Dissertations / Theses on the topic 'Hormones, Sex – Receptors'

Dans le cadre de la production de nouvelles espèces aquacoles, le sandre, Sander lucioperca, est devenu, depuis plusieurs années, une espèce d’intérêt piscicole en raison de sa valeur économique potentielle. Pour développer et pérenniser sa production aquacole, il est nécessaire de comprendre et maîtriser son cycle de reproduction ainsi que les mécanismes physiologiques mis en jeu afin d’obtenir des œufs et des juvéniles viables tout au long de l’année. Dans cet optique d’optimisation du contrôle du cycle, la dopamine apparaît, chez de nombreux téléostéens dont certains perciformes, comme un inhibiteur de l’axe gonadotrope, via les récepteurs de la famille D2, en bloquant le pulse ovulatoire de LH et l’ovulation. Chez le sandre, le rôle de la dopamine et de ses récepteurs, notamment les récepteurs de la famille D1, est inconnu. L’objet de cette thèse est de déterminer le rôle du système dopaminergique lors des phases finales de l’ovogénèse chez le sandre à travers trois axes principaux : (1) déterminer l’effet du blocage des récepteurs de la dopamine, D1 ou D2, sur la régulation de l’axe gonadotrope et l’induction de l’ovulation en absence et en présence d’une molécule de sGnRHa, (2) définir le répertoire et le profil d’expression des récepteurs dopaminergiques par l’étude du transcriptome cérébral du sandre en période pré-ovulatoire et (3) établir le rôle de la dopamine et de ses différents récepteurs (familles D1 et D2) dans la régulation directe et locale de l’axe gonadotrope aux niveaux cérébral et ovarien. La première partie de ce travail a permis pour la première fois, par l’utilisation d’antagonistes spécifiques des familles de récepteurs D1 et D2, de mettre en évidence un rôle potentiel de la dopamine sur la sécrétion de certains stéroïdes sexuels en période pré-ovulatoire chez le sandre par l’intermédiaire des récepteurs de la famille D1. L’identification de l’ensemble des récepteurs de la dopamine existant chez le sandre nous a permis de confirmer leur expression à tous les niveaux de l’axe gonadotrope (cerveau, hypophyse et ovaires) étayant l’hypothèse d’un rôle de la dopamine dans la reproduction du sandre. Enfin, la dernière partie de ce projet a permis de montrer un rôle régulateur du système dopaminergique, directement au niveau ovarien, sur la production de testostérone par l’intermédiaire des deux familles de récepteurs de la dopamine. L’implication des deux familles de récepteurs a également été mise en évidence dans la production ovarienne de la 17β-estradiol. Au niveau cérébral, seule la famille des récepteurs D2 a été montrée impliquée dans la régulation de l’expression du gène de la GnRH-3. De façon générale, cette étude a permis de mettre en évidence l’implication des récepteurs de la dopamine dans la régulation de l’axe gonadotrope lors des phases finales de l’ovogenèse. Toutefois, des travaux ultérieurs devront être menés pour approfondir les mécanismes physiologiques mis en jeu. D’un point de vue aquacole, les traitements hormonaux à base d’antagonistes des récepteurs de la dopamine ont été inefficaces pour améliorer les performances de reproduction du sandre ce qui n’est pas en faveur de leur utilisation future pour induire l’ovulation chez cette espèce. Ainsi, la mise au point d’autres méthodes d’optimisation sera nécessaire pour continuer à développer la production aquacole du sandre
Pikeperch, Sander lucioperca, is a potential valuable economic fish, making it a species of interest for aquaculture diversification. In the domestication process, controlling and understanding the reproductive cycle is a crucial step in order to produce viable offspring in a synchronous and predictable way. In many teleosts including some perciforms, dopamine inhibits the ovulatory pulse of LH and the ovulation step through D2 dopamine receptors family. In pikeperch, the roles of dopamine and its receptors, especially those belonging to the D1 receptors family, are unknown. For the purpose of the optimization of pikeperch reproduction, we investigated the role of the dopaminergic system during the final stages of oogenesis in this species: (1) by determining the effects of D1 or D2 receptor antagonists alone or in association with sGnRHa on the regulation of the reproductive axis and on the induction of ovulation, (2) by determining the repertoire and the expression profile of the dopamine receptors using a brain transcriptome analysis during the pre-ovulatory period and (3) by evaluating the role of dopamine and its receptors (D1 and D2 families) in the direct and local regulation of the gonadotropic axis at the brain and ovarian levels. For the first time, we showed that the dopamine/D1 receptors complex regulates the sex-steroids release during the pre-ovulatory period, suggesting that dopamine is involved in pikeperch reproduction. Also, we support its involvement thanks to the identification of the dopamine receptors gene expression at the brain, pituitary and ovarian levels. Finally, we showed that the dopaminergic system directly regulates the ovarian testosterone production, through both D1 and D2 receptor families. The involvement of both dopamine receptor families was also highlighted on ovarian 17β-estradiol production. Only the D2 receptor family was shown to be involved on the brain GnRH-3 gene expression. In conclusion, we point out a dopamine receptors implication on the gonadotropic axis regulation during the final stages of oogenesis in pikeperch. However, further studies should be performed to pinpoint the physiological mechanisms behind this phenomenon. From an aquaculture point of view, hormonal treatments with dopamine receptor antagonists appear to be ineffective to improve pikeperch reproductive performances. Therefore, their use to induce pikeperch ovulation should be put into question and the development of alternative methods is necessary to further promote pikeperch production

The aim of this investigation was to determine the effect of a nicotine-conditioned context on locomotor hyperactivity in an animal model of D2-priming, and whether conditioned hyperactivity could be blocked by the D2 antagonist eticlopride or the D3 antagonist nafadotride. D2-primed male rats showed enhanced nicotine sensitization as evidenced by statistically significant differences in horizontal activity. D2-primed female rats administered nicotine demonstrated an increased hypoactive response after initial sensitization and increased stereotypy. Eticlopride and nafadotride blocked sensitization to nicotine in both D2-primed and non D2-primed males and females. Eticlopride blocked conditioned hyperactivity in females but not in males. D2-primed female rats administered nicotine demonstrated significantly higher conditioned-hyperactivity as compared to non D2-primed females and controls, and this increase was more effectively blocked by nafadotride as compared to eticlopride. These results suggest differential roles of the dopamine D2 and D3 receptors in both adolescent nicotine sensitization and conditioned activating effects of nicotine.

Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-mediated metabolism of some androgen receptor ligands, indicating that CYP7B1 can be involved also in the regulation of androgenic signaling. CYP7B1 substrates and metabolites were found to exert androgenic effects in a cell line-specific manner. Furthermore, cell line differences were observed in the expression pattern for androgen receptor comodulators. This thesis reports that 1α,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of CYP21A2 and CYP17A1 leading to suppressed production of aldosterone, dehydroepiandrosterone and androstenedione in adrenocortical cells. These are novel findings on vitamin D action. A mechanism is reported for the vitamin D-mediated regulation of the CYP21A2 gene. Data indicate that vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) are key comodulators in this novel vitamin D receptor (VDR)-mediated mechanism. Furthermore, the results indicate that altered expression levels of VDIR and WSTF can shift the suppressing effect of vitamin D to a stimulatory effect. Also, epigenetic components were found to be involved in the effects of vitamin D on CYP21A2 transcriptional rate. In addition, a functional vitamin D response element was identified in the CYP21A2 promoter. This study also reports that 1α,25-dihydroxyvitamin D3 affects sex hormone production in a tissue-specific way. Gene expression and enzyme activity of aromatase were found to be downregulated in cells derived from breast, but not in cells derived from prostate and adrenal cortex. The production of estradiol and dihydrotestosterone was altered in a tissue-selective manner following vitamin D treatment. These findings are of importance for the discussion on vitamin D as a potential anti-breast cancer agent.

Three point mutations have been found in the hormone-binding domain (HBD) of the human androgen receptor (hAR): one in the N-terminal end (Ile663Asn in a family with partial androgen insensitivity syndrome (PAIS)); one in the middle, (Leu820Val in a family with PAIS); and one in the C-terminal end (Pro903Ser, in a family with complete AIS). The positions 663 and 903 were the most terminal mutation sites in the HBD found to date. The three mutant hARs have been previously characterized biochemically in genital skin fibroblasts. In the family with the Leu820Val substitution, the mother and the grandmother were found to be carriers for the same mutation. To prove their pathogenicity, each of the three mutations has been reproduced in an hAR expression vector that was transfected into COS-1 cells. In COS-1 cells, the complexes from Pro903Ser and Leu820Val had: increased thermolability; increased dissociation rates; decreased affinity; and abnormal transactivation. There was a hierarchy in the severity of the mutations expressed in kinetic and transactivation assays that correlated with the severity of the clinical phenotype. The pathogenicity of the Pro903Ser and the Leu820Val mutations was thereby confirmed. In COS-1 cells, the AR with Ile663Asn had normal thermolability, normal dissociation rates, and normal transactivation, but a decreased affinity. Although this sequence alteration has only been found in a PAIS patient, its pathogenicity is not considered to be proven. More sensitive assays are needed for this purpose.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009.
Disputationsordförande;Professor Eva Brittebo, Inst. för Biovetenskap, Avd. för Toxikologi, Uppsala Universitet, UppsalaBetygsnämndens ledamöten; Docent Lena Ekström, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeDocent Ulf Diczfaluzy, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeProfessor Agneta Oskarsson, Inst. BVF, Avd. för farmakologi och toxikologi, SLU, Uppsala. Härtill 4 uppsatser.

Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

Le récepteur de la progestérone (PR) est un facteur de transcription hormono-régulé qui joue un rôle crucial dans la coordination de tous les aspects de la fonction de reproduction chez la femme. Pour activer ses gènes cibles, PR recrute de façon dynamique, séquentielle et combinatoire différents partenaires moléculaires : les corégulateurs transcriptionnels. Les coactivateurs de la famille p160 (Steroid Receptor Coactivators : SRC-1, -2, -3), dont l’expression est augmentée dans certains cancers hormono-dépendants, sont les partenaires privilégiés de PR. Au cours de ce travail, nous avons étudié les mécanismes d’interaction entre le récepteur de la progestérone et ses corégulateurs ainsi que leurs conséquences fonctionnelles sur l’activité de PR. Nous avons ainsi pu mettre en évidence l’importance de la dégradation des complexes PR/SRC-1 par le protéasome sous l’effet du ligand agoniste de PR, et le caractère nécessaire de cette régulation négative pour l’activation de la transcription des gènes cibles de PR. Nous avons également identifié un candidat possiblement impliqué dans la dégradation des complexes PR/coactivateurs p160 : le corégulateur transcriptionnel Jab1. En effet, il a été décrit comme un coactivateur des complexes PR-SRC-1 au laboratoire, et nous avons pu observer que, hors du cadre de l’activation par l’hormone, Jab 1 régule les niveaux d’expression de SRC-1 et SRC-2. En revanche, ce corégulateur reste sans effet sur SRC-3. Enfin, nous avons mis au point les conditions expérimentales de l’étude de la dynamique des interactions entre PR et ses corégulateurs par la techninique de FRET.Les évidences croissantes de l’implication de PR et de ses cofacteurs (SRC-1, SRC-3, Jab1) dans le développement et les métastases des cancers du sein font de la compréhension de leurs mécanismes d’action un élément important dans la recherche de nouvelles thérapies. La détermination du rôle exact des corégulateurs de PR dans ces processus permettra une éventuelle redéfinition des cibles pharmacologiques dans le traitement de ces maladies, qui représentent un véritable enjeu de santé publique
The progesterone receptor (PR) is a ligand-activated transcription factor playing a crucial role in female reproduction. To regulate gene expression, PR recruits several coregulators to target gene promoters, in a cyclic and combinatorial manner. Among these coregulators, PR recruits most notably members of the p160 family coactivators (Steroid Receptor Coactivators SRC-1, -2 and -3) which have recently been implicated in several hormono-dependent cancers.Here, we studied the mechanisms of interaction between PR and its coregulators as well as their functional consequences on PR transcriptional activity. We have demonstrated that PR activity is paradoxically coupled to the agonist ligand-dependent down-regulation of PR/SRC-1 complexes. Two degradation motifs found in SRC-1 were identified as signals involved in this proteasome- and ubiquitin-mediated process. We also identified a putative candidate implicated in the degradation of these complexes, namely the transcriptional coregulator Jab1. Indeed, Jab1 has previously been described in our laboratory as a coactivator of PR/SRC-1 complexes. We observed that it can specifically regulate SRC-1 and SRC-2 expression in absence of hormone. Finally, we optimized the experimental conditions of FRET experiments to get new insights on the dynamic interactions between PR and its coregulators. Collectively our findings are consistent with the emerging role of proteasome-mediated proteolysis in the gene-regulating process. Understanding PR mechanisms of action is an important step in the development of new therapies, due to growing evidences of PR and its coregulators implication in breast carcinogenesis and metastasis. Deciphering precisely the role of PR coregulators in these processes will permit to define new pharmacological targets for the treatment of these diseases, which represent a serious public health problem

Orientadores: Munir S. Skaf, Igor Polikarpov
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Mestrado

Uma das marcantes características morfológicas e funcionais dos crustáceos e de outros artrópodes é a presença de um exoesqueleto que cria uma barreira física para o crescimento desses animais. Nos crustáceos, a muda é um evento cíclico, dividido em 5 estágios, e um deles compreende a troca desse exoesqueleto, permitindo o aumento de tamanho. O início, período e a frequência do ciclo de muda dependem da idade e do sexo do animal e de fatores ambientais e fisiológicos. Hormônios como os ecdiesteróides e o hormônio inibidor da muda produzidos e secretados pelos órgãos Y e X, respectivamente, atuam diretamente no ciclo de muda, porém outros hormônios podem regular, de forma positiva ou negativa, este processo. A melatonina é um hormônio encontrado amplamente no reino animal, porém em crustáceos, diferentemente do que ocorre nos vertebrados, a sua síntese e secreção não estão relacionadas com a presença ou ausência de luz, e seu papel na muda tem sido pouco investigado. Os animais foram aclimatados no laboratório à temperatura 22±2 °C e ciclo claro-escuro 12h:12h LD, sendo os experimentos realizados nesta mesma condição. Considerando o acima exposto, os objetivos do presente trabalho foram (1) verificar a produção de melatonina no siri azul Callinectes sapidus, através da investigação da expressão das enzimas AANAT e ASMT no pedúnculo óptico e hepatopâncreas, bem como os níveis hemolinfáticos da indolamina; (2) avaliar se existe um perfil oscilatório diário na expressão gênica dos fatores relacionados com a muda, CasMIH e CasEcR1; (3) verificar se a manipulação com melatonina exógena influencia essa expressão. Para isso, técnicas de imunohistoquímica, citometria de fluxo, ensaio imunoenzimático e PCR quantitativo foram empregadas. Nossos resultados demonstraram uma oscilação dos níveis hemolinfáticos de melatonina em siris em pré-muda, com pico às 8 horas; entretanto, no estágio de intermuda os níveis deste hormônio foram menores e constantes ao longo de 24 horas. Não pudemos comprovar a presença das enzimas da via de síntese da melatonina, uma vez que os anticorpos utilizados não apresentaram homologia às proteínas de C. sapidus. Quanto à expressão gênica, uma oscilação diária semelhante nos transcritos dos genes CasMIH e CasEcR1 ocorreu no hepatopâncreas, independente do estágio de muda. No pedúnculo óptico a oscilação dos genes em questão também foi semelhante, mas apenas na pré-muda; na intermuda houve entre eles uma relação de anti-fase. A administração de melatonina exógena (10-7 mol/siri) levou à inibição da expressão dos genes em relação ao controle: no caso de CasMIH foi de 99,7% no pedúnculo óptico e 100% no hepatopâncreas e o CasEcR1 sofreu inibição de 77% no pedúnculo óptico e 99% no hepatopâncreas. A presença de melatonina na hemolinfa é um forte indício de que o animal a sintetiza e pode estar atuando no ciclo de muda, uma vez que a administração deste hormônio inibiu a transcrição dos genes relacionados ao processo. Diante disso, fica mais clara a relevância de entender a flutuação de hormônios que não estão classicamente envolvidos no ciclo de muda, essencial para o crescimento dos crustáceos, mas que podem apresentar a função de regular este processo, como a melatonina. Ademais, a melatonina poderá ser uma boa ferramenta a ser utilizada no cultivo do siri-azul, como agente indutor da redução do período de intermuda levando à uma ecdise precoce
One of the remarkable morphological and functional features of crustaceans and other arthropods is the presence of an exoskeleton that creates a physical barrier for the animal growth. In crustaceans, molting is a cyclic event usually divided into five stages, one of them comprising the exoskeleton exchange what thus allows the increase in size. The onset, period, and frequency of the molt cycle depend on the animal age and sex, as well as on environmental and physiological factors. Hormones such as ecdysteroids and the molt-inhibiting hormone produced and secreted by the Y- and X- organ, respectively, exert direct effects on the molt cycle. Nevertheless, other hormones are known to positively or negatively regulate this process, such as melatonin. Melatonin is a hormone widely found in the animal kingdom, but in crustaceans, differently from what happens in vertebrates, its synthesis and secretion are not regulated by the presence or absence of light. In fact, its role in the molting process has been poorly investigated. The animals were acclimated in the laboratory at 22±2 °C and light-dark cycle 12h:12h LD, and the experiments were performed under the same condition. Considering the above, the objectives of this study were to: 1) verify the production of melatonin in the blue crab Callinectes sapidus, through the evaluation of the expression of key enzymes involved in the synthesis of melatonin, AANAT and ASMT, in the eyestalk and hepatopancreas, as well as melatonin levels in the hemolymph; 2) evaluate whether there exists a daily oscillatory profile in gene expression of the related molt factors, CasMIH and CasEcR1; (3) whether the exogenous melatonin influences the expression of the latter genes. To achieve these goals, immunohistochemistry, flow cytometry, immunoenzymatic assay, and quantitative PCR techniques were used. Our results demonstrated an oscillation of the hemolymphatic levels of melatonin in premolt crabs, peaking at 8 AM; however, in the intermolt stage, the levels of this hormone were smaller and constant along 24 hours. We were not able to show the presence of the enzymes involved in melatonin synthesis, since the antibodies used had no homology with C. sapidus proteins. We also demonstrated a daily oscillatory profile of CasMIH and CasEcR1 transcripts in hepatopancreas independently of the molt stage. In the eyestalk the oscillatory profile of both genes was also similar, but only in the premolt stage; in intermolt, an antiphase relationship between both genes was found. The exogenous administration of melatonin (10-7 mol/crab) inhibited the expression of CasMIH by 99.7 and 100% in eyestalk and hepatopancreas, respectively, whereas CasEcR1 was inhibited by 77% and 99%, in the eyestalk and hepatopancreas, respectively, compared to saline-treated animals. The presence of melatonin in the hemolymph is a reliable indicator that the animal synthesizes the hormone, and thus melatonin may influence the molt cycle since it inhibited the expression of molt-related genes. Therefore, the relevance of understanding the oscillation of hormones that are not classically involved in the molt cycle - essential for crustacean growth - but which can regulate the process, becomes evident. From an economic standpoint, melatonin may be a useful tool in culturing blue crab, which ultimately can shorten the intermolt stage period leading to an early ecdysis

Multiple Sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system, characterized by axonal demyelination and multifocal inflammation. Like many autoimmune diseases, it is a sexually dimorphic disease, being 3-4 times more common in females than in males. p38α MAP kinase (MAPK) has an integral role in modulating inflammatory processes in autoimmunity. Conditionally ablating p38α MAPK in myeloid cells in B6 mice shows a sex difference in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In the absence of sex hormones, this sex difference was reversed, suggesting a role for sex hormones in modulating p38α MAPK signaling in EAE. Based on these findings, we hypothesized that pro-inflammatory functions in EAE is p38-indepdendent in the presence of androgens and p38-dependent in the presence of estrogens. For the purposes of this project, the role of androgens was evaluated. Both in vivo and in vitro techniques were used to assess how androgen receptor (AR) signaling: 1) impacts EAE pathogenesis, and 2) impacts the role of p38α in EAE pathogenesis and macrophage function. To this end, using Cre-Lox technology, we generated mice deficient in: 1) AR globally or conditionally in macrophages, as well as 2) mice doubly deficient in AR and p38α. In vivo results from p38α-sufficient global AR knockout mice show no effect of global AR deletion on EAE pathogenesis. Surprisingly, results from p38α-sufficient conditional AR knockout mice showed significant worsening in disease compared to WT counterparts, suggesting that AR signaling in myeloid cells has a protective role in EAE pathogenesis. These findings implicate a protective role for AR signaling in EAE. Studies with mice doubly deficient in p38α and AR to determine whether AR regulates the role of p38α in EAE are ongoing, but so far show no effect on AR deletion on the role of p38α MAPK. Further studies with larger cohorts of mice are needed elucidate the relationship between AR and p38α MAPK signaling in myeloid cells in EAE pathogenesis. In vitro studies using the immortalized macrophage cell line RAW 264.7 showed that pharmacologic inhibition of p38 MAPK after stimulation with LPS reduced the production of classic pro-inflammatory cytokines IL-6 and TNFα, and effect that was not affected by treatment with 5-dihydrotestosterone, suggesting that the AR does not modulate the role of p38α in cytokine production. These findings implicate no direct role of AR signaling on the functional role of p38α MAPK in the myeloid cell lineage in inflammatory and autoimmune responses.

Host defense against infection and disease relies on the reciprocal communication between the immune and neuroendocrine systems where sex hormones exert negative and positive feedback actions on immune functions. Indeed, sex hormones have been implicated in gender dimorphic immune response and in the potentiation of immune-related disorders. The female hormone estrogen plays a role as an immunomodulator and may exert immunosuppressive and immunostimulatory effects. Though many studies focus on estrogen’s role in immunity within the female reproductive tract and autoimmunity, the modulatory effects of estrogen on vaccine responses are largely unexplored. The insufficient efficacy of some vaccines in certain target populations, as for example the elderly population, is well recognized. Hormones fluctuate throughout an individual’s life, and females in particular undergo several necessary reproductive (pregnancy and menopause) and lifestyle (oral contraceptive use) changes which involve sex hormones. Vaccine efficacy might be influenced by endogenous estrogen levels or by exogenous estrogen administration. Therefore, in the pursuit of improved vaccine efficacy, it is necessary to consider such hormonal factors and their contribution to immune status. We have studied estrogen’s role in modulation of vaccine responses using a mouse ovariectomy model where exogenous estrogen delivery can be controlled. Our studies included two different types of vaccines, a bacterial toxoid formulation and a bacterial secreted protein formulation. Results from these studies indicate that estrogen enhances vaccine-specific antibody production by likely supporting a general TH2 pathway and also modulates expression of genes encoding molecules critical in innate immune signaling and required for development of proper adaptive immune responses and antigen clearance through antibody-mediated mechanisms. The level at which estrogen modulates antibody responses appears to be dependent on the route of vaccine administration. The enhancement of specific humoral responses may involve mechanisms involving TLR2 and antibody Fc receptor expression on macrophages, cells that link innate and adaptive immune responses. Advances in our understanding of the relationship between sex hormones and the immune system may provide new insights into the mechanisms by which hormones act and thus may be exploited to guide the design of future vaccine strategies.

Les oestrogènes peuvent favoriser la croissance, la promotion et la progression des cancers hormono-dépendants tels que le cancer du sein, de la prostate et des ovaires. Certains métabolites secondaires de plantes constituent une source importante de phyto-œstrogènes, capables de réduire le risque du cancer en antagonisant les fonctions des hormones. La férutinine (FRT), est une phyto-œstrogène biologiquement active extraite des racines de Ferula hermonis, espèce endémique du Liban. Plusieurs études ont été menées sur l'activité œstrogénique et anticancéreuse de la FRT, cependant son activité cytotoxique sur les lignées de cancer œstrogéno-dépendantes n'a pas été élucidée. La FRT est connue pour son effet agoniste vis-à-vis des récepteurs aux œstrogènes a (REa) et agoniste/antagoniste vis-à-vis des REß. Dans un premier temps, la production de la FRT a été optimisée par une hémisynthèse à partir de l'hydrolyse basique de l'extrait brut. Son activité anticancéreuse sur des lignées du sein MCF-7 (REa+, REß+) et MDA-MB-231(REa-, REß-), de la prostate PC-3 (REa+,REß+), DU 145 (REa-, REß+) et 22Rv1 (REa-, REß-) et des ovaires OVCAR-3 (REa+, REß+), SKOV-3 (REa muté, REß+) et IGROV-1 (REa-, REß-) a été testée. Un effet biphasique a été observé sur la prolifération des cellules du carcinome mammaire MCF-7, où son activité proliférative a été corrélée avec la stimulation des REs. La FRT est capable à des concentrations élevées d'inhiber la prolifération des lignées cellulaires cancéreuses et d'induire un arrêt du cycle cellulaire au niveau de la phase pré G0/G1 agissant ainsi via un mécanisme pro-apoptotique. L'efficacité de la férutinine réside dans son pouvoir à cibler spécifiquement les cellules souches/progénitrices cancéreuses chez les lignées cellulaires étudiées qui sont à la base de la réémission tumorale. Cependant, son activité antiproliférative est considérée moyenne puisqu'elle ne peut pas déclencher à des faibles concentrations une activité pure antagoniste des œstrogènes. Les travaux de thèse se sont ensuite focalisés sur la synthèse de composés analogues de la FRT en conservant sa structure indispensable à son activité. Nous avons élaboré un filtre in silico de chimiothèques d'antagonistes potentiels des REs. Ce nouvel outil d'arrimage moléculaire a guidé la synthèse des analogues de la FRT en améliorant leur fixation en position antagoniste dans le site de liaison des REs. L'arrimage moléculaire de la férutinine dans le site actif des REs a confirmé structuralement sa double compétence œstrogénique (agoniste/antagoniste) observée in vitro. Une douzaine d'analogues de la FRT a été synthétisée et testée pour son pouvoir antiprolifératif sur les lignées cancéreuses considérées. Des résultats prometteurs ont été obtenus notamment pour trois analogues sur les lignées cancéreuses mammaires (3c' et 2c'), prostatiques (2c') et ovariennes (3b). Ces composés ont montré une sélectivité vis-à-vis des lignés cancéreuses. Ces analogues constitueront alors des candidats pour une éventuelle plateforme de développement d'agents anticancéreux
Estrogens are key regulators of cell growth in hormone-dependent cancers such as breast, prostate and ovarian. Phyto-estrogens are a diverse group of plant-derived compounds, exhibiting potential benefits for chemoprevention by antagonizing the function of estrogens. Ferutinin (FRT) is the main active phyto-chemical extracted from the endemic plant of Lebanon, Ferula hermonis. Several studies were conducted on the estrogenic and anti-proliferative activities of FRT; nevertheless, its cytotoxic activity against estrogen-dependent cancers is not yet elucidated. FRT has been reported as agonist to estrogen receptor a (ERa) and agonist/antagonist to ERß. FRT production was first optimized by hemi-synthesis from basic hydrolysate of crude root extract. The anticancer properties of FRT was assessed against breast (MCF-7, MDA-MB-231), prostate (PC-3, DU 145, 22Rv1) and ovarian (OVCAR-3, SKOV-3, and IGROV-1) cancer cell lines. A biphasic effect was observed on the proliferation of mammary MCF-7 cell line, where the proliferative activity was correlated to ER stimulation. FRT inhibited the proliferation of all studied cell lines at high concentrations and induced a pre G0/G1 cell cycle arrest via a pro-apoptotic mechanism of action. FRT targeted the enriched population of cancer stem cells/progenitors which is responsible for tumor reemission. However, its antiproliferative activity is considered weak since it cannot trigger at low concentrations pure estrogen antagonistic activity. This project emphasizes next on the synthesis of FRT analogues by preserving its active structure. We have created an in silico filter of chemical compounds that might act as potential antagonists to ERs. This novel molecular docking tool was used to design FRT derivatives by enhancing their antagonist position inside the binding cavity of ERs. Docking results of FRT in the binding site of ERs confirmed structurally the dual potency that exerts this molecule (agonist/antagonist) in vitro. A list of FRT analogues were synthesized and tested for their anti-proliferative activity against the studied cell lines. Promising results were obtained with three ferutinin analogues on the proliferation of breast (3c' and 2c'), prostate (2c') and ovarian (3b) cancer cell lines. These compounds were also shown to be more selective to cancer cell lines. The cytotoxic properties of these analogues suggest that they could be promoted as potential candidates for useful anticancer therapy

A ocitocina (OT) é um neuropeptídio hipotalâmico, que dentre suas funções na fêmea destaca-se a contração uterina durante o parto e a ejeção do leite. No entanto, estudos vêm demonstrando importantes funções endócrinas e parácrinas no trato reprodutivo masculino. Evidenciando a possível ação conjunta entre OT e a Globulina ligadora de hormônios sexuais (SHBG). Entretanto, em cães não existem informações disponíveis quanto sua atuação. Assim, estudos direcionados aos receptores de ocitocina (OTR) e SHBG e suas funções no sistema reprodutor masculino, mais especificamente na fisiologia espermática, são de suma importância para os conhecimentos da fisiologia reprodutiva para posterior aplicação em biotecnologias reprodutivas em pequenos animais e humanos, fomentando também novas perspectivas para a utilização terapêutica da ocitocina em enfermidades reprodutivas. Portanto, o objetivo deste estudo é verificar a expressão gênica e proteica do OTR e SHBG no testículo e epidídimo de cães, correlacionando tais dados com a qualidade espermática e dosagem de testosterona. Para tal, foram coletados testículos e epidídimos de 26 cães em idade reprodutiva (1 a 5 anos). Após a orquiectomia, foi realizada a coleta dos espermatozoides provenientes da cauda do epidídimo e então, as amostras foram analisadas quanto à motilidade computadorizada do sêmen (CASA), integridade de membrana plasmática (Eosina/Nigrosina), integridade de membrana acrossomal (Fast Green / Rosa Bengala) e atividade mitocondrial (3´3 Diaminobenzidine). A imunolocalização do OTR e SHBG foi realizada através de imunoistoquímica e imunofluorescência. E a análise de expressão gênica, através da Reação em cadeia da polimerase em tempo real (qRT PCR). E da expressão proteica, através do Western Blotting. Foram encontradas correlações significantes e positivas entre as expressões gênicas do OTR e do SHBG, tanto no testículo como no epidídimo. Além disto, a expressão do OTR no testículo correlacionou-se positivamente com espermatozoides com membrana acrossomal íntegra e negativamente com a porcentagem de células com baixa atividade mitocondrial. Já o SHBG do testículo, correlacionou-se positivamente com a concentração de espermatozoides, porcentagens de células com membrana plasmática e acrossomal íntegras, motilidade, motilidade progressiva e velocidade rápida, e negativamente com a porcentagem de células com baixa atividade mitocondrial. Por outro lado, no epidídimo, a expressão gênica do SHBG apresentou correlação positiva com a porcentagem de células com membrana plasmática íntegra e expressão proteica de SHBG no testículo. Quanto a expressão proteica, o OTR no testículo obteve correlação positiva com testosterona e negativa com atividade mitocondrial nula, já no epidídimo, ocorreu correlação positiva com integridade de membrana acrossomal e negativa também com atividade mitocondrial nula. Em relação ao SHBG, houve correlação positiva com a expressão gênica do SHBG no epidídimo, células normais e padrões de velocidade. E na imunoistoquímica foi possível observar a imunomarcação do OTR e SHBG na musculatura lisa e células de Leydig do testículo e OTR na musculatura lisa do epidídimo. No entanto, não houve imunomarcação do SHBG no epidídimo, assim como expressão proteica. Nossos resultados demonstraram que o OTR e SHBG são expressos nos testículos e epidídimos de cães e que estão relacionados a funções espermáticas importantes, sendo essenciais para o sucesso reprodutivo
Oxytocin (OT) is a hypothalamic neuropeptide that plays important and well known roles in the female such as uterine contraction during childbirth and milk ejection. Notwithstanding, studies have shown important endocrine and paracrine functions also in the male reproductive tract, highlighted by the possible joint action between OT and sex hormone-binding globulin (SHBG). In dogs, however, there is no information available with regards to the role of these hormones in the reproductive function. Thus, studies directed to oxytocin (OTR) and SHBG receptors and their functions in the male reproductive system, specifically with regards to sperm physiology. Such knowledge is essential to understand the reproductive physiology for the subsequent use in reproductive biotechnologies in small animals and humans, especially by providing new perspectives for the therapeutic use of oxytocin in reproductive disorders. Therefore, the aim of this study is to assess the gene and protein expression of OTR and SHBG in the testis and epididymis of dogs, correlating these data with sperm quality and testosterone dosage. To this end, testis and epididymis were collected from 26 dogs in reproductive age (1 to 5 years). After orchiectomy, collection of sperm from the cauda epididymis was carried out and then the samples were analyzed for computerized motility of semen (CASA), plasma membrane integrity (eosin / nigrosine), acrosome membrane integrity (Fast Green / rose Bengal) and mitochondrial activity (3\'3 Diaminobenzidine). The immunolocalization of OTR and SHBG was performed by immunohistochemistry and immunofluorescence. Gene expression analysis was performed by real time polymerase chain reaction (qRT - PCR). The protein expression was further assessed by Western Blotting. Significant positive correlations were found between the gene expressions of OTR and SHBG in both the testis and epididymis. Furthermore, the OTR expression in testis was positively correlated to sperm with intact acrosome membrane and negatively to the percentage of cells with low mitochondrial activity. On the other hand, testicular SHBG was positively correlated with sperm concentration, percentage of sperm with intact plasma membrane and acrosome, motility, progressive motility and the percentage of RAPID sperm. Also, negative correlation was found between testicular SHBG and the percentage of cells with low mitochondrial activity. Furthermore, in the epididymis, SHBG gene expression was positively correlated to the percentage of cells with intact plasma membrane and protein expression of SHBG in the testis. In relation to the protein expression, the OTR in the testis correlated positively with blood plasma testosterone and negatively with sperm with no mitochondrial activity. In the epididymis, OTR protein expression correlated positively with sperm showing intact acrosome and negatively with cells with no mitochondrial activity. With regards to SHBG proteins expression, there was a positive correlation to SHBG gene expression in the epididymis, normal cells and some patterns of sperm velocity. In the immunohistochemistry, we observed the OTR and SHBG immunostainings in the smooth muscle and Leydig cells of the testis while, in the epididymis, the OTR immunostaining could be observed only in the smooth muscle. Interestingly, there was no immunostaining or protein expression of SHBG in the epididymis. Our results demonstrated that OTR and SHBG are expressed in the testis and epididymis of dogs and are related to important sperm functions, essential for reproductive success

Investigamos a distribuição de ghrelina e de seu receptor (GHS-R) na mucosa gástrica de ratos durante a terceira semana de vida pós-natal e avaliamos o efeito do desmame precoce sobre estas moléculas. Estudamos também a participação da ghrelina no controle da proliferação celular do epitélio gástrico, e para tanto utilizamos a administração de um antagonista. Detectamos o aumento do número de células imunomarcadas para ghrelina nos animais desmamados precocemente e observamos que nem a expressão de GHS-R nem a concentração proteica deste receptor foram alteradas pela mudança da dieta. O uso do antagonista [D-Lys-3]-GHRP-6 resultou na diminuição do índice de síntese de DNA no epitélio gástrico. Concluímos que a ghrelina e o GHS-R estão distribuídos no estômago durante a terceira semana de vida pós-natal e que o desmame precoce aumenta os níveis de ghrelina no epitélio gástrico, sem comprometer seu receptor. Por fim, sugerimos que esta modulação pode estar envolvida no controle da proliferação celular que é fundamental para o desenvolvimento do estômago.
In the present study, we investigated the distribution of ghrelin and growth hormone secretagogue receptor (GHS-R) in the rat gastric mucosa during the third postnatal week, and evaluated the effects of early weaning on these molecules. In addition, we studied whether ghrelin is part of cell proliferation control in gastric epithelium, and to that we used an antagonist. We detected an increase of ghrelin immunolabelled cells in animals submitted to early weaning and observed that GHS-R expression and protein levels of this receptor were not altered by dietary change. The antagonist [D-Lys-3]-GHRP-6 reduced DNA synthesis index. We concluded that ghrelin and GHS-R are distributed in the gastric mucosa during the third postnatal week and that early weaning increases hormone levels in the gastric epithelium, without changing its receptor. We can suggest that such modulation might be involved in the control of cell proliferation, which is essential for stomach development.

La glande uropygienne de la caille male est un organe cible des androgenes, elle possede des recepteurs aux androgenes qui ont ete mis en evidence. Mise au point d'une technique qui permet de mesurer a la fois les recepteur actives et non actives. La glande uropygienne est androgeno-dependante: la testosterone entraine une activation rapide des recepteurs des androgenes suivie d'une augmentation de leur concentration cellulaire, c'est le compose le plus efficace de tous les androgenes. L'oestradiol et la progesterone se comportent comme des anti-androgenes par competition au niveau du recepteur. Le ru 38882 est un anti-androgene pur qui utilise en application locale garde une action strictement locale (traitement de choix des affections cutanees androgeno-dependantes)

La ghréline, ligand endogène du Growth Hormone Secretagogue Receptor (GHSR), constitue la seule hormone orexigène connue à ce jour et stimule fortement la sécrétion d’hormone de croissance (GH). Ces propriétés font du GHSR une cible potentielle dans le traitement des maladies de la croissance et de la balance énergétique. Le séquençage du gène GHSR chez 290 patients présentant un déficit isolé en GH ou une petite taille idiopathique a permis d’identifier 2 mutations tronquantes (W2X, c. 370delG) et 6 variations faux-sens (L42V, A204E, V216A, R237W, L311F, A358T) au sein de 8 familles indépendantes. L’analyse fonctionnelle comparative de ces variants in vitro a évalué leur affinité pour la ghréline, leur localisation cellulaire et leur activité transcriptionnelle sur des gènes cibles. L’étude des variations W2X, R237W, A204E et c. 370delG a permis de mettre en évidence une perte de fonction des mutations tronquantes et un mécanisme original de perte totale (A204E) ou partielle (R237W) de l’activité constitutive des récepteurs avec conservation de leur capacité à répondre à la ghréline. Ces mutations sont responsables d’une petite taille, avec ou sans altération des tests de sécrétion de GH, se transmettant selon un mode récessif ou dominant à pénétrance incomplète. Ce travail nous a donc permis de caractériser une nouvelle étiologie de retard de croissance chez l’homme et de confirmer l’importance du GHSR et de son activité constitutive dans la croissance. Une meilleure compréhension des mécanismes moléculaires et physiopathologiques des anomalies du GHSR chez l’homme devrait fournir des connaissances importantes pour le développement de molécules ciblant l’axe ghréline/GHSR.

Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico
Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH

A tendência da indústria em produzir alimentos mais saudáveis, com menos gordura vem aumentando sensivelmente. O desafio do melhoramento nos dias atuais é melhorar a qualidade da carcaça dos frangos juntamente com a redução nos teores de gordura, que é uma exigência do mercado consumidor. A leptina é um hormônio polipeptídico secretado principalmente pelo tecido adiposo com um importante papel na regulação da ingestão de alimentos, metabolismo de energia e reprodução. O gene da leptina e seu receptor vem sendo objeto de intensa investigação representando excelentes genes candidatos para deposição de gordura na carcaça nos programas de seleção assistida por marcadores. A função desses genes tem sido intensamente estudada em mamíferos, porém, em aves poucos estudos foram realizados. O presente trabalho teve como objetivo investigar a ocorrência de polimorfismos no gene da leptina e de seu receptor em galinhas, e avaliar os efeitos desses polimorfismos sobre características de desempenho e carcaça. Duas linhagens de aves, uma de corte (TT) e uma de postura (CC) foram utilizadas para desenvolver a população referência F2 empregada no presente estudo. A polução referência foi desenvolvida no sistema de Melhoramento Genético de Aves da Embrapa Suínos e Aves, em Concórdia/SC. Foram desenhados primers para amplificar o gene da leptina e os íntrons 8 e 19 do do gene do receptor da leptina. Entretanto, após diversas tentativas não foi possível amplificar o DNA genômico e cDNA do gene da leptina. No íntron 8 do gene do receptor da leptina o SNP C352T foi genotipado por seqüenciamento em 247 animais da população F2. O alelo T foi associado ao maior rendimento de carcaça (P=0,0165), rendimento de peito (P=0,0137), gramas de proteína bruta (P=0,0112) e cinza na carcaça (P=0,0150), enquanto o alelo C foi associado somente ao rendimento de fígado (0,0102). No íntron 19 o SNP G915A foi genotipado por PCR-RFLP em 137 animais da população F2. O alelo A foi associado ao consumo de ração (P=0,0339)), o alelo G ao rendimento de pulmão (P=0,287) e os alelos A e G quando em homozigose foram associados com rendimento de coxas e sobrecoxas (0,0302). Por ocorrerem em região de íntron, provavelmente esses SNPs não estão envolvidos diretamente com as características associadas, mas podem estar ligados a outra mutação localizada na região regulatória do gene do receptor da leptina ou em outro gene próximo. No futuro outros polimorfismos deverão ser explorados, em regiões codificadoras desse gene, para serem utilizados como marcadores associados a características de desempenho e carcaça em aves. Em adição o gene do receptor da leptina foi localizado no mapa de ligação do cromossomo 8 da população de aves da Embrapa, o próximo passo será realizar a análise de QTL incluindo os SNPs estudados como marcadores.
The trend of industry in producing healthier nourishment, with less fat has raised sensibly. The challenge of animal breeding in the current days is to improve carcass quality of chickens with concomitant reduction in the fat content, witch is a consumer\'s demand. Leptin is a polypeptide hormone secreted mainly by adipocytes with an important role in the regulation of food ingestion, energy metabolism energy and reproduction. The leptin (LEP) and its receptor (LEPR) genes are being subject of intense investigation representing excellent candidate genes for fat deposition in the carcass in marker assisted selection programs. The function of those genes has been intensely studied in mammals, however, in birds few studies were accomplished. The present work aimed to investigate the occurrence of polymorphisms in the LEP and LEPR genes in Chickens, and also evaluate the effects of those polymorphisms on performance and carcass traits. Two chicken lines, a broiler (TT) and a layer line (CC) were used to develop an F2 reference population used in the present study. The reference population was developed in the Poultry Genetic Breeding System at Embrapa Suínos e Aves, in Concórdia/SC. Primers were designed for the PCR amplification of the LEP gene leptina and introns 8 and 19 of the LEPR gene. However, after several attempts it was not possible to amplify the genomic DNA or cDNA of the leptin gene. In the intron 8 of the LEPR gene, SNP C352T was genotyped by DNA sequencing of 247 animals of the F2 population. The T allele was associated with greater carcass yield (P=0,0165), breast yield (P=0,0137), grams of crude protein (P=0,0112) and ashes in the carcass (P=0,0150), while the C allele was associated only to liver yield (0,0102). The SNP G915A in the intron 19 was genotyped by PCR-RFLP of 137 animals from the F2 population, the allele A was associated to the feed intake (P=0,0339), the allele G to the lung yield (P=0,287), and the alleles A and G, when in homozigose, were associated with drumstick and thigh yield (0,0302). Because these SNP are located within an intron area, they are not likely directly involved with the associated characteristics, but they can be linked the other mutation located in the regulatory area of the LEPR gene. In the future other polymorphisms should be explored, in the coding regions of this gene, so they can be used as markers associated to performance and carcass characteristics in poultry. In addition, the LEPR gene was located in the ligation map of the chromosome 8 for the Embrapa population. The next step will be to accomplish the analysis of QTL including the SNPs studied as markers.

Le fipronil, insecticide largement utilisé, est un perturbateur thyroïdien chez le rat modulant le catabolisme hépatique des hormones thyroïdiennes. Ses effets chez le mouton, considéré comme un modèle plus pertinent que le rat pour étudier une régulation de la fonction thyroïdienne chez l'Homme, sont plus limités. Le but de cette thèse était de caractériser au niveau hépatique le mode d'action du fipronil sur la fonction thyroïdienne en s'intéressant 1) au rôle potentiel du principal métabolite du fipronil formé in vivo, le fipronil sulfone, et 2) aux différences interspécifiques de métabolisme du fipronil et/ou de sensibilité à la perturbation thyroïdienne qui peuvent préjuger de la pertinence des différents modèles animaux pour l'analyse du risque du fipronil pour la santé humaine. L'efficacité du fipronil sulfone à induire l'expression et/ou l'activité d'enzymes responsables du métabolisme hépatique des hormones thyroïdiennes ou du fipronil était la même que celle du fipronil autant in vivo chez le rat que in vitro sur hépatocytes. L'utilisation d'un modèle de souris déficientes pour des récepteurs nucléaires xénosenseurs suggérait fortement une implication des récepteurs nucléaires Constitutive Androstane Receptor et/ou Pregnane X Receptor dans la perturbation thyroïdienne induite par le fipronil
The widely used insecticide fipronil is a thyroid disruptor in rat acting on thyroid hormone hepatic metabolism. In sheep, a more relevant species for the human thyroid regulation, fipronil-induced thyroid-disruption is much more limited. The goal of this thesis was to characterize the mode of action of fipronil on thyroid function at the hepatic level focusing on 1) the potential role of fipronil sulfone, the main fipronil metabolite formed in vivo, and on 2) interspecific differences in terms of fipronil metabolism and/or sensitivity to thyroid disruption that can prejudge of the relevance of the different animal models for the risk assessment of fipronil for human health. Fipronil sulfone was as efficient as fipronil to induce the expression and/or activity of enzymes involved in thyroid hormone or fipronil hepatic metabolism both in vivo in rat and in vitro on hepatocytes. The use of knock-out mice for xenosensors nuclear receptors strongly suggested an implication of the nuclear receptor Constitutive Androstane Receptor and/or Pregnane X Receptor on fipronil-induced thyroid disruption

La mise en evidence des recepteurs de la progesterone (rp) dans les cellules tumorales mammaires revele a l'echelon cellulaire une heterogeneite d'expression. Les travaux presentes dans cette these ont pour objectif l'etude des origines possibles de cette heterogeneite. Deux lignees tumorales mammaires mcf-7 et t47-d ont ete utilisees ainsi que des empreintes de tumeurs. La quantification par analyse d'images de plusieurs immunomarquages fluorescents (rp-ki-67 ou brdu-adn) a montre que l'expression des rp est, en partie, liee a la cinetique de proliferation des cellules sur les trois modeles etudies ainsi que dans un sous clone de la lignee mcf-7. D'autre part, l'etude de sous populations clonales de la lignee mcf-7 revele l'existence de sous-clones soit rp positifs soit rp negatifs qui indique que l'expression des rp est egalement liee a l'information genetique. Dans un deuxieme temps, nous avons envisage la combinaison de l'immunomarquage des rp et de l'hybridation in situ de leurs arnm mais nous avons rencontre certaines difficultes. De ce fait nous avons synchronise les cellules d'un sous-clone de la lignee mcf-7 et realise les differents marquages cellulaires. Avec l'aide de la technique de rt-pcr (polymerase chain reaction) nous avons etabli un modele d'expression des rp (arnm, proteine) au cours des differentes phases du cycle cellulaire pour le meme sous-clone. Des anomalies numeriques du chromosome 11 (porteur du gene rp) existent dans les cellules tumorales de la lignee t47-d. L'hybridation in situ de ce chromosome et l'immunomarquage des rp sur cellules interphasiques a montre une absence de correlation entre le nombre de chromosomes 11 et la positivite en rp, on l'explique par l'absence de correlation entre le nombre de chromosome 11 et le nombre de copies du gene des rp, detecte par double hybridation des chromosomes 11 et du gene des rp

La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l’intermédiaire d’un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d’être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l’efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d’interaction du RGnRH, jusqu’alors inconnus, avec l’idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d’identifier le premier partenaire d’interaction du RGnRH : la protéine SET. Par des expériences de « GST pull down », nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d’AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l’AMPc la première démonstration d’un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l’interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l’activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l’induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l’expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d’amplifier l’action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l’expression de SET est fortement augmentée dans l’hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d’une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien
Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle

A plethora of naturally-produced steroid hormones, or artificial homologues of them, are being introduced into the aquatic and terrestrial environments each year. Two examples of these are the natural oestrogen 17-oestradiol (E2) and the oestrogen receptor antagonist, Bisphenol A (BPA), both of which target the ribonucleoprotein telomerase through upregulation of its telomerase reverse transcriptase component, TERT. The main objectives of this study were firstly to isolate and characterize the actual mRNA sequence for the telomerase catalytic subuninit, Tert, in rainbow trout (Oncorhynchus mykiss) (Walbaum, 1792) and European purple sea urchin (Paracentrotus lividus) (Lamarck, 1816), with the aim of developing qPCR assays for the amplification and quantification of Tert. Further objectives were to use these assays in controlled exposure studies to establish whether and to what extent the aforementioned chemicals regulate Tert transcription and by doing so further understand the mechanism of Telomerase gene expression and the extent to which environmental oestrogen can interfere. The initial step of sequence characterization and assay devlopment was successful in the case of rainbow trout where two possible splice variants of Tert mRNA are identified, omTertShort and omTertLong. Two qPCR assays were developed for the relative quantification of both of these splice variants in rainbow trout samples, the latter of these successfully amplifying its target in test samples. In order to demonstrate in vitro and in vivo modulation of telomerase activity and mRNA expression, early life-stages of rainbow trout and purple sea urchin, as well as rainbow trout hepatocytes, were exposed to a range of concentrations of E2 and BPA. Purple sea urchin embryos were exposed to 200, 20 and 2 ng E2/ml for 28 hours until they had reached the stage of pluteus larvaes. Rainbow trout embryos were exposed to 500, 20 and 0.1 ng E2/ml and 600 and 150 ng BPA/ml for 167 days from immediately after fertilization. Rainbow trout hepatocytes were exposed to 20 and 2 ng E2/ml for 48 hours. The results from this study show that telomerase activity as well as TERT mRNA expression can be significantly modulated by exposure to oestrogens and other oestrogenic chemicals. E2 concentrations as low as 20 ng/ml lead to an increase in telomerase activity early-life stages of purple sea urchin and upregulation in the transcription of Tert mRNA in unhatched rainbow trout embryos. BPA induced similar response (600 ng/ml) in hatched rainbow trout alevins larvae. Very high exposures to E2 (500 ng/ml) do however lead to downregulation of Tert mRNA in hatched alevins larvae. Differential regulatory response can be observed between different tissue types of 167 day old fry, with an upregulatory response observed at 0.1 ng E2/ml in liver and muscle tissues, but not in brain. Similarly, brain tissues were observed expressing significantly less mRNA than liver and muscle samples when exposed to BPA (150 ng/ml). It is evident that the previously observed link between environmental oestrogens and telomerase is also present in the two test species examined; purple sea urchin and rainbow trout.

Introduction. L’étiopathogénie de l’hypospadias reste largement discutée, faisant intervenir des facteurs endocriniens, génétiques, vasculaires et environnementaux. Objectifs. Étudier les mécanismes génétiques impliquées dans la différenciation sexuelle masculine. Matériels et méthodes. Une cohorte de 284 patients a été établie depuis 2011 pour séquençage de l’ADN lymphocytaire, préputial et urothélial de trois gènes candidats (NR5A1, MAMLD1, AR). Une analyse pangénomique (puce à ADN et Whole Exome Sequencing (WES)) a été réalisée. Le registre REMAPAR a été utilisé pour analyser l’association entre hormone chorionique gonadotrophique (hCGb) maternelle et hypospadias. Résultats. Des polymorphismes dans AR ont une fréquence allélique significativement plus élevée (p<0,01). Des variations non décrites des trois gènes ne semblent pas susceptibles d’altérer l’épissage. Une duplication hétérozygote intragénique de NR5A1 a été retrouvée chez un individu. Par ailleurs, 35 anomalies (15,7%) pouvant être en lien avec l’hypospadias ont été identifiées par puces à ADN englobant 25 gènes candidats. L’analyse WES a mise en évidence 22 nouveaux variants. Enfin, une association a été constatée pour l’hCGb surtout en cas de forme sévère et après ajustement pour dysfonction placentaire (p<0,03). Conclusion. L’étude des trois gènes candidats n’a pas vérifié l’hypothèse selon laquelle les séquences lymphocytaires et sur les tissus cibles seraient différentes. Cependant, l’analyse pangénomique a trouvé 15,7% anomalies et 47 gènes potentiellement candidats. L’utilisation des nouveaux outils d’analyse génétique semble essentielle à la compréhension des mécanismes conduisant à l’hypospadias
Background. Hypospadias is the most common malformation affecting the male genitalia. Although the causes remain often unknown, endocrine, vascular and environmental factors have been implicated. However, the genetic basis is probably underestimated. Objectives. To study the genetic factors implicated in sexual differentiation. Methods. A cohort of 284 children born with hypospadias has been established since 2011. DNA was extracted from blood lymphocytes and fibroblasts obtained after a foreskin biopsy. Sanger sequencing of AR, MAMLD1 and NR5A1, SNP-array analysis, and Whole Exome Sequencing (WES) were performed. A second cohort of 1,311 pregnant women was established to evaluate an association between maternal fhCGb and hypospadias. Results. No mutation in AR and MAMLD1 was found. Non-described variations of AR, MAMLD1 and NR5A1 were identified without any splicing activity. Some polymorphisms in AR gene had a MAF significantly higher (p<0.01). A heterozygous intragenic duplication of NR5A1 was found. Pangenomic analysis included 35 anomalies (15.7%) that could be a potential cause of isolated, distal, non-hereditary hypospadias encompassing 25 candidate genes. 22 variants was classified as pathogenic on WES. Finally, a significant difference of fhCGb for severe types was identified even after adjustment for placenta dysfunction (p<0.03). Conclusion. Our hypothesis about “somatic” mutations in 3 candidate genes was not ascertained. However, pangenomic analysis found 15.7% anomalies which could be likely linked to hypospadias. The use of WES could be an attractive method for exploring further these results as we identified 22 variants in 30 cases of familial hypospadias

Les glandes salivaires sont des glandes mixtes : la salive (produit exocrine) estimpliquée dans le maintien de l'homéostasie buccale alors que les secrétions endocrines (ex :facteurs de croissance) ont un rôle physiologique (gamétogénèse, ostéogenèse,hypertension...) Chez les mammifères, elles affichent un dimorphisme sexuel qui laisseentrevoir une sensibilité éventuelle à des xeno-hormones.Ce mémoire présente l'action de la génistéine (phyto-oestrogène) et/ou de la vinclozoline(anti-androgène) sur la glande submandibulaire (SM) de rat lors d'une exposition précoce viala mère (gestation-lactation) et lors d'une exposition pendant la période de croissance (dusevrage à l'âge adulte). Les glandes SM, prélevées au stade immature et jeune adulte, ont faitl'objet d'une analyse histologique et d'une étude de marqueurs moléculaire des fonctionsendocrines et exocrines associées aux processus gustatifs. L'exposition précoce ralenti ledéveloppement de la glande SM et augmente sélectivement la préférence au sucré des malesimmatures mais pas des adultes ; l'analyse moléculaire révèle une action sélective sur lesfonctions exocrines corrélée à celle sur les préférences, ainsi qu'une action sur les fonctionsendocrines (facteurs de croissances) qui s'inverse avec l'âge. L'exposition à partir du sevrageperturbe seulement les mâles qui présentent des altérations des structures sécrétrices coupléesà des modifications d'expression des récepteurs hormonaux et facteurs de croissance, maisaussi au taux sérique de l'EGF.Cette étude identifie la glande submandibulaire comme cible de perturbateurs endocriniens etpose la question des conséquences physiologiques à terme

The choroid plexuses (CPs) are highly vascularized structures constituted by a single layer of epithelial cells that project into the brain ventricles. The CPs are the main site of cerebrospinal fluid (CSF) production and constitute the Blood-CSF Barrier (BCSFB), holding high relevance in the surveillance of the CSF chemical composition. These structures contribute for the synthesis of biological compounds essential for the functioning and protection of the central nervous system (CNS) against neurotoxic insults. The expression of taste signalling pathway components in the CPs and its regulation by sex hormones was previously determined in a cDNA microarray study previously performed by our group. Moreover, the taste signalling pathway was determined as one as the top five pathways regulated by female sex hormones. The ectopic expression of sweet, umami and bitter taste signalling in extra oral organs have been extensively studied. In these organs, the taste receptors seems to behave as sensors to assess the composition of body fluids. The expression of the taste molecular machinery and its putative regulation by sex hormones in the CP raised the hypothesis that the taste signalling pathway could be one of the mechanisms involved in the monitoring of the chemical composition of blood and CSF at the BCSFB that my differ with gender. Considering this, we aimed to evaluate the presence and the functionality of the taste signalling pathway, as well as its regulation by the female sex hormones 17β-estradiol (E2) and progesterone (P4) in rat CP. In the first study, the presence and functionality of the taste signalling pathway was assessed. Transcripts for the taste-related genes Tas1r1, Tas1r2, Tas1r3, Tas2r109, Tas2r144, Gustducin, Plcb2, Ip3R3 and TrpM5 were found in CPs from adult Wistar rats. The expression of Tas1r1, Tas1r2, Tas2r144, Gustducin, Plcb2 and TrpM5 proteins was confirmed by Western blot, immunohistochemistry and immunocytochemistry. As umami and sweet receptors are heterodimeric receptors, we performed double labelling immunofluorescence, that showed the co-expression of T1R1 and T1R3 proteins that form the umami receptor, as well as, the coexpression of T1R2 and T1R3 proteins that form the sweet receptor. Having established the cellular localization of the taste machinery in CP epithelial cells (CPEC) we further evaluated the subcellular expression of taste proteins. For that, CPs were double labelled with antibodies for each of the taste-related proteins studied and a fluorescent marker of glycosylated surfaceexpressed proteins, revealing that taste-related proteins are located in the plasma membrane. After confirming the presence of the taste pathway molecular machinery, we proceed with functional assays. Considering that most of toxic/noxious compounds are bitter compounds that may exist in the CSF, we turned our attention to the bitter taste signalling pathway. Thus, to evaluate the functionality of the bitter pathway in primary cultures of CPEC we performed single cell calcium imaging assays using D-Salicin as the bitter stimulus. We observed an increase in intracellular Ca2+ evoked by D-Salicin that was diminished in the presence of the bitter taste receptors (T2Rs) blocker Probenecid, suggesting that T2R in the CPs are capable of sensing bitter compounds in the CSF and/or blood. An analysis in silico of our previous cDNA microarray data revealed that the decline of hormone levels in female rats upon ovariectomy clearly induced an up-regulation of the T2Rs Tas2r109, Tas2r124, Tas2r134, and Tas2r144, and the downstream effector molecules Plcb2 and Trpm5. Moreover, Tas2r109 and Tas2r144 were differentially expressed between female and male, showing a higher expression in males. This data led us to the second study of these thesis where the regulation of the taste pathway by female sex hormones was analyzed. For that, we compared the expression of taste-related genes in the CPs of sham and ovariectomized female Wistar rats and in CPs explants from newborn rats incubated with different concentrations of E2 and/or P4. Our results confirmed the cDNA microarray data, corroborating the regulation of taste-related genes by E2 and P4. The bitter receptors Tas2r109, Tas2r144, and the tasterelated genes Plcb2 and Trpm5 were down-regulated by ovarian hormones both in vivo and ex vivo. Functional implications of female sex hormones regulation was assessed, by single cell Ca2+ imaging, with the bitter compound, Denatonium Benzoate (DB), which is a known ligand of Tas2r144. Single cell Ca2+ imaging was performed in the immortalized CP epithelial cell line Z310 incubated with E2 and/or P4 in the presence of the respective hormone receptor blocker (fulvestrant or mifepristone, respectively). Intracellular Ca2+ variation, observed by single cell calcium imaging, was diminished in the presence of female sex hormones. However, while E2 effects were mediated via the nuclear E2 receptor, P4 effects were not abolished by the blocker of nuclear P4 receptor. Knocking-down Tas2r144 with a specific siRNA effectively reduced the Ca2+ response to the bitter compound DB, in a similar manner to E2 and P4, suggesting that female sex hormones down-regulated the responses of CPEC to chemical stimuli by reducing Tas2r144. In summary, our results confirmed and characterized the presence and functionality of the taste signalling machinery in CPs showing its regulation by female sex hormones. These results suggest that the taste signalling pathway may be one of the mechanisms by which the CP surveys the chemical composition of the CSF and elicit responses to modulate and maintain brain homeostasis. The achievements reached with this work will contribute to a better understanding of the mechanisms underlying the sensor/protective role of CPs in the CNS.
Os plexos coróides (PCs) são estruturas altamente vascularizadas localizadas nos ventrículos cerebrais. São constituídos por uma monocamada de células epiteliais cuboides assentes numa membrana basal que está em contacto direto com um estroma altamente vascularizado, com tecido conjuntivo rico em fibroblastos e com células do sistema imunitário. O lado apical destas estruturas está em contacto direto com o líquido cefalorraquidiano e apresenta microvilosidades e alguns cílios. O lado oposto, o lado basal, está em contacto com vasos sanguíneos altamente fenestrados. Os PCs formam vilosidades de modo a aumentar as superfícies de contacto entre as células epiteliais do lado apical com o líquido cefalorraquidiano, e com o fluído intersticial do lado basal. Os PCs são o principal local de síntese de líquido cefalorraquidiano, estão envolvidos na vigilância imunológica do sistema nervoso central, são estruturas ativas na neurogénese e são ainda responsáveis pela remoção de compostos tóxicos e metabolitos do líquido cefalorraquidiano resultantes de processos metabólicos do sistema nervoso central. A sua ultra estrutura é composta por células unidas através de junções de oclusão que permite que este órgão forme uma barreira entre o sangue e o líquido cefalorraquidiano, a barreira sanguelíquido cefalorraquidiano, que previne o movimento de substâncias para dentro e fora do cérebro pelos espaços intercelulares. Tendo em conta a sua estrutura e localização, os PCs têm um papel crucial na monotorização da composição química do líquido cefalorraquidiano e do sangue, contribuindo para a síntese e/ou transporte de compostos essenciais para um normal funcionamento e proteção do sistema nervoso central contra agentes neurotóxicos. A expressão de genes relacionados com a via de transdução de sinal do paladar no PC foi detetada num estudo de microarrays de DNA complementar, realizado previamente pelo nosso grupo de trabalho, em PCs de ratos castrados Wistar Han. Nesse estudo, verificou-se ainda que a via de sinalização do paladar foi uma das cinco vias mais afetadas pelas hormonas sexuais femininas, estando sobre expressa em ratos fêmea ovariectomizados. A expressão da via do paladar tem sido amplamente estudada, fora da cavidade oral, em órgãos como o estômago, intestino, pulmões, coração, testículos, artérias, entre outros. Nestes órgãos, os recetores do paladar parecem funcionar como sensores que monitorizam a composição química dos fluidos biológicos circundantes que, quando ativados, desencadeiam respostas metabólicas defensivas. A via de transdução de sinal do paladar, descrita originalmente nas células sensoriais nos botões gustativos, inicia-se com a ligação das moléculas do sabor ao respetivo recetor do paladar (Tas1r2/Tas1r3 e Tas1r1/Tas1r3, respetivamente para o doce e umami, e Tas2r para o amargo) ativando uma via de transdução de sinal que resulta na despolarização da célula. Os ensaios experimentais apresentados nesta tese tiveram como objetivo geral investigar o papel da via do paladar na capacidade de monitorização da composição química do líquido cefalorraquidiano e/ou sangue por parte dos PCs. Deste modo, o presente estudo teve como objetivos específicos a análise da expressão dos diferentes componentes da via de sinalização do paladar e o estudo da sua funcionalidade no PC de rato, bem como a avaliação da sua regulação pelas hormonas sexuais. No primeiro trabalho desenvolvido, avaliou-se a expressão e a funcionalidade da via do paladar no PC de rato, por RT-PCR e singel cell calcium imagingI, respectivamente. Identificaram-se transcritos de genes da via de sinalização do paladar tais como os recetores do paladar Tas1r1, Tas1r2, Tas1r3, que formam os recetores do doce e umami, os recetores Tas2r109 e Tas2r144 que detetam compostos amargos, e moléculas da maquinaria da via de sinalização (Gustducina, fosfolipase beta 2, inositol tri-fosfato e o membro 5 da subfamília M do canal recetor de catiões com potencial transitório). A expressão das respetivas proteínas foi confirmada por Western blot, imunofluorescência, imunohistoquímica e imunocitoquímica. Uma análise imunocitoquímica de explantes de PC com marcação dupla da proteína alvo (Tas1r3 e Tas2r144) e do marcador de células epiteliais de PC, a transtirretina, revelou que os recetores do paladar estão localizados nas células epiteliais de PC. Uma vez confirmada a expressão de toda a maquinaria da via de transdução de sinal do paladar no PC, procedemos aos estudos funcionais. Sabendo que a maioria dos compostos tóxicos e/ou nocivos correspondem a compostos amargos, selecionámos como alvo do nosso estudo os recetores do amargo, dado que a sua presença no PC poderá estar associada à deteção de compostos neurotóxicos. De modo a avaliar a sua funcionalidade, utilizámos culturas primárias de células epiteliais de PC onde realizámos a técnica de single cell calcium imaging utilizando a D-Salicina como composto amargo. O estímulo com D-Salicina provocou um aumento nos níveis intracelulares do ião cálcio que na presença de um bloqueador de recetores do amargo, o Probenecid, diminuíram de intensidade. Uma vez que a presença de um bloqueador dos recetores do amargo diminui a resposta observada, é muito provável que esta se deva à ativação dos recetores do amargo. Deste modo, podemos afirmar que os recetores do amargo presentes no PC são funcionais, podendo detetar compostos amargos presentes no líquido cefalorraquidiano e/ou no sangue. O segundo estudo desenvolvido, teve por base uma análise de dados de microarrays de DNA complementar de PC de ratos castrados, realizado previamente pelo nosso grupo. A análise in silico destes dados mostrou que o declínio das hormonas sexuais femininas aumentava significativamente os níveis de expressão dos recetores do amargo Tas2r109, Tas2r124, Tas2r134 e Tas2r144, e as moléculas da via de sinalização do paladar Plcb2 e Trpm5. Mostrou também que os recetores do amargo Tas2r109 e Tas2r144 são diferencialmente expressos entre machos e fêmeas, apresentando uma expressão mais elevada nos machos. Deste modo, no segundo estudo apresentado nesta tese, analisámos a regulação da via de transdução do paladar pelas hormonas sexuais femininas. Assim, estudámos a expressão dos genes Tas2r109, Tas2r144, Plcb2 e Trpm5 no PC de ratos fêmea castrados e não castrados e em explantes de PC de ratos recém-nascidos (5-6 dias de idade) incubados com diferentes concentrações de estradiol e progesterona. Os resultados obtidos, in vivo e ex vivo, corroboram os resultados dos microarrays de DNA complementar comprovando a regulação hormonal dos genes da via de transdução de sinal do paladar no PC. O efeito das hormonas sexuais na resposta das células do PC a um estímulo amargo foi avaliado por single cell calcium imaging com o composto Benzoato de Denatónio, um ligando do recetor do amargo Tas2r144, numa linha celular imortalizada de células epiteliais de PC (Z310) em presença de estradiol e/ou progesterona e também na presença dos bloqueadores dos recetores nucleares das hormonas. Observámos que a presença de estradiol e/ou progesterona diminuiu a amplitude de resposta das células Z310. O estudo com os bloqueadores hormonais permitiu-nos concluir que o efeito do estradiol parece ocorrer via recetor nuclear, e os resultados com progesterona sugerem o envolvimento dos recetores membranares da progesterona. Com o objetivo de analisar se as respostas observadas por single cell calcium imaging ocorrem via recetor Tas2r144, realizaram-se ainda ensaios de cálcio com o recetor Tas2r144 silenciado com um RNAi específico. Os ensaios de cálcio após silenciamento do Tas2r144 mostraram uma redução significativa da resposta ao Benzoato de Denatónio, de maneira semelhante aos efeitos do estradiol e progesterona, sugerindo que as hormonas sexuais femininas regulam negativamente as respostas do PC a estímulos químicos, reduzindo a Tas2r144. A regulação exercida pelas hormonas sexuais na resposta a compostos amargos no PC, eventualmente neurotóxicos, poderá estar envolvida na diferença no aparecimento e progressão de doenças do sistema nervoso central entre géneros. Em suma, os nossos resultados confirmam a presença da maquinaria molecular da via de transdução de sinal do paladar no PC de rato, mostrando que a via do amargo está funcional, e revelam a sua regulação pelas hormonas sexuais femininas. Os resultados obtidos neste trabalho suportam a hipótese de que a via de transdução do paladar poderá ser um dos mecanismos utilizado pelo PC para monitorizar o conteúdo químico do líquido cefalorraquidiano e/ou sangue e responder de modo a modular e manter a homeostasia do sistema nervoso central. No futuro, os resultados apresentados poderão contribuir para uma melhor compreensão dos mecanismos por detrás da função do PC em monitorizar/proteger o sistema nervoso central.

Polycystic ovary syndrome is a complex, heterogeneous disease that affects 5-10% of reproductive-aged women. It is characterized by clinical or biochemical hyperandrogenism, oligo-anovulation, and polycystic ovary morphology. Instigating endocrine findings include aberrantly rapid gonadotropin-releasing hormone pulsatile secretion, elevated luteinizing hormone, sub-optimal levels of follicle-stimulating hormone, and hyperandrogenism. Metabolic symptoms are also present including hyperinsulinemia, insulin resistance, and dyslipidemia. These endocrine and metabolic findings are also accompanied by ovarian dysfunction and improper folliculogenesis. Because aberrant functioning of the hypothalamic-pituitary-gonadal axis is central to the pathophysiology of polycystic ovary syndrome, it is beneficial to examine it for abnormalities. Polycystic ovary syndrome has been shown to have both genetic and environmental components. Its strong genetic component has been demonstrated in twin studies, family association studies, candidate gene studies, and genome-wide association studies. Previous genome-wide association studies have found many candidate genes including those for DENND1A (differentially expressed in normal and neoplastic cells domain containing 1A), THADA (thyroid adenoma-associated protein), FSHR (follicle-stimulating hormone receptor), and LHR (luteinizing hormone receptor). This, together with the central endocrine abnormalities, prompted this review on polymorphisms of receptors of the hypothalamic-pituitary-gonadal axis, including those for gonadotropin-releasing hormone, luteinzing hormone, follicle-stimulating hormone, estrogen, and progesterone, as well as anti-müllerian hormone. Studies on single-nucleotide polymorphisms of these receptors were found on PubMed, Web of Science, and Google Scholar and subsequently analyzed. Many different single-nucleotide polymorphisms were studied, but only a handful of them have been subjected to repeated studies. Only rs2293275 of the luteinizing hormone receptor and rs2349415 of the follicle-stimulating hormone receptor, both at 2p16.3, were found to have a possible role in the etiology of polycystic ovary syndrome, but all eight were found to have a possible phenotypic role: rs13405728, rs2293275, and rs4539842 of the luteinizing hormone receptor; rs6165, rs6166, rs2268361, and rs2349415 of the follicle-stimulating hormone receptor; and rs2002555 of the anti-müllerian hormone receptor. The limitations most affecting the results of these studies include small sample sizes, heterogeneous study populations, lack of generalizability due to ethnicity, and lack of control or adjustment for confounders. It is necessary to develop a standardized study protocol and separate polycystic ovary syndrome patients based on phenotype in order to obtain stronger results in the future.

碩士
慈濟大學
生理暨解剖醫學碩士班
99
It is well known that there is sexual difference on stress responses. Studies indicate that testosterone (T) has an inhibitory effect on stress-induced hypothalamic-pituitary-adrenal (HPA) activation. Androgen receptor (AR) is found throughout the central nervous system, but the paraventricular nucleus in the hypothalamus (PVH), the integrator of HPA activity, expresses little. Evidence shows that medial preoptic neucleus (MPN), an AR bearing nucleus, may play a critical role on modulating the effects of T on stress-induced HPA axis activation. The mechanism, however, is not clear. Male Spraque-Dawley rats (250-300g) were used in this project. We used 30-min restraint (RST) as the stressor, and the cFos expression by immunohitochemistry (IHC) as the index of neuronal activation. The numbers of cFos-immunoreactivity (-ir) cells at both MPN and PVH in gonadectomized (GDX) and intact rats were similar under non RST condition. But GDX rats showed more cFos-ir cells at both nuclei than intact control after RST challenge. T supplement reversed the promotive effect of GDX on stress-induced neuronal activation. These results indicated that T might tonically inhibit stress-induced HPA activation through inhibiting MPN neurons. Surppissingly, AR blockage did not mimic the phenomena seen in stress suffering GDX rats. It indicated that AR may not be important on T regulating stress response. Moreover, T can be transformed into estradiol (E) by neurons in the MPN. We found that E supplement could reverse the promotive effect of GDX like T supplement. So, T may inhibit stress response, after it is converted into E.

Thesis (M.S.)--Michigan State University. Dept. of Physiology, 2008.
"Committee members, Weiming Li, Richard Miksicek, and Cheryl Sisk"--Acknowledgments. Title from PDF t.p. (viewed on Aug. 4, 2009) Includes bibliographical references (p. 51-54). Also issued in print.