Recently, the European Union published regulations regarding the welfare of horses during transport requiring that horses be transported in individual stalls separated by partitions. The objective of this study was to determine if concentrations of cortisol, corticosterone, or dehydroepiandrosterone (DHEA) differed among horses with no prior transport experience when transported in individual stalls versus loose groups. Twenty na�ve yearlings were assigned to either individual stalls or a loose group, then transported for 6 hours. Ten horses were transported per day (5 in stalls and 5 in a loose group) over a two day trial. The experiment was replicated with a second trial 35 days later, and utilized a switchback design where the horses exchanged treatments between trials. Blood samples were collected and analyzed for cortisol, corticosterone, and DHEA concentrations at pre-transport, 2, 4, and 6 h of transport, and at 2 and 4 h after unloading. After Trial 2, the horses? changed housing from group paddocks to stalls and a follow-up experiment was conducted. The data were analyzed using a mixed model repeated measures ANOVA with the animal as the subject, with trial, treatment, sample time, and treatment-sample time interaction in the model with unstructured covariance (SAS 9.1). Differences between sample times within each trial, and pre-transport concentrations between trials, were analyzed using paired t-tests (SPSS 12.0.1). No significant differences were found in hormone concentrations for horses transported in individual stalls versus in loose groups. Horses exhibited a significant elevation in cortisol and corticosterone during transport which returned to pre-transport concentrations by 2 hr after transport (P < 0.01). Mean pre-transport cortisol concentrations rose significantly in Trial 3 (7.87 ng/ml) from Trials 1 (2.71 ng/ml) and 2 (2.84 ng/ml) (P < 0.001). Pre-transport concentrations of DHEA in Trials 1 (482 pg/ml) and 2 (392 pg/ml) also rose significantly in Trial 3 (1607 pg/ml) (P < 0.01). Changes in cortisol and DHEA indicated that transportation was a significant stressor for horses, however, being transported in a loose group versus individual stalls was not different. Also, housing changes from paddocks to stalls resulted in significant increases in pre-transport concentrations of stress-related compounds.

Recently, the European Union published regulations regarding the welfare of horses during transport requiring that horses be transported in individual stalls separated by partitions. The objective of this study was to determine if concentrations of cortisol, corticosterone, or dehydroepiandrosterone (DHEA) differed among horses with no prior transport experience when transported in individual stalls versus loose groups. Twenty na�ve yearlings were assigned to either individual stalls or a loose group, then transported for 6 hours. Ten horses were transported per day (5 in stalls and 5 in a loose group) over a two day trial. The experiment was replicated with a second trial 35 days later, and utilized a switchback design where the horses exchanged treatments between trials. Blood samples were collected and analyzed for cortisol, corticosterone, and DHEA concentrations at pre-transport, 2, 4, and 6 h of transport, and at 2 and 4 h after unloading. After Trial 2, the horses? changed housing from group paddocks to stalls and a follow-up experiment was conducted. The data were analyzed using a mixed model repeated measures ANOVA with the animal as the subject, with trial, treatment, sample time, and treatment-sample time interaction in the model with unstructured covariance (SAS 9.1). Differences between sample times within each trial, and pre-transport concentrations between trials, were analyzed using paired t-tests (SPSS 12.0.1). No significant differences were found in hormone concentrations for horses transported in individual stalls versus in loose groups. Horses exhibited a significant elevation in cortisol and corticosterone during transport which returned to pre-transport concentrations by 2 hr after transport (P < 0.01). Mean pre-transport cortisol concentrations rose significantly in Trial 3 (7.87 ng/ml) from Trials 1 (2.71 ng/ml) and 2 (2.84 ng/ml) (P < 0.001). Pre-transport concentrations of DHEA in Trials 1 (482 pg/ml) and 2 (392 pg/ml) also rose significantly in Trial 3 (1607 pg/ml) (P < 0.01). Changes in cortisol and DHEA indicated that transportation was a significant stressor for horses, however, being transported in a loose group versus individual stalls was not different. Also, housing changes from paddocks to stalls resulted in significant increases in pre-transport concentrations of stress-related compounds.

Com o envelhecimento observa-se redução da massa muscular, aumento da resistência à insulina e queda nos níveis séricos de alguns hormônios, entre eles o DHEA. Ratos com 12-14 meses de idade apresentam regulação músculo-específica em vias da insulina e IGF 1 em músculos com populações distintas de tipos de fibras e como indicador de sarcopenia apresentam redução da proporção e tamanho de fibras tipo 2b. Dose única de DHEA não modula as vias da insulina e IGF 1 no músculo esquelético e reduz tamanho de fibras tipo 1 no EDL. / Reductions in skeletal muscle mass, increased insulin resistance, and decrements in several hormones serum levels, including DHEA, are features observed in aging. Twelve to fourteen months old rats present specific regulation in the insulin and the IGF-1 intracellular pathway in distinct skeletal muscle fiber composition and show reductions in size and proportion of muscle composed predominantly by fiber type 2. DHEA treatment had no effect in these intracellular pathways but induces reduction in the size of type 1 fiber in the EDL muscle.

Made available in DSpace on 2014-12-17T15:36:56Z (GMT). No. of bitstreams: 1 RodrigoPAF.pdf: 243624 bytes, checksum: 7ee4c45834008c3d9373cfa3bf41f6cf (MD5) Previous issue date: 2009-04-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Fibromyalgia (FM) is a non-inflammatory rheumatic syndrome characterized by widespread musculoskeletal pain with palpable tender points, muscle stiffness, fatigue, and sleep disturbances. Patients with FM have hormonal changes that are directly correlated with symptoms of the syndrome. The neuroendocrine regulation may be impaired, with abnormalities in the hypothalamus-pituitary-adrenal (HPA) axis with various hormones showing changes in their levels. In women in fertile period, various gonadal hormones are associated with symptoms of the syndrome, but studies focusing only a population of women in post-menopausal period who do not use hormone replacement are rare. We developed an analytical cross sectional study to assess the plasma levels of cortisol and dehidroepiandrosterona sulfate (DHEA-S) with quimioluminescence method in a group of 17 women with FM and 19 healthy women in post-menopause who do not use hormone replacement and observe the correlation with the symptoms of pain through algometry, depression and physical functional capacity measured from the Beck Depression Index (BDI) and the Fibromyalgia Impact Questionnaire (FIQ). Three blood samples were collected in the morning (between 8:00 9:30) with an interval of 24 hours for the measurements of hormonal levels and biochemical profile. There were no immunological or lipid changes in patients with FM. Comparing the two groups, there is no difference in levels of cortisol and a tangential effect for DHEA-S (p=0,094) with the lowest levels in the FM. DHEA-S also correlated with pain threshold (r=0,7) and tolerance (r=0,65) in group FM. We found the presence of depressive state and low physical functional capacity in FM. It was also evident that women in post-menopausal period, DHEA-S should influence the symptoms of increased sensitivity to pain, but not the presence of depressive status and low physical functional / A fibromialgia (FM) ? uma s?ndrome reum?tica n?o inflamat?ria caracterizada por fadiga, dist?rbios do sono e dor muscular difusa com presen?a de s?tios dolorosos a palpa??o denominados Terder Points. Pacientes com FM apresentam altera??es hormonais que est?o diretamente correlacionados com os sintomas da s?ndrome. A regula??o neuroend?crina pode ser considerada prejudicada, sendo encontradas anormalidades no eixo hipot?lamo-pituit?ria-adrenal (HPA) com diversos horm?nios apresentando altera??es em seus n?veis. Em mulheres no per?odo f?rtil, diversos horm?nios gonadais est?o associados com os sintomas da s?ndrome, por?m estudos enfatizando unicamente uma popula??o de mulheres no per?odo p?s-menopausa que n?o fazem uso de reposi??o hormonal s?o raros. Com base nesse fato foi desenvolvido este estudo anal?tico transversal para avaliar os n?veis plasm?ticos de cortisol e dehidroepiandrosterona sulfato (DHEA-S) atrav?s da quimioluminesc?ncia em um grupo de 17 mulheres com FM e 19 mulheres saud?veis e observar se h? correla??o com os sintomas de dor atrav?s da algometria, estado de humor com o ?ndice de Depress?o de Beck (BDI) e grau de funcionalidade com o Question?rio de Impacto de FM (Fibromyalgia Impact Questionnaire FIQ). Foram realizadas tr?s coletas de sangue com um intervalo de 24 horas entre elas, no per?odo da manh? para as dosagens hormonais e estudo do perfil bioqu?mico das pacientes. N?o foram observadas altera??es imunol?gicas ou lip?dicas nas pacientes com FM. Quando comparados os dois grupos, n?o houve diferen?a significativa nos n?veis de cortisol, mas foi encontrado um efeito tangencial para o DHEA-S (p=0,094) com n?veis mais baixos no grupo FM. O DHEA-S apresentou correla??o significativa positiva com o limiar (r=0,7) e a toler?ncia (r=0,65) ? dor no grupo FM. Foi encontrada presen?a de estado depressivo e baixa funcionalidade no grupo FM. Estes resultados sugerem que nas mulheres no per?odo p?s-menopausa o DHEA-S deve influenciar os sintomas de maior sensibilidade ? dor, mas n?o a presen?a de estado depressivo e a baixa funcionalidade

Com o envelhecimento observa-se redução da massa muscular, aumento da resistência à insulina e queda nos níveis séricos de alguns hormônios, entre eles o DHEA. Ratos com 12-14 meses de idade apresentam regulação músculo-específica em vias da insulina e IGF 1 em músculos com populações distintas de tipos de fibras e como indicador de sarcopenia apresentam redução da proporção e tamanho de fibras tipo 2b. Dose única de DHEA não modula as vias da insulina e IGF 1 no músculo esquelético e reduz tamanho de fibras tipo 1 no EDL. / Reductions in skeletal muscle mass, increased insulin resistance, and decrements in several hormones serum levels, including DHEA, are features observed in aging. Twelve to fourteen months old rats present specific regulation in the insulin and the IGF-1 intracellular pathway in distinct skeletal muscle fiber composition and show reductions in size and proportion of muscle composed predominantly by fiber type 2. DHEA treatment had no effect in these intracellular pathways but induces reduction in the size of type 1 fiber in the EDL muscle.

Made available in DSpace on 2014-12-17T15:37:05Z (GMT). No. of bitstreams: 1 PauloJFC_DISSERT.pdf: 1001139 bytes, checksum: 32624b3dc04255ae3e75e4e16600e75d (MD5) Previous issue date: 2011-08-08 / A disfun??o sexual corresponde a altera??es em uma ou mais fases da resposta sexual humana e apresenta maior preval?ncia na popula??o feminina. Ademais, a participa??o de alguns fatores como obesidade e n?veis dos horm?nios esteroidais na disfun??o sexual feminina (DSF) permanece incerta. O presente estudo deteve-se na an?lise da ocorr?ncia de DSF numa popula??o de mulheres portadoras de obesidade, cadastradas no Ambulat?rio de Cirurgia Bari?trica do Hospital Universit?rio Onofre Lopes, da Universidade Federal do Rio Grande do Norte, no munic?pio de Natal, RN. O estudo foi realizado em uma amostra composta por trinta e uma mulheres, com idade entre 20 e 50 anos, com ?ndice de massa corp?rea (IMC) > 30 Kg/m2. A todas as pacientes foi aplicado um question?rio composto por uma se??o com dados socio-econ?micos, e outra abordando a sa?de sexual feminina, sendo esta ?ltima correspondente ao Female Sexual Function Index (FSFI), para diagn?stico de DSF. A partir dessa caracteriza??o, as pacientes foram reunidas nos grupos CD (pacientes com disfun??o, n= 9) e SD (sem disfun??o, n= 22). Para a an?lise do efeito da obesidade na DSF, as pacientes foram reunidas nos grupos 1 (6 pacientes com IMC grau I e II: entre 30 e 40 Kg/m2) e 2 (25 com IMC grau III: acima de 40). Para o estudo da participa??o dos horm?nios esteroidais foram determinadas as concentra??es s?ricas de cortisol, estradiol e dehidroepiandrosterona (DHEA) pelo m?todo de quimiluminesc?ncia. A an?lise estat?stica dos dados foi realizada usando os testes ANOVA, MANOVA (Pillai), al?m de an?lise de Cluster. Para identificar as diferen?as entre os dom?nios do FSFI, foi usado o teste T de Student. A signific?ncia considerada para todos os testes foi para p< 0,01. Das pacientes estudadas, 25,8% apresentaram DSF de acordo com o escore total do FSFI. A an?lise estat?stica posterior evidenciou que as diferen?as ocorreram para os dom?nios desejo, excita??o e orgasmo. N?o foi encontrada rela??o da presen?a de DSF com os diferentes graus de obesidade ou com os n?veis hormonais dos ester?ides cortisol, estradiol ou DHEA. Contudo, foi encontrado aumento significativo nos n?veis s?ricos de estradiol para o grupo 1, que corresponde ao de menor ?ndice de IMC. Estes resultados mostram que a preval?ncia de DSF n?o diferiu entre os graus I,II e III de obesidade das pacientes deste estudo mas, quando presente, a disfun??o ocorre nos dom?nios desejo, excita??o e orgasmo. A maior concentra??o de estradiol encontrada nas pacientes de menor ?ndice de IMC sugere uma poss?vel rela??o entre as duas vari?veis que precisa ser investigada em estudos futuros. / Sexual dysfunction corresponds to changes in one or more phases of human sexual response and it has major prevalence in women. At the same time, the participation of some factors such as obesity and levels of steroid hormones in female sexual dysfunction (FSD) remains uncertain. This study analyzed the occurrence of DSF in a population of obese women part of the Bariatric surgery clinic at the Onofre Lopes Academic Hospital of the Federal University of Rio Grande do Norte, Natal, RN. The study was conducted in a sample of thirty-one women, aged between 20 and 50 years with body mass index (BMI) > 30 kg/m2. All patients answered a questionnaire consisting of two sections addressing socioeconomic conditions and female sexual health, the latter corresponding to the Female Sexual Function Index (FSFI) for the diagnosis of FSD. Using this characterization the patients were divided in groups CD (nine patients with dysfunction) and SD (twenty-two patients with no sexual dysfunction). To analyze the effect of obesity on the DSF, the patients were divided in groups 1 (6 patients with obesity grade I and II: BMI between 30 and 40 kg/m2) and 2 (22 patients with obesity grade III: BMI above 40). To investigate the role of steroid hormones, serum levels were determined for cortisol, estradiol, and dehydroepiandrosterone (DHEA), using chemiluminescence method. The statistical analysis was performed using ANOVA, MANOVA (Pillai) and cluster analysis. To identify the differences between the domains of the FSFI, was used the Student t test. The significance level for all tests was p <0.01. Of the patients studied, 25.8% had DSF according to the total FSFI score. The statistical analysis showed that the differences occurred for the domains desire, arousal and orgasm. No relationship was found for the presence of DSF with different degrees of obesity and steroid hormone levels. However, was found significant increase in estradiol serum levels for group 1, which corresponds to the lowest BMI. These results show that the presence of FSD did not differ between grades I, II and III of obesity in this study, but, when present, dysfunction occurs in the domains desire, arousal and orgasm. The highest estradiol concentration found in patients with the lowest BMI suggests a possible relationship between the two variables, and should be investigated in future studies.

Le narval (Monodon monoceros) est une espèce emblématique de l'Arctique. Les narvals sont de plus en plus exposés à des perturbations anthropiques, pouvant augmenter leur niveau de stress et, par conséquent, avoir des impacts inconnus sur la dynamique de la population. La validation et l’étude de biomarqueurs de stress chronique pourraient donc améliorer les efforts de conservation chez cette espèce. La déhydroépiandrostérone (DHEA) et son métabolite sulfaté, la DHEA-S, sont collectivement appelés DHEA(S). Lorsque combinés sous forme de ratios avec le cortisol (cortisol/DHEA(S)), ils se sont révélés prometteurs dans l'évaluation du stress chronique chez les humains et certaines espèces animales domestiques ou sauvages. Au cours de projets de pose d’émetteurs en 2017 et 2018 dans la baie de Baffin, Nunavut, Canada, des narvals (n = 14) ont été échantillonnés au début et à la fin des manipulations. La DHEA(S) sérique a ensuite été mesurée à l’aide de deux techniques immuno-enzymatiques (ELISAs) développées pour les humains et disponibles commercialement. Une validation partielle des deux ELISAs a pu être réalisée par détermination du coefficient de variation intra-essai, confirmation de la linéarité de dilution de la DHEA(S) et calcul du pourcentage de récupération. La DHEA était également bien conservée dans le sérum de narvals suite à un stockage prolongé de 12 et 24 mois à -80°C, soulignant le potentiel d'analyse d'échantillons archivés. Les valeurs sériques moyennes (ng/ml ± SEM) de cortisol, de DHEA(S) et des ratios cortisol/DHEA(S) au début et à la fin des manipulations respectivement étaient les suivantes : cortisol = 30,74 ± 4,87 et 41,83 ± 4,83; DHEA = 1,01 ± 0,52 et 0,99 ± 0,50; DHEA-S = 8,72 ± 1,68 et 7,70 ± 1,02; cortisol/DHEA = 75,43 ± 24,35 et 84,41 ± 11,76; cortisol/DHEA-S = 4,16 ± 1,07 et 6,14 ± 1,00). Le cortisol sérique et le ratio cortisol/DHEA-S étaient statistiquement plus élevés à la fin des manipulations (P = 0,024 et P = 0,035 respectivement). De plus, le cortisol sérique à la fin des manipulations était positivement corrélé à la longueur totale du corps de l’animal (P = 0,042) et avait tendance à être plus élevé chez les mâles (P = 0,086). Cette étude confirme que ces ELISAs sont une méthode d’analyse facile à réaliser, rapide et appropriée pour mesurer la DHEA(S) sérique chez le narval. La DHEA(S) sérique et les ratios cortisol/DHEA(S) représentent des biomarqueurs potentiels pour évaluer le stress chronique chez les narvals et possiblement chez d'autres espèces de cétacés. / Narwhals (Monodon monoceros) are an iconic Arctic species and are increasingly being exposed to anthropogenic disturbances that may increase their stress levels with unknown consequences for the overall population dynamics. The validation and measurement of chronic stress biomarkers could contribute towards an improved understanding and conservation efforts for this species. Dehydroepiandrosterone (DHEA), and its sulfated metabolite DHEA-S, are collectively referred to as DHEA(S). When serum DHEA(S) concentrations are combined in a ratio with cortisol (cortisol/DHEA(S)), it has shown promise for evaluating chronic stress in humans, domestic animals, and wildlife. During field tagging in 2017 and 2018 on Baffin Bay, Nunavut, Canada, wild narwhals (n = 14) were sampled at the beginning and end of capture-tagging procedures (acute stressor). Serum DHEA(S) were measured with commercially available competitive enzyme-linked immunosorbent assays (ELISA) developed for humans. A partial validation of the assays was performed by determination of the intra-assay coefficient of variation, confirmation of the DHEA(S) dilutional linearity, and the calculation of the percentage of recovery. In addition, DHEA was conserved following extended storage at -80°C, highlighting the potential to analyze archival samples. Mean values (ng/mL ± SEM) of narwhal serum cortisol, DHEA(S), and cortisol/DHEA(S) ratios at the beginning and at the end of handling respectively are reported (cortisol = 30.74 ± 4.87, 41.83 ± 4.83; DHEA = 1.01 ± 0.52, 0.99 ± 0.50; DHEA-S = 8.72 ± 1.68, 7.70 ± 1.02; cortisol/DHEA = 75.43 ± 24.35, 84.41 ± 11.76, and cortisol/DHEA-S = 4.16 ± 1.07, 6.14 ± 1.00). Serum cortisol and cortisol/DHEA-S were statistically higher at the end of the capture (P = 0.024 and P = 0.035 respectively). Moreover, serum cortisol at the end of handling and prior to release was positively correlated to total body length (P = 0.042) and tended to be higher in males (P = 0.086). This study showed that these assays are easy to perform, rapid, and suitable for measuring serum DHEA(S) of narwhals and that serum DHEA(S) and calculated cortisol/DHEA(S) are potential biomarkers for chronic stress in narwhals and possibly other species of cetaceans, but this requires additional study.

The comorbidity between female sexual dysfunction (FSD) and major depressive disorder (MDD) is well documented; however, the mechanism(s) underlying the relationship between these disorders has not been defined. The literature has associated the adrenal hormones cortisol and dehydroepiandrosterone (DHEA) with FSD and MDD, suggesting a biological mechanism that may elucidate the comorbidity between these disorders. Based on evidence pointing to a high cortisol/DHEA ratio (C/D Ratio) in MDD and low DHEA in FSD, this study investigated if the potential association between a high C/D Ratio and FSD would be greater for women with a history of MDD when compared to women without a history of MDD. Two groups of women (MDD history group; control group), each with a range of sexual function, collected saliva samples, completed questionnaires, and participated in a clinical interview and a psychophysiological assessment. Results did not support the hypothesis that the relationship between the C/D Ratio and sexual function would be greater for women with a history of MDD. Relevant to the effects of hormones on sexual function, a higher C/D Ratio was associated with lower frequency of sexual activity and lower sexual assertiveness. Results also showed DHEA positively associated with overall frequency of sexual activity, while cortisol was associated with lower subjective assessment of sexual desire/arousal prior to erotic stimuli. Lastly, secondary analyses revealed a positive association between DHEA and frequency of sexual activity, which was mediated by women's sexual desire. These results suggest that the effects of the C/D Ratio on FSD are not associated with a history of MDD. Results also point to contrasting roles for C/D Ratio and DHEA in FSD. In particular, a high C/D Ratio may have inhibitory effects on frequency of sexual activity and sexual assertiveness, while high DHEA may have facilitatory effects on sexual activity frequency through heightened sexual desire. Lastly, high cortisol may predispose women to have a negative assessment of sexual stimuli. These findings contribute to a further understanding of the roles of the C/D Ratio, DHEA, and cortisol in female sexuality and offer support for future studies investigating the role of these hormones in FSD.

The comorbidity between female sexual dysfunction (FSD) and major depressive disorder (MDD) is well documented; however, the mechanism(s) underlying the relationship between these disorders has not been defined. The literature has associated the adrenal hormones cortisol and dehydroepiandrosterone (DHEA) with FSD and MDD, suggesting a biological mechanism that may elucidate the comorbidity between these disorders. Based on evidence pointing to a high cortisol/DHEA ratio (C/D Ratio) in MDD and low DHEA in FSD, this study investigated if the potential association between a high C/D Ratio and FSD would be greater for women with a history of MDD when compared to women without a history of MDD. Two groups of women (MDD history group; control group), each with a range of sexual function, collected saliva samples, completed questionnaires, and participated in a clinical interview and a psychophysiological assessment. Results did not support the hypothesis that the relationship between the C/D Ratio and sexual function would be greater for women with a history of MDD. Relevant to the effects of hormones on sexual function, a higher C/D Ratio was associated with lower frequency of sexual activity and lower sexual assertiveness. Results also showed DHEA positively associated with overall frequency of sexual activity, while cortisol was associated with lower subjective assessment of sexual desire/arousal prior to erotic stimuli. Lastly, secondary analyses revealed a positive association between DHEA and frequency of sexual activity, which was mediated by women's sexual desire. These results suggest that the effects of the C/D Ratio on FSD are not associated with a history of MDD. Results also point to contrasting roles for C/D Ratio and DHEA in FSD. In particular, a high C/D Ratio may have inhibitory effects on frequency of sexual activity and sexual assertiveness, while high DHEA may have facilitatory effects on sexual activity frequency through heightened sexual desire. Lastly, high cortisol may predispose women to have a negative assessment of sexual stimuli. These findings contribute to a further understanding of the roles of the C/D Ratio, DHEA, and cortisol in female sexuality and offer support for future studies investigating the role of these hormones in FSD.

The thesis focuses on selected, yet unsolved question of the role of less common steroids and SHBG as a junction of three endocrine systems. Answering these questions may help to understand the complex mechanism of action of these hormones on the human organism. This thesis is based on five author and co-written studies mostly published in foreign scientific journals. In the case of studies of metabolites 16α-OH-DHEA and 7-oxo-DHEA (Zamrazilová et al., 2007; Kazihnitková et al., 2007) were focused on the development of appropriate methodological approach because recently used methods showed a low sensitivity and specificity. We developed and statistically evaluated new RIA methods which are rapid, sensitive and inexpensive. Both of them can be used in other research even in routine practice. New methods were also used to determine the metabolites in a statistically significant sample of healthy human population. In another study (Zamrazilová et al., 2010) we examined the relationships of selected steroid metabolites and SHBG in patients with CAH. We assumed that SHBG may act as a non-steroidal laboratory parameter reflecting the effectiveness of substitution therapy in these patients. Our assumption was not confirmed. We observed lower levels of SHBG which at least in women reflect effects of...