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La recherche de nouvelles molécules pour combattre Pseudomonas.aeruginosa est d'une grande importance. L'utilisation des antibiotiques a spectre large a grandement accru la résistance de P.aeruginosa aux antibiotiques. Malgré cette situation, aucune nouvelle drogue active sur P.aeruginosa n'a été introduite en pratique clinique durant les 2 dernières décennies. Le monoxyde de carbone (CO) pourrait agir comme un inhibiteur efficace de la chaîne respiratoire de P.aeruginosa mais l'utilisation pratique de ce gaz comme molécule antibactérienne est gênée par sa toxicité et les difficultés de manipulation. Une avancée fondamentale récente dans le domaine de la recherche sur le CO a été la découverte des « carbon monoxide releasing molecules » (CO-RMs), qui servent de transporteur et délivre des quantités contrôlées de CO aux systèmes biologiques.Nous montrons ici que les CO-RMs possèdent des propriétés antibactériennes contre P.aeruginosa. Cet effet antibactérien des CO-RMs à lieu à des concentrations non toxiques pour les cellules eucaryotes et passe par une interaction du CO libérer par le transporteur avec la chaîne respiratoire bactérienne. Nous présentons des résultats in vivo montrant que les CO-RMs diminuent l'inoculum bactérien et augmentent la survie des souris après une bactériémie à P.aeruginosa. La comparaison de 4 CO-RMs ayant différente structures chimiques suggère que la précence d'un métal de transition joue un rôle important dans l'activité antibactérienne des CO-RMs. Une autre découverte importante présentée dans ce travail est l'inhibition de l'activité antibactérienne de certain CO-RMs par les molécules contenant des résidus thiols. Cette découverte limite la possibilité d'utiliser les CO-RMs concernés comme des agents anti-infectieux.En considérant les résultats présentés dans ce travail, l'inhibition de la chaîne respiratoire pourrait être considérée comme un nouveau mécanisme prometteur pour la recherche de nouveaux agents pharmaceutique pour combattre les infections à P.aeruginosa. / The search of new molecules to fight Pseudomonas.aeruginosa is of paramount importance. The use of broad spectrum antibiotics has greatly increased the antibiotic resistance of P.aeruginosa. In spite of this situation, no new drug against P.aeruginosa has been successfully introduced into the clinic in the past 2 decades. Carbon monoxide (CO) could act as an effective inhibitor of the respiratory chain in P. aeruginosa but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. A recent fundamental development in the field of CO research has been the discovery of carbon monoxide-releasing molecules (CO-RMs), which serve as carriers for the delivery of controlled amounts of CO in biological systems.Here, we show that CO-RMs possesse bactericidal properties against P.aeruginosa. This antimicrobial effect of CO-RMs occurs at non toxic concentrations for eukaryotic cells and is mediated by an interaction of CO liberated by the carrier with bacterial respiratory chain. We present in vivo results showing that CO-RMs decrease bacterial inoculum and increase survival in mice following P.aeruginosa bacteraemia. A comparison of 4 CO-RMs with different chemical structures suggests that the presence of a transition metal center plays an important role in the antibacterial activity of CO-RMs. Another important finding presented in this work is the inhibition of the antibacterial activity of some CO-RMs by thiol containing molecules. This finding could deserve the possibility to use concerning CO-RMs as anti-infective agent.Considering results presented in this work, inhibition of respiratory chain could be considered as a promising new mechanism for the research in new pharmaceutical agent to fight P.aeruginosa infections.

L’ATP est synthétisée par les mitochondries à partir de réactions d’oxydoréduction catalysées les complexes de la chaîne respiratoire. Ces réactions impliquent des transferts d’électrons intra-protéine. Une capacité de production de l’anion superoxyde, formé par la réaction de l’oxygène avec un électron, a été identifiée pour les complexes I et III. Les espèces actives de l’oxygène (EAOs) sont des molécules dérivées de l’anion superoxyde. Si elles ne sont pas correctement régulées par les défenses antioxydantes de la cellule, ces EAOs peuvent réagir avec les composants de la cellule et nuire à son fonctionnement : ce déséquilibre est appelé stress oxydatif. L’altération d’un ou plusieurs complexes respiratoires associée à un stress oxydatif cellulaire est un mécanisme commun à de nombreuses maladies neurodégénératives. Dans ce travail nous nous intéressons plus particulièrement à l’atrophie optique autosomique dominante de type 1 (ADOA-1). L’ADOA-1 est une maladie neurodégénérative principalement causée par des mutations du gène codant la protéine mitochondriale OPA1 impliquée dans la dynamique mitochondriale. Les tableaux cliniques et l’âge de début de la maladie sont variables. Il n’existe pas de corrélation claire entre génotypes et phénotypes permettant d’expliquer cette variabilité ni de traitement à cette pathologie. L’hypothèse d’un stress oxydatif a été proposée pour expliquer la variabilité de ces symptômes. C’est pourquoi notre objectif est d’améliorer la compréhension des mécanismes physiopathologiques impliqués dans cette maladie en développant des modèles mathématiques de la production des EAOs par la chaîne respiratoire. Nous avons utilisé deux méthodes de modélisation. Dans le premier cas, nous modélisons l’activité des complexes respiratoires et la production d’anion superoxyde par les complexes I et III par des équations de vitesse que nous construisons en trois étapes. Nous analysons d’abord les données biochimiques disponibles dans la littérature. Nous proposons ensuite des interprétations physiques à ces comportements et les traduisons sous forme de règles floues. Nous modélisons enfin ces règles en utilisant des fonctions données par le formalisme de Michaelis-Menten. Les équations de vitesse sont fonction de variables chimiques telles que la concentration des espèces chimiques impliquées dans les réactions des complexes respiratoires et ne prennent pas en compte le détail des réactions intra-protéine impliquées dans le fonctionnement des complexes. Cette méthode permet de construire un modèle simple, permettant de simuler l’activité des complexes I et III et leur production de superoxyde dans différentes conditions, et qui est facilement modifiable ou intégrable dans un modèle plus complet de la mitochondrie. Le modèle du complexe I que nous avons créé, est capable de simuler l’activité catalytique et la production des EAOs en mode direct par le complexe I pour différentes configurations et concentrations de substrats et produits. / Mitochondria are cellular organelles involved in ATP (adenosine triphosphate) supply to cells. Mitochondrial ATP is produced by the oxidative phosphorylation which involves redox reactions catalysed by the four protein complexes of the mitochondrial respiratory chain. These redox reactions require intra-protein electron transfers. The complex I and complex III of the respiratory chain are able to generate superoxide anion, which is formed by the reaction of oxygen with one electron. Reactive oxygen species (ROS) are molecules derived from the superoxide anion. ROS which are not regulated by cellular antioxidant defences can react with the components of the cells and disturb its functioning: this imbalance between ROS and antioxidant defences has been termed “oxidative stress”. Dysfunctions of one or several respiratory complexes associated to an oxidative stress is a mechanism common to numerous neurodegenerative diseases. In this work, we focus on autosomal dominant optic atrophy 1 (ADOA-1 or DOA-1). DOA-1 is a neurodegenerative pathology mainly caused by mutations in the gene OPA1 which codes for a mitochondrial protein involved in mitochondrial dynamics. The symptoms and ages of onset of the disease are variable. There is no clear correlation between genotypes and phenotypes which can explain this variability and to date, there is no established medical treatment for the disease. The hypothesis of an oxidative stress has been proposed to explain the variability of symptoms observed in patients. Indeed, the mitochondrial energetic metabolism is altered in biological models (cell cultures and animal models) of DOA-1 and low levels of antioxidant defences have been measured in cells from patients suffering from severe forms of the pathology. Hence, our objective is to improve the understanding of the physio-pathological mechanisms involved in this disease by developing mathematical models of ROS production by the respiratory chain. We use two modelling methods. The first method consists in modelling the activities of respiratory complexes and the superoxide production by complexes I and III with rate equations that we build in three steps. We first analyse the biochemical data available in the literature. We subsequently interpret this data physically and translate them in the form of fuzzy rules. We then model these rules with mathematical functions provided by the formalism of Michaelis-Menten. The rate equations depend on chemical variables such as the concentrations of chemical species involved in the reactions catalysed by the respiratory complexes. They do not include the details of intra-protein electron transfers, occurring during the catalysis performed by the complexes. This method enables us to build a simple model simulating the activities and superoxyde productions of complexes I and III in different conditions and that can easily be modified or integrated in a more comprehensive model of the mitochondrion. Our model of complex I can simulate the forward and reverse activities and ROS productions of the enzyme for different concentrations of substrates and products.

The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention. We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the ¡§life-and-death¡¨ process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM. Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; ¡§pro-life¡¨ phase) and a secondary decrease (Phase III; ¡§pro-death¡¨ phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered. Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. Conclusion The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

No description available.

[Truncated abstract] Mitochondria are semi-autonomous organelles found in almost all eukaryotic cells to contain more than 1000 different proteins. The majority of these proteins are encoded in the nucleus, translated in the cytosol and imported into mitochondria. The overall aim of this study was to characterise the regulation of nuclear-encoded mitochondrial proteins (NEMP). This was carried out in the plant, Arabidopsis thaliana, using the alternative oxidase (AOX) as a model. Specifically, the aims were to i) determine how regulation of NEMP interact with known regulatory pathways/mechanisms; ii) determine if the pattern of coexpression observed for NEMP are due to co-regulation, and iii) to determine whether mitochondrial retrograde regulatory pathways interact with known chloroplast regulatory pathways. AOX1c is one of five genes encoding AOX in Arabidopsis. It is expressed in a variety of organs and is not induced by stress. Thus, its regulation was characterised in order to gain insight into the regulation of NEMP under normal growth conditions. Analysis of the promoter of AOX1c revealed cis-acting regulatory elements (CAREs) common to both AOX1c from Arabidopsis and AOX2b from soybean. Additionally, Site II elements, previously shown to be involved in the regulation of the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were shown to be strong negative regulators of AtAOX1c expression. AOX1a is a gene encoding AOX that is induced at a transcript level, by many stress treatments. BA signalling and provide evidence of at least one common factor between chloroplastic and mitochondrial retrograde regulatory pathways, i.e. ABI4. ... The above results reveal that the regulation of NEMP are integrated with the mainstream regulatory pathways that control gene expression for a variety of proteins in various locations. Although this is not unexpected, it does raise the question of how mitochondrial function impacts, or feeds back, to alter these pathways, i.e. how mitochondrial retrograde signals affects the regulation of genes encoding proteins in a variety of locations. The observed interaction of mitochondrial and plastid retrograde regulatory pathways at the level of ABI4, suggests that mitochondrial signals have the potential to act as a powerful regulators of many cellular functions. Although interaction between mitochondrial and other organelles at a cellular level has been known for some time, there is still much work left to be done to define the network of molecular interactions that exists to regulate and integrate the expression of NEMP with all other proteins in the cell. This study reveals that interactions also occur at regulatory steps that have to potential to regulate many function in organelles, even if no direct metabolic link exists. However, this study has only begun to uncover these interactions at a molecular level.

Le coenzyme Q (ubiquinone ou Q) est une molécule organique lipophile composée d'une benzoquinone substituée et d'une chaîne polyisoprényle contenant 6 unités chez Saccharomyces cerevisiae (Q6), 8 chez Escherichia coli (Q8) et 10 chez l'homme (Q10). Q a un rôle bien connu de transporteur d'électrons dans les chaînes respiratoires et fonctionne également comme un antioxydant membranaire. La déficience primaire en Q10 a maintenant été attribuée à des mutations dans 6 gènes de la biosynthèse de Q10 et cause des pathologies sévères. La biosynthèse de Q6 est mitochondriale et nécessite au moins 9 protéines organisées au sein d'un complexe multiprotéique chez la levure (Coq1-Coq9). L'acide 4-hydroxybenzoique (4-HB) et l'acide para-aminobenzoique (pABA) sont les deux précurseurs connus du noyau aromatique de Q6. Malgré de nombreuses recherches et l'importance cruciale de Q dans le métabolisme eucaryote, certaines étapes de la voie de biosynthèse de Q ne sont pas connues. L'étude présentée dans ce manuscrit a permis de montrer l'implication de la protéine Coq6, proposée comme étant une mono-oxygénase à flavine, dans une seule des trois réactions d'hydroxylation que compte la voie de biosynthèse de Q6: l'hydroxylation en C5. De plus, notre étude sur Coq8, une protéine kinase dont sa surexpression stabilise le complexe multiprotéique, nous a permis de confirmer les fonctions de certaines protéines Coq (Coq5, Coq7), de découvrir la fonction de Coq6 et d'éclaircir le rôle des autres (Coq4, Coq9). Nous rapportons également que des analogues hydroxylés ou méthoxylés de 4-HB et du pABA peuvent court-circuiter des étapes déficientes des mutants particuliers conduisant ainsi à la synthèse du coenzyme Q6 dans ces derniers. Ce résultat ouvre de nouvelles perspectives pour traiter les déficiences en coenzyme Q10 qui jusqu'à présent sont traitées par supplémentation en Q. Finalement, la réaction de déamination, essentielle à la biosynthèse de Q6 à partir du pABA, reste incomprise mais nos résultats suggèrent fortement l'implication de Coq6 dans cette étape. / Coenzyme Q (ubiquinone or Q) is a lipophilic organic molecule composed of a substituted benzoquinone and a polyisoprenyl chain containing 6 units in Saccharomyces cerevisiae (Q6), 8 in Escherichia coli (Q8) and 10 in humans (Q10). Q has a well known role as an electron carrier in the mitochondrial respiratory chain and also functions as a membrane soluble antioxidant. Primary Q10 deficiency has now been linked to mutations in six genes of Q biosynthesis and results in severe pathologies. The biosynthesis of Q is mitochondrial and requires at least nine proteins in yeast (Coq1-Coq9). 4-hydroxybenzoate (4-HB) and para-aminobenzoic acid (pABA) are the long-known aromatic precursors of the benzoquinone ring of Q. Despite intensive research efforts and the biological importance of Q, some biosynthetic steps are still uncharacterized. Herein we report that Coq6, a predicted flavin-dependent monooxygenase, is involved exclusively in the C5-hydroxylation reaction. We also demonstrate that the overexpression of the protein Coq8, which is proposed to be a kinase, in Δcoq strains restores steady state levels of the unstable Coq proteins. Moreover, we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. The overexpression of Coq8 helped us clarify the role of some proteins (Coq4, Coq9). We also show that using synthetic analogues of 4-HB and pABA allows bypassing deficient biosynthetic steps in some mutants. This result opens new perspectives to address the deficiencies in coenzyme Q which until now are processed by Q supplementation. Finally, the deamination reaction, which is essential for Q6 biosynthesis from pABA remains misunderstood but our results strongly suggest the involvement of Coq6 in this step.

Pour développer des biomarqueurs permettant d'estimer l'état de santé des animaux et de prévoir les effets des contaminants sur les niveaux d'organisation biologique supérieurs, le métabolisme énergétique apparaît comme un candidat de choix. En effet, il participe à la mise en place de grandes fonctions comme par exemple la reproduction ou la croissance. L'altération des processus d'allocation énergétique par la contamination peut avoir des conséquences sur le devenir des individus et de leurs populations. L'objectif de ce travail est d'analyser en laboratoire les impacts potentiels de xénobiotiques (cuivre et éthofumésate) sur le métabolisme énergétique d'un cyprinidé, le gardon Rutilus rutilus, à travers l'étude des processus participant à la formation d'énergie cellulaire(ATP). L'effet des xénobiotiques sur l'état de santé général des juvéniles de gardons a d'abord été appréhendé, et peu d'impacts ont pu être observés sur les indices généraux mesurés. L'effet des contaminants sur la formation aérobie et anaérobie de l'énergie (glycolyse, ses substrats et produits) a ensuite été évalué. Si des différences d'effet des deux contaminants ont été observées au niveau moléculaire, ils ont tous deux induit une augmentation du métabolisme anaérobie au niveau biochimique. Enfin, l'impact des contaminants sur les fonctions mitochondriales a été appréhendé.Des atteintes ont été observées au niveau de la chaîne respiratoire et des ultra-structures des mitochondries, impliquant un effet sur l'énergie cellulaire disponible. Ce travail constitue la base du développement de nouveaux marqueurs précoces de troubles physiologiques des organismes utilisables en biosurveillance. / The energy metabolism constitutes an appropriate approach for the development of biomarkers allowing to estimate the health status of animals and to predict the effects of contaminants on higher levels of biological organization. Indeed, it participates in the establishment of key functions such as reproduction or growth. Alterations of energy allocation process by the contamination can affect the fate of individuals and populations. The aim of this study was to determine in laboratory the potentials effects of chemicals (copper and ethofumaste) on energy metabolism of a cyprinid species, the roach Rutilus rutilus, through the study of processes involved in the synthesis of cellular energy (ATP). The effect of chemicals was first performed on general health status of juvenile roach, and few impacts were found on general indexes measured. Secondly, the study of aerobic and anaerobic energy production (glycolysis, substrates and products) was performed. Significant differences were observed at the molecular regulation level, depending on chemicals. However, at the biochemical level, an increase in anaerobic metabolism was observed with both contaminants. Finally, the effects of contaminants on mitochondrial functions were assessed. Mitochondrial alterations were observed either in the respiratory chain and in the ultra-structure of mitochondria;these results involved an effect on the availability of cellular energy. This work constitutes the basis for the development of new early markers of physiological disorders in organisms used in biomonitoring studies.

Isolated deficiency of F1FO-ATPsynthase is a soubgroup of mitochondrial diseases caused by mutations in nuclear and mitochondrial-encoded structural subunits, or nuclear-encoded assembly factors of F1FO-ATPsynthase. The most often mutations are found in a MTATP6 gene localized in the mitochondrial DNA and a TMEM70 gene, localized in the nuclear DNA. A MTATP6 gene encodes subunit a of F1FO-ATPsynthase and its mutation usually leads to reduced phosphorylation activity of F1FO-ATPsynthase. A TMEM70 gene encodes a 21 kDa mitochondrial protein of the inner mitochondrial membrane of not completely explained function and its mutation results in the decrease in a content of fully assembled F1FO- ATPsynthase. The aim of this thesis was to investigate the impact of isolated F1FO- ATPsynthase deficiency on the oxidative phosphorylation system (complex I-IV), other selected mitochondrial proteins, and mitochondrial network in two cell lines of primary human skin fibroblasts with an isolated deficiency of F1FO-ATPsynthase (mutation m.8851T>C in MTATP6 and mutation c.317-2A>G in TMEM70) during the first days of their cultivation in media containing galactose or glucose as a carbohydrate source with a presence or absence of L-glutamine. The control cell line was found to have higher amounts of respiratory chain...