The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...

Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...

ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...

The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...

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.

The role of αPhe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.

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Mitochondria are cell organelles which play an essential role in the cell energy supply providing the universal high energetic molecule ATP which is used in numerous energy consuming processes. The core of the ATP production, oxidative phosphorylation (OXPHOS) consists of four enzyme complexes (respiratory chain) which establish, driven by redox reactions, a proton gradient over the inner mitochondrial membrane. The ATP-synthase uses this electrochemical gradient to phosphorylate ADP to ATP. Dysfunctioning of an OXPHOS complex can have severe consequences for the energy metabolism and cause rare but incurable dysfunctions in particular tissues with a high energy demand such as brain, heart, kidney and skeleton muscle. Moreover mitochondria are linked to widespread diseases like diabetes, cancer, Alzheimer and Parkinson. Further, reactive oxygen species which are a by-product of the respiratory chain, are supposed to play a crucial role in aging. The aim of this work is to provide a realistic model of OXPHOS which shall help understanding and predicting the interactions within the OXPHOS and how a local defect (enzyme deficiency or modification) is expressed globally in mitochondrial oxygen consumption and ATP synthesis. Therefore we chose a bottom-up approach. In a first step different types of rate equations were analyzed regarding their ability to describe the steady state kinetics of the isolated respiratory chain complexes in the absence of the proton gradient. Here Michaelis-Menten like rate equations were revealed to be appropriate for describing their behavior over a wide range of substrate and product concentrations. For the validation of the equations and the parameter estimation we have performed kinetic measurements on bovine heart submitochondrial particles. The next step consisted in the incorporation of the proton gradient into the rate equations, distributing its influence among the kinetic parameters such that reasonable rates were obtained in the range of physiological electrochemical potential differences. In the third step, these new individual kinetic rate expressions for the OXPHOS complexes were integrated in a global model of oxidative phosphorylation. The new model could fit interrelated data of oxygen consumption, the transmembrane potential and the redox state of electron carriers. Furthermore, flux inhibitor titration curves can be well reproduced, which validates its global responses to local effects. This model may be of great help to understand the increasingly recognized role of mitochondria in many cell processes and diseases as illustrated by some simulations proposed in this work. / Les mitochondries sont l’usine à énergie de la cellule. Elles synthétisent l’ATP à partir d’une succession de réactions d’oxydo-réduction catalysées par quatre complexes respiratoires qui forment la chaîne respiratoire. Avec la machinerie de synthèse d’ATP l’ensemble constitue les oxydations phosphorylantes (OXPHOS). Le but de ce travail est de bâtir un modèle des OXPHOS basé sur des équations de vitesse simples mais thermodynamiquement correctes, représentant l’activité des complexes de la chaîne respiratoire (équations de type Michaelis- Menten). Les paramètres cinétiques de ces équations sont identifiés en utilisant les cinétiques expérimentales de ces complexes respiratoires réalisées en absence de gradient de proton. La phase la plus délicate de ce travail a résidé dans l’introduction du gradient de protons dans ces équations. Nous avons trouvé que la meilleure manière était de distribuer l’effet du gradient de proton sous forme d’une loi exponentielle sur l’ensemble des paramètres, Vmax et Km pour les substrats et les produits. De cette manière, j’ai montré qu’il était possible de représenter les variations d’oxygène, de ΔΨ et de ΔpH trouvés dans la littérature. De plus, contrairement aux autres modèles, il fut possible de simuler les courbes de seuil observées expérimentalement lors de la titration du flux de respiration par l’inhibiteur d’un complexe respiratoire donné.Ce modèle pourra présenter un très grand intérêt pour comprendre le rôle de mieux en mieux reconnu des mitochondries dans de nombreux processus cellulaires, tels que la production d’espèces réactives de l’oxygène, le vieillissement, le diabète, le cancer, les pathologies mitochondriales etc. comme l’illustrent un certain nombre de prédictions présentées dans ce travail.

Adaptation to chronic hypoxia provides cardioprotective effects. Molecular mechanism of this phenomenon is not yet completely understood, but it is known that cardiac mitochondria play an essential role in induction of protective effects. The purpose of this diploma thesis is to study effects of continuous normobaric hypoxia (CNH; 10 % O2, 21 days) on spontaneously hypertensive rats (SHR) and conplastic strain that is derived from SHR. These animals have nuclear genome of SHR strain and mitochondrial genome of Brown Norway (BN) strain. Cardiac homogenate was used to measure enzymatic activity of malate dehydrogenase (MDH), citrate synthase (CS), NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase and cytochrome oxidase (COX). Using Western blot procedure the protein amount of antioxidant enzymes was measured - manganese superoxide dismutase and copper-zinc superoxide dismutase (MnSOD, Cu/ZnSOD), catalase and chosen subunits of oxidative phosphorylation complexes (Ndufa9, Sdha, Uqcrc2, COX-4, MTCO1, Atp5a1). Under normoxic conditions the conplastic strain has lower amount of complex IV subunit MTCO1 in comparison with SHR. This subunit is encoded by mitochondrial DNA and it is one of the seven protein-coding genes in conplastic strain that differ from SHR. Adaptation to hypoxia causes an...