22–5. Study sponsor h Charité—Universitätsmedizin Berlin. Prof. Dr. Friedemann Paul (Principle Investigator), Charité—Universitätsmedizin Berlin. Prof. Dr. Friedemann Paul (Principle Investigator), 5. Pawlitzki M, Neumann J, Kaufmann J, Heidel J, Stadler E, Sweeney-Reed C, et al. Loss of corticospinal tract integrity in early MS disease stages. Neurol Neuroimmunol Neuroinflamm. 2017;4(6):e399. g Charitéplatz 1, 10117 Berlin, Germany; email: friedemann.paul@charite.de g Charitéplatz 1, 10117 Berlin, Germany; email: friedemann.paul@charite.de Charitéplatz 1, 10117 Berlin, Germany; email: friedemann.paul@charite.de 6. Azevedo CJ, Overton E, Khadka S, Buckley J, Liu S, Sampat M, et al. Early CNS neurodegeneration in radiologically isolated syndrome. Neurol Neuroimmunol Neuroinflamm. 2015;2(3):e102. Supplementary information y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-019-3928-9. Charité—Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1, D-10117 Berlin, Germany. 9Department of Internal and Integrative Medicine, Immanuel Krankenhaus Berlin, Berlin, Germany. 10DZHK (German Centre for Cardiovascular Research), Berlin, Germany. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 2. Model informed consent form. Additional file 3. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 2. Model informed consent form. Additional file 3. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 2. Model informed consent form. Additional file 3. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 1. Approval of institutional review board of Charité - Universitätsmedizin Berlin. Additional file 2. Model informed consent form. Additional file 2. Model informed consent form. Additional file 3. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. Ethics approval and consent to participate This study was approved by the institutional review board of Charité—Universitätsmedizin Berlin and written informed consent was obtained from all study participants before study entry. 12. Forsythe CE, Phinney SD, Fernandez ML, Quann EE, Wood RJ, Bibus DM, et al. Comparison of low fat and low carbohydrate diets on circulating fatty acid composition and markers of inflammation. Lipids. 2008;43(1):65–77. 12. Forsythe CE, Phinney SD, Fernandez ML, Quann EE, Wood RJ, Bibus DM, et al. Comparison of low fat and low carbohydrate diets on circulating fatty acid composition and markers of inflammation. Lipids. 2008;43(1):65–77. Page 9 of 9 Page 9 of 9 Page 9 of 9 Bahr et al. Trials (2020) 21:3 Bahr et al. Trials (2020) 21:3 Bahr et al. Trials 13. Oliveira SR, Kallaur AP, Lopes J, Colado Simao AN, Reiche EM, de Almeida ERD, et al. Insulin resistance, atherogenicity, and iron metabolism in multiple sclerosis with and without depression: associations with inflammatory and oxidative stress biomarkers and uric acid. Psychiatry Res. 2017;250:113–20. 37. Kafami L, Raza M, Razavi A, Mirshafiey A, Movahedian M, Khorramizadeh MR. Intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in C57BL/6 mice. Avicenna J Med Biotechnol. 2010;2(1):47–52. 38. Razeghi Jahromi S, Ghaemi A, Alizadeh A, Sabetghadam F, Moradi Tabriz H, Togha M. Effects of intermittent fasting on experimental autoimune encephalomyelitis in C57BL/6 mice. Iran J Allergy Asthma Immunol. 2016; 15(3):212–9. 14. Oliveira SR, Simao AN, Kallaur AP, de Almeida ER, Morimoto HK, Lopes J, et al. Disability in patients with multiple sclerosis: influence of insulin resistance, adiposity, and oxidative stress. Nutrition. 2014;30(3):268–73. 39. Riccio P, Rossano R. Diet, gut microbiota, and vitamins D + A in multiple sclerosis. Neurotherapeutics. 2018;15(1):75–91. 15. Zuniga LA, Shen WJ, Joyce-Shaikh B, Pyatnova EA, Richards AG, Thom C, et al. IL-17 regulates adipogenesis, glucose homeostasis, and obesity. J Immunol. 2010;185(11):6947–59. 40. Miyake S, Kim S, Suda W, Oshima K, Nakamura M, Matsuoka T, et al. Dysbiosis in the gut microbiota of patients with multiple sclerosis, with a striking depletion of species belonging to Clostridia XIVa and IV clusters. PLoS One. 2015;10(9):e0137429. 16. Bakshi R, Miletich RS, Kinkel PR, Emmet ML, Kinkel WR. High-resolution fluorodeoxyglucose positron emission tomography shows both global and regional cerebral hypometabolism in multiple sclerosis. J Neuroimaging. 1998;8(4):228–34. 41. Chen J, Chia N, Kalari KR, Yao JZ, Novotna M, Paz Soldan MM, et al. Multiple sclerosis patients have a distinct gut microbiota compared to healthy controls. Ethics approval and consent to participate Neurology. 1983;33(11):1444–52. 47. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an Expanded Disability Status Scale (EDSS). Neurology. 1983;33(11):1444–52. 23. Ortiz GG, Macias-Islas MA, Pacheco-Moises FP, Cruz-Ramos JA, Sustersik S, Barba EA, et al. Oxidative stress is increased in serum from Mexican patients with relapsing–remitting multiple sclerosis. Dis Markers. 2009;26(1):35–9. 48. Abdi H. The Bonferonni and Šidák corrections for multiple comparisons. Encyclopedia of measurement and statistics. Volume 1. London: SAGE Publications; 2007. 24. Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF Jr. Ketone bodies, potential therapeutic uses. IUBMB Life. 2001;51(4):241–7. 49. Sv B. Flexible imputation of missing data. 2nd ed. New York: CRC Press, Taylor and Francis Group, LLC; 2018. 25. Gasior M, Rogawski MA, Hartman AL. Neuroprotective and disease-modifying effects of the ketogenic diet. Behav Pharmacol. 2006;17(5–6):431–9. 50. Courchesne-Loyer A, Croteau E, Castellano CA, St-Pierre V, Hennebelle M, Cunnane SC. Inverse relationship between brain glucose and ketone metabolism in adults during short-term moderate dietary ketosis: a dual tracer quantitative positron emission tomography study. J Cereb Blood Flow Metab. 2017;37(7):2485–93. 50. Courchesne-Loyer A, Croteau E, Castellano CA, St-Pierre V, Hennebelle M, Cunnane SC. Inverse relationship between brain glucose and ketone metabolism in adults during short-term moderate dietary ketosis: a dual tracer quantitative positron emission tomography study. J Cereb Blood Flow Metab. 2017;37(7):2485–93. 26. Pinto A, Bonucci A, Maggi E, Corsi M, Businaro R. Anti-oxidant and anti- inflammatory activity of ketogenic diet: new perspectives for neuroprotection in Alzheimer's disease. Antioxidants (Basel). 2018;7(5). https://doi.org/10.3390/antiox7050063. 27. Martinez-Lopez N, Tarabra E, Toledo M, Garcia-Macia M, Sahu S, Coletto L, et al. System-wide benefits of intermeal fasting by autophagy. Cell Metab. 2017;26(6):856–71 e5. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 28. Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging. Cell. 2011; 146(5):682–95. 29. Wang BH, Hou Q, Lu YQ, Jia MM, Qiu T, Wang XH, et al. Ketogenic diet attenuates neuronal injury via autophagy and mitochondrial pathways in pentylenetetrazol-kindled seizures. Brain Res. 2018;1678:106–15. 29. Wang BH, Hou Q, Lu YQ, Jia MM, Qiu T, Wang XH, et al. Ketogenic diet attenuates neuronal injury via autophagy and mitochondrial pathways in pentylenetetrazol-kindled seizures. Brain Res. 2018;1678:106–15. 30. Kossoff EH. More fat and fewer seizures: dietary therapies for epilepsy. Lancet Neurol. 2004;3(7):415–20. 30. Kossoff EH. More fat and fewer seizures: dietary therapies for epilepsy. Lancet Neurol. 2004;3(7):415–20. 31. Winesett SP, Bessone SK, Kossoff EH. The ketogenic diet in pharmacoresistant childhood epilepsy. Expert Rev Neurother. 2015;15(6):621–8. 31. Winesett SP, Bessone SK, Kossoff EH. The ketogenic diet in pharmacoresistant childhood epilepsy. Expert Rev Neurother. 2015;15(6):621–8. 32. Reger MA, Henderson ST, Hale C, Cholerton B, Baker LD, Watson GS, et al. Effects of beta-hydroxybutyrate on cognition in memory-impaired adults. Neurobiol Aging. 2004;25(3):311–4. 32. Reger MA, Henderson ST, Hale C, Cholerton B, Baker LD, Watson GS, et al. Effects of beta-hydroxybutyrate on cognition in memory-impaired adults. Neurobiol Aging. 2004;25(3):311–4. 33. Vanitallie TB, Nonas C, Di Rocco A, Boyar K, Hyams K, Heymsfield SB. Treatment of Parkinson disease with diet-induced hyperketonemia: a feasibility study. Neurology. 2005;64(4):728–30. 33. Vanitallie TB, Nonas C, Di Rocco A, Boyar K, Hyams K, Heymsfield SB. Treatment of Parkinson disease with diet-induced hyperketonemia: a feasibility study. Neurology. 2005;64(4):728–30. 34. Kim DY, Hao J, Liu R, Turner G, Shi FD, Rho JM. Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis. PLoS One. 2012;7(5):e35476. 34. Kim DY, Hao J, Liu R, Turner G, Shi FD, Rho JM. Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis. PLoS One. 2012;7(5):e35476. 35. Choi IY, Piccio L, Childress P, Bollman B, Ghosh A, Brandhorst S, et al. A diet mimicking fasting promotes regeneration and reduces autoimmunity and multiple sclerosis symptoms. Cell Rep. 2016;15(10):2136–46. 35. Choi IY, Piccio L, Childress P, Bollman B, Ghosh A, Brandhorst S, et al. A diet mimicking fasting promotes regeneration and reduces autoimmunity and multiple sclerosis symptoms. Cell Rep. Ethics approval and consent to participate Sci Rep. 2016;6:28484. 17. Blinkenberg M, Rune K, Jensen CV, Ravnborg MH, Kyllingsbaek S, Holm S, et al. Reduced metabolism in cerebral cortex correlates with MRI changes and cognitive dysfunction in patients with disseminated sclerosis. Ugeskr Laeger. 2001;163(27):3788–92. 42. Swidsinski A, Dorffel Y, Loening-Baucke V, Gille C, Goktas O, Reisshauer A, et al. Reduced mass and diversity of the colonic microbiome in patients with multiple sclerosis and their improvement with ketogenic diet. Front Microbiol. 2017;8:1141. 18. Blinkenberg M, Jensen CV, Holm S, Paulson OB, Sorensen PS. A longitudinal study of cerebral glucose metabolism, MRI, and disability in patients with MS. Neurology. 1999;53(1):149–53. 43. Cignarella F, Cantoni C, Ghezzi L, Salter A, Dorsett Y, Chen L, et al. Intermittent fasting confers protection in CNS autoimmunity by altering the gut microbiota. Cell Metab. 2018;27(6):1222–35 e6. 19. Tenney JR, Rozhkov L, Horn P, Miles L, Miles MV. Cerebral glucose hypometabolism is associated with mitochondrial dysfunction in patients with intractable epilepsy and cortical dysplasia. Epilepsia. 2014;55(9):1415–22. 44. Brenton JN, Banwell B, Bergqvist AGC, Lehner-Gulotta D, Gampper L, Leytham E, et al. Pilot study of a ketogenic diet in relapsing–remitting MS. Neurol Neuroimmunol Neuroinflamm. 2019;6(4):e565. 20. Storoni M, Plant GT. The therapeutic potential of the ketogenic diet in treating progressive multiple sclerosis. Mult Scler Int. 2015;2015:681289. 45. Yadav V, Marracci G, Kim E, Spain R, Cameron M, Overs S, et al. Low-fat, plant-based diet in multiple sclerosis: a randomized controlled trial. Mult Scler Relat Disord. 2016;9:80–90. 45. Yadav V, Marracci G, Kim E, Spain R, Cameron M, Overs S, et al. Low-fat, plant-based diet in multiple sclerosis: a randomized controlled trial. Mult Scler Relat Disord. 2016;9:80–90. 21. Amorini AM, Nociti V, Petzold A, Gasperini C, Quartuccio E, Lazzarino G, et al. Serum lactate as a novel potential biomarker in multiple sclerosis. Biochim Biophys Acta. 2014;1842(7):1137–43. 46. Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 2018;17(2):162–73. 46. Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 2018;17(2):162–73. 22. Karlik M, Valkovic P, Hancinova V, Krizova L, Tothova L, Celec P. Markers of oxidative stress in plasma and saliva in patients with multiple sclerosis. Clin Biochem. 2015;48(1–2):24–8. 47. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an Expanded Disability Status Scale (EDSS). Publisher’s Note 2016;15(10):2136–46. 36. Piccio L, Stark JL, Cross AH. Chronic calorie restriction attenuates experimental autoimmune encephalomyelitis. J Leukoc Biol. 2008;84(4):940–8. 36. Piccio L, Stark JL, Cross AH. Chronic calorie restriction attenuates experimental autoimmune encephalomyelitis. J Leukoc Biol. 2008;84(4):940–8.
https://openalex.org/W3007476026	https://bmcwomenshealth.biomedcentral.com/track/pdf/10.1186/s12905-020-0890-3	English	null	Dual contraception method utilization and associated factors among sexually active women on antiretroviral therapy in Gondar City, northwest, Ethiopia: a cross sectional study	BMC women's health	2,020	cc-by	6,965	Open Access Dual contraception method utilization and associated factors among sexually active women on antiretroviral therapy in Gondar City, northwest, Ethiopia: a cross sectional study Fewuze Abay1, Hedija Yenus Yeshita1, Fantahun Ayenew Mekonnen2 and Mekonnen Sisay3* * Correspondence: mekudesu@gmail.com * Correspondence: mekudesu@gmail.com 3Department of Human Nutrition, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia Full list of author information is available at the end of the article * Correspondence: mekudesu@gmail.com 3Department of Human Nutrition, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Mother to child transmission is responsible for 90% of child infection with human immune deficiency virus (HIV). Dual contraceptive use is one of the best actions to prevent mother’s human immune deficiency virus transmission to her child and partner. This study aimed at assessing the prevalence and factors associated with dual contraceptive use among sexually active women on antiretroviral therapy in Gondar City, northwest, Ethiopia. Methods: An institution based cross sectional study was conducted in Gondar City public health facilities from December 1 to 31, 2018. Systematic random sampling technique was utilized to include 563 study participants. Data were collected by interview using a structured questionnaire. Descriptive analysis was made to compute mean, median and proportion. Finally, multivariable logistic regression model was fitted to identify the factors associated with dual contraceptive method utilization. Analysis was performed by using Statistical Package for Social Sciences (SPSS) software version 20. Results: The overall prevalence of dual contraceptive method utilization among sexually active women on antiretroviral therapy was 28.8% (95% CI: 24.9, 32.7). Women aged 35–49 years (Adjusted odds ratio (AOR): 6.99; 95% CI: 3.11, 15.71)), who lived in urban areas (AOR: 4.81; 95% CI: 2.04, 11.31), attended secondary and above education (AOR: 4.43; 95% CI: 1.92, 10.22), and disclosed HIV status to sexual partners (AOR: 9.84; 95% CI: 3.48, 27.81) were more likely to use dual contraceptive method. Conclusion: In this study, the proportion of women who utilized dual contraceptive method was low. Age, place of residence, educational status and disclosure of HIV status were factors associated with dual contraceptive use. Therefore, providing education about the advantages of disclosing HIV status to sexual partners and strengthening of counseling about the advantages of dual contraceptive use will be helpful in enhancing the use of dual contraceptive method among sexually active women on antiretroviral therapy. Keywords: Dual contraceptive utilization, Women on antiretroviral therapy, Gondar, Ethiopia Abay et al. BMC Women's Health (2020) 20:26 https://doi.org/10.1186/s12905-020-0890-3 Abay et al. BMC Women's Health (2020) 20:26 https://doi.org/10.1186/s12905-020-0890-3 Background A study done at the University of Gondar hospital, north- west Ethiopia, a study conducted in selected public hospi- tals of northern Ethiopia and another study done in Gebretsadik Shawo hospital, southwest Ethiopia found out that about 13.2, 15.7 and 19.8% of HIV positive women who were attending antiretroviral therapy (ART) clinics used dual contraceptive method, respectively [4, 29, 30]. Previous studies have reported that unmarried women [31], women who disclosed their HIV status to their partners, sero-discordant and no desire for more chil- dren [32–34] were more likely to use dual contraceptive method. In Sub Saharan Africa increasing the contraceptive prevalence rate (CPR) has been estimated to reduce the proportion of infants infected with HIV by 35–55% through reduction in primary HIV infection and unin- tended pregnancies in HIV infected women [12]. This reduction rate can be possible through exercising con- sistent use of male condom alone which can protect both unwanted pregnancies and sexually transmitted in- fections (STIs)/HIV at the same time [13]. However, using it alone as a contraceptive method is not a guaran- tee to be protected from unwanted pregnancy since in- consistent and improper use is the common practice [14]. For example, about 15% of a one-year cumulative incidence of unintended pregnancy had occurred as a re- sult of using it alone [15]. To add more, among those who had routine use of condom alone, about 14 to 21% of them got pregnant during the first year [14, 16]. Currently, in Ethiopia, HIV transmission is high and is a public health problem. Many HIV positive women are still encountering unintended pregnancy with a con- comitant risk of mother to child transmission (MTCT) of HIV, pregnancy related morbidity and mortality, and transmission of HIV and new strain of HIV virus to their sexual partners due to low dual contraceptive method utilization. Ethiopian national health policy and strategy encourage dual contraceptive method use as a key inter- vention to reduce HIV transmission and unintended pregnancy. In addition, Ministry of health of Ethiopia in collaboration with partners also developed the post 2015 objective of prevention of mother to child transmission (PMTCT) to consolidate & sustain the elimination of MTCT and reduce the vertical transmission to less than 2% by 2020. Background unwanted pregnancies and STIs/HIV simultaneously [18, 19]. According to 2015 Global statistics, 36.7 million people were living with human immune deficiency virus (HIV) worldwide [1], of whom, reproductive age group contrib- uted 75% of the burden [2]. The problem is highest among people residing in Sub Saharan Africa where, 60% of people reported to live with human immune defi- ciency virus/acquired immune deficiency syndrome (HIV/AIDS) and more than half of them were females [3]. In Ethiopia, the total number of people living with HIV was documented to be 769, 600 in 2014. Of these, about 60% were females [4]. Dual contraceptive method use is the use of two differ- ent types of contraceptive methods, a barrier contracep- tive along with another family planning method which can reduce transmission of STIs/HIV and prevent preg- nancy respectively [20, 21]. Evidences show that 40 and 80% of unplanned pregnancies and abortions would be prevented if half and all of them who were using one type of contraceptive alone started using dual methods [22]. An intervention based study had also demonstrated that participants who adhered to dual method use had lower rates of unintended pregnancy at 24 months [23]. HIV/AIDS continues to have disastrous medical, eco- nomic, social, and physical impacts on individuals, fe- males, nations and global community at large [5]. Globally, more than 2 million HIV positive women be- come pregnant every year due to poor contraceptive utilization and unsafe sex practices, out of which, 600, 000 die due to pregnancy related complications [6]. Un- intended pregnancies accounted for 21.3% of new paedi- atric HIV infections [7], 90% were from Sub Saharan Africa [8]. Ethiopia is one of the countries severely af- fected by the disease with over 100, 000 pregnancies tested positive for HIV and over 12, 000 HIV positive births annually [9]. Unintended pregnancies among HIV infected women contribute to poor maternal and child outcomes [10, 11]. Apart from its effectiveness, its utilization is low particu- larly in developing countries including Ethiopia [24–28]. Several recent studies conducted in different parts of Ethiopia have examined dual contraceptive method use among HIV positive women and reported low utilization. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abay et al. BMC Women's Health (2020) 20:26 Abay et al. BMC Women's Health (2020) 20:26 Page 2 of 9 Page 2 of 9 Sample size determination and sampling procedure The sample size was determined by using single popula- tion proportion formula taking the assumptions of 32% dual contraceptive proportion (P) (28), 95% confidence interval (CI), 4% margin of error (d) and a 10% nonre- sponse rate using Epi-Info software version 7 which yielded 574 participants. Sexually active A client who engaged in sexual activity within 1 month prior to the survey [40]. Background To ensure virtual elimination, one of the main focuses is improving the use of dual contraceptive method among HIV positive women by integrating fam- ily planning services with PMTCT [35]. However, there is no adequate evidence in Ethiopia, particularly in our study area, Gondar City. Even though, HIV-positive women can use other hor- monal and non-hormonal contraceptive methods rather than male condom for preventing pregnancy, these methods do not protect partners against the transmis- sion of both STIs/HIV between them [15, 17]. Conse- quently, it is true that there is no a single type of contraceptive method which is effective at preventing both unintended pregnancies and STIs/HIV at a time. Thus, dual-contraceptive method use is the most effect- ive approach and strategy to effectively prevent both Therefore, the results of the current work would be helpful by providing updated evidences regarding the prevalence of dual contraceptive method utilization and Page 3 of 9 Abay et al. BMC Women's Health (2020) 20:26 Abay et al. BMC Women's Health (2020) 20:26 from the sampling frame until the total sample size was achieved. its associated factors which hinder and/or enhance its utilization among sexually active women on antiretro- viral therapy (ART), in Gondar City, northwest Ethiopia to public health practitioners and policy makers to cre- ate programs and restructure the system to reach the target audiences. its associated factors which hinder and/or enhance its utilization among sexually active women on antiretro- viral therapy (ART), in Gondar City, northwest Ethiopia to public health practitioners and policy makers to cre- ate programs and restructure the system to reach the target audiences. Data collection procedures and quality control A structured questionnaire was used to collect the data (Additional file 1). It was composed of socio demographic and economic factors (age, marital status, residence, reli- gion, woman’s educational level, partner’s educational level, woman’s occupation, partner’s occupation and in- come), sexual and reproductive factors (number of chil- dren, woman’s desire to have children, partner’s desire to have children and knowledge about contraceptive), and factors related to HIV and STI (CD4 cell count, duration of ART, viral load and history of STI, knowledge of part- ner’s HIV status, HIV status disclosure to partner). The questionnaire was originally prepared in English and translated to Amharic and back to English to ensure consistency. Five diploma and two-degree holder nurses were recruited as data collectors and supervisors, respect- ively. The data were collected after a one-day training was given regarding interview techniques and ethical concerns. Chart review was done for medical records of participants, including confirmation of highly active antiretroviral ther- apy (HAART), duration of ART, STI history, CD4cell and viral load counts. The questionnaire was pretested on 5% of the total sample from similar participants outside of Gondar City. Then, necessary modifications were done to the questionnaire. The data collection process was closely supervised by the supervisors and principal investigator on a daily basis. Study population All randomly selected sexually active HIV positive women of reproductive age (15–49), who had follow up in the ART clinics of Gondar City health facilities were included in this study. Women who were pregnant, had hearing difficulties and/or with confirmed infecundity were excluded. Study design and setting An institutional based cross sectional study was con- ducted from December 1 to 31, 2018 in Gondar City public health facilities with ART clinics. Gondar town is located in the Amhara regional State, Ethiopia at a dis- tance of 747 km from Addis Ababa, the capital of Ethiopia. The City has a total population of 333, 103. About 2, 919 and 2, 152 reproductive age women were enrolled in ART clinics of Gondar referral hospital and Gondar City health centers, respectively [36, 37]. Measurement of variables and operational definitions Dual contraceptive method use Utilization of any hormonal, intrauterine contraceptive device or permanent modern contraceptive method along with male or female condom for the last 1 month prior to the study period [38]. The estimated sample size was allocated to each health facility proportion to size of ART user women in the re- spective health facility. During the data collection period, there were a total of 1148 sexually active HIV positive women, of whom, 655 were from Gondar referral hos- pital and 295, 114, 47 and 37 were from Gondar, Azezo, Maraki and Teda health centers, respectively. Accord- ingly, 327 (655574/1148), 148 (295574/1148), 57 (114574/1148), 23 (47574/1148) and 19 (37574/1148) samples were proportionally taken from Gondar referral hospital, Gondar, Azezo, Maraki and Teda health cen- ters, respectively. A systematic random sampling tech- nique was used to select participants from each health facilities. Sampling interval (K) was determined by divid- ing the total number of pregnant women who were visit- ing the ART clinics by the estimated sample size; K = 1148/574 = 2. The first participant was identified by lot- tery method from 1 and 2, and 2 was drawn as the first participant. Then, every 2 participants were selected Knowledge of contraceptive Respondents, who have heard of at least one contracep- tive method [4, 39]. Ethical considerations h l l Ethical clearance was obtained from the Institutional Re- view Board of the University of Gondar. Permission let- ter was obtained from Gondar City Administration Health Office. Each health facility head was communi- cated through formal letter obtained from Gondar City Administration Health Office. Objectives of the study were explained to study participants. Written informed consent was obtained from participants, or parents/ guardians of participants for those whose ages were less than 18 years old. The confidentiality of information was guaranteed by using code numbers rather than personal identifiers and by keeping the data protected by locking. Participants were told that they could decline at any time if they feel uncomfortable, even after the interview has started. Results Socio, demographic characteristics of respondents Socio, demographic characteristics of respondents A total of 563 women on ART participated in this study with a response rate of 98%. The remaining participants provided incomplete responses and were rejected from the analysis. The median age of the respondents was 31 (IQR; 26, 37) years. Half of the participants were found in the age category of 25–34 years. Below one fifth, 97 (17.2%) and 184 (32.7%) of the participants were in the age category of 35 and above and 15–24 years, respect- ively. About half, 282 (50.1%) of them were also between 25 and 34 years. Regarding to their monthly income, 185 (32.9%), 207 (36.8%), 162 (28.8%), and 9 (1.6%) had > 2500, 1501–2500, 501–1500, and < =500 Ethiopian Birr, respectively. Four hundred seventy-six (84.5%) women were urban residents. The majority, 514 (91.3%) and 546 (97%), of the participants belong to Orthodox Christian religion and Amhara ethnic, respectively. Four hundred eighty- six (86%) women were married (Table 1). Data processing and analysis Data were coded, cleaned, and entered in to Epi-Info version 7, and then exported to SPSS software version 20 for analysis. Summary statistics of variables such as percentage, median and Inter Quartile Range (IQR) were calculated. Multivariable analysis was used to identify as- sociated factors of dual contraceptive utilization. Effect size was presented by odds ratio (OR) with 95% Abay et al. BMC Women's Health (2020) 20:26 Page 4 of 9 Table 1 Socio- demographic characteristics of sexually active women on ART in public health facilities of Gondar City, northwest, Ethiopia, 2018 (n = 563) Variables Frequency (n) Percent (%) Age 15–24 97 17.2 25–34 282 50.1 > =35 184 32.7 Residence Urban 476 84.5 Rural 87 15.5 Religion Orthodox 514 91.3 Muslim 49 8.7 Marital status Married 486 86.3 Never married 51 9.1 Divorced 17 3.0 Separated 9 1.6 Ethnicity Amhara 546 97.0 Tigray 10 1.8 Othera 7 1.2 Women’s education No education 245 43.5 Primary education 183 32.5 Secondary education 94 16.7 Above secondary 41 7.3 Husband’s education No education 226 40.1 Primary education 212 37.7 Secondary education 107 19.0 Above secondary 18 3.2 Woman’s occupation Student 16 2.8 Merchant 100 17.8 Government employee 49 8.7 Housewife 184 32.7 Daily laborer 82 14.6 Private employee 102 18.1 Otherb 30 5.3 Husband’s occupation Student 3 0.5 M h t 122 21 7 confidence interval and p-value. A predictor variable with p-value less than 0.05 was considered as having a statistically significant association with the dependent variable. Furthermore, model fitness was checked by Hosmer and Lemeshow goodness of fit-test (p = 0.35). Factors associated with dual contraceptive use Both bivariable and multivariable binary logistic regres- sion models were used to conduct the crude and ad- justed odds ratios with their 95% confidence intervals (CI) and p-values. Accordingly, the result of bivariable analysis showed that age, residence, and educational sta- tus of the woman, occupation of partner, knowledge of partner’s HIV status, HIV status of the partner and dis- closure of HIV status to sexual partner were associated with dual contraceptive utilization. However, in multi- variable logistic regression analysis, age, residence, edu- cational status of the woman, and disclosure of HIV status to sexual partner were statistically significantly as- sociated with dual contraceptive utilization. Dual contraceptive method utilization This study showed that 162 (28.8%) of the study partici- pants (sexually active HIV positive women), used dual contraceptive method in the past 1 month prior to the survey. All, 563 (100%) of the study participants reported using at least one modern contraceptive method. Of them, 386 (68.6%) used condom and 200 (35.5%) used dual contraceptive method. Twenty-five (4.4%) used condom only. The most common methods used along with condom were, injectable 112 (69%), pills 25 (15%), implants 22 (14%) and Intra Uterine Contraceptive De- vice (IUCD) 3 (2%). were urban residents were nearly 5 times more likely to use dual contraceptive method compared to their rural resident counterparts (AOR: 4.80; 95% CI: 2.04, 11.31). Likewise, higher odds of dual contraceptive method utilization were observed among women who attended secondary and above education than those who attended lower education (AOR: 4.43; 95% CI: 1.92, 10.22). Again, the odds of dual contraceptive method utilization were nearly 10 times higher among those who disclosed their HIV status to their sexual partners (AOR: 9.84; 95% CI: 3.48, 27.81) (Table 3). Clinical and HIV related factors Clinical and HIV related factors Concerning their sexual partners’ HIV status, more than two third, 403 (71.6%) of them reported to aware their partners’ HIV status. The majority, 393 (69.8%) of women disclosed their HIV status to their sexual part- ners. Of all participants, only 34 (6%) of them had his- tory or symptom of other STIs in the past 1 year, 30 (88.2%) of whom received treatment (Table 2). Sexual and reproductive characteristics Regarding women’s knowledge about contraceptive method, all, 563 (100%) of study participants knew at least one method of contraception. The majority, 465 (82.6%) of study participants and 359 (63.8%) of women’s sexual partners had no desire to have more children in the future. Abay et al. BMC Women's Health (2020) 20:26 Page 5 of 9 Table 2 Clinical and HIV related characteristics of sexually active women on ART in public health facilities of Gondar City, northwest Ethiopia, 2018 Variables Frequency (n) Percent (%) STI history No 529 94.0 Yes 34 6.0 Know HIV status of partner No 160 28.4 Yes 403 71.6 Partner’s HIV status Negative 164 40.7 Positive 239 59.3 Disclosed HIV status No 170 30.2 Yes 393 69.8 Duration on ART (Years) 0, 1 44 7.8 2, 3 134 23.8 4, 5 75 13.3 6, 7 142 25.2 8, 9 111 19.7 > =10 57 10.1 Current CD4 cell count < 250 97 17.2 250, 349 109 19.4 > =350 357 63.4 Current viral load Not high viral load 526 93.4 High viral load 37 6.6 Table 1 Socio- demographic characteristics of sexually active women on ART in public health facilities of Gondar City, northwest, Ethiopia, 2018 (n = 563) (Continued) Variables Frequency (n) Percent (%) Private employee 185 32.9 Otherc 31 5.5 Income < =500 9 1.6 501, 1500 162 28.8 1501, 2500 207 36.8 > 2500 185 32.9 Note: Other aOromo, bFarmer, No occupation, cWaitress, Farmer Discussion This study was conducted to assess the proportion of dual contraceptive utilization and its associated factors among sexually active women on ART in Gondar City health facilities, northwest Ethiopia. The study revealed that, 28.8% (95% CI: 24.9–32.7) of sexually active women Consequently, women in the age group of 35–49 years were 7 times more likely to use dual contraceptive method than those in the age group of 15 to 24 years (AOR: 6.99; 95% CI: 3.11, 15.71). Similarly, women who Abay et al. Discussion BMC Women's Health (2020) 20:26 Page 6 of 9 Table 3 Factors associated with dual contraceptive use among sexually active women on ART in public health facilities of Gondar City, northwest Ethiopia, 2018 Variable Dual contraceptive use (n = 563) Crude OR (95% CI) Adjusted OR (95% CI) P-value Yes (%) No (%) Age (years) 15, 24 11 (11.3) 86 (88.7) 1 1 25, 34 65 (23) 217 (77) 2.34 (1.18, 4.65) 2.11 (0.97, 4.55) 0.058 35, 49 86 (46.7) 98 (53.3) 6.86 (3.44, 13.71)* 6.99 (3.11, 15.71)** < 0.001 Residence Urban 154 (32.4) 322 (67.6) 4.72 (2.23, 10.02)* 4.8 (2.04, 11.31)** < 0.001 Rural 8 (9.2) 79 (90.8) 1 1 Educational status of woman No formal education 51 (20.8) 194 (79.2) 1 1 Primary education 44 (24) 139 (76) 1.2 (0.76, 1.91) 1.03 (0.54, 1.98) 0.921 Secondary and above 46 (48.9) 48 (51.1) 3.65 (2.19, 6.06)* 4.43 (1.92, 10.22)** < 0.001 Partner’s occupation Student 1 (33.3) 2 (66.7) 0.53 (0.04, 6.51) 2.5 (0.14, 46.11) 0.537 Merchant 31 (25.4) 91 (74.6) 0.36 (0.16, 0.82)* 0.72 (0.26, 1.98) 0.53 Government employee 21 (26.9) 57 (73.1) 0.39 (0.17, 0.93)* 0.59 (0.20, 1.73) 0.337 Daily labor 41 (28.5) 103 (71.5) 0.43 (0.19, 0.94)* 1.05 (0.37, 2.88) 0.918 Private employee 53 (28.6) 132 (71.4) 0.43 (0.21, 0.93)* 0.63 (0.24, 1.65) 0.348 Other* 15 (48.4) 16 (51.6) 1 1 Aware of partner’s HIV status No 7 (4.4) 153 (95.6) 1 1 Yes 155 (38.5) 248 (61.5) 13.66 (6.24, 29.91)* 2.69 (1.01, 7.31) 0.051 Disclosure of HIV status No 6 (3.5) 164 (96.5) 1 1 Yes 156 (39.7) 237 (60.3) 17.99 (7.77, 41.65)* 9.84 (3.48, 27.81)** < 0.001 Note: Crude OR, Significant at P value < 0.05, *Adjusted OR, Significant at P value < 0.05 Table 3 Factors associated with dual contraceptive use among sexually active women on ART in public health facilities of Gondar City, northwest Ethiopia, 2018 The proportion of women using dual method is lower in the present study compared to the findings of studies done in Tigray (59.9%), Ethiopia, Kenya (38.5%), and Nigeria (45%) [32, 44, 45].. The discrepancy between study reports could probably be due to socio- demographic and cultural differences of the study popu- lations, presence of quality, integrated sexual and repro- ductive health and ART services in Kenya and Nigeria. on ART used dual contraceptive method in the last month prior to the study. Discussion The factors identified to be significantly associated with dual contraceptive use were women’s age, residence, educational status and disclos- ure of HIV status to sexual partner. The proportion of dual contraceptive use in this study is consistent with the results of studies done in Fitche (32%) and Addis Ababa (31%), Ethiopia, and in Nigeria (27.2%), and India (30%) [10, 34, 41, 42]. But, the per- centage of women using dual method is higher in our study compared to the findings of studies conducted in Tigray region (14%), Mekelle public hospitals (15.7%), and Gimbie town (17%), Ethiopia and in Zambia (17.7%) [4, 39, 40, 43]. The observed variation between the stud- ies might be due to the differences in socio demographic characteristics of participants included. Again, the incon- sistency noted with studies from other countries may be attributable to socio demographic and cultural charac- teristics of study participants, study period and settings. According to our study results, older age women (35 to 49 years) were more likely to use dual contraceptive method than younger age women (15 to 24 years). The possible reason could be older age women might have bet- ter understanding the advantage of using dual contracep- tives, in turn, making them use the method. In addition, there is an established evidence that as age advances the desire for more children in the future decreases. This find- ing suggested that providing due attention for younger age women is crucial to improve the utilization of dual contra- ceptive method among HIV positive women. This finding Page 7 of 9 Abay et al. BMC Women's Health (2020) 20:26 Page 7 of 9 is in line with studies reported from southern part of Ethiopia, Uganda and Brazil [30, 33, 46]. status to sexual partner in particular and the advantage of using dual contraceptive in general is suggested for the prevention of the transmission of HIV to the child and sexual partner. Furthermore, encouraging and pro- viding more attention for younger age, illiterate and rural women by health care providers and other con- cerned bodies is recommended. Place of residence was one of the key determinants of dual contraceptive method utilization. Conclusion The proportion of sexually active HIV positive women who were using dual contraceptive method was low. Women’s age, educational status, place of residence, and disclosure of HIV status to sexual partners were found to be significantly associated with dual contraceptive method utilization. Strengthening education of women on ART about the importance of disclosing own HIV Acknowledgements We are very thankful for women for their willingness and time to participate in the study. We also would like to appreciate Gondar City Administration health office, health facility heads and health care providers for their support during data collection. Supplementary information Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12905-020-0890-3. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12905-020-0890-3. Additional file 1. Questionnaire (English Version Questionnaire). Additional file 1. Questionnaire (English Version Questionnaire). This study also showed that disclosing own HIV status to sexual partner has been found to significantly increase utilization of dual contraceptive method. This may be due to the fact that women have common understanding about the importance of using dual contraceptive method and easily reach at consensus to use it. Moreover, disclos- ing HIV status fosters more open discussion with their sexual partners with regard to safe sexual practice and limiting number of children. As the study showed, disclos- ing their HIV status to their respective sexual partners is crucial to increase the coverage of dual contraceptive method among HIV positive women. This finding is sup- ported by studies conducted in Mekelle and Addis Ababa, Ethiopia, Ghana, and Kenya [32, 40, 47, 48]. Abbreviations AIDS: Acquired immune deficiency syndrome; AOR: Adjusted Odds Ratio; ART: Antiretroviral therapy; CI: Confidence Interval; COR: Crude Odds Ratio; CPR: Contraceptive Prevalence Rate; HAART: Highly active antiretroviral therapy; HIV: Human immune deficiency virus; IQR: Inter Quartile Range; IUCD: Intra uterine contraceptive device; MTCT: Mother to child transmission; PMTCT: Prevention of mother to child transmission; SPSS: Statistical Package for Social Sciences; STI: Sexually transmitted infection Authors’ contributions FA and KYY conceived the study, developed the tool, coordinated data collection, and carried out the statistical analysis and drafted the manuscript. FA, KYY, FAM and MS conceived the study, participated in the statistical analysis, and drafted the manuscript. FA, MS and FAM conceived the study and review the drafted manuscript. All authors read and approved the final manuscript. Funding Th h The authors received no specific funding for this work. Discussion This can be ex- plained by the fact that urban residents are more exposed for better health information and technologies which might enable them to have a better decision power and free discussion with their sexual partners about utilization of family planning services than their rural counterparts. Furthermore, women living in urban areas are believed to have better physical access to contraceptives. Such explan- ation suggested the importance of enhancing counselling services focussing on rural women to increase the uptake of dual contraceptive method utilization. This evidence is supported by a study done in southern part of Ethiopia [30] which revealed that HIV positive urban women were more likely to use dual contraceptive method than rural women. Limitations of the study Th d d This study tried to generate evidences on dual contra- ceptive utilization among HIV positive women. How- ever, it cannot be free from some limitations. Due to the cross-sectional nature of the study design, temporal rela- tionship could not be assessed. Since the issue is a sensi- tive one, this result may be prone to social desirability bias. In addition, variables such as social support aware- ness towards pregnancy and STDs, financial support, availability of services/supplies and involvement of asso- ciation of people living with HIV/AIDS that might have been associated with dual contraceptive use were not in- cluded. Furthermore, the current study was health facil- ity based, the result of this study may not be generalizable to those HIV positive mothers who were attending health institutions outside the study area and those who were in the community. Higher proportion of dual contraceptive method utilization was observed among women who attended secondary and above education. This can be explained as women with high level of education might appreciate the advantages of dual contraceptive use, have higher levels of HIV/AIDS knowledge, able to discuss freely with their sexual partners and are less likely to have stigma towards HIV/AIDS, which, in turn, assists them to easily change their risky sexual behaviour. This result implied that the paramount contribution of women’s education to improve dual contraceptive method utilization. Similar results were reported from Brazil and Uganda [33, 46]. Availability of data and materials Availability of data and materials Data will be available upon request from the corresponding author. Data will be available upon request from the corresponding author. Page 8 of 9 Page 8 of 9 Page 8 of 9 Abay et al. BMC Women's Health (2020) 20:26 Abay et al. BMC Women's Health Received: 21 September 2019 Accepted: 24 January 2020 Received: 21 September 2019 Accepted: 24 January 2020 25. Beyeza-Kashesya J, Ekstrom AM, Kaharuza F, Mirembe F, Neema S. My partner wants a child. A cross-sectional study of the determinants of the desire for children among mutually disclosed sero-discordant couples receiving care in Uganda. BMC Public Health. 2010;10:247. Consent for publication Not applicable. 21. Wilson TE, Koenig LJ, Walter E, Fernandez I, Ethier K. Perinatal Guidelines Evaluation P. Dual contraceptive method use for pregnancy and disease prevention among HIV-infected and HIV-uninfected women: the importance of an event-level focus for promoting safer sexual behaviors. Sex Transm Dis. 2003;30(11):809–12 PubMed Epub 2003/11/07. eng. Competing interests The authors declare that they have no competing interests. References 1. UNAIDS, Fact sheet: global statistics. 2015. 2. Carpenter LM, Kamali A, Ruberantwari A, Malamba S, Whitworth JAG. Rates of HIV-1 transmission within marriage in rural Uganda in relation to HIV sero-status in rural Uganda in relation to the HIV sero-status of the partners. AIDS Behav. 1999;13:1083–9. 26. Elul B, Delvaux T, Munyana E. Pregnancy desires and contraceptive knowledge and use among prevention of mother-to-child transmission clients in Rwanda. AIDS. 2009;23:S19–26. https://doi.org/10.1097/01.aids. 0000363774.91376.dc. 3. UNAIDS, AIDS. Epidemic update in united nation joint programme on HIV/ AIDS. Geneva: WHO/UNAIDS; 2011. 27. Hoffman IF, Martinson FEA, Powers KA. The year-long effect of HIV-positive test results on pregnancy intentions contraceptive use and pregnancy incidence among Malawian women. J Acquir Immune Defic Syndr. 2008; 47(4):477–83. 4. Melaku Y, Zeleke EG. Contraceptive utilization and associated factors among HIV positive women on chronic follow up care in Tigray Region, Northern Ethiopia: a cross sectional study. PLoS One. 2014;9(4). 28. Kakaire O, Kaye DK, Osinde MO. Contraception among persons living HIV with infection attending an HIV care and support Centre in Kabale Uganda. J Public Health Epidemiol. 2010;2(8):180–8. 5. UNAIDS WHO. Global Summary of AIDS Epidemic Report. Geneva: WHO; 2007. p. 23–5. 6. Preventing HIV and unintended pregnancies: strategic framework 2011– 2015 The Inter-agency Task Team for Prevention and Treatment of HIV Infection in Pregnant Women, Mothers, and their Children. 2012. 29. Reta MM, Tessema GA, Shiferaw G. Prevalence of dual contraceptive use and associated factors among HIV positive women at University of Gondar Hospital, Northwest Ethiopia. BMC Res Notes. 2019;12:36 https://doi.org/10. 1186/s13104-019-4053. 7. The Gap Report children and pregnant women living with HIV. 2015, USAID. 8. Global Update on the Health Sector Response to HIV. 2014, World Health Organization (WHO). 30. Erashi MWM, Tesso FY, Beyene TT. Dual-Contraceptive Method Utilization and Associated Factors among HIV Positive Women Attending Art Clinic in Gebretsadik Shawo Hospital SNNPR South West Ethiopia. J Womens Health Care. 2015;4(6). https://doi.org/10.4172/2167-0420.1000267. 9. Reynolds H. Contraception to prevent HIV-positive births: current contribution and potential cost savings in PEPFAR countries. Sex Transm Infect. 2008;84:49–53. 31. Pazol K, Kramer M, Hogue C. Condoms for dual protection patterns of use with highly effective contraceptive methods, in Public Health Report; 2010. p. 208–17. 10. Lawani L, Onyebuchi AK, Iyoke C. Dual method use for protection of pregnancy and disease prevention among HIV-infected women in South East Nigeria. BMC Womens Health. 2014;14(1):39. 10. Lawani L, Onyebuchi AK, Iyoke C. Author details 1 f 22. Pazol K, Kramer MR, Hogue CJ. Condoms for dual protection: patterns of use with highly effective contraceptive methods. Public Health Rep. 2010; 125:208–17 [PubMed: 20297747]. 1Department of Reproductive Health, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. 2Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, E hi i 3D f H N i i I i f P bli H l h C ll 1Department of Reproductive Health, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. 2Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. 3Department of Human Nutrition, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. 23. Peipert JF, Redding CA, Blume JD, et al. Tailored intervention to increase dual-contraceptive method use: a randomized trial to reduce unintended pregnancies and sexually transmitted infections. Am J Obstet Gynecol. 2008; 198:630.e1–8 [PubMed: 18395692]. Ethiopia. 3Department of Human Nutrition, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia. 24. Magwali Thulani SM. Toms Harold, Brown Joelle, how are condoms used in a family planning setting: evidence from Zimbabwe. Cent Afr J Med. 2005; 51(7–8):79–84. Ethics approval and consent to participate 16. Fu H, Darroch JE, Haas T, Ranjit N. Contraceptive failure rates: new estimates from the 1995 National Survey of Family Growth. Fam Plann Perspect. 1999; 31(2):56–63 PubMed Epub 1999/05/04. eng. Ethical clearance was obtained from the Institutional Review Board of the University of Gondar. Permission letter was obtained from Gondar City Administration Health Office. Each health facility head was communicated through formal letter obtained from Gondar City Administration Health Office. Objectives of the study were explained to study participants. Written informed consent was obtained from participants, or parents/guardians of participants for those whose ages were less than 18 years old. The confidentiality of information was guaranteed by using code numbers rather than personal identifiers and by keeping the data protected by locking. Participants were told that they could decline at any time if they feel uncomfortable, even after the interview has started. 17. Cates W Jr, Steiner MJ. Dual protection against unintended pregnancy and sexually transmitted infections: what is the best contraceptive approach? Sex Transm Dis. 2002;29(3):168–74. 18. Crosby RA, DiClemente RJ, Windgood GM, et al. Correlates of using dual methods for sexually transmitted diseases and pregnancy prevention among high-risk African–American female teens. J Adolesc Health. 2001;28: 410–4 [PubMed: 11336871]. 19. Family Health International. Dual Protection: Best approach to recommend may vary. Network. 2003;22(4) Available at: http://www.fhi.org/en/RH/Pubs/ Network/v22_4/nt2244.htm. Retrieved November 6, 2010. 20. Dual protection against unwanted pregnancy and HIV / STDs. Sexual health exchange. 1998 (3):8. PubMed.. 20. Dual protection against unwanted pregnancy and HIV / STDs. Sexual health exchange. 1998 (3):8. PubMed.. References Fertility desires and unmet need for family planning among HIV infected individuals in two HIV clinics with differing models of family planning service delivery. BMC Women's Health. 2015;15:5. 10.1186/s12905-014-0158-x. 48. Rhoda W, Joseph M, Moses K, Nazarius T, Maria N. Fertility desires and unmet need for family planning among HIV infected individuals in two HIV clinics with differing models of family planning service delivery. BMC Women's Health. 2015;15:5. 10.1186/s12905-014-0158-x. References Desire and family planning demand among HIV positive men and women in follow up care in Addis Ababa antiretroviral treatment units; 2006. p. 65–72. 41. Tamene W. Desire and family planning demand among HIV positive men and women in follow up care in Addis Ababa antiretroviral treatment units; 2006. p. 65–72. 42. Chakrapani Venkatesan KT, Murali S, Peter N, Deborah C. Prevalence of and barriers to dual-contraceptive methods use among married men and women living with HIV in India. Infect Dis Obstet Gynecol. 2011. 42. Chakrapani Venkatesan KT, Murali S, Peter N, Deborah C. Prevalence of and barriers to dual-contraceptive methods use among married men and women living with HIV in India. Infect Dis Obstet Gynecol. 2011. 43. Chibwesha C, Matoba CK, Mbewe R, Stringer J, Stringer E. Modern Contraceptive and Dual Method Use among HIV-Infected Women in Lusaka Zambia; 2011. p. 1–8. 43. Chibwesha C, Matoba CK, Mbewe R, Stringer J, Stringer E. Modern Contraceptive and Dual Method Use among HIV-Infected Women in Lusaka Zambia; 2011. p. 1–8. 44. Lawrence A, Joanne M, Eugene W, Grace D, Jumoke O. Promoting dual protection planning clinics in Ibadan Nigeria. Int Fam Plan Perspect. 2002; 28(2):87–95. 44. Lawrence A, Joanne M, Eugene W, Grace D, Jumoke O. Promoting dual protection planning clinics in Ibadan Nigeria. Int Fam Plan Perspect. 2002; 28(2):87–95. 45. Berhane Y. Utilization of modern contraceptives among HIV positive reproductive age women in Tigray Ethiopia. A Cross Sectional Study. ISRN AIDS. 2013;2013. https://doi.org/10.1155/2013/319724. 45. Berhane Y. Utilization of modern contraceptives among HIV positive reproductive age women in Tigray Ethiopia. A Cross Sectional Study. ISRN AIDS. 2013;2013. https://doi.org/10.1155/2013/319724. 46. Jolly B-K, Frank K, Mia EA, Stella N, Asli K, Florence M. To use or not to use a condom a prospective cohort study comparing contraceptive practices among HIV-infected and HIV-negative youth in Uganda. BMC Infect Dis. 2011;11:144. 46. Jolly B-K, Frank K, Mia EA, Stella N, Asli K, Florence M. To use or not to use a condom a prospective cohort study comparing contraceptive practices among HIV-infected and HIV-negative youth in Uganda. BMC Infect Dis. 2011;11:144. 47. Mekonnen AH, Gashe FE. Contraceptive use and method preference among HIV positive women in Addis Ababa Ethiopia a cross sectional survey. PubMed. 2014;14(1):566. 48. Rhoda W, Joseph M, Moses K, Nazarius T, Maria N. References Dual method use for protection of pregnancy and disease prevention among HIV-infected women in South East Nigeria. BMC Womens Health. 2014;14(1):39. 32. Mulongo AM, Lihana RW, Githuku J, Gura Z. Factors associated with uptake of dual contraception among HIV-infected women in Bungoma County. Pan Afr Med J. 2017;28(Supp 1):2. https://doi.org/10.11604/pamj.supp.2017. 28.1.9289. 11. Heidi R, et al. The value of contraception to prevent perinatal HIV transmission. PubMed. 2006;33(6):350–6. 12. Chris O. Contraception in the context of HIV/AIDS. Afr J Reprod Health. 2012;16:1–25. 12. Chris O. Contraception in the context of HIV/AIDS. Afr J Reprod Health. 2012;16:1–25. 33. Kiyomi Tsuyuk RB, de Araujo Pinho A. Dual Protection and Dual Methods in Women Living with HIV. The Brazilian Context. J Sex Transm Dis. 2013;2013: 8. 13. United Nations Population Fund and World Health Organization (WHO). Sexual and Reproductive Health ofWomen Living with HIV/AIDS: Guidelines on Care, Treatment and Support for Women Living with HIV/AIDS and Their Children in Resource-Constrained Settings. Geneva: WHO; 2006. 34. Demissie DB, Girma T, Abdissa G. Dual contraceptive utilization and associated factors among people living with HIV attending ART clinic in Fitche Hospital Ethiopia. J Health Med Nurs. 2015;20:2422–8419. 14. Trussell J. Contraceptive failure in the United States. Contraception. 2011; 83(5):397–404 PubMed Pubmed Central PMCID: 3638209, Epub 2011/04/12. eng. 35. The Federal Democratic Republic of Ethiopia, Ministry of Health. HIV/AIDS strategic plan 2015-2020 in an investment case approach, vol. 2014. Addis Ababa: Federal HIV/AIDS prevention and control office. 15. Trussell J. Choosing a contraceptive: efficacy, safety, and personal considerations. In: Hatcher RA, Trussell J, Nelson AL, Cates Jr W, Stewart FH, Kowal D, editors. Contraceptive Technology. 19th ed. New York: Ardent Media; 2008. 36. Gondar City Administration Office Report, 2018. 37. Health facilities ART follow up registration, 2018. Page 9 of 9 Abay et al. BMC Women's Health (2020) 20:26 Abay et al. BMC Women's Health (2020) 20:26 38. Federal Democratic Republic of Ethiopia Country Progress Report on HIV/ AIDS response. HIV/AIDS Prevention and Control Office (HAPCO). 2012. 39. Polisi A, Gebrehanna E, Tesfaye G, Asefa F. Modern contraceptive utilization among female ART attendees in health facilities of Gimbie town West Ethiopia. Reprod Health. 2014;11(30). 40. Gebrehiwot SW, Azeze GA, Robles CC, Adinew YM. Utilization of dual contraception method among reproductive age women on antiretroviral therapy in selected public hospitals of Northern Ethiopia. Reprod Health. 2017;14:125. 41. Tamene W. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3043325149	https://www.revistas.usp.br/rbefe/article/download/170706/161151	English	null	Moderate/high resistance exercise is better to reduce blood glucose and blood pressure in middle-aged diabetic subjects	Revista Brasileira de Educação Física e Esporte	2,020	cc-by	7,430	Moderate/high resistance exercise is better to reduce blood glucose and blood pressure in middle-aged diabetic subjects Ricardo Augusto Leoni de SOUSA* Karinn Faro HAGENBECK Gisela ARSA* Emerson PARDONO* http://dx.doi.org/10.11606/1807-5509202000010165 http://dx.doi.org/10.11606/1807-5509202000010165 Abstract Type 2 diabetes (T2D) main feature is insulin resistance. Hypertension is a comorbidity linked to T2D. Resistance exercise (RE) is an important non-pharmacological tool to contribute to managing blood glucose and blood pressure (BP), but there is not a common sense about acute effects. The aim of this study was to evaluate the acute physiological effects after two different RE sessions using different intensities at middle-aged hypertensive T2D subjects. There were 40 middle-aged men (20 nondiabetics; 20 diabetics), who underwent an exercise protocol with the same volume at 60% or 75% of the one maximum repetition test (1RM), consisting in: bench press, triceps pulley, rowing machine, barbell curl, lateral raise with dumbbells and barbell squat. Physiological changes were evaluated through BP, glycemia, creatine kinase, lactate dehydrogenase, C reactive protein (C-RP), testosterone and cortisol. For non-diabetics, both intensities promoted blood glucose uptake (8.2% to 11.1%, p<0.05), and only the 75%1RM session induced blood glucose uptake in 5.7% in the diabetics. Post-exercise hypotension was significant after RE at 75%1RM for systolic BP (SBP) and after both intensities for diastolic BP (DBP) in non-diabetics, while the SBP and DPB reduced after both intensities for diabetics. RE at 75%1RM resulted in better blood glucose uptake, and both intensities reduced the BP in diabetic subjects. After 75%1RM there was a higher indirect muscle damage result. The alterations in hormones, C-RP, and indirect muscle damage markers indicated an adequate acute anabolic recovery with no significant inflammation in both intensities. Acute RE at 60%1RM or 75%1RM can used as an extra tool to manage both pathologic conditions. Keywords: Type 2 Diabetes Mellitus; Hypertension; Resistance Exercise; Health. Departamneto de Educação Física, Uni versidade Federal de Sergipe, São Cristó vão, SE, Brazil. Colégio de Medicina da Bahia, Salvador, BA, Brazil. *Federal University of Mato Grosso, Cuiabá, MT, Brazil. Introduction There are different types of diabetes, but type 2 diabetes mellitus (T2D) is the most common, affecting 95% of subjects with diabetes worldwide1. T2D main characteristic is insulin resistance and presents two clinical outcomes: hyperglycemia and hyperinsulinemia2. Diabetes can lead to different types of comorbidities such as hypertension. At least 70% of individuals with T2D have uncontrolled hypertension or have been treated for elevated blood pressure (BP)3. The maintenance of BP within the normal level is important in order to not only promote greater and better control of hypertension but also of the blood glucose levels and physical exercise is an essential tool to make it happen4. The absence or low practice of physical exercise, as well as a sedentary lifestyle, can contribute to the development of T2D and hypertension2,5. Physical exercise can induce post-exercise hypotension (PEH) and reduce glycemic level, especially during the first 24h6,7. Thus, aerobic exercise is usually recommended in T2D and hypertension, but resistance exercise (RE) can also induce physiological benefits6. RE movements are interspaced, and the muscle contraction is executed by a particular muscle Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 165 Sousa RAL, et al. Sousa RAL, et al. or a muscle group which is opposed to a force, and sometimes, is against to the movement6,8. Low T:C ratio may indicate the beginning of the development of insulin resistance, obesity, and cardiovascular dysfunction10. Physiological alterations are observed in RE such as muscle damage and hormonal changes immediately after RE. Creatine kinase (CK) and lactate dehydrogenase (LDH) increase are well- known indirect muscle damage markers and are important tools to define the effectiveness of the physical training. The high concentration differences in pre and post-workout for CK and LDH are the basis of muscle damage and C reactive protein (C-RP) can be used as a complementary marker to determine if the damage is harmless or not9. It has been shown12 that the blood glucose control obtained with the use of chronic high- intense RE is more effective in controlling glycemia levels than the moderate and light RE. However, moderate exercise intensity, in a chronic model, is more indicated for safety reasons13 to groups with a history of diabetes and hypertension, as recommended by American College of Sports Medicine and American Heart Association14. Introduction The moderate RE is performed at any intensity between 60% and 80% of the maximum weight workload carried out by an individual15, however, it is not clear in which percentage of the maximum intensity, lower or higher, the moderate RE would provide a better acute physiological response when performed by hypertensive T2D individuals. The confirmation of changes in physiological results after different moderate intensities of RE to this population can lead to advances in the prescription and evaluation of physical exercise training. Hormonal changes are also part of the physiological changes during and immediately after RE sessions. Testosterone levels are usually reduced in T2D. Changes in testosterone levels can influence insulin sensitivity, amplifying or not the glucose uptake and its transport to different tissues10. Alterations in the levels of cortisol can induce modifications in the recruitment of different types of muscular fibers during RE and may contribute to loss of muscle mass (sarcopenia), and to the reduction of muscular strength (dynapenia)11. Positive change in the testosterone and cortisol ratio (T:C) indicates a good physiological recovery and represents the relation between anabolic and catabolic systems. The aim of this study was to evaluate the acute effects of two different moderate RE intensities on the glycemia, blood pressure and markers of stress and muscle damage at middle-aged hypertensive type 2 diabetic subjects. Method individuals each, separated according to the percentage of the maximum intensity session. The groups performed the RE protocols at 60% (ND60 and D60) or at 75% (ND75, and D75) of the 1 repetition maximum test (1RM). The main features of the groups are shown in TABLE 1. These intensities are moderate and they were chosen due safety reasons, being 60% of 1RM closer to lowest moderate exercise zone, and 75% of 1RM is still moderate zone, closer to 80% of 1RM, considered the threshold zone between moderate and intense zone16. The National Committee for Ethics in Research and the Ethics Committee of the Federal University of Sergipe approved this study under protocol 387.704. All participants signed a consent form authorizing the participation and the use of the data collected in this study, according to Resolution 466/12 of the National Health Council of Brazil and followed the Declaration of Helsinki. Subjects The sample was composed of 40 male volunteers. There were initially two groups: nondiabetics (ND; control group) and hypertensive diabetics (D), who were later subdivided into 4 groups, consisting of 10 The including criteria adopted to participate in the exercise protocol was: to be a male between 40 and 60 years old, and could not be doing regular physical activity during the last six months was a must. It was not considered regular 166 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 High intensity exercise is better for diabetics Five minutes after the warm-up the test protocol was initiated. physical activity if the exercise or sport was performed less than 3 times a week in a shorter period than 30 minutes13. Diabetic hypertensive volunteers included in the study had the disease diagnosed clinically by a physician for at least one year and the glucose levels and blood pressure controlled by diet and/or medication. It would be excluded from the study anyone who was making use of exogenous insulin, or if they had chronic complications such as cardiovascular disease (excluding hypertension), diabetic foot, retinopathy, neuropathy, and nephropathy6. The experimental protocol was performed 72 hours after the 1RM test for each group. All subjects performed the RE session just once and the four groups executed the same exercise sequence, with intensities at 75% or 60% of 1RM. All sessions were performed consisting of 3 sets of 10 repetitions each. The resting time between sets was 1 minute13 and the rate of contraction of the exercise was 2s for the concentric phase and 2s for the eccentric18. Exercises used for all groups were an attempt to portray a traditional session for beginners at gyms, consisting of 6 exercises and working out the main muscular groups. The exercises chosen to be performed were: bench press, triceps pulley, rowing machine, barbell curl, lateral raise with dumbbells and barbell squat, and always in the same order for all participants. It is important to highlight that the 1RM was performed in each of these six exercises. The total volume was calculated to each intensity of RE from the sum the results of the multiplication of the load of each exercise by repetitions and series. An individualized breakfast was suggested to all participants of the experimental protocol. Subjects This procedure was adopted because a rich breakfast in fats or carbohydrates may influence the blood glucose level at pre- and post-exercise protocol. Water was freely consumed before and after the procedure. All procedures were conducted by the same professional at all times. Exercise Sessions One week prior to the 1RM, there was a habituation session where the participants had contact with the exercises they would perform and execute them without weights. The 1RM test was performed to establish the weight load of two moderate RE intensities used during exercise sessions. 1RM consisted of one single repetition using the maximum of the weight load that can lifted during the execution of a specific exercise. It was performed with alternation between the concentric and eccentric phases taking 2s for each phase17. After the first trial, participants had a passive interval of 180s. When the execution of the movement was successful, 10% of the work load used was added, and in case of failure, 10% was removed by the weight used. A maximum of 3 attempts, increasing or decreasing the load, was established to determine the maximal strength. Results TABLE 1 shown the rest values for different variables in the present study, divided between nondiabetic (ND) and diabetic (D) groups in each RE intensity. Only heart rate, testosterone, cortisol, CK and LDH levels did not differ between groups. All three sets were completed for all volunteers in all attempts.h 60% and 75% 1RM for nondiabetic groups (8.2% and 11.1%, respectively), however only RE at 75% 1RM resulted in significant reduction of 5.7% on glycemia level in the diabetic group (FIGURE 1A). After exercise protocols, significant reductions (p≤0.05) were observed for SBP to ND75, D60, and D75 (FIGURE 1B), and for DBP to ND and D groups (FIGURE 1C). The RE at 75% 1RM revealed a better reduction for SBP and DBP for ND and D groups, especially for ND group (p<0.05). The total volume of the exercise sessions was not differ between groups and RE intensities (ND60: 20952 ± 6603; ND75: 22680 ± 7500; D60: 21168 ± 6891; D75: 22896 ± 5778, p> 0.05). Glycemia was reduced (p<0.05) after RE at BMI: Body Mass Index; BP: Blood pressure; C-RP: C reactive protein (Po sitive results if the levels had caused severe inflammation or Negative if C-RP level was under the inflammation stan dards); CK: Creatine kinase; LDH: Lac tate dehydrogena se. Different letters indicate differences (p≤ 0.05) between groups. TABLE 1 - Rest values of all variables are presented and compared between the presence or not of type 2 diabetes mellitus (n=40). Statistical Analyses Mean and standard deviation (SD) were used for statistical analysis. Normality of the data was evaluated through Shapiro-Wilk test. Analysis of variance (ANOVA) for repeated measures between the four groups and two moments (with a syntax of 4x2) with multiple’s pairs comparisons of Bonferroni was applied for glycemia, BP, testosterone, cortisol, CK, and LDH. The significance level was set at 5% (p<0.05), and all analysis were carried out using the SPSS 15.0 version. Disposable syringes and materials were used in all samples collected by the same professional at all times. One Huma Star 300 spectrophotometer, Pre- and post-exercise procedures The method used to dosage of the C-RP was the high sensitivity turbidimetry21. C-RP analysis was performed in an automatic spectrophotometer and when “positive”, it was considered as an inflammatory zone. Testosterone and cortisol analyses were performed through chemiluminescence method. Blood samples were centrifuged for 10 min at 3000 rpm and an automatic biochemical analyzer was used. Bio Advance Kits were used during the dosages. The T: C ratio was obtained by dividing the testosterone concentrations by cortisol concentrations. Pre- and post-exercise procedures Blood pressure and heart rate were measured before and 1h after the exercise session by an oscillometric tensiometer (branch Omron, automatic arm equipment, Hem 7200 model). The BP was measured on the left arm of the participants that were keeping in a seated position. Blood capillary samples from fingers were collected and placed in tapes to evaluate glycemia (Acku-Check Advantage), as well as blood samples, were collected by antecubital vein for 4 ml per tube from each subject. Blood samples collections occurred between 7:00 and 9:00 am, before experimental protocols and 10 minutes after exercise sessions to analyze glycemia, as well as, CK, LDH, C-RP, testosterone, and cortisol. Blood capillary samples from fingers were collected and placed in tapes to evaluate glycemia (Acku-Check Advantage), as well as blood samples, were collected by antecubital vein for 4 ml per tube from each subject. Blood samples collections occurred between 7:00 and 9:00 am, before experimental protocols and 10 minutes after exercise sessions to analyze glycemia, as well as, CK, LDH, C-RP, testosterone, and cortisol. When the systolic and/or diastolic blood pressure and/or glycemia presented values ≥ 180 mmHg and/or 105 mmHg19 and/or ≤100mg/ dL or ≥300mg/dL20, the experimental session was rescheduled, and the volunteer was aimed at finding a basic health unit. These situations did not occur. Each group performed a single session of the exercise protocol. After the resting period, stretches were performed for 5 minutes to the upper limb. Right after the stretching period, the subjects performed a warm-up for the main muscle groups of their body (chest, back, and legs) using two series with the minimum weight on the machines and barbells, executing 20 repetitions of specific exercises for these muscle groups (described in the experimental protocol below). The interval between sets was 45 seconds. When the systolic and/or diastolic blood pressure and/or glycemia presented values ≥ 180 mmHg and/or 105 mmHg19 and/or ≤100mg/ dL or ≥300mg/dL20, the experimental session was rescheduled, and the volunteer was aimed at finding a basic health unit. These situations did not occur. CK and LDH were measured in the plasma, through the kinetic colorimetric method at Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 167 Sousa RAL, et al. Sousa RAL, et al. Human brand, was used for biochemical analyses. A centrifugal of the CELM brand model was also used. multiple time points9. 168 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 Results RE - 60% 1RM RE - 75% 1RM Non Diabetic (n=10) Diabetic (n=10) Non Diabetic (n=10) Diabetic (n=10) Age (years) 49 ± 8.5 54.1 ± 6.9 45 ± 3.4 54.1 ± 10.5 BMI (kg/m2) 27.1 ± 2.8a 32.6 ± 3.2b 27.6 ± 2.9a,c 32.3 ± 4.1b,d Glycemia (mg/dL) 104.1 ± 11.6a 127.4 ± 14.6b 102.9 ± 10.9a,c 135.6 ± 11.7b,d Heart Rate (bpm) 73.2 ± 11 77.0 ± 8.5 77.6 ± 11 77.0 ± 5.9 Systolic BP (mmHg) 118.2 ± 8.9a 141.1 ± 8.2b 122.3 ± 8.3a,c 147.9 ± 15b,d Diastolic BP (mmHg) 79.1 ± 3.8a 82.3 ± 2.7b 78.6 ± 5.1a,c 82.6 ± 2.4b,d Testosterone (ng/dL) 308.6 ± 122.8 299.3 ± 75.1 366.2 ± 64.5 317.2 ± 52.2 Cortisol (ug/dL) 19.7 ± 3.8 16.4 ± 2.9 22.1 ± 4.9 17.3 ± 1.5 C-RP Negative Negative Negative Negative CK (U/mL) 99.1 ± 40.8 95.2 ± 22.2 104.1 ± 21.7 98 ± 27.2 LDH (U/L) 238.8 ± 36.2 280.9 ± 65.1 221.9 ± 23.7 294.9 ± 41.6 values of all variables are presented and compared between the presence or not of type 2 etes mellitus (n=40). 168 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 High intensity exercise is better for diabetics FIGURE 1 -Glycemia (A), systolic (B) and diastolic blood pressure (C) for all groups pre- and post-resistance exercise sessions. a p<0.05 in rela tion to each other; b p<0.05 in relation to Pre from both D groups; c p<0.05 in relation to Post from both D groups; d p<0.05 in relation to Post from other groups. FIGURE 1 -Glycemia (A), systolic (B) and diastolic blood pressure (C) for all groups pre- and post-resistance exercise sessions. than pre-RE for both groups and intensities, except for ND60. In relation to hormonal variables, there was significant elevation (p<0.05) of the testosterone for ND75, but not for ND60, however, both RE sessions resulted in increasing of the testosterone (p<0.05) for D groups (FIGURE 2A). There was a reduction (p<0.05) of the cortisol, but not to D60 (FIGURE 2B). The ratio T:C post-RE was higher CK and LDH presented significant changes in different moments and groups but is relevant to highlight the significant increase (p<0.05) for those variables after RE at 75%1RM, (FIGURES 3A and B). FIGURE 2 -Testosterone (A), cortisol (B) concentrations and ratio T:C (C) for all groups pre- and post-resistance exercise sessions. Discussion The main finding in this study was the fact of highest moderate intensity (75%1RM) showed a better glucose uptake from the diabetic group. In addition, independently of RE intensity, the moderate exercises sessions were capable to reduce BP of diabetic groups with a notable tendency to a better reduction in the higher RE intensity. The hormonal response after exercises was suitable for a good exercise recovery, mainly for exercise performed at 75%1RM, which resulted in an elevated indirect marker of muscle damage. a single exercise session, is one of the most non- pharmacological treatments used to prevent and/ or reduce the advancement of hypertension and T2D27,28. In this sense, one useful method that can be used to achieve the benefits for diabetic subjects is the RE since it promotes cardio-metabolic and neuromuscular adaptations29. In this context, stimulation and a very good prescription of the physical exercise are necessary to this population. The knowledge about the best moderate RE intensity that should be prescribed to improve physiological benefits can help to control blood glucose and BP levels. In the present study, the diabetics presented higher BMI, glycemia, SBP, and DPB than nondiabetic groups. Normally, diabetic individuals present many physiological dysfunctions such as cardiovascular disease and hypertension, what increase morbidity and mortality22. T2D is associated with the gain of fat mass and can generate obesity, particularly central obesity. This may lead to an increased release of free fatty acids due to its high sensitivity through the action of catecholamines, which can produce a greater vasoconstriction and fluid retention, leading to hypertension23,24. Moreover, it is known that obese individuals usually have more difficulty to move around, join mobility reduction, and/or difficult to execute simple tasks of daily life such as climbing stairs25. The lack of regular physical activity in these pathological groups during their lives26 could have contributed to a worse quality of strength, higher BMI (obese) and BP levels observed in the present study (TABLE 1). Resistance exercise at 75% 1RM performed by D group resulted in a significant reduction of its glycemic level. This result was in agreement with previous findings where exercise with higher intensity was better to control blood glucose level30,31, even existing another study showing that regardless of intensity (low or moderate), an acute blood glucose uptake will be observed after a resistance circuit exercise32. Results a p<0.05 in rela tion to each other; b p<0.05 in relation to Post from ND75 and D75; c p<0.05 in relation to Pre from both D groups; d p<0.05 in relation to Pre from both ND groups; e p<0.05 in relation to Post from D75. FIGURE 2 -Testosterone (A), cortisol (B) concentrations and ratio T:C (C) for all groups pre- and post-resistance exercise sessions. Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 169 Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 169 Sousa RAL, et al. Sousa RAL, et al. FIGURE 3 -Creatine kinase (A) and lactate dehydrogenase (B) for all groups pre- and post-resistance exercise sessions. a p<0.05 in relation to each other; b p<0.05 in relation to Post from ND75 and D75; c p<0.05 in relation to Pre from both D groups; d p<0.05 in relation to Pre from both ND groups; e p<0.05 in relation to Post from D75. FIGURE 3 -Creatine kinase (A) and lactate dehydrogenase (B) for all groups pre- and post-resistance exercise sessions. 170 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 Discussion The exercise sessions applied in the present study resulted in acute BP benefits for all groups (FIGURE 2) and PEH has been observed in normotensive, hypertensive37,38, and diabetic individuals being well described in the literature35,36,39. In relation to hormonal kinetics, Kim et al.46 demonstrated that healthy individuals who performed a high-intensity RE presented elevated testosterone levels as observed in our present study, even in different intensities (FIGURE 3A). However, it is also described that testosterone can be reduced in RE10. Our results revealed an increase of the testosterone plasma level in D groups after both moderate intensities. Higher testosterone levels in response to RE is associated with a necessary adaptation of the organism, especially at an increased intensity47.i Acute resistance circuit exercise is better than aerobic exercise to control BP during the 24h after exercise in diabetic subjects6, an acute circuit of RE is better than aerobic exercise to control BP during the 24h after exercise in diabetic subjects. Asano et al.22 in a review study suggest as the exercise intensity increases it is possible to observe a higher PEH. Basically, the studies reviewed by these authors were conducted utilizing exercises at light and/or moderate intensities. A greater PEH for the highest intensities when analyzed light and moderate intensities, independently if the exercise was aerobic or resistance31,39,40. Part of these results can explained by the nitric oxide levels since a high- intensity exercise protocol is capable to induce nitric oxide release in hypertensive older women41. Cortisol levels were significantly reduced in ND groups and in D group who performed RE at 75% 1RM (FIGURE 3B). Cortisol reduction was also observed in the elderly population after performing RE at 50% and 80% 1RM48, as reported by Uchida et al.49 in women. On the other hand, Motta et al.42 did not observe significant PEH for diabetics, suggesting a possible positive relationship between PEH and plasma kallikrein activity, since there was less kallikrein activity for diabetics. However, the exercise model was not RE and it was performed 10% below of their anaerobic threshold. According to Dutra et al.43, the PEH can influenced by many variables, including the exercise intensity. Our results reveal a positive relation of T:C ratio in moderate RE at both intensities for D groups, indicating anabolic parameter was immediately active. Discussion This data of the present study indicated that with more intensive stimulation (until the upper limit of the moderate intensity), more blood glucose uptake is obtained, corroborating others studies31,32. Some hormones such as catecholamine, cortisol, and glucagon increase as the exercise intensity raised, what may result in an increase of the liver glycolysis, increasing the blood glucose availability33. However, even with this hormonal pattern, the high-moderate exercise intensity performed at 75% 1RM resulted in an adequate stimulus for increasing blood glucose The regular practice of physical exercise, or even High intensity exercise is better for diabetics at 80% 1RM37,44. The ACSM suggests RE at 60% 1RM for hypertensive and diabetic individuals, and American Heart Association describe it should be over 60% of the maximum intensity14. uptake by the active muscles, resulting in a reduction of BP after exercise. Moreover, the cortisol levels after exercise performed at 75% 1RM presented a significant reduction.h As observed in the present study, good results in both moderate exercise intensities used were found, but the results revealed to be better at 75% 1RM. A new division or subdivision in moderate RE could be proposed and further studies to confirm our findings are necessary to assess whether the changes in RE intensity affects the results. The insulin sensibility increases after exercise due to an improvement of PI3-K and AKT activity, among others possibilities such as interleukins activation34. The high-moderate intensity probably resulted in more vigorous muscle contraction than low-moderate intensity, resulting in a higher blood glucose uptake as observed in the present study, what can explain that the muscle is considered the major body tissue capable to use blood glucose to generate biological energy30,35. It is important to note that the total volume of the resistance exercise at 60% and 75% 1RM were not differed (p>0.05) between the four groups (ND60, ND75, D60, D75), revealing the effects of intensity on metabolic and hemodynamic variables. Similar to the present study, an acute RE session of higher intensity followed by an oral glucose tolerance presented lower glycemia and insulinemia than lower intensity, even with a similar total volume of exercise sessions45. BP level is associated with many health risk factors and diseases including T2D36. Discussion It might be higher at 75%1RM to help to repair the physiological stress observed through a higher muscle damage at this intensity, even they were not obtaining at their peak level after exercise. T:C ratio was increased in our study, showing a prevalence of anabolic conditions over catabolic, corroborating the data found by Uchida et al.49.l In the present study, the acute exercises performed at 75% and 60% 1RM were capable to induce PEH in D groups with a better BP kinesis after exercise performed at 75% 1RM, which is considered a moderate exercise since a few relevant scientific studies have shown that the high RE intensity starts T2D pathology influences and promotes the increase of serum markers of muscle damage50 for rest values. In the present study, there was only Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 171 Sousa RAL, et al. Sousa RAL, et al. concomitant and significant increase of CK and LDH in higher amounts of moderate RE, revealing a greater muscle damage in T2D hypertensive individuals at 75%1RM. It is possible that the greatest amount of CK found after RE was due to a constant trial of the organism to restore the energy reserves during the exercise protocols9,51,52. The largest amount of LDH could be justified by an increase of the anaerobic work, disposing of more lactate in blood, once the exercise performed at a higher intensity can activate three times more LDH, contributing to a possible greater expression of glucose transporters and improving, on this way, insulin sensitivity53,54.h 79% of the maximum intensity, could divided into two or more sub sessions such as lower moderate and upper moderate training zones. Further studies, diagnosing physiological responses at 60%, 65%, 70%, 75% and 79% 1RM, for example, would give us a better idea about the differences in these physiological changes at the moderate training zone. Our data reinforce the importance of RE as a non-pharmacological treatment for T2D, mainly because of the improvement in the blood glucose uptake and the BP reduction after one single session of RE at 75%1RM. The moderate resistance exercise at 75% 1RM promotes better blood glucose uptake and BP management in diabetic subjects. Moderate exercise at 60% or 75% 1RM is capable to reduce BP after RE to diabetic and hypertensive subjects. Discussion The testosterone and cortisol hormones after these RE in moderate intensities suggest the existence of an appropriated exercise recovery, especially after 75% 1RM that results in higher values of indirect muscle damage markers. There is no acute increase in C-RP after moderate RE intensities at 60 and 75% 1RM. The analysis of muscle damage associated with the elevation or the lowering of inflammatory proteins, such as C-RP, may help to diagnose the best type of RE to be developed. The C-RP is release in greater quantities in the body when there is a proinflammatory activity in progress that can be caused by bacteria, viruses or simple metabolic stress provided by physical activity, for example55,56. The acute RE at any level of moderate intensity in this study was not able to generate a severe pro-inflammatory activity to increase beyond the point of reference standards C-RP levels. This result indicates that there is no contraindication for using moderate RE at 60% or 75% 1RM in an RE program for diabetics or non-diabetics. Acute RE brings benefits to diabetics who are hypertensive. Thus, higher intensity of RE targeting over 75% 1RM can performed without risk to the individual. Besides, the results found in this study, it is always necessary that diabetic hypertensive individuals to be supervised during all period of their training sessions for safety reasons. Acute RE at 60% or 75% of 1RM can be used as an extra tool to help the management of both pathologic conditions combined (diabetes and hypertension). Physiological changes at 75% 1RM occurred more than in 60% 1RM to diabetics and healthy people in the present study. In spite of that, moderate training zone, which goes from 60% to References 1. Ogurtsova K, da Rocha Fernandes JD, Huang Y, Linnenkamp U, Guariguata L, Cho NH, et al. IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract. 2017;128:40-50. 2. Snel M, Gastaldelli A, Ouwens DM, Hesselink MK, Schaart G, Buzzigoli E, et al. Effects of adding exercise to a 16- week very low-calorie diet in obese, insulin-dependent type 2 diabetes mellitus patients. J Clin Endocrinol Metab. 2012;97(7):2512-20. 3. Suh DC, Kim CM, Choi IS, Plauschinat CA, Barone JA. Trends in blood pressure control and treatment among type 2 diabetes with comorbid hypertension in the United States: 1988-2004. J Hypertens. 2009;27(9):1908-16. 4. Roden M. Exercise in type 2 diabetes: to resist or to endure? Diabetologia. 2012;55(5):1235-9.hh 5. Maiorana AJ, Naylor LH, Exterkate A, Swart A, Thijssen DH, Lam K, et al. The impact of exercise training on conduit artery wall thickness and remodeling in chronic heart failure patients. Hypertension. 2011;57(1):56-62. 6. Morais PK, Campbell CS, Sales MM, Motta DF, Moreira SR, Cunha VN, et al. Acute resistance exercise is more effective than aerobic exercise for 24h blood pressure control in type 2 diabetics. Diabetes Metab. 2011;37(2):112-7. 6. Morais PK, Campbell CS, Sales MM, Motta DF, Moreira SR, Cunha VN, et al. Acute resistance exercise is more effective than aerobic exercise for 24h blood pressure control in type 2 diabetics. Diabetes Metab. 2011;37(2):112-7. 7. Tompkins CL, Soros A, Sothern MS, Vargas A. Effects of physical activity on diabetes management and lowering risk for type 2 diabetes. J Health Educ. 2009;40(5):286-90. 7. Tompkins CL, Soros A, Sothern MS, Vargas A. Effects of physical activity on diabetes management and lowering risk for type 2 diabetes. J Health Educ. 2009;40(5):286-90. 8. Roma MFB, Busse AL, Betoni RA, Melo ACd, Kong J, Santarem JM, et al. Effects of resistance training and aerobic exercise in elderly people concerning physical fitness and ability: a prospective clinical trial. Einstein. 2013;11(2):153-7. exercise in elderly people concerning physical fitness and ability: a prospective clinical trial. Einstein. 2013;11(2):153-7. 9. Santos WOC, Brito CJ, Júnior EAP, Valido CN, Mendes EL, Nunes MAP, et al. Cryotherapy post-training reduces muscle damage markers in jiu-jitsu fighters. J Hum Sport Exerc. 2012;7(3):629-38. 9. Santos WOC, Brito CJ, Júnior EAP, Valido CN, Mendes EL, Nunes MAP, et al. Cryotherapy post-training reduces muscle damage markers in jiu-jitsu fighters. J Hum Sport Exerc. 2012;7(3):629-38. 10. High intensity exercise is better for diabetics High intensity exercise is better for diabetics p<0,05), e apenas a sessão 75%1RM induziu captação de glicose sanguínea em 5,7% nos diabéticos. A hipotensão pós-exercício foi significativa após ER a 75%1RM na PA sistólica (PAS), e após ambas intensi dades na PA diastólica (PAD) em não-diabéticos, enquanto a PAS e PAD reduziram após ambas intensidades nos diabéticos. ER a 75%1RM resultou em melhor captação de glicose sanguínea, e ambas intensidades reduziram a PA nos diabéticos. Após 75%1RM, houve maiores danos musculares indiretos. As alterações nos hormônios, PCR e marcadores indiretos de dano muscular indicaram adequada recuperação anabólica aguda sem inflamação significativa em ambas intensidades. O ER agudo a 60%1RM ou 75%1RM pode ser usado como uma ferramenta extra para gerenciar ambas condições patológicas. p<0,05), e apenas a sessão 75%1RM induziu captação de glicose sanguínea em 5,7% nos diabéticos. A hipotensão pós-exercício foi significativa após ER a 75%1RM na PA sistólica (PAS), e após ambas intensi dades na PA diastólica (PAD) em não-diabéticos, enquanto a PAS e PAD reduziram após ambas intensidades nos diabéticos. ER a 75%1RM resultou em melhor captação de glicose sanguínea, e ambas intensidades reduziram a PA nos diabéticos. Após 75%1RM, houve maiores danos musculares indiretos. As alterações nos hormônios, PCR e marcadores indiretos de dano muscular indicaram adequada recuperação anabólica aguda sem inflamação significativa em ambas intensidades. O ER agudo a 60%1RM ou 75%1RM pode ser usado como uma ferramenta extra para gerenciar ambas condições patológicas. Palavras-chave: Diabetes Mellitus Tipo 2; Hipertensão; Exercício Resistido; Saúde. Palavras-chave: Diabetes Mellitus Tipo 2; Hipertensão; Exercício Resistido; Saúde. Resumo Exercício resistido de intensidade moderada/alta é melhor para reduzir a glicose sanguínea e pressão arterial em indivíduos diabéticos na meia-idade Exercício resistido de intensidade moderada/alta é melhor para reduzir a glicose sanguínea e pressão arterial em indivíduos diabéticos na meia-idade A principal característica do diabetes tipo 2 (DM2) é a resistência à insulina. A hipertensão é uma comor bidade relacionada à DM2. O exercício resistido (ER) é uma importante ferramenta não-farmacológica para o controle da glicemia e pressão arterial (PA), mas não existe consenso sobre os efeitos agudos. O objetivo do estudo foi avaliar o efeito agudo fisiológico após duas sessões de ER em diferentes intensidades em indivíduos de meia-idade hipertensos e com DM2. Foram 40 homens de meia-idade (20 não-diabéticos; 20 diabéticos), submetidos a um protocolo de exercícios de mesmo volume a 60% ou 75% do teste de repetição máxima (1RM), consistindo em: supino reto, tríceps no pulley, remada no aparelho, rosca direta com barra, elevação lateral com halteres e agachamento com barra. As alterações fisiológicas foram avaliadas através da PA, glicemia, creatina quinase, lactato desidrogenase, proteína C-reativa (PCR), testosterona e cortisol. Para os não-diabéticos, ambas intensidades promoveram captação de glicose sanguínea (8,2% a 11,1%, 172 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 References Acute effects of physical exercise in type 2 diabetes: A review. World J Diabetes. 2014;5(5):659‐665. doi:10.4239/wjd.v5.i5.659. 23. Ferrao FM, Lara LS, Lowe J. Renin-angiotensin system in the kidney: What is new? World J Nephrol. 2014;3(3):64- 76. 24. Sullivan PW, Ghushchyan V, Ben-Joseph RH. The effect of obesity and cardiometabolic risk factors on expenditures and productivity in the United States. Obesity (Silver Spring). 2008;16(9):2155-62. 25. Akpinar TS, Tayfur M, Tufan F, Sahinkaya T, Kose M, Ozsenel EB, et al. Uncomplicated diabetes does not accelerate age-related sarcopenia. Aging Male. 2014;17(4):205-10. 26. Mendes R, Sousa N, Almeida A, Subtil P, Guedes-Marques F, Reis VM, et al. Exercise prescription for patients with type 2 diabetes—a synthesis of international recommendations: narrative review. Br J Sports Med. 2015;50(22):1379- 81. 27. Mendes TAB, Goldbaum M, Segri NJ, Barros MBA, César CLG, Carandina L. Factors associated with the preva lence of hypertension and control practices among elderly residents of São Paulo city, Brazil. Cad Saude Publica. 2013;29(11):2275-86. 28. Cavalcante PAM, Rica RL, Evangelista AL, Serra AJ, Figueira Jr A, Pontes Jr FL, et al. Effects of exercise intensity on postexercise hypotension after resistance training session in overweight hypertensive patients. Clin interv Aging. 2015;10:1487. 29. Cadore EL, Pinto RS, Bottaro M, Izquierdo M. Strength and endurance training prescription in healthy and frail elderly. Aging Dis. 2014;5(3):183-95. 30. van Dijk J-W, Manders R, Tummers K, Bonomi A, Stehouwer C, Hartgens F, et al. Both resistance-and endurance-type exercise reduce the prevalence of hyperglycaemia in individuals with impaired glucose tolerance and in insulin-treated and non-insulin-treated type 2 diabetic patients. Diabetologia. 2012;55(5):1273-82. 31. Hiyane WC, de Sousa MV, Moreira SR, do Valle G, de Oliveira RJ, Arsa G, et al. Blood glucose responses of type-2 diabetics during and after exercise performed at intensities above and below anaerobic threshold. Brazilian J Kinanthrop Human Perfor. 2008;10(1):8-11. 32. Moreira SR, Simoes GC, Moraes JF, Motta DF, Campbell CS, Simoes HG. Blood glucose control for individuals with type-2 diabetes: acute effects of resistance exercise of lower cardiovascular-metabolic stress. J Strength Cond Res. 2012;26(10):2806-11. 33. Canali ES, Kruel LFM. Respostas hormonais ao exercício. Rev Paul Educ Fís. 2001;15(2):141-53. 34. Folli F, Saad M, Backer JM, Kahn CR. Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat. J Biol Chem. 1992;267(31):22171-7.i 35. Arsa G, Lima L, Almeida S, Moreira SR, Campbell CSG, Simões HG. References Cheung KK, Luk AO, So WY, Ma RC, Kong AP, Chow FC, et al. Testosterone level in men with type 2 diabetes mellitus and related metabolic effects: A review of current evidence. J Diabetes Investig. 2015;6(2):112-23. 11. Kraemer WJ, Ratamess NA. Hormonal responses and adaptations to resistance exercise and training. Sports Med. 2005;35(4):339-61. 12. Adams OP. The impact of brief high-intensity exercise on blood glucose levels. Diabetes Metab Syndr Obes. 2013;6:113- 22. 13. Haskell WL, Lee IM, Pate RR, Powell KE, Blair SN, Franklin BA, et al. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Med Sci Sports Exerc. 2007;39(8):1423-34. 14. de Freitas Brito A, de Oliveira CVC, do Socorro Brasileiro-Santos M, da Cruz Santos A. Resistance exercise with different volumes: blood pressure response and forearm blood flow in the hypertensive elderly. Clin Interv Aging. 2014;12:2151-8. 15. Gonzalez-Badillo JJ, Gorostiaga EM, Arellano R, Izquierdo M. Moderate resistance training volume produces more favorable strength gains than high or low volumes during a short-term training cycle. J Strength Cond Res. 2005;19(3):689-97. 16. Colberg SR, Albright AL, Blissmer BJ, Braun B, Chasan-Taber L, Fernhall B, et al. Exercise and type 2 diabetes: American College of Sports Medicine and the American Diabetes Association: joint position statement. Exercise and type 2 diabetes. Med Sci Sports Exerc. 2010;42(12):2282-303. 17. Brown LE, Weir JP. ASEP Procedures recomendation I: Accurate assessment of muscular strenght and power. JEPonline. Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 173 Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 173 Sousa RAL, et al. 2001;4(3):1-21. 18. Koch AJ, Pereira R, Machado M. The creatine kinase response to resistance exercise. J Musculoskelet Neuronal Interact. 2014;14(1):68-77. 19. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, et al. The Seventh Report of the Joint Na tional Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. JAMA. 2003;289(19):2560-72. 20. Pescatello LS, Franklin BA, Fagard R, Farquhar WB, Kelley GA, Ray CA, et al. Exercise and Hypertension. Med Sci Sports Exerc. 2004;36(3):533-53. 21. Kelly AS, Bergenstal RM, Gonzalez-Campoy JM, Katz H, Bank AJ. Effects of exenatide vs. metformin on endothelial function in obese patients with pre-diabetes: a randomized trial. Cardiovasc Diabetol. 2012;11:64. 22. Asano RY, Sales MM, Browne RAV, Nova JFV, Moraes HJCJ, Moraes MR, et al. 174 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 High intensity exercise is better for diabetics influenced by exercise intensity in type-2 diabetic and nondiabetic individuals. J Strength Cond Res. 2010;24(5):1277- 84. influenced by exercise intensity in type-2 diabetic and nondiabetic individuals. J Strength Cond Res. 2010;24(5):1277- 84. 41. Santana HA, Moreira SR, Asano RY, Sales MM, Cordova C, Campbell CS, et al. Exercise intensity modulates nitric oxide and blood pressure responses in hypertensive older women. Aging Clin Exp Res. 2013;25(1):43-8. 42. Motta DF, Lima LC, Arsa G, Russo PS, Sales MM, Moreira SR, et al. Effect of type 2 diabetes on plasma kallikrein activity after physical exercise and its relationship to post-exercise hypotension. Diabetes Metab. 2010;36(5):363-8. 43. Dutra MT, Lima RM, Mota MR, de Oliveira PFA, Veloso JHCL. Hipotensão pós-exercício resistido: uma revisão da literatura. . Rev Educ Fis/UEM. 2013;24(1):145-57. 44. Pollock ML, Franklin BA, Balady GJ, Chaitman BL, Fleg JL, Fletcher B, et al. AHA Science Advisory. Resistance exercise in individuals with and without cardiovascular disease: benefits, rationale, safety, and prescription: An advisory from the Committee on Exercise, Rehabilitation, and Prevention, Council on Clinical Cardiology, American Heart Association; Position paper endorsed by the American College of Sports Medicine. Circulation. 2000;101(7):828-33. 45. Miller AD, Ruby BC, Laskin JJ, Gaskill SE. Effects of high intensity / low volume and low intensity / high volume isokinetic resistance exercise on postexercise glucose tolerance. J Strength Cond Res. 2007;21(2):330-5.f 46. Kim HH, Kim YJ, Lee SY, Jeong DW, Lee JG, Yi YH, et al. Interactive effects of an isocaloric high-protein diet and resistance exercise on body composition, ghrelin, and metabolic and hormonal parameters in untrained young men: A randomized clinical trial. J Diabetes Investig. 2014;5(2):242-7. 47. Sgro P, Romanelli F, Felici F, Sansone M, Bianchini S, Buzzachera CF, et al. Testosterone responses to standardized short-term sub-maximal and maximal endurance exercises: issues on the dynamic adaptive role of the hypothalamic -pituitary-testicular axis. J Endocrinol Invest. 2014;37(1):13-24. 48. Oliveira RJ, Lima RM, Gentil P, Simões HG, Ávila WRdM, Silva RW, et al. Acute hormonal responses to different intensities of resistance exercises in older women. Rev Bras Med Esporte. 2008;14(4):367-71. 49. Uchida MC, Bacurau RFP, Navarro F, Pontes Jr FL, Tessuti VD, Moreau RL, et al. Alteration of testosterone: cortisol ratio induced by resistance training in women. Rev Bras Med Esporte. 2004;10(3):165-8. 50. Passarella S, de Bari L, Valenti D, Pizzuto R, Paventi G, Atlante A. Mitochondria and L-lactate metabolism. FEBS Lett. 2008;582(25-26):3569-76. 51. References Diabetes Mellitus tipo 2: Aspectos fisiológicos, genéticos e formas de exercício físico para seu controle. Rev Bras Cineantropom Desempenho Hum. 2009;11(1):103- 11. 36. Costanzi CB, Halpern R, Rech RR, Bergmann MLdA, Alli LR, Mattos APd. Associated factors in high blood pressure among schoolchildren in a middle size city, southern Brazil. J Pediatr. 2009;85(4):335-40. 37. Rezk CC, Marrache RC, Tinucci T, Mion D, Forjaz CL. Post-resistance exercise hypotension, hemodynamics, and heart rate variability: influence of exercise intensity. Eur J Appl Physiol. 2006;98(1):105-12. l en CY, Bonham AC. Postexercise hypotension: central mechanisms. Exerc Sport Sci Rev. 2010;38(3):122-7. 39. Sales MM, Russo P, Moreira SR, Santana H, Moraes JF, Asano RY, et al. Resistance exercise elicits acute blood pressure reduction in type 2 diabetics. J Exerc Physiol Online. 2012;15:98-109. 40. Simoes GC, Moreira SR, Kushnick MR, Simoes HG, Campbell CS. Postresistance exercise blood pressure reduction is 40. Simoes GC, Moreira SR, Kushnick MR, Simoes HG, Campbell CS. Postresistance exercise blood pressure reduction is 174 • Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 High intensity exercise is better for diabetics Submitted: 20/03/2017 1st. Review: 06/07/2017 2nd. Review: 15/09/2017 Accepted: 28/07/2017 High intensity exercise is better for diabetics Ostler JE, Maurya SK, Dials J, Roof SR, Devor ST, Ziolo MT, et al. Effects of insulin resistance on skeletal muscle growth and exercise capacity in type 2 diabetic mouse models. Am J Physiol Endocrinol Metab. 2014;306(6):E592- 605. 52. Christ CY, Hunt D, Hancock J, Garcia-Macedo R, Mandarino LJ, Ivy JL. Exercise training improves muscle insulin resistance but not insulin receptor signaling in obese Zucker rats. J Appl Physiol. 2002;92(2):736-44. 53. Goodwin ML, Harris JE, Hernandez A, Gladden LB. Blood lactate measurements and analysis during exercise: a guide for clinicians. J Diabetes Sci Technol. 2007;1(4):558-69.h 54. Billat VL, Sirvent P, Py G, Koralsztein JP, Mercier J. The concept of maximal lactate steady state: a bridge between biochemistry, physiology and sport science. Sports Med. 2003;33(6):407-26. 55. Dunstan DW, Zimmet PZ, Welborn TA, De Courten MP, Cameron AJ, Sicree RA, et al. The rising prevalence of diabetes and impaired glucose tolerance: the Australian Diabetes, Obesity and Lifestyle Study. Diabetes Care. 2002;25(5):829- 34. 56. American Diabetes A. Standards of medical care in diabetes--2014. Diabetes Care. 2014;37(Suppl. 1): Corresponding author: Gisela Arsa Avenida Fernando Corrêa da Costa, sn - Boa Esperança Cuiabá – MT - BRAZIL CEP: 78060900 E-mail: gisarsa@gmail.com Corresponding author: Gisela Arsa Avenida Fernando Corrêa da Costa, sn - Boa Esperança Cuiabá – MT - BRAZIL CEP: 78060900 E-mail: gisarsa@gmail.com Corresponding author: Gisela Arsa Avenida Fernando Corrêa da Costa, sn - Boa Esperança Cuiabá – MT - BRAZIL CEP: 78060900 E-mail: gisarsa@gmail.com Submitted: 20/03/2017 1st. Review: 06/07/2017 2nd. Review: 15/09/2017 Accepted: 28/07/2017 Submitted: 20/03/2017 1st. Review: 06/07/2017 2nd. Review: 15/09/2017 Accepted: 28/07/2017 Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 175 Rev Bras Educ Fís Esporte, (São Paulo) 2020 Jan-Mar; 34(1):165-175 • 175
https://openalex.org/W2946857083	https://europepmc.org/articles/pmc6543604?pdf=render	English	null	Sex steroid hormone modulation of neural stem cells: a critical review	Biology of sex differences	2,019	cc-by	8,513	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract While numerous in vivo experiments have sought to explore the effects of sex chromosome composition and sex steroid hormones on cellular proliferation and differentiation within the mammalian brain, far fewer studies as reviewed here, have explored these factors using a direct in vitro approach. Generally speaking, in vivo studies provide the gold standard to demonstrate applicable findings in regards to the role hormones play in development. However, in the case of neural stem cell (NSC) biology, there remain many unknown factors that likely contribute to observations made within the developed brain, specifically in regions where there are abundant sex steroid hormone receptors. For these reasons, using a NSC in vitro model may provide a more controlled and refined system to explore the direct effects of sex and hormone response, limiting the vast array of other influences on NSCs occurring during development and within adult cellular niches. These specific cellular models may have the ability to greatly improve the mechanistic understanding of changes occurring within the developing brain during the hormonal organization process, in addition to other modifications that may contribute to neuro- psychiatric sex-biased diseases. Sex steroid hormone modulation of neural stem cells: a critical review Matthew S. Bramble, Neerja Vashist and Eric Vilain* Introduction While the vast majority of the aforementioned studies have drawn conclusions based on analyses of gross brain tissue, other studies have looked at the direct effects of chromosomal composition and sex steroid influence on specific cells comprising the central nervous system (CNS). Studies have highlighted the effects of testoster- one and estrogens on various types of neurons and as- trocytes [11–15]; however, few studies to date have explored these effects and the epigenetic consequences of such, on cultured neural stem cells isolated from the embryonic and adult mammalian brains. Investigating the phenomenon of hormonal organization, or the enduring effects of sex steroid hormone exposure on the brain, became a focal point within the field of neu- roendocrinology since the seminal findings of Phoenix et.al was first published in 1959 [1]. While these findings elegantly demonstrated that exposing female fetuses to an- drogenic compounds resulted in altered adult sexual be- havior, the exact mechanisms behind this organization process remain to be fully elucidated. Significant works have built on the hormonal organization theory, and as such, have identified numerous sex-differences in addition to behavior that are set in motion by sex steroid hormone exposures in utero and during the perinatal period [2]. In addition to hormone exposure on the developing brain, it also appears that genetic composition [3, 4] [5, 6] and epi- genetic modifications [7–9] significantly contribute to developing adult sexual behavior, sexually dimorphic brain structures, and other sex differences within rodents and humans [10]. Neural stem cells (NSCs) by definition are multipotent populations capable of giving rise to all of the main cell types that comprise the CNS, in addition to having self- renewal capacity [16]—the hallmark of any “stem” cell. There are two general groupings of neural stem cells, those present during early development which will be re- ferred to as embryonic neural stem cells (eNSCs) and those that are maintained during/throughout adulthood (aNSCs). Embryonic neural stem cells are abundant, rapidly dividing, and differentiating during early devel- opment, providing sufficient cellular numbers for proper brain formation. These embryonic cells are subject to estrogen and androgen exposures during early develop- ment, predominately in utero. Adult NSCs, however, are * Correspondence: evilain@childrensnational.org; evilain@CNMC.org Center for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington, D.C., USA Bramble et al. Biology of Sex Differences (2019) 10:28 https://doi.org/10.1186/s13293-019-0242-x Bramble et al. Biology of Sex Differences (2019) 10:28 https://doi.org/10.1186/s13293-019-0242-x (2019) 10:28 Bramble et al. Biology of Sex Differences https://doi.org/10.1186/s13293-019-0242-x Open Access Page 2 of 10 Bramble et al. Biology of Sex Differences (2019) 10:28 Page 2 of 10 protein expression level of aromatase, an enzyme re- sponsible for the conversion of testosterone into estra- diol, which plays a vital role in hormonal organization of mammalian brains [28, 29]. Using 8–10-week-old adult murine NSCs isolated from the SVZ [30] and NSCs from the SVZ of 3-month-old Long-Evans rats [31], aro- matase expression was quantified using both a GFP reporter assay and total protein analysis. Those two in- dependent studies found that aromatase expression in the absence of gonadal hormones displayed a significant male bias with regard to protein expression in adult NSCs. However, our recent study utilizing RNA- sequencing did not identify any expressed aromatase transcripts in either XX or XY murine eNSCs [32] (Fig. 1c). While this discrepancy would typically seem contradictory, our group used NSCs isolated from the telencephalons of E-13.5 C57/B6/J mice, whereas the other two groups used adult-isolated NSCs from both the rat and mouse SVZ. This difference in aromatase expression between adult and embryonic NSCs raises an interesting possibility that the effects of androgenic hor- mone exposure on this cell type may have markedly dif- ferent consequences depending on developmental stage. If aromatase is not expressed in murine embryonic NSCs, then during the in utero testosterone surge, the effects on these cells are likely due to direct testosterone signaling. On the other hand, since adult mouse and rat NSCs do express aromatase, there could be downstream effects of pubertal androgenic exposure on these cells. Whether these effects are activational or organizational could be modulated either by direct testosterone action on the androgen receptor (AR) [33] or estrogen signal- ing, through its various receptors (ERα, ERβ, GPR30) once locally converted into estradiol by aromatase [34]. As earlier mentioned, our group conducted a global tran- restricted to specific regions within the mature brain and remain under complex regulatory control within their respective niches [17–19]. aNSCs in theory are ex- posed to pubertal surges of testosterone and/or estrogen depending on gonadal composition, which remain in abundant circulation for most of adult life. Areas rich in quiescent NSCs during adulthood include the sub- ventricular zone (SVZ) and the sub-granular zone (SGZ) of the dentate gyrus (DG) [20]. Both types of NSCs re- tain stem properties; however, they appear to have differ- ent cellular features and protein expression patterns [16, 20]. This raises the notion that there are intrinsic and extrinsic distinctions to be made between adult NSCs and those present during early brain development, which will be particularly relevant to this review. Research focusing on neural stem cells and adult neuro- genesis has seen an explosion in the past two decades, which has been thoroughly described by Gage and Temple [21]. As noted, despite intense investigation, few studies have sought to explore inherent sex differences and the role that sex steroids have in shaping neural stem cell biol- ogy, although studies indicate that such hormones influ- ence adult neurogenesis within the DG [22, 23]. The intent of this review is to highlight the in vitro work that has investigated these aspects in mammalian NSCs, exposing a novel role of sex steroid hormone influence during early brain development and throughout adult- hood. Despite being outside the scope of this review, it should be noted that much of our understanding of sex steroid influence on the mammalian brain has been built on studies using the songbird as a research model organ- ism, as reviewed elsewhere [24–27]. Basal sex-differences in cultured NSCs As earlier mentioned, our group conducted a global tran- scriptional analysis using RNA-sequencing on eNSCs to de- termine if there were inherent sex differences with regard to gene expression. To our surprise, we identified 103 tran- scripts that were differentially expressed between XX and During analysis of neural stem cells, there have been several studies that have identified inherent basal sex dif- ferences between XX and XY NSCs, independent of active androgen or estrogen exposure. One such sex- difference that has been found by several groups is the Fig. 1 a, b Upon stimulation with retinoic acid during the differentiation process, XX and XY NSCs show variable differential outcomes, as highlighted post differentiation. Astrocytes and oligodendrocytes are enriched in XX backgrounds post differentiation whereas XY NSCs display a pro-neuronal differentiation pattern. c General distributions of aromatase expression found in adult and embryonic NSCs Fig. 1 a, b Upon stimulation with retinoic acid during the differentiation process, XX and XY NSCs show variable differential outcomes, as highlighted post differentiation. Astrocytes and oligodendrocytes are enriched in XX backgrounds post differentiation whereas XY NSCs display a pro-neuronal differentiation pattern. c General distributions of aromatase expression found in adult and embryonic NSCs Page 3 of 10 Bramble et al. Biology of Sex Differences (2019) 10:28 Page 3 of 10 Bramble et al. Biology of Sex Differences (2019) 10:28 XY murine eNSCs (FDR = 0.10) at a time prior to gonadal- derived hormonal surges [32] The vast majority of these transcriptional sex differences were enriched in pathways predominately involved in cellular replication, possibly indi- cating that XX and XY eNSCs differently regulate prolifera- tive states. While many of the identified differential transcripts were novel, this was the first study utilizing RNA-seq to probe for sex differences of global gene expres- sion in eNSCs. The identified basal sex differences within eNSCs are quite interesting and provide a unique gene set for further exploration, and it should also be evident that these findings represent a brief snapshot of developmental time, identifying differences that are likely transient. The developing body is highly dynamic and rapidly changing, as are neural stem cells [35]. When assessing early sex differ- ences, it is vital to keep in mind that such findings have spatial and temporal fluctuations and are unlikely to remain static throughout the life of the animal. been exposed to endogenous testosterone and estrogens prior to isolation. In vitro effects of estrogenic compounds on NSCs In vitro effects of estrogenic compounds on NSCs Estrogen receptor(s) expression: In order to determine the effects exerted by estrogens, predominantly 17β- estradiol (E2), on NSCs, it first had to be established that these cell types expressed the appropriate receptors. In one of the first studies assessing the role of E2 on NSC physiology in vitro, it was determined that both embry- onic NSCs isolated from E15–E20 Wistar rats and NSCs isolated from the lateral ventricles of adults of the same species, expressed both ERα and ERβ [38] (Fig. 2a) (Table 1). These findings successfully demonstrated that ERα was most expressed in eNSCs during earlier time points of development, when cells were isolated at E15 and E17, and such expression was greatly reduced by E20 and within aNSC populations. The reverse was true for ERβ, which showed increased expression over the E15–E20 developmental period, as well as into adult- hood [38]. In studies using aNSCs isolated from the SVZ of 3- and 20-month-old male and female Long-Evans rats, ERα and ERβ were again found to be expressed in both age groups (Fig. 2a) (Table 1), with expression levels being significantly higher in aNSCs isolated at 20 months of age. The same group also claims that ERα and ERβ exhibit different expression patterns based on sex chromosome composition in aNSCs at 3 months of age, but not at the 20-month stage [37]. In a more re- cent study, the expression of estrogen receptors was again confirmed in eNSCs isolated from E14.5–E16.5 Sprague-Dawley rat embryos. This study however also probed for a newly identified membrane-bound ER re- ceptor, GPR30, and found that in addition to ERα and β, GPR30 protein was also strongly expressed [39] (Fig. 2a) (Table 1). While receptors for estrogens appear to be present in both embryonic and adult NSCs isolated from the rat, the same does not appear to be true for NSCs isolated from embryonic mouse brain tissue. When assaying nuclear hormone receptor expression within NSCs isolated from E-13.5 murine embryos using RT- PCR analysis, it was concluded that both ERα/β were not expressed or were transcriptionally undetectable at that particular time point [40]. Basal sex-differences in cultured NSCs This endogenous exposure may have al- tered epigenetic programming or specific protein expres- sion, and the conclusions found may not be due to inherent differences based on sex and age, per se, but ra- ther to this possible programming of NSCs induced by en- dogenous T exposure. This would seem like a possible explanation, as the same group attributed the findings of variances in differentiation potentials to the differences in expression of CYP19 (p-450 aromatase), which was un- covered in their later finding [31]. In vitro effects of estrogenic compounds on NSCs Similar findings demon- strating low/no detection of estrogen receptors α/β was Additional research focusing on improving NSC trans- plantation therapy for various neurological conditions, which still has significant hurdles to overcome [36], identi- fied that both age and sex of NSCs are contributing factors that should be taken into consideration [37]. Using adult NSCs isolated from the SVZ of 3- and 20-month-old Long-Evans rats revealed that upon differentiation in retin- oic acid, XY and XX NSCs displayed altered neurogenic and gliogenic potentials, which were both sex and age- dependent. When measuring protein levels, it was determined that neuronal markers such as MAP2 (micro- tubule-associated protein2), GAP43 (GAP43 growth asso- ciated protein 43), and DCX (doublecortin) along with the oligodendrocyte marker CNPase displayed an increased expression pattern in those differentiated NSCs of XY ori- gin [37] (Fig. 1b). The same study found a reverse trend when looking at an astrocyte marker, GFAP (glial fibrillary acidic protein), which showed a very significant enrich- ment in expression on a XX background (Fig. 1a). The early neuronal maker βIII tubulin did not show sex differ- ences in this study; however, in a later assessment using immunofluorescence, it did appear that βIII tubulin may show sex differences in expression post-differentiation [31]. It was identified that the expression of βIII tubulin, along with those proteins that showed sex differences (GFAP, MAP2, GAP43), were age-dependent and were sig- nificantly reduced in differentiated NSCs isolated from 20- month-old rats as compared to those isolated at 3 months of age [37]. The authors concluded that XY NSCs may have inherent potential to differentiate into neuronal lin- ages, while XX NSCs preferentially differentiate down a glia pathway when stimulated with retinoic acid, with cel- lular age being a large contributing factor. It should be noted, however, that while their differentiation process contained no exogenous gonadal hormones, the XY NSCs from both the 3-month and 20-month-old rats would have Bramble et al. Biology of Sex Differences (2019) 10:28 Page 4 of 10 Fig. 2 a Estrogen receptor expression profiles of NSCs across time and rodent species. b The general cellular proliferation affects as a result of estrogen exposure on NSCs isolated from various species of rodents at different time points of development. c The effects of estrogen exposure on NSCs during cellular differentiation, highlighting indicates the preferential differential cell outcomes, during or after estrogenic treatment Fig. In vitro effects of estrogenic compounds on NSCs 2 a Estrogen receptor expression profiles of NSCs across time and rodent species. b The general cellular proliferation affects as a result of estrogen exposure on NSCs isolated from various species of rodents at different time points of development. c The effects of estrogen exposure on NSCs during cellular differentiation, highlighting indicates the preferential differential cell outcomes, during or after estrogenic treatment proliferation, as well as differentiation potentials of NSC populations. In the Brännvall et.al study, which de- scribed the presence of estrogen receptors within these cell types, also assessed the physiological effects of estro- gen exposure. Using eNSCs and aNSCs from Wistar rats, it was shown that when 10 nM of 17β-estradiol was introduced to their culture media in the absence of the mitogen EGF (epidermal growth factor), there was a sig- nificant (7%) proliferation increase of eNSCs, as mea- sured by BrdU-positive cells [38] (Fig. 2b) (Table 1). This effect was not seen in their aNSC population, or when the ER antagonist ICI-182,780 was present. When the same assay was conducted in the presence of EGF, there also corroborated by our groups complete transcrip- tomic analysis of E.13.5 mouse NSCs using RNA-Seq [32] (Fig. 2a) (Table 1). While these findings demon- strate that estrogen receptors are at least present in rat NSCs, they also highlight the notion that all NSCs should not be grouped together and the possible response to gonadal hormones such as E2 may be species-specific and variable over both developmental time and in different brain regions. The effects of estrogens on NSC cellular proliferation Studies focusing on the effects of estrogens have predominately investigated how E2 affects cellular The effects of estrogens on NSC cellular proliferation Table 1 Summary of in vitro studies assessing estrogenic response in neural stem cells Table 1 Summary of in vitro studies assessing estrogenic response in neural stem cells Table 1 Summary of in vitro studies assessing estrogenic response in neural stem cells Table 1 Summary of in vitro studies assessing estrogenic response in neural stem cells Page 5 of 10 Bramble et al. Biology of Sex Differences (2019) 10:28 Page 5 of 10 was actually decreased proliferation of both eNSCs and aNSCs (Fig. 2b) (Table 1). This decrease in proliferation in the presence of EGF was attributed to the upregula- tion of the cyclin-dependent kinase (CDK) P21, as it was shown that protein expression of this cell-cycle regulator was significantly increased in the presence of E2. The authors note that while E2 can moderately increase pro- liferation of embryonic derived NSCs, this effect is greatly dependent on other growth factors, as in their case, EGF. While a 2008 study investigating the effects of E2 on rat eNSCs was unable to replicate these initial findings, using a modified media composition still dem- onstrated that estradiol alone was capable of increasing proliferation of eNSCs isolated from the telencephalons of E15 Wistar rat embryos [41]. The same conclusions were also drawn from both a 2010 [42] and 2016 study; however, it was also determined that increased doses of E2 (50 nM) actually had the opposite effect, resulting in a reduced proliferative state [39] (Table 1). stimulated differentiation to proceed down a neural lineage as determined by the ratio of Tuj-1 (βIII tubulin marker) to GFAP signal (Fig. 2c) (Table 1). These find- ings again demonstrated that 10 nM of E2 seems to be the most optimal dose for stimulating this differentiation outcome, as 1, 20, and 50 nM concentrations of E2 did not result in altered neuron/glia ratios [39]. There also appears to be no sex differences in the effects of E2 on NSC differentiation in cultured adult murine cells, where both XX and XY lines showed increased neuronal staining patterns when allowed to differentiate in the presence of 10 nM of E2 [30]. p Two studies conducted by Okada et.al using embryonic- derived rat NSCs were unable to replicate the findings showing preferential neuron differentiation upon stimula- tion with E2 [41, 42] (Table 1). The effects of estrogens on NSC cellular proliferation While this group did not ob- serve increased neuronal potential, they did however determine that E2 increased differentiation of both CNPase-positive oligodendrocytes and NG-2-positive oligo- dendrocyte precursor cells (Fig. 2c). The authors concluded (but did not demonstrate) that this effect was not a result of classical ER signaling, but rather membrane ER signaling, because when NSCs were pre-treated with ICI-182,780, the ratios of oligodendrocytes and precursor cells were still in- creased during differentiation in the presence of E2 [41, 42]. It should also be noted, that while all studies are not in agreement with regard to pro-neuronal outcomes when NSCs are stimulated with E2, there were also technical dif- ferences between experiments that may have played a fac- tor. The studies that identified E2 as a pro-neuronal stimulant [38, 39] grew their NSCs in the presence of EGF, whereas the studies that were unable to replicate these find- ings and saw pro-oligodendrocyte differentiation [41, 42] grew their NSCs with the mitogen FGF-2 (fibroblast growth factor 2) (Table 1). It has been established that murine em- bryonic NSCs express FGF and EGF receptors in a tem- poral and spatial manner, and as such, respond to these mitogens in different ways [43], highlighting the importance of what otherwise may seem like a minor technical differ- ence between studies. From these independent studies, it can be concluded that while E2 can significantly increase proliferation of rat eNSCs through ER action (Fig. 2b), these outcomes are greatly dependent on both the dose of estrogen and the site of NSC isolation. To our knowledge, only one study has assessed the effects of estradiol exposure on adult- derived mouse NSCs and also found that E2 significantly increased cellular proliferation, using a Ki67 proliferation marker, regardless of chromosomal sex [30] (Fig. 2b) (Table 1). This work did not show estrogen receptor ex- pression per se and as previously stated such transcripts have not been identified in murine eNSCs by other groups. However, given their results, it can be inferred that perhaps estrogen receptor expression, while not present at the embryonic stages, becomes expressed by aNSCs in the murine SVZ and stimulation with E2 elicits proliferative effects in the adult mouse also. The effects of estrogens on NSC differentiation In addition to proliferation, many of the studies asses- sing those effects also investigated the role that E2 plays during the differentiation process. When eNSCs isolated from the rat were treated with 10 nM of E2 while under- going a 4-day differentiation, it was found that there was a significant increase in the ratio of βIII tubulin-positive neurons over GFAP-expressing cells (Fig. 2c) (Table 1). The same outcome was not seen when aNSCs were treated with E2 during differentiation, indicating again that embryonic and adult cells may have completely dif- ferent responses to estrogens (Fig. 2c) This pro-neuron effect was attenuated when the ER antagonist ICI-182, 780 was added to the media, demonstrating that these observations during differentiation were modulated in part by ERα/β signaling [38]. Recent publications came to similar conclusions and determined that 10 nM of E2 In vitro effects of androgenic compounds on NSCs Androgen receptor expression While several in vitro approaches have provided evidence supporting the role of estrogens in proliferation and differ- entiation of cultured NSCs, few studies have addressed how such cells respond to androgenic compounds, despite known androgen response in other types of stem cells [52]. Brännvall et.al demonstrated that both embryonic and adult rat NSCs derived from the SVZ of Wistar rats expressed the androgen receptor (AR). Using RT-PCR and western blots, the group showed that AR expression was most abundant in embryonic NSCs as compared to aNSCs, with peaks of expression occurring between gesta- tional days E15 to E17 [53] (Table 2). Androgen receptor also appears to be expressed in both XX and XY aNSCs isolated from the adult mouse SVZ and does not appear to show a sex difference in protein expression. Unlike the results demonstrating low/no estrogen receptor gene ex- pression in mouse eNSCs, this same study [40], along with our groups RNA-seq findings, did identify that AR was transcriptionally detectable and non-dimorphic in both male and female murine NSCs isolated from gestational day E13.5 embryos [32] (Table 2). While limited, these in- dependent studies identified that both embryonic and adult NSCs isolated from the rat and mouse express AR and therefore should be responsive to various types of androgen exposure. g These reviewed in vitro studies have shown that NSCs isolated from the adult mouse SVZ show increased pro- liferation in the presence of E2, but in vivo approaches have observed the opposite, indicating that estrogens de- crease proliferation within the SVZ [48]. When focusing on cellular propagation within the mouse DG, It was found neither sex nor estrogens stimulated adult neuro- genesis or increased cellular proliferation in the C57/ BL6 mouse strain [49]. This again shows that there are important distinctions to be made between the site of NSC isolation and the mammalian species from which they arose, grouping NSCs of different ages or from dif- ferent regions not recommended. Based on these few studies, it can be concluded that estrogen exposure on NSCs isolated from embryonic re- gions elicits altered differentiation potentials, increasing the ratios of neurons over GFAP expressing glia cell types. This effect appears to be restricted to embryonic stages, as estrogen stimulation on aNSCs in vitro did not have the same outcome, an effect also observed within the adult DG post-estrogen treatment [50]. Conclusions of the effects of estrogens on NSCs Despite a growing body of evidence now indicating that estrogen stimulation can have dramatic epigenetic ef- fects within the brain [8, 51], no study to date has dir- ectly assessed these effects on neural stem cells or progenitor cells within the brain. Considering that these cells respond to estrogens, these NSC in vitro models will prove to be extremely useful for the study of epigen- etic changes ensuing post-estrogen stimulation. These methods will limit the numerous variables occurring within the mammalian brain and allow for a more direct assessment of estrogen influence on epigenetic alter- ations such as DNA methylation, histone modifications, and the ensuing gene expression outcomes. Conclusions of the effects of estrogens on NSCs Conclusions of the effects of estrogens on NSCs Prior to these in vitro applications using NSCs, groups have demonstrated the effects of sex and estrogens on eliciting both cellular proliferation and increased neuro- genesis within the adult rodent brain, predominantly within the DG region of the hippocampus [23]. In vivo assessments have shown that cycling female adult SD rats display altered states of cellular proliferation within the DG, in accordance with various time points during the estrous cycle. Increases of cell proliferation were ob- served in the DG during proestrus, when circulating levels of estrogens were at their peak and subsequently declined during estrus, when estrogens were at lower Bramble et al. Biology of Sex Differences (2019) 10:28 Bramble et al. Biology of Sex Differences (2019) 10:28 Page 6 of 10 Page 6 of 10 Page 6 of 10 levels [44]. However, if cellular proliferation is assessed prior to the onset of the estrous cycle, it appears that sex differences exist, showing a male bias increase in cel- lular proliferation within the same brain region, as mea- sured by BrdU labeling [45, 46]. These reviewed in vitro applications (Table 1) have also drawn similar conclu- sions, in at least cells isolated from embryonic stage SD rats, where E2 alone can elicit increased cellular prolifer- ation. Interestingly, these proliferation increases were not seen in adult-isolated NSCs from the SVZ, which may indicate that while estrogens elicit proliferation in the DG, the same events may not occur within the SVZ. Additionally, the same strain of rat was not used for all studies, and data indicates that cellular proliferation within the DG is variable between strains [45], a factor that should be taken into consideration. In vitro evidence also supports that both doses of estrogen as well as other modulators such as EGF and FGF contrib- ute to the observed effects, as E2 in the presence of such mitogens actually causes a reduction in proliferation. Similar outcomes have also been observed in proliferat- ing cells within the DG, where E2 dosage was a contrib- uting factor, as well as the presence of other variables, such as progesterone, which essentially reverses the in- creased proliferation effects of estrogen [47]. In vitro effects of androgenic compounds on NSCs Androgen receptor expression However, as noted, not all groups observed this difference in neuron/ glia outcome; those that did not detect a pro-neuronal outcome as a result of estrogenic stimulation did detect an increase in oligodendrocyte differentiation. The effects of androgens on NSC cellular proliferation When both embryonic and rat aNSCs were cultured in the presence of the androgen nandrolone (19-nortestosterone), along with the mitogen EGF, proliferation was decreased by 30% in eNSCs and 20% in aNSCs (Fig. 3a) (Table 2). However, if cultured in the absence of EGF with nandro- lone, there was roughly a 7% increase in proliferation of eNSCs, but this effect was not observed in adult female- derived NSC populations (Fig. 3c). It was determined that these alterations in proliferation were modulated in part by androgen receptor action, as treatment with the AR antag- onist flutamide quelled some, but not all of nandrolone’s Generally speaking, both the in vivo studies and in vivo studies have drawn similar conclusions when asses- sing the effects of estrogen stimulation on NSCs in cul- ture or progenitor cells within the adult rodent brain. Bramble et al. Biology of Sex Differences (2019) 10:28 Page 7 of 10 Table 2 Summary of in vitro studies assessing androgenic effects on neural stem cells Table 2 Summary of in vitro studies assessing androgenic effects on neural stem cells Table 2 Summary of in vitro studies assessing androgenic effects on neural stem cells effects [53] (Fig. 3a) (Table 2). Again, the authors probed for differences in various cell-cycle regulators that may be responsible for proliferation changes as a result of andro- gen exposure and determined that unlike their findings using E2 [38], CDK P-21 was not upregulated as a result. As concluded by the group, despite similar proliferation differences in response to E2 and nandrolone in the pres- ence of EGF, the pathways responsible for such outcomes appear to be modulated by different mechanisms of action. This demonstrates that the conversion of T into E2 via aromatase can also modulate cellular proliferation in XY but not XX cells, highlighting the fact that male and female adult NSCs have differential response mechanisms when exposed to specific hormones. These two studies indicate that androgenic compounds can have significant effects on NSC proliferation, albeit in opposite directions, perhaps again revealing that NSCs isolated from different species respond differently to stimulations by sex-steroid hor- mones. In vitro effects of androgenic compounds on NSCs Androgen receptor expression Cellular proliferation also appears to be increased in human-derived XY neural stem cells (hNSCs), when treated with the more potent androgen, dihydrotestoster- one (DHT). The mechanism of action to increase prolifera- tion in hNSCs is AR-dependent, and it was demonstrated that when siRNA was used to ablate AR signaling, prolifer- ation differences were not observed in the presence of DHT [54]. It remains unclear if there are sex differences in this hNSC response, as this particular study utilized only the XY line for proliferation experimentation (Table 2). After measuring the effects of various concentration of testosterone (T) on the proliferation of murine aNSCs, Ransome et.al demonstrated that while 1 nM T did not produce proliferative differences, 10 and 50 nM of T sig- nificantly increased proliferation of XX and XY aNSCs, even in the presence of both EGF and FGF-2 (Fig. 3a) (Table 2). The increased proliferation as a result of T exposure was determined to result from Erk phosphoryl- ation induced by MEK-1, as sex hormone influences were ablated in the presence of U0126, a MEK1 phosphorylation inhibitor [30]. This particular study also found interesting sex differences in how testosterone elicited these responses in aNSCs. It was determined that while T induced prolifer- ation in both sexes, XX aNSC growth was inhibited by flu- tamide, but XY aNSCs were still capable of increased proliferation even when AR was actively subdued [30]. The effects of androgen exposure on the transcriptome and epigenome of NSCs Findings published by our group aimed to explain many of the unanswered questions regarding the development of the sex differences in the brain and the role of andro- gens, mainly testosterone on such events. To model the organization of the prenatal brain, we assessed the global transcriptional and epigenetic changes that occur as a re- sult of testosterone propionate (TP) exposure on murine eNSCs. We determined that 20 nM of TP resulted in 2854 transcriptional differences on a XX background, and 792 gene transcript expression differences in XY eNSCs, using a false discovery rate of 10% (FDR = 0.10) [32]. While TP had more robust effects on a XX genetic background, 600 of these differentially expressed transcripts were mutually shared between the XX and XY cells, indicating that there are both sex-chromosome independent as well as dependent effects of TP exposure on eNSCs gene tran- scription (Table 2). Interestingly, it was recently shown that the more potent form of testosterone, dihydrotestos- terone (DHT), exposure on human-derived NSCs also elicited differences in gene expression. Many of the observed changes in transcription post-DHT exposure on a human XY background were enriched in genes that have been associated with autism spectrum disorders [54]. These changes in gene expression were ablated when AR was inhibited, indicating that the observations were due to direct androgen signaling. In addition to detecting gene expression differences as a result of androgen exposure, TP significantly reduced global levels of 5-methylcytosine during active exposure, an epigenetic modification that was apparently transmissible to daughter cells in the ab- sence of androgen. Another epigenetic modification that showed androgen sensitivity was acetylation levels of his- tone tails, which were found to be modified in a sex- dependent manner [32] (Table 2). Although additional studies will be necessary to solidify these findings, these data indicate that androgens not only have the capacity to Differentiation outcomes appear similar to those found with estrogen stimulation, showing that androgenic treatment can increase the ratio of neurons to glia dur- ing cultured differentiation of embryonic rat NSCs and adult murine NSCs from the SVZ (Fig. 3b). Despite in vivo studies showing that androgen did not increase pro- liferation in the DG, prolonged androgen exposure did increase neurogenesis by enhancing the survival of adult-born neurons within this region [55]. Conclusions Unlike studies involving estrogen stimulation on NSCs, very few studies have demonstrated the effects of andro- gens on the same cells. The limited evidence supports that androgens can induce proliferation of embryonic rat NSCs in the absence of growth mitogens; however, if present in culture, androgen stimulation reduces prolif- eration (Fig. 3a). This effect was not observed in adult- isolated NSCs from the rat SVZ, indicating that andro- gen stimulation on embryonic populations are markedly different than when assayed using adult NSCs (Fig. 3a) In vivo studies have also found that that androgenic stimulation does not increase proliferation in the DG of adult rats nor does there appear to be AR expression within that region [55, 56]. This indicates that AR is expressed and responsive to androgens within adult cells isolated from the SVZ, however, not from other neuro- genic niches such as the DG. The same also appears to be true for NSCs isolated from the SVZ from the mouse, where testosterone exposure can increase proliferation in both XX and XY NSCs. The effects of androgens on NSC differentiation Like E2, it appears that androgens also have a pro-neuronal influence during differentiation of embry- onic NSCs from the rat and adult NSCs from the mouse. Due to lack of experimental evidence, it remains unclear if the same differentiation outcome occurs in embryoni- cally derived murine NSCs. The effects of androgens on NSC differentiation The groups that have assessed proliferative effects of tes- tosterone on NSCs also addressed the role of androgens on differential outcomes, post-NSC differentiation. If nandrolone was present during a 5-day differentiation Fig. 3 a General trends for cellular proliferation outcomes when NSCs are treated with androgens and various growth factors. b General observed trends for final cellular outcomes when NSCs are treated with androgenic stimulation during differentiation, highlighting indicates the cell types that were preferentially favored post-treatment with androgenic compounds drogens and various growth factors. b General observed trends rentiation highlighting indicates the cell types that were Fig. 3 a General trends for cellular proliferation outcomes when NSCs are treated with androgens and various growth factors. b General observed trends for final cellular outcomes when NSCs are treated with androgenic stimulation during differentiation, highlighting indicates the cell types that were preferentially favored post-treatment with androgenic compounds Bramble et al. Biology of Sex Differences (2019) 10:28 Page 8 of 10 greatly influence gene transcription, but also various epigenetic modifications within murine eNSCs. process of rat eNSCs, the proportion of βIII tubulin- expressing neurons was higher than GFAP-expressing cells, an effect not seen in adult NSCs (Fig. 3b) (Table 2) . This effect was ablated when flutamide was present, in- dicating that this effect was modulated by androgens binding AR [53]. The Ransome et.al study also showed that the presence of 10 nM T during a 2-day murine aNSC differentiation resulted in an increase of βIII tubulin-positive cells, in both XX and XY lines [30] (Fig. 3b). Like E2, it appears that androgens also have a pro-neuronal influence during differentiation of embry- onic NSCs from the rat and adult NSCs from the mouse. Due to lack of experimental evidence, it remains unclear if the same differentiation outcome occurs in embryoni- cally derived murine NSCs. process of rat eNSCs, the proportion of βIII tubulin- expressing neurons was higher than GFAP-expressing cells, an effect not seen in adult NSCs (Fig. 3b) (Table 2) . This effect was ablated when flutamide was present, in- dicating that this effect was modulated by androgens binding AR [53]. The Ransome et.al study also showed that the presence of 10 nM T during a 2-day murine aNSC differentiation resulted in an increase of βIII tubulin-positive cells, in both XX and XY lines [30] (Fig. 3b). Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 24. Chen Z, Ye R, Goldman SA. Testosterone modulation of angiogenesis and neurogenesis in the adult songbird brain. Neuroscience. 2013;239:139–48. Received: 3 January 2019 Accepted: 13 May 2019 Received: 3 January 2019 Accepted: 13 May 2019 Received: 3 January 2019 Accepted: 13 May 2019 25. Doupe AJ. Songbirds and adult neurogenesis: a new role for hormones. Proc Natl Acad Sci U S A. 1994;91(17):7836–8. Acknowledgements Not applicable 16. Kornblum HI. Introduction to neural stem cells. Stroke. 2007;38(2 Suppl):810–6. 16. Kornblum HI. Introduction to neural stem cells. Stroke. 2007;38(2 Suppl):810–6 The effects of androgen exposure on the transcriptome and epigenome of NSCs Our group’s findings indicating that testosterone exposure can sig- nificantly reduce DNA methylation and alter histone tail acetylation within murine eNSCs indicate that andro- genic exposure also has significant consequences on the NSC epigenome. While in vivo findings have shown that testosterone can alter DNA methylation [7] in gross brain regions, our group showed that these effects are also observed in cells at early time points of brain development. This raises the possibility that early androgen exposure may elicit significant developmental, as well as behavioral, outcomes by reprogramming the epigenome of NSCs. These outcomes may contribute significantly to normal male behavioral development, as XY rodents with in- active AR (Tfm model) display reduced male-typical ag- gressive and sexual behaviors in adulthood, despite functional estrogen receptors [57]. Though still in the very early stages, it is likely that deeper investigations into the roles of estrogens and androgens on neural stem cells will significantly improve our knowledge of sex steroid hormone response on the embryonic and adult Bramble et al. Biology of Sex Differences (2019) 10:28 Bramble et al. Biology of Sex Differences Page 9 of 10 Page 9 of 10 brains. These future investigations will likely lead to a more complete and comprehensive understanding of the early mechanisms behind hormonal organization of the mammalian brain and some of the factors that lead to sex differences in neuro-psychiatric disease. 10. Bramble MS, Lipson A, Vashist N, Vilain E. Effects of chromosomal sex and hormonal influences on shaping sex differences in brain and behavior: lessons from cases of disorders of sex development. J Neurosci Res. 2017; 95(1–2):65–74. 11. Reddy RC, Amodei R, Estill CT, Stormshak F, Meaker M, Roselli CE. Effect of testosterone on neuronal morphology and neuritic growth of fetal lamb hypothalamus-preoptic area and cerebral cortex in primary culture. PLoS One. 2015;10(6):e0129521. Funding 19. Hsieh J. Orchestrating transcriptional control of adult neurogenesis. Genes Dev. 2012;26(10):1010–21. Not applicable Author contributions 17. Shen Q, Wang Y, Kokovay E, Lin G, Chuang SM, Goderie SK, Roysam B, Temple S. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell. 2008;3(3):289–300. MSB and NV conceived, designed, and wrote the manuscript; EV oversaw the final manuscript and content for publication. All authors read and approved the final manuscript. 18. Tavazoie M, Van der Veken L, Silva-Vargas V, Louissaint M, Colonna L, Zaidi B, Garcia-Verdugo JM, Doetsch F. A specialized vascular niche for adult neural stem cells. Cell Stem Cell. 2008;3(3):279–88. Consent for publication Consent for publication Consent from all authors to publish was received. 23. Galea LA, Wainwright SR, Roes MM, Duarte-Guterman P, Chow C, Hamson DK. Sex, hormones and neurogenesis in the hippocampus: hormonal modulation of neurogenesis and potential functional implications. J Neuroendocrinol. 2013;25(11):1039–61. Availability of data and materials Not applicable 20. Urbán N, Guillemot F. Neurogenesis in the embryonic and adult brain: same regulators, different roles. Front Cell Neurosci. 2014;8:396. 21. Gage FH, Temple S. Neural stem cells: generating and regenerating the brain. Neuron. 2013;80(3):588–601. Abbreviations 12. Spritzer MD, Galea LA. Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats. Developmental neurobiology. 2007;67(10):1321–33. aNSCs: Adult neural stem cells; AR: Androgen receptor; CNS: Central nervous system; DCX: Doublecortin; DG: Dentate gyrus; DHT: Dihydrotestosterone; E2: 17β-estradiol; EGF: Epidermal growth factor; eNSCs: Embryonic neural stem cells; ERα: Estrogen receptor alpha; ERβ: Estrogen receptor beta; FGF: Fibroblast growth factor; GAP43: GAP43 growth associated protein 43; GFAP: Glial fibrillary acidic protein; MAP2: Microtubule-associated protein2; NSCs: Neural stem cells; SGZ: Sub-granular zone; SVZ: Sub-ventricular zone; T: Testosterone; TP T t t i t 13. Woolley CS. Acute effects of estrogen on neuronal physiology. Annu Rev Pharmacol Toxicol. 2007;47:657–80. 14. Gu F, Hata R, Toku K, Yang L, Ma YJ, Maeda N, Sakanaka M, Tanaka J. Testosterone up-regulates aquaporin-4 expression in cultured astrocytes. J Neurosci Res. 2003;72(6):709–15. TP: Testosterone propionate 15. McCarthy MM, Wright CL. Convergence of sex differences and the neuroimmune system in autism spectrum disorder. Biol Psychiatry. 2017; 81(5):402–10. References 26. Wingfield JC. Historical contributions of research on birds to behavioral neuroendocrinology. Horm Behav. 2005;48(4):395–402. 1. Phoenix CH, Goy RW, Gerall AA, Young WC. Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology. 1959;65:369–82. 27. Goodson JL, Saldanha CJ, Hahn TP, Soma KK. Recent advances in behavioral neuroendocrinology: insights from studies on birds. Horm Behav. 2005;48(4):461–73. behavior in the female guinea pig. Endocrinology. 1959;65:369–82. 2. Arnold AP. The organizational-activational hypothesis as the foundation for a unified theory of sexual differentiation of all mammalian tissues. Horm Behav. 2009;55(5):570–8. 28. Roselli CE, Liu M, Hurn PD. Brain aromatization: classical roles and new perspectives. Semin Reprod Med. 2009;27(3):207–17. 3. Ngun TC, Ghahramani N, Sanchez FJ, Bocklandt S, Vilain E. The genetics of sex differences in brain and behavior. Front Neuroendocrinol. 2011;32(2): 227–46. 29. Boon WC, Chow JD, Simpson ER. The multiple roles of estrogens and the enzyme aromatase. Prog Brain Res. 2010;181:209–32. 30. Ransome MI, Boon WC. Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression. Front Cell Neurosci. 2015;9:253. 4. Ngun TC, Ghahramani NM, Creek MM, Williams-Burris SM, Barseghyan H, Itoh Y, Sanchez FJ, McClusky R, Sinsheimer JS, Arnold AP, et al. Feminized behavior and brain gene expression in a novel mouse model of Klinefelter syndrome. Arch Sex Behav. 2014;43(6):1043–57. 31. Waldron J, McCourty A, Lecanu L. Neural stem cell sex dimorphism in aromatase (CYP19) expression: a basis for differential neural fate. Stem Cells Cloning. 2010;3:175–82. 5. Arnold AP, Chen X. What does the “four core genotypes” mouse model tell us about sex differences in the brain and other tissues? Front Neuroendocrinol. 2009;30(1):1–9. 32. Bramble MS, Roach L, Lipson A, Vashist N, Eskin A, Ngun T, Gosschalk JE, Klein S, Barseghyan H, Arboleda VA, et al. Sex-specific effects of testosterone on the sexually dimorphic transcriptome and epigenome of embryonic neural stem/progenitor cells. Sci Rep. 2016;6:36916. 6. Arnold AP. A general theory of sexual differentiation. J Neurosci Res. 2017; 95(1–2):291–300. 7. Ghahramani NM, Ngun TC, Chen P-Y, Tian Y, Krishnan S, Muir S, Rubbi L, Arnold AP, de Vries GJ, Forger NG, et al. The effects of perinatal testosterone exposure on the DNA methylome of the mouse brain are late-emerging. Biol Sex Differ. 2014;5:8–8. 7. Ghahramani NM, Ngun TC, Chen P-Y, Tian Y, Krishnan S, Muir S, Rubbi L, Arnold AP, de Vries GJ, Forger NG, et al. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable 22. Mahmoud R, Wainwright SR, Galea LA. Sex hormones and adult hippocampal neurogenesis: regulation, implications, and potential mechanisms. Front Neuroendocrinol. 2016;41:129–52. References The effects of perinatal testosterone exposure on the DNA methylome of the mouse brain are late-emerging. Biol Sex Differ. 2014;5:8–8. 33. Bennett NC, Gardiner RA, Hooper JD, Johnson DW, Gobe GC. Molecular cell biology of androgen receptor signalling. Int J Biochem Cell Biol. 2010;42(6):813–27. 34. Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M, Strom A, Treuter E, Warner M, et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev. 2007;87(3):905–31. 8. McCarthy MM, Nugent BM. At the frontier of epigenetics of brain sex differences. Front Behav Neurosci. 2015;9:221. 8. McCarthy MM, Nugent BM. At the frontier of epigenetics of brain sex differences. Front Behav Neurosci. 2015;9:221. 9. Nugent BM, Wright CL, Shetty AC, Hodes GE, Lenz KM, Mahurkar A, Russo SJ, Devine SE, McCarthy MM. Brain feminization requires active repression of masculinization via DNA methylation. Nat Neurosci. 2015;18(5):690–7. 9. Nugent BM, Wright CL, Shetty AC, Hodes GE, Lenz KM, Mahurkar A, Russo SJ, Devine SE, McCarthy MM. Brain feminization requires active repression of masculinization via DNA methylation. Nat Neurosci. 2015;18(5):690–7. 35. Ohtsuka T, Shimojo H, Matsunaga M, Watanabe N, Kometani K, Minato N, Kageyama R. Gene expression profiling of neural stem cells and Page 10 of 10 Page 10 of 10 Bramble et al. Biology of Sex Differences (2019) 10:28 extranuclear sites in the rat hippocampal formation. Neuroscience. 2005; 130(1):151–63. identification of regulators of neural differentiation during cortical development. Stem Cells. 2011;29(11):1817–28. identification of regulators of neural differentiation during cortical development. Stem Cells. 2011;29(11):1817–28. identification of regulators of neural differentiation during cortical development. Stem Cells. 2011;29(11):1817–28. 57. Sato T, Matsumoto T, Kawano H, Watanabe T, Uematsu Y, Sekine K, Fukuda T, Aihara K, Krust A, Yamada T, et al. Brain masculinization requires androgen receptor function. Proc Natl Acad Sci U S A. 2004;101(6):1673–8. 57. Sato T, Matsumoto T, Kawano H, Watanabe T, Uematsu Y, Sekine K, Fukuda T, Aihara K, Krust A, Yamada T, et al. Brain masculinization requires androgen receptor function. Proc Natl Acad Sci U S A. 2004;101(6):1673–8. 36. Reekmans K, Praet J, Daans J, Reumers V, Pauwels P, Van der Linden A, Berneman ZN, Ponsaerts P. Current challenges for the advancement of neural stem cell biology and transplantation research. Stem Cell Rev. 2012; 8(1):262–78. 37. Waldron J, McCourty A, Lecanu L. Aging differentially affects male and female neural stem cell neurogenic properties. Stem Cells Cloning. References 2010; 3:119–27. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 38. Brannvall K, Korhonen L, Lindholm D. Estrogen-receptor-dependent regulation of neural stem cell proliferation and differentiation. Mol Cell Neurosci. 2002;21(3):512–20. 39. Zhang L, Yulong M, Qin P, Deng Y, Zhang Z, et al. The effects of various estrogen doses on the proliferation and differentiation of cultured neural stem cells. J Cell Sci Ther. 2016;7:247. https://doi.org/10.4172/2157-7013. 1000247. 40. Androutsellis-Theotokis A, Chrousos GP, McKay RD, DeCherney AH, Kino T. Expression profiles of the nuclear receptors and their transcriptional coregulators during differentiation of neural stem cells. Horm Metab Res. 2013;45(2):159–68. 41. Okada M, Murase K, Makino A, Nakajima M, Kaku T, Furukawa S, Furukawa Y. Effects of estrogens on proliferation and differentiation of neural stem/ progenitor cells. Biomed Res. 2008;29(3):163–70. 42. Okada M, Makino A, Nakajima M, Okuyama S, Furukawa S, Furukawa Y. Estrogen stimulates proliferation and differentiation of neural stem/ progenitor cells through different signal transduction pathways. Int J Mol Sci. 2010;11(10):4114–23. 43. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol. 1999;208(1):166–88. 44. Tanapat P, Hastings NB, Reeves AJ, Gould E. Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. J Neurosci. 1999;19(14):5792–801. 45. Perfilieva E, Risedal A, Nyberg J, Johansson BB, Eriksson PS. Gender and strain influence on neurogenesis in dentate gyrus of young rats. J Cereb Blood Flow Metab. 2001;21(3):211–7. 46. Bowers JM, Waddell J, McCarthy MM. A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol. Biol Sex Differ. 2010;1(1):8. 47. Tanapat P, Hastings NB, Gould E. Ovarian steroids influence cell proliferation in the dentate gyrus of the adult female rat in a dose- and time-dependent manner. J Comp Neurol. 2005;481(3):252–65. 48. Brock O, Keller M, Veyrac A, Douhard Q, Bakker J. Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice. Neuroscience. 2010;166(2):368–76. 49. Lagace DC, Fischer SJ, Eisch AJ. Gender and endogenous levels of estradiol do not influence adult hippocampal neurogenesis in mice. Hippocampus. 2007;17(3):175–80. 50. Barker JM, Galea LA. Repeated estradiol administration alters different aspects of neurogenesis and cell death in the hippocampus of female, but not male, rats. Neuroscience. Publisher’s Note S N 2008;152(4):888–902. 51. McCarthy MM, Auger AP, Bale TL, De Vries GJ, Dunn GA, Forger NG, Murray EK, Nugent BM, Schwarz JM, Wilson ME. The epigenetics of sex differences in the brain. J Neurosci. 2009;29(41):12815–23. 52. Huang CK, Luo J, Lee SO, Chang C. Concise review: androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal, and hematopoietic lineages. Stem Cells. 2014;32(9):2299–308. 53. Brannvall K, Bogdanovic N, Korhonen L, Lindholm D. 19-Nortestosterone influences neural stem cell proliferation and neurogenesis in the rat brain. Eur J Neurosci. 2005;21(4):871–8. 54. Quartier A, Chatrousse L, Redin C, Keime C, Haumesser N, Maglott-Roth A, Brino L, Le Gras S, Benchoua A, Mandel JL, et al. Genes and pathways regulated by androgens in human neural cells, potential candidates for the male excess in autism spectrum disorder. Biol Psychiatry. 2018;84(4):239–52. https://doi.org/10.1016/j.biopsych.2018.01.002. Epub 2018 Jan 9. 55. Hamson DK, Wainwright SR, Taylor JR, Jones BA, Watson NV, Galea LA. Androgens increase survival of adult-born neurons in the dentate gyrus by an androgen receptor-dependent mechanism in male rats. Endocrinology. 2013;154(9):3294–304. 56. Tabori NE, Stewart LS, Znamensky V, Romeo RD, Alves SE, McEwen BS, Milner TA. Ultrastructural evidence that androgen receptors are located at

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