Outre son rôle dans le métabolisme du cycle de l’urée, la citrulline possède de nombreuses propriétés notamment celle de stimuler la synthèse protéique musculaire. Or, la synthèse protéique est un poste de dépense énergétique important de la cellule. Nos travaux ont ainsi exploré, à la fois in vivo et in vitro, les effets de la citrulline sur le métabolisme énergétique afin de comprendre comment l’activation de la synthèse protéique musculaire par cet acide aminé est coordonnée avec le métabolisme énergétique. Nos résultats ont permis de mettre en évidence que la citrulline module bien le métabolisme énergétique, notamment via une réorientation des flux énergétiques au profit de la synthèse protéique. De plus, nos travaux ont précisé les effets de la citrulline sur le métabolisme protéique avec notamment un effet synergique de la citrulline et de l’exercice sur la synthèse protéique et sur la performance. Enfin, ces travaux ont permis d’explorer pour la première fois in vitro les effets de la citrulline (et de la leucine) sur le sécrétome musculaire. Nous avons ainsi démontré que la citrulline module le sécrétome musculaire et mis en évidence la complexité de régulation des protéines sécrétées par les acides aminés. En conclusion, nos travaux sont une contribution à la meilleure compréhension de la régulation musculaire du métabolisme protéino-énergétique par la citrulline. / Besides his role in the metabolism of the urea cycle, citrulline has many properties including the ability to stimulate muscle protein synthesis. However, protein synthesis is an important item of cell energy expenditure. Our work has explored both the in vivo and in vitro effects of citrulline on energy metabolism in order to understand how the activation of muscle protein synthesis by this amino acid is coordinated with energy metabolism. Our results have shown that citrulline modulates energy metabolism via a reorientation of energy flux in favor of protein synthesis. Moreover, our work has clarified citrulline effects on protein metabolism with a synergistic effect of citrulline and exercise on protein synthesis and performance. Finally, this work allowed to explore for the first time citrulline (and leucine) effects on muscle secretome. We have thus demonstrated that citrulline modulates muscle secretome and highlighted how complex is the regulation of secreted proteins by amino acids. In conclusion, our work contributes to a better understanding on muscle regulation of protein-energy metabolism by citrulline.

The main objective of this work is to determine the atomic structure of mammalian respiratory complex I. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) is one of the central enzymes in the oxidative phosphorylation pathway. It couples electron transfer between NADH and ubiquinone to proton translocation across the inner mitochondrial membrane, contributing to cellular energy production. Complex I is the largest and most elaborate protein assembly of the respiratory chain with a total mass of 970 kilodaltons. It consists of 14 conserved ‘core subunits’ and 31 mitochondria-specific ‘supernumerary subunits’. Together they form a giant, Lshaped molecule, with one arm buried in the mitochondrial membrane and another protruding into the mitochondrial matrix. Here, a novel method for the purification of ovine (Ovis aries) complex I was developed and suitable conditions for cryo-EM imaging established, after extensive screening of detergents and additives. Cryo-EM images were acquired with the recently developed direct electron detector and processed using the latest software. This allowed the solution of the nearly complete atomic model of mitochondrial complex I at 3.9 Å resolution. The membrane part of the complex contains 78 transmembrane helices, mostly contributed by conserved antiporter-like subunits responsible for proton translocation. These helices are stabilized by tightly bound lipids (including cardiolipins). The hydrophilic arm harbours flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer. Supernumerary subunits build a scaffold around the conserved core, strongly stabilizing the complex. Additionally, subunits containing cofactors (NADPH, zinc ion and phosphopantetheine) may play a regulatory role. Two distinct conformations of the complex are observed, which may describe the active and deactive states or reflect conformations occurring during the catalytic cycle of the enzyme. Currently this is the most detailed model of this molecular machine, providing insight into the mechanism, assembly and dysfunction of mitochondrial complex I. It also allows molecular analysis of numerous disease-causing mutations, and so the structure may serve as a stepping-stone for future medical developments.

Orientador: Anibal Eugenio Vercesi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:52:03Z (GMT). No. of bitstreams: 1 Paim_BrunoAlves_D.pdf: 3170914 bytes, checksum: d4f5cbbb9bfc262aef0879eaf9d377d6 (MD5) Previous issue date: 2008 / Resumo: Neste trabalho, o estresse oxidativo em mitocôndrias isoladas de fígados de camundongos knockout para o receptor de LDL (LDLr-/-) foi caracterizado por apresentar um menor conteúdo de isocitrato mitocondrial, uma reduçãoda razão GSH/GSSG e um maior conteúdo de proteínas oxidadas quando comparados a camundongos controles. A maior produção de EROs pelas mitocôndrias de camundongos LDLr-/- foi prevenida quando as mesmas foram incubadas na presença de isocitrato exógeno, o qual é capaz de manter NADPH completamente reduzido. Resultados semelhantes foram obtidos quando os animais foram tratados com solução de citrato solução de citrato de sódio 55 mM/ ácido cítrico 67 mM durante duas semanas, onde houve uma redução na produção de EROs pelas mitocôndrias isoladas de fígado assim como uma recuperação parcial no consumo de oxigênio mantido por substratos endógenos bem como no perfil de oxidação dos nucleotídeos de piridina. Os linfócitos de camundongos LDLr-/- também apresentaram elevação na produção de EROs, celular e mitocondrial bem como uma elevação nos níveis de Ca2+, citosólico e mitocondrial, quando comparados a camundongos controles. Uma vez que [Ca2+] intracelular tem demonstrado participar na modulação da resposta Th1 e Th2 e no processo de ativação linfocitária, decidimos estudar algumas citocinas em camundongos LDLr-/-. As citocinas Th1 (IL-2 e TNF- a) e Th2 (IL-4 e IL-10) se mostraram elevadas quando comparadas a camundongos controles. A elevação das citocinas Th1 e Th2, a geração de EROs e as elevações das concentrações de Ca2+ foram parcialmente inibidas após tratamento dos camundongos com verapamil, um antagonista de canal de cálcio tipo L. Os resultados nos levam a concluir que o estresse oxidativo observado em mitocôndrias isoladas de fígados de camundongos LDLr-/- ocorre em resposta a uma redução no conteúdo de substratos endógenos redutores de NADPH e que pode, ao menos em parte, ser corrigido após suplementação oral com solução de citrato. Além do mais, foi demonstrado que o Ca2+, em linfócitos isolados de baços de camundongos LDLr-/-, possui papel fundamental modulando a geração de EROs e a produção de citocinas. Estas alterações são provavelmente relacionadas com o processo de ativação linfocitária, em razão das lesões ateroscleróticas observadas no arco aórtico dos camundongos LDLr-/-, formadas espontaneamente / Abstract: In this work, oxidative stress in liver mitochondria isolated from LDLr-/- mice was further characterized by showing a lower mitochondrial isocitrate content, lower matochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to control mice. The higher rate of ROS production in LDLr -/- mitochondria was also prevented by the presence of exogenous isocitrate which maintains NADP+ fully reduced. Similar results were obtained when the annimals were treated with drinking water containing 55 mM sodium citrate/67 mM citric acid during two weeks, when was observed lower levels of ROS production, an improve in the rate of oxygen consumption supported by NADP-linked substrates as well as a higher capacity to sustain reduced NAD(P)H in liver mitochondria. The spleen lymphocytes isolated from k/o mice also showed higher ROS generation by lymphocyte mitochondria and whole cell, and higher levels of cytosolic and mitochondrial Ca2+ compared to control mice. As intracellular Ca2+ has been shown to affect Th1 and Th2 priming and lymphocyte activation we evaluated some cytokines in LDL receptor k/o mice compared to controls. Th1 cytokines (interleukin-2 and TNF-a), as well as Th2 cytokines (interleukin-10 and interleukin-4) were higher in k/o mice than in control. The increase of Th1 and Th2 cytokines, ROS generation, and Ca2+ concentration were partially inhibited by treatment of the k/o mice with verapamil, an L-type Ca2+ channel antagonist. In conclusion, the data demonstrate that the liver mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate. Also, we demonstrate that Ca2+ presents a central role in spleen mononuclear cell from hypercholesterolemic LDL receptor knockout mice modulating ROS generation and cytokines production. These alterations are probably related to lymphocyte activation due the expontaneous atherosclerotic lesion observed in aortic arch of k/o mice / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Les maladies mitochondriales sont les anomalies congénitales du métabolisme les plus fréquentes. Elles sont caractérisées par une très grande hétérogénéité clinique et génétique, et le gène responsable de la maladie n’a pu être identifié que pour seulement 30% d’entre elles. Malgré l’hétérogénéité de ces maladies, il est possible d’identifier des groupes de patients cliniquement homogènes. C’est notamment le cas des atteintes hépatiques mitochondriales, qui peuvent se présenter sous une forme syndromique ou isolée. Les patients ayant une forme isolée ont soit une déplétion de l’ADNmt, soit une quantité d’ADNmt normale. Les patients avec déplétion de l’ADNmt sont très bien caractérisés génétiquement et sont mutés dans les gènes DGUOK, POLG, PEO1 ou MPV17, alors que les atteintes hépatiques sans déplétion de l’ADNmt n’ont commencé à l’être que plus récemment et montrent une très grande hétérogénéité génétique.Nous avons dans ce travail de recherche constitué une cohorte cliniquement homogène de 70 patients provenant des hôpitaux Necker-Enfants Malades et du Kremlin-Bicêtre présentant une atteinte hépatique mitochondriale isolée ou syndromique, sans déplétion de l’ADNmt, dont nous disposions de matériel (fibroblastes, ADN) nécessaire à leur étude. Nous avons tout d’abord identifié des mutations dans le gène TRMU, codant pour une enzyme de modification des ARNt mitochondriaux, responsables d’une anomalie de la traduction mitochondriale. Nous avons par ailleurs établi l’hétérogénéité génétique de ce groupe de patients, puisque nous avons pu exclure la présence de mutations dans les gènes TRMU, TSFM, GFM1 et LARS chez 40 patients, démontrant qu’il n’y a pas de gènes majeurs associé aux atteintes hépatiques sans déplétion de l’ADNmt. Pour deux familles multiplex pour lesquels l’ADN de plusieurs membres de la famille était disponible, nous avons réalisé une cartographie génétique combinée avec un séquençage exome et une étude du transcriptome, qui n’a pas permis de mettre en évidence de gène causal. Pour 38 autres patients, essentiellement des cas sporadiques, nous avons utilisé les stratégies du transcriptome et du séquençage exome, ce qui nous a permis d’identifier des variations robustes dans de nouveaux gènes MRPS5, ALDH1B, NOX5, MTUS1, AARS2, PPA2, MTHFD1, ALDH6A1, NME4 et GLDC pour 17 patients. Enfin, nous avons étudié particulièrement les mutations identifiées dans le gène NOX5, retrouvées chez 3 patients de la cohorte. Ce gène code pour une protéine NADPH de fonction inconnue, que pensons être impliquée dans la traduction mitochondriale. / Genetic characterization of mitochondrial liver damage

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

Les cellules tumorales sont caractérisées par un métabolisme hautement glycolytique à l’inverse des cellules normales qui utilisent préférentiellement un métabolisme oxydatif. Cette adaptation métabolique permet aux cellules d’être moins dépendantes de l’oxygène et favorise les processus d’invasion. D’autres mécanismes associés aux espèces réactives de l’oxygène qui induisent des dommages de l’ADN contribuent à la transformation cellulaire et à l’initiation tumorale. Le transporteur mitochondrial UCP2 est le deuxième membre identifié dans la famille des UCP dont la fonction mitochondriale reste encore mal comprise. Cependant, UCP2 a été impliquée dans un grand nombre de fonctions biologiques telles la régulation de la production de radicaux libres, l’inflammation, la prolifération cellulaire ou encore le métabolisme énergétique. UCP2 est donc un bon candidat pour comprendre le dialogue entre ces événements connus pour favoriser l'initiation, la progression et l'invasion du cancer. Au cours de ma thèse, dans une première partie, nous avons montré que la surexpression d’UCP2 dans différentes lignées de cellules cancéreuses entraîne une diminution de leur prolifération. En effet, les cellules cancéreuses surexprimant UCP2 changent leur métabolisme de la glycolyse vers l’oxydation phosphorylante et deviennent peu tumorigènes. La modification de l’expression des enzymes de la glycolyse et de la phosphorylation oxydative contribue à ce changement métabolique. De plus, la surexpression d’UCP2 augmente la signalisation d’AMPK et diminue l’expression de HIF. UCP2 agit donc dans le contrôle du routage des substrats mitochondriaux. Dans une seconde partie, nous avons étudié si UCP2 joue un rôle dans le développement des tumeurs in vivo en modulant le métabolisme énergétique cellulaire et la production des ROS. Ainsi, nous avons montré in vivo l’impact de l’invalidation du gène Ucp2 (souris Ucp2-/-) dans deux modèles murins de cancer colorectal : un modèle transgénique (souris APCmin/+) et un modèle chimique (azoxyméthane + dextran disulfate (AOM-DSS)). Chez des souris APCmin/+ Ucp2+/+ ou des souris Ucp2+/+ sous traitement AOM-DSS, les tumeurs présentent une augmentation d’expression d’UCP2 par rapport au tissu adjacent non tumoral. L’invalidation du gène Ucp2 dans le modèle APCmin/+ diminue la survie des animaux. Une augmentation du nombre total de tumeurs est observée dans les deux modèles. Ces résultats suggèrent que l’initiation tumorale pourrait être augmentée en absence d’UCP2. / Dysregulation of cellular metabolism has been associated with malignant transformation. Switching from oxidative phosphorylation (OXPHOS) to glycolysis for ATP production allows cancer cells to be less oxygen dependent, thus favoring invasion processes. Effects on metabolism, and more particularly mitochondria metabolism, thus represent a potential therapeutic target for cancer therapy. Uncoupling protein 2 (UCP2) is a member of UCPs, a subfamily of the mitochondrial carriers. The function of UCP2 is still controversial but we recently showed its role in the modulation of cell metabolism. Therefore, UCP2 is a good candidate to address the crosstalk between metabolic alteration and promotion of cancer progression and invasion. We show that cancer cells overexpressing UCP2 shift their metabolism from glycolysis toward oxidative phosphorylation and become poorly tumorigenic. Altered expression of glycolytic and oxidative enzymes underlies the cell metabolic shift. Moreover, UCP2 overexpression is associated with an increased adenosine monophosphate-activated protein kinase (AMPK) signaling together with a downregulation of hypoxia-induced factor (HIF) expression. In line with our previous observations, UCP2 does not function as an uncoupling protein but rather controls mitochondrial substrate routing. To address UCP2 role in cancer in vivo, we investigate the impact of Ucp2 deletion in two colorectal cancer mice models: a transgenic mice model APCmin/+ and a chemical cancer mice model (azoxymethane + dextran disulfate (AOM-DSS)). These two models are complementary because they allow us to determine if the role of UCP2 in cancer differs in only one genetic background (APC) compared with an inflammatory model (AOM + DSS). We found in those two colorectal cancer models that UCP2 is more expressed in tumors instead of the adjacent healthy mucosa. Deletion of Ucp2 in APCmin/+ mice leads to decrease in animal survival and Ucp2 deletion is associated in both mice models with an increased number of tumors. Altogether the results suggest that tumor initiation could be increased with Ucp2 deletion. UCP2 thus appears as a critical regulator of cellular metabolism with a relevant action against tumor maintenance and malignancy.

M.Tech. / Low Level Laser Therapy (LLLT), also known as photo-biostimulation or simply phototherapy, has widely been used in the treatment of wounds, with its history dating back to the early 1960s (Ohshiro and Calderhead, 1991). Despite some literature reporting negative and non-existent cellular responses to LLLT, a growing body of literature reports the positive and beneficial effects of LLLT. LLLT has proved to be efficient in speeding and improving the quality of wound healing. Stressed cells respond more favourably to LLLT by recovering to their most natural state and functional capability (Bernett, 1998; Karu, 1998). When healing appears to be impaired, these tissues respond positively to the appropriate doses of light, especially light that is within 600 to 1,000 nm wavelengths (Enwemeka et al., 2004). Cellular responses to LLLT include changes in mitochondrial intracellular calcium ion (Ca2+) levels, Mitochondrial Membrane Potential (MMP), Adenine Triphosphate (ATP) concentration, and cyclic 5’, 3’ Adenosine Monophosphate (cAMP) (Karu, 1998). The mitochondrion is the power house of a cell and the major location of cellular ATP synthesis (Bayens and Dominiczak, 1999). ATP is an energy rich molecule that drives processes responsible for cell growth or proliferation (Klug et al., 2003). LLLT alters intracellular pH which is related to activation of ATPase leading to an increase in ATP production in the mitochondria of the cell (Alexandratou et al., 2002; Karu, 1998). However the mechanisms by which the beneficial effects are attained by cells in stress or injury state are not clear.

Neuropathic pain is a common and dose-limiting adverse effect of several cancer chemotherapeutic agents including paclitaxel. Current treatments for chemotherapy-induced peripheral neuropathy (CIPN) are largely ineffective and the pain can persist long after the cessation of the chemotherapy regimen. Whilst the specific underlying mechanisms are not fully understood, oxidative stress and mitochondrial damage are thought to be involved in the development of CIPN. Antioxidants which protect mitochondria may inhibit oxidative stress and protect mitochondrial function more effectively than antioxidants which do not specifically act within mitochondria, and may attenuate CIPN. The overall aim of the study was therefore to determine the effects of mitochondrial-targeted antioxidants on CIPN. This was addressed in two main parts. Firstly, in vitro studies aimed to determine the effects of paclitaxel alone and in combination with mitochondrial-targeted antioxidants melatonin and MitoVitE, and a non-targeted antioxidant, Trolox, on oxidative stress and mitochondrial function in cells. In vivo studies aimed to determine the effects of melatonin, MitoVitE and Trolox in a preclinical rat model of paclitaxel neuropathic pain. In vitro studies used a dorsal root ganglion (DRG) cell line (50B11). Cells were cultured with a range of concentrations of paclitaxel, with or without the addition of melatonin, MitoVitE or Trolox. Several measures of oxidative stress including free radical production, and glutathione levels, and measures of mitochondrial function, including mitochondrial metabolic rate, membrane potential, mitochondrial pore opening and ATP production were made. In vivo studies used a rat model of paclitaxel-CIPN, and assessed the effects of melatonin, MitoVitE and Trolox on behavioural measures of pain. In vitro studies showed that paclitaxel induced oxidative stress and caused mitochondrial damage in the DRG cell line. Compared to paclitaxel alone, cells co-treated with melatonin and MitoVitE had reduced oxidative stress and mitochondrial damage. Co-treatment of cells with paclitaxel and Trolox did not differ from conditions with paclitaxel only. In vivo studies demonstrated that melatonin and MitoVitE attenuated paclitaxel-induced mechanical hypersensitivity, whilst Trolox did not affect behavioural measures of CIPN. These studies suggest that mitochondrial-targeted antioxidants may be useful as a potential treatment strategy for CIPN.

Philosophiae Doctor - PhD / Cancer is one of the leading causes of morbidity and mortality in developed and developing nations. It is estimated that 86% of new cases and 64% of death due to cancer are from Africa and 13.1 million deaths are estimated to occur worldwide by the year 2030. Cancer death rates have not subsided despite recent advances in cancer drug development and treatment. Present cancer drug regimens are limited due to unpredictable efficiency, severe side effects, resistance and high cost. Plants provide a vast array of natural compounds such as terpenoids, phenolics and alkaloids with antiproliferative pro-apoptotic and antioxidant effects. Plants are principal sources of compounds for drug discovery and development of several clinically proven useful anticancer drugs. The present study focused on the isolation of compounds from the Holarrhena floribunda (H. floribunda) leaves for their potential anticancer activities. Standard methods were employed to assess the antiproliferative potential, apoptosis, cell cycle analysis and reactive oxygen species of the methanolic leaf extract (MLE) of H. floribunda. The standard methods of isolation such as column chromatography, thin layer chromatography, high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) were used to isolate and purify bioactive compounds from the leaves. To elucidate the mechanism of cytotoxicity of the isolated compounds, apoptosis effect was studied by flow cytometry analysis using the ApopercentageTM dye, Annexin-V/PI stain, induction of caspase-3 using the Caspase-3/7 Glo assay kit and PARP-1 deactivation using Western blot analysis. The mode of action was further assessed by evaluating reactive oxygen species (ROS), mitochondrial toxicity, light and fluorescent microscopic morphological evaluations of F-actin and topoisomerase-I relaxation assay. In addition, potential cancer prevention of the plant was also evaluated by assessing the antioxidant activity of the flavonoids compounds isolated from the MLE.

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