Vários estudos têm sugerido que o estresse pode ser um dos fatores relacionados com à grande variabilidade cognitiva observada em idosos. Esta associação se explica porque o cortisol, principal classe de hormônios do estresse em humanos, apresenta alta afinidade por receptores específicos localizados no hipocampo, amígdala e região pré-frontal, estruturas associadas ao aprendizado e à memória. Concentrações cronicamente elevadas de cortisol estão associadas à atrofia hipocampal e baixo desempenho cognitivo. Entretanto, o efeito do estresse agudo no desempenho da memória ainda se encontra inconclusivo em idosos. Isto é particularmente relevante, uma vez que, idosos com comprometimento cognitivo patológico apresentam concentração elevada de cortisol, que por sua vez, está associada com rápida progressão da doença. Assim, o objetivo do estudo foi analisar a relação entre desempenho da memória e resposta neuroendócrina e cardiovascular de estresse em idosos saudáveis. Foram selecionados aleatoriamente 100 idosos alfabetizados, predominantemente do sexo feminino, sem prejuízo cognitivo e funcional, moradores da cidade de São Paulo. A resposta neuroendócrina de estresse foi avaliada a partir concentração de cortisol salivar enquanto que a reação cardiovascular a partir da pressão arterial e frequência cardíaca antes, durante e após a exposição do participante a um estressor psicossocial agudo (Trier Social Stress Test TSST). O TSST envolve duas tarefas: falar em público e realizar cálculos aritméticos mentalmente diante de uma banca examinadora. O desempenho da memória foi avaliado mediante aplicação do teste Pares de Palavras (PP) 20 minutos antes do TSST para evocação imediata e aprendizado e 15 minutos após o fim do TSST para evocação tardia. Foi observado aumento de 96% na concentração de cortisol 15 minutos após o TSST, bem como elevação da pressão arterial em relação à situação basal. Ademais, observamos redução significativa do escore do teste PP após o TSST e correlação negativa entre concentração de cortisol, evocação imediata e tardia dos PP. Os resultados revelam influência do estresse agudo no desempenho da memória, particularmente da evocação tardia, de idosos, destacando a vulnerabilidade destes indivíduos aos efeitos neurotóxicos do cortisol na memória e, consequentemente ao desenvolvimento de transtornos cognitivos. / Several studies have suggested that stress may be a factor related to cognitive variability observed in the elderly. This association exists because cortisol, the main class of stress hormones in humans, has a high affinity to specific receptors located in the hippocampus, amygdala and prefrontal regions, structures associated with learning and memory. Chronically elevated cortisol concentrations are associated with hippocampal atrophy and low cognitive performance. However, the effect of acute stress on memory performance is still inconclusive in the elderly. This is particularly relevant, since elderly patients with pathological cognitive impairment present high cortisol level, which in turn is associated with rapid disease progression. The objective of the study was to analyze the relationship between memory performance and neuroendocrine as well as cardiovascular response stress in healthy elderly. One hundred elderly randomly selected, literate, predominantly female, with no cognitive impairment and functional, residents of the city of São Paulo were included. The neuroendocrine response to stress was evaluated using salivary cortisol while the cardiovascular reactivity was assessed through blood pressure and heart rate measured before, during and after exposure to a participant\'s acute psychosocial stressor (\"Trier Social Stress Test\" - TSST). The TSST involves two tasks: public speaking and performing mental arithmetic in front of an examining board. The memory performance was evaluated by the Pairs of Words test (PW) 20 minutes before the TSST for immediate recall and learning and 15 minutes after the end of TSST for delayed recall. It was observed an increase of 96% in the cortisol concentration 15 minutes after the TSST, as well as increased blood pressure compared to baseline. Furthermore, we observed significant reduction in the PP score after TSST and negative correlation between cortisol concentration, immediate and delayed recall of PP. The results revealed influence of acute stress on memory performance, particularly to delayed recall, of older adults, highlighting the vulnerability of older adults to the neurotoxic effects of cortisol on memory and, therefore, to the development of cognitive disorders.

Les hyperplasies micronodulaires pigmentées de la surrénale (PPNAD) sont les tumeurs endocrines les plus fréquentes d’un syndrome multinéoplasique d’origine génétique, le Complexe de Carney. Ces hyperplasies bilatérales sont associées à des mutations inactivatrices de PRKAR1A, le gène codant la sous-unité régulatrice R1 de la protéine kinase dépendante de l’AMPc (PKA). Ces tumeurs bénignes conduisent à une activation constitutive de la PKA responsable d’un hypercortisolisme indépendant de l’ACTH (syndrome de Cushing), associant diverses comorbidités telles que l’obésité centrale, le diabète, l’ostéoporose, des troubles de l’humeur ou encore des complications cardiovasculaires. Cependant, les mécanismes de cette tumorigenèse restent mal compris. Afin d’évaluer les conséquences de l’activation de la signalisation PKA sur l’induction tumorale et l’activité endocrine, l’équipe a précédemment généré un modèle de souris transgéniques reproduisant l’inactivation de Prkar1a dans la corticosurrénale. Ces souris développent une hyperplasie bilatérale composée de cellules présentant des caractéristiques fœtales naturellement absente d’une surrénale adulte. L’objectif général de ce travail de thèse était d’identifier l’origine des cellules constituant cette hyperplasie retrouvée dans le cortex interne des souris mutantes. Nous avons utilisé pour cela une approche génétique de lignage cellulaire afin de tracer chez la souris, l’origine de ces tumeurs après invalidation de Prkar1a dans les précurseurs du cortex définitif ou dans ceux du cortex fœtal. Les résultats montrent que l’activation de la signalisation PKA dans le cortex surrénalien adulte est suffisante pour promouvoir le développement d’hyperplasies surrénaliennes associées à la mise en place d’un syndrome de Cushing. L’invalidation de Prkar1a dans les précurseurs du cortex fœtal ne conduit à aucune anomalie endocrine ni tumorale. En revanche, l’activation de la signalisation PKA dans le cortex adulte favorise le renouvellement cellulaire centripète, l’identité fasciculée et sa conversion en identité réticulée dans la partie interne. L’activation de la signalisation PKA, conjointement à la croissance corticale, apparait donc comme un moteur possible de l’adrénarche, normalement restreinte aux grands primates.L’analyse transcriptomique des surrénales et les expériences de lignages cellulaires montrent que la prédisposition des femelles au syndrome de Cushing et au développement d’hyperplasies « pseudo-réticulée » pourraient reposer sur un dimorphisme sexuel des capacités de recrutement des cellules progénitrices et sur le métabolisme du cholestérol.En parallèle de ces travaux, l’exploration des mécanismes conduisant à la présence inappropriée de cellules « pseudo-réticulée » nous a amené à tester l’implication de la SUMOylation dans les défauts de zonation observés. Nos résultats montrent que l’activation de la signalisation PKA in vitro et in vivo exerce une hypoSUMOylation globale, d’origine transcriptionnelle. En accord avec cet effet, les nodules présents chez les patients atteints de PPNAD sont hypoSUMOylés. Enfin nous montrons chez les deux espèces, un gradient de SUMOylation régionalisé dans le cortex qui suggère l’implication de cette modification dans la zonation de la glande surrénale. / Primary Pigmented Nodular Adrenal Disease (PPNAD) is the most frequent endocrine manifestation of a rare, dominantly inherited multiple endocrine neoplasia syndrome, the Carney Complex. These bilateral hyperplasia are associated with inactivating mutations of PRKAR1A, the gene encoding the R1 regulatory subunit of cAMP-dependent protein kinase (PKA). These benign tumors lead to constitutive activation of PKA, responsible for an ACTH-independent hypertcortisolism (Cushing's syndrome), associating various comorbidities such as central obesity, diabetes, osteoporosis, mood disorders or cardiovascular complications. However, the mechanisms of tumorigenesis remain poorly understood. In order to evaluate the consequences of activation of PKA signaling on tumor induction and endocrine activity, the team previously generated a model of transgenic mice reproducing the inactivation of Prkar1a in the adrenal cortex. These mice develop bilateral hyperplasia composed of cells with fetal characteristics naturally absent from an adult adrenal gland.The general objective of this thesis was to identify the origin of the cells constituting this hyperplasia found in the internal cortex of the mutant mice. We used a genetic approach of cell lineage to trace the origin of these tumors after deletion of Prkar1a in the precursors of the adult or fetal cortex.The results show that activation of PKA signaling in the adult cortex is sufficient to promote the development of adrenal hyperplasia associated with Cushing's syndrome. The ablation of Prkar1a in the precursors of the fetal cortex does not lead to any endocrine or tumor abnormalities. On the contrary, activation of PKA signaling in the adult cortex promotes centripetal cell renewal, fasciculata identity and its conversion into reticularis identity in the internal part of the gland. The activation of PKA signaling, together with cortical growth, therefore appears to be a possible motor of adrenarche, normally restricted to human and some primates.Transcriptomic analysis of the adrenals and cell lineage experiments show that female predisposition to Cushing's syndrome and development of "pseudo-reticularis" hyperplasia may be based on sexual dimorphism of progenitor cell recruitment capacities and on metabolism of cholesterol.In parallel, the exploration of the mechanisms leading to the inappropriate presence of "pseudo-reticularis" cells led us to test the involvement of SUMOylation in the observed zonation defects. Our results show that the activation of PKA signaling in vitro and in vivo exerts a global hypoSUMOylation, of transcriptional origin. In agreement with this effect, the nodules present in patients with PPNAD are hypoSUMOylated. Finally, we show in both species a regionalized SUMOylation gradient in the cortex that suggests the implication of this modification in the zonation of the adrenal gland.

Cortisol, the principal glucocorticoid secreted from the adrenal glands, is essential for life. Healthy cortisol levels are maintained through negative feedback on the central nervous system (CNS) – pituitary stimulatory apparatus which regulates production of adrenocorticotropin (ACTH) and contains a light–entrained intrinsic CNS driven diurnal rhythm. Cortisol participates in a regulatory mechanism where inflammatory cytokines stimulate cortisol release and cortisol in turn suppresses cytokine release. The effects of cortisol in inflammatory states include elevating blood pressure and metabolic regulation. This thesis contains three exploratory studies examining circulating cortisolaemia using the best available methodologies (total and free cortisol and corticosteroid-binding globulin (CBG)) in clinical states characterized by immune activation/ inflammation and altered blood pressure. These clinical states include: (1) septic shock, (2) hypertensive disorders of pregnancy and (3) obesity-induced hypertension. Prior to the studies described here, little was know about cortisolaemia in these common pathological states. Septic shock is a life threatening condition that complicates severe infection and is characterized by systemic inflammation and refractory hypotension. High plasma total cortisol levels and attenuated responses to synthetic ACTH stimulation are associated with increased mortality. The use of corticosteroids in septic shock has been highly controversial for decades, however recent trials have reported haemodynamic and survival benefits associated with the use of physiologic steroid replacement in patients with relative adrenal insufficiency (RAI) – currently defined as a total cortisol increment of 248 nmol/L or less following ACTH (250 μg) stimulation. However, CBG and albumin levels fall by around 50% with an increase in plasma free cortisol in critical illness. Hence, total cortisol may not reflect the biologically active free (unbound) cortisol, suggesting that standard assays for plasma cortisol (which measure total plasma cortisol) underestimate HPA axis activity. In this study, we have showed that plasma free cortisol is a better guide to circulating glucocorticoid activity in systemic infection than total cortisol. We have also validated the use of Coolens’ method in estimating free cortisol in systemic infection, using plasma total cortisol and CBG measurements as plasma free cortisol is not performed in clinical laboratories. Free cortisol measurement allows better categorization of RAI and non-RAI groups with a free cortisol increment of 110 nmol/L as cut-off. Moreover, we have shown that survivors of RAI have normal adrenocortical function on follow-up testing suggesting a lack of functional adrenal reserve rather than adrenal damage during critical illness. Larger randomized controlled trials will be required to redefine RAI using free cortisol measurements and relate that to clinical outcomes and responses to corticosteroid therapy. Nitric oxide (NO) is normally produced in the endothelium by the constitutive form of the NO synthase and this physiologic production is important for blood pressure regulation and blood flow distribution. Studies have shown that an overproduction of NO by the inducible form of NO synthase (iNOS) may contribute to the hypotension, cardiodepression and vascular hyporeactivity in septic shock. Clinical studies of non-selective inhibitors of the L-arginine nitric oxide pathway showed increased mortality from cardiovascular complications. However, glucocorticoids, which improve vasopressor sensitivity, may act by partially suppressing NO synthesis through selective direct inhibition of iNOS, and suppression of inflammatory cytokine synthesis. Hence, plasma nitrate/ nitrite (NOx) levels may provide a titratable end point to individualize glucocorticoid therapy in sepsis. The NOx study in this thesis showed that cortisol (total and free), CBG and NOx correlated to illness severity. Free cortisol, and to a lesser extent total cortisol, but not NOx levels, predicted septic shock. NOx levels were characteristically stable within individuals but inter-individual differences were only partly accounted for by illness severity or renal dysfunction. NOx levels correlated weakly with cortisol, did not relate to the need for vasopressors and were not suppressed by hydrocortisone treatment. Thus, NOx is not a suitable target for glucocorticoid therapy in septic shock. Pregnancy is the only sustained physiologic state of hypercortisolism in humans. A large body of data suggests that excessive foetal and prenatal glucocorticoid exposure leads to reduced birth weight and adverse health in offspring such as elevated blood pressure and insulin resistance. Pre-eclampsia and gamete donor pregnancies are associated with immune activation, elevated inflammatory cytokines as well as elevated blood pressure. Prior to the study described in this thesis however, there was no prospective data on maternal cortisolaemia in these complicated pregnancies. My study has demonstrated for the first time that there was a substantial fall in plasma CBG levels in the last few weeks of gestation with a corresponding rise in free cortisol in normal pregnancy, a finding obscured for methodological reasons in past studies. This free cortisol elevation in late pregnancy may facilitate organ maturation in the foetus and perhaps prepare the mother for the metabolic demands of labour. In pre-eclampsia and gestational hypertension, plasma CBG, total and free cortisol levels were lower in late third trimester; and in IUGR, plasma CBG levels were suppressed from 28 weeks gestation until delivery but with no significant difference in plasma total and free cortisol. Women with assisted reproduction using donor gametes/ embryos had significantly lower plasma CBG, total and free cortisol levels even in those with normal pregnancy outcomes. Low CBG may be due to reduced synthesis or enhanced inflammation-driven degradation. Low maternal cortisol may be due to a lack of placental corticotropin-releasing hormone, or reduced maternal ACTH, driving cortisol production. This unanticipated maternal hypocortisolism in complicated pregnancies may trigger precocious activation of the foetal HPA axis and could have implications for postnatal and adult health. Speculatively, since excess prenatal GCs increase HPA axis activity, we proposed that maternal hypocortisolism may predispose to the hypocortisolaemic state characterized by fatigue, pain and stress sensitivity, in offspring. The third state of immune/ inflammatory activation associated with blood pressure dysregulation studied in this thesis is obesity. The epidemiologic relationship between obesity and hypertension is widely recognised. Central obesity in particular has been associated with exaggerated HPA responses to stimuli. Studies of severe dieting and starvation resulted in hypercortisolism and a significant decrease in CBG. The HPA axis and the renin-angiotensin-aldosterone system (RAAS) have been implicated in the pathophysiology of obesity-induced hypertension. However, there is little data on the effect of moderate weight loss (30% caloric restriction) on adrenocortical function, and the relation of adrenal hormones to altered blood pressure with weight loss. In this study, measures of HPA axis and RAAS and blood pressure monitoring were performed in twenty-five obese subjects before and after a 12-week diet program (6000kJ/day). Short-term, moderate weight loss (mean 8.5 kg) was associated with a small reduction in blood pressure (mean arterial pressure 6 mmHg) and significantly reduced levels of aldosterone and renin but not cortisol levels. These findings suggest that aldosterone may have an important role in the blood pressure reduction with weight loss via a renin mediated mechanism, perhaps involving renal sympathetic tone. In contrast to severe caloric restriction, HPA axis activation does not occur with moderate weight loss. This suggests a threshold effect of weight loss on the HPA axis where greater caloric restriction is required for HPA stimulation, or a counterbalancing of central and direct adrenal effects on HPA axis function. Overall, these three exploratory studies have provided novel data on HPA axis function in systemic infection, pregnancy and in diet-induced weight loss. Each study offers a basis for further studies of HPA axis function in these disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1289330 / Thesis(Ph.D.)-- School of Medicine, 2007.

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

Cortisol, the principal glucocorticoid secreted from the adrenal glands, is essential for life. Healthy cortisol levels are maintained through negative feedback on the central nervous system (CNS) – pituitary stimulatory apparatus which regulates production of adrenocorticotropin (ACTH) and contains a light–entrained intrinsic CNS driven diurnal rhythm. Cortisol participates in a regulatory mechanism where inflammatory cytokines stimulate cortisol release and cortisol in turn suppresses cytokine release. The effects of cortisol in inflammatory states include elevating blood pressure and metabolic regulation. This thesis contains three exploratory studies examining circulating cortisolaemia using the best available methodologies (total and free cortisol and corticosteroid-binding globulin (CBG)) in clinical states characterized by immune activation/ inflammation and altered blood pressure. These clinical states include: (1) septic shock, (2) hypertensive disorders of pregnancy and (3) obesity-induced hypertension. Prior to the studies described here, little was know about cortisolaemia in these common pathological states. Septic shock is a life threatening condition that complicates severe infection and is characterized by systemic inflammation and refractory hypotension. High plasma total cortisol levels and attenuated responses to synthetic ACTH stimulation are associated with increased mortality. The use of corticosteroids in septic shock has been highly controversial for decades, however recent trials have reported haemodynamic and survival benefits associated with the use of physiologic steroid replacement in patients with relative adrenal insufficiency (RAI) – currently defined as a total cortisol increment of 248 nmol/L or less following ACTH (250 μg) stimulation. However, CBG and albumin levels fall by around 50% with an increase in plasma free cortisol in critical illness. Hence, total cortisol may not reflect the biologically active free (unbound) cortisol, suggesting that standard assays for plasma cortisol (which measure total plasma cortisol) underestimate HPA axis activity. In this study, we have showed that plasma free cortisol is a better guide to circulating glucocorticoid activity in systemic infection than total cortisol. We have also validated the use of Coolens’ method in estimating free cortisol in systemic infection, using plasma total cortisol and CBG measurements as plasma free cortisol is not performed in clinical laboratories. Free cortisol measurement allows better categorization of RAI and non-RAI groups with a free cortisol increment of 110 nmol/L as cut-off. Moreover, we have shown that survivors of RAI have normal adrenocortical function on follow-up testing suggesting a lack of functional adrenal reserve rather than adrenal damage during critical illness. Larger randomized controlled trials will be required to redefine RAI using free cortisol measurements and relate that to clinical outcomes and responses to corticosteroid therapy. Nitric oxide (NO) is normally produced in the endothelium by the constitutive form of the NO synthase and this physiologic production is important for blood pressure regulation and blood flow distribution. Studies have shown that an overproduction of NO by the inducible form of NO synthase (iNOS) may contribute to the hypotension, cardiodepression and vascular hyporeactivity in septic shock. Clinical studies of non-selective inhibitors of the L-arginine nitric oxide pathway showed increased mortality from cardiovascular complications. However, glucocorticoids, which improve vasopressor sensitivity, may act by partially suppressing NO synthesis through selective direct inhibition of iNOS, and suppression of inflammatory cytokine synthesis. Hence, plasma nitrate/ nitrite (NOx) levels may provide a titratable end point to individualize glucocorticoid therapy in sepsis. The NOx study in this thesis showed that cortisol (total and free), CBG and NOx correlated to illness severity. Free cortisol, and to a lesser extent total cortisol, but not NOx levels, predicted septic shock. NOx levels were characteristically stable within individuals but inter-individual differences were only partly accounted for by illness severity or renal dysfunction. NOx levels correlated weakly with cortisol, did not relate to the need for vasopressors and were not suppressed by hydrocortisone treatment. Thus, NOx is not a suitable target for glucocorticoid therapy in septic shock. Pregnancy is the only sustained physiologic state of hypercortisolism in humans. A large body of data suggests that excessive foetal and prenatal glucocorticoid exposure leads to reduced birth weight and adverse health in offspring such as elevated blood pressure and insulin resistance. Pre-eclampsia and gamete donor pregnancies are associated with immune activation, elevated inflammatory cytokines as well as elevated blood pressure. Prior to the study described in this thesis however, there was no prospective data on maternal cortisolaemia in these complicated pregnancies. My study has demonstrated for the first time that there was a substantial fall in plasma CBG levels in the last few weeks of gestation with a corresponding rise in free cortisol in normal pregnancy, a finding obscured for methodological reasons in past studies. This free cortisol elevation in late pregnancy may facilitate organ maturation in the foetus and perhaps prepare the mother for the metabolic demands of labour. In pre-eclampsia and gestational hypertension, plasma CBG, total and free cortisol levels were lower in late third trimester; and in IUGR, plasma CBG levels were suppressed from 28 weeks gestation until delivery but with no significant difference in plasma total and free cortisol. Women with assisted reproduction using donor gametes/ embryos had significantly lower plasma CBG, total and free cortisol levels even in those with normal pregnancy outcomes. Low CBG may be due to reduced synthesis or enhanced inflammation-driven degradation. Low maternal cortisol may be due to a lack of placental corticotropin-releasing hormone, or reduced maternal ACTH, driving cortisol production. This unanticipated maternal hypocortisolism in complicated pregnancies may trigger precocious activation of the foetal HPA axis and could have implications for postnatal and adult health. Speculatively, since excess prenatal GCs increase HPA axis activity, we proposed that maternal hypocortisolism may predispose to the hypocortisolaemic state characterized by fatigue, pain and stress sensitivity, in offspring. The third state of immune/ inflammatory activation associated with blood pressure dysregulation studied in this thesis is obesity. The epidemiologic relationship between obesity and hypertension is widely recognised. Central obesity in particular has been associated with exaggerated HPA responses to stimuli. Studies of severe dieting and starvation resulted in hypercortisolism and a significant decrease in CBG. The HPA axis and the renin-angiotensin-aldosterone system (RAAS) have been implicated in the pathophysiology of obesity-induced hypertension. However, there is little data on the effect of moderate weight loss (30% caloric restriction) on adrenocortical function, and the relation of adrenal hormones to altered blood pressure with weight loss. In this study, measures of HPA axis and RAAS and blood pressure monitoring were performed in twenty-five obese subjects before and after a 12-week diet program (6000kJ/day). Short-term, moderate weight loss (mean 8.5 kg) was associated with a small reduction in blood pressure (mean arterial pressure 6 mmHg) and significantly reduced levels of aldosterone and renin but not cortisol levels. These findings suggest that aldosterone may have an important role in the blood pressure reduction with weight loss via a renin mediated mechanism, perhaps involving renal sympathetic tone. In contrast to severe caloric restriction, HPA axis activation does not occur with moderate weight loss. This suggests a threshold effect of weight loss on the HPA axis where greater caloric restriction is required for HPA stimulation, or a counterbalancing of central and direct adrenal effects on HPA axis function. Overall, these three exploratory studies have provided novel data on HPA axis function in systemic infection, pregnancy and in diet-induced weight loss. Each study offers a basis for further studies of HPA axis function in these disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1289330 / Thesis(Ph.D.)-- School of Medicine, 2007.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 2 uppsatser.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / "Response to stress" is the designation for the set of metabolic and hormonal changes that take place after any traumatic situation. The stress response is intended to maintain and / or restore the homeostasis of the body injured. The major endocrine response to stress is characterized as the increased secretion of pituitary hormones and activation of the sympathetic nervous system. Among the pituitary hormones is ACTH, which will sensitize the cells of the adrenal gland to secrete cortisol. The biochemical measurements, especially cortisol and glucose has been shown to be important references for the evaluation of pain and stress by indirect means. Therefore, the objective of this research is to measure cortisol, the glucose and triglyceride metabolic energy, in evaluating the neuroendocrine response of dogs with tibial fractures. For the present study, we used 12 dogs with tibial fractures. Were colected five venous blood samples, at the time of patient care, during the surgical procedure after induction of anesthesia, five minutes after stimulation by periosteal implant, 24 hours after the first collection during surgery and after seven days of the surgery. There were no statistically significant differences between times and between the values of cortisol, glucose and triglycerides, but there was a numerical difference between the mean times analyzed, we observed that the cortisol showed an average decrease between times. The mean glucose values had increased in the pre and trans-surgical times, and with a decrease in mean postoperative periods. Mean triglyceride values varied widely between periods. It is possible to conclude that cortisol is an important mediator in the neuroendocrine response of dogs with acute tibial fractures, the mean values of glucose did not respond similarly to cortisol during surgical procedures and that the triglyceride is not a good parameter for evaluating the neuroendocrine stress dogs with acute tibial fractures. / Resposta ao estresse é a designação para o conjunto de alterações hormonais e metabólicas que decorrem após qualquer situação de trauma. A resposta ao estresse tem como finalidade manter e/ou restaurar a homeostase do organismo lesado. A principal resposta endócrina ao estresse é caracterizada como o aumento da secreção de hormônios pituitário e ativação do sistema nervoso simpático. Dentre os hormônios pituitários está o ACTH, que irá sensibilizar as células da adrenal para secretar cortisol. As mensurações bioquímicas, especialmente o cortisol e a glicose tem se mostrado importantes referencias para a avaliação da dor e do estresse por meio indireto. Sendo assim, o objetivo desta pesquisa é medir o cortisol, os metabólicos energéticos glicose e triglicerídeo, na avaliação da resposta neuroendócrina de cães com fratura tibial. Para o presente estudo, foram utilizados 12 cães com fratura tibial. Foram realizadas cinco coletas de sangue venoso, no momento do atendimento do paciente, durante o procedimento cirúrgico logo após a indução anestésica, cinco minutos após a estimulação periosteal pelo implante, 24 horas após a primeira coleta durante o procedimento cirúrgico e após sete dias do procedimento cirúrgico. Não foram observadas diferenças estatísticas significativas entre os tempos e entre os valores de cortisol, glicose e triglicerídeo, porém houve diferença numérica entre as médias dos tempos analisados, sendo observado que o cortisol apresentou uma média decrescente entre os tempos. As médias dos valores de glicose apresentaram acréscimo nos períodos pré e trans cirúrgicos com decréscimo das médias nos períodos pós-cirúrgicos. As médias dos valores de triglicerídeo apresentaram grande variação entre os períodos. É possível concluir que o cortisol é um importante mediador na resposta neuroendócrina aguda de cães com fratura tibial, os valores médios de glicose não responderam de forma similar ao cortisol durante os procedimentos cirúrgicos e que o triglicerídeo não é um bom parâmetro de avaliação do estresse neuroendócrino agudo de cães com fratura tibial.

Introdução: A vulnerabilidade dos profissionais de enfermagem ao estresse está associada à exposição crônica aos estressores cotidianos de trabalho e, consequentemente, aos efeitos cumulativos dos mediadores primários e secundários do estresse. Embora vários estudos tenham relatado o elevado nível de estresse dos profissionais de enfermagem, pouco se sabe a respeito das implicações biológicas do estresse no trabalho, expressas na carga alostática e no ritmo de secreção dos hormônios de cortisol um dos principais hormônios do estresse. Isto particularmente é importante visto que a carga alostática aumenta o risco do indivíduo desenvolver transtornos relacionados ao estresse como síndromes cardiovasculares, metabólicas, endócrinas, emocionais e cognitivas. Neste sentido, questiona-se se os profissionais de enfermagem apresentam sobrecarga do sistema adaptativo de reação do estresse (carga alostática), bem como alterações no ritmo de secreção de cortisol (hiper ou hipocortisolemia) ao longo do dia e o risco para o desenvolvimento de doenças relacionadas ao estresse. Objetivo: Descrever a frequência de profissionais de enfermagem com carga alostática elevada e ritmo atípico de secreção de cortisol. Analisar se a carga alostática elevada está associada ao ritmo atípico de secreção de cortisol. Método: Foram incluídos 142 profissionais de enfermagem do turno diurno randomicamente selecionados, alocados nas unidades ambulatório, clínica médica, clínica cirúrgica, centro cirúrgico, pronto socorro infantil e adulto, unidade de terapia intensiva adulto e pediátrica de um hospital universitário. Para avaliação do padrão diurno de secreção de cortisol foram coletadas amostras de saliva em dois dias úteis consecutivos de trabalho e, para a análise dos biomarcadores foram coletadas, em um único dia, amostras de sangue de todos os participantes, bem como, verificada sua pressão arterial e medidas antropométricas. A carga alostática foi analisada por mediadores neuroendócrinos, metabólicos, cardiovasculares e imunológicos. Os dados foram armazenados e analisados utilizando o programa estatístico SPSS versão 14.0 e o nível de significância adotado foi de 5%. Resultados: 31% dos profissionais de enfermagem apresentaram padrão atípico (inconsistente ou flat) de secreção de cortisol e 47,2% carga alostática elevada. Não houve associação entre o ritmo de secreção de cortisol e a carga alostática. Conclusão: Quase metade dos profissionais de enfermagem apresentaram sinais de desgaste do sistema biológico regulador da resposta de estresse, sugerindo que o trabalho possa estar associado a esta sobrecarga e destacando a vulnerabilidade destes trabalhadores ao desenvolvimento de doenças relacionadas ao estresse / Background: The vulnerability of nursing professionals to stress is associated with chronic exposure to everyday stressors and, therefore, with the cumulative effects of primary and secondary stress mediators. Despite the fact that several studies have reported the high stress level of nursing professionals, little is known about the biological implications of stress at work, expressed in the allostatic load and in the rhythm of cortisol secretion, one of the main hormones of stress. This is especially important since the allostatic load increases the risk of an individual to develop stress-related disorders like cardiovascular syndromes, metabolic, endocrine, cognitive and emotional. In this aspect, there are questions whether the nursing professionals present overload on the stress adaptive reaction system (allostatic load), as well as, changes in the rhythm of cortisol secretion (hyper or hypocortisolemia) throughout the day and the risk of developing stress-related diseases. Objective: To describe the frequency of nursing professionals with high allostatic load and atypical rhythm of cortisol secretion. To analyze whether the high allostatic load is associated with atypical rhythm of cortisol secretion. Methods: We included 142 nursing professionals day shift randomly selected, allocated in the outpatient units, medical clinic, surgical clinic, surgery, children and adults emergency room and adult intensive care unit in a pediatric teaching hospital. To evaluate the daytime pattern of cortisol secretion, saliva samples were collected in two work day and, for the analysis of biomarkers, were collected in a single day, blood samples from all participants, as well as, checked his blood pressure and anthropometric measurements. Allostatic load was analized by neuroendocrine, metabolic, cardiovascular and immune mediators. The data was stored and analyzed using the program SPSS version 14.0 and the statistical significance level adopted was 5%. Results: 31% of nursing professionals showed atypical pattern (inconsistent or \"flat\") of secretion of cortisol and 47.2% showed high allostatic load. There was no association between the rate of secretion of cortisol and the allostatic load. Conclusion: Nearly half of nursing professionals showed signs of wear in the biological system stress response regulator, suggesting that the job can be associated with this overload and highlighting the vulnerability of these workers to the development of stress-related diseases.

As massas adrenocorticais são majoritariamente (70-80%) adenomas adrenocorticais (ACA). Os carcinomas adrenocorticais (ACC) são mais raros e de prognóstico restrito, com incidência de 1-2 casos por milhão, com alta taxa de reincidência (70-80%). Apesar dos tumores adrenocorticais (ACT) serem raros, no Brasil a incidência desses tumores em crianças é cerca de 10 vezes superior ao do restante do mundo, devido a uma mutação do gene TP53. Atualmente, o diagnóstico de massas adrenocorticais é realizado através dos critérios de Weiss, que possuem limitações, e por isso é intensa a busca de novos marcadores moleculares que facilite o diagnóstico de ACTs. POD-1/ TCF21 é um fator de transcrição do tipo helix-loop-helix básica (bHLH) expresso nos sítios de interação mesênquima-epitélio durante o desenvolvimento embrionário. Em ACTs, POD-1 regula a expressão endógena de SF-1 através da ligação na sequencia E-box da região promotora de SF-1, e nesses tumores parece estar relacionado negativamente com genes reguladores do ciclo celular, como BUB1B. BUB1B é um gene que codifica uma quinase com funções importantes durante o checkpoint mitótico. A expressão de BUB1B é considerada fator de prognóstico em diferentes tipos de tumores, inclusive em ACTs humanos, nos quais a expressão combinada de BUB1B e PINK1 (ΔCtBUB1B - ΔCtPINK1) mostrou-se um bom marcador de sobrevida em pacientes com ACC. PINK1, quinase 1 induzida por PTEN, é regulada principalmente pela mitocôndria, e em ACTs sua expressão está reduzida em ACC mais agressivos. Temos como hipótese que a expressão de POD-1 pode ter valor diferencial no diagnóstico de massas adrenocorticais, e que a análise da expressão combinada de POD-1, BUB1B e PINK1 pode ter valor diferencial para prognóstico de pacientes com ACT. Nesse trabalho foram analisados, por reação de qPCR com sondas Taqman, o cDNA obtido de 130 amostras de tumores: 79 adultos (44 ACAs e 35 ACCs), 35 crianças com menos de 5 anos de idade (27 ACAs e 8 ACCs) e 16 crianças de 5 a 18 anos de idade (6 ACAs e 10 ACCs). Nossos resultados mostram que POD-1 e BUB1B tem valor diferencial em ACT adulto e que a expressão combinada de POD-1 e BUB1B pode ser um marcador de prognóstico em pacientes com carcinoma adulto. Enquanto que, a expressão combinada de POD-1 e SF-1 pode ter valor de diagnóstico em pacientes pediátricos com menos de 5 anos. Em resumo, concluímos que estudos experimentais devem ser realizados para comprovar a relação entre os genes estudados, para que os resultados sejam sólidos o suficiente para serem utilizados no diagnóstico e prognóstico dos tumores adrenocorticais. / The adrenocortical masses are mostly (70-80%) adrenocortical adenomas (ACA). Adrenocortical carcinomas (ACC) are scarce and have limited prognosis, with an incidence of 1-2 cases per million and high recurrence rate (70-80%). Despite adrenocortical tumors (ACT) are rare in Brazil the incidence of these tumors in children is about 10 times higher than the rest of the world, due to a mutation of the TP53 gene. Currently, the diagnosis of adrenocortical mass is carried through Weiss criteria that have limitations, so it is intensive the search for new molecular markers that facilitate the diagnostic of ACTs. TCF21/POD-1 is a transcription factor helix-loop-helix type expressed in mesenchymal-epithelial sites of interaction during embryonic development. In ACTs, POD-1 regulates the expression of endogenous SF-1 through binding the E-box sequence of SF-1 promoter region, and seems to be negatively relates with cell cycle regulatory genes such as BUB1B. BUB1B is a gene encoding a kinase-with important function during mitotic checkpoint. The expression of BUB1B is considered a prognostic factor in different types of tumors, including ACTs. Combined expression of BUB1B and PINK1 (ΔCtBUB1B - ΔCtPINK1) has been shown to be a good marker of survival in adults with ACC, whereas the PINK1 expression is reduced in the most aggressive ACC. We hypothesized that the POD-1 expression may have differential value in the diagnosis of adrenocortical masses, and that the analysis of the combined expression of POD-1, BUB1B and PINK1 may have differential value for the prognosis of patients with ACT. In this work were analyzed by PCRq Taqman probes the cDNA obtained from 130 tumor samples: 79 adults (44 ACAs and 35 ACCs), 35 children under 5 years old (27 ACAs and 8 ACCs) and 16 children 5-18 years of age (6 ACAs and 10 ACCs). Our results show that POD-1 and BUB1B has differential value in adult ACT, and the combined expression of POD-1 and BUB1B may have a prognostic value in patients with adult carcinoma. In addition, the combined expression of POD-1 and SF-1 might have diagnostic value in pediatric patients younger than 5 years. In summary, we conclude that experimental studies should be conducted to confirm the relationship between the genes studied, so that the results are solid enough to be used in the diagnosis and prognosis of adrenocortical tumors.

Vários estudos têm sugerido que o estresse pode ser um dos fatores relacionados com à grande variabilidade cognitiva observada em idosos. Esta associação se explica porque o cortisol, principal classe de hormônios do estresse em humanos, apresenta alta afinidade por receptores específicos localizados no hipocampo, amígdala e região pré-frontal, estruturas associadas ao aprendizado e à memória. Concentrações cronicamente elevadas de cortisol estão associadas à atrofia hipocampal e baixo desempenho cognitivo. Entretanto, o efeito do estresse agudo no desempenho da memória ainda se encontra inconclusivo em idosos. Isto é particularmente relevante, uma vez que, idosos com comprometimento cognitivo patológico apresentam concentração elevada de cortisol, que por sua vez, está associada com rápida progressão da doença. Assim, o objetivo do estudo foi analisar a relação entre desempenho da memória e resposta neuroendócrina e cardiovascular de estresse em idosos saudáveis. Foram selecionados aleatoriamente 100 idosos alfabetizados, predominantemente do sexo feminino, sem prejuízo cognitivo e funcional, moradores da cidade de São Paulo. A resposta neuroendócrina de estresse foi avaliada a partir concentração de cortisol salivar enquanto que a reação cardiovascular a partir da pressão arterial e frequência cardíaca antes, durante e após a exposição do participante a um estressor psicossocial agudo (Trier Social Stress Test TSST). O TSST envolve duas tarefas: falar em público e realizar cálculos aritméticos mentalmente diante de uma banca examinadora. O desempenho da memória foi avaliado mediante aplicação do teste Pares de Palavras (PP) 20 minutos antes do TSST para evocação imediata e aprendizado e 15 minutos após o fim do TSST para evocação tardia. Foi observado aumento de 96% na concentração de cortisol 15 minutos após o TSST, bem como elevação da pressão arterial em relação à situação basal. Ademais, observamos redução significativa do escore do teste PP após o TSST e correlação negativa entre concentração de cortisol, evocação imediata e tardia dos PP. Os resultados revelam influência do estresse agudo no desempenho da memória, particularmente da evocação tardia, de idosos, destacando a vulnerabilidade destes indivíduos aos efeitos neurotóxicos do cortisol na memória e, consequentemente ao desenvolvimento de transtornos cognitivos. / Several studies have suggested that stress may be a factor related to cognitive variability observed in the elderly. This association exists because cortisol, the main class of stress hormones in humans, has a high affinity to specific receptors located in the hippocampus, amygdala and prefrontal regions, structures associated with learning and memory. Chronically elevated cortisol concentrations are associated with hippocampal atrophy and low cognitive performance. However, the effect of acute stress on memory performance is still inconclusive in the elderly. This is particularly relevant, since elderly patients with pathological cognitive impairment present high cortisol level, which in turn is associated with rapid disease progression. The objective of the study was to analyze the relationship between memory performance and neuroendocrine as well as cardiovascular response stress in healthy elderly. One hundred elderly randomly selected, literate, predominantly female, with no cognitive impairment and functional, residents of the city of São Paulo were included. The neuroendocrine response to stress was evaluated using salivary cortisol while the cardiovascular reactivity was assessed through blood pressure and heart rate measured before, during and after exposure to a participant\'s acute psychosocial stressor (\"Trier Social Stress Test\" - TSST). The TSST involves two tasks: public speaking and performing mental arithmetic in front of an examining board. The memory performance was evaluated by the Pairs of Words test (PW) 20 minutes before the TSST for immediate recall and learning and 15 minutes after the end of TSST for delayed recall. It was observed an increase of 96% in the cortisol concentration 15 minutes after the TSST, as well as increased blood pressure compared to baseline. Furthermore, we observed significant reduction in the PP score after TSST and negative correlation between cortisol concentration, immediate and delayed recall of PP. The results revealed influence of acute stress on memory performance, particularly to delayed recall, of older adults, highlighting the vulnerability of older adults to the neurotoxic effects of cortisol on memory and, therefore, to the development of cognitive disorders.