Le métabolisme énergétique cérébral via l’utilisation du glucose est fortement impliqué dans la production d’énergie nécessaire au fonctionnement du neurone en situation basale et d’activation. Des travaux précédents ont mis en évidence chez le rat chroniquement déficient en acides gras polyinsaturés (AGPI) de la série n-3 une altération de ce métabolisme en situation basale (diminution de l’utilisation cérébrale du glucose et de la densité des transporteurs de glucose GLUT1). Pour cerner les différentes étapes du métabolisme énergétique pouvant être modifiées par les AGPI n-3, l’expression des gènes clés a été mesurée par approche transcriptomique (cartes microfluidiques) chez l’animal déficient en AGPI n-3 ou supplémenté en acide docosahexaénoïque (DHA, 22 :6n-3). Ces mesures ont été réalisées sur deux zones cérébrales (cortex fronto-pariétal et couche CA1 de l’hippocampe) chez les animaux en situation basale et soumis à un environnement enrichi activant ces deux zones cérébrales. Pour ces 2 situations, le niveau d’utilisation cérébrale de glucose a été quantifié par la technique du fluoro-2-déoxyglucose couplée à l’imagerie de tomographie par émissions de positons (18FDG-TEP) chez les animaux déficients en AGPI n-3. L’analyse de la teneur cérébrale en AGPI membranaire a été réalisée par chromatographie en phase gazeuse et une approche in vitro sur culture primaire d’astrocytes a été développée pour apprécier l’impact du DHA sur les paramètres métaboliques de ces cellules.Les principaux résultats montrent que :- la déficience en n-3 diminue de 67% la teneur membranaire en DHA dans les deux zones cérébrales étudiées. Si la déficience induit principalement une diminution spécifique de l’expression de GLUT1 (-33%) dans le cortex fronto-pariétal en situation basale et d’activation, en revanche elle perturbe la neurotransmission glutamatergique dans l’hippocampe en augmentant l’expression des 2 transporteurs de glutamate (GLAST et GLT1). Par ailleurs, les données d’imagerie TEP mettent en évidence un hypométabolisme général du glucose chez les animaux déficients en n-3 en situation basale. Les données recueillies sur le modèle astrocytes soulignent un effet direct du DHA sur l’utilisation du glucose et l’expression de GLUT1 ;- La supplémentation en DHA ne modifie pas de façon appréciable la teneur membranaire en DHA dans les deux zones cérébrales étudiées. Au contraire de la déficience, il apparaît clairement pour la couche CA1 de l’hippocampe que l’expression de l’ensemble des gènes codant pour les complexes enzymatiques du cycle de krebs et de la voie de phosphorylation oxydative est significativement augmentée.Ces résultats originaux laissent ainsi entrevoir la possibilité que les acides gras de cette famille d’AGPI puissent intervenir sur l’énergétique et le fonctionnement de la synapse glutamatergique en modulant 1) le métabolisme glucidique (captage de glucose) et du glutamate en situation de déficit d’apport et 2) la production d’ATP (phosphorylation oxydative) en situation de supplémentation en DHA. L’altération de ces paramètres métaboliques au cours du vieillissement et dans certains désordres neurologiques, liée à un déficit de statut en DHA, mettent en avant les potentialités nutritionnelles des AGPI n-3 comme facteur préventif. / Cerebral energy metabolism via glucose utilization is heavily involved in the production of energy required to the neuron in basal conditions and activation. Previous work has shown in rats chronically deficient in n-3 polyunsaturated fatty acids (PUFA) altered the metabolism in basal condition (decrease of cerebral glucose use and density of glucose transporters GLUT1). To identify the different stages of energy metabolism may be modified by n-3 PUFA, the expression of key genes was measured by transcriptomic approach (Taqman Low Density Arrays) in animals deficient in n-3 PUFA or docosahexaenoic acid supplementation (DHA, 22:6n-3). These measurements were performed on two brain areas (fronto-parietal cortex and layer CA1 of the hippocampus) in animals in basal condition or submit to an enriched environment. For these two situations, the level of cerebral glucose utilization was quantified by the technique of fluoro-2-deoxyglucose imaging coupled with positron emission tomography (18FDG-PET) only in deficient n-3 PUFA animals. Analysis of brain PUFA content of membrane was performed by gas chromatography and an in vitro approach to primary culture of astrocytes was developed to assess the impact of DHA on metabolic parameters of these cells.The main results show that: - n-3 Deficiency decreases from 67% in membrane DHA content in both brain areas studied. If the deficiency induces mainly a decrease in the specific expression of GLUT1 (-33%) in the fronto-parietal cortex in basal and activation conditions, however it disrupts glutamatergic neurotransmission in the hippocampus by increasing the expression of two glutamate transporters (GLAST and GLT1). In addition, PET data show a general hypometabolism of glucose in animals deficient in n-3 in basal situation. Data collected on the model astrocytes point to a direct effect of DHA on glucose utilization and expression of GLUT1; - DHA supplementation does not alter significantly the membrane content of DHA in both brain areas studied. Unlike the n-3 deficiency, it is clear for the layer CA1 of the hippocampus that the expression of all genes encoding the enzyme complexes of the Krebs cycle and oxidative phosphorylation pathway is significantly increased. These original results suggest the possibility that the fatty acids of the n-3 PUFAs family can act on the energy and functioning of the glutamatergic synapse by modulating 1) glucose metabolism (glucose uptake) and glutamate in deficit intake situation and 2) the production of ATP (oxidative phosphorylation) in DHA supplementation. The alteration of these metabolic parameters during aging and certain neurological disorders, related to a deficit of DHA status, highlight the potential of dietary n-3 PUFA as a preventive factor.

We can meet pathological hypoxia in the cases of hearth attack, ischemic stroke, but also during tumor invasion, thanks to insufficient angiogenesis. The activation of HIF- 1 factor during hypoxic conditions is crucial for the cell survival. This factor modulates energetic metabolism in favor of fast progressing glycolysis (with the contribution of glutaminolysis) which provides to cell enough ATP and "building blocks", while suppressing Krebs cycle and respiration because of shortage of oxygen. The thesis studies energetic metabolism of HepG2 cells (derived from liver carcinoma) which are cultivated in the media with various energetic substrates, i. e. glucose or galactose (always together with glutamine and pyruvate) under the hypoxic conditions (5% O2). HepG2 cells use particularly oxidative metabolism for ATP and "building blocks" production under the normoxic conditions while hypoxic environment causes metabolic shift in glycemic condition. Interestingly, cells cultured in galactose (glutamine) didn't switch the energy metabolism from oxidative to aerobic glycolysis such as cells cultivated in glucose, although HIF-1 factor was stabilized. We found that enhanced activity and integrity of mitochondria, enhanced maximal capacity and reserve capacity of respiration chain correlates with...

Les cardiopathies ischémiques sont une cause majeure de mortalité cardiovasculaire pouvantaboutir à la mort subite par fibrillation ventriculaire (FV). Un des objectifs thérapeutiqueschez les coronariens est de diminuer la demande en O2 par réduction de la fréquencecardiaque (RFC). Plusieurs médicaments dont les bêta-bloquants et inhibiteurs calciquesrépondent à cet objectif, mais peuvent être responsable d’altération de la contractilitécardiaque et d’effets secondaires limitant leur utilisation. Nous nous sommes intéressés auxeffets d’un inhibiteur sélectif du courant pacemaker If, l’ivabradine (IVA) dontl’administration induit une RFC sinusale. L’objectif de ce travail a été d’évaluer l’impact dela RFC sur la propension à la FV et de comprendre les mécanismes fonctionnels etmétaboliques impliqués. Un premier travail a été réalisé sur un modèle d’ischémiemyocardique aigue. Dans un second travail l’impact de la RFC sur la sélection des substratspour la production d’énergie a été évalué sur coeur isolé et perfusé en mode travaillant. Nosrésultats démontrent que l’IVA dans les deux modèles a induit une RFC qui était associée invivo à une amélioration de la perfusion coronaire, une préservation de la structuremitochondriale et du statut énergétique myocardique, réduisant la propension à la FV. Exvivo, la RFC n’a pas modifié la sélection des substrats et le statut énergétique. Enthérapeutique, il est important d’améliorer les conditions tissulaires et de réduire la perte desubstrats lors d’ischémie myocardique, l’IVA semble répondre à cette attente. Son effetspécifique sur l’automaticité sinusale lui confère la possibilité d’agir en préventif et en curatif / Ischemic cardiopathies are a major cause of cardiovascular mortality potentially leading tosudden death by ventricular fibrillation (VF). In patients with coronary disease, onetherapeutic goal is to reduce oxygen requirements via slowing of heart rate (SHR). Severalmedicinal drugs, such as beta-blockers and calcium inhibitors can achieve this goal, but canresult in altered cardiac contractility and various adverse effects, which restricts their use. Wefocused our interest on the effects of a selective inhibitor of the pacemaker current If, namelyivabradine (IVA), which can reduce sinusal heart rate. The aim of the present work was toevaluate the impact of SHR on the propensity to VF and to better understand thephysiological and metabolism mechanisms involved. The first study was performed on a pigmodel of acute myocardial ischemia leading to VF. In the second study, the impact of SHRwas evaluated using a mouse model of isolated perfused working heart. Our resultsdemonstrated that IVA induced SHR in both models and this was associated in vivo withenhanced coronary flow, conservation of mitochondrial structure and myocardial energeticstatus, thus reducing the risk of triggering ischemia-induced VF. Moreover, ex vivo, theobserved SHR did not change the selection of substrates for energy production. From atherapeutic point of view, it is critical to improve the conditions within tissues and to reducethe loss of substrates during myocardial ischemia. IVA apparently met this goal. Indeed, viaspecific effects on sinusal automaticity, it can act both preventively and curatively

O pacu, Piaractus mesopotamicus, é um caracídeo neotropical autóctone, de hábito alimentar onívoro, largamente utilizado na aqüicultura brasileira. No entanto, alguns aspectos da sua nutrição permanecem controversos ou desconhecidos. O presente trabalho visou determinar a exigência em proteína, energia e aminoácidos para juvenis de pacu por meio da avaliação de parâmetros de desempenho, composição corporal e hematológicos em dois ensaios distintos. No primeiro ensaio, juvenis de pacu (15.5±0.4 g) foram alimentados durante 10 semanas, duas vezes ao dia, com dietas contendo teores de proteína bruta (PB) entre 22 e 38% (incremento de 4%) e energia digestível (ED) entre 2600 e 3400 kcal kg-1 (incremento 200 kcal kg-1), em um delineamento totalmente aleatorizado, em esquema fatorial 5 × 5 (n=3). O ganho de peso (GP) e a taxa de crescimento específico (TCE) aumentaram significativamente com a elevação da PB dietética. A retenção de nitrogênio (RN) e a taxa de eficiência protéica (TEP) diminuíram (p<0,05) com o aumento da proteína das dietas em todos os níveis de ED testados. As concentrações de ED afetaram (p<0,05) a concentração de umidade, proteína, gordura, matéria mineral e energia bruta corporal e o fator de condição. A taxa de eficiência econômica foi influenciada pela proteína (p<0,05) e energia digestível (p<0,05) das dietas e pela interação entre ambas (p<0,05). Entretanto, o índice de lucratividade econômica somente foi influenciado pela concentração de proteína bruta (p<0,05) das dietas. Efeito significativo (p<0,05) dos tratamentos nos parâmetros hematológicos foi registrado para taxa de hemoglobina, concentração de hemoglobina corpuscular média, proteína plasmática total e glicose plasmática. A exigência mínima para ganho de peso de juvenis de pacu é 27% de PB, com relação PB:ED ótima de 92,9 mg PB kcal-1 ED. As concentrações protéicas e energéticas estudadas não causaram prejuízos a saúde dos peixes. A exigência em lisina dietética para juvenis do pacu Piaractus mesopotamicus, com peso inicial de 4,3 g, foi determinada fornecendo cinco dietas experimentais isonitrogenadas (32% proteína bruta) contendo caseína, gelatina e L-aminoácidos cristalinos com níveis crescentes de lisina (0,90; 1,17; 1,44; 1,69 e 1,96% da matéria seca da dieta) por 74 dias. Cada dieta foi aleatoriamente designada para grupos (n=4) de 18 peixes alimentados três vezes ao dia até a aparente saciedade. Não foram observados mortalidade ou sinais de deficiência além da redução no desempenho dos peixes alimentados com dietas deficientes ou com excesso de lisina. O peso final (PF), GP, TCE, índice de eficiência alimentar (IEA), TEP, VPP, composição corporal, morfometria e hematologia foram afetados (p<0,05) pelas concentrações de lisina dietética. A análise polinomial quadrática indicou a exigência em lisina em 1,45, 1,51 e 1,43% da matéria seca da dieta, respectivamente para GP, IEA e VPP. Foram obtidos valores de lipídios e proteína corporais significativamente mais altos nos peixes alimentados com as dietas com 0,9 e 1,96% de lisina. Foi então determinado que a exigência em lisina para juvenis de pacu é de 1,5% da matéria seca da dieta. / Pacu, Piaractus mesopotamicus, a neotropical, omnivorous native characin, widely used in Brazilian aquaculture. However, some aspects of your nutrition are controversial or ignored. This work aimed at determining dietary protein, energy and amino acids requirements of juvenile pacu through the evaluation of performance, body composition and hematological parameters. In a first trial, pacu juveniles (15.5±0.4 g) were fed twice a day for 10 weeks until apparent saciety with diets containing crude protein (CP) from 22 to 38 % (intervals of 4%) and digestible energy levels among 2600 and 3400 kcal kg-1 (intervals of 200 kcal), in a totally randomized experimental design, 5 × 5 factorial scheme (n=3). Weight gain (WG) and specific growth rate (SGR) increased significantly with amount dietary CP. Nitrogen retention (NR) and protein efficiency ratio (PER) decreased (p<0.05) with increasing dietary protein at all tested levels of dietary energy. DE levels affected (p<0.05) whole body moisture, protein, fat, ash, gross energy and condition factor. Economic efficiency ratio was influenced by dietary protein (p<0.05) and digestible energy (p<0.05), and by interaction between the two factors. However, income was affected by dietary protein level (p<0.05) alone. Significant effect of treatments in hematological parameters were registered for mean corpuscular hemoglobin concentration, total plasma protein and plasma glucose. Crude protein requirements and optimum protein:energy ratio for weight gain of juveniles of pacu were 271 g kg-1 and 92.9 mg kcal-1, respectively. All dietary crude protein and digestible energy levels studied did not pose harms to fish health. Dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (4.3 g) was determined by feeding five isonitrogenous (32% crude protein) amino acid test diets containing casein, gelatin and Lcrystalline amino acids with graded levels of lysine (0.90, 1.17, 1.44, 1.69 and 1.96% of dry diet) for 74 days, three times a day until apparent satiation, to groups (n=4) of 18 fish in a totally randomized design trial. No mortality or nutritional deficiency signs were observed; reduced growth was recorded for fish fed diets with either the lower or the higher lysine contents. Final weight (FW), WG, SGR, feed efficiency (FE), PER, NR, proximate whole body composition, morphometry and hematology were affected (p<0.05) by dietary lysine concentrations. Seconddegree polynomial regression analysis of WG and PER data indicated dietary lysine requirement of 1.45, 1,51 and 1.43% of dry diet, respectively to WG, FE and PPV. Significantly higher lipid and protein contents values were obtained for whole body of fish fed the diets with 0.9 and 1.96% of lysine. Lysine requirement of juvenile pacu was determined as being 1.5% of dry diet.

O diabetes tipo 1 (DT1) é uma doença metabólica associada a uma inflamação sistêmica de baixo grau, responsável por importantes co-morbidades associadas. Nosso grupo demonstrou que esta inflamação depende dos níveis plasmáticos aumentados de leucotrieno-B4 (LTB4), o qual estimula o eixo Myd88/STAT1 amplificando a resposta dos receptores TLR/IL1&#946 em macrófagos. Isso caracteriza o programa pró-inflamatório M1 nestas células, que requer energia proveniente da glicólise e produz substancias tóxicas como espécies reativas de oxigênio (ROS) e óxido nítrico (NO). Isto pode ser minimizado pela respiração mitocondrial desacoplada à síntese de ATP no metabolismo lipídico. Assim, na primeira parte do trabalho os macrófagos de camundongos (C57Bl/6) DT1, expressaram níveis elevados de marcadores de oxidação de ácidos graxos, o que não foi observado em macrófagos de camundongos diabéticos tratados com o antagonista de LTB4 (u75302). Além disso, o u75302 também reduziu a expressão aumentada de CD36, receptor envolvido na captação de lipídios, assim como aumento de lipídios intracelulares nestas células. Os elevados níveis de triglicérides e ácidos graxos presentes no plasma dos diabéticos também foram reduzidos pelo antagonista u75302, e isso foi consistente com o aparecimento de marcadores de lipólise (Prdm16 e Fgf21) no tecido adiposo branco destes animais. Da mesma forma, u75302 reduziu o consumo de oxigênio dos macrófagos de animais diabéticos. Isso foi consistente com os resultados obtidos em macrófagos de camundongos diabéticos deficientes da UCP1, os quais apresentaram maior peso corporal, maior massa de gordura e metabolismo mitocondrial mais baixo do que diabéticos WT. A perda de gordura também foi recuperada pelo tratamento com u75302 indicando o envolvimento do LTB4 na perda de adiposidade e dislipidemia em DT1. Na segunda parte do trabalho, confirmamos a participação dos leucotrienos na inflamação sistêmica em DT1 induzida por estreptozotocina. Camundongos (129SvE) diabéticos apresentaram níveis sistêmicos elevados das citocinas e este aumento não ocorreu em 129Sve deficientes da enzima 5-lipoxigenase (5LO-/-), responsável pela síntese de leucotrienos. A freqüência de monócitos pró-inflamatórios (CD11b&#43Ly6ChighLy6G-) circulantes estava aumentada em camundongos diabéticos WT mas não nos 5LO-/-. Macrófagos peritoneais residentes de camundongos diabéticos também apresentaram um fenótipo semelhante ao M1 classicamente ativado (alta expressão de Nos2 e Stat1, e alta produção de NO), que não foi revertido com o estímulo de IL4 in vitro ou in vivo. Por outro lado, os macrófagos dos 5LO-/- diabéticos apresentaram o fenótipo de macrófagos M2 alternativamente ativados (alta expressão de Ym1 e Arg1, e alta atividade de arginase). Os animais WT diabéticos tiveram cicatrização deficiente que se correlacionou com uma baixa freqüência de macrófagos M2 (CD45&#43F4/80&#43CD206&#43) nas lesões cutâneas comparado com os demais grupos. Juntos, estes dados sugerem que no DT1 os leucotrienos contribuem para a inflamação sistêmica e reprogramação dos monócitos e macrófagos para perfil inflamatório e isto está associado com aumento do metabolismo energético nestas células. Estas alterações induzidas nos macrófagos pelos níveis elevados de leucotrienos, particularmente LTB4, se correlacionam com a cicatrização deficiente, com a perda de gordura e hiperlipidemia nos camundongos DT1, sugerindo que o LTB4 possa ser um alvo terapêutico no diabetes. / Type 1 diabetes (T1D) is a metabolic disease associated to systemic low grade inflammation, which has an important role in co-morbidities. Our group showed that this inflammation depends on the high systemic levels of leukotriene-B4 (LTB4), which stimulateds MyD88/Stat1 axis, amplifying TLR/IL1&#946 response in macrophages. This characterizes pro inflammatory M1 program, which requires energy from glycolysis, and produces harmful molecules, such as reactive oxygen species (ROS) and nitric oxide (NO). This can be mitigated by mitochondrial respiration uncoupled to ATP synthesis in lipid metabolism. Therefore, in the first part of this study, macrophages from T1D mice (C57Bl/6) expressed high levels of fatty acid oxidation markers, which was not observed in macrophages from T1D mice treated with LTB4 receptor antagonis, u75302. Moreover, u75302 also reduced the high expression od CD36, a receptor involved in lipids uptake, and also reduced intracellular lipids in these cells. The high levels of triglycerides in diabetic plasma were reduced by u75302, and this is consistent with lipolysis markers (Prdm16 and Fgf21) in white adipose tissue of these mice. This was also consistent with results obtained in macrophages from diabetic UCP1 deficient mice, which had higher body weight and lower mitochondrial metabolism then WT diabetics. Fat loss was also recovered by u75302 treatment, indicating an involvement of LTB4 in adiposity loss and dyslipidemia in T1D. In the second part of this study, we confirmed leukotrienes participation in systemic inflammation in T1D streptozotocin-induced. T1D mice (129SvE) increased systemic levels of cytokines, which was not observed in T1D 5-lipoxygenase deficient mice (5LO-/-).The frequency of pro inflammatory monocytes (CD11b&#43Ly6ChighLy6G-) was increased in WT diabetic mice, but not in 5LO-/-. Resident peritoneal macrophages in diabetics had a phenotype similar to M1 classically activated (Nos2 and Stat1 highly expressed, and high production of NO), which was not reversed by IL-4 stimulation in vitro and in vivo. On the other hand, macrophages from diabetic 5LO-/- had a phenotype M2 alternatively activated (Ym1 and Arg1 highly expressed, and high arginase activity). WT diabetic mice had a defective wound healing, which was related to low frequency of M2 (CD45&#43F4/80&#43CD206&#43) macrophages in cutaneous wounds, compared to the other groups. All together, our data suggest that in T1D leukotrienes induce systemic inflammation, and reprogram pro inflammatory phenotype in monocytes and macrophages, and this is related to increased energetic metabolism in these cells. These alteration in macrophages due to leukotrienes effects, mainly LTB4, are correlated to defective healing, with fat loss, and dyslipidemia in T1D mice, suggesting that LTB4 can be a therapeutic target in diabetes.

L’absence de réponse et la résistance des cellules cancéreuses mammaires aux thérapies actuelles justifient de développer de nouveaux traitements. Une stratégie prometteuse consiste à cibler le métabolisme énergétique des cellules cancéreuses. Dans ce contexte, des thiazolidinediones (TZD) présentent des effets antiprolifératifs qui pourraient résulter d’une atteinte du métabolisme énergétique. Notre laboratoire étudie des TZD dérivées de la troglitazone (TGZ). Durant cette thèse, nous avons cherché à déterminer si la Δ2-Troglitazone (Δ2-TGZ) modifie le métabolisme énergétique des cellules cancéreuses mammaires. Jusqu’à présent, les expériences menées au laboratoire étaient réalisées dans un milieu de culture contenant 1% de sérum de veau fœtal (SVF) qui crée un stress peu propice à l’étude du métabolisme. Nous avons donc d’abord caractérisé les effets de la Δ2-TGZ dans un milieu de culture contenant 10% SVF. Dans ces conditions, la Δ2-TGZ diminue toujours la prolifération des cellules cancéreuses mammaires, mais les doses requises sont plus élevées. En outre, la Δ2-TGZ induit des effets cytostatiques plutôt que l’apoptose. Nous avons ensuite montré que la Δ2-TGZ induit une perturbation du métabolisme énergétique, consistant en un blocage de la respiration mitochondriale que les cellules semblent compenser en stimulant la glycolyse. En parallèle, nous avons caractérisé le mode d’action du 2-désoxyglucose dont l’action antiproliférative dans les cellules cancéreuses mammaires est due à l’inhibition de la glycolyse et à la perturbation de la N-glycosylation des protéines. Il reste à déterminer la part des altérations métaboliques dans l’action anti-cancéreuse de la Δ2-TGZ / The absence of response and the resistance of cancer cells to therapies are strong arguments for the development of new therapeutic strategies. Data from the literature suggest that it could be interesting to target energy metabolism of cancer cells. In this context, thiazolidinediones (TZDs) display antiproliferative effects that could be the result of energy metabolism alteration. During this PhD, we aimed at determining if Δ2-Troglitazone (Δ2-TGZ) could modify energy metabolism of breast cancer cells. The experiments performed previously used a culture medium containing 1% of fetal calf serum (FCS) that is rather a stress inducing condition that can disturb cell metabolism. Thus, we first characterized the effects of Δ2-TGZ in a 10% FCS containing medium. In this case, Δ2-TGZ still decreases cell proliferation of breast cancer cells, but it requires high doses. Besides, Δ2-TGZ induces cell cycle arrest instead of apoptosis. Then, we have shown that Δ2-TGZ induced modifications of energy metabolism, which are due to a decrease in oxidative phosphorylation. We also observed an increase in glycolytic activity that is probably a compensatory mechanism. During this part of our work, we have also characterized the mechanisms involved in the anticancer activity of 2-deoxyglucose. We have shown that in breast cancer cells, this compound acts not only by glycolysis inhibition but also by protein N-glycosylation alteration. We have now to determine the part of metabolic alterations that are involved in the anti-cancer effects of Δ2-TGZ

La protéine kinase activée par AMP (AMPK) est un senseur ubiquitaire du statut énergétique de la cellule eucaryote. Elle est exprimée sous la forme d'un complexe hétérotrimèrique comprenant les sous unités catalytique (α) et régulatrices (β et γ). Ce large complexe protéique (130kDa), fonctionne comme un hub central de la signalisation cellulaire, régulateur du métabolisme énergétique et au-delà. La (dé)régulation de l'AMPK est impliquée dans de nombreuses pathologies et l'AMPK apparait comme une cible de choix pour développer de nouveaux médicaments contre le diabète de type 2. Une fois activée, l'AMPK va restaurer l'homéostasie énergétique en diminuant le métabolisme demandeur d'énergie (anabolisme) et en stimulant le métabolisme produisant le l'énergie (catabolisme). In vivo, l'AMPK est activée par des mécanismes multiples et complexes permettant la fine régulation de son activité lors de différentes situations de stress métaboliques. Premièrement, l'activité de l'AMPK est modulée de manière systémique par phosphorylation et déphosphorylation de la sous unité α (par des kinases et phosphatases en amont respectivement). De plus, l'attachement d'AMP et d'ADP à la sous unité γ augmente la phosphorylation de l'AMPK. Deuxièmement, l'AMPK est activée de manière allostérique par l'AMP qui se lie à sous unité γ lors de chutes du ratio ATP/AMP. Tous ces mécanismes requièrent une communication entre les sous unités α et γ, mais un modèle consensus complet de l'activation de l'AMPK est toujours manquant. Se basant sur différentes études structurales, d'autres et nous-mêmes avons proposé un changement de conformation induit par AMP au sein de l'hétérotrimère AMPK. Afin de mieux élucider ce mécanisme, nous avons tiré profit de ces changements conformationels pour imaginer et créer un hétérotrimère d'AMPK permettant de suivre directement et en temps réel l'état de conformation de l'AMPK par FRET. Une limite importante lors du développement de complexes multiprotéiques est l'augmentation exponentielle de la quantité de travail liée à la modification et la combinaison de nombreux gènes hétérologues lors du remaniement de ces complexes protéiques et de leurs productions. Nous avons utilisé la technologie ACEMBL, qui exploite des techniques de recombinaisons homologues, pour faciliter la révision rapide et itérative de la production et de l'analyse fonctionnelle, après ingénierie, de complexes multi protéiques. Le senseur fluorescent génétiquement codé ainsi crée, et nommé AMPfret, a la propriété de rapporter les changements de conformation induits par les nucléotides ayant lieu au sein de l'AMPK. De plus, les changements de signal FRET corrèlent avec l'activation allostérique de l'AMPK. Le senseur répond à de faible concentrations en AMP (micromolaire) et a démontré la capacité exclusive qu'a l'ATP, et non l'ATP-Mg, à concurrencer l'AMP. De plus, son utilisation a permis une meilleure compréhension du rôle des sites CBS lors de l'activation allostérique. AMPfret peut aussi être considérer comme un outil de choix pour le criblage de molécules ciblant l'AMPK, et pour le monitoring de l'état énergétique intracellulaire. / AMP-activated protein kinase (AMPK) is a ubiquitous sensor of cellular energy and nutrient status in eukaryotic cells. It is expressed as heterotrimeric complexes comprising catalytic (α) and regulatory (β and γ) subunits. This large protein complex (130kDa), conserved from yeast to plants and mammals, functions as a central signaling hub and master regulator of energy metabolism and beyond. (Dys)regulation of AMPK signaling has been implicated in various pathologies. In particular, AMPK emerged as a suitable target to develop novel drugs for type II diabetes. Once activated AMPK will attempt to restore the energy homeostasis by down-regulating energy demanding pathways (anabolism) and up-regulating the energy producing ones (catabolism). AMPK is activated in vivo by multiple, complex mechanisms allowing fine tuning of AMPK activity in different situations of metabolic stress. First, AMPK activity is systemically modulated via activating phosphorylation at the α-subunit (by upstream kinases) and inactivating dephosphorylation (by upstream phosphatases). In addition, AMP and ADP binding to the γ-subunit increase AMPK phosphorylation. Second, AMPK is allosterically activated by AMP binding to the γ-subunit when the ATP/AMP ratio is falling. All these mechanisms require close communication between the γ- and α subunits, but a complete consensus model for AMPK activation is still lacking. We and others have proposed an AMP-induced conformational switch within the full-length heterotrimeric AMPK complex based on different, complementary structural studies. To further elucidate this mechanism, we have profited from these structural rearrangements to imagine and engineer an AMPK complex that allows a direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). A definite bottleneck in engineering multiprotein complexes is the exponential increase in work-load if several heterologous genes need to be altered, engineered and combined for revised protein complex production experiments. We used the ACEMBL technology which harnesses site-specific and homologous recombination techniques in tandem to facilitate rapid, iterative revision of multi-protein complex expressions after engineering and functional analysis of multiprotein complex. The resulting genetically encoded fluorescent biosensor, named AMPfret, can report conformational changes within the AMPK heterotrimer induced by nucleotide binding and the monitored FRET correlates with AMPK allosteric activation. The sensor responds to low micromolar concentrations of AMP, shows the exclusive ability of ATP, but not Mg-ATP, to compete with AMP, and allows insight into the role of CBS domains for allosteric AMPK activation. It may also be a tool of choice for AMPK targeted drug screening, and reporting the intracellular energy state.

Orientador: Ana Angélica Henrique Fernandes / Resumo: A obesidade é considerada um dos maiores problemas de saúde pública em muitos países, uma vez que está associada queda da qualidade de vida. Embora existam vários fatores que corroboram com o desenvolvimento para tal fato, os hábitos alimentares seja o fator relevante. Os transtornos metabólicos podem resultar em alterações na funcionalidade do fígado, podendo desenvolver Doença Hepática Gordurosa Não Alcoólica (DHGNA). Como o número de obesos e as co-morbidades associadas ao sobrepeso vêm aumentando abruptamente nas últimas décadas, vários modelos de obesidade experimental têm sido propostos para investigar os distúrbios metabólicos envolvendo suas causas e consequências. O glutamato monossódico é amplamente utilizado na culinária e também por indústrias alimentícias, contudo atua no sistema nervoso central e promove a degeneração de áreas importantes do hipotálamo que leva a distúrbios da saciedade e, consequentemente acúmulo excessivo de gordura abdominal. Com a finalidade de estudar substâncias que apresentem potencial atividade terapêutica no controle dos distúrbios metabólicos, o N-acetil-cisteína possui propriedades antioxidantes e exerce hepatoproteção. Desta forma, o objetivo do presente estudo foi evidenciar a indução da obesidade pelo glutamato monossódico e determinar o efeito do N-acetil-cisteína sobre os parâmetros calorimétricos, metabolismo energético, atividade dos complexos respiratórios e estresse oxidativo no tecido hepático. Foram utilizados 32 ratos Wins... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity is considered one of the greatest public health problems in many countries, since it is associated with a drop in quality of life. Although there are several factors corroborating with the development for this fact, eating habits are the relevant factor. Metabolic disorders can result in changes in liver function, and can develop Non-Alcoholic Fatty Liver Disease (NAFLD). As the number of obese and co-morbidities associated with overweight have increased steeply in recent decades, several models of experimental obesity have been proposed to investigate metabolic disorders involving their causes and consequences. Monosodium glutamate is widely used in cooking and also in food industries, but it acts on the central nervous system and promotes the degeneration of important areas of the hypothalamus which leads to satiety disorders and consequently excessive accumulation of abdominal fat. In order to study substances that present potential therapeutic activity in the control of metabolic disorders, N-acetyl-cysteine has antioxidant properties and exerts hepatoprotection. Thus, the objective of the present study was to evidence the induction of obesity by monosodium glutamate and to determine the effect of Nacetyl-cysteine on calorimetric parameters, energy metabolism, respiratory complex activity and oxidative stress in hepatic tissue. Thirty-two Winstar male mice were used at 21 days of age. Initially the animals were distributed in two experimental groups (n = 16). Grou... (Complete abstract click electronic access below) / Doutor

Cette thèse présente une étude du métabolisme énergétique du spermatozoïde d’huître creuse, Crassostrea gigas. Les principaux objectifs sont (1) d’établir la dynamique des caractéristiques cellulaires et biochimiques du spermatozoïde de cette espèce durant la phase de mouvement, (2) d’identifier les voies de synthèse d’ATP et leurs substrats et (3) de déterminer l’implication du métabolisme énergétique dans le succès à la fécondation du spermatozoïde de cette espèce. Les résultats de ce travail montrent la longue durée du mouvement (de 24 à 72h) du spermatozoïde d’huître creuse et le maintien d’une concentration élevée en ATP intracellulaire, jusqu'à la fin du mouvement. Le rôle de la phosphorylation oxydative (OXPHOS) dans la synthèse d’ATP a été mis en évidence. Le spermatozoïde d’huître creuse montre une grande plasticité dans l’utilisation de ses voies de production d’énergie, assurant la synthèse d’ATP par d’autres voies qu’OXPHOS lorsque la mitochondrie est inhibée. L’accumulation de phosphoarginine en fin de mouvement montre une capacité de stockage de l’ATP synthétisé. La nature des substrats endogènes utilisés pour la synthèse d’ATP reste cependant à déterminer. La forte capacité de régulation du métabolisme énergétique du spermatozoïde d’huître creuse, associée à la possibilité de synthétiser de l’énergie au cours de sa longue phase de nage, contribuent probablement au succès du peuplement observé chez cette espèce. Ce travail apporte une première description du métabolisme énergétique du spermatozoïde d’huître creuse, améliorant la connaissance fine de cette cellule. / The present study aims at describing the energy metabolism of Pacific oyster, Crassostrea gigas, spermatozoon. The main objectives are: (1) describing the kinetic of cellular and biochemical characteristics of Pacific oyster spermatozoon during its movement phase, (2) identifying the metabolic pathways to produce energy and their substrates, (3) determining the involvement of energy metabolism in fertilization success. The results showed the long duration (ranging 24 to 72h) of the movement of Pacific oyster spermatozoon and the maintenance of a high intracellular ATP content until the end of the movement phase. The role of oxidative phosphorylation (OXPHOS) in ATP production was highlighted. Alternative ATP producing pathways might contribute to sustain energy production when OXPHOS is impaired, showing the high adaptation capacity of Pacific oyster spermatozoa. An increase of phosphoarginine content observed at the end of the movement phase suggests energy storage capacity. Endogenous substrates involved in ATP synthesis must be determined. Both the high regulation capacity of the energy metabolism of Pacific oyster spermatozoa and their ability to restore energy stores catabolized during movement likely contribute to the successful settlement observed in the wild. In conclusion, this work provided a fine description of energetic metabolism of Pacific oyster spermatozoon.

Resumo: Foram realizados dois experimentos com objetivo de avaliar os efeitos da temperatura de criação, dos consumos de ração e de energia sobre o desempenho, composição bromatológica da carcaça e concentrações de alguns componentes do metabolismo intermediário no plasma e fígado de frangos com potencial genético de crescimento diferenciado. No primeiro experimento, 600 pintos de corte, machos, foram alojados em esquema fatorial 2 x 3, correspondendo a dois grupos genéticos: um da Embrapa Suínos e aves (PCLC), sob acasalamento ao acaso, desde 1984, e outro comercial (ROSS 308). O outro fator foi representado por três grupos de manejo, diferenciados pela prática de fornecimento de ração e temperatura de criação, sendo dois com ração à vontade e submetidos a duas temperaturas distintas (uma termoneutra, 23RA, e outra de estresse por calor, 32RA), e um terceiro grupo utilizado como controle (23RR) com temperatura termoneutra e consumo de ração equivalente, em pair-feed diário com o grupo 32RA. No segundo experimento, 600 pintos de corte, machos, foram alojados em esquema fatorial 2 x 3, correspondendo a dois grupos genéticos, os mesmos da descrição para o primeiro experimento, e três níveis de energia metabolizável da ração (2.950, 3.200 e 3.450 kcal/EM/kg). Frangos de corte mantidos sob altas temperaturas e consumo de ração à vontade apresentam carcaças com menor quantidade de proteína e maior deposição de gordura. Linhagens de maior potencial genético para crescimento são mais adequadas para produzir carcaças com maior rendimento e quantidade de proteína, menor deposição de gordura e maior rendimento de peito. As diferenças observadas nos níveis dos metabólitos permitem concluir que tanto a temperatura elevada quanto a redução do consumo de ração provocam alterações significativas no direcionamento de nutrientes para o metabolismo relacionado com a deposição de gordura. / Abstract: The objective of this study was to evaluate the effect of temperature, feed consumption, and metabolizable energy consumption on performance, carcass yield, carcass composition and levels of intermediate metabolites in plasma and liver of broiler chickens. Two experiments were performed. In the first trial, 600 male-chicks from two broiler strains (ROSS 308 and PCLC, a control line kept by Embrapa since 1984) were housed considering a factorial design 2 x 3 (strains x management practices). The management consisted of two groups submitted to ad libitum feed consumption, with a temperature difference (neutral, 23RA or heat stres, 32RR), and a third group (23RR) under neutral temperature and controlled feed consumption (daily pair-feed to 32RR). In the second trial, 600 male broiler-chicks were housed considering a factorial design 2 x 3 (strains, as in trial 1 x diet energy levels (2,950; 3,200 and 3,450 kcal/EM/kg). At 42 days of age, the birds were processed in order to access body composition. The results showed that birds raised at high temperatures and ad libitum feed consumption produce a carcass with lower protein content and higher abdominal fat deposition. The use of high growth potential strains showed to be the choice in order to obtain carcasses with higher protein concentration, lower fat deposits, and higher breast yield. It is possible to conclude that both high temperature and reduced feed consumption do produce significant effects on the direction of nutrients for fat deposition. / Orientador: Renato Luis Furlan / Coorientador: Poliana Fernanda Giachetto / Banca: Vera Maria Barbosa de Moraes / Banca: Danísio Prado Munari / Banca: Fabiano Dahlke / Banca: Ricardo de Albuquerque / Doutor