The majority of our physiological and metabolic processes are coordinated by an internal clock, which has evolved as an adaptive response to the daily light-dark cycles. Thus, several physiological and behavioral activities display an oscillatory rhythmic period of 24 hours. This highly conserved molecular mechanism is achieved through a specific program of gene expression, characterized by a complex interaction between clock-core proteins, chromatin remodelers and epigenetic events associated with the oscillatory nature of circadian transcriptional activity in the genome. Clock disruption leads to a wide spectrum of severe health problems including chronic metabolic disorders, muscle waste and cardiopathies. Previous studies revealed that each cell and organ possess an intrinsic clock and that coordination between central versus peripheral clocks is key for health. Furthermore, it has been found that under nutritional challenge such as High Fat Diet (HFD), the circadian transcriptome and metabolome are rapidly remodeled in the mouse model. Surprisingly, metabolome and gene expression analysis on various tissues revealed that skeletal muscle is the most affected under HFD. Mechanisms that regulate circadian cycle and stress induced rapid adaptation and in particular metabolic stress at the chromatin level are largely unknown. In this study, we investigated the role of Polycomb proteins group (PcG) mediate cell memory system by maintaining transcriptional gene silencing, in particular the PRC2-Ezh1. We hypothesized that Ezh1 could play an important role in circadian clock regulation in post-mitotic skeletal muscle, and this pathway has never been explored in this context. We explored the circadian role of PRC2-Ezh1 in the mouse skeletal muscle. Intriguingly, we found that the oscillatory profile of a novel isoform of Ezh1 (Ezh1beta), localized specifically in the cytoplasm and controlling stress induced nuclear PRC2 activity, was completely disrupted under HFD. More interestingly, the circadian pattern of core clock components was impaired in Ezh1 depleted cells. Our data unveils an interesting physiological role of the PcG memory system, from cytoplasm to chromatin, which could indicate a new link between the chromatin remodeler Polycomb proteins and the endogenous clock in adaptation mechanism in skeletal muscle.

Nous avons mis au point un fermenteur continu multi-étagé (MSCF) dans le but de reproduire les conditions de la fermentation alcoolique en conditions œnologiques. Ce bioréacteur permet de maintenir les levures dans un milieu stable et contrôlé tout en découplant la croissance et la phase stationnaire. Le système offre donc la possibilité d'obtenir des levures non croissantes dans un milieu de composition stable. Dans un premier temps, nous avons validé la pertinence du MSCF pour reproduire les conditions de fermentation du batch, par approche intégrée (des paramètres cinétiques, des métabolites intra et exo cellulaire et de l'expression génique). Nous avons ensuite utilisé ce bioréacteur pour étudier les mécanismes d'adaptation métabolique des microorganismes suite à un ajout d'azote, pratique largement répandue en œnologie. Plusieurs résultats originaux ont été obtenus concernant, notamment, la réorganisation du cycle TCA, le transport des sources azotées et la synthèse des alcools supérieurs et esters. La fiabilité de l'outil mis au point et l'originalité des données obtenues ouvrent des perspectives à l'utilisation du MSCF pour la compréhension du métabolisme et des mécanismes d'adaptation des levures. / We set up a multi-stage continuous fermentor (MSCF) to mimic the conditions of alcoholic fermentation. In this bioreactor, the yeasts are in a steady and well controlled state representative of the growth and stationary phases of the batch. The ability of the MSCF to reproduce batch fermentation was assessed using an integrated approach (measurement of kinetic parameters, intra and exo-cellular metabolites and gene expression). We then used the MSCF to study the impact of nitrogen supplementation performed during the stationary phase, on yeasts metabolism. Several original results were obtained, concerning the TCA cycle, the transport of nitrogenous sources and the synthesis of higher alcohols and esters. This work points out the interest of using the MSCF to assess the effect of medium perturbations during alcoholic fermentation, especially during the stationary phase. More generally, the accuracy of the MSCF and the originality of the data obtained open new prospects for a better understanding of yeasts metabolism and regulation mechanisms.

It is well established that improved nutritional strategies can enhance both health and exercise performance. Scientific developments in recent years have furthered our understanding of cellular metabolism, which in turn, has provided an additional platform to investigate the impact of diet on health and adaptation. The overall aim of this research programme was to build on the current understanding of dietary intake in athletes and the impact dietary manipulation has on cellular and metabolic adaptation at rest and in combination with endurance training. It is postulated that nutrition is the most controllable risk factor impacting long-term health and chronic disease (World-Health-Organization, 2003), and enhanced knowledge of nutrition has been associated with improved dietary choices. A number of nutrition knowledge questionnaires have been developed to assess this; however the validity of each tool is reduced if implemented outside the target population. A valid and reliable general and sport nutrition knowledge questionnaire had not yet been developed. Using a parallel groups repeated measures study design (N = 101) the aim of the first experimental Chapter (Chapter 4) was to develop a new tool to measure general and sport nutrition knowledge in UK track and field athletes. Following the questionnaire design 53 nutrition educated and 48 non-nutrition educated participants completed the questionnaire on two occasions separated by three weeks. The results of the process demonstrated face and construct validity from the development of the question pool, content validity (the nutrition educated group scored > 30% higher that the non-nutrition educated group), reliability (test - retest correlation of 0.98, p < 0.05) and internal consistency (Chronbach's alpha value > 0.7) as such establishing a new tool (Nutrition knowledge Questionnaire for Athletes (NKQA)) for the assessment of general and sport nutrition knowledge in track and field athletes. Athletes' diets are commonly reported as inadequate and previous work has demonstrated a weak positive relationship between diet quality and nutrition knowledge. Additionally a commercially available tool, the metabolic typing questionnaire, claims to identify individual metabolic function and subsequently prescribe a personalised diet to optimise health. Thus the aim of the second experimental Chapter (Chapter 5) was to quantify nutrition knowledge (using the questionnaire developed in Chapter 4), measure diet intake and quality and investigate the efficacy of the metabolic typing questionnaire in UK track and field athletes. Using a parallel groups repeated measures design participants (UK track and field athletes n = 59, and non-athletic control group n = 29) completed a food diary, the NKQA and the metabolic typing questionnaire at two time points through the year (October and April) to investigate seasonal change. The results of the metabolic typing questionnaire concluded that 94.3% of the participants were the same dietary type and would subsequently have been prescribed the same diet. Athletes possess greater general and sport nutrition knowledge the non-athletes (60.4 ± 2.0 % vs. 48.6 ± 1.5 %) and also had better diet quality (76.8 ± 10.5 % vs. 67.6 ± 2.6 %). However no relationship was observed between individual nutrition knowledge score and diet quality (r2 = 0.003, p = 0.63). No difference in dietary intake was observed between power and endurance athletes; average diet intake consisted of 57.0% carbohydrate, 17.1% protein and 25.9% fat. The metabolic typing diet is based around three different diets: high carbohydrate, high protein and mixed diet. The results from Chapter 5 identified that the metabolic typing questionnaire was not able to differentiate between metabolic function in healthy individuals. Additionally all athletes, independent of event (power vs. endurance), consumed similar diets. With such similarities a clearer understanding of the impact such diets have at a cellular level is required. Therefore for the remainder of the thesis it was decided to investigate the impact of dietary manipulation utilising more robust measures. Mitochondria are responsible for energy production; their quantity and density have been associated with improved health and endurance performance. External stressors such as energy reduction, carbohydrate restriction and exercise are potent stimulators of transcription markers of mitochondrial biogenesis. Thus manipulating carbohydrate and energy availability in vivo may enhance cellular adaptation and limited literature exists on the impact increased protein intake has on this. The aim of Chapter 6 was to investigate the impact of acute (7-day) continuous dietary manipulation on metabolic markers, body composition and resting metabolic rate (RMR). Using a repeated measures parallel group (N = 45) design, participants were randomly assigned one of four diets: high protein hypocaloric, high carbohydrate hypocaloric, high protein eucaloric or high carbohydrate eucaloric. The macronutrient ratio of the high protein diets was 40% protein, 30 % carbohydrate and 30% fat, the high carbohydrate diets were 10% protein, 60% carbohydrate and 30% fat. Energy intake in the hypocaloric diets was matched to resting metabolic rate (RMR). Participants consumed habitual diet for 7-days then baseline measures were collected (skeletal muscle biopsy, dual energy X-ray absorptiometry scan (DXA) and RMR, habitual diet was consumed for a further 7-days and repeat testing was completed (these time points were used as a control), the intervention diet was then consumed for 7-days and post measures were collected. The results of the skeletal muscle biopsy demonstrated no group x time interaction in any marker, however a pre-post time difference subsequent to the high protein hypocaloric diet (the diet which induced the greatest metabolic stress) was observed in four transcriptional markers of mitochondrial biogenesis (pre-post intervention fold increase: PCG1-α 1.27, AMPK 2.09, SIRT1 1.5, SIRT3 1.19, p < 0.05). The results of the DXA scan demonstrated that the high protein hypocaloric group lost significantly more fat mass than the high carbohydrate eucaloric group (-0.99 kg vs. -0.50 kg, p < 0.015). Irrespective of macronutrient ratio, no energy-matched between group difference was observed in lean mass (LM) loss. However when matched for macronutrient ratio the high protein diet attenuated LM loss to a greater extent that the high carbohydrate diet, suggesting an important role of increased protein intake in the maintenance of lean mass. No time point or group difference in RMR was observed. This data suggests that a high protein low carbohydrate hypocaloric diet may provide a stimulus to promote skeletal muscle metabolic adaptation. The aim of the final experimental Chapter (Chapter 7) in this thesis was to explore the impact exercise in combination with a high protein diet on metabolic adaptation, substrate utilisation and exercise performance in well trained runners. Using a parallel groups repeated measures study design the participants (well-trained endurance runners, N = 16) consumed normal habitual diet for 7-days, then 7-days intervention diet (high protein eucaloric or high carbohydrate eucaloric, same dietary ratios as Chapter 6) and finally returned to habitual diet for 7-days, training was consistent throughout. A pre exercise muscle biopsy was taken subsequent to each diet and immediately followed by a 10 km sub-maximal run and a time to exhaustion run (TTE) at 95% of velocity at maximal aerobic capacity (vV̇O2max). Post intervention the high protein group presented significant changes in sub-maximal substrate utilisation with 101% increase in fat oxidation (0.59 g·min-1, p = 0.0001). No changes were observed in substrate utilisation in the high carbohydrate group. A trend towards a reduction in average weekly running speed was observed in the PRO group (-0.9 km·h-1), the high carbohydrate group maintained the same training speed. TTE was decreased (-23.3%, p = 0.0003) in the high protein group subsequent to the intervention, no change was observed in subsequent to the high carbohydrate diet. / The high carbohydrate group demonstrated preferential increases in markers of metabolic adaptations (fold increase: AMPK = 1.44 and PPAR = 1.32, p < 0.05) suggesting that training intensity, rather than carbohydrate restriction, may be a more profound driver of metabolic adaptation. All performance measures, in both groups, returned to pre intervention levels once habitual diet was returned; however the increased gene expression observed in the high carbohydrate group remained elevated 7-days post intervention. The increased metabolic stress imposed by reducing carbohydrate intake did not increase transcriptional markers of mitochondrial biogenesis. For continuous endurance training and high intensity endurance performance a high carbohydrate diet is preferential to a high protein diet.

Dans son environnement, la bactérie Escherichia coli (E. coli) fait face à d’importantes fluctuations des ressources carbonées. Une capacité d’adaptation métabolique lui permet de coloniser ou de subsister en fonction des substrats disponibles. Cette adaptation est régie par un réseau complexe de régulations de l’expression génique. Le régulateur global post-transcriptionnel CSR (Carbon Storage Regulator) régule la stabilité et/ou l’initiation de la traduction d’ARNm par l’intermédiaire de la protéine CsrA. Ce système, essentiel en présence de glucose et est également supposé être impliqué dans la régulation d’une transition métabolique glycolyse vers gluconéogenèse. Le caractère essentiel de CSR est à ce jour inexploré, tout comme son implication dans la régulation d’une adaptation métabolique. Une approche de biologie intégrative menée pour différents mutants du système CSR a permis d’avancer pour la première fois, une explication de l’essentialité de CSR lors d’une croissance exponentielle sur glucose et de caractériser son implication dans la régulation de la transition métabolique glucose-acétate. Des approches de transcriptomique et de stabilomique utilisées pour une souche sauvage au cours d’une adaptation métabolique ont mis en évidence l’importance des régulations de la stabilité des ARNm au cours de l’adaptation. En conclusion, ces travaux approfondissent grandement les connaissances concernant le système CSR et son implication dans la régulation du métabolisme d’E. coli. Ce système, indispensable à la régulation du métabolisme durant une phase de croissance sur glucose s’ajoute de façon indéniable au réseau déjà complexe de régulations du métabolisme d’E. coli / In its natural environment, Escherichia coli (E. coli) faces strong fluctuations of the nutrient availability. A complex gene regulatory network makes the bacterium able to switch between a state of growth in the presence of an appropriate carbon source and a non-growth state in its absence. Within this network, the global post-transcriptional regulator CSR (Carbon Storage Regulator) modifies mRNA stability and/or translation initiation by the CsrA protein. This system has been shown to be essential for cells to grow on glucose and is hypothesized to be involved in the regulation of metabolic transitions. However both observations remained unexplored so far. An integrative approach has been used to investigate for the first time the essentiality of CSR on glucose as well as its involvement in the regulation of the glucose-acetate transition. Molecular and phenotypic data for different mutants of the CSR system have been produced and integrated into mathematical models. Transcriptomic and Stabilomic approaches have been used eventually to characterize the importance of the control of mRNA stability during the metabolic adaptation. mRNA stability regulations appear to be of particular importance in gene expression regulation during metabolic adaptation. To conclude, this work shed a new light upon CSR’s involvement in the regulation of E. coli’s metabolism. CSR is definitely essential to regulate glycolysis and thus constitutes another regulator to be integrated into the already complex regulations network of E.coli’s metabolism

Made available in DSpace on 2016-06-02T19:22:06Z (GMT). No. of bitstreams: 1 3723.pdf: 1219093 bytes, checksum: 799005c02c5059316a25fc3dd92fd55d (MD5) Previous issue date: 2011-05-24 / Universidade Federal de Sao Carlos / The composition of the diet, the metabolic adaptations and variations in the activity of the fish are the main factors responsible for seasonal changes in physiological variables. It is known that aerobic exercise can improve the fish growth due to: the enhancement of feed conversion, reduced dominance, increased protein synthesis and lipid and glucose catabolism. The diet must satisfy the nutritional requirements of each species. Knowledge of the appropriate protein level is critical because maximizes feed conversion efficiency, lowers costs and reduces the load of nutrients released to the aquatic ecosystem. In Brazil, the pacu, Piaractus mesopotamicus, is an important fish in aquaculture, especially for hardiness, fast growth, excellent meat quality. This species may respond to exercise with lower levels of protein catabolism and for directing them to growth. Thus, a balanced diet and a well established aerobic exercise protocol could promote better growth for pacu. This study aimed to evaluate the effects of the combination of different levels of crude protein in the diet and sustained exercise in pacu through growth performance, hepatosomatic and viscerosomatic indexes, metabolic and hematological parameters. The experiment followed a completely randomized 2 x 3 factorial arrangement, consisting of two swimming activities (exercised-E, non-exercising) and three levels of dietary protein percentage (24, 28 and 32). The pacu exercised and fed 24 % crude protein (CP) showed better growth. The hematologic variables were responsive to nutritional status and exercise. When comparing the exercised groups all together, it was observed that the high levels of CP in the diet increased protein catabolism and lipogenesis in the liver and white muscle and lipid catabolism in red muscle. High levels of CP in the diet of non-exercised pacu increased protein catabolism in white muscle and lipogenesis in the liver. Sustained swimming activity increased the use of liver glycogen at all levels of protein studied, and protein anabolism and lipogenesis in pacu fed higher-protein diet. Based on the responses obtained in this study we can infer that for exercised pacu, the appropriate level of protein in the diet is 24 % of crude protein and levels above that promote the use of protein as an energy source. / A dieta deve atender as necessidades de cada especie. O conhecimento do teor de proteina adequado e fundamental, pois maximiza a eficiencia da conversao alimentar, reduz os custos e a carga de nutrientes lancados para o ecossistema aquatico. Sabe-se que o exercicio aerobico pode contribuir para a criacao mais eficiente de peixes aumento as taxas de crescimento devido a aumento da eficiencia alimentar, diminuicao da dominancia, aumento da sintese proteica e catabolismo lipidico e glicidico. No Brasil, o pacu, Piaractus mesopotamicus, destaca-se pela rusticidade, crescimento rapido, carne de excelente qualidade. Essa especie pode responder ao exercicio utilizando menores niveis de proteinas para o catabolismo e direcionando-as para o crescimento. Assim, uma dieta balanceada e o exercicio aerobico bem estabelecido poderiam promover maior crescimento para o pacu. Esse estudo teve como objetivo avaliar os efeitos da associacao de teores diferentes de proteina bruta da dieta e exercicio aerobico em pacu atraves das respostas de crescimento, indices hepatosomatico e viscerosomatico, variaveis hematologicas e metabolicas. O experimento obedeceu a um delineamento inteiramente casualizado em esquema fatorial 2 x 3, constituido de duas atividades natatorias (exercitado- E; nao-exercitado- SE), e tres niveis percentuais de proteina na dieta (24, 28 e 32). Os pacus exercitados e alimentados com 24 % proteina bruta (PB) apresentaram melhor crescimento. As variaveis hematologicas foram responsivas a condicao nutricional e ao exercicio. Quando comparados os pacus exercitados entre si, observou-se que os altos teores de PB na dieta aumentaram o catabolismo proteico e lipogenese no figado e musculo branco e o catabolismo lipidico no musculo vermelho. Altos niveis de PB na dieta de pacus nao-exercitados aumentaram o catabolismo proteico no musculo branco e a lipogenese no figado. A atividade natatoria sustentada aumentou o uso de glicogenio hepatico em todos os niveis de proteina avaliados e o anabolismo proteico e lipogenese nos pacus que receberam o maior teor de proteina na dieta. Com base nas respostas obtidas neste estudo podemos inferir que para pacus exercitados, o nivel adequado de proteina na dieta foi 24 % PB e que niveis acima promovem o uso da proteina como fonte energetica.

Made available in DSpace on 2014-08-20T14:37:57Z (GMT). No. of bitstreams: 1 dissertacao_maikel_alan_goulart.pdf: 244034 bytes, checksum: 458e594d86ecd076dc3f12b7d9e46913 (MD5) Previous issue date: 2009-04-02 / The aim of this study was to evaluate the effects of feed restriction and of recombinant bovine somatotropin (bST) on energetic, proteic, enzymatic and mineral metabolic parameters of prepartum dairy heifers. Forty four pregnant heifers were used for this study. The experimental period ranged from 35 days prepartum to partum and from 60 days to 160 postpartum. These heifers were divided in four groups with 11 animals each: Control+bST: fed to allow 100% of the dry matter intake (DMI) plus bST injections; Restriction+bST: fed to allow 80% of the DMI plus bST injection; Control: fed to allow 100% of the DMI plus placebo injections and Restriction: fed to allow 80% of the DMI plus placebo injections. The heifers receive three injections in the prepartum period 14 days apart. Measurements of body condition score, weight, productive and reproductive performance were conducted during pre and postpartum periods, while blood parameters were measured only in the prepartum period. Glucose concentrations were higher (P<0.05) in the Control+bST groups compared to Control and Restriction groups, while urea concentrations were higher in Restriction group compared to Control and Control+bST. Non-esterified fatty acids concentrations were higher (P<0.05) for Restriction group compared to Control and Control+bST groups. There was not observed difference (P > 0.05) in the concentrations of calcium (Ca), phosphorus (P), Ca:P ratio, ketone bodies and liver enzymes, aspartate aminotransferase (AST) and gamma glutamil transferase (GGT) between groups. Regarding performance evaluations, no difference was observed for calf weight at birth, retention of fetal membranes, body condition score and reproductive performance, but a tendency (P = 0.06) to higher production in the Control+bST group compared to restriction group was observed. These results shown that prepatum management, including feed restriction and bST injection, could be efficient to allow metabolic and physiologic adaptation to the postpartum period. / O objetivo deste estudo foi determinar os efeitos da restrição alimentar e da somatotropina bovina recombinante (bST) sobre parâmetros do metabolismo energético, protéico, enzimáticos e mineral de novilhas leiteiras no pré-parto. Foram utilizadas no experimento 44 novilhas prenhes da raça holandês. O período experimental variou de 35 dias do pré-parto até o parto e dos 60 até 160 dias pós-parto. Estas novilhas foram divididas em 4 tratamentos com 11 animais cada: Controle+bST: alimentadas com 100% da ingestão de matéria seca (IMS) com aplicações de bST; Restrição+bST - alimentadas com 80% da IMS com aplicações de bST; Controle: alimentadas com 100% da IMS com aplicações de placebo; Restrição: alimentadas com 80% da IMS com aplicações de placebo. Foram realizadas no período pré-parto 3 aplicações de bST ou placebo a cada 14 dias. Realizaram-se avaliações de escore de condição corporal, peso ao nascer dos neonatos e desempenho produtivo e reprodutivo durante o pré e pós-parto e determinações dos parâmetros metabólicos energético, protéico, mineral e enzimático, somente no período pré-parto das novilhas. Os níveis de glucose foram maiores (P<0,05) para o grupo Controle+bST, comparado aos Grupos Controle e Restrição. Quanto aos níveis de ácidos graxos não esterificados, estes foram maiores (P<0,05) para o grupo Restrição+bST em comparação aos grupos Controle e Controle+bST. Em relação aos níveis de uréia, estes foram superiores (p<0,05) para o grupo Restrição comparado ao Controle e Controle+bST. Não foram observadas diferenças (P>0,05) entre os grupos experimentais quanto aos metabólitos, cálcio (Ca), fósforo (P), a relação Ca:P e corpos cetônicos, além das enzimas hepáticas, aspartato aminotransferase (AST) e gama-glutamiltransferase (GGT). Em relação às avaliações zootécnicas não foram observadas diferenças (P>0,05) entre os grupos quanto as variáveis, peso ao nascer dos neonatos, retenção de placenta, escore de condição corporal e desempenho reprodutivo, sendo observado tendências (P=0,06) de maior produção leiteira para vacas do grupo Controle+bST em comparação a vacas do grupo Restrição. Estes resultados demonstraram que as estratégias adotadas no pré-parto, utilizando a restrição alimentar e a administração de bST em novilhas leiteiras, podem ser eficientes na adaptação metabólica e fisiológica para o período pós-parto destes animas.

L’anorexie mentale (AN), un trouble du comportement alimentaire multifactoriel, se traduit par une perte de poids. La sévère dénutrition retrouvée dans l’AN est associée à des altérations métaboliques induisant une dérégulation de l’axe intestin cerveau. Les mécanismes physiopathologiques sont encore mal connus. Le travail de cette thèse était de mieux appréhender les dysfonctions de l’axe intestin cerveau en évaluant le métabolisme protéique de divers tissus (hypothalamus, côlon et estomac) dans un modèle murin d’anorexie par une approche protéomique. Le premier travail a permis de mieux caractériser le modèle d’anorexie nommé activity-based anorexia (ABA) en fonction du sexe. Puis les différentes analyses protéomiques ont permis de constater une adaptation tissu dépendant des mécanismes régulant l’équilibre énergétique, avec une activité cérébrale potentiellement augmentée au détriment des fonctions digestives. Chez les souris femelles ABA, il a été constaté une augmentation d’expression de protéines mitochondriales au niveau de l’hypothalamus et à l’inverse, une diminution du métabolisme protéino-énergétique au niveau colique avec un rôle de la voie de signalisation mTOR. L’autophagie était augmentée dans ces deux tissus. Ensuite, nous avons démontré un ralentissement de la vidange gastrique secondaire à la dénutrition, et l’analyse protéomique a permis de constater une augmentation du stress oxydant au niveau de l’antre des souris ABA femelles. Ces altérations peuvent contribuer aux troubles fonctionnels gastro intestinaux. En conclusion, nos études soulignent des mécanismes d’adaptation tissu dépendants dans l’anorexie, qui devront être ultérieurement approfondis. / Anorexia nervosa, a multifactorial eating disorder, is a major public health problem and results in a severe body weight loss. The severe malnutrition observed in anorectic patients is associated with metabolic alterations inducing disturbance of the gut-brain axis. However, involved mechanisms remained poorly understood. The aim of the present thesis was to better understand the alterations of the gut-brain axis in the activity-based anorexia (ABA) model by evaluating the protein metabolism of various tissues (hypothalamus, colon and stomach) by proteomic approach. Firstly, we have better characterized the response to ABA model according to sex. Then, different proteomic analyses were performed using female C57BL/6 mice. Our results revealed a tissue-dependent adaptation of protein and energy metabolism with an increased hypothalamic activity and a decrease in the gastrointestinal tract. Indeed, ABA mice exhibited an increased expression of proteins involved in mitochondrial metabolism at the level of the hypothalamus, and conversely a decrease of proteins involved in protein and energy metabolism in colonic mucosa with a key role of the mTOR signaling pathway. Both in hypothalamus and colon, autophagy was increased. We were also able to show that gastric emptying was delayed in ABA mice that is mainly due to malnutrition. In addition, proteomic analysis revealed an increase in gastric oxidative stress in female ABA mice. These alterations may contribute to the gastrointestinal functional disorders frequently described in anorexia nervosa. In conclusions, our study underlined tissue-dependent adaptive metabolic process during anorexia that should be further explored.

Le but d’un traitement anticancéreux est d’être spécifique et efficacité dans la durée vis-à-vis des cellules tumorales. De nombreux agents chimiothérapeutiques ont rencontré des obstacles quant à leur utilisation en raison de leur toxicité pour les cellules saines ou de la résistance développée par les cellules cancéreuses. Cela souligne la nécessité de développer des médicaments alternatifs. Notre laboratoire a développé une classe d'inhibiteurs de réparation de l'ADN, Dbait, qui agissent en détournant et en hyperactivant les protéines de la réparation de l’ADN, telles que la protéine PARP et DNA-PK. Cela conduit en conséquence à des modifications de la chromatine, visualisées par la phosphorylation pan-nucléaire de l’histone H2AX, et en l’inhibition du recrutement aux sites des dommages de plusieurs protéines de réparation. AsiDNA, une forme active de Dbait, sensibilise les tumeurs aux radiations, à la chimiothérapie, à la thérapie ciblée, sans effet sur les cellules non tumorales et les tissus sains. Dans la mesure où la chimiothérapie consiste en des traitements cycliques de l'agent anti-cancéreux, l'objectif de cette étude était d'étudier in vitro les conséquences d’un traitement répété d’AsiDNA sur les cellules tumorales et non tumorales, plus particulièrement pour ce qui concerne l’émergence de clones tumoraux résistants ou inversement de clones non tumoraux devenus sensibles au traitement. Dans un premier temps, nous avons conçu des expériences dans le but d'isoler des clones résistants au traitement par AsiDNA. Nous montrons que des traitements cycliques ne conduisent pas à des clones résistants, mais au contraire à la sélection de cellules tumorales caractérisées par une hyper sensibilité à l'AsiDNA. Cette sensibilité acquise est stable dans le temps et n'a jamais été observée en traitant des cellules non tumorales. Afin d’identifier le(s) mécanisme(s) responsable(s) de cette sensibilité acquise, nous avons comparé des cellules de sein non tumorales (MCF-10A) et tumorales triples négatives (MDA-MB-231) après 3 trois cycles de traitement par AsiDNA. Nous montrons que les traitements cycliques d'AsiDNA causent une inhibition de l'expression génique, essentiellement au niveau de gènes impliqués dans la réparation de l'ADN, le cycle cellulaire et la prolifération. Néanmoins, aucune différence dans la capacité de réparation de l'ADN, la progression du cycle cellulaire et le taux de prolifération n'est observable. Les cellules cancéreuses augmentent les voies métaboliques énergétiques pour produire d’énergie nécessaire à leur prolifération. En tenant compte du fait que l’expression de certains gènes impliqués dans les voies métaboliques sont aussi dérégulées par le traitement cyclique d’AsiDNA, nous avons émis l’hypothèse que l’épuisement métabolique pouvait être responsable de la sensibilisation des cellules tumorales à l’AsiDNA. Une étude du métabolome a révélé une dérégulation de plusieurs métabolites incluant NAD+. Nous montrons que cette dérégulation bioénergétique est responsable de l'hypersensibilité acquise des cellules cancéreuses suite au traitement par AsiDNA. Une étude bioénergétique des cellules tumorales non traitées et sélectionnées après les traitements cycliques par AsiDNA confirment une diminution de glycolyse aérobique et de la phosphorylation oxydative dans ces dernières. En conséquence de cette réduction énergétique, les cellules cancéreuses ont perdu leur caractère malin, ce qui est démontré par une inhibition de la migration et de la formation de tumeur. Nous montrons que les cellules tumorales dérivées de traitements cycliques par AsiDNA sont dépourvues de cellules souches cancéreuses dont les caractéristiques sont leur résistance aux drogues et leur phénotype invasif. En conclusion, à côté de son rôle dans l'inhibition de la réparation de l'ADN, AsiDNA interfère également avec le métabolisme énergétique des cellules cancéreuses. / The goal of anti-cancer treatment is long term specificity and efficacy towards cancer cells. Many of the clinically available chemotherapy have encountered obstacles due to their toxicity towards healthy cells or to development of resistance by the cancer cells. This emphasizes the need for development of alternative drugs. Our laboratory developed an original class of DNA repair inhibitor, Dbait, that acts by hijacking and hyper activating DNA repair proteins involved in repairing DNA breaks, such as PARP and DNA-PK. Consequently, this leads to chromatin modification, as revealed by pan-nuclear phosphorylation of H2AX, and inhibition of the recruitment at the damage site of several DNA repair proteins at the damage site. AsiDNA, an active form of Dbait linked to a cholesterol moiety, sensitizes tumours, and not non-tumour cells, to radiation, chemotherapy, targeted therapy. As most of clinical protocols of chemotherapy involve cyclic treatments, the aim of this study was to investigate consequences of cyclic AsiDNA treatment in vitro on non-tumor and tumor cells, conditions that experience cancer patients during chemotherapy. Particular emphasis was paid to emergence of resistant clones during cyclic AsiDNA treatment of tumour cells and emergence of toxicity toward normal cells. At first, various tumor and non-tumor cells were exposed to cyclic treatments consisting of one week of treatment and one week of drug-free recovery. After few cycles of treatment, we didn’t observe toxicity toward normal cells and we failed to isolate resistant clones to AsiDNA from tumor cells. Importantly, this treatment protocol induced resistance of MDA-MB-231 cells to imatinib or PARPi. Unexpectedly, we observed that sensitivity to AsiDNA increased with repeated cycles in tumor cells. This acquired sensitization was stable over time and was never observed in non-tumor cells. In an attempt to understand the specific and acquired sensitization of tumor cells along treatment, we compared non-tumor (MCF-10A) and triple-negative breast cancer (MDA-MB-231) cells that were exposed (3CAsiDNA) or not (3CMT) to 3 rounds of AsiDNA. Transcriptome analysis of MDA-MB-231 revealed global downregulation of transcription after cyclic AsiDNA treatment. Although the expression of genes involved in DNA repair, cell cycle and proliferation, was highly affected, strikingly no clear difference in DNA repair capacity, cell cycle or proliferation rate was observed between MDA-MB-231_3CAsiDNA and MDA-MB-231_3CMT. In contrary, modification of gene expression was weakly affected in non-tumor cells.As impaired DNA repair capacity or cell cycle deregulation couldn’t explain this acquired sensitivity, therefore alternative mechanisms should account for the higher mortality of cyclic treated AsiDNA cells. Cancer cells upregulate energy metabolic pathways to produce enough energy for cell proliferation and repair. Noteworthy, AsiDNA is a PARP activator requiring NAD+ consumption. Based on the fact that metabolic pathways were also deregulated at the transcriptional level, we hypothesized that metabolic exhaustion may be responsible for AsiDNA induced sensitization. Metabolome study revealed deregulation of several metabolites including NAD+. We showed that this bioenergetics deregulation is responsible for increasing sensitivity to AsiDNA. Bioenergetics study confirmed low metabolic activity after repeated AsiDNA treatment due to deregulating aerobic glycolysis and oxidative phosphorylation. As a consequence of energetic deprivation, cancer cells deregulated their malignant behavior by inhibition of migration and tumor formation. We showed that 3CAsiDNA tumor cells are depleted of cancer stem cells, which features are responsible of drug resistance and cancer invasive phenotype. Altogether, we demonstrated that AsiDNA, beside its role in DNA repair inhibition, also interferes with energy metabolism in cancer cells.