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Cloning and characterization of neuropeptide Y receptors of the Y1 subfamily in mammals and fishStarbäck, Paula January 2000 (has links)
Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors. In rat a receptor called PP1, later renamed Y4, was cloned and characterized. It displays the highest amino acid sequence identity to the Y1 receptor. Rat Y4 differs extensively from human Y4, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y4 are the most diverged orthologues in the NPY receptor family. In guinea pig, the y6 receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig. In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y1, Y4 and y6, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. The discoveries of the rat Y4 and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.
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Neuronale Grundlagen appetitiven und konsumatorischen Verhaltens: Die Funktion des Neuropeptids SIFamid bei Drosophila melanogaster / The neuronal basis of appetitive and consumatory behavior: The function of the neuropeptide SIFamide in Drosophila melanogasterKobbenbring, Simon 09 September 2013 (has links)
Für alle Tiere ist die Nahrungsaufnahme ein überlebenswichtiger Vorgang. Das konsumatorische Verhalten ist Teilaspekt eines homöostatischen Prozesses. Ausgelöst wird konsumatorisches und appetitives Verhalten durch interne, motivationale Zustände. Die internen Zustände „gesättigt“ und „ungesättigt“ werden bei vielen Tierarten durch ein Wechselspiel zwischen orexigen- und anorexigen-wirkenden Neuropeptiden vermittelt. Um die zentralnervöse Steuerung von Appetit und Sättigung genauer zu klären, wurde in dieser Studie die schwarzbäuchige Taufliege Drosophila melanogaster als Modellorganismus genutzt. Dabei konnte gezeigt werden, dass durch eine artifizielle, thermogenetisch induzierte Aktivierung von Neuronen, welche das Neuropeptid SIFamid exprimieren, das Verhalten der Insekten verändert wird. Die Tiere zeigen vermehrte Nahrungsaufnahme und sind motivierter, auf appetitive gustatorische und olfaktorische Reize zu reagieren. Des Weiteren wurden Hinweise gesammelt, dass das Neuropeptid SIFamid keinen inhibitorischen Einfluss auf das Balzverhalten der Taufliegen ausübt. Mit immunhistochemischen Färbungen konnte gezeigt werden, dass die Dendriten der SIFamidergen Neurone in unmittelbarer Nähe zu den Axonendigungen von Neuronen, die orexigen- oder anorexigen-wirkende Neuropeptide produzieren, liegen. Dieser Befund lässt auf ein mögliches Zusammenspiel zwischen den diversen peptidergen Neuronen schließen. Mit Hilfe der split-GFP-Technik konnte das peptiderge Netzwerk der SIFamidergen Neurone detaillierter untersucht werden. Es wurde gefunden, dass die SIFamidergen Neurone in enger räumlicher Nachbarschaft zu Neuronen, die in die Nahrungsaufnahme sowie die nervöse Steuerung des Metabolismus involviert sind, stehen. In Kombination mit einem zweiten, unabhängigen Expressionssystem konnten die SIFamidergen Neurone thermogenetisch depolarisiert werden und die neuronale Antwort der olfaktorischen Rezeptorneurone auf Duftstimuli in den Antennalloben mit Hilfe von in-vivo Calcium Imaging untersucht werden. Es konnte dadurch gezeigt werden, dass die neuronale Aktivität in den Duftsinneszellen erhöht ist. Aufgrund vorliegender Daten aus Anatomie, Verhaltensexperimenten und in-vivo Calcium Imaging lässt sich schlussfolgern, dass SIFamid ein bis dato unbekannter „Mitspieler“ bei der Steuerung der Nahrungsaufnahme ist und modulierend auf sensorische neuronale Schaltkreise sowie insgesamt appetitfördernd auf die Taufliege wirkt.
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Rôles fonctionnels de neuropeptides chez Drosophila melanogaster : développement d'outils génétiques et exemples d'études physiologique et comportementaleSellami Chakroun, Azza 25 June 2010 (has links)
Dans le but d’étudier le rôle fonctionnel des neuropeptides chez la drosophile, nous avons essayé d’utiliser le récepteur µ aux opioïdes (MOR) afin d’inhiber temporairement la libération de neuropeptides. Cependant, quand nous l’avons exprimé dans les cellules endocrines produisant l’hormone adipokinétique (AKH), le récepteur MOR a présenté une activité constitutive empêchant tout contrôle dans le temps. Nous proposons d’utiliser les récepteurs RASSL (Receptors Activated Solely by Synthetic Ligands) développés chez les vertébrés. Ces récepteurs, activés uniquement par des ligands synthétiques, ont été modifiés afin qu’ils soient dénués d’activité constitutive. Afin de tester rapidement l’efficacité d’un récepteur RASSL inhibiteur chez la drosophile, nous avons cherché une alternative à la mesure des taux de tréhalose et de glycogène (démontrant la libération d’AKH) qui en plus d’être laborieuse produit des résultats variables. L’hormone GPA2/GPB5, récemment découverte, est particulièrement intéressante, en effet, elle semble avoir un rôle antidiurétique. Nous avons généré des lignées transgéniques exprimant la protéine Gal4 dans les cellules GAP2/GPB5 et nous avons montré que l’ablation génétique de ces cellules compromet la survie suggérant qu’elles pourraient représenter un bon système pour tester l’efficacité du RASSL inhibiteur. Ensuite nous nous sommes intéressés au contrôle de la libération du neuropeptide AKH qui a un rôle fonctionnel homologue à celui du glucagon des vertébrés. Malgré quelques résultats encourageants, nous n’avons pas pu confirmer le rôle inhibiteur de l’AstA sur la libération de l’AKH. Enfin nous nous sommes intéressés au réseau qui contrôle le comportement de cour. Nous avons généré des lignées transgéniques exprimant la protéine Gal4 sous la dépendance du promoteur potentiel du récepteur du neuropeptide SIFamide (SIFR) et nous avons démontré que ce neuropeptide contrôle le comportement de cour via son récepteur SIFR en agissant, du moins en partie, sur les neurones fruitless. / In order to study the functional roles of neuropeptides in Drosophila, I attempted to use the µ opioïd receptor (MOR) to temporarily inhibit liberation of neuropeptides. However, when expressed in the AKH (adipokinetic hormone) endocrine cells of Drosophila, MOR turned out to be constitutively active, making it impossible to use it as envisioned. Others have encountered and subsequently solved similar problems for the so-called RASSL (Receptors Activated Solely by Synthetic Ligands) in vertebrates. As the bioassay for testing release of AKH is cumbersome, time-consuming and variable, I searched for an alternative neuroendocrine system to test the efficiency of an inhibitory RASSL in Drosophila. The recently discovered GPA2/GPB5 seemed particularly attractive, as it is likely an antidiuretic hormone. Transgenic flies expressing Gal4 in the GPA2/GPB5 were produced and genetic ablation of these cells severely compromised survival, suggesting that this might indeed be good model system. Attempts so demonstrate a physiological role of allatostatin A in the release of AKH yielded inconclusive results. In order to study the role of the neuropeptide SIFamide, which modulates sexual behavior in Drosophila, I generated transgenic flies expressing Gal4 under dependence of potential promoter of this neuropeptide receptor (SIFR) and showed that SIFamide controls courtship at least in part by acting directly on fruitless neurons.
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Circuits neuronaux sous-tendant la régulation émotionnelle par le système ocytocinergique / Neuronal circuits underlying the regulation of emotions by the oxytocinergic systemGoyon, Stéphanie 10 September 2018 (has links)
L’ocytocine (OT) est un neuropeptide synthétisé au sein de l’hypothalamus. On sait aujourd’hui que l’OT est fortement impliquée dans la modulation de nombreux comportements et émotions. Pourtant, il reste encore difficile d’expliquer comment s’organise le système ocytocinergique, par exemple en sous-ensembles spécifiques. De même, les circuits neuronaux impliqués dans leur recrutement restent obscures, tout comme leur potentielle plasticité. C’est pourquoi, au cours de ma thèse, je me suis attachée à mieux comprendre ces différents points. Les résultats obtenus ont montré que i) un sous-ensemble spécifique de neurones OT est recruté par la peur ; ii) le système OT fait preuve d’une grande plasticité après une exposition à un contexte effrayant ; iii) le neuropeptide S est capable de recruter une sous-population de neurones OT afin d’exercer une action anxiolytique ; iv) les neurotransmetteurs monoaminergiques sont eux-mêmes capables de recruter différents sous-ensembles de neurones OT. En conclusion, mon travail a mis en évidence la plasticité de ce système peptidergique et différentes manières de recruter de manière spécifiques certains sous-ensembles existants de neurones OT. / Oxytocin (OT) is a peptide synthesized within the hypothalamus. We now know that OT is strongly involved in the modulation of many behaviors and emotions. However, it is still difficult to explain how the oxytocinergic system is organized, for example in specific sub-populations. Similarly, the neuronal circuits involved in their recruitment remain obscure, like their potential plasticity. That is why, during my thesis, I tried to better understand these different points. The results obtained showed that i) a specific sub-population of OT neurons is recruited by fear; ii) the OT system exhibits great plasticity after exposure to a scary context; iii) the neuropeptide S is able to recruit an OT neuron sub-population in order to exert an anxiolytic effect; iv) monoaminergic neurotransmitters are themselves able to recruit different sub-populations of OT neurons. In conclusion, my work has highlighted the plasticity of this peptidergic system and different ways to recruit in a specific manner some existing sub-populations of OT neurons.
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Neuropeptide Y Receptors in Human, Guinea pig and Chicken : Cloning, <i>in vitro</i> Pharmacology and <i>in situ</i> HybridizationHolmberg, Sara January 2001 (has links)
<p>Neuropeptide Y (NPY) is known to influence a vast number of physiological and behavioral processes such as vasoconstriction, circadian rhythms, feeding, anxiety and memory. Peptides of the NPY family bind to five different cloned G-protein coupled receptor subtypes (Y1, 2, 4-6). The studies compiled in this thesis present inter-species comparisons of sequence similarities, binding properties and expression patterns among receptors of the NPY family.</p><p>Cloning of Y1 and Y2 receptor subtypes from guinea pigs revealed strong binding profile similarity to the corresponding human receptors. Previously demonstrated atypical binding profiles in the caval vein of guinea pigs were concluded to result from other receptors than the cloned Y1 and Y2 receptors, or possibly combinations of distinct receptor subtypes.</p><p>The guinea pig Y5 receptor was found to be expressed in regions of the brain that have been indicated as important for regulation of food intake. Expression in the hypothalamus, amygdala and brain stem was noticed, similar to studies in rats and humans. In other brain regions, such as the striatum and hippocampus, some species differences were observed.</p><p>Mutagenesis studies of the human Y1 receptor indicated sites important for binding both of endogenous agonists and synthetic antagonists. Putative new sites of interaction with the Y1 antagonists BIBP3226 and/or SR120819A were recognized. The data were used to construct a three-dimensional structure model, based on a high-resolution bovine rhodopsin model.</p><p>Cloning of the chicken (<i>Gallus gallus</i>) Y1, Y2 and Y5 receptors revealed high sequence similarities with mammalian receptors. Most endogenous ligands bound with similar affinities as to mammalian receptors. The strongest exception was the discovery of high-affinity binding to chicken Y2 of [Leu<sup>31</sup>, Pro<sup>34</sup>]NPY, which was previously considered to bind non-Y2 receptors only. </p><p>The new human Y1 receptor model provides a basis for further investigations of ligand-receptor interactions which will be aided by information on NPY receptors from other taxa. Guinea pigs are concluded to be a good complement to rats and mice for studying NPY signaling. These results demonstrate the benefits of species comparisons for pharmacological studies.</p>
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Neuropeptide Y Receptors in Human, Guinea pig and Chicken : Cloning, in vitro Pharmacology and in situ HybridizationHolmberg, Sara January 2001 (has links)
Neuropeptide Y (NPY) is known to influence a vast number of physiological and behavioral processes such as vasoconstriction, circadian rhythms, feeding, anxiety and memory. Peptides of the NPY family bind to five different cloned G-protein coupled receptor subtypes (Y1, 2, 4-6). The studies compiled in this thesis present inter-species comparisons of sequence similarities, binding properties and expression patterns among receptors of the NPY family. Cloning of Y1 and Y2 receptor subtypes from guinea pigs revealed strong binding profile similarity to the corresponding human receptors. Previously demonstrated atypical binding profiles in the caval vein of guinea pigs were concluded to result from other receptors than the cloned Y1 and Y2 receptors, or possibly combinations of distinct receptor subtypes. The guinea pig Y5 receptor was found to be expressed in regions of the brain that have been indicated as important for regulation of food intake. Expression in the hypothalamus, amygdala and brain stem was noticed, similar to studies in rats and humans. In other brain regions, such as the striatum and hippocampus, some species differences were observed. Mutagenesis studies of the human Y1 receptor indicated sites important for binding both of endogenous agonists and synthetic antagonists. Putative new sites of interaction with the Y1 antagonists BIBP3226 and/or SR120819A were recognized. The data were used to construct a three-dimensional structure model, based on a high-resolution bovine rhodopsin model. Cloning of the chicken (Gallus gallus) Y1, Y2 and Y5 receptors revealed high sequence similarities with mammalian receptors. Most endogenous ligands bound with similar affinities as to mammalian receptors. The strongest exception was the discovery of high-affinity binding to chicken Y2 of [Leu31, Pro34]NPY, which was previously considered to bind non-Y2 receptors only. The new human Y1 receptor model provides a basis for further investigations of ligand-receptor interactions which will be aided by information on NPY receptors from other taxa. Guinea pigs are concluded to be a good complement to rats and mice for studying NPY signaling. These results demonstrate the benefits of species comparisons for pharmacological studies.
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Conception, synthèse et activité anti-hyperalgésique de dérivés de guanidine comme antagonistes des récepteurs du neuropeptide FF (NPFF) / Design, synthesis and anti-hyperalgesic activity of guanidine derivatives as antagonists of neuropeptide FF (NPFF)Hammoud, Hassan 30 March 2012 (has links)
Le traitement des douleurs sévères repose dans la plupart des cas sur l’utilisation d’analgésiques opiacés. Cependant après administration répétée d’analgésique morphinique, on observe une tolérance aux agents morphiniques. Au niveau moléculaire, l’hypothèse physiopathologique met en avant la participation de systèmes ‘’dits anti-opiacés’’, comme le système du neuropeptide FF (NPFF). Dans un premier temps, une méthode de synthèse a été développée pour introduire divers α-aminoacides sur les chlorures d’α-hétéroaryle correspondants. Cette méthode originale, faisant appel aux conditions et catalyseurs décrits par Buchwald, a conduit à la découverte de ligands puissants de ces récepteurs. D’autre part, notre travail s’est basé sur la conception, la synthèse et l’étude des relations structure-activité d’autres familles de ligands pour les récepteurs du NPFF. Plusieurs séries de molécules ont été étudiées, parmi elles deux grandes familles : les aminoguanidines hydrazones et les quinolin-2-yl guanidines. Cette étude nous a permis d’identifier plusieurs ligands actifs in vitro (nanomolaire) et in vivo sur les récepteurs du NPFF. Ainsi nous disposons actuellement de ligands sélectifs vis-à-vis des deux récepteurs du NPFF. Une difficulté de synthèse des aryl/hétéroaryl guanidines a été rencontrée, en particulier avec les anilines appauvries en électrons. Pour cela nous avons développé une méthode de guanidinylation directe des halogénures d’aryle/hétéroaryle catalysée par le cuivre. Cette méthode de synthèse de dérivés de guanidines N-monosubstituées s’est montré très efficace, généralisable à quasiment tous les systèmes aromatiques et hétéroaromatiques. Enfin, quelques observations inattendues au cours de la réalisation de la réaction de Buchwald ont été décrites. / The treatment of severe pain involves in most cases the use of opioid analgesics such as morphine or fentanyl. However, after repeated administration of narcotic analgesics, a tolerance to opioid agents is observed. This result needs to gradually increase the dose of drug to obtain the desired analgesic effect. At the molecular level, the pathophysiological hypothesis emphasizes the participation of so-called ''anti-opioid'' systems, like the neuropeptide FF (NPFF) system. Initially, a synthetic method was developed to introduce various α-amino acids on various α-heteroaryl chlorides. This original method, using the conditions and catalysts described by Buchwald, led to potent NPFF receptor ligands. Then, our work was based on the design, synthesis and study of structure-activity relationships of other ligands for NPFF receptors. Several series of molecules have been studied. Among them two large families were selected: aminoguanidine hydrazones and quinolin-2-yl guanidines. This study allowed us to identify several potent ligands in vitro (nanomolar Ki) and active in vivo. So we have now selective ligands towards the two NPFF receptors. A loss of reactivity during the synthesis of aryl / heteroaryl guanidines was observed, in particular with electron deficient anilines. For these compounds we developed a new method of direct guanidinylation of aryl and heteroaryl halides catalyzed by copper. This method of synthesis of N-monosubstituted guanidines is very effective, generalizable to almost all aromatic and heteroaromatic systems. Finally, some unexpected observations during the completion of Buchwald reaction were described.
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Efeito da leptina e da nutrição sobre o perfil de expressão de genes hipotalâmicos em novilhas zebuínas (Bos taurus indicus) no início da puberdade / Leptin and nutrition effect on gene expression profile of hypothalamic genes in Bos taurus indicus heifers on the onset of puberty: an experimental studyMagalhães, Juliane Diniz 25 June 2010 (has links)
Investigou-se o efeito da leptina exógena e do maior consumo de energia, sobre o padrão da expressão de genes no hipotálamo de novilhas zebuínas; de modo a elucidar o mecanismo de sinalização da leptina no hipotálamo e os genes responsáveis pela obtenção da puberdade. Trinta e seis novilhas não púberes, e com idade entre 18 e 20 meses, foram divididas em três grupos experimentais: baixa energia (BAIXA), alta energia (ALTA), baixa energia com administração de leptina recombinante ovina (BAIXA+LEP), totalizando 56 dias de tratamento. Vinte e quatro novilhas foram abatidas ao apresentar sinais de puberdade, sendo eles: concentração de progesterona no soro superior a 1 ng/mL por duas amostras seguidas e presença de corpo lúteo detectável por ultra-sonografia. O hipotálamo foi colhido e armazenado a -80ºC. As amostras foram submetidas à extração do RNA total, tratadas com DNAse I e submetidas à síntese de cDNA. A quantificação relativa de quatro genes candidatos reconhecidamente envolvidos com a sinalização hipotalâmica da leptina em bovinos: NPY, NPY-Y1, NPY-Y4 e SOCS-3, foi feita através de PCR quantitativo (tempo real). Não houve efeito da administração de leptina sobre a expressão do NPY (P=0,70), ou de seus receptores: NPY-Y1 (P=0,27) e NPY-Y4 (P=0,92) no início da puberdade. A expressão de SOCS-3 foi reduzida (P=0,05) no hipotálamo de novilhas tratadas com leptina, o que sugere menor ação inibitória sobre a leptina. Em novilhas alimentadas com dieta de alta energia, a expressão do NPY-Y1 foi reduzida (P=0,04), o que indica que o hipotálamo estaria menos sensível à ação do NPY, permitindo a entrada precoce em puberdade. Nos demais genes estudados, NPY (P=0,75), NPY-Y4 (P=0,92) e SOCS-3 (P=0,24), a dieta não alterou significativamente suas expressões hipotalâmicas. Estudo mais abrangente do efeito da nutrição e da administração de leptina foi realizado através de hibridização em microarranjos de DNA, objetivando a identificação de possíveis genes candidatos, expressos no hipotálamo, que influenciam na obtenção da puberdade em novilhas Nelore tratadas com leptina ou submetidas à dieta de alta energia. Foram encontrados 78 genes cuja expressão foi alterada pela densidade energética da dieta (P=0,05) no hipotálamo das novilhas Nelore, destes foram selecionados os que apresentaram razões de expressão da ordem de 1,4 ou mais, totalizando 20 genes. Entre esses se destaca o gene da β-arrestina 1 (ARRB1), que foi 1,40 vezes mais expresso (P=0,04) em novilhas submetidas à dieta de alta energia, pois atua na mediação da dessensibilização dos receptores acoplados à proteína-G-(GPCRs)1, como os receptores de NPY. Foram encontrados 134 genes diferencialmente expressos (P=0,05) devido a aplicação de leptina. Dentre os 80 genes que apresentaram razões superiores a 1,4, 18 genes tiveram a expressão reduzida, e 62 tiveram a expressão aumentada pela aplicação de leptina. Destes, alguns estão envolvidos na regulação da sinalização da leptina. O gene SRC foi menos expresso (1,64 vezes; P=0,04) em novilhas tratadas com leptina, o que sugere menor ação inibitória pela SHP-2. A proteína SOCS-2 foi 1,43 vezes (P=0,01) mais expressa no hipotálamo de novilhas tratadas com leptina. Sabe-se que, ao contrário de SOCS-1 e SOCS-3, CIS e SOCS-2 não se ligam, ou inibem, as janus kinases. O STAT-3 foi 2,14 vezes (P=0,03) mais expresso em novilhas tratadas com leptina, e sua ativação possibilita a ligação hipotalâmica da leptina com seu receptor (Ob-Rb). As IGFPB-1 e -2 foram mais expressas no hipotálamo de novilhas tratadas com leptina que em novilhas não tratadas, sendo IGFPB-1 1,78 vezes (P=0,04) mais expressa e IGFPB-2 1,89 vezes (P=0,05). As IGFPBs podem desempenhar função de potencialização da ação do IGF-1, ou exercer ação inibitória. Conclui-se que tanto o consumo de energia quanto a aplicação com leptina influenciaram o padrão de expressão gênica no hipotálamo de novilhas Nelore. A modulação da quantidade do receptor do NPY, NPY-Y1, no hipotálamo pode ser uma via importante pela qual a nutrição afeta o início da puberdade em novilhas. E ainda que estudos mais aprofundados de expressão dos genes encontrados nas hibridizações por microarranjo poderão revelar interações mais concisas entre os genes, a nutrição e a leptina na obtenção da puberdade. / It was investigated the effect of exogenous leptin and the high energy intake on gene expression pattern in the hypothalamus of zebuine heifers; in a way to elucidate the mechanism of leptin signaling in hypothalamus and the responsible genes for puberty. Thirty six heifers not in puberty at 18 and 20 months of age were divided in three experimental groups: low energy diet (LOW), high energy diet (HIGH), low energy diet with administration of recombinant ovine leptin (LOW+LEP), totalizing 56 days of treatment. Twenty four heifers were slaughtered when presented the signals of puberty: progesterone serum concentration above 1 ng/mL for two followed weeks and the presence of detectable corpus luteum by ultrasonography. The hypothalamus was collected and stored at -80ºC. Samples were submitted to total RNA extraction, treated with DNAse I and submitted to cDNA synthesis. The relative quantification of four candidate genes admittedly involved with hypothalamic leptin signaling in bovine: NPY, NPY-Y1, NPY-Y4 and SOCS-3, was evaluated through quantitative PCR (real time). There was no effect of leptin administration on NPY expression (P=0.70), or on its receptors: NPY-Y1 (P=0.27) and NPY-Y4 (P=0.92) in the onset of puberty. The expression of SOCS-3 was reduced (P=0.05) in the hypothalamus of heifers treated with leptin, what suggests lower inhibitory action over leptin. In heifers fed high energy diets, the expression of NPY-Y1 was reduced (P=0.04), which indicates that the hypothalamus would be less sensitive to the action of NPY, allowing the precocious onset of puberty. In other studied genes, NPY (P=0.75), NPY-Y4 (P=0.92) and SOCS-3 (P=0.24), the diet did not significantly altered their hypothalamic expressions. A more comprehensive study regarding the effect of nutrition and leptin administration was performed through the hybridization in DNA microarrangements, aiming the identification of possible candidate genes, expressed in hypothalamus that influence in the onset of puberty in Nelore heifers treated with leptin or submitted to high energy diets. It was found 78 genes whose expression was altered by the energy density of the diet (P<0.05) in the hypothalamus of Nelore heifers. From them, it was selected those genes which presented rates of expression in the order of 1.4 or more, totalizing 20 genes. From them, the highlight gene was β-arrestin 1 (ARRB1) which was 1.40 more expressed (P=0.04) in heifers fed high energy diet due to its action in the mediation of receptors desensibilization coupled to protein-G-(GPCRs)1, as the receptors of NPY. It was found 134 genes differently expressed (P<0.05) due to leptin administration. From the 80 genes that presented rates of expression higher than 1.4, 18 genes had their expression reduced and 62 had their expression increased by leptin administration. Some of these 62 genes are involved in the regulation of leptin signaling. The gene SRC was the less expressed (1.64 times; P=0.04) in heifers treated with leptin what suggests lower inhibitory action by SHP-2. The protein SOCS-2 was 1.43 times (P=0.01) more expressed in the hypothalamus of heifers treated with leptin. It is known that on the contrary of SOCS-1 and SOCS-3, CIS and SOCS-2 do not bind or inhibit, as janus kinases. The STAT-3 was 2.14 times (P=0.03) more expressed in heifers treated with leptin and its activation enables the hypothalamic binding of leptin and its receptor (Ob-Rb). The IGFPB-1 and -2 were more expressed in the hypothalamus of heifers treated with leptin than the animals not treated, being the IGFPB-1 1.78 times (P=0.04) more expressed and the IGFPB-2 1.89 times (P=0.05). The IGFPBs could play a function of IGF-1 action enhancer or exert an inhibitory action. It is concluded that both energy intake and leptin administration influenced gene expression pattern in the hypothalamus of Nelore heifers. The modulation of the receptor quantity of NPY, NPY-Y1 in hypothalamus could be an important route in which nutrition affects the onset of puberty in heifers. Moreover, more detailed studies regarding gene expression in hybridization by microarrangement could reveal more concise interactions between genes, nutrition and leptin in the onset of puberty.
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Estudo do mecanismo molecular da progesterona e do estradiol sobre o início da puberdade em novilhas Nelore / Study of the molecular mechanism of progesterone and estradiol on the onset of puberty in Nellore heifersMagalhães, Juliane Diniz 08 September 2014 (has links)
A elucidação dos mecanismos moleculares pelos quais tratamentos hormonais alteram o início da puberdade é de fundamental importância para o desenvolvimento de estratégias que reduzam a idade ao primeiro parto, e consequentemente a taxa de desfrute do rebanho Nelore. Foram investigados os efeitos do uso de dispositivos de progesterona, e do estradiol endógeno, sobre mecanismos moleculares controlando a obtenção da puberdade de novilhas Nelore peripúberes. Especificamente, como as diferenças na expressão de genes relativos à reprodução em duas áreas do hipotálamo. Trinta e cinco novilhas Nelores não púberes, e com idade entre 13 e 14 meses, foram divididas em quatro tratamentos experimentais (nove ou oito por tratamento): dispositivo de P4 sem estradiol (SP); dispositivo de P4 com estradiol (PE); sem dispositivo de P4 e sem estradiol (SS); e sem dispositivo de P4 e com estradiol (SE). As novilhas foram alimentadas no cocho pós desmame até atingirem 295 ± 11 kg, com fornecimento de água à vontade. Ao término do tratamento hormonal as novilhas foram abatidas e as porções de hipotálamo colhidas para processamento e armazenagem a -80 ºC. O RNA total do tecido hipotalâmico foi extraído, tratado com DNAse I e submetido à síntese de cDNA para estudo da expressão gênica por PCR em tempo real (qRT-PCR). Foram formados pools de RNA para a realização de um estudo abrangente da administração de progesterona e do efeito do estradiol endógeno e das diferenças entre áreas do hipotálamo, realizado por sequenciamento de nova geração (RNA-Seq), de forma a identificar possíveis genes candidatos no hipotálamo. Foram encontrados genes diferencialmente expressos alterados pelos tratamentos e entre as áreas do hipotálamo relativos à obtenção da puberdade. / The understanding of the molecular mechanisms by which nutrition, genetics and hormonal treatments affect the beginning of puberty is of great importance for developing strategies aiming to reduce the age at first calving, and therefore increase the slaughter rate in Nellore cattle. The effects of progesterone device and of endogenous estradiol on the molecular mechanisms controlling the attainment of puberty in Nellore heifers were investigated. Specifically, the molecular pathways of progesterone and estradiol were studied in the hypothalamus. Thirty five non-pubertal heifers, between 13 and 14 months of age, were divided into four treatment (nine or eight per treatment): P4 device without estradiol (SP), P4 device with estradiol (PE), without P4 device and without estradiol (SS), and without P4 device and with estradiol (SE). The heifers were fed after weaning until reach 295 ± 11 Kg, with water access. At the end of the hormonal treatments all heifers were slaughtered and the hypothalamus areas were harvested, processed and then also stored at -80°C. Total RNA of hypothalamus were extracted, treated with DNase I and submitted to cDNA synthesis for gene expression quantification by real time PCR (qRT-PCR). RNA samples were pooling to realize a comprehensive study of the effects of progesterone administration and endogenous estrogen on attainment of puberty by next-generation sequencing (RNA-Seq), in order to identify possible candidate genes in the hypothalamus. Genes diffentially expressed between hypothalamic areas and affected by treatments were found.
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Evolution of the Neuropeptide Y System in Vertebrates with Focus on FishesLarsson, Tomas January 2007 (has links)
<p>Gene families in vertebrates often contain more dulicates (paralogs) than in invertebrates. This has been attributed to genome duplications, i.e., tetraploidizations. Two of the gene families that have expanded in vertebrate evolution are the neuropeptide Y (NPY) family of peptides and the neuropeptide Y receptors (NPYR) that are involved in many brain functions including appetite regulation.</p><p>Two NPYR genes, Y2 and Y7, were cloned in the rainbow trout. Although they arose from a common ancestral gene in early vertebrate evolution, their ligand-binding properties are very similar. Two NPYR genes were cloned in the coelacanth <i>Latimeria chalumnae</i> and found to be orthologs of Y5 and Y6 discovered in mammals.</p><p>Analyses of gene families close to the NPYR genes in the pufferfishes <i>T. nigroviridis</i> and <i>T. rubripes</i> showed that at least 25 additional gene families had an evolutionary history similar to the NPYR family, thereby providing evidence for fish specific-duplications of these chromosomes. Cloning and phylogenetic analysis of 22 NPYR gene fragments from several ray-finned fishes showed that basal species seem to have the same repertoire as tetrapods. Despite the tetraploidization in the teleost fish lineage, many teleosts seem to have fever genes than the gnathostome ancestor due to gene loss. Only one duplicate seems to have survived.</p><p>The NPY peptide family was found to have expanded in the teleost tetraploidization with duplicates of both NPY and PYY (peptide YY) in some teleosts. Fourteen neighboring gene families were found to have evolved in a similar manner as the NPY-family genes. Positional information fascilitated orthology assignment of peptide genes in teleost fishes and allowed correction of previously misidentified genes.</p><p>In summary, the evolutionary history of the NPY and NPYR gene families involve large-scale duplication events coinciding with the proposed tetraploidizations. The appearance of new genes in early vertebrates and in teleost fishes probably had important implications for the evolution of new functions in this system.</p>
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