Die oxidative Phosphorylierung (OXPHOS) spielt eine zentrale Rolle im Energiestoffwechsel der Zelle. Sie besteht aus der Atmungskette, deren vier Enzymkomplexe einen Protonengradienten über die innere mitochondriale Membran aufbauen, und der ATP-Synthase, die diesen Gradienten zur Phosphorylierung von ADP zu ATP, der zelluläre Energieeinheit, nutzt. In der vorliegenden Arbeit wurde ein thermodynamisch konformes OXPHOS Modell erstellt, welches auf Differentialgleichungen basiert. Dazu wurden Gleichungen entwickelt, welche die Kinetiken jedes OXPHOS-Komplexes über weite Bereiche von Substrat- und Produktkonzentrationen sowie unterschiedlichster Werte des elektrochemischen Gradientens wiedergeben. Zunächst wurden für jeden Komplex der Atmungskette kinetische Messungen in Abwesenheit des Protonengradientens durchgeführt. Für deren Beschreibung erwiesen sich Gleichungen vom Typ Michaelis-Menten als adäquat; hierbei wurden verschiedene Gleichungstypen verglichen. Anschließend wurde der Einfluss des Protonengradientens auf die kinetischen Parameter so modelliert, dass physiologisch sinnvolle Raten in dessen Abhängigkeit erzielt werden konnten. Diese neuen Ratengleichungen wurden schließlich in ein OXPHOS Modell integriert, mit dem sich experimentelle Daten von Sauerstoffverbrauch, elektrischem Potential und pH-Werten sehr gut beschreiben ließen. Weiter konnten Inhibitor-Titrationskurven reproduziert werden, welche den Sauerstoffverbrauch in Abhängigkeit der relativen Hemmung eines OXPHOS-Komplexes darstellen. Dies zeigt, dass lokale Effekte auf globaler Ebene korrekt wiedergeben werden können. Das hier erarbeitete Modell ist eine solide Basis, um die Rolle der OXPHOS und generell von Mitochondrien eingehend zu untersuchen. Diese werden mit zahlreichen zellulären Vorgängen in Verbindung gebracht: unter anderem mit Diabetes, Krebs und Mitochodriopathien, sowie der Bildung von Sauerstoffradikalen, die im Zusammenhang mit Alterungsprozessen stehen. / Oxidative phosphorylation (OXPHOS) plays a central role in the cellular energy metabolism. It comprises the respiratory chain, consisting of four enzyme complexes that establish a proton gradient over the inner mitochondrial membrane, and the ATP-synthase that uses this electrochemical gradient to phosphorylate ADP to ATP, the cellular energy unit. In this work a thermodynamically consistent OXPHOS model was built based on a set of differential equations. Therefore rate equations were developed that describe the kinetics of each OXPHOS complex over a wide concentration range of substrates and products as well for various values of the electrochemical gradient. In a first step, kinetic measurements on bovine heart submitochondrial particles have been performed in the absence of the proton gradient. An appropriate data description was achieved with Michaelis-Menten like equations; here several types of equations have been compared. The next step consisted in incorporating the proton gradient into the rate equations. This was realized by distributing its influence among the kinetic parameters such that reasonable catalytic rates were obtained under physiological conditions. Finally, these new individual kinetic rate expressions for the OXPHOS complexes were integrated in a global model of oxidative phosphorylation. This new model could fit interrelated data of oxygen consumption, the transmembrane potential and the redox state of electron carriers. Furthermore, it could well reproduce flux inhibitor titration curves, which validates its global responses to local perturbations. This model is a solid basis for analyzing the role of OXPHOS and mitochondria in detail. They have been linked to various cellular processes like diabetes, cancer, mitochondrial disorders, but also to the production of reactive oxygen species, which are supposed to be involved in aging.

Introdução: A lesão de isquemia/reperfusão do fígado é caracterizada pelo agravamento da lesão isquêmica hepatocelular quando o órgão é revascularizado, podendo originar, nos casos mais graves, uma reação inflamatória sistêmica com lesão de órgãos à distância. O controle desse fenômeno é importante no transplante de fígado, nas cirurgias de ressecção hepática e no choque hemorrágico. A administração de soluções salinas hipertônicas têm se mostrado eficaz no tratamento do choque hemorrágico, pois melhora as alterações hemodinâmicas e, possivelmente, apresenta uma ação antiinflamatória. Neste trabalho foram avaliados os efeitos locais e sistêmicos da administração da solução salina hipertônica na lesão de isquemia/reperfusão hepática em ratos. Métodos: Enquanto 14 ratos Wistar machos, dos 56 utilizados no estudo, compuseram o grupo controle, grupo C; os demais, que foram submetidos à uma hora de isquemia hepática e 4 horas de reperfusão, compuseram outros grupos de 14 animais: o grupo ST, animais que não receberam tratamento; o grupo SSF, animais que receberam 34 mL/kg de NaCl 0,9%, por via endovenosa, 15 minutos antes da reperfusão; o grupo SSH, animais que receberam 4 mL/kg de NaCl 7,5%, 15 minutos antes da reperfusão. Após 4 horas de reperfusão, os materiais foram coletados para análise. Foram realizadas as dosagens das transaminases AST e ALT, a avaliação das funções oxidativas e fosforilativas mitocondriais, a dosagem das interleucinas IL-6 e IL-10, as análises teciduais pulmonares e a análise histológica do fígado isquêmico e não isquêmico. Resultados: Quando comparado aos grupos ST e SSF, o grupo SSH apresentou elevação dos níveis de AST e ALT significantemente menores; preservação da função mitocondrial tanto dos lobos isquêmicos, como dos não isquêmicos, significantemente melhor; elevação dos níveis de IL-6 e IL-10 sem significância estatística; aumento da permeabilidade vascular pulmonar significantemente menor; e elevação da atividade da mieloperoxidase pulmonar sem significância estatística. Em relação à análise histológica da lesão de isquemia/reperfusão hepática, o escore da lesão do grupo SSH foi significantemente menor que o da lesão do grupo C; entretanto, quando comparados os três grupos submetidos à isquemia hepática ST, SSH e SSF não se observaram diferenças significantes estatisticamente. Conclusão: A administração da solução salina hipertônica a 7,5% na isquemia/reperfusão hepática normotérmica melhorou as lesões hepáticas locais e as lesões à distância, principalmente pulmonares / Introduction: During liver ischemia, the drop in mitochondrial energy production leads to cellular damage, which is aggravated during restoration of blood supply. Besides local hepatic injury, the ischemia/reperfusion process can trigger a systemic inflammatory syndrome producing remote organ damage. To control these alterations in clinical conditions like liver transplantation, liver resections and hypovolemic shock, is crucial to achieve proper patient management. Aim: To evaluate the effect of the sodium chloride hypertonic solution on prevention of local and systemic injury during partial liver ischemia/reperfusion. Methods: Animals underwent partial warm liver ischemia/reperfusion. Fity six Wistar male rats were randomly allocated into four groups. Fourteen animals were submitted to sham operation and allocated to C group; 42, submitted to one hour of liver ischemia followed by 4 hours of reperfusion, were allocated in three additional groups: ST group, 14 animals that received no treatment; SSF group, 14 animals that received NaCl 0.9%, 34 mL/kg, intravenously; SSH group, animals that received NaCl 7.5%, 4 mL/kg, intravenously. Blood and tissue samples were collected four hours after reperfusion, when animals were killed. Blood samples were collected to determinate AST, ALT, IL-6 and IL-10 levels. Liver and pulmonary tissues were assembled for liver histology and for liver mitochondrial phosphorylation, pulmonary vascular permeability and myeloperoxidase analyzes. Results: Hypertonic saline solution showed beneficial effects in the treatment of liver ischemia/reperfusion injury. SSH group presented elevation of AST and ALT plasma levels significantly lower than ST and SSF groups. A significant reduction on mitochondrial dysfunction was observed in SSH group compared with ST and SSH groups. Elevation in serum IL-6 and IL-10 was similar among ST, SSF and SSH groups. Pulmonary vascular permeability was significantly lower in group SSH compared with ST and SSF groups. No differences in myeloperoxidase activity were observed among these three groups. Histological score for liver ischemia/reperfusion injury was significantly lower in SSH group compared to ST group, however no differences were observed between SSH and SSF groups. Conclusion: Administration of hypertonic saline solution in an experimental rat model of liver ischemia-reperfusion ameliorated local and systemic injuries

Le postconditionnement cardiaque est paradoxal chez le rat. Certains auteurs ont montré que cet animal pouvait être protégé par le postconditionnement alors que d’autres ont montré qu’il était inefficace. L’objectif de notre travail était d’éclaircir cette situation et d’établir un lien entre la régulation des fonctions mitochondriales et la réussite ou l’échec du postconditionnement. Nous avons montré sur un modèle in vivo que le rat est sensible au postconditionnement cardiaque sous certaines conditions. Il semble que la réussite de cette thérapie nécessite à la fois une préservation de la phosphorylation oxydative, une inhibition de l’ouverture du mPTP et une diminution de la production de ROS. Nous avons également montré que le complexe I de la chaîne respiratoire régule l’ouverture du mPTP en liaison avec l’état de la Cyp D. L’ensemble de nos résultats montrent que le rat n’est pas réfractaire au postconditionnement, mais pour que celui-ci soit efficace, il est nécessaire de préserver l’intégrité des différentes fonctions mitochondriales. La cardioprotection et la régulation des fonctions mitochondriales sont donc spécifiquement liées au modèle utilisé / Cardiac postconditioning is paradoxical in rat. Certain authors showed that this animal could be protected by postconditioning whereas others showed that it was ineffective. The objective of our work was to clarify this situation and to establish a link between the regulation of the mitochondrial functions and the success or the failure of postconditioning. We showed on an in vivo model that the rat is sensitive to cardiac postconditioning under certain conditions. It seems that the success of this therapy requires at the same time a safeguarding of oxidative phosphorylation, an inhibition of the mitochondrial permeability transition pore opening and a reduction in the ROS production. We also showed that complex I of the respiratory chain controls the opening of the mPTP in relation with the state of Cyp D. Our results show that the rat is not insensitive to postconditioning, but it is necessary to preserve the integrity of the mitochondrial functions to be effective. The cardioprotection and the regulation of the mitochondrial functions are specifically related to the model used

Nous avons étudié les relations entre les différentes voies du métabolisme énergétique lors de la mise en place des effets Crabtree et Warburg. L’effet du glucose sur le métabolisme énergétique de S. cerevisiae se traduit dans un premier temps par une inhibition cinétique du métabolisme oxydatif (effet Crabtree). Après l’ajout de glucose aux cellules, nous avons mis en évidence l’accumulation d’un intermédiaire de la glycolyse, le F1,6bP. Ceci induit une diminution drastique du rapport G6P/F1,6bP. Or, il a été montré que le G6P stimule et le F1,6bP inhibe l’activité de la chaine respiratoire mitochondriale « in-situ ». L’utilisation de mutants et la modulation de ce rapport nous a permis de montrer que l’induction de l’effet Crabtree chez la levure Saccharomyces cerevisiae est dû à une diminution du rapport G6P/F1,6bP. Parallèlement, le glucose induit un réarrangement génétique qui à terme conduit à un effet Warburg. Nous avons mis en évidence une diminution, au cours du temps du contenu mitochondrial par effet de dilution, suite à un arrêt de la biogenèse mitochondriale (répression de HAP4). Nous avons pu montrer que cette diminution quantitative des OXPHOS est sans effet sur la synthèse d’ATP cellulaire. Ceci est dû à une augmentation du flux de synthèse d’ATP glycolytique. L’utilisation de mutants HAP4", nous a permis de montrer qu’il n’y a pas de lien simple entre prolifération et répression des OXPHOS. Bien que le flux glycolytique diminue dans les conditions de maintien des OXPHOS, ceci est sans effet notoire sur la vitesse de prolifération. Ceci est un rare exemple d’une situation biologique ou l’on observe un découplage entre métabolisme énergétique et prolifération. / We used the yeast Crabtree (+) model to study the relationships between the energy metabolism pathways during the implementation of the Warburg effect. The effect of glucose on S. cerevisiae energetic metabolism results initially in a kinetic inhibition of the oxidative metabolism (Crabtree effect). Rapidly after the addition of glucose, we found an accumulation of F1, 6bP. This induces a drastic reduction in the ratio G6P / F1,6bP. Moreover, it has been shown that G6P stimulates and F1,6bP inhibits the activity of the respiratory chain "in-vitro". Mutants and the modulation of this ratio allowed us to show that the induction of the Crabtree effect is due to a decrease in the G6P / F1,6bP ratio. In parallel with the implementation of the Crabtree effect, glucose induces a genetic rearrangement that leads to a Warburg effect. We showed a decrease over time of mitochondrial enzymatic equipment by dilution effect, due to a halt of mitochondrial biogenesis (transcriptional repression of HAP4). We have been able to show that this decrease in respiratory capacity has no effect on the cellular capacity for ATP synthesis. This is due to the increase in glycolytic ATP synthesis flux. Furthermore, the use of mutants where there is no repression of mitochondrial metabolism upon glucose addition allowed us to show that there is no simple link between OXPHOS activity and cell proliferation. i.e. Mitochondrial metabolism repression/high glycolytic flux is not mandatory to allow a rapid cell proliferation. This is a rare example where energetic metabolism and cell proliferation are uncoupled.

Introdução: A lesão de isquemia/reperfusão do fígado é caracterizada pelo agravamento da lesão isquêmica hepatocelular quando o órgão é revascularizado, podendo originar, nos casos mais graves, uma reação inflamatória sistêmica com lesão de órgãos à distância. O controle desse fenômeno é importante no transplante de fígado, nas cirurgias de ressecção hepática e no choque hemorrágico. A administração de soluções salinas hipertônicas têm se mostrado eficaz no tratamento do choque hemorrágico, pois melhora as alterações hemodinâmicas e, possivelmente, apresenta uma ação antiinflamatória. Neste trabalho foram avaliados os efeitos locais e sistêmicos da administração da solução salina hipertônica na lesão de isquemia/reperfusão hepática em ratos. Métodos: Enquanto 14 ratos Wistar machos, dos 56 utilizados no estudo, compuseram o grupo controle, grupo C; os demais, que foram submetidos à uma hora de isquemia hepática e 4 horas de reperfusão, compuseram outros grupos de 14 animais: o grupo ST, animais que não receberam tratamento; o grupo SSF, animais que receberam 34 mL/kg de NaCl 0,9%, por via endovenosa, 15 minutos antes da reperfusão; o grupo SSH, animais que receberam 4 mL/kg de NaCl 7,5%, 15 minutos antes da reperfusão. Após 4 horas de reperfusão, os materiais foram coletados para análise. Foram realizadas as dosagens das transaminases AST e ALT, a avaliação das funções oxidativas e fosforilativas mitocondriais, a dosagem das interleucinas IL-6 e IL-10, as análises teciduais pulmonares e a análise histológica do fígado isquêmico e não isquêmico. Resultados: Quando comparado aos grupos ST e SSF, o grupo SSH apresentou elevação dos níveis de AST e ALT significantemente menores; preservação da função mitocondrial tanto dos lobos isquêmicos, como dos não isquêmicos, significantemente melhor; elevação dos níveis de IL-6 e IL-10 sem significância estatística; aumento da permeabilidade vascular pulmonar significantemente menor; e elevação da atividade da mieloperoxidase pulmonar sem significância estatística. Em relação à análise histológica da lesão de isquemia/reperfusão hepática, o escore da lesão do grupo SSH foi significantemente menor que o da lesão do grupo C; entretanto, quando comparados os três grupos submetidos à isquemia hepática ST, SSH e SSF não se observaram diferenças significantes estatisticamente. Conclusão: A administração da solução salina hipertônica a 7,5% na isquemia/reperfusão hepática normotérmica melhorou as lesões hepáticas locais e as lesões à distância, principalmente pulmonares / Introduction: During liver ischemia, the drop in mitochondrial energy production leads to cellular damage, which is aggravated during restoration of blood supply. Besides local hepatic injury, the ischemia/reperfusion process can trigger a systemic inflammatory syndrome producing remote organ damage. To control these alterations in clinical conditions like liver transplantation, liver resections and hypovolemic shock, is crucial to achieve proper patient management. Aim: To evaluate the effect of the sodium chloride hypertonic solution on prevention of local and systemic injury during partial liver ischemia/reperfusion. Methods: Animals underwent partial warm liver ischemia/reperfusion. Fity six Wistar male rats were randomly allocated into four groups. Fourteen animals were submitted to sham operation and allocated to C group; 42, submitted to one hour of liver ischemia followed by 4 hours of reperfusion, were allocated in three additional groups: ST group, 14 animals that received no treatment; SSF group, 14 animals that received NaCl 0.9%, 34 mL/kg, intravenously; SSH group, animals that received NaCl 7.5%, 4 mL/kg, intravenously. Blood and tissue samples were collected four hours after reperfusion, when animals were killed. Blood samples were collected to determinate AST, ALT, IL-6 and IL-10 levels. Liver and pulmonary tissues were assembled for liver histology and for liver mitochondrial phosphorylation, pulmonary vascular permeability and myeloperoxidase analyzes. Results: Hypertonic saline solution showed beneficial effects in the treatment of liver ischemia/reperfusion injury. SSH group presented elevation of AST and ALT plasma levels significantly lower than ST and SSF groups. A significant reduction on mitochondrial dysfunction was observed in SSH group compared with ST and SSH groups. Elevation in serum IL-6 and IL-10 was similar among ST, SSF and SSH groups. Pulmonary vascular permeability was significantly lower in group SSH compared with ST and SSF groups. No differences in myeloperoxidase activity were observed among these three groups. Histological score for liver ischemia/reperfusion injury was significantly lower in SSH group compared to ST group, however no differences were observed between SSH and SSF groups. Conclusion: Administration of hypertonic saline solution in an experimental rat model of liver ischemia-reperfusion ameliorated local and systemic injuries

Dans un contexte environnemental anthropisé, fragmenté et soumis à un changement climatique rapide, l’appréhension des processus d'adaptation locale des organismes marins par l'étude de zones de contact entre taxa proches constitue une approche privilégiée. Dans ces zones, des génotypes hybrides persistent malgré un état de maladaptation liée à des incompatibilités génétiques endogènes et/ou des barrières exogènes. L'histoire biogéographique complexe de la telline baltique Macoma balthica fait émerger quatre zones hybrides européennes, dont l'une, localisée autour de la Pointe Finistère (France), est le résultat d’un contact entre deux stocks génétiques ayant divergé en allopatrie. Ces divergences sont susceptibles de rompre la coadaptation entre génomes nucléaire et mitochondrial en raison de l'émergence d'incompatibilités mitonucléaires (MNIs). Ainsi, les sous-unités protéiques des cinq complexes de la chaine OXPHO sont codées à la fois par des gènes nucléaires et mitochondriaux, et une coévolution inter-génomique étroite est requise pour maintenir la production énergétique cellulaire. De précédentes données de transcriptomique dévoilent de probables MNIs chez M. balthica au niveau des complexes respiratoires I et V. Afin d’apporter des éléments de compréhension aux mécanismes de maintien des zones hybrides dans un contexte de pression anthropique, le présent travail se propose de tester l'hypothèse de putatives MNIs dans cette zone de contact. Pour cela, (i) six mitogénomes correspondant à cinq lignées haplotypiques divergentes en Europe ont été séquencés et l'architecture génomique a été étudiée conjointement à une cartographie des mutations des 13 gènes mitochondriaux, (ii) le niveau de transcription de 5 gènes nucléaires et 8 gènes mitochondriaux (complexe I à V) des individus hybrides a été comparé à celui des lignées parentales après détermination du statut d'hybridation de chaque individu (six populations françaises). A défaut d'apporter des éléments de réponses concrets quant à l'existence de MNIs chez M. balthica, et ses répercussions évolutives en terme de dépression d'hybridation, ce travail constitue un tremplin vers une étude approfondie de la zone hybride française en développant de nouveaux outils moléculaires, et de solides techniques expérimentales pour la conduite de futurs croisements artificiels. / In the anthropized, fragmented environmental context subject to rapid climate change, understand local adaptation processes of marine organisms by studying the contact zones between close taxa is a preferred approach. In these areas, hybrid genotypes persist despite a maladaptive state related to endogenous genetic incompatibilities and/or exogenous barrier. The complex biogeographic history of the Baltic tellin Macoma balthica leads to emergence of several European hybrid zones, one of which, located around the Pointe Finistère (France), is the result of the confluence of two genetic stocks that have diverged in allopatric. These divergences may have lead to the co-adaptation breakdown between nuclear and mitochondrial genomes due to the emergence of mito-nuclear incompatibilities (MNIs). Thus, the different protein subunits of the five OXPHO chain complexes are encoded by both nuclear and mitochondrial genes, and a tight inter-genomic coevolution is required to maintain the cellular energy production. Recent transcriptomic data unveil the existence of underlying MNIs in M. balthica, bearing by the respiratory complexes I and V. In order to provide some understanding clues of mechanisms in hybrid zone's maintenance in the context of human pressure, the present work aims to test the assumption of putative MNIs in this area. For this purpose, (i) six mitogenomes corresponding to five divergent haplotype lineages in Europe were sequenced and the genomic architecture has been studied jointly to a mapping mutation of the 13 mitochondrial genes, (ii) the level of transcription of 5 nuclear and 8 mitochondrial genes (complex I to V) of hybrid individuals was compared to the parental lineages after identification of the hybrid status of each individual (six french populations). For the lack of bringing concrete answers concerning the existence of MNIs in M. balthica, and its evolutionary effects in term of hybrid breakdown, this work is a springboard for a comprehensive study of the French hybrid zone by developing new molecular tools, and stable experimental technics for the realisation of future artificial crosses.

Mitochondrial disorders, with incidence 1:5000 live births children, are one of the most common metabolic diseases. Clinically, it is heterogeneous group of disorders caused by mutations in more than 250 genes. Diagnostic of patients with suspected mitochondrial disorder relies on broad spectrum of biochemical analysis. One of them is a measurement of Mitochondrial Energy Generating Capacity (MEGC). The principle of MEGC analysis is measuring oxidations rate of 14 C - labeled substrates in 10 different incubations. These incubations contain [1-14 C]pyruvate, [U-14 C]malate or [1,4-14 C]succinate, donors and acceptors of Acetyl-CoA and inhibitors of TCA cycle. The results of MEGC analysis provide a variety of information about mitochondrial energy metabolism (MEM) of individual in particular tissue. In diagnostic of patients with suspected mitochondrial disorder is MEGC routinely determined in skeletal muscle. The aim of this study is to optimize MEGC analysis for its use in cultures skin fibroblasts. In sum, MEGC analysis was performed in 23 patients with primary deficiency of oxidative phosphorylation (OXPHOS), in 7 patients with secondary deficiency of OXPHOS and in 15 controls cell lines. The results of MEGC in cultured skin fibroblasts were then compared with results of spectrophotometric...

Abstract NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13–14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function. Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G>A and m.3865A>G mutations and the ND6-affecting m.14498T>C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype. Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G>A and m.14498T>C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A>G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1. The results corroborate the pathogenicity of the m.14498T>C and m.3376G>A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme. / Tiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen.

Tout d’abord considérés comme simples composants d’une barrière imperméable, il a été démontré que les lipides membranaires auraient en fait un rôle biologique bien plus important, pouvant modifier l’environnement des enzymes membranaires et moduler l’activité de ces dernières. Dans la thèse présentée ici, ces relations ont été étudiées dans les mitochondries de l’huître creuse Crassostrea gigas. Les bivalves subissent d’importants changements environnementaux et l’adaptation à ces changements peut passer par un remodelage des membranes, ce qui fait de ces animaux des modèles intéressants pour les études des relations entre la structure des membranes et les activités d’enzymes associées. Des huîtres ont été nourries en écloserie avec deux régimes d’algues monospécifiques, T-Iso et Chaetoceros gracilis, et un mélange équilibré de ces deux algues. Malgré d’importantes modifications de composition en acides gras induites par les différents régimes alimentaires, une grande stabilité des processus membranaires mitochondriaux a été observée. D’un autre côté, la comparaison entre des huîtres élevées en écloserie et des huîtres élevées dans leur milieu naturel a révélé d’importantes modifications de capacités mitochondriales, qui pourraient être liées à une modulation des classes de phospholipides et de leur insaturation. Ces différences ne peuvent pas s’expliquer par une influence des cycles tidaux dans la mesure où, malgré un changement de production d’ATP, l’activité des mitochondries a été montrée comme étant similaire chez les huîtres collectées en émersion et en immersion. L’homéostasie mitochondriale observée dans cette étude pourrait être un moyen pour les huîtres de faire face aux variations biotiques (disponibilité en nourriture) et abiotiques (disponibilité en oxygène) de l’environnement naturel de C. gigas, et de maintenir leurs fonctions physiologiques malgré ces variations. / First considered as simple components of an impermeable barrier, it has been shown that membrane lipids would have a more important biological role. These lipids could modify the environment of membrane enzymes and modulate their activity. In this thesis, these relationships have been studied in mitochondria of the oyster Crassostrea gigas. Bivalves undergo major environmental changes and adaptation to these changes may require a membrane remodelling, which makes these animals interesting models to study the relationship between membrane structure and membrane processes. In this study oysters were fed in hatchery with two monospecific algal diets, T-Iso and Chaetoceros gracilis, and an equilibrated mix of both algae. Despite significant changes in fatty acid composition induced by these diets, mitochondrial capacities remained stable. On the other hand, the comparison between hatchery-reared oysters and oysters reared in their natural environment revealed significant changes in mitochondrial capacity, which could be related to modulation of phospholipid class composition and unsaturation. These differences can not be explained by the influence of tidal cycles. Indeed, despite a change in ATP production, mitochondrial activity was shown to be similar in oysters collected during emersion and immersion.Mitochondrial homeostasis observed in this study could be a way for oysters to cope with biotic (food availability) and abiotic (oxygen availability) variations in the natural environment of C. gigas, and to maintain their physiological functions despite these variations.

Le récepteur aux cannabinoïdes de type 1 (CB1) est un récepteur couplé aux protéines G, abondamment exprimé dans le cerveau et régulant plusieurs processus physiologiques. Cependant, les mécanismes cellulaires par lesquels les CB1 régulent ces processus n’ont été que peu analysés. Bien que les CB1 localisés dans les membranes plasmiques sont connus pour induire la transduction de signal; une partie de ces récepteurs sont aussi fonctionnels au niveau des mitochondries (mtCB1), où leur stimulation réduit la respiration mitochondriale. L’objectif de cette thèse fut d’évaluer l’impact de l’activation des récepteurs mtCB1 du cerveau sur les effets connus des cannabinoïdes. Afin de distinguer la fonction des mtCB1 de celle des autres populations de récepteurs, nous avons développé des outils basés sur la signalisation induite par les mtCB1. Dans les mitochondries isolées de cerveau, l’activation des protéines Gαi/o, dépendante des mtCB1 diminue l’activité de l’adénylyl cyclase soluble (sAC). L'inhibition locale de l’activité de sAC prévient l’amnésie, la catalepsie et partiellement l’hypolocomotion induite par les cannabinoïdes. De plus, nous avons généré une protéine fonctionnelle mutante CB1 (DN22-CB1) dépourvue des 22 premiers acides aminés des CB1 ainsi que de sa localisation mitochondriale. Contrairement aux CB1, l'activation des DN22-CB1 n’affecte pas l'activité mitochondriale. Enfin, l’expression des DN22-CB1 dans l’hippocampe bloque à la fois la diminution de la transmission synaptique et l’amnésie induites par les cannabinoïdes. Ces travaux démontrent l’implication des mtCB1 dans certains effets des cannabinoïdes et le rôle clé des processus bioénergétiques contrôlant les fonctions cérébrales. / Type-1 cannabinoid receptor CB1 is a G protein-coupled receptor (GPCR), widely expressed in the brain, which regulates numerous physiological processes. However, the cellular mechanisms of CB1-mediated control of these functions are poorly understood. Although CB1 are known to signal at the plasma membrane, a portion of these receptors are also present in mitochondria (mtCB1), where mtCB1 activation decreases mitochondrial activity. The goal of this thesis was to dissect the impact of brain mtCB1 signaling in known behavioral effects induced by cannabinoids. To distinguish the functions of mtCB1 from other receptor pools, we developed tools based on the characterization of the intra-mitochondrial molecular cascade induced by mtCB1 receptors. In isolated brain mitochondria, we found that intra-mitochondrial decrease of soluble-adenylyl cyclase (sAC) activity links mtCB1- dependent activation of Gαi/o proteins to decrease cellular respiration. Local brain inhibition of sAC activity blocks cannabinoid-induced amnesia, catalepsy and contributes to the hypolocomotor effect of cannabinoids. In addition, we generated a functional mutant CB1 protein (DN22-CB1) lacking the first 22 amino acid of CB1 and its mitochondrial localization. Differently from CB1, activation of DN22-CB1 does not affect mitochondrial activity. Hippocampal in vivo expression of DN22-CB1 abolished both cannabinoid-induced impairment of synaptic transmission and amnesia in mice. Together, these studies couple mitochondrial activity to behavioral performances. The involvement of mtCB1 in the effects of cannabinoids on memory and motor control highlights the key role of bioenergetic processes as regulators of brain functions.