Adipose tissue plays a crucial role in nutrient and energy homeostasis. At the time of worldwide pandemy of obesity and consequent metabolic syndrome, a great effort is made to find new treatments with potential to preserve insulin sensitivity, or even counteract development of obesity and type 2 diabetes. There are three principal possibilities how the adipose tissue biology can contribute to this goal: 1) induction of UCP1-dependent energy dissipation in brown adipose tissue; 2) conversion of white adipose depots to brown-like tissue (i.e. "browning"); and 3) stimulation of UCP1-independent thermogenesis in white adipose tissue. This thesis is based on two published works and one article under preparation. Generaly, it is focused on three different approaches targeting the above mentioned processes in adipose tissue of laboratory mouse: 1) diet supplementation with bile acids; 2) combination treatment of ω-3 polyunsaturated fatty acids and calorie restriction; and 3) cold exposure. In the experiments with administration of bile (specifically chenodeoxycholic) acid to mice, we confirm specific induction of UCP1 in both brown and subcutaneous white adipose tissue, as well as reversion of obesity in the response to the treatment. Nevertheless, most of the acute beneficial effects are mediated by...

Cancer-associated cachexia is a condition defined by a sustained net-negative energy imbalance. Although the different types of adipose tissue – white, beige, and brown – have been implicated in contributing to cancer-associated cachexia, the mechanisms of these maladaptive changes and their impact on whole-body energy expenditure have not been fully elucidated. Using breast cancer as our model, we demonstrate white adipose tissue browning in murine and human breast cancer; furthermore, we demonstrate that this effect is extremely localized and takes place early in tumor progression. We utilized in vitro cell culture techniques and demonstrate that cancer secreted factors and cross-talk with white adipocytes decrease expression of classic white adipose tissue-related genes. We also demonstrate in murine and human culture models that cancer secreted factors reduce white adipocyte lipid droplet size, and cross-talk between cancer cells and adipocytes results in an increase in lipolysis-related gene expression. Interestingly, our results strongly suggest that in mice, neither cancer secreted factors nor cross talk with adipocytes can induce white adipose tissue browning, indicate that this process likely occurs independently of direct cancer interactions with local white adipocytes. We demonstrate that interleukin 6, a cytokine with previous implications in white adipose tissue browning, induces interleukin 6-mediated signaling; however, that signaling alone is not enough to directly induce white adipose tissue browning. We present preliminary data suggesting that immune cell population shifts within the white adipose tissue of mice with breast cancer tumors may be source of white adipose tissue browning. We show that the Virginia Commonwealth University Health System has an identifiable population of patients with cancer with what we hypothesize as maladaptive thermogenic adipose tissue activity, and discuss ongoing experiments aimed at understanding the implications of these changes on whole body energy expenditure in human patients. Lastly, in a case of autoimmune diabetes mellitus in the setting of an extra-adrenal paraganglioma, we demonstrate that the interaction between cancer and whole-body metabolism is multifaceted. Together, these experiments demonstrate that adipose tissue plasticity occurs in breast cancer (and other cancers), and that different drivers for individual changes exist within the tumor microenvironment. We predict that further exploration of the exact mechanisms and translational implications will provide useful information to lead to new therapeutic treatments for patients with cancer-associated cachexia.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cloroplasto à uma organela caracterÃstica dos organismos fotossintetizantes sendo seu papel primordial na geraÃÃo de energia a partir de gÃs carbÃnico e Ãgua. Essa organela pode ter seu funcionamento comprometido quando submetida a estresses ambientais devido a fragilidade e complexidade do sistema. Para evitar perdas provocadas pelo estresse existem vÃrios mecanismos de adaptaÃÃo e regulaÃÃo das reaÃÃes que ocorrem no cloroplasto. Recentemente caracterizou-se mais um desses provÃveis mecanismos que foi chamado de clororespiraÃÃo. A clororespiraÃÃo foi esclarecida com a descoberta de uma enzima similar a oxidase alternativa da mitocondria que chamou-se de oxidase terminal do plastÃdeo (PTOX). A funÃÃo dessa respiraÃÃo do cloroplasto permanece incerta, mas uma das hipÃteses mais aceitas à que o funcionamento da clororespiraÃÃo poderia prevenir a formaÃÃo de espÃcies reativas de oxigÃnio atravÃs da reciclagem dos intermediÃrios redutores do cloroplasto. No presente trabalho foi caracterizado a presenÃa de dois genes que codificam para a oxidase terminal do plastÃdeo em plantas de Zea mays. Estudou-se tambÃm a expressÃo diferencial de ambos genes da PTOX em resposta ou estresse hÃdrico, alÃm da caracterizaÃÃo da clororespiraÃÃo atravÃs da atividade da NADH desidrogenase plastidial (NDH) em gel de poliacrilamida. A caracterizaÃÃo molecular dos genes da PTOX mostrou homologia de 60% quando comparadas as sequÃncias dos genes e de 79% quando comparadas as prÃ-proteÃnas traduzidas. Os genes dessa proteÃna tÃm estruturas similares, sendo compostos por oito introns e 9 Ãxons. Um estudo das regiÃes dos promotores dos genes mostrou que existiam elementos comuns porÃm a presenÃa de elementos diferentes como, o elementos cis MBS que à responssivo à seca, poderia revelar uma regulaÃÃo diferencial dos genes. A resposta diferencial foi confirmada atravÃs de RT-PCR semiquantitativo. O gene chamado de ptox1 teve sua expressÃo estÃvel, podendo ser considerado um gene constitutivo, enquanto que o gene chamado de ptox2 teve um aumento da expressÃo proporcional ao estresse aplicado tanto em folhas como em raÃzes de plantas de milho. A anÃlise da atividade da NDH em gel (zimograma) revelou a presenÃa dessa enzima em cloroplastos de milho confirmando a presenÃa das enzimas da clororespiraÃÃo. O estudo filogenÃtico de sequencias de cDNA de bancos de dados mostraram que milho e sorgo pertencentes ao grupo das monocotiledÃneas, sÃo espÃcies muito prÃximas e que compartilham dois genes ortÃlogos da PTOX identificados como ptox1 e ptox2. Concluiu-se pela primeira vez a presenÃa de dois genes da PTOX no genoma do milho, uma monocotiledÃena de metabolismo C4. Os genes foram denominados de ptox1 e ptox2. Eles foram encontrados em raÃzes e folhas e apenas o gene da ptox2 pareceu ser induzido em resposta ao estresse osmÃtico. / The chloroplast is an organelle characteristic of photosynthetic organisms and their role in generating energy from carbon dioxide and water. This organelle may be functionally compromised when subjected to environmental stress due to the fragility and complexity of the system. To avoid losses caused by stresses plants have evolved various coping mechanisms, as well as, regulation of the reactions that occur in the chloroplast. Most recently it was characterized one of these mechanisms that was called chlororespiration. The chlororespiration was bring to light with the discovery of an enzyme, similar to the alternative oxidase of mitochondria, that was called the plastid terminal oxidase (PTOX). The function of this chloroplast respiration remains uncertain, but one of the most accepted hypothesis is that the operation of chlororespiration could prevent the formation of reactive oxygen species by recycling the reducing intermediates of the chloroplast. The present study characterized the presence of two genes encoding the plastid terminal oxidase in plants of Zea mays., and its differential expression in response to water stress. It was also characterized the chlororespiration through the activity of plastidial NADH dehydrogenase (NDH) in polyacrylamide gel. The molecular characterization of PTOX genes showed 60% homology when compared sequences of genes, but 79% when compared to pretranslated proteins. The genes of this protein have similar structures, being composed of nine exons and eight introns. A study of regions of the promoters of the genes showed that there were common elements, but the presence of different elements such as the cis elements that MBS responsive to drought, could reveal a differential regulation of genes. The differential response was confirmed by semiquantitative RT-PCR. The gene called ptox1 had its expression level stable and could be considered a constitutive gene, while the gene called ptox2 had an increased expression proportional to the applied stress in both leaves and roots of maize plants. The analysis of NDH activity gel (zimograms) revealed the presence of this enzyme in maize chloroplasts suggesting the existence of the chlororespiratory pathway. The phylogenetic analysis of cDNA sequences from NCBI databases showed that maize and sorghum, being closely related species, share two genes )identified as orthologs of PTOX (ptox1 and ptox2). It was confirmed for the first time the presence of two PTOX genes in the genome of maize, a C4-metabolism monocotyledon and its differential expression under drought stress.

L’attention scientifique s’est récemment portée sur l’identification des mécanismes proximaux sous-tendant les compromis évolutifs;tels que les compromis existant entre croissance/reproduction et longévité. La production d’espèces réactives de l’oxygène (ROS )a été suggérée comme un candidat potentiel ,de par sa liaison étroite au métabolisme énergétique (sous-­produits du fonctionnement mitochondrial) et son caractère inévitable. Si la production de ROS excède le niveau des défenses antioxydantes, une situation de stress oxydant va en résulter et a été associé au vieillissement . Puisque la mitochondrie n’est pas uniquement la centrale énergétique de la cellule mais aussi le principal producteur de ROS, cette thèse s’est attachée à clarifier les relations entre métabolisme énergétique , fonctionnement mitochondrial et stress oxydant ; avec des études concernant l’impact d’activités coûteuses en énergie (croissance, reproduction, thermogénèse) sur l’équilibre de la balance oxydative. / In recent years, scientific attention has turned towards the identification of the mechanisms underlying the trade-­‐offs occurring between growth rate/reproductive investment and longevity. Amongst these mechanisms, the production of reactive oxygen species (ROS) appears to be a key factor due both to its universal and inevitable nature. ROS are by-­‐products of energy processing by the mitochondria. If ROS production exceeds the capacity of the various antioxidant systems, oxidative stress will occur, and the accumulation of oxidative damage over time is thought to be a potential cause of ageing. Since mitochondria are not only the powerhouse of animal cells but also the main producer of ROS, this PhD thesis aimed to clarify the relationships between mitochondrial uncoupling state (i.e. efficiency to produce ATP), energy metabolism and oxidative stress. I investigated the impact of energy-­‐demanding activities such as thermogenesis, reproduction and growth on oxidative homeostasis.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A FotossÃntese e a respiraÃÃo sÃo bem conhecidos como sendo responsÃveis pela produÃÃo de energia no interior das cÃlulas de plantas. Acoplados à estes processos bioenergÃticos, a transferÃncia de elÃtrons que ocorre nas membranas do cloroplasto e mitocÃndria, tambÃm sÃo considerados com tendo potencial de produÃÃo de ROS. PossÃveis interaÃÃes no funcionamento dessas organelas permanecem pouco conhecidas, especialmente em resposta a condiÃÃes de estresse. Sob estas condiÃÃes, sugere-se que as vias de transferÃncia de elÃtrons ligadas à AOX, à pUCP ou à PTOX poderia limitar a formaÃÃo de ROS reduzindo assim danos oxidativos Ãs organelas celulares. Em nosso trabalho, foram selecionadas duas cultivares de Vigna unguiculata, inicialmente caracterizados como sensÃvel (cv 1183) ou tolerante (cv EPACE1) à seca. Sob condiÃÃes experimentais, as duas cultivares nÃo apresentaram diferenÃas significativas na sensibilidade à seca. Entretanto o cultivar EAPCE1 foi mais tolerante ao ozÃnio com base no desenvolvimento de necrose e de vÃrios parÃmetros fisiolÃgicos (ϕPSII) e bioquÃmicos (teor de glutationa). Para os perfis de expressÃo de genes que codificam AOX, PUCP e PTOX duas respostas foram claramente identificadas no cultivar EPACE1. Em um tempo curto, a expressÃo destas proteÃnas à geralmente estimulada. Em um perÃodo mais longo (14 dias), a resposta à diferente, dependendo da tipo de estresse. Sob o ozÃnio, um aumento da expressÃo das proteÃnas mitocondriais à mantida enquanto o gene que codifica para a PTOX à sub-regulada. Sob seca a proteÃna desacopladora do plastÃdio (PTOX) à superexpressa. Sob condiÃÃes de combinaÃÃo stress, seca combinada com ozÃnio, o efeito da seca sobre o fluxo de ozÃnio nas folhas nÃo foi alterado, e os genes VuPTOX e VuUCP1b sÃo super-expressos depois de 3 e 7 dias de stress. Este aumento da expressÃo, jà observado em resposta à seca por aplicada separadamente poderia desempenhar um papel na prevenÃÃo da formaÃÃo de ROS tanto em mitocÃndrias e cloroplastos prevenindo assim os danos causados pelo ozÃnio. / Possible interactions between chloroplast and mitochondria remain poorly known, particularly in response to stress conditions. Under these conditions, it is suggested that the electron transfer pathways linked to AOX or pUCP (mitochondrial uncoupling proteins) and PTOX (plastidial uncoupling protein) could limit the formation of ROS to reduce oxidative damage in cellular organelles. In our work, we selected two cultivars of Vigna unguiculata, cv 1183 and cv EPACE. Under our experimental conditions, both cultivars showed no real differences in sensitivity to drought. However cv EPACE was more tolerant to O3 based on the development of necrosis and several physiological parameters (Fv/Fm, ϕPSII) and biochemical (glutathione content). For the expression profiles of genes encoding AOX, PUCP and PTOX two responses were clearly identified in the cultivar EPACE. On a shortterm, expression of these proteins is generally stimulated. On a longer term (14 days), the answer differs depending on the treatment. Under O3, the strongest expression of mitochondrial proteins is maintained while the gene encoding the PTOX is own-regulated. Under drought only the plastid protein (PTOX) is upregulated. Under conditions of stress combination, drought has little effect on the influx of O3 in the leaves, and the VuPTOX and VuUCP1b genes are up-regulated after 3 and 7 days of stress. This ver-regulation, already observed in response to drought alone could play a role in preventing the formation of ROS in both mitochondria and chloroplasts.