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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.

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.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T09:47:46Z No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T10:03:57Z (GMT) No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2015-12-10T10:03:57Z (GMT). No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Central Giant Cell Lesion (CGCL) is an intraosseous lesion that can be classified into non aggressive and aggressive. Due to the aesthetic and functional defects of surgical treatment of CGCL, therapies with drugs have been reported, such as glucocorticoid injections and calcitonin. The studies reported in the literature support the use of these drugs through the investigation of the presence of glucocorticoid receptors (RGC) and calcitonin (RCT) in CGCL; however there is no consensus if all lesions express these receptors and if there is any difference between non aggressive and aggressive lesion. In addition, there are no studies that evaluated the bone formation potential through the investigation of Osteocalcin (OC) in aggressive and non-aggressive lesions. The aim of this study was to compare, using immunohistochemistry, the GR and CTR and osteocalcin protein (OC) expression in non aggressive (n = 20) and aggressive (n = 11) CGCL, and the correlation between the OC expression and these receptors determined in both groups of lesions. The number of mononuclear cells in mitosis (MOC), and the number of multinucleated giant cells (MGC) were also investigated using immunohistochemical techniques (hematoxylin and eosin). Our results show that all the cases express the GR and CTR and that there is no difference in the expression of these receptors or the number of mitosis between non aggressive and aggressive lesions. The OC expression was rare and higher in non aggressive lesions, however, not statistically significant (p> 0.05). There was a correlation between the CTR expression in MOC and MGC (r = 0.45; p <0.01). Considering the different variants of CGCL, there was a correlation between CTR expression in MOC and MGC in non aggressive lesions (r = 0.66; p <0.01) and between the CTR and OC expression in MGC (r = 0.718; p = 0.01). There was a higher number of MGC in aggressive lesions (p = 0.01). The results indicate that all cases express GR and CTR and that there are no differences between non aggressive and aggressive CGCL lesions of these receptors expression, these results strengthens CGCL treatment with glucocorticoids and calcitonin. Aggressive lesions have a higher number of MGC. The CGCL express glucocorticoid and calcitonin receptors and this finding give biological basis to the CGCL treatment with intralesional glucocorticoid and calcitonin either in non aggressive and aggressive cases. It was also identified osteocalcin positive cells, that may be related to bone repair, it is believed that these cells may also serve as a therapeutic target. / A Lesão Central de Células Gigantes (LCCG) é uma lesão intraóssea que pode ser classificada em não agressiva e agressiva. Devido aos defeitos estéticos e funcionais do tratamento cirúrgico da LCCG, terapias medicamentosas tem sido relatadas, como injeções de glicocorticoide e calcitonina. Há na literatura estudos que suportam o uso desses medicamentos através da investigação da presença de receptores de glicocorticoides (RGC) e de calcitonina (RCT) em LCCG. No entanto não existe consenso se todas as LCCG expressam esses receptores e se existe alguma diferença entre lesões agressivas e não agressivas. Além disso, não existem estudos sobre a avaliação do potencial de formação óssea através da Osteocalcina (OC) em lesões agressivas e não agressivas. O propósito deste estudo foi avaliar comparativamente, por meio de imunohistoquímica, a expressão de RGC e RCT e da OC em LCCG não agressivas (n= 20) e agressivas (n= 11) e a correlação entre a expressão da OC e desses receptores nos dois grupos de lesões estudados. O número de mitoses nas células mononucleares e o número de células gigantes multinucleadas também foram investigados, utilizando técnica histoquímica (hematoxilina e eosina). Nossos resultados mostram que todos os casos analisados expressam o RGC e RCT e que não existe diferença na expressão do RGC, RCT ou do número de mitoses entre lesões não agressivas e agressivas. A expressão de OC em células mononucleares foi rara e maior em lesões não agressivas, no entanto, sem diferenças estatisticamente significantes (p>0,05). Houve correlação entre a expressão do RCT em células mononucleares e células gigantes multinucleadas (r=0,45; p<0,01). Considerando as diferentes variantes foi verificada correlação do RCT entre o componente mononuclear e as células gigantes multinucleadas nas lesões não agressivas (r=0,66; p<0,01) e entre a expressão de OC e RCT em células gigantes multinucleadas (r= 0,718; p=0,01). Houve maior número de células gigantes em lesões agressivas (p= 0,01). Os resultados indicam que todos os casos expressam RGC e RCT e que não há diferenças entre lesões agressivas e não agressivas de LCCG quanto à expressão desses receptores, fortalecendo a recomendação o tratamento da LCCG com o uso de glicocorticoide e calcitonina. Lesões agressivas apresentam maior número de CGM. As células da LCCG expressam o RGC e RCT e esse achado pode fornecer bases biológicas para o tratamento com injeções intralesionais de glicocorticoides e o uso de calcitonina, seja em lesões não agressivas ou agressivas. Adicionalmente, foram identificadas células expressando OC, que podem estar relacionadas ao reparo ósseo, acredita-se que essa linhagem celular também pode se tornar um alvo terapêutico.

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.

Les glucocorticoïdes (GC), généralement sécrétés par le cortex surrénalien, exercent de très nombreuses fonctions dans l’organisme, via leur liaison au récepteur des glucocorticoïdes (GR). Les rares mutations inactivatrices du GR déjà décrites, sont responsables d’un syndrome de résistance aux GC et peuvent conduire à une hypertension artérielle (HTA), une hyperplasie surrénalienne (HBS), un hirsutisme et une obésité. Dans ce travail, nous avons caractérisé fonctionnellement 13 variants hétérozygotes du GR (expression, transactivation, localisation subcellulaire,...). Six variants du GR, découverts par séquençage à haut débit (NGS) ne sont pas pathogènes alors que 7 mutations hétérozygotes originales délétères ont été identifiées dans le cadre du protocole hospitalier de recherche clinique (Muta-GR). Ce PHRC a permis de préciser une prévalence à 5% de mutations inactivatrices du GR dans une cohorte de 100 patients avec HBS associée à une HTA et/ou un hypercortisolisme biologique sans signe clinique de Cushing.Une haploinsuffisance du GR, démontrée par la diminution d’induction par la déxamethasone du gène cible FKBP5, a été mise en évidence dans les fibroblastes cutanés de certains patients porteurs de mutations inactivatrices du GR. Ces patients présentent souvent un hypercorticisme avec hypokaliémie, aldostérone et rénine basse, signant un pseudohyperaldostéronisme. Nous avons démontré que le gène HSD11B2 codant pour l’enzyme 11β-HSD2, assurant l’inactivation des GC, est une cible directe du GR comme démontré par transfection transitoire de gène-rapporteur, RT-qPCR, LC/MSMS et ChIP. L’établissement des modèles de knock-in de mutations GR par stratégie Crispr/cas9 dans des lignées cellulaires préadipocytaires ou corticosurrénaliennes humaines s’est soldé par un échec. Ce travail devrait faciliter la sélection des patients chez qui la recherche de mutation inactivatrice du GR doit être faite et invite à un suivi régulier de ces patients. / Glucocorticoids (GC) regulate many essential biological functions by activating the glucocorticoid receptor (GR). GR loss-of function mutations are responsible for GC resistance syndrome, often associated with high blood pressure, hirsutism, bilateral adrenal hyperplasia (BAH) and obesity. Herein, functional characterization of 13 GR variants is presented (expression and binding studies, transactivation assays, subcellular localization) 6 variants were discovered with next-generating sequencing and had no functional impact on GR signaling while 7 GR loss-of-function mutations were mainly discovered during the National Clinical Hospital Research Program, Muta-GR. This PHRC discloses a 5% prevalence of GR loss-of-function mutations in a cohort of 100 patients with BAH, biological hypercortisolism and/or hypertension without Cushing signs. A GR haploinsuffisiency was demonstrated by a reduced dexamethasone-induced FKBP5 expression in skin fibroblasts of some patients harbouring GR loss-of-function mutations. These patients often presented with hypercorticism, hypokalemia, low renin and aldosterone levels, consistent with a pseudohypermineralocorticism. We showed that HSD11B2 encoding the 11β-HSD2 enzyme inactivating GC, is a direct GR target gene by transient transfection of reporter gene, RT-qPCR, LC/MSMS and ChIP. We failed to introduce GR loss-of-function mutations in human preadipocytes and adrenocortical cells by Crispr/Cas 9 technology. This work should facilitate selection of patients in whom GR mutation may be search, enabling an appropriate follow-up.

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