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Agradecemos a compreensão. on 2017-09-28T12:44:18Z (GMT) / Submitted by ANA REBECA CASTRO LIMA null (anarebecalima@hotmail.com) on 2017-09-30T14:34:04Z No. of bitstreams: 1 Tese_rebeca_com_certificado.pdf: 1529070 bytes, checksum: c56d5fa5c704e63a65dc06b2c95fb45d (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-10-02T16:59:38Z (GMT) No. of bitstreams: 1 lima_arc_dr_jabo.pdf: 1529070 bytes, checksum: c56d5fa5c704e63a65dc06b2c95fb45d (MD5) / Made available in DSpace on 2017-10-02T16:59:38Z (GMT). No. of bitstreams: 1 lima_arc_dr_jabo.pdf: 1529070 bytes, checksum: c56d5fa5c704e63a65dc06b2c95fb45d (MD5) Previous issue date: 2017-08-07 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Objetivou-se com o presente estudo determinar a influência da temperatura ambiente sobre o metabolismo basal e a partição de energia em cabras Saanen e Anglo-Nubiana. Dois experimentos foram conduzidos utilizando-se seis cabras da raça Saanen e seis cabras da raça Anglo-Nubiana, em mantença, a partir da técnica da calorimetria indireta de circuito aberto, com máscaras faciais. No experimento 1, após um período de alimentação de 3 dias, os animais foram submetidos a jejum e a medição das trocas gasosas foi realizada durante 30 min a 0, 12, 20, 36, 44, 60 e 68 horas após jejum. A produção de metano foi igual a zero após 31 horas de jejum para cabras Saanen e 40 horas para as Anglo-Nubiana. Os resultados sugerem que o período ideal para medir a produção de calor em jejum (PCJ) deve ser entre 40 horas e 60 horas de jejum para cabras em mantença. No experimento 2, as cabras foram submetidas a três diferentes temperaturas: 10 ºC ± 0,23; 20 ºC ±0,41 e 35 ºC ±1,05. Após um período de alimentação de 4 dias, os animais foram submetidos a jejum por 60 horas e a medição da troca gasosa foi realizada a partir da metodologia da calorimetria indireta utilizando as máscaras faciais durante 30 min em cada animal aleatoriamente. Para ambas as raças, houve uma diminuição linear na ingestão de matéria seca (MS) e matéria orgânica (MO; P<0,10) a medida que a temperatura aumentou de 10 para 35ºC. A digestibilidade aparente da MS, da MO, da proteína bruta e da fibra em detergente neutro apresentaram comportamento quadrático (P < 0,10), com menores valores na temperatura de 20ºC. Aos 35ºC, a digestibilidade da proteína bruta das cabras Anglo-Nubiana foi maior que a das cabras Saanen. A frequência respiratória (FR) e volume respiratório aumentaram em taxas crescentes com o aumento da temperatura ambiente. Aos 35ºC, tanto a FR quanto o volume respiratório das cabras Saanen foram maiores que o das cabras Anglo-Nubiana. A metabolizabilidade e a energia metabolizável da dieta das cabras Saanen, aos 35ºC, foram menores que as das cabras Anglo-Nubiana. Em ambas as raças, para cada 1ºC de variação abaixo de 20ºC houve um aumento na produção de calor (PC) na ordem de 6,7 kJ/ kg0,75 PV, enquanto que para cada 1ºC de variação acima de 20ºC houve um aumento na PC na ordem de 7,9 kJ/ kg0,75 PV. Por outro lado, para PCJ indicou que para cada 1ºC de variação abaixo de 20ºC houve um decréscimo na PCJ na ordem de 5,4 kJ/ kg0,75 PV, enquanto que para cada 1ºC de variação acima de 20ºC houve um decréscimo na ordem de 8,7 kJ/ kg075 PV, em temperatura ambiente entre 10 e 35ºC. As cabras da raça Saanen e Anglo-Nubiana são capazes de manter a homeostasia em ambientes entre 10 e 35ºC. Por outro lado, o aumento da temperatura ambiental induz a um menor consumo, tendendo a balanços energéticos negativos. Acima de 20ºC, as cabras tendem a diminuir a produção de calor em jejum, como uma tentativa de conservação de energia em ambientes quentes. / The aim of this study was to determine the influence of the ambient temperature on the basal metabolism and the energy partition in Saanen and Anglo-Nubian goats. Two experiments were carried out using six Saanen goats and six Anglo-Nubian goats, in maintenance, using the indirect calorimetry technique with open-circuit facemask respirometry. In experiment 1, after a three-day feed period, the animals were fasted and gas exchange measurements were performed for 30 min at 0, 12, 20, 36, 44, 60 and 68 hours after fasting. Methane production approached zero after 31 hours of fasting for Saanen goats and 40 hours for Anglo-Nubian goats. The results suggest that the period for measuring fasting heat production (FHP) should be between 40 hours and 60 hours of fasting for maintenance goats. In experiment 2, goats were submitted to three different temperatures: 10 ºC ± 0.23, 20 ºC ± 0.41 and at 35 ºC ± 1.05. After a feeding period of four days, goats were fasted for 60 hours and gas exchange measurement was performed for 30 min in each animal randomly. For both breeds, dry matter (DM) and organic matter intake (MO; P <0.10) decrease linearly as the temperature increased from 10 to 35ºC. The apparent digestibility of DM, OM, crude protein and neutral detergent fiber presented quadratic behavior (P <0.10), with the lowest values at 20ºC. At 35ºC, the crude protein digestibility of Anglo-Nubian goats was higher than that of Saanen goats. Respiratory rate (RR) and respiratory volume increased with increasing ambient temperature. At 35ºC, both RR and respiratory volume of Saanen goats were higher than that of Anglo-Nubian goats. The diet metabolizability and metabolizable energy of Saanen goats, at 35ºC, were lower than those of the Anglo-Nubian goats. In both breeds, for each 1ºC of variation below 20ºC there was an increase in HP of 6.7 kJ/kg0.75 BW, whereas for each 1ºC of variation above 20ºC there was an increase in HP of 7.9 kJ/kg0.75 BW. On the other hand, FHP indicated that for each 1ºC of variation below 20ºC there was a decrease in FHP of 5.4 kJ/kg0.75 BW, while for each 1ºC of variation above 20ºC there was a decrease of 8.7 kJ/kg0.75 BW, between 10 and 35 °C. Saanen and Anglo-Nubian goats are able to maintain homeostasis in environments between 10 and 35ºC. On the other hand, the increase in ambient temperature leads to lower consumption, tending to negative energy balances. Above 20°C, goats tend to decrease fasting heat production, as an attempt to conserve energy in hot environments. / CNPq: 157525/2014-7

La ghréline acylée, une hormone produite par l’estomac, favorise la prise de poids corporel, majoritairement sous forme de masse grasse, par le biais de divers mécanismes centraux et périphériques via le récepteur sécrétagogue de l’hormone de croissance (GHSR). Le GHSR est un récepteur couplé aux protéines G qui, en plus de répondre à la ghréline acylée, possède une signalisation indépendante de la ghréline par le biais de son activité constitutive ou par une modulation de réponses dopaminergiques via oligomérisation du GHSR avec des récepteurs dopaminergiques. Malgré les puissantes réponses pharmacologiques à la ghréline acylée, des modèles animaux capables d’appréhender la complexité du système ghréline acylée-GHSR in vivo manquent, ce qui a considérablement ralenti l’élucidation des rôles physiologiques de cette hormone et de son récepteur. En effet, les modèles génétiques murins générés jusqu’à présent manquent de spécificité au niveau de l’hormone (incapacité à discriminer la ghréline acylée de la ghréline désacylée), et/ou au niveau du GHSR (incapacité à discriminer les différents modes de signalisation du GHSR). Dans ce contexte, de nouveaux modèles qui impacteraient de façon différentielle les voies de signalisation du GHSR seraient des outils pertinents pour contribuer au déchiffrage du système ghréline acylée-GHSR in vivo. Nous nous sommes ainsi attachés à caractériser un modèle de rats porteur d’une mutation ponctuelle dans le Ghsr qui prédit la délétion d’un domaine régulateur dans l’extrémité C-terminale du GHSR (GhsrQ343X). Dans des modèles cellulaires, nous avons montré que cette mutation découple le GHSR des processus d’internalisation du récepteur et de recrutement de la β-arrestine induits par la ghréline acylée, tout en augmentant la réponse aux agonistes du GHSR dans la voie des protéines G. Conformément à ce mécanisme, les rats mutants homozygotes GhsrM/M ont une réponse accrue à l’administration d’agonistes du GHSR sur le plan de la libération d’hormone de croissance, de la prise alimentaire ou de l’activité locomotrice. L’exploration physiologique et comportementale des rats GhsrM/M indique que la mutation GhsrQ343X est associée à une augmentation du poids et de l’adiposité indépendamment de la prise alimentaire, une diminution de l’oxydation globale des acides gras, de la flexibilité métabolique et de la tolérance au glucose, sans impact critique sur la prise alimentaire homéostatique. En outre, étant donné que la mutation GhsrQ343X n’augmente pas les niveaux circulants de ghréline, le phénotype métabolique général des rats GhsrM/M est en accord avec une sensibilité augmentée du GHSR en réponse au tonus endogène de ghréline acylée. Enfin, des résultats préliminaires suggèrent que la mutation GhsrQ343X pourrait être associée à des altérations relatives aux fonctions de récompense et de mémoire dont les mécanismes sous-jacents restent à décrypter. En résumé, nous proposons le modèle de rat mutant GhsrQ343X comme un nouvel outil, plus spécifique que les modèles murins d’invalidation génétique, pour explorer in vivo la signalisation du GHSR dans diverses fonctions biologiques, et à plus long terme aider au design de composés pharmacologiques ciblant le GHSR efficaces dans un cadre clinique. / The stomach-derived hormone acyl ghrelin promotes body weight gain, mostly in the form of fat mass, by means of several central and peripheral mechanisms mediated by the growth hormone secretagogue receptor (GHSR). The GHSR is a G protein-coupled receptor that, in addition to respond to acyl ghrelin, displays agonist-independent signaling through high constitutive activity and possibly heteromerization with dopamine receptors. Despite the potent biological properties of exogenous acyl ghrelin, the lack of animal models able to apprehend the complexity of the acyl ghrelin-GHSR system in vivo has been hampering the elucidation of its physiological roles. Indeed, genetic mouse models generated so far lack specificity either at the level of the hormone (not able to discriminate between acyl ghrelin versus desacyl ghrelin) and/or at the level of the GHSR (not able to discriminate between GHSR signaling modes). In this context, new models differentially affecting GHSR signaling pathways would represent valuable tools to decipher the acyl ghrelin-GHSR system in vivo. We therefore aimed at characterizing a new rat model carrying a point mutation in Ghsr that predicts truncation of a regulatory domain in the C-terminus, the GhsrQ343X mutation. In cellular models, this mutation was found to uncouple the GHSR from agonist-dependent receptor internalization and β-arrestin recruitment, while enhancing GHSR responsiveness in the G protein pathway. Accordingly, homozygous mutant GhsrM/M rats show enhanced responsiveness to exogenous GHSR agonists in terms of growth hormone release, food intake and locomotor activity. Physiological and behavioral exploration of GhsrM/M rats supports that the GhsrQ343X mutation is associated with increased body weight gain and adiposity independently of calorie intake, reduced whole-body fat oxidation, metabolic flexibility and glucose tolerance, without any critical impact on homeostatic feeding behavior. Moreover, given that circulating ghrelin levels are not increased by the GhsrQ343X mutation, the overall metabolic phenotype of GhsrM/M rats is consistent with enhanced GHSR sensitivity to the endogenous tone of acyl ghrelin. Furthermore, preliminary results suggest that the GhsrQ343X mutation could be associated with behavioral alterations related to reward and memory functions, through mechanisms that remain to be elucidated. Altogether, we propose the GhsrQ343X mutant rat model as a novel tool, more specific than knockout mouse models in its mechanism-of-action, to explore GHSR signaling across biological functions in vivo, and ultimately help in the design of efficient GHSR-targeting drugs.