O estresse provoca alterações significativas no metabolismo dos animais, provocando a liberação de hormônios glicocorticoides. Estas alterações do metabolismo têm efeito anabólico sobre o metabolismo proteico muscular, podendo afetar os processos bioquímicos de transformação do músculo em carne. O presente trabalho teve como objetivo geral (i) verificar as relações entre variáveis endócrinas e metabólicas associadas ao estresse e características indicadoras de qualidade da carne, em animais castrados e não-castrados; (ii) avaliar a expressão gênica dos receptores mineralocorticoide (MR) e glicocorticoide (GR) em variáveis endócrinas, metabólicas e relacionadas à qualidade da carne de bovinos Nelore castrados e não-castrados. Para tal, 130 animais foram abatidos entre os anos de 2009 e 2011. Amostras de sangue foram coletadas antes e depois do abate para mensuração das concentrações de ACTH e cortisol. Amostras do músculo Longissimus dorsi foram coletadas durante os abates para mensuração do glicogênio e lactato, bem como, para análises de expressão gênica (RT-qPCR). Para as análises de maciez, foram coletadas amostras maturadas por um, sete e 14 dias. Para expressão gênica foram determinados os genótipos dos animais para três marcadores relacionados ao MR (MR1_1, MR1_2 e MR1_3) e dois ao GR (GR2_1 e GR2_2), por meio de PCR em tempo real. Foi verificado que animais castrados apresentam pH 24 horas menores e carnes mais macias ao sétimo e 14º dias de maturação, bem como, concentrações de cortisol (in vivo e post mortem) e lactato significativamente superiores aos animais não-castrados. O marcador MR1_3 apresenta expressão gênica significativamente diferenciada. Os animais com genótipo GA apresentaram 57,27% mais transcritos quando comparados aos animais GG. A expressão gênica do MR e GR foi significativamente relacionada às concentrações de cortisol in vivo e post mortem, porém não influenciou as concentrações de ACTH (in vivo e post mortem), glicogênio e lactato. A expressão gênica do MR e GR não foi relacionada às características indicadoras da qualidade da carne. / The stress causes significant changes in the metabolism of the animals causing the release of glucocorticoid hormones. These metabolic changes have anabolic effect on muscle protein metabolism, affecting the biochemical processes of transformation of muscle on meat. This study aimed to (i) examine relationships between endocrine and metabolic variables associated with stress and meat quality characteristics in castrated and non-castrated animals, (ii) evaluate mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in endocrine and metabolic characteristics and related this to meat quality of Nellore castrated and non-castrated animals. To this end, 130 animals were slaughtered between the years 2009 and 2011. Blood samples were collected before and after slaughter to measure concentrations of ACTH and cortisol. Longissimus dorsi muscle samples were collected during slaughter for measurement of glycogen and lactate, as well for gene expression analyzes (RT-qPCR). For the shear force analyzes, samples were aged for one, seven and 14 days. For gene expression analysis, genotypes of three markers related to MR (MR1_1, MR1_2 and MR1_3), and the two related to GR (GR2_1 and GR2_2) were determined via real-time PCR. It was observed that castrated have lower pH value at 24 hours than non-castrated animals, and tender meat on the seventh and 14th day of aging, such as cortisol (in vivo and post mortem) and lactate concentrations significantly superior to non-castrated animals. Gene expression of MR1_3 was significantly different. Animals with GA genotype had 57.27% more transcripts than GG genotype. The gene expression of MR and GR was significantly related to cortisol concentrations in vivo and post mortem, but did not influence the concentrations of ACTH (in vivo and post mortem), glycogen and lactate. The MR and GR gene expression was not related to the meat quality characteristics.

Endocrine Disrupting Chemicals (EDC) are exogenous agents that mimic endogenous hormone activity in the body. EDC exposure during the critical period of neonatal development can potentially cause life-long neurological, behavioural and physiological disease. This thesis focuses on the EDC Bisphenol A (BPA), a synthetic xenoestrogen widely prevalent in everyday materials that has significant environmental relevance given its ubiquitous presence in humans around the world. The central research question of my thesis is: Does perinatal exposure to BPA affect rat pup development? A rodent model was selected to study the effects of BPA on the adrenal component of the hypothalamic-pituitary-adrenal axis (HPA axis) stress pathway, which has not been extensively studied. Rat dams were divided into five groups (vehicle control (VEH), positive control diethylstilbestrol (DES), BPA 5, BPA 50 and BPA 500 μg/kg bw/day) and dosed daily throughout gestation and for four days of lactation. Rat pups were sacrificed at two time-points at the beginning and the end of the stress hyporesponsive period (SHRP), at postnatal day (PND) 5 and PND 15. Changes in three morphometric parameters (bodyweight, crown-rump (CR) length and anogenital distance (AGD) were assessed based on the factors of Treatment and Sex. Adrenal gland glucocorticoid receptor (GR) and 18SrRNA expression was determined by qPCR in male pups at PND 5 and PND 15. At PND 5, compared to the VEH group, the BPA 50 pups were significantly heavier (ANOVA, Dunnett’s post-hoc) and the DES and BPA 50 pups had significantly longer CR lengths (ANOVA, Dunnetts’ post-hoc). At PND 15, xenoestrogen treatment significantly influenced CR length (ANOVA). At both time-points, males had significantly longer AGD than females, as physiologically expected (ANOVA). Adrenal gland GR expression in male pups was not significantly affected by treatment, but there was an effect of treatment in18SrRNA gene expression at PND 5 (Kruskal-Wallis). Using the Ct method to determine GR and 18SrRNA fold changes, we cautiously suggest that our experimental doses resulted in a non-monotonic dose response to BPA in the PND 5 animals and a monotonic dose response to BPA exposure in the PND 15 animals. This study highly values the importance of investigating the effects of environmentally relevant, low dose BPA exposure during the critical window of development, given the little that is known about potentially permanent alterations to the stress pathway due to exposure during this delicate period of development.

Dans le système nerveux central (SNC), l'hippocampe est une structure majeure pour les fonctions cognitives et comportementales. Le Brain-Derived Neurotrophic Factor (BDNF), un acteur clé dans ces fonctions neuronales, est fortement exprimé dans l'hippocampe. La structure du gène Bdnf murin est complexe, comportant 8 exons non codants (I à VIII), chacun avec un promoteur spécifique (1 à 8) et un exon IX codant commun. Les glucocorticoïdes (GC) exercent des actions pleiotropes sur ces processus neuronaux en se liant et en activant le récepteur des glucocorticoïdes (GR), et le récepteur des minéralocorticoïdes (MR). Le GR est un facteur de transcription, modulant la transcription de ses gènes cibles, en se liant directement aux éléments de réponse des glucocorticoïdes ou en interagissant indirectement sur d’autres facteurs de transcription. Il a été suggéré que l'expression de Bdnf est régulée par le stress et les concentrations élevées de GC. Cependant, il reste à définir si BDNF est un gène cible du GR et quels sont les mécanismes moléculaires impliqués. Dans ce travail, nous avons démontré que les fortes concentrations de GC diminuent l'expression de l'ARNm de Bdnf via le GR dans divers modèles cellulaires neuronaux. Dans des cultures primaires de neurones hippocampiques de souris et dans les cellules BZ, les transcrits de BDNF contenant l’exon IV et VI sont reprimés par le GR. Par ailleurs les transfections transitoires démontrent que l’activité du promoteur 4 est diminuée par GR. Les expériences de mutagenèse et de ChIP ont révélé que la répression induite par le GR sur l'expression et l’activité transcriptionnelle de Bdnf implique un petit fragment de 74 bp situé dans le promoteur en amont de l'exon IV. La localisation précise de l’interaction génomique du GR et les facteurs de transcription potentiels mis en jeu restent à identifier. Ce travail a contribué à une meilleure compréhension des mécanismes impliqués dans la régulation de l’expression de Bdnf par GR. Il apporte de nouveaux éléments sur les interactions moléculaires et fonctionnelles entre la signalisation GC et celle de BDNF dans les neurones, d’importance majeure dans la physiopathologie du SNC. / In the central nervous system (CNS), the hippocampus is a structure of major importance for cognitive and behavioral functions. The brain-derived neurotrophic factor (BDNF), a key player in such neuronal functions is highly expressed in the hippocampus. Rodent Bdnf gene structure is relatively complex, composed of 8 noncoding exons (I to VIII), each one with a specific promoter (1 to 8), and one common coding exon IX. Glucocorticoids (GC) exert pleiotropic actions on neuronal processes by binding to and activating the glucocorticoid receptor (GR), as well as the mineralocorticoid receptor (MR). GR functions as a transcription factor, directly by interacting to glucocorticoid response elements or indirectly by interacting with other transcription factors, leading to the regulation of target gene transcription. It has been suggested that Bdnf expression is regulated by stress and high GC concentrations. However, it remains to define whether Bdnf is a GR target gene and what are the underlying molecular mechanisms. Herein, we demonstrate that high GC levels downregulate total Bdnf mRNA expression via GR in various in vitro neuron-like cellular models. In primary cultures of mouse hippocampal neurons and BZ cells, BDNF IV- and VI-containing transcripts are involved in this regulatory mechanism. Moreover, in transient transfections, promoter 4 activity was reduced by activated GR. Furthermore, ChIP analysis and mutagenesis experiments demonstrate that the GR-induced repression on Bdnf expression and transcriptional activities occurs through GR binding to a small 74 bp promoter sequence upstream of exon IV. The exact GR binding site on DNA and its putative transcription factor partners are currently under investigation. Altogether, these findings contribute to a better understanding of the mechanisms by which GR represses BDNF expression. Our study brings new insights into the molecular interactions between GC signaling and BDNF signaling in neurons, both important pathways in the pathophysiology of the CNS.

No description available.

Ainda são escassos estudos que avaliem biomarcadores para diferenciação de transtorno depressivo maior (TDM) e transtorno bipolar (TB), principalmente relativo à etiologia desses transtornos e sua relação com os receptores glicocorticoides (GR) e, principalmente, com os receptores mineralocorticoides (MR). Objetivo: Encontrar biomarcadores genéticos e/ou hormonais e observar sua associação entre si e/ou a fatores externos (estresse precoce - EP) para compreender melhor sua fisiopatogenia e auxiliar no diagnóstico diferencial entre TDM e TB. Material e Métodos: Participaram deste estudo N=273 sujeitos, sendo n=113 controles, n=78 unipolares e n=82 bipolares. A triagem diagnóstica de todos os sujeitos foi realizada por meio do MINI PLUS, checagem de história de trauma na infância pela CTQ, avaliação de sintomas depressivos pela GRID-HAM-D21, e demais comorbidades pela BAI, BHS e BSI. Na busca de biomarcador genético, observou-se as frequências genotípicas e alélicas de 3 polimorfismos de receptor de glicocorticoide (GR) (N363S, R22/23K e BclI) e de 2 polimorfismos de MR (MI180V e -2G/C) após realizada a discriminação alélica por reação em cadeia da polimerase quantitativa (qPCR). Foram avaliados de forma intragrupo as variáveis genéticas e endócrinas (e combinadas) e o efeito do EP sobre tais variáveis. Também, as variáveis polimorfismos, níveis hormonais e exposição a EP foram comparadas entre grupos para avaliar se havia diferença de prevalência, de perfil endócrino, ou se havia suscetibilidade maior por parte dos unipolares ou bipolares para alteração dos níveis hormonais e/ou intensidade do quadro depressivo frente a EP ou a determinado genótipo. Resultados: Todos os sujeitos unipolares e bipolares mostraram piora de seus sintomas depressivos frente a EP e seus subtipos, sendo eles unipolares ou bipolares. Como biomarcador hormonal, comparando-se controles x unipolares x bipolares, ou apenas unipolares x\' bipolares, foi possível observar que os níveis de cortisol e os níveis de aldosterona apresentaram-se os altos em unipolares e os baixos mais em bipolares, quando estes pacientes estavam com depressão grave ou gravíssima. Também, bipolares expostos a EP global, abuso físico e emocional mostraram níveis mais baixos de aldosterona que bipolares que não foram expostos. Frente a exposição a esses EP global e abuso físico, os bipolares tenderam a se mostrar mais suscetíveis que os unipolares a alteração dos níveis de aldosterona. Para biomarcador genético, frequência de genótipos ou alelos não diferenciaram unipolares de bipolares. Entretanto, houve maior prevalência do genótipo heterozigoto AG de GR N363S em pacientes depressivos uni e bipolares quando comparados com controles. Combinando-se os biomarcadores genéticos e hormonais, unipolares apresentaram níveis mais baixos de cortisol e de aldosterona quando carregavam genótipo variante GG de MR -2G/C, enquanto bipolares mostraram tendência a redução de cortisol quando carregavam o alelo variante G de MR MI180V. Quando comparados os genótipos por si só, intragrupo, novamente o polimorfismo MR -2G/C mostra influência sobre o fenótipo unipolar. Em unipolares, presença do alelo variante G de MR -2G/C piora significativamente o quadro depressivo, mas o alelo variante G de MI180V mostrou-se protetor frente a EP. Tanto os unipolares frente aos outros 4 polimorfismos, quanto os bipolares frente a todos os polimorfismos estudados, apresentaram piora significativa de seu quadro depressivo se expostos a EP. Bipolares mostraram uma tendência a ser mais suscetíveis que unipolares a alterações endócrinas (aldosterona) quando expostos a EP global e abuso físico. Conclusão: Tendo em vista os vários achados significativos a cerca dos polimorfismos de MR, tanto para unipolar quanto para bipolar, sua influência sobre os níveis de aldosterona e cortisol basais, reforça-se a importância do papel dos receptores MR dentro da etiologia dos transtornos depressivos unipolares e bipolares, e a forma diferente de funcionamento do MR para a distinção entre TDM e TB / There are still few studies assessing biomarkers for differentiation of major depressive disorder (MDD) and bipolar disorder (TB), mainly related to the etiology of these disorders and its relationship with glucocorticoid receptors (GR) and, manily, with mineralocorticoid receptors (MR). Aim: Finding genetic and / or hormonal biomarkers and observing their association to each other and / or external factors (early-life stress - ELS) for better comprehend their pathophysiology and, then, assisting in differential diagnosis between MDD and TB. Material and Methods: A total of N = 273 subjects composed the study sample, being n = 113 control, n = 78 unipolar, and n = 82 bipolar subjects. The diagnostic screening of all subjects was performed applying MINI PLUS, for history of ELS, CTQ; assessment of depressive symptoms, GRID-HAM-D21; and assessment of other comorbidities, BAI, BHS, and BSI. Researching for genetic biomarker, genotypic and allelic frequencies of 3 GR polymorphisms (N363S, R22 / 23K and BclI) and 2 MR polymorphisms (MI180V and -2G/C) were evaluated after allelic discrimination by quantitative polymerase chain reaction (qPCR). Genetic and endocrine variables (and their combination), and the effect of ELS over these variables were assessed intragrups. Also, polymorphisms, hormonal levels and history to ELS were compared between groups to assess whether there was difference in prevalence, endocrine profile, or whether there was greater susceptibility on the part of unipolar or bipolar for alteration of hormonal levels and / or severity of depressive symptoms considering history of ELS and/or a specific genotype. Results: All unipolar and bipolar subjects showed worsening of their depressive symptoms in the presence of ELS and its subtypes. As hormonal biomarker, comparing unipolar x bipolar x control subjects, or comparing unipolar x bipolar, cortisol and aldosterone levels were higher in unipolar subjects, and lower in bipolar subjects, when these patients presented severe or very severe depressive symptoms. Also, bipolar subjects\' exposed to global ELS, physical and emotional abuse showed lower basal levels of aldosterone than did bipolar who were not exposed to ELS. Concerning global ELS and physical abuse, bipolar tended to be more susceptible than unipolar for aldosterone levels to change. For genetic biomarker, frequency of genotypes or alleles did not distinguished unipolar from bipolar sample. However, there was a higher prevalence of GR N363S heterozygous genotype (AG) in unipolar and bipolar depressive patients when compared to controls. Combining the genetic and hormonal biomarkers, unipolar had lower levels of cortisol and aldosterone when carrying GG variant genotype of MR-2G / C, while bipolar showed tendency to reduce cortisol when carrying the variant G allele of MR MI180V. When comparing the genotypes (intragroup), again, MR-2G/C polymorphism shows influence on the unipolar phenotype. In unipolar, the presence of the variant G allele of MR-2G / C significantly worsens the depressive condition, unlike variant G allele of MI180V has shown to be protective against ELS. Both the unipolar compared to the other 4 polymorphisms, and the bipolar ones against all polymorphisms studied, presented a significant worsening of their depressive condition if exposed to ELS. Bipolar tend to be more susceptible than unipolar to endocrine changes (aldosterone) when exposed to global ELS and physical abuse. Conclusion: Considering the several significant findings regarding MR polymorphisms, for both unipolar and bipolar subjects, and their influence on basal aldosterone and cortisol levels, we highlight importance of the role of MR receptors within the etiology of depressive unipolar and bipolar disorders, and different way of MR functioning in each disorder for assisting the distinction between MDD and TB

Aktivator Protein-1 (AP-1) ist ein kollektiver Terminus für dimerische Transkriptionsfaktoren, die sich aus Fos- und Jun- Proteinen zusammensetzen. Diese Untereinheiten binden an eine gemeinsame, spezifische DNA-Sequenz, die AP-1 Bindungsstelle. Zusätzlich zu der gut dokumentierten Rolle des c-Fos Proteins in der Tumorgenese, wo dieses Gen als ein Aktivator beschrieben ist, übt AP-1 einen Einfluss auf mesenchymale Stromazellen und Immunzellen aus. Mesenchymale Knochenmarkszellen sind die Vorläuferzellen für Adipozyten, Osteoblasten, Chondrozyten, Myozyten und Fibroblasten. Die molekularen Mechanismen, welche die Differenzierungen regeln, sind noch weitgehend unerforscht. Der heterodimere Transkriptionsfaktor AP-1 übt eine wichtige Rolle in der Kontrolle der Zelldifferenzierung aus. Verschieden genetisch veränderte Mausmodelle untermauerten dies. Mäuse, welche das Fos-related antigen-1 (Fra1) oder eine kürzere Protein-Isoform von FosB (deltaFosB) überexpremieren, entwickelten, durch eine beschleunigte Differenzierung der Osteoblasten, eine Osteosklerose. Interessanterweise konnte gezeigt werden, dass die transgenen deltaFosB Mäuse weniger Fett haben. Die Stabilität und Aktivität von Fos Proteinen kann durch post-transkriptionale Modifizierungen geregelt werden. Basierend auf knockout Mausmodellen, wurde eine tragende Rolle für das wachstumsregulierende Enzym Rsk2 postuliert. Rsk2 spielt eine mögliche Rolle bei der Ausdifferenzierung von mesenchymalen Vorläuferzellen zu Osteoblasten und Adipozyten. Das Ziel dieser Arbeit war es molekulare Mechanismen zu finden, welche die unterschiedlichen Phänotypen (wild typ, fra1-tg, rsk2-defizient und fra1-tg/rsk2-defizient) charakterisieren. Die Knochenuntersuchungen der verschiedenen Genotypen zeigten, dass Fra1 und Rsk2, unabhängig voneinander, tragende Rollen im Knochenmetabolismus spielen. Quantitative Analysen von Adipozytenmarker, wie PPARgamma und C/EBPalpha zeigten, dass das Protein Fra1 die Adipozytenreifung in vivo und in vitro reguliert. Zusätzlich entwickelten die „doppel-mutierten“ fra1-tg/rsk2-/y Mäuse einen Lipodystrophy. Ein milderer Phänotyp wurde in den fra1-tg Tieren beobachtet, jedoch nicht in den Rsk2-knockout Mäusen. Zusätzlich wurde beobachtet, dass mesenchymale Zellen, welche Fra1 überexprimieren, gegen Glucocorticoid-induzierte Wachstumshemmung resistent waren. Diese Wirkung kann am wahrscheinlichsten durch die Fra1-vermittelte Suppression des Glucocorticoidrezeptors erklärt werden. Außerdem beeinflusste die Überexpression von Fra1 die Milzentwicklung. Leber und Herzanalysen zeigten, dass Fra1 kollagenhaltiges Gewebe induziert. Krankheiten wie Cholangitis und Fibrosen waren die Folge. / AP-1 transcription factor is a general name for multiple dimers formed by the association of Fos (or ATF) and Jun proteins. AP-1 acts as a sensor of changes in the cellular environment and thus, it is implicated in the modulation of cell proliferation, differentiation, transformation and cell death. Besides the well-documented role of c-Fos protein in oncogenesis, where this gene can function as a tumor promoter, AP-1 proteins are being recognized as regulators for mesenchymal stromal cell development and as regulators of immune cells. The mesenchymal stromal cells are the common progenitors for various mesenchymal lineages such as adipocytes, osteoblasts, chondrocytes, myocytes and fibroblasts. AP-1 seems to play a key role in the control of mesenchymal cell fate decision and differentiation. This is suggested by phenotypes of mice with a genetic modifications in either the Jun or the Fos component of AP-1. In particular, mice overexpressing the Fos-related antigen-1 (Fra1) or the short isoform of FosB (deltaFosB) have been found to develop osteosclerosis due to an accelerated differentiation of osteoblasts. Interestingly, mice overexpressing deltaFosB also developed less fat tissue. The activity of Fos proteins can be regulated by post-transcriptional modification. Based on knockout mouse model, a role for the growth factor regulated kinase Rsk2 was proposed in the differentiation of mesenchymal stromal cells to osteoblasts as well as in fat tissue development. Goal in this study was to identify the molecular mechanisms explaining the differences between the wild type, fra1-tg, rsk2-deficient and fra1-tg/rsk2-deficient phenotypes. The comparison of the bones of the different mice genotypes revealed, that Fra1 and Rsk2 were independently regulating bone metabolism. Quantitative analysis of adipocyte markers expressions, like PPARgamma and C/EBPalpha revealed, that Fra1 overexpression was blocking adipocyte maturation in vivo and in vitro. Moreover, the in vivo results show that the fra1-tg/rsk2-/y mice develop a severe lipodystrophy. A milder phenotype was observed in the parental fra1-tg strain but not in the Rsk2 knockout strain. Additionally, it was been observed, that mesenchymal cells overexpressing Fra1 were resistant to glucocorticoid-induced growth inhibition. This effect can most likely be explained by Fra1-mediated downregulation of the glucocorticoid receptor. Furthermore, Fra1 overexpression influenced spleen development. Liver and heart analyses showed that Fra1 overexpression induced collagen tissue. Diseases like cholangitis and fibrosis were the outcome.

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.

O estado nutricional influencia o crescimento gástrico, e enquanto a proliferação celular é estimulada pelo jejum em filhotes, ela é inibida em ratos adultos. A corticosterona também age no desenvolvimento, e seus efeitos são regulados pela corticosterone binding globulin (CBG) e receptores de glicocorticóides (GR). Para investigar se a atividade da corticosterona responde ao jejum e como possíveis mudanças poderiam controlar o ciclo celular epitelial gástrico, nós avaliamos diferentes parâmetros durante a progressão do jejum em ratos de 18 e 40 dias de vida pós-natal. A restrição alimentar induziu um aumento de corticosterona no plasma em ambas as idades, mas apenas em filhotes o binding da CBG se elevou após o jejum curto, permanecendo alto até o final do tratamento. O jejum aumentou a atividade transcricional do GR na mucosa gástrica e os níveis protéicos, porém o efeito foi mais pronunciado em adultos. Além disso, observamos que nos filhotes, o GR é principalmente citoplasmático, enquanto em animais adultos, o receptor é acumulado no núcleo durante o jejum. As proteínas HSP 70 e HSP 90 foram diferencialmente reguladas, e podem contribuir para a estabilidade do GR no citoplasma em filhotes, e para o trânsito de GR para o núcleo em animais adultos. Quanto ao ciclo celular epitelial, observamos que em filhotes, ciclina D1 e p21 aumentaram durante o jejum, enquanto em ratos adultos, a ciclina E diminuiu e a p27 aumentou muito. Assim, nós demonstramos que a atividade da corticosterona é diferencialmente regulada pelo jejum em filhotes e ratos adultos, e que as variaçõe observadas poderiam atenuar um possível efeito supressor durante o desenvolvimento pós-natal. Sugerimos que este mecanismo pode estimular a proliferação celular e possibilitar o crescimento da mucosa gástrica durante condições nutricionais adversas. / The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-d-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-d-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas in adults, it accumulated in the nucleus after the onset of fasting. HSP 70 and HSP 90 were differentially regulated, and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18- and 40-d-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

Lors de la liaison d’un glucocorticoïde (GC) naturel ou synthétique (par exemple, la Dexaméthasone) au récepteur des glucocorticoïdes (GR), les GCs régulent l’expression de gènes cibles soit par (i) transactivation par liaison ‘’directe’’ à un élément de liaison à l’ADN de type ‘’(+)GRE’’, (ii) transrépression ‘’directe’’ par liaison à un élément de type ‘’nGRE’’ ou (iii) transrépression ‘’indirecte’’ par interaction physique directe avec des facteurs de transcription pro-inflammatoires tels que AP-1 et NF-κB. Les effets anti-inflammatoires bénéfiques des GCs sont généralement attribués à la transrépression indirecte, alors que nombre de leurs effets secondaires pathologiques indésirables paraissent liés à la transactivation et/ou à la transrépression directe. Notre laboratoire a récemment découvert qu’un composé non-stéroïdien dénommé CpdX ainsi que ses dérivés deutérés, ne présentent ni la fonction de transactivation, ni celle de transrépression directe du GR, tout en stimulant son activité bénéfique de transrépression indirecte. Notre projet a consisté à caractériser un composé non-stéroïdien dit CpdX, ainsi que ses dérivés, quant à leurs activités thérapeutiques et à démontrer qu’elles sont semblables à celles des glucocorticoïdes anti-inflammatoires, couramment utilisés, tout en étant débarrassés de leurs effets pathologiques secondaires, tels que l’ostéoporose, l’atrophie cutanée et le syndrome métabolique. Pour atteindre nos objectifs, nous avons utilisés des modèles de souris présentant soit les affections cutanées (dermatites de contact ou atopique, psoriasis), l'asthme, l’arthrite rhumatismale, la colite ulcérative ou la conjonctivite allergique, associés à des études d’immunologie et de biologie moléculaire et cellulaire. Mon travail de thèse a démontré que CpdX, et certains de ses dérivés deutérés, présentent une activité anti-inflammatoire dans le traitement de ces modèles ‘’souris’’ (Partie I). Nous avons aussi montré que le traitement par CpdX et ses dérivés n’induit pas les effets secondaires pathologiques des glucocorticoïdes (Partie II), ouvrait ainsi la vue à une nouvelle ère dans le traitement à long-terme de maladies inflammatoires, sans provoquer les effets pathologiques indésirables des traitements actuels aux glucocorticoïdes. / Upon binding of natural or synthetic glucocorticoids (GCs) (e.g. Dexamethasone) to their glucocorticoid receptor (GR), GCs regulate the expression of target genes either by (i) direct transactivation through direct binding to “(+)GRE” DNA binding sites (DBS), (ii) direct transrepression through binding to “IR nGRE” DBSs or (iii) tethered indirect transrepression mediated through interaction with transactivators, such as NFkB, AP1, or STAT3 bound to their cognate DBSs. The beneficial anti-inflammatory effects of GCs have been generally ascribed to tethered transrepression, whereas many of their long-term undesirable side-effects could be due to transactivation and/or direct transrepression. Our laboratory recently reported that a non-steroidal compound, named CpdX, selectively lacks both direct transactivation and direct transrepression functions, while still exerting an indirect transrepression activity. The goal of our project was to characterize CpdX and some of its derivatives as effective anti-inflammatory drugs similar to glucocorticoids, but lacking their main deleterious side-effects, e.g. osteoporosis, skin atrophy and metabolic disorders. To this end, we have used experimental mouse model for skin disorders (atopic dermatitis, contact dermatitis, and psoriasis), asthma, rheumatoid arthritis, ulcerative colitis and allergic conjunctivitis, combined with immunology, molecular and cellular biology. My thesis studies have demonstrated that in mouse models, CpdX and its derivatives exhibit anti-inflammatory activities, which are similar to those of glucocorticoids (Part I). Importantly, we further show that CpdX and its derivatives do not exhibit the long-term debilitating side-effects of glucocorticoids (Part II). Thereby paving the way to a new era in the long-term therapy of major inflammatory diseases.

Approximately 1 in 6 infants are born prematurely each year. Typically, these infants spend 25 days in the Neonatal Intensive Care Unit (NICU) where they experience 10-18 painful and inflammatory procedures each day. Remarkably, pre-emptive analgesics and/or anesthesia are administered less than 30% of the time. Unalleviated pain during the perinatal period is associated with permanent decreases in pain sensitivity, blunted cortisol responses and high rates of neuropsychiatric disorders. To date, the mechanism(s) by which these long-term changes in stress and pain behavior occur, and whether such alterations can be prevented by appropriate analgesia at the time of injury, remains unclear. We have previously reported in rats that inflammation experienced on the day of birth permanently upregulates central opioid tone, resulting in a significant reduction in adult pain sensitivity. However, the impact on early life pain on anxiety- and stress-related behavior and HPA axis regulation is not known. Therefore the goal of this dissertation was to determine the long-term impact of a single neonatal inflammatory pain experience on adult anxiety- and stress-related responses. Neuroanatomical changes in stress-associated neurocircuits were also examined. As the endogenous pain control system and HPA axis are in a state of exaggerated developmental plasticity early in postnatal life, and these systems work in concert to respond to noxious or aversive stimuli, this dissertation research aimed to answer the following questions: (1) Does neonatal injury produce deficits in adult stress-related behavior and alter stress-related neuroanatomy through an opioid-dependent mechanism? (2) Does neonatal injury alter receptor systems regulating the activation and termination of the stress response in adulthood? (3) Are stress- and pain-related neurotransmitters altered within the first week following early life pain? (4) Is early activation of the pain system necessary for the long-term changes in anxiety- and stress-related behavior? Together these studies demonstrate the degree, severity and preventability of the long-term deficits in stress responding associated with a single painful experience early in life. The goal of this research is to promote change in the treatment of infant pain in the NICU to reduce long-term sensory and mental health complications associated with prematurity.