Physical activity results in various types of stress in skeletal muscle including energetic, oxidative, and heat stress. Acute exposure to stress impairs skeletal muscle mitochondrial function. In contrast, chronic intermittent exposure to mild stress through exercise training results in increased mitochondrial content and respiratory capacity. While oxidative and energetic stress have received much attention regarding their long-term effect on skeletal muscle mitochondria, heat stress is not well understood. The purpose of this work was to investigate the effects of localized heat therapy on human skeletal muscle mitochondria, and to compare these effects to those of high-intensity interval exercise training. To accomplish this purpose, 35 subjects were assigned to receive 6 weeks of sham therapy, heat therapy, or exercise training; all localized to the quadriceps muscles of the right leg. Two-hour sessions of short-wave diathermy were used for the heat therapy, and identical sessions were used for sham therapy, but the diathermy units were not activated. Forty-minute sessions of single-leg extension, high-intensity interval training were used for the exercise intervention. All interventions took place three times per week. Muscle biopsies were performed at baseline, and after three and six weeks of intervention. Muscle fiber bundles were isolated and permeabilized for measurement of oxygen consumption via high-resolution respirometry. The primary finding of this work was that heat therapy improves mitochondrial respiratory capacity by 24.8 ± 6.2% compared to a 27.9 ± 8.7% improvement following exercise training. Both heat and exercise significantly increased mitochondrial respiration compared to baseline measures (p<0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p<0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. Neither heat nor exercise training increased mitochondrial respiratory protein content. Overall these results suggest that heat therapy significantly improves mitochondrial function, but not to the same degree as exercise training.

Le virus Influenza de type A (IAV) est un agent pathogène hypervariable responsable d’une infection respiratoire aiguë appelée la grippe. L’hyper-variabilité de ce virus IAV lui permet d’être résistant aux traitements antiviraux et est responsable de l’apparition des épidémies de grippe saisonnières. Il est donc essentiel d’établir de nouveaux traitements curatifs « à spectre large » insensibles aux variations du virus de la grippe. Les nanoparticules d’argent (NPs-Ag) sont les nanomatériaux métalliques les plus présents dans le secteur de la santé. En effet, leurs propriétés physico-chimiques leur confèrent de nombreuses capacités telles que la modulation des réponses immunitaires au niveau du poumon et des effets antimicrobiens. Quelques études ont démontré le potentiel anti-IAV des NPs-Ag lorsqu’elles sont placées directement en contact avec le virus IAV. Cependant, aucune de ces études ne porte sur les effets des NPs-Ag dans un contexte physiologique constitué d’une infection grippale suivie d’un traitement. D’autre part, au jour d’aujourd’hui, on ignore les mécanismes d’action mis en place par ces NPs-Ag et les effets induits par l’interaction de ces NPs-Ag avec le système immunitaire dans le contexte d’une infection par l’IAV. Dans ce travail de thèse, l’objectif est d’identifier les mécanismes d’action mis en place par les NPs-Ag au cours de l’infection par le virus IAV et également d’identifier si ces NPs-Ag pourraient être utilisées comme traitement curatif.Dans ce manuscrit de thèse, nous avons pu identifier, dans les cellules épithéliales pulmonaires, un nouveau mécanisme de modulation des NPs-Ag sur la réponse anti-IAV précoce médiée, entre autres, par la sécrétion de la chimiokine CCL5 et de l’IFN-. En effet, les NPs-Ag ciblent spécifiquement la voie de signalisation RIG-I-MAVS-IRFs, activée suite à l’infection par l’IAV et qui est liée à la mitochondrie. Ces NPs-Ag ciblent également en parallèle, à la fois le réseau mitochondrial et le flux autophagique. L’ensemble de ces effets conduit à une redistribution des facteurs de régulation des IFNs (IRFs), les empêchant potentiellement d’interagir avec d’autres facteurs de la voie de signalisation RIG-I/MAVS, ce qui pourrait expliquer l’inhibition de la sécrétion de CCL5 et de l’IFN-b, induite par le virus influenza de type A, par les nanoparticules d’argent. / The Influenza A virus (IAV) is a hyper-variable pathogen causing acute respiratory infection known as Flu. Its hyper-variability allows it to be resistant to antiviral treatment. It is therefore essential to establish new curative "broad spectrum" treatments. Silver nanoparticles (NPs-Ag) are the most metallic nanomaterials present in the health sector and are potent microbicidal agents with major concerns about their use on humans because of their toxicity. Some studies have shown the antiviral effect of NPs-Ag against IAV, but not in a physiological context of Flu. Moreover, the antiviral and immunomodulation mechanisms of NPs-Ag during infection by IAV is still unclear. Here, we show that intra-tracheal administration of AgNPs to influenza infected mice or treatment of human lung epithelial cells with AgNPs resulted in exacerbated inflammation, reduced viral clearance and enhanced mortality associated to different regulation of KC (pro-inflammatory cytokine functionally homologue to human IL-8) and CCL-5 (interferon-related cytokine) in the lung. In this PhD thesis, we identified in lung epithelial cells, a new mechanism explaining dampening of mitochondrial antiviral immunity by AgNPs through alteration of the mitochondrial network leading to redistribution of IFNs regulatory factors 7, which prevents nuclear translocation of these factors. Finally, AgNPs increased LC3 positive vesicles and p62 expression, indicating that AgNPs modify the autophagy flux in lung epithelial cells. Thus, the NPs-Ag Ag inhibited the early anti-IAV response by specifically targeting the RIG-I/MAVS/IRFs signaling pathway resulting in down- regulation of CCL-5 and IFN-ß expression induced by IAV.

Les mitochondries sont des organites dynamiques dont la morphologie dépend de l’équilibre fusion/fission de leurs membranes. Ce processus essentiel à la survie cellulaire est nommé dynamique mitochondriale et sa dérégulation est associée à des troubles neurologiques. Cependant les mécanismes précis régissant la dynamique mitochondriale ne sont pas élucidés. Cette thèse porte sur la protéine Fzo1p, une grande GTPase de la superfamille des Dynamin-related-Protein. C’est un élément clé impliqué dans la fusion mitochondriale de la membrane externe de la levure. Sa structure et sa dynamique ont été étudiées par modélisation et simulations de dynamiques moléculaires tout-atome dans une bicouche lipidique solvatée. Le modèle structural obtenu tient compte de données expérimentales, de template structuraux, et de modèles ab initio du domaine transmembranaire de Fzo1p. Ce modèle a été validé expérimentalement par mutagenèse dirigée. Des permutations de charges ont confirmé des ponts salins à longue distance prédits dans le modèle. En outre, des mutations ont montré que les domaines coiled-coil de Fzo1p, contrairement à sa partie N-terminale, sont indispensables à sa fonction. L’ensemble des résultats expérimentaux et in silico met en évidence l’implication des domaines charnières dans le changement conformationnel de Fzo1p, ainsi que des résidus critiques affectant sa stabilité. Les précisions atomiques obtenues sur l’interaction de Fzo1p avec le GDP permet de formuler des hypothèses sur le mécanisme moléculaire de la catalyse du GTP pour la fusion membranaire; voire à la compréhension de la dynamique mitochondriale. / Mitochondria are dynamic organelles whose morphology is determined by fusion and fission of their membranes. This essential process is known as mitochondrial dynamics. Defects in mitochondrial dynamics are associated with neurological disorders making the investigation of physiological relevance. However, the precise sequence of events that lead mitochondrial dynamics are still not well characterised. Fzo1p, a large GTPase of the Dynamin-Related Proteins superfamily, is a key component in mitochondrial outer membrane fusion in yeast. During this PhD project I built a model of the protein Fzo1p. The structure and dynamics of the model was investigated through molecular modelling and all-atom molecular dynamics simulation in a fully hydrated lipid bilayer environment. The Fzo1p structural model integrates information from several template structures, experimental knowledge, as well as ab initio models of the transmembrane segments. The model is validated experimentally through directed mutagenesis, for instance charge-swap mutations confirm predicted long-distance salt bridges. A series of mutants indicate that coiled-coil domains are required for protein function at variance with its N-terminal region. Overall, the experimental and in silico approaches pinpoint the hinge domains involved in the putative conformational change and identifies critical residues affecting protein stability. Finally, key Fzo1p-GDP interactions provide insights about the molecular mechanism of membrane fusion catalysis. The model provides insight on atomic level and proposes a structure that will be instructional to understanding mitochondrial membrane fusion.

Indiana University-Purdue University Indianapolis (IUPUI) / Friedreich’s Ataxia (FRDA) is a rare and poorly understood autosomal recessive disease caused by a pathological deficiency of the mitochondrial protein frataxin. Patients suffer neurodegeneration, ataxia, diabetes, and heart failure. In an effort to understand the mechanisms of heart failure in FRDA, we investigated the role of the protein modification acetylation, which is highly abundant on mitochondrial proteins and has been implicated in regulating intermediary metabolism. Using mouse models of FRDA, we found that cardiac frataxin deficiency causes progressive hyperacetylation of mitochondrial proteins which is correlated with loss of respiratory chain subunits and an altered mitochondrial redox state. Mitochondrial protein hyperacetylation could be reversed by the mitochondria-localized deacetylase SIRT3 in vitro, suggesting a defect in endogenous SIRT3 activity. Consistently, frataxin-deficient cardiac mitochondria showed significantly decreased rates of fatty acid oxidation and complete oxidation to carbon dioxide. However, the degree of protein hyperacetylation in FRDA could not be fully explained by SIRT3 loss. Our data suggested that intermediary metabolites and perhaps acetyl-CoA, which is required for protein acetylation, are accumulating in frataxin-deficient mitochondria. Upon testing the hypothesis that mitochondrial protein acetylation is non-enzymatic, we found that the minimal chemical conditions of the mitochondrial matrix are sufficient to cause widespread non-enzymatic protein acetylation in vitro. These data suggest that mitochondrial protein hyperacetylation in FRDA cardiomyopathy mediates progressive post-translational suppression of mitochondrial oxidative pathways which is caused by a combination of SIRT3 deficiency and, likely, an accumulation of unoxidized acetyl-CoA capable of initiating non-enzymatic protein acetylation. These findings provide novel insight into the mechanisms underlying a poorly understood and fatal cardiomyopathy and highlight a fundamental biochemical mechanism that had been previously overlooked in biological systems.

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Purpose of the Review Mitochondrial dysfunction has long been proposed to play a crucial role in the pathogenesis of a considerable number of disorders, such as neurodegeneration, cancer, cardiovascular, and metabolic disorders, including obesity-related insulin resistance and non-alcoholic fatty liver disease (NAFLD). Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify their formation through biogenesis and the opposite processes of fission and fusion, the fragmentation, and connection of mitochondrial network areas respectively. Herein, we review and discuss the current literature on the significance of mitochondrial adaptations in obesity and metabolic dysregulation, emphasizing on the role of hepatocyte mitochondrial flexibility in obesity and NAFLD. Recent Findings Accumulating evidence suggests the involvement of mitochondrial morphology and bioenergetics dysregulations to the emergence of NAFLD and its progress to non-alcoholic steatohepatitis (NASH). Summary Most relevant data suggests that changes in liver mitochondrial dynamics and bioenergetics hold a key role in the pathogenesis of NAFLD. During obesity and NAFLD, oxidative stress occurs due to the excessive production of ROS, leading to mitochondrial dysfunction. As a result, mitochondria become incompetent and uncoupled from respiratory chain activities, further promoting hepatic fat accumulation, while leading to liver inflammation, insulin resistance, and disease’s deterioration. Elucidation of the mechanisms leading to dysfunctional mitochondrial activity of the hepatocytes during NAFLD is of predominant importance for the development of novel therapeutic approaches towards the treatment of this metabolic disorder.

CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015