Ces dernières décennies ont mis à jour une implication pathologique nouvelle del’aldostérone, via le récepteur aux minéralocorticoïdes (RM) dans le coeur. L’ensemble desdonnées issues des études expérimentales et des essais cliniques suggère une association délétèreentre l’aldostérone et la survenue d’arythmies. L’utilisation d’antagonistes du RM prévient cesarythmies. Cependant, les voies de signalisations, comme les mécanismes moléculaires soustendantces effets bénéfiques du blocage des RM demeurent incertains. Nous avons accumulésdes preuves d’une modulation de la signalisation calcique dans le cardiomyocyte, et en particulierde l’influx calcique (Ca2+) au travers du canal Ca2+ de type L (LTCC). Celui-Ci pourrait être unecible primaire de l’aldostérone et du RM dans les cardiomyocytes ventriculaires. Toutefois, lesmécanismes par lesquels l’aldostérone et le RM régulent l’expression du LTCC restent à définir.Au cours de ces travaux menés sur cardiomyocytes de rats nouveau-Nés, nous avonsétudiés les évènements moléculaires par lesquels l’aldostérone exerce ses effets sur le CaV1.2,qui correspond à la sous-Unité principale du LTCC formant le pore du canal ; cette protéine estcodée par le CACNA1C. Par microscopie confocale, nous avons suivi en temps réel le traffickingnucléo-Cytoplasmique du RM couplé à la GFP en réponse à l’aldostérone, démontrant ainsi queles RM cardiaques sont fonctionnels. Le traitement durant 24 heures des cardiomyocytes avec del’aldostérone montre une augmentation dose-Dépendante des protéines et de l’ARN messager duCaV1.2. L’utilisation de la technique du gène rapporteur de la luciférase permet l’analyse del’activité du promoteur du CaCNA1C. Celui-Ci montre une activité transcriptionnelle dose ettemps dépendante en réponse à l’aldostérone. De plus, ces effets sont dépendant des RM carinhibés en présence de RU28318, un antagoniste sélectif du RM, ou par l’utilisation de siRNAdirigés contre le RM. L’analyse in silico de la séquence du promoteur du CaCNA1C nous a permisd’identifier cinq séquences putatives correspondant à des éléments de réponse auxglucocorticoïdes (GRE). La mutation du site le plus lointain du site d’initiation de la transcriptionne révèle aucun changement dans les réponses transcriptionnelles induites par un RM humainconstitutivement actif (hMRΔ5,6) ou dans les réponses doses-Dépendantes de l’aldostérone ou dela déxaméthasone, un glucocorticoïde de synthèse. La mutation des trois sites GRE putatifssuivants provoque une diminution des réponses au hMRΔ5,6 comme à l’aldostérone, alors que lesréponses à la déxaméthasone sont soit inchangées, soit augmentées. En contraste, la mutation dusite le plus proximal du promoteur augmente de façon importante l’activité transcriptionnelle dupromoteur en réponse au hMRΔ5,6, à l’aldostérone comme à la déxaméthasone.Ces résultats démontrent que le LTCC cardiaque constitue une cible directe del’aldostérone et du RM, et apportent de nouvelles perspectives quant aux conséquencesmoléculaires et fonctionnelles engendrées par l’activation délétère du système minéralocorticoïdedans la défaillance cardiaque. / During the past decades, major novel pathogenic roles of the steroid hormone,aldosterone, via the Mineralocorticoid Receptor (MR) have been identified in heart. Collectively,experimental studies and clinical trials, suggest a detrimental association between aldosteroneand life threatening arrhythmias that may be prevented by MR blockade. However, the signalingpathways and underlying mechanisms still remain elusive. We have accumulated evidence thatmodulation of Ca2+ signaling, especially Ca2+ influx via L-Type Ca2+ channel (LTCC), might bethe primary aldosterone/MR target in ventricular cardiomyocytes. Yet, the molecularmechanisms by which MR regulates expression of LTCC remain to be defined. Here, weinvestigated, in primary cultures of neonatal rat ventricular myocytes, the molecular eventscritical for aldosterone-Mediated cardiac effects on CaV1.2, the pore-Forming main subunit ofLTCC, which is encoded by the CaCNA1C gene.We showed that cardiac MR are functional as demonstrated by aldosterone-Induced MRnucleocytoplasmic trafficking observed by time-Lapse imaging of transfected GFP-Labeled MRusing confocal microscopy. Aldosterone exposure for 24 hours, induced a dose-Dependentincrease in CaV1.2 expression at both mRNA and protein levels. Analysis of the CaCNA1Cpromoter activity using luciferase reporter assays, revealed a dose- and time-Dependent activationby aldosterone. These effects were inhibited in the presence of either RU28318, a selective MRantagonist, or MR siRNA. In silico analyze enabled us to identify five putative GlucocorticoidResponse Elements (GRE) within the CaCNA1C promoter sequence. The mutation of the mostdistal GRE from Transcription Start Site (TSS) did not altered responses either elicited by theconstitutively active human MR (hMRΔ5,6) or dose-Dependent effects of aldosterone anddexamethasone (a synthetic glucocorticoïd with minimal MR effect). Mutations of the three nextones decreased responses to hMRΔ5,6 and aldosterone, whereas dexamethasone responses wereeither unchanged or increased. In sharp contrast, the mutation of the most proximal GRE fromTSS, increased responses to hMRΔ5,6, aldosterone and dexamethasone.These results provide new insights into the molecular mechanisms associated with cardiacMR activation, and suggest that LTCC is a primary MR target, with subsequent molecular andfunctional consequences that could lead to MR-Related cardiac dysfunction.

En dehors de son rôle dans la régulation de la balance hydrosodée, le récepteur minéralocorticoïde (MR) est un facteur de transcription hormono-dépendant qui exerce des effets pro-adipogéniques et anti-thermogéniques in vitro, mais son rôle dans la régulation du métabolisme énergétique in vivo n’a jamais été précisément étudié. Dans ce travail, nous avons montré que les souris surexprimant le MR humain (Tg) ont une résistance à l’obésité induite par le régime hyperlipidique. Ceci s’accompagne d’un défaut de développement de la masse adipeuse comme en témoignent des surfaces adipocytaires plus petites en histomoprhométrie et une diminution de l’expression de gènes impliqués dans l’adipogenèse tels que PPARγ2. Ce défaut d’adipogenèse n’est pas dû à une altération de la capacité intrinsèque des préadipocytes surexprimant le MR, isolés de la fraction stroma vasculaire, mais probablement à une modification de la polarisation macrophagique analysée par la technique du FACS. Ces résultats soulignent un impact immuno-métabolique de la surexpression du MR in vivo. Par ailleurs, dans notre modèle adipocytaire brun, nous démontrons que les corégulateurs du MR ont un profil d’expression différentiel pouvant rendre compte d’une coopération moléculaire au cours de la différenciation adipocytaire des cellules T37i. De plus, nous confirmons in vitro l’effet inhibiteur de l’aldostérone sur l’expression de UCP1 (Uncoupling protein 1). Enfin nous démontrons in vivo que la surexpression du MR dans le tissu adipeux brun des souris Tg induit une diminution de l’induction l’expression de UCP1 par une exposition au froid. L’ensemble de ces résultats apporte une meilleure compréhension du rôle du MR dans la régulation du métabolisme énergétique et devrait ouvrir des nouvelles perspectives thérapeutiques innovantes tels que l’utilisation de modulateurs sélectifs du MR dans le traitement des troubles métaboliques / Besides its role in the regulation of sodium homeostasis, the mineralocorticoid receptor (MR) is a hormone-dependent transcription factor that exerts pro-adipogenic and anti- thermogenic effects in vitro, but its role in vivo in the regulation of energy balance has never been precisely studied. In this study, we show that human MR overexpressing mice (Tg) were resistant to high fat diet-induced obesity. This was associated with a defect of fat mass as evidenced by smaller adipocyte size analyzed by histomorphometric study and a decreased expression of genes involved in adipogenesis such as PPARγ2. This alteration in adipogenesis was not related to a defect of the intrinsic capacity of MR overexpressing preadipocytes to differentiate into adipocytes, but probably to a change in macrophage polarization studied by FACS analysis. These results indicate an immuno-metabolic impact of MR overexpression in vivo. Moreover, in our brown adipocyte model, we demonstrate that MR coregulators have a differential expression profile, consistent with a coordinated and physiologically relevant cooperation occuring during brown adipogenesis. In addition, we confirm in vitro the inhibitory effect of aldosterone on UCP1 expression (Uncoupling protein 1). Finally, we demonstrate in vivo that MR overexpression in brown adipose tissue of Tg mice induced a decrease in the cold-induced UCP1 expression. Taken together, these results provide a better understanding of MR involvement in the regulation of energy metabolism and should open new therapeutic oportunities such as the use of selective MR modulators in the management of metabolic disorders.

Hypertension is the chronic elevation in blood pressure that is regulated in part through the retention and regulation of sodium retention and excretion in the kidneys. Hence the kidney has been considered the organ that regulates blood pressure. There are a cohort of patients that suffer with high blood pressure due to lack of 11β-hydroxysteroid dehydrogenase-type 2 (11β-HSD2) expression (which inactivates glucocorticoids (GCs), allowing selective activation of mineralocorticoid receptors (MR) by aldosterone) that results in hypertensive and increased salt appetite phenotypes - a condition known as syndrome of apparent mineralocorticoid excess (SAME). This disorder can be recapitulated in the mouse through the global deletion of 11β-HSD2, which results in over activation of the MR driving an elevation in blood pressure. However, the distinction between blood pressure elevation because of kidney dysfunction with loss of 11β-HSD2 or increased salt appetite due to loss of brain 11β-HSD2 expression is not clear from the global 11β-HSD2 knockout model. Salt appetite is regulated by regions of the brain out-with the blood-brain barrier, known as circumventricular organs. In the mouse, salt appetite is controlled by aldosterone-sensitive cells in the nucleus of the solitary tract (NTS) in the brain stem, where 11β-HSD2 is expressed to provide mineralocorticoid selectivity. However, in the fetal brain, 11β-HSD2 is widely expressed, protecting against adverse GC action that alters brain development and increases susceptibility to psychiatric disorders as adults. 11β-HSD2 deletion solely in the brain from embryonic day 12 resulting in GC fetal programming (HSD2BKO) causes effects on both behaviour and salt appetite. To determine the role of developmental versus adult expression of brain 11β- HSD2, mice with deletion of brain 11β-HSD2 from mid gestation (HSD2BKO) and mice with adult deletion of 11β-HSD2 in the NTS using lentivirus (HSD2.v- BKD) were compared. The phenotypes (salt appetite, blood pressure (BP), baroreceptor response (BRR) and cognition), can be categorised as either due to GC fetal programming (as indicated by HSD2BKO groups), or increased activation of MR in adult 11β-HSD2 expressing neurons (recapitulated in the HSD2.v-Cre groups). Salt appetite increased in both HSD2BKO and HSD2.v-BKD cohorts (mean percentage increase 65% n=8 and 46% n=6, compared to their respective controls), leading to an increased BP in both groups (+12% and +8%, respectively) as well as an impaired BRR, indicating all phenotypes are mediated by adult NTS neurons. However, spatial recognition memory (Object-in-Place task) is abolished in HSD2BKO mice, whereas, HSD2.v-BKD mice still retain short-term memory. Our data suggest that neural 11β-HSD2 protects against inappropriate activation of MR by corticosterone to regulate salt appetite and salt-induced rises in blood pressure. However, spatial recognition memory is not influenced by deletion of 11β-HSD2 in the adult brain, confirmation that this phenotype is underpinned by developmental programming by GCs, which is observed in the 11β-HSD2 brain KO. Salt appetite has been shown to be centrally regulated through the adult deletion of 11β-HSD2. From this, our data suggest that an increased salt appetite is due to adult loss of function of 11β-HSD2 rather than GC programming during development. Highlighting the NTS as a region for drug delivery to try and control salt appetite in salt sensitive individuals who struggle with administering a recommended change in diet. To develop this further, minimally invasive modes of delivery of viruses and drugs into the brain were investigated. In so doing, a non-invasive and reversible method to temporarily disrupt the blood brain barrier (BBB) was optimised. The technique required acoustic insonation of ultrasonic contrast agents (CAs) (gas microbubbles) adjacent to the BBB. These microbubbles (SonoVueTM, Bracco) were delivered via tail vein injection into the vasculature. To target the BBB, an ultrasonic transducer was suspended and focused through coupling gel onto the area of interest in the brain with skull the intact. The optimisation of this technique required determination of the focal position of the 3.5MHz transducer that was utilised, in addition to optimisation of the pulse length, pulse repetition frequency and power output of the ultrasound beam to enable the BBB to be disrupted. In addition, measurement of the attenuation of the ultrasound beam through ex vivo mouse skulls were measured. These results showed a 50% reduction in pressure amplitude from the baseline of 335.2mV (Baseline mean = 100% +/-SEM 0 n=3 (No skull), five regions across the skull averaged 47.79% +/-SEM 1.913 n=25 (using 5 different animals). In in vivo mice, after co-injection of the microbubbles with Evans Blue and insonation of the brain, disruption of the BBB was confirmed by the presence of Evans Blue dye in the brain, with no measurable damage occurring in the brain. This was confirmed by cell and nuclear morphology with no red blood cell extravasation into the surrounding tissue. The parameters used to open the BBB used a peak negative pressure of 2.1MPa (single pulse), transducer frequency 3.5MHz, 35,000 cycles over a 10ms burst at a pulse repetition frequency of 10Hz. The technique when applied in vivo in recovery animals is speculated to work by the focused ultrasound causing the microbubbles to oscillate within the vasculature adjacent to the BBB, resulting in high-shear stresses being generated on the tight junctions within the BBB. The resultant gaps in the BBB allow free circulating compounds (e.g. large dye molecules (Evans Blue - 960.8g/mol molecular weight) and adeno-associated-viruses (25nm with a packing capacity of 4.5kb) within the blood to pass into the brain, but there is no penetration of red blood cells (7μm). Longitudinal mouse experiments demonstrated that within 12-hours these gaps close with no long-term damage observed. Currently, utilising this technique, successful passage of an adeno-associated virus expressing GFP (as a marker) has been shown to pass into the brain (n=6 for each cohort including control) - indicating that the virus requires the ultrasound and microbubbles to facilitate its movement into the brain. Further technique optimisation is being explored looking at the role of CAs used in the opening and disruption of the BBB, comparing composition and size of the CAs. Microbubbles (2-3μm) and nanobubbles (200nm) were compared as well as lipid and non-ionic surfactant surface compositions, using volume of drug delivery and degree of disruption as outputs. Using this technique, the hydrophilic drug mimic calcein was delivered into the brain (n=5 non-ionic surfactant nanobubble, n=5 lipid nanobubble). Results have indicated that the delivery of calcein is most efficient when using non-ionic surfactant nanobubbles as opposed to lipid nanobubbles - with a greater volume of the drug being delivered into the cerebral tissue. Furthermore, the concentration and surface composition of the nanobubble have an effect as to the size and potential damage to the brain when opening the BBB. In conclusion, it has been shown that it is possible to non-invasively open the BBB and deliver viruses and dye into the brain. In addition, this thesis has investigated the use of nanobubbles as both facilitators to opening the BBB and delivery vectors for potentially therapeutic drugs. Finally, a non-invasive opening of the BBB has been achieved using focused ultrasound. Ultimately this non-invasive opening of the BBB can be used to achieve delivery of larger molecules (such as antibodies and viruses) into the brain to target treatments. Focused ultrasound brain targeting can be applied to the potential treatment of salt appetite regulation in the NTS. For the individuals who suffer from salt sensitive hypertension, the NTS can be targeted to reduce the drive to ingest high salt diets. Furthermore, the continuation of research into the central control of BP, salt appetite and baroreceptor reflex control can become better understood, using less invasive delivery techniques to the brain.

Introdução: Crescentes evidências indicam que o abandono e o abuso infantis são fatores de risco para transtornos psiquiátricos. Estudos realizados tanto em animais como em humanos sugerem que o estresse nas fases iniciais de desenvolvimento pode induzir alterações persistentes na capacidade do eixo HPA em responder ao estresse na vida adulta e que esse mecanismo pode levar a uma maior suscetibilidade à depressão. Esta desregulação do eixo HPA parece estar relacionada às mudanças na capacidade dos glicocorticóides circulantes em exercer seu feedback negativo na secreção dos hormônios do eixo HPA por meio da ligação aos receptores de mineralocorticóides (MR) e glicocorticóides (GR) nos tecidos do eixo HPA. Objetivo: O objetivo deste trabalho foi avaliar a resposta do eixo HPA frente aos agonistas e antagonistas dos GR e MR em pacientes depressivos com e sem estresse precoce (EP) e controles. Metodologia: Selecionamos uma amostra total de 75 sujeitos composta por um grupo de pacientes com diagnóstico de episódio depressivo atual (n=47) e um grupo de controles saudáveis (n=28). Os pacientes foram divididos em 2 grupos de acordo com o estresse precoce: um grupo de pacientes depressivos com EP (n=33) e um grupo de pacientes depressivos sem estresse precoce (n=14). Os pacientes foram avaliados por meio da Mini Entrevista Neuropsiquiátrica Internacional (MINI-Plus), para a confirmação do diagnóstico. Para avaliação da gravidade dos sintomas depressivos foi aplicada a Escala de Depressão GRID de Hamilton (GRID-HAM-D21), sendo incluídos apenas pacientes com HAM-D21>=16. Para a avaliação do estresse precoce foi aplicado o Questionário Sobre Traumas na Infância (CTQ). Utilizamos também a Escala de Avaliação de Depressão de Montgomery-Asberg (MADRS), o Inventário de Depressão de Beck (BDI-II), o Inventário de Ansiedade de Beck (BAI), a Escala de Desesperança de Beck (BHS), a Escala de Ideação Suicida de Beck (BSI), a Escala de Impulsividade de Barratt (BIS-11) e o Questionário de Qualidade de Sono de Pittsburg (PSQI), para a avaliação dos sintomas psiquiátricos. A avaliação endócrina foi controlada por placebo, cego por parte dos controles e pacientes, não randomizada, onde os efeitos da fludrocortisona (0.5 mg), da prednisolona (5 mg), da dexametasona (0.5 mg) e da espironolactona (400mg) foram avaliados através do hormônio adrenocorticotrópico (ACTH) plasmático, do cortisol plasmático e salivar, da prolactina plasmática e do sulfato de desidroepiandrosterona (DHEA-S) plasmático. A secreção de cortisol salivar e dos hormônios plasmáticos foi avaliada em todos os sujeitos, após terem tomado no dia anterior às 22h: uma cápsula de placebo, fludrocortisona, prednisolona, dexametasona e espironolactona. A secreção de cortisol salivar foi avaliada às 22h após a tomada da medicação ou do placebo, ao acordar, 30 e 60 min após acordar e às 9h (antes da coleta plasmática), para avaliação da resposta do cortisol ao acordar (CAR) e do ritmo circadiano do cortisol (RC). Foi realizado também uma coleta plasmática as 9h nos dias seguintes após os desafios para medir o cortisol plasmático, o ACTH, o DHEA-S e a prolactina. Resultados: Os pacientes depressivos apresentaram níveis basais menores de cortisol salivar, de prolactina e de DHEA-S e níveis maiores na relação cortisol/DHEA-S. Não foram encontradas diferenças entre os pacientes depressivos e os controles nos níveis basais de ACTH, de cortisol plasmático, na CAR e no RC. Os pacientes depressivos apresentaram níveis menores de ACTH e de DHEA-S após a dexametasona e a fludrocortisona e tenderam a apresentar níveis menores de cortisol salivar após a fludrocortisona. Após a espironolactona encontramos níveis menores de ACTH, de cortisol salivar e de DHEA-S e níveis maiores no índice cortisol/DHEA-S nos pacientes depressivos. Os pacientes depressivos apresentaram também níveis menores de DHEA-S após a prednisolona, porém não foram encontradas diferenças entre os grupos nos demais hormônios avaliados após a prednisolona. Não foram encontradas diferenças no cortisol plasmático e na prolactina após os desafios entre os pacientes depressivos e os controles. Com relação à avaliação do estresse precoce nas medidas hormonais, encontramos uma tendência dos pacientes com EP apresentarem níveis menores basais de prolactina e após a fludrocortisona, a prednisolona, a dexametasona e a espironolactona do que os pacientes sem EP. No entanto, não foram encontradas diferenças entre os grupos nas demais medidas hormonais basais e após os desafios avaliadas neste estudo. Conclusão: Nossos achados fornecem evidências de que existem diversas alterações nas medidas hormonais relacionadas ao funcionamento do eixo HPA e de seus receptores GR e MR nos pacientes depressivos, associado à hipocortisolemia e um aumento do feedback inibitório mediado pelos GR e MR. Sugerem também o envolvimento da prolactina no desenvolvimento de quadros depressivos com estresse precoce, porém mais estudos são necessários para elucidarmos melhor a importância dos demais hormônios do eixo HPA e dos seus receptores em quadros depressivos com estresse precoce / Introduction: There are evidences indicating that child neglect and abuse are risk factors for psychiatric disorders. Studies that had as subjects animals or human suggest that stress in early phases of development may induce persistent changes in HPA axis response to stress in adulthood, which can lead to a greater susceptibility of developing depression. These abnormalities appear to be related to changes in the ability of circulating glucocorticoids and negative feedback on the secretion of HPA hormones through binding to glucocorticoid (GR) and mineralocorticoid receptors (MR) in HPA tissue. Aim: The aim of the present study was to assess HPA response after ingestion of GR and MR agonists and antagonists by depressive patients with and without early life stress (ELS) and controls. Methods: The sample was composed by a group of patients in current depressive episode (n=47), and a healthy control group (n=28). The depressed patients were divided in 2 groups, according to the presence or absence of ELS - a group with ELS (n=33) and a group without ELS (n=14). For diagnostic assessment, MINI International Neuropsychiatric Interview (MINI-Plus) was used. To assess the intensity of depressive symptoms, GRID-Hamilton Depression Rating Scale (GRIDHAM-D21) was applied, and for being included in the patient\'s group, subjects had to score >=16 in GRID-HAM-D21. To assess ELS, Childhood Trauma Questionnaire (CTQ) was applied. Other instruments were also used in the present study to assess psychiatric symptoms: Montgomery-Åsberg Depression Rating Scale (MADRS), Beck Depression Inventory II (BDI-II), Beck Anxiety Inventory, Beck Hopelessness Scale (BHS), Beck Scale for Suicide (BSI), Barratt Impulsiveness Scale (BIS-11), and Pittsburg Sleep Quality Index (PSQI). The neuroendocrine assessment was controlled using placebo, blind to subjects, and non-randomized. The effects of fludrocortisone (0.5 mg), prednisolone (5 mg), dexamethasone (0.5 mg), and spironolactone (400mg) were assessed by measuring plasmatic adrenocorticotropic hormone (ACTH), plasmatic and salivary cortisol, plasmatic prolactin, and plasmatic dehydroepiandrosterone sulfate (DHEA-S). The secretion of all plasmatic hormones was assessed in all subjects in blood collection sample at 9AM, after they took a pill containg placebo or fludrocortisone or prednisolone or dexamethasone or spironolactone, the day before, at 10 PM. The secretion of salivary cortisol assessed the day before 10 PM (after the ingestion of the pill), upon awakening, 30 minutes and 60 minutes after awakening, and at 9AM (before plasmatic collection), for assessed the cortisol awakening response (CAR) and the cortisol circadian rhythm (CR). At 9 AM there was a blood sample collection to assess plasmatic cortisol, ACTH, DHEA-S and prolactin. Results: Depressive patients presented lower basal levels of salivar cortisol, plasmatic prolactin and DHEA-S, and higher levels in the ratio cortisol/DHEA-S. There were no differences between depressive patients and healthy controls in basal levels of ACTH, plasmatic cortisol, in CAR, and in CR. Depressive patients had lower levels of ACTH and DHEA-S after dexamethasone and fludrocortisone, and there was a tendency of having lower salivary cortisol levels after fludrocortisone. After spironolactone, lower levels of ACTH, salivary cortisol, DHEA-S were found, and higher levels in ratio cortisol/DHEA-S were found in depressive patients. These patients also presented lower levels of DHEA-S after prednisolone, although there were no differences between groups concerning the levels of other hormones assessed after prednisolone. There were no differences found in plasmatic cortisol and prolactin levels after all challenges between depressive patients and controls. Considering ELS and hormonal level assessment, there was a tendency of patients with ELS of presenting lower levels of prolactin after placebo, fludrocortisone, prednisolone, dexamethasone, and spironolactone than patients without ELS. Nevertheless, there were no differences between these groups concerning the other hormonal basal levels and after the pharmachological challenges. Conclusion: Our findings provide evidence that there are several changes in hormonal levels related to the functioning of the HPA axis and its receptors GR and MR in depressive patients associated to hypocortisolism and the increase of negative feedback MR- and GR- mediated. Our data also suggest the role of prolactin in the development of depressive disorder with ELS, however, more studies are needed to better highlight the importance of other hormones of HPA axis and its receptors in depressive disorders with ELS

La grossesse induit de profonds changements hémodynamiques et métaboliques de l’organisme maternel qui ont des conséquences sur le cœur. L’adaptation du cœur à cette condition physiologique nécessite un remodelage de sa structure et par conséquent des ajustements de sa fonction. Les mécanismes responsables de ces adaptations sont en grande partie inconnus. Cependant, ces connaissances sont essentielles pour la compréhension des complications cardiovasculaires, telle que l’hypertension gestationnelle (HG), qui constituent un risque pour la santé de la mère et du fœtus. Afin de caractériser les adaptations du cœur lors de la grossesse, l’originalité de notre approche expérimentale consistait à étudier le remodelage à l’échelle des cardiomyocytes du ventricule gauche. Ainsi, notre premier objectif était de déterminer les modifications structurales et fonctionnelles des cardiomyocytes chez la rate en vue d’identifier les altérations lors de l’HG. Chez les rates gestantes, le remodelage structural des cardiomyocytes se caractérise par une hypertrophie cellulaire avec une augmentation proportionnelle des dimensions. L’HG a été induite par un supplément sodique (0.9% NaCl) dans la diète. L’inadaptation structurale lors de l’HG se traduit par une diminution du volume cellulaire. L’étude des modifications fonctionnelles a révélé que lors de la gestation le fonctionnement contractile des cellules est dépendant de l’adaptation du métabolisme maternel. En effet, les substrats énergétiques, lactate et pyruvate, induisent une augmentation de la contractilité des cardiomyocytes. Cet effet est plus faible dans les cellules des rates hypertendues, ce qui suggère des anomalies du couplage excitation-contraction, dans lequel les courants calciques de type L (ICa-L) jouent un rôle important. Paradoxalement, le lactate et le pyruvate ont induit une augmentation de la densité des courants ICa-L seulement chez les rates hypertendues. Le récepteur aux minéralocorticoïdes (RM) est connu pour son implication dans le remodelage structuro-fonctionnel du cœur dans les conditions pathologiques mais pas dans celui induit par la grossesse. Notre deuxième objectif était donc de déterminer le rôle du RM dans l’adaptation de la morphologie et de la contractilité des cardiomyocytes. Des rates gestantes ont été traitées avec le canrénoate de potassium (20 mg/kg/jr), un antagoniste des RM. L’inhibition des RM pendant la gestation empêche l’hypertrophie cellulaire. De plus, l’inhibition des RM bloque l’effet du lactate et du pyruvate sur la contractilité. Chez la femme, la grossesse est associée à des changements des propriétés électriques du cœur. Sur l’électrocardiogramme, l’intervalle QTc est plus long, témoignant de la prolongation de la repolarisation. Les mécanismes régulant cette adaptation restent encore inconnus. Ainsi, notre troisième objectif était de déterminer le rôle du RM dans l’adaptation de la repolarisation. Chez la rate gestante, l’intervalle QTc est prolongé ce qui est corroboré par la diminution des courants potassiques Ito et IK1. L’inhibition des RM pendant la gestation empêche la prolongation de l’intervalle QTc et la diminution des courants Ito. Les travaux exposés dans cette thèse apportent une vision plus précise du remodelage cardiaque induit par la grossesse, qui est permise par l’étude à l’échelle cellulaire. Nos résultats montrent que lors de la gestation et de l’HG les cardiomyocytes subissent des remodelages morphologiques contrastés. Notre étude a aussi révélé que lors de la gestation, la fonction contractile est tributaire des adaptations métaboliques et que cette relation est altérée lors de l’HG. Nos travaux montrent que la régulation de ces adaptations gestationnelles fait intervenir le RM au niveau de la morphologie, de la relation métabolisme/fonctionnement contractile et de la repolarisation. En faisant avancer les connaissances sur l’hypertrophie de la grossesse, ces travaux vont permettre d’améliorer la compréhension des complications cardiovasculaires gestationnelles. / Pregnancy is characterized by marked hemodynamic and metabolic changes, which have consequences on the heart. The adaptation of the heart to this physiological situation requires a remodeling of its structure, and consequently functioning adjustments. Mechanisms responsible for these adaptations are largely unknown. However, this knowledge is essential for the understanding of cardiovascular complications, such as gestational hypertension (GH), which represents a risk for the mother and the fœtus. To characterize cardiac adaptations to pregnancy, our experimental approach consisted in studying this remodelling at the level of left ventricle cardiomyocytes. Therefore, our first objective was to determine structural and functional modifications of cardiomyocytes in pregnant rats to be able to identify their variations in GH. In pregnant rats, structural remodelling of cardiomyocytes was characterized by a proportional volume expansion. GH was induced by a high sodium supplement (0.9% NaCl). In hypertensive rats, we observe significant cell volume shrinkage. The study of functional modifications elicited a strong relationship between metabolic adaptations and cell contractility. According to our results, in pregnant rats cardiomyocyte contractility was increased in presence of energy substrates lactate and pyruvate. This effect was weaker in the cells from hypertensive rats. This suggested modifications of the excitation-contraction coupling, in which L-type calcium currents (ICa-L) play an important role. Unexpectedly, lactate and pyruvate induced a significant increase in ICa-L only in hypertensive rats. In pathological conditions, mineralocorticoid receptors (MR) have been shown to mediate structural as well as functional remodelling of the heart. Our study is the first to investigate MR involvement in cardiac remodelling during pregnancy. Thus, our second objective was to determine MR involvement in cardiomyocyte remodelling. For this study, pregnant rats were treated with potassium canrenoate of (20 mg / kg / day), a MR antagonist. Our results revealed that MR inhibition during the pregnancy elicited a significant decrease of cell volume. MR inhibition has also affected metabolism and cellular functioning relationship. Indeed, plasma concentration of lactate was lower, which was in correlation with its blunted effect on cell contractility. In women, pregnancy-induced hypertrophy is associated with changes in electrical properties of the heart. Indeed, repolarisation is prolonged, which is characterised by a longer duration of QTc interval on the electrocardiogram. Regulation mechanisms involved in this adaptation are still largely unknown. Our third objective was therefore to determine the role of MR in the adaptation of repolarisation to pregnancy. Pregnancy induced a prolongation in QTc interval, which correlates with a decrease in potassium currents Ito and IK1. MR inhibition prevented QTc interval prolongation and the lowering of Ito. Our study gives a new insight of pregnancy-induced cardiac hypertrophy, which is provided by investigations at the cellular level. Our results demonstrate that pregnancy and GH are characterised by opposite remodellings. Moreover, in pregnancy the contractile function is dependent on metabolic adaptations. This is all the more glaring in GH as metabolic alterations induced modifications of electric properties to maintain contractile functioning. Furthermore, our work reveals MR involvement in the regulation of morphology, metabolism/contractility relationship, and repolarisation. By improving the knowledge of hypertrophy during pregnancy, this work contributes to improve the understanding of pregnancy-induced cardiac complications.

No description available.

Teneurin C-terminal associated peptides (TCAP) are a family of bioactive peptides found on the terminal exon of the four teneurin genes. TCAP-1 is found within brain regions that modulate the activity of corticotropin-releasing factor (CRF), which is the principal neuropeptide regulator of the hypothalamic-pituitary-adrenal (HPA) axis. TCAP-1 has suppressive effects on CRF-induced anxiety behaviours in rats. However, previous studies determined that TCAP-1 does not act directly on the CRF receptors (CRFR). Thus, I postulate that TCAP-1 may act centrally to modify elements of the HPA axis. Using an immortalized mouse hippocampal cell line, I tested the hypothesis that TCAP acts either downstream of CRFR activation, or on the regulation of the glucocorticoid receptors (GCR), which modulate CRF actions. These studies indicate that TCAP-1 represents a novel peptide in the regulation of stress related systems, which acts independently of either CRF-, or glucocorticoid- mediated signal transduction and transcription.

Teneurin C-terminal associated peptides (TCAP) are a family of bioactive peptides found on the terminal exon of the four teneurin genes. TCAP-1 is found within brain regions that modulate the activity of corticotropin-releasing factor (CRF), which is the principal neuropeptide regulator of the hypothalamic-pituitary-adrenal (HPA) axis. TCAP-1 has suppressive effects on CRF-induced anxiety behaviours in rats. However, previous studies determined that TCAP-1 does not act directly on the CRF receptors (CRFR). Thus, I postulate that TCAP-1 may act centrally to modify elements of the HPA axis. Using an immortalized mouse hippocampal cell line, I tested the hypothesis that TCAP acts either downstream of CRFR activation, or on the regulation of the glucocorticoid receptors (GCR), which modulate CRF actions. These studies indicate that TCAP-1 represents a novel peptide in the regulation of stress related systems, which acts independently of either CRF-, or glucocorticoid- mediated signal transduction and transcription.

Introdução: Diversos estudos sugerem que o estresse nas fases iniciais de desenvolvimento pode induzir alterações persistentes na capacidade do eixo Hipotálamo-Pituitária-Adrenal (HPA) em responder ao estresse na vida adulta. O desequilíbrio do cortisol tem sido identificado como um correlato biológico dos transtornos depressivos. Essas anormalidades parecem estar relacionadas às mudanças na capacidade dos glicocorticóides circulantes em exercer seu feedback negativo na secreção dos hormônios do eixo HPA por meio da ligação aos receptores mineralocorticóides (RM) e glicocorticóides (RG) nos tecidos do eixo HPA. Devido à grande variedade de estressores, assim como os diferentes subtipos de depressão, os achados dos estudos atuais têm sido inconsistentes. Dessa forma, necessitando de mais estudos para que se possa elucidar os mecanismos envolvidos na associação entre o Estresse Precoce (EP) e o desenvolvimento de quadros depressivos. Objetivo: O objetivo deste estudo é avaliar a correlação entre Estresse Precoce e alterações no eixo Hipotálamo-Pituitária-Adrenal e na função dos receptores glicocorticóides e mineralocorticóides em pacientes depressivos. Metodologia: Foram recrutados inicialmente 30 sujeitos divididos em dois grupos: grupo de pacientes com diagnóstico de episódio depressivo atual (n=20) e grupo de controles (n=10). Posteriormente os pacientes foram divididos em outros dois grupos de acordo com o EP, compondo a amostra final por três grupos: grupo de pacientes depressivos com presença de EP (n=13), grupo de pacientes depressivos com ausência de EP (n=7) e grupo de controles (n=10). Os pacientes foram avaliados por meio de Entrevista Clínica de acordo com os critérios diagnósticos do DSM-IV, para a confirmação do diagnóstico. Para avaliação da gravidade dos sintomas depressivos foi aplicada a Escala de Depressão de Hamilton (HAM-D21), sendo incluídos apenas pacientes com HAM-D21 17. A presença de EP foi confirmada através da aplicação do Questionário Sobre Traumas na Infância (QUESI). Foram utilizados também a Escala de Avaliação de Depressão de Montgomery-Asberg (MADRS), o Inventário de Depressão de Beck (BDI), o Inventário de Ansiedade de Beck (BAI), a Escala de Ideação Suicida de Beck (BSI), a Escala de Desesperança de Beck (BHS), a Escala Hospitalar de Ansiedade e Depressão (HAD) e a Escala de Impulsividade de Barratt (BIS-11) para a avaliação de sintomas psiquiátricos. A avaliação endócrina foi controlada por placebo, cego por parte dos controles e pacientes, não randomizado, com desenho de medidas repetidas, onde os efeitos da Fludrocortisona (0.5 mg) e da Dexametasona (0.5 mg) foram avaliados através do cortisol salivar e plasmático. A secreção de cortisol plasmático e salivar foi avaliada nos sujeitos, após a administração de uma cápsula de Placebo, Fludrocortisona e Dexametasona às 22hs do dia anterior. O cortisol salivar foi coletado às 22h, ao acordar, 30 e 60 minutos após acordar e antes da coleta plasmática, nos dias seguintes após os desafios. Resultados: Na amostra de pacientes depressivos e controles, encontramos níveis significativamente menores de cortisol salivar ao acordar após a administração de Placebo nos pacientes depressivos comparados aos controles. Encontramos também uma tendência dos pacientes apresentarem níveis maiores de cortisol salivar ao acordar do que os controles após a administração de Dexametasona. Quando avaliado o cortisol após a administração de Fludrocortisona, os pacientes apresentaram níveis significativamente menores de cortisol salivar 30 minutos após acordar e na Área Sob a Curva (AUC) do que os controles. Além disso, encontramos também uma tendência dos pacientes depressivos apresentarem níveis menores de cortisol salivar 60 minutos após acordar do que os controles. Quando comparados entre pacientes depressivos com presença e ausência de EP e controles, encontramos uma tendência dos pacientes depressivos com ausência de EP apresentarem níveis menores de cortisol salivar ao acordar após Placebo do que os controles. As médias dos níveis de cortisol salivar ao acordar não diferiram entre os pacientes com presença de EP e os controles e entre os pacientes do grupo presença e ausência de EP. Com relação aos níveis de cortisol salivar após a administração de Dexametasona entre pacientes depressivos com presença e ausência de EP e controles, os pacientes depressivos com ausência de EP apresentaram níveis significativamente maiores de cortisol salivar ao acordar do que os controles. Encontramos também uma tendência dos pacientes com ausência de EP apresentarem níveis maiores de cortisol salivar ao acordar do que os pacientes com presença de EP, porém não foram encontradas diferenças significativas entre os pacientes com presença de EP e os controles. Conclusão: Nossos dados demonstram uma hipoatividade do eixo HPA nos pacientes depressivos. Além disso, estes achados sugerem que esta desregulação do eixo HPA se deva em parte a uma diminuição da sensibilidade dos RG e uma hiperativação dos RM nos pacientes depressivos. No entanto, quando comparados pacientes depressivos com presença e ausência de Estresse Precoce, os desafios com agonistas seletivos como a Dexametasona (agonista RG) e a Fludrocortisona (agonista RM) não foram capazes de detectar esta diferença fisiopatológica e distinguir entre os diferentes tipos de psicopatologia. Dessa forma, estes resultados sugerem que estudos com um agonista misto (RG/RM) como a Prednisolona teriam potencial para distinguir os pacientes depressivos com presença de Estresse Precoce. / Introduction: Several studies suggest that stress in early stages of development can induce persistent changes in the ability of the Hypothalamic-Pituitary-Adrenal (HPA) axis to respond to stress in adulthood. The imbalance of cortisol has been identified as a biological correlate of depressive disorders. These abnormalities seem to be related to changes in the ability of circulating glucocorticoids to practice their negative feedback on the secretion of HPA axis hormones through connecting to the mineralocorticoid receptor (MR) and glucocorticoid (GR) in the tissues of HPA axis. Due to the wide variety of stressors, as well as the different subtypes of depression, the findings of current studies have been inconsistent. Thus, more studies need to be able to elucidate the mechanisms involved in the association between Early Life Stress (ELS) and the development of depression. Objective: The objective this study is to evaluate the correlation between of Early Life Stress and changes in Hypothalamic-Pituitary-Adrenal axis and at receptors function glucocorticoid and mineralocorticoid in depressive patients. Methodology: We recruited 30 subjects initially divided into two groups: patients with current depressive episode (n =20) and control group (n = 10) Subsequently, patients were divided into two groups according to the ELS, making the final sample of three groups: depressive patients with ELS (n =13) group of depressive patients without ELS (n=7) and control group (n=10). Patients were evaluated by clinical interview according to the diagnostic criteria of DSM-IV to confirm the diagnosis. To evaluate the severity of depressive symptoms was applied to the Hamilton Depression Scale (HAM-D21), and included only patients with HAM-D21 17. The presence of ELS was confirmed by the Childhood Trauma Questionnaire (CTQ). We also used the Depression Rating Scale Montgomery-Asberg (MADRS), the Beck Depression Inventory (BDI), the Beck Anxiety Inventory (BAI), the Scale for Suicide Ideation Beck (BSI), the Scale Beck Hopelessness (BHS), the Hospital Anxiety and Depression Scale (HADS) and the Barratt Impulsiveness Scale (BIS-11) for the assessment of severity psychiatric symptoms. Endocrine evaluation was placebo-controlled, blinded by the patients and controls, non-randomized design with repeated measures, where the effects of Fludrocortisone (0.5 mg) and dexamethasone (0.5 mg) were assessed using salivary cortisol and plasma. The secretion of plasma cortisol and salivary was evaluated in the subjects, after administration of a capsule of Placebo, Fludrocortisone and Dexamethasone to 22hs the previous day. The salivary cortisol was collected at 22h, on waking, 30 and 60 minutes after waking and before plasma collection in the following days after the challenges. Results: In these sample of depressed patients and controls, we found significantly lower levels of salivary cortisol around waking after administration of Placebo in depressed patients than controls. We also found a trend for patients to have higher levels of salivary cortisol than controls on awakening after administration of Dexamethasone. When measured cortisol after administration of Fludrocortisone, patients showed significantly lower levels of salivary cortisol 30 minutes after waking and the Area Under the Curve (AUC) than controls. In addition, we also found a tendency for depressed patients showed lower levels of salivary cortisol 60 minutes after awakening than controls. When compared between depressed patients with and without ELS and controls, we found a tendency for depressed patients without ELS presented lower levels of salivary cortisol on awakening after Placebo than controls. The mean salivary cortisol levels on waking did not differ between patients with ELS and controls and between patients with and without ELS. The levels of salivary cortisol after Dexamethasone administration between depressed patients with and without ELS and controls, depressed patients without ELS had significantly higher levels of salivary cortisol on awakening than controls. We also found a trend for patients without Early Life Stress have higher levels of salivary cortisol upon waking than patients with Early Life Stress, but there were no significant differences between patients with Early Life Stress and controls. Conclusion: Our data show a hypoactivity of the HPA axis in depressed patients. Moreover, these findings suggest that this dysregulation HPA axis is partly due to a decrease the sensitivity of RG and a hyperactivation of MR in patients depressive. However, when compared depressed patients with and without Early Life Stress, the challenges with selective agonists as the Dexamethasone (agonist GR) and Fludrocortisone (agonist MR) were not able to detect this difference pathophysiological and distinguish between the different types of psychopathology. Thus, these results suggest that studies with a mixed agonist (GR/MR) such as Prednisolone have potential to distinguish of depressive patients with Early Life Stress.

Alterações no sistema glutamatérgico e mudanças no funcionamento do córtex pré-frontal medial (CPFm) têm sido associadas a diversos distúrbios psiquiátricos, dentre os quais a ansiedade. Também é reconhecido que alterações nas concentrações circulantes de glicocorticóides podem induzir alterações nas sinapses e circuitos glutamatérgicos e, consequentemente, modificar a reatividade emocional dos animais. Embora se saiba que os glicocorticóides influenciam a liberação de glutamato no CPFm, a interação entre os efeitos mediados pelos receptores mineralocorticóides (MR) ou glicocorticóides (GR) e o sistema glutamatérgico, na expressão da resposta condicionada de medo, ainda não está elucidada. Nesse sentido, os objetivos do presente estudo foram investigar (i) a influência dos glicocorticóides na expressão do medo condicionado contextual e seus efeitos sobre a atividade do CPFm em ratos, (ii) o papel dos receptores MR e GR localizados no córtex prelímbico (PrL) na expressão da resposta condicionada de congelamento e (iii) a interação entre os mecanismos mediados pelos glicocorticoides e o sistema glutamatérgico, via receptores do tipo NMDA, na expressão dessa resposta. Ratos Wistar machos foram tratados com veículo ou metirapona, um bloqueador de síntese de corticosterona, e expostos a um contexto previamente pareado com choque nas patas. Foram avaliados o tempo de medo contextual (comportamento de congelamento) e a expressão de proteína Fos em diferentes regiões do CPFm. Os resultados mostraram que a exposição ao contexto aversivo levou a um aumento significativo da expressão de congelamento e de proteína Fos no PrL, nas áreas do córtex cingulado anterior 1 e 2 (Cg1 e Cg2), mas não no córtex infralímbico. A administração de metirapona levou a uma diminuição da expressão de congelamento e de proteína Fos no PrL, Cg1 e Cg2. A administração bilateral de espironolactona, um antagonista de receptores MR, no PrL antes do teste diminuiu as respostas de medo e o pré-tratamento com RU38486, um antagonista de receptores GR, aboliu este efeito. Os resultados também mostraram que a diminuição da resposta de congelamento induzida por injeções intra-PrL de corticosterona foi abolida pela administração prévia de RU38486, mas não por espironolactona, indicando que a corticosterona recruta preferencialmente os receptores GR para produzir esses efeitos. A administração prévia do antagonista de receptor NMDA também preveniu os efeitos induzidos pelo tratamento com corticosterona sugerindo que, no PrL, parte dos efeitos rápidos do glicocorticóides sobre a expressão do medo condicionado se dá por uma interação com o sistema glutamatérgico. A administração de NMDA no PrL, antes do teste, induziu efeitos similares ao tratamento com corticosterona nessa região. De modo geral, os resultados sugerem que a liberação de corticosterona durante a apresentação de um estímulo condicionado aversivo influencia a atividade do CPFm de maneira que, uma mudança no equilíbrio das atividades mediadas por MR e GR, por meio de um aumento da atividade de GR, interage com o sistema glutamatérgico via aumento da atividade dos receptores NMDA influenciando a expressão da resposta de medo condicionado contextual. Sugere-se que a redução na expressão do medo condicionado observada após a administração local de corticosterona no PrL também seja decorrente de mudanças no equilíbrio entre MR e GR em direção a um aumento de suas ações mediadas por GR, assim como um aumento na liberação de glutamato e maior atividade de receptores NMDA nessa região. / Changes in the glutamatergic system and in the functioning of the medial prefrontal cortex (mPFC) have been associated with different psychiatric disorders, including anxiety. It is also recognized that changes in circulating levels of glucocorticoids can induce changes in glutamatergic synapses and circuits and therefore alter the emotional reactivity of animals. Although is known that glucocorticoids can influence the release of glutamate in the mPFC, the interaction between mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) activation and the glutamatergic activity on the expression of conditioned fear response is not yet elucidated. The aims of the present study were to investigate (i) the influence of glucocorticoids on the expression of contextual conditioned fear and its effects in the activity of the mPFC in rats, (ii) the role of MR and GR in the prelimbic cortex (PrL) on expression of conditioned freezing response and (iii) a possible interaction between the effects mediated by the glucocorticoids and the glutamatergic system, via NMDA receptors on the expression of this response. Male Wistar rats were treated with vehicle or metyrapone, a corticosterone synthesis blocker, and exposed to a context previously paired with footshock. The time of contextual fear (freezing behavior) and Fos protein expression in different regions of mPFC were evaluated. The results showed that exposure to the aversive context induced a significant increase in freezing and Fos protein expression in the PrL, in the anterior cingulate cortex, areas 1 and 2 (Cg1 and Cg2), but not in the infralimbic cortex. The administration of metyrapone induced a decrease on the expression of freezing and Fos in PrL, Cg1 and Cg2. Bilateral administration of spironolactone (a MR antagonist) in PrL before the test, decreased conditioned fear response and the pretreatment with RU38486 (a GR antagonist) abolished this effect. The results also showed that the decrease of freezing response induced by intra-PrL corticosterone injections was abolished by prior administration of RU38486, but not by spironolactone, indicating that corticosterone recruits preferentially GR to produce the observed effects. Prior administration of the NMDA receptor antagonist also prevented the effects induced by corticosterone treatment in the PrL, suggesting that part of rapid effects of glucocorticoids on the expression of conditioned fear occurs by an interaction with the glutamatergic system. Additionally, NMDA administration in the PrL prior to the test induced similar effects to corticosterone treatment in this region. Overall, the results suggest that the release of corticosterone during the presentation of a conditioned aversive stimulus influences the mPFC activity so that a change in the balance of the activities mediated by MR and GR through an increase in GR activity interacts with the glutamatergic system by increasing the activity of NMDA receptors influencing the expression of contextual fear conditioning response. It is suggested that the reduction in the expression of conditioned fear observed after local administration of corticosterone in the PrL is also due to changes in the balance between MR and GR towards an increase in the actions mediated by GR, as well as an increase in the release of glutamate and a greater NMDA receptor activity in this region.