No description available.

Familial risk and environmental stress promote the development of alcohol dependence. We investigated whether a positive family history of alcoholism affects the neuroendocrine response to a standardized laboratory stress test in healthy subjects without alcohol use disorders. Twenty-four high-risk subjects with a paternal history of alcoholism (PHA) and 16 family history negative (FHN) controls were evaluated. Psychosocial stress was induced by having subjects deliver a 5-min speech and mental arithmetics in front of an audience on separate days, after drinking either placebo or ethanol (0.6 g/kg) in a randomized sequence. Adrenocorticotropin (ACTH) was measured in 10 plasma samples covering up to 75 min after the stress test. Plasma arginine vasopressin (AVP) was determined before the stressor, at the time of maximum ACTH secretion, and at 75 min after stress onset. The stress test induced a phasic increase in ACTH secretion. At the time of maximum ACTH, AVP was significantly increased in relation to baseline. Compared to placebo, alcohol administration significantly attenuated maximum ACTH concentration in PHA but not FHN subjects, and decreased AVP measured in the same samples in PHA but not FHN subjects. We conclude that activation of the hypothalamic–pituitary–adrenal system by psychosocial stress is accompanied by an increase in peripheral plasma AVP levels. Secretion of both ACTH and AVP suggest that alcohol attenuates the stress response selectively in PHA but not FHN subjects. This might imply some short-term positive alcohol effect in sons of alcoholics, but also constitute a mechanism by which their risk to develop alcohol use disorders is increased.

Background: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Methods: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. Results: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. Conclusions: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

One of the few attributes common to almost all living organisms is an ability to generate and maintain endogenous rhythms, which are controlled by a biological clock. The processes, which recur with a period of about 24 hours, are known as the circadian rhythms. The circadian clock controls rhythms of molecular, physiological as well as behavioral processes and adapts their activity to regularly appearing changes in day and night or season. In case of mammals, central oscillator is located in the hypothalamic suprachiasmatic nuclei (SCN). The SCN clock entrains rhythms of peripheral oscillators located in cells of other tissues. The central oscillator itself is synchronized with external environment mainly by a light-dark cycle, however, other cues can entrain the SCN clock as well. For example, during prenatal development, entrainment of a fetal clock is entirely dependent on non-photic cues derived from maternal organism. This study aimed to investigate a mechanism of the communication between the maternal and fetal central oscillators. A hypothesis was tested whether maternal melatonin may play a role in entrainment of the circadian clock in the fetal SCN. Furthermore, a mechanism, how melatonin may entrain the fetal clock was investigated at molecular level. The results provided evidence, that...

Le noyau paraventriculaire (PVN) de l'hypothalamus régule une série de phénomènes physiologiques incluant l'équilibre énergétique et la pression artérielle. Nous avons identifié une cascade de facteurs de transcription qui contrôle le développement du PVN. SIM1 et OTP agissent en parallèle pour contrôler la différenciation d'au moins cinq types de neurones identifiables par la production d'OT, AVP, CRH, SS et TRH. Ces Facteurs de transcriptions contrôlent le développement des lignées CRH, AVP et OT en maintenant l'expression de Brn2 qui à son tour est nécessaire pour la différenciation terminale de ces neurones. L'analyse du transcriptome du PVN nous a permis d'identifier plusieurs gènes qui ont le potentiel de contrôler le développement du PVN. Nous voulons développer un paradigme de perte de fonction qui permettrait l'étude de ces gènes candidats sur une grande échelle. Le but de ce projet est de caractériser le PVN en développement de l'amphibien en vue de l'utilisation de ce modèle pour des études fonctionnelles. Nous avons cloné des fragments de cDNA de Sim1, OTP, Brn2, Sim2, CRH, Ot, AVP et TRH à partir de l'ARN total de Xenopus Laevis. Nous avons adapté notre technique d'hybridation in situ pour caractériser l'expression de ces gènes chez l'amphibien aux stades 33-39, 44, 51, 54, 60, et chez l'adulte. Résultats. Les Facteurs de transcription Sim1, OTP, et Brn2 commencent à être exprimés dans le PVN prospectif au stade 33. L'expression des marqueurs de différenciation terminale devient détectable entre les stades 37 et 39. De façon intéressante, le PVN occupe initialement un domaine de forme globulaire puis à partir du stade 44 s'allonge le long de l’axe dorso-ventral. Cet allongement se traduit par une organisation en colonnes des cellules du PVN que nous n'avons pas observée chez les rongeurs. Le développement du PVN est conservé chez l'amphibien dans la mesure où la relation entre l'expression des facteurs de transcription et des marqueurs de différenciation terminale est conservée. Il existe par ailleurs des différences entre la topographie des PVN des mammifères et de l'amphibien. L'organisation en colonnes de cellules pourrait correspondre à des mouvements de migration tangentielle. Nous sommes maintenant en mesure de tester la fonction des facteurs de transcription dans le PVN par l'approche d'invalidation par morpholinos. / The paraventricular nucleus PVN of the hypothalamus regulates a series of physiological phenomena including the maintenance of energetic balance and arterial blood pressure. We have previously identified a cascade of transcription factors that control the development of the PVN. Sim1 and OTP act in concert to mediate the terminal differentiation of at least five types of neurons identifiable by their production of OT, AVP, CRH, SS and TRH. These transcription factors control the development of the OT, AVP and CRH producing neurons by maintaining the expression of Brn2, which is in turn required for the terminal differentiation of these cell lines. The transcriptome analysis of the PVN allowed us to identify a handful of genes that are potentially implicated in the development of this brain structure. Our goal is to develop a loss of function paradigm that would allow a high troughput study of these candidate genes. The main goal of this project is to characterize the developing PVN in the amphibian in order to use this model in our functional studies of these genes. We have cloned fragments of cDNA of Sim1, OTP, Brn2, Sim2, CRH, TRH, AVP and OT using Xenopus laevis total RNA. We have also adapted our in situ hybridization technique to characterize the expression of these genes in stage 33-39, 44, 51, 54, 60 and adult amphibian brain. Sim1, OTP and Brn2 are expressed in the prospective PVN as soon as stage 33. The expression of the terminal differentiation markers become detectable between stages 37-39. Interestingly, the PVN is initially restricted to a more globular domain and begins to extend along the dorso-ventral axis at around stage 44. This vertical extension translates into a column organization that we do not observe in rodents. The development of the PVN is well conserved in the amphibian in the sense that the relation between the expression of the different transcription factors and the terminal differentiation markers is conserved. We can also observe some topographical differences between the mammalian and amphibian PVN. The column organization the different PVN cell types might correspond to the tangential migration that is observed in the mouse. We are now well equipped to test the function in the PVN of the known transcripton factors as well as the candidate genes previously identified in our lab using a morpholino-mediated gene knock down.

La vasopressine (AVP), ou hormone antidiurétique, a deux effets majeurs : 1°) des effets sur la perméabilité à l'eau du canal collecteur rénal, médiés par les récepteurs V2, qui permettent la formation d'urine hyperosmotique et donc une économie d'eau ; 2°) des effets vasoconstricteurs sur le muscle lisse vasculaire, médiés par les récepteurs V1a, qui peuvent induire un effet presseur. L'idée que l'AVP puisse jouer un rôle dans l'hypertension artérielle par ses effets vasculaires est souvent avancée, mais les travaux explorant cette hypothèse n'ont pas été concluants. Par contre, peu de travaux ont été consacrés au fait que l'AVP puisse contribuer à l'hypertension de façon indirecte, par son action sur le rein. Pourtant, on sait que l'AVP stimule la réabsorption de sodium dans le canal collecteur en augmentant l'activité du canal sodium épithélial. Il est donc concevable que, dans certains cas, les effets de l'AVP puissent produire une rétention hydrosodée susceptible d'augmenter la pression artérielle. Le but de nos travaux a été d'étudier les relations entre l'excrétion d'eau et de sodium et la pression artérielle, en tenant compte notamment du débit urinaire, de la concentration de l'urine, de l'AVP et du rythme nycthéméral d'excrétion d'eau et de sodium en relation avec celui de la pression artérielle. Nous avons réalisé des travaux expérimentaux chez le rat normal, conscient, et chez des sujets participant à diverses investigations cliniques. Les principaux résultats obtenus sont les suivants. (1) En utilisant des agonistes et des antagonistes sélectifs des récepteurs V1a et V2 in vivo chez le rat, nous avons pu montrer que l'influence de l'AVP sur l'excrétion du sodium était biphasique, du fait d'effets opposés médiés par les récepteurs V1a et V2 et de leurs seuils de réponse différents. (2) Chez l'Homme, nous avons montré que des changements d'apport sodé entraînent des changements correspondants de la concentration urinaire de sodium (qui ne sont pas immédiats), sans modification du débit urinaire, contrairement à l'idée généralement acceptée. (3) Nous avons également montré que le niveau de concentration de l'urine est très variable d'un individu à l'autre mais qu'en moyenne, les hommes ont une osmolalité urinaire plus élevée que les femmes. D'autre part, les afro-américains ont une urine significativement plus concentrée et un volume urinaire plus faible que les caucasiens et un rythme nycthéméral atténué. Chez les hommes jeunes normotendus, la pression pulsée est positivement corrélée à la concentration de l'urine. Ceci suggère que l'AVP a une action plus intense chez certains sujets et pourrait jouer un rôle dans le contrôle de la pression artérielle par ses effets V2. (4) Parmi des sujets présentant des caractéristiques du syndrome métabolique, ceux qui ont un rythme nycthéméral d'excrétion d'eau et de sodium perturbé ont une chute nocturne de pression artérielle plus faible (or, on sait que c'est d'un mauvais pronostic). Ce travail a permis pour la première fois de mettre en évidence des relations entre l'excrétion d'eau et de sodium et la pression artérielle, relations qui sont variables selon la période du nycthémère considérée et dans lesquelles l'AVP est probablement impliquée. Nos études ouvrent ainsi de nouvelles pistes de recherche sur le rôle potentiel de cette hormone dans certaines pathologies cardiovasculaires et rénales qui pourraient, à terme, conduire à l'utilisation d'antagonistes des récepteurs V2 de l'AVP dans le traitement de ces pathologies.

Le noyau paraventriculaire (PVN) de l'hypothalamus régule une série de phénomènes physiologiques incluant l'équilibre énergétique et la pression artérielle. Nous avons identifié une cascade de facteurs de transcription qui contrôle le développement du PVN. SIM1 et OTP agissent en parallèle pour contrôler la différenciation d'au moins cinq types de neurones identifiables par la production d'OT, AVP, CRH, SS et TRH. Ces Facteurs de transcriptions contrôlent le développement des lignées CRH, AVP et OT en maintenant l'expression de Brn2 qui à son tour est nécessaire pour la différenciation terminale de ces neurones. L'analyse du transcriptome du PVN nous a permis d'identifier plusieurs gènes qui ont le potentiel de contrôler le développement du PVN. Nous voulons développer un paradigme de perte de fonction qui permettrait l'étude de ces gènes candidats sur une grande échelle. Le but de ce projet est de caractériser le PVN en développement de l'amphibien en vue de l'utilisation de ce modèle pour des études fonctionnelles. Nous avons cloné des fragments de cDNA de Sim1, OTP, Brn2, Sim2, CRH, Ot, AVP et TRH à partir de l'ARN total de Xenopus Laevis. Nous avons adapté notre technique d'hybridation in situ pour caractériser l'expression de ces gènes chez l'amphibien aux stades 33-39, 44, 51, 54, 60, et chez l'adulte. Résultats. Les Facteurs de transcription Sim1, OTP, et Brn2 commencent à être exprimés dans le PVN prospectif au stade 33. L'expression des marqueurs de différenciation terminale devient détectable entre les stades 37 et 39. De façon intéressante, le PVN occupe initialement un domaine de forme globulaire puis à partir du stade 44 s'allonge le long de l’axe dorso-ventral. Cet allongement se traduit par une organisation en colonnes des cellules du PVN que nous n'avons pas observée chez les rongeurs. Le développement du PVN est conservé chez l'amphibien dans la mesure où la relation entre l'expression des facteurs de transcription et des marqueurs de différenciation terminale est conservée. Il existe par ailleurs des différences entre la topographie des PVN des mammifères et de l'amphibien. L'organisation en colonnes de cellules pourrait correspondre à des mouvements de migration tangentielle. Nous sommes maintenant en mesure de tester la fonction des facteurs de transcription dans le PVN par l'approche d'invalidation par morpholinos. / The paraventricular nucleus PVN of the hypothalamus regulates a series of physiological phenomena including the maintenance of energetic balance and arterial blood pressure. We have previously identified a cascade of transcription factors that control the development of the PVN. Sim1 and OTP act in concert to mediate the terminal differentiation of at least five types of neurons identifiable by their production of OT, AVP, CRH, SS and TRH. These transcription factors control the development of the OT, AVP and CRH producing neurons by maintaining the expression of Brn2, which is in turn required for the terminal differentiation of these cell lines. The transcriptome analysis of the PVN allowed us to identify a handful of genes that are potentially implicated in the development of this brain structure. Our goal is to develop a loss of function paradigm that would allow a high troughput study of these candidate genes. The main goal of this project is to characterize the developing PVN in the amphibian in order to use this model in our functional studies of these genes. We have cloned fragments of cDNA of Sim1, OTP, Brn2, Sim2, CRH, TRH, AVP and OT using Xenopus laevis total RNA. We have also adapted our in situ hybridization technique to characterize the expression of these genes in stage 33-39, 44, 51, 54, 60 and adult amphibian brain. Sim1, OTP and Brn2 are expressed in the prospective PVN as soon as stage 33. The expression of the terminal differentiation markers become detectable between stages 37-39. Interestingly, the PVN is initially restricted to a more globular domain and begins to extend along the dorso-ventral axis at around stage 44. This vertical extension translates into a column organization that we do not observe in rodents. The development of the PVN is well conserved in the amphibian in the sense that the relation between the expression of the different transcription factors and the terminal differentiation markers is conserved. We can also observe some topographical differences between the mammalian and amphibian PVN. The column organization the different PVN cell types might correspond to the tangential migration that is observed in the mouse. We are now well equipped to test the function in the PVN of the known transcripton factors as well as the candidate genes previously identified in our lab using a morpholino-mediated gene knock down.