Positive-strand RNA virus group are the most abundant among viruses affecting plants and animals. To successfully achieve replication, these viruses usurp or co-opt host proteins. To facilitate the discovery of host factors involved in Tomato bushy stunt virus (TBSV), yeast has been developed as a surrogate model host. Genome-wide approaches covering 95% of yeast genes, has revealed approximately hundred factors that could affect virus replication. Among the identified host factors, there are fourteen yeast genes, which affect/regulate lipid metabolism of the host. One of the identified host gene is ERG25, which is an important factor for sterol biosynthesis pathway, affecting viral replication. Sterols present in eukaryotes affect the lipid composition of membranes, where tombusviruses, similar to other plus-strand viruses of tobacco, replicate. Since potent inhibitors of sterol synthesis are known, I have tested their effects on tombusvirus replication. We demonstrated that these sterolsynthesis inhibitors reduced virus replication in tobacco protoplasts. Virus replication is resumed to the wild type level by providing phytosterols in tobacco protoplasts confirming the role of sterols in RNA virus replication in tobacco. We have also identified INO2, a transcription factor for many phospholipid biosynthetic genes, reduces virus replication in its deletion background. When we provided this gene product in the mutant background, viral replication was back to normal, confirming the role of Ino2p in tombusvirus replication. Further biochemical assays showed that the viral inhibition is because of alteration in the formation of the viral replicase complex. Using confocal microscopy, we showed that the viral replication protein, termed p33, is forming large and few punctate structures rather than the small and many by overexpressing Ino2p in the wild type yeast cells. Over-expression of Opi1, an inhibitor of Ino2p led to greatly reduced viral replication, further supporting the roles of the phospholipid pathway in tombusvirus replication. One of the phospholipid, which is regulated by this pathway, is cardiolipin an important component of the mitochondrial as well as peroxisomal membranes. We further characterized how cardiolipin is playing an important role for tombusvirus replication by using different biochemical approaches.

Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes

Les cardiolipines (CL), phospholipides spécifiques des membranes mitochondriales, sont impliquées dans différentes fonctions mitochondriales. Il a été précédemment démontré que l’accumulation des CL entraînait une augmentation du gaspillage énergétique mitochondrial hépatique et une réduction du rendement de la synthèse d’ATP. Ces travaux de thèse montrent qu’à l’inverse, une diminution modérée de la quantité de CL (- 45 %) induit une réduction des capacités oxydatives mitochondriales sans diminuer la synthèse d’ATP et donc une augmentation de l’efficacité de la synthèse d’ATP. Nous démontrons également les mécanismes conduisant à l’accumulation des cardiolipines hépatiques en situation de cachexie cancéreuse. Le TNFa, cytokine proinflammatoire impliquée dans la cachexie cancéreuse, induit une surexpression spécifique de la phosphatidylglycérolphosphate synthase. La surexpression de cette enzyme impliquée dans la synthèse de novo des CL entraîne une accumulation de CL, responsable du gaspillage énergétique mitochondrial. / Cardiolipin (CL), a specific mitochondrial phospholipid, is involved in various mitochondrial functions. It has been shown that CL accumulation led to increased mitochondrial hepatic energy wasting and reduced ATP synthesis efficiency. This work showed, on the opposite, that moderate reduction in CL content (-45%) induced a decrease in mitochondrial oxidative capacity without decreasing ATP synthesis rate and thus an increased ATP synthesis efficiency. Then we demonstrated mechanisms responsible for hepatic cardiolipin accumulation during cancer cachexia. TNFa, proinflammatory cytokine involved in cancer cachexia, induced a specific overexpression of phosphatidylglycerolphosphate synthase. Overexpression of this enzyme involved into CL de novo biosynthesis led to CL accumulation, responsible for energy wasting during cancer.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T12:57:27Z No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T12:57:43Z (GMT) No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) / Made available in DSpace on 2016-02-04T12:57:43Z (GMT). No. of bitstreams: 1 Iukary Takenami Avaliação de biomarcadores....2015.pdf: 2420884 bytes, checksum: b10235421512081a9897b20afdf98354 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A tuberculose (TB), doença crônica infecciosa causada por Mycobacterium tuberculosis, é considerada um grave problema de saúde pública no país. A caracterização de antígenos protéicos e/ou lipídios que induzem uma resposta imunológica no hospedeiro, torna-se um importante passo para o desenvolvimento de novas ferramentas de diagnóstico e resposta terapêutica. Dentre os diferentes antígenos, em especial a mammalian cell entry protein 1A (proteína Mce1A), e os fosfolipídios da parede celular do bacilo como a cardiolipina (CL), os fosfatidilinositol (FI), fosfatidilcolina (FC), fosfatidiletanolamina (FE) e o sulfatide (SL), são, em sua maioria altamente imunogênicos, podendo então ser úteis no sorodiagnóstico. Portanto, o objetivo do estudo é avaliar a produção de anticorpos anti- Mce1A e anti-fosfolipídios como biomarcadores no diagnóstico e no monitoramento do tratamento da TB pulmonar. Além disso, o estudo também objetivou avaliar o perfil de citocinas e quimiocinas produzidas em sobrenadantes de cultura após estímulo in vitro com a proteína Mce1A. PACIENTES E MÉTODOS: O estudo foi conduzido no 6º Centro de Saúde Rodrigo Argolo e no Instituto Brasileiro para Investigação da Tuberculose (IBIT). A população de estudo foi composta por pacientes recém diagnosticados com TB pulmonar, seus respectivos comunicantes domiciliares (infectados por M. tuberculosis e saudáveis) e pacientes diagnosticados com outras doenças pulmonares. RESULTADOS: Pacientes com TB produzem uma forte e consistente resposta de anticorpos anti-Mce1A e anti-fosfolipídios (anti-CL, anti-FE, anti-FI e anti-FC) quando comparados com os indivíduos do grupo controle. Além disso, após início do tratamento os níveis de anti-Mce1A e anti-fosfolipídios diminuem significativamente. O sobrenadante de culturas dos pacientes TB, após cultura com Mce1A, induzem uma acentuada produção de TNF, o que não se observa nas demais citocinas e quimiocinas avaliadas. CONCLUSÃO: Estes resultados sugerem que os anticorpos anti- Mce1A e anti-fosfolipídios desempenham potencial papel como biomarcadores sorológicos no diagnóstico da TB pulmonar. Além disso, a proteína Mce1A parece desempenhar um papel importante na produção de TNF, que pode contribuir com a indução de necrose pelo bacilo, permitindo sua evasão das respostas imunes e favorecendo a dispersão do bacilo para outras células não infectadas. / INTRODUCTION: Tuberculosis (TB), chronic infectious disease caused by Mycobacterium tuberculosis, is still a serious public health problem in the country. The characterization of protein and/or lipids antigens that induce an immune response in the host, it is an important step in the development of new diagnostic tools and monitoring TB treatment response. Among the different antigens, particularly mammalian cell entry protein 1A (Mce1A protein), and phospholipids from the cell wall of bacillus such as cardiolipin (CL), phosphatidylinositol (PI), phosphatidylcholine (PTC), phosphatidylethanolamine (PE) and sulfatide (SL), are highly immunogenic and can be used for improvement of the serodiagnosis. Therefore, the aim of the study is to evaluate the production of anti-Mce1A and anti-phospholipids as biomarkers for diagnosis and monitoring of TB treatment response. In addition, the study also aimed to evaluate the profile of cytokines and chemokines produced in vitro after stimulation with Mce1A protein in culture supernatants. PATIENTS AND METHODS: The study was conducted on the 6º Centro de Saúde Rodrigo Argolo and the Instituto Brasileiro para Investigação da Tuberculose (IBIT). The study population consisted of newly diagnosed pulmonary TB patients, their household contacts (infected by M. tuberculosis and healthy) and patients diagnosed with other lung diseases. RESULTS: Patients with TB produce a strong and consistent response to anti-Mce1A and anti-phospholipids (anti-CL, anti-PE, anti- PI and anti-PTC) than those in the control groups. Furthermore, after the beginning of the treatment, anti-Mce1A and anti-phospholipid levels were significantly decreased compared with TB patient at baseline. Culture supernatants of TB patients after stimulation with Mce1A induce a strong TNF production, which is not observed in the other evaluated cytokines and chemokines. CONCLUSION: These results suggest that anti-Mce1A and anti-phospholipids play role as potential serum biomarker in the diagnosis of pulmonary TB. Furthermore, Mce1A protein appears to play an important role in TNF production, which may contribute to the induction of necrosis by M. tuberculosis, allowing for avoidance of immune responses and facilitating the dispersion of bacillus to other uninfected cells.

Orientadores: Maria Helena Andrade Santana, Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-11T17:04:37Z (GMT). No. of bitstreams: 1 Martins_MoysesOstDamm_M.pdf: 2259859 bytes, checksum: 461e16fd565ac08cc88d2c5c24c42ac1 (MD5) Previous issue date: 2007 / Resumo: Neste trabalho, foi estudada a detecção da IgG-anticardiolipina presente em soros de pacientes com doenças autoimunes, utilizando lipossomas polimerizados. A vantagem desse sistema é o reconhecimento molecular e transdução de sinal em uma única etapa. O uso de soros reais, ao invés de soros de referência, objetivou avaliar a potencialidade dos lipossomas polimerizados para aplicação em diagnóstico clínico-laboratorial. Os estudos foram realizados com "pools" de soros de pacientes com doenças auto-imunes, os quais foram previamente caracterizados através da dosagem de três níveis de IgG-específica designados como alta (5152,01 mg/mL), média (2030,49 mg/mL) e baixa concentração (13,39 e 14,03 mg/mL). Soro sadio, com concentração de IgG anti-cardiolipina 5,58 mg/mL foi usado como controle. Os lipossomas monoméricos foram compostos do ácido diacetilênico 10,12-tricosadiinóico e cardiolipina. A polimerização foi feita por irradiação de luz ultravioleta com a máxima absorção na região do azul. As interações foram estudadas com a cardiolipina livre e na superfície dos lipossomas polimerizados. A discriminação entre os soros autoimunes e soro sadio foi estudada em lipossomas polimerizados, através da diluição dos soros ou através da sua purificação com gel de sepharose- proteína G, o grau de polimerização e a concentração de cardiolipina. Os resultados mostram que as ligações específicas são predominantes em relação às inespecíficas, porém os componentes não específicos exercem interferência expressiva. Nos lipossomas polimerizados, os padrões de absorbâncias do vermelho e do azul com o tempo, são diferentes para os soros autoimunes e sadio, resultando em sinais espectrais maiores para o soro sadio. A diluição ou purificação muda o padrão de absorbância do azul, a qual decresceu com a interação, intensificando o sinal colorimétrico do vermelho resultante. A polimerização e a concentração de cardiolipina intensificam os sinais colorimétricos, porém não discriminaram a olho nu a diferença entre soro sadio e autoimune. O estudo dos efeitos da diluição e purificação apontam condições onde a discriminação do sinal pode ser maximizada. Esses resultados mostram a potencialidade dos lipossomas polimerizados para a detecção de anticorpos anticardiolipina em soros reais em ensaio de etapa única, e demonstram a factibilidade da análise espectral no estudo de interações moleculares em sistemas complexos. / Abstract: This work studies the detection of anticardiolipin IgG present in the sera o patients with autoimmune diseases , using polymerized liposomes. The advantage of this system is the molecular recognition and transduction of signal in a single step. The use of real sera, instead reference sera, aimed to evaluate the potentiality of polymerized liposomes to application in clinical-laboratorial diagnosis. The studies were carried out using pools of sera from patients with autoimmune diseases, which were previously characterized through the dosage of specific IgG level. Three levels of specific-IgG were selected, which were classified as high (5152,01 mg/mL), medium (2030,49 mg/mL) and low concentration (13,39 e 14,03 mg/mL). Serum from healthy individuals, with 5,58 mg/mL IgG anti-cardiolipin was used as control. The monomeric liposomes were composed by the diacetylenic acid 10,12-tricosadiynoic and cardiolipin. The polymerization was perfomed by irradiation in the UV wave lenght, with the maximum absorption in the blue region. The interactions were studied on the free cardiolipin and on the surface of polymerized liposomes. The discrimination between autoimmune and health sera was studied in polymerized liposomes through the dilution of sera, purification of sera using sepharose- protein G gel, the polymerizatio level and the cardiolipin concentration. The results show that the specific binding are predominant in related to the inspecific ones, but the interference of non-specific components is significant. The patterns of absorbance in red and blue along time in polymerized liposomes are different for the autoimmune sera and healthy serum. The dilution or purification chages the absorbance pattern in the blue, which decreasing as a consequence of the interaction, intensifying the final red signal. The polymerization and the cardiolipin concentration in liposomes intensified the colorimetric signals, but they don't discriminate by naked eye the dfferences between autoimmune and health sera. The study of the effects of dilution and purification pointed out to conditions where the signal discrimination may be maximized. These results show the potentialities of polymerized liposomes to detection of anticardiolipin antibodies in real sera using a single step assay. Furthermore, they demonstrate the factibility of the spectral analysis on the study of molecular interactions in complex systems. / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

No description available.

Wie auch andere bakterielle Pathogene überträgt C. trachomatis Effektorproteine in die Wirtszelle, um deren Funktionen zu manipulieren. Das während der Invasion sekretierte Effektorprotein Tarp besitzt N-terminale SH2-Bindungsstellen und eine C-terminale SH3-Bindungsstelle für die Interaktion mit Wirtszellproteinen. Zur Bestimmung dieser Interaktionen wurden Protein-Microarrays mit nahezu alle humanen SH2- und SH3-Domänen verwendet. Zahlreiche neue Interaktionen wurden detektiert, wobei das Adaptorprotein SHC1 eine der stärksten SH2-abhängigen Interaktionen mit Tarp zeigte. Mittels Transkriptionsanalyse SHC1-abhängiger Genregulation während der Infektion konnten Gene identifiziert werden, welche an der Apoptose- und Zellwachstumskontrolle beteiligt sind. Infizierte Wirtszellen mit SHC1-Knockdown wiesen eine erhöhte Apoptoserate nach Stimulation mit TNF-alpha auf. Diese Ergebnisse offenbaren eine wichtige Rolle von SHC1 im Kontext des frühen, Chlamydien-induzierten Wirtszellüberlebens und deuten darauf hin, dass Tarp als vielseitige Signaltransduktionsplattform dient. Um Wirtszelllipide aufzunehmen, nutzt C. trachomatis Transportrouten der Wirtszelle und modifiziert Lipide bei der Aufnahme. Zur Bestimmung der Lipidzusammensetzung der Wirtszelle wurde diese mittels MALDI-TOF-Massenspektrometrie analysiert. Dabei hatte die Infektion den stärksten Einfluss auf Phosphatidylinoslitol (PI)- und Cardiolipin (CL)-Spezies. Des Weiteren konnten im Infektionsverlauf PI- und CL-Spezies mit einem Massenunterschied von 14 Da detektiert werden, was auf verzweigtkettige Fettsäurereste chlamydialen Ursprungs und eine Beteiligung der cytosolischen Phospholipase A2 (cPLA2) hindeutet. Entsprechend zeigten infizierte Zellen mit einem Knockdown von cPLA2 oder der Cardiolipinsynthase 1 eine signifikant reduzierte Bildung infektiöser Bakterien. Dies unterstreicht die Bedeutung von CL und einer funktionellen Nährstoffversorgung für die erfolgreiche Vermehrung von C. trachomatis. / Like many bacterial pathogens, C. trachomatis translocates effector proteins into the host cell to manipulate host cell functions. The early phase effector protein Tarp harbors N-terminal SH2 binding sites and a C-terminal SH3 binding site for the interaction with host cell proteins. To assess these interactions, protein microarrays comprising virtually all human SH2 and SH3 domains were used. Numerous novel interactions were discovered, while the adaptor protein SHC1 was among Tarp’s strongest SH2-dependent interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to TNF-alpha-induced apoptosis. These findings reveal a critical role for SHC1 in early Chlamydia-induced cell survival and suggest that Tarp functions as an important multivalent signaling hub. To acquire host-derived lipids, C. trachomatis hijacks cellular trafficking pathways and modifies lipids during the acquisition. To assess infection-dependent changes of the host cell lipid composition, cells were analyzed by MALDI-TOF mass spectrometry. Phosphatidylinositol (PI) and cardiolipin (CL) levels were most prominently influenced by C. trachomatis infection. Furthermore, PI and CL species with a 14 Da mass difference were detected during the course of infection, indicating the presence of Chlamydia-derived branched chain fatty acids and a role of cytosolic phospholipase A2 (cPLA2) in this process. Accordingly, infection of cPLA2 or cardiolipin synthase 1 knockdown cells resulted in a significantly reduced formation of infectious particles. These data demonstrate the importance of cardiolipin and a functional nutrient supply for the successful propagation of C. trachomatis.

Ce travail a porté sur une nouvelle protéine impliquée dans l’apoptose, la mitogaligine, et plus particulièrement sur le fragment interne [31-53] responsable de son adressage à la mitochondrie. L’objectif général du projet est de comprendre au niveau atomique son mécanisme d’action sur les membranes mitochondriales. Le fragment d’adressage est à lui seul cytotoxique. C’est pourquoi j’ai concentré l’essentiel de mon travail de thèse sur son étude. Nous avons défini sa toxicité sur des cellules humaines et montré qu’il perturbait l’intégrité membranaire, excluant certaines protéines de la mitochondrie. Ce phénomène concorde avec le relargage de cytochrome c, à l’origine du déclenchement de l’apoptose par la voie mitochondriale. Pour mieux comprendre le mode d’action du fragment d’adressage et le rôle joué par la cardiolipine, lipide caractéristique des membranes mitochondriales, j’ai étudié par différentes techniques biophysiques complémentaires l’effet du milieu membranaire sur la structuration du peptide et l’effet du peptide sur la membrane. Le peptide a une très forte affinité (13nM) pour des membranes contenant de la cardiolipine. Il se place à plat sur la membrane, s’enfouissant dans l’interface, sans induire d’organisation particulière des lipides. De plus, nous avons mis en évidence que le peptide était capable d’induire une courbure positive de la membrane, ce qui va interférer avec de nombreux processus vitaux pour la cellule. Enfin, pour réaliser les études structurales et fonctionnelles de la protéine entière, j’ai participé aux premières étapes de production de mitogaligine, qui s’est avéré très délicate aussi bien par voie d’expression que par synthèse chimique. / This work is about a new protein of apoptosis, mitogaligin, and more particularly about the internal fragment [31-53] responsible for its mitochondrial targeting. General aim of the project is to understand at the atomic scale its mechanism of action on mitochondrial membranes. The addressing fragment is cytotoxic by itself. That is the reason why I focused the main part of my work on this peptide. We defined its toxicity on human cells and showed that it was capable of disrupting the membrane integrity, excluding some proteins from mitochondrion. This phenomenon agrees with the release of cytochrom c, which induces apoptosis by the mitochondrial pathway. In order to better understand the mode of action of the addressing fragment and the role played by Cardiolipin, a specific lipid of mitochondrial membranes, I studied by various and complementary biophysics techniques the effect of membrane environments on the peptide structuration and the effect of the peptide on the membrane. The peptide has a very high affinity (13nM) for cardiolipin-containing membranes. It takes place parallel to the membrane, standing at the interface, without leading to a particular lipids organization. Furthermore, we highlighted that the peptide was capable of inducing a positive curvature of the membrane, what is going to interfere with numerous vital processes for the cell. Finally, to realize the structural and functional studies of the whole protein, I was involved in the first stages of mitogaligin’s production, which has proved to be very tricky either by recombinant pathway or by chemical synthesis.

Dans les pays industrialisés, une alimentation riche en lipides et en sucres et le manque d'exercice sont responsables d'une épidémie d'obésité, d'insulino-résistance (IR) et de stéatose hépatique non-alcoolique (NAFLD). L'alimentation apporte différents types de lipides, et non seulement la quantité mais également la qualité des lipides alimentaires module le métabolisme lipidique et joue un rôle important dans le développement de ces pathologies. La nature des acides gras (AG) ingérés peut également influencer la composition des membranes biologiques, et ainsi leurs fonctions comme la fluidité membranaire, la signalisation cellulaire, la translocation des protéines vers la membrane ou à travers la membrane, et des activités enzymatiques variées. Le fonctionnement de la cellule toute entière dépend donc de la composition membranaire en lipides. Par ailleurs, certains microconstituants alimentaires, tels les polyphénols, pourraient moduler le métabolisme lipidique, prévenant ainsi la NAFLD et l'IR.L'objectif de ce travail de thèse est d'explorer, en même temps, l'impact potentiellement protecteur des polyphénols lors d'une surcharge en graisses et en saccharose d'un côté et l'effet de différents profils lipidiques nutritionnels d'un autre sur la composition en acides gras et les taux des différents lipides complexes (triglycérides (TG) tissulaires et phospholipides (PL) membranaires) et sur le métabolisme lipidique en s'intéressant au tissu tout entier et à la mitochondrie en particulier ; puisque de plus en plus d'arguments expérimentaux et cliniques suggèrent qu'un déficit de la chaîne respiratoire mitochondriale joue un rôle physiopathologique important dans la NAFLD et l'IR. Ainsi, ce travail de thèse nous a permis de montrer qu'une supplémentation en polyphénols à dose nutritionnelle modulerait différemment le métabolisme lipidique au niveau des tissus, (1) en activant l'oxydation des AG et prévenant l'accumulation des TG intra-hépatiques et la stéatose au niveau du foie ; et (2) en modulant la composition membranaire en AG des cellules musculaires ainsi que l'expression de certains transporteurs prévenant ainsi l'accumulation cytosolique des lipides et améliorant le transport du glucose ce qui préviendrait une IR au niveau du muscle. De plus, nous avons pu montrer que la qualité et la quantité des AG apportés par l'alimentation affecteraient de façon significative la composition en acide gras de l'ensemble des PL membranaires de la mitochondrie du foie, particulièrement le cardiolipide; altérant ainsi la fonction de la mitochondrie et le métabolisme lipidique au niveau du foie, ce qui pourrait jouer un rôle dans le développement de la NAFLD. / High fat and high sugar diets and lack of physical activity are believed to contribute to the increasing rates of obesity in wealthy societies. This trend is associated with a parallel increase in the prevalence of insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). Diets contains different types of lipids, and not only the quantity but also the quality of dietary lipids modulates lipid metabolism and are involved in these nutritional associated pathologies. Moreover, the quality of dietary lipids influences the biological membrane composition and thus their functions. So, membrane fluidity, cellular signalisation pathways, protein translocation into and across the membrane and many enzymatic activities, which are crucial for cell functions, are influenced by membrane lipid composition. However, some dietary nutrients as polyphenols may also modulate lipid metabolism and thus prevent against hepatic steatosis and/or IR. In that aim, this work was designed to determine, firstly, the preventive effect of polyphenols in rats fed a high fat high sucrose diet and, secondly, the impact of the quantity and the quality of dietary lipids, on triglycerides (TG) and membranes phospholipids (PL) content and/or fatty acid composition and on lipid metabolism in the whole tissue and particularly in mitochondria. Indeed, mitochondria are both a major site for fat metabolism and the main source of reactive oxygen species in hepatocytes and they are postulated to play a central role in the pathogenesis of NAFLD and IR. Our results showed clearly that polyphenols modulate differently lipid metabolism in tissues. In liver, polyphenols prevent lipid accumulation and hepatic steatosis by activating fatty acid oxidation. In skeletal muscle, polyphenols regulate membrane fatty acids composition and fatty acid and glucose transporters expression, thus preventing lipid accumulation and enhancing glucose transport. These modifications may prevent IR in skeletal muscle. In addition, dietary fatty acids quantity and quality influenced significantly fatty acids composition of membrane phospholipids from liver mitochondria, particularly cardiolipin; and thus altered mitochondria functions and liver lipid metabolism which could play a role in the NAFLD pathogenesis.

L’AMPK est un senseur métabolique qui phosphoryle de nombreux substrats afin de maintenir l’homéostasie énergétique cellulaire. Dans le cœur, un rôle protecteur de l’AMPK a été montré dans des modèles pathologiques, néanmoins la spécificité cardiaque, métabolique et sexuelle des effets protecteurs de l’AMPK n’a pas été complètement élucidée. Nous avons donc généré un modèle de souris invalidées pour l’AMPKalpha2, l’isoforme majoritaire dans le cœur. Cette délétion est induite spécifiquement dans le cœur et à l’âge adulte après injection de tamoxifène. A l’état basal, la délétion de l’AMPKalpha2 a induit une dysfonction systolique progressive du ventricule gauche et le développement d’une fibrose cardiaque uniquement chez les souris mâles. Seize semaines après induction de la délétion, ces altérations cardiaques sont associées à une diminution de la respiration mitochondriale initiée par le complexe I de la chaîne respiratoire qui pourrait être liée au réarrangement des espèces de cardiolipides et à l’augmentation de la proportion de complexe I non intégré dans des supercomplexes observés chez ces souris mâles KO. Ces effets induits par la délétion de l’AMPKalpha2 ne sont pas observés chez les femelles KO mais la fibrose cardiaque et le remodelage des cardiolipides ont été retrouvés chez des souris femelles KO ayant subi une ovariectomie. Ces résultats montrent aussi une implication de l’AMPK dans la régulation de la fibrose cardiaque et de la composition en cardiolipides de la membrane mitochondriale et mettent également en avant un dimorphisme sexuel qui pourrait être en partie dû aux hormones femelles. Une étude comparable dans un modèle pathologique est en cours d’analyse afin de préciser les effets cardioprotecteurs de l’AMPK. / AMPK is a metabolic sensor which phosphorylates a various number of substrates in order to maintain cellular energetic homeostasis. In the heart, a protective role of AMPK has been demonstrated in pathological models, nevertheless the tissue, the metabolic and the sexual specificity of those effects has not been fully investigated. Thus, we generated a mice model of AMPKalpha2 deletion, the major cardiac isoform, specifically induced in the heart and at adult age after tamoxifen injection. At basal condition, AMPKalpha2 deletion lead to a progressive systolic dysfunction of the left ventricle and to the development of fibrosis only in males. Sixteen weeks after the deletion induction, these alterations were associated to a decrease of the mitochondrial respiration initiated by complex I of the observed in these KO male mice respiratory chain which could be linked to a cardiolipin species remodeling and to an increase of complex I proportion which is not integrated in supercomplexes. These effects induced by AMPKalpha2 deletion were not observed in KO female mice; however the cardiac fibrosis and the cardiolipins remodeling were found in KO female mice with ovariectomy. These last results tend to confirm an involvement of AMPK in fibrosis regulation and membrane cardiolipin composition and highlight a sexual dimorphism which could be due to female hormones. Analysis of a similar study in a pathological model is ongoing in order to specify the AMPK cardioprotective effects.