Despite over a century of advancement, tuberculosis remains a grave threat to world health. In particular, third world countries continue to struggle with the crushing weight of the disease. Furthermore, the emergence of drug resistance in TB strains poses a significant threat to the first world where incidence and mortality is low. The dwindling efficacy of current drug regimens necessitates research into new small molecules capable of arresting the growth and spread of TB. The capuramycin family of nucleoside antibiotics shows strong potential to become part of this new generation of anti-TB small molecules. Indeed, their ability to inhibit Translocase I, a key enzyme in the biosynthesis of bacterial cell walls, makes them exciting targets for medicinal chemistry efforts. The synthesis of the family focused on dividing the molecules into three congruent, synthetically separate parts: the variable amide linked tail, the hexauronic acid linker, and the uridine "head". Construction of the ubiquitous core structure comprised of the hexauronic acid and uridine would allow rapid diversification while the variable tail would allow SAR studies and development of novel new members of the family.

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Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...

La découverte de nouveaux agents antipaludiques est primordiale. A travers le monde, les chercheurs se sont focalisés sur plusieurs stratégies. Les plus développées sont : soit les tests de molécules issues de bibliothèques chimiques dans une recherche phénotypique (comme le test direct d’agents sur des cultures de parasites in vitro), soit la recherche de nouvelles molécules agissant sur l’activité d’une cible ou d’une voie spécifique et essentielle. Cette thèse est centrée sur le second type d’approche. Nous nous sommes intéressés aux transporteurs membranaires de P. falciparum. Pour cela, nous exprimons les protéines d’intérêt dans la levure et nous les purifions. Nous optimisons les tests fonctionnels, dans le but de : a) déterminer l’effet des molécules sur les cibles spécifiques ; b) tester leur effet sur les cultures d’érythrocytes infectés par P. falciparum in vitro ; c) vérifier leur toxicité sur des cellules de mammifères ; et d) réaliser le test des molécules les plus efficaces in vivo dans un modèle de paludisme murin. Notre travail actuel est focalisé sur l’ATPase6 de P. falciparum (PfATP6) et l’adénylate translocase (PfAdT), deux protéines membranaires essentielles localisées respectivement sur le réticulum endoplasmique et la membrane mitochondriale. Nous exprimons de manière hétérologue PfATP6 dans les membranes de levure, nous purifions la protéine et mesurons une activité ATPase spécifique. Nous avons ainsi pu tester une bibliothèque chimique importante et identifier des inhibiteurs spécifiques. Ces derniers ont ensuite été testés pour évaluer leur effet sur les stades érythrocytaires du parasite in vitro et leur cytotoxicité sur des cellules de mammifères. Pour le transporteur PfAdT, nous procédons comme pour PfATP6, mais nous avons choisi un autre type de test fonctionnel dans lequel la protéine est directement exprimée sur la membrane plasmique d’E. coli. Cela devrait permettre de mesurer le transport d’ATP radiomarqué, et l’identification d’inhibiteurs spécifiques dont les effets pourront être évalués sur des cultures de parasites in vitro et dans des essais de cytotoxicité. / New drug discovery for malaria treatment urges, now more than ever. There is no optimal solution to the search for new antimalarials. Worldwide, researchers have focused their energies on several strategies. The most commonly employed are: either by screening molecules issued from chemical libraries in a phenotypic way (i.e., direct testing of drugs on in vitro parasite cultures), or by searching for new molecules acting upon the activity of a specific essential target or pathway. This PhD thesis centers on the second type of approach. We are interested in targeting membrane transporters of P. falciparum. For this, we plan to express proteins of interest in yeast and proceed to their isolation. With optimized functional tests, we aim to: a) Determine the effect of molecules upon specific targets; b) Test their effect on P. falciparum in vitro erythrocytes cultures; c) As well as verify their toxicity on mammalian cells; and d) Perform in vivo testing of the best molecules on a rodent model for malaria. Our actual work is focused on the P. falciparum Ca2+ - ATPase 6 (PfATP6) and adenylate translocase (PfAdT), two essential membrane proteins localized on the endoplasmic-reticulum and the mitochondrial membrane, respectively. We were able to express heterologously PfATP6 in yeast membranes, purify the protein and measure a specific ATPase activity. With this, we have tested a large chemical library and identified specific inhibitors. These were then tested for their effect on in vitro blood stages of P. falciparum and for their cytotoxicity on mammalian cells. For the ATP/ADP carrier PfAdT, we proceeded as previously done with PfATP6 but we have also chosen another type of functional test where we express directly this protein in the plasma membrane of E. coli. This will enable in the future the measurement of radiolabelled ATP uptake, and the identification of specific inhibitors that could then be tested for their effect on P. falciparum in vitro cultures and for their cytotoxicity.

Long-chain fatty acids (LCFA) are the primary energy source in the myocardium and an imbalance in the LCFA and glucose utilization could cause cardiovascular diseases. More than 50% of LCFA uptake by the heart is mediated by the fatty acid translocase CD36 and disruption of its function has been shown to impair cardiovascular functions. The spontaneously hypertensive rat (SHR) harbors a deletion variant of the Cd36 gene that results in reduced LCFA transport into myocytes. Therefore, the main aim of this thesis was to investigate the importance of a functional CD36 to sustain normal physiological functions of the heart. We used SHR and two genetic modified SHR strains, the congenic SHR-4 and the transgenic SHR-Cd36, with fully functional CD36. They differ in the CD36 expression and in the manner how they were derived from the SHR. CD36 has been proven to play a role in the pathogenesis of insulin resistance. Therefore we analyzed the effect of a functional CD36 on insulin resistance and protein kinase C (PKC) expression, which is known to be involved in the mechanism of insulin resistance, in the heart of SHR-4 and SHR. We showed that the SHR-4 had lower serum free fatty acids (FFA) and triacylglycerols (TAG) concentrations, indicating improved insulin sensitivity. Furthermore, SHR-4 had increased...

Die vorliegende Arbeit beschäftigt sich mit den molekularen Prozessen der Lipidanreicherung in der Gallenflüssigkeit. Leberzellen (Hepatozyten) sind polare Zellen, die für die Sekretion der Gallenflüssigkeit verantwortlich sind. Die Anbindung an den Blutkreislauf besteht über die basolaterale Membran. Durch die gegenüberliegende, sogenannte apikale Membran werden zwischen benachbarten Leberzellen tubuläre Stukturen (bile canaliculi, BC) gebildet, in die die Gallenflüssigkeit abgesondert wird. Daher wird diese Membran auch als Canalicularmembran (CM) bezeichnet. Die Gallenflüssigkeit besitzt hinsichtlich ihrer Lipidzusammensetzung eine bemerkenswerte Spezifität. Obwohl der Anteil von Phosphatidylcholin (PC) an den Phospholipiden der CM nur 35% beträgt, macht es 95% der Phospholipide der Gallenflüssigkeit aus. Mögliche Mechanismen, die zur Spezifität der Lipidsekretion in die Gallenflüssigkeit führen, werden untersucht und diskutiert. Phospholipide werden aus der äußeren Lamelle der CM durch Gallensalze herausgelöst. Die Wechselwirkung von Gallensalzen mit Phospholipiden ist kopfgruppenunspezifisch. Eine Solubilisierung von Phosphatidylserin (PS) und Phosphatidylethanolamin (PE) durch Gallensalze könnte durch die Wirkung einer Aminophospholipidtranslokase (APLT) verhindert werden, die diese Lipide aktiv auf die zytoplasmatische Seite der Membran pumpt. Zur Überprüfung dieser Hypothese wurden Versuche durchgeführt, um die Aktivität einer APLT in der CM nachzuweisen. Dabei wurde die Hepatomazelllinie HepG2 eingesetzt, die in der Lage ist, Canalicularvakuolen (BC) zu bilden. Zunächst wurde die Einwärtsbewegung einer Reihe fluoreszierender Lipidanaloga mit unterschiedlicher Affinität zur APLT charakterisiert. Dies geschah an der basolateralen Membran von HepG2 Zellen, wo eine APLT-Aktivität bereits bekannt ist. Die Aufnahme geeigneter APLT-Substrate konnte durch den APLT-Inhibitor Suramin reduziert werden. Ebenso wurde die Affinität eines Paares von PS-Analoga bestätigt, von denen Diether PS ein "schlechtes" und Diacyl PS ein "gutes" APLT-Substrat darstellt. Im zweiten Schritt wurde die Anreicherung der gleichen Analoga in BC von HepG2 Zellen untersucht. Es ergab sich eine auffallende Korrelation zwischen einer APLT vermittelten Aufnahme von Phospholipidanaloga an der basolateralen Membran und dem Fehlen dieser Analoga im Lumen der BC. Wenn Zellen mit Phospholipiden markiert wurden, die keine oder nur "schlechte" APLT-Substrate darstellen, erschienen die BC stark fluoreszierend. Diese Beobachtungen zeigen, dass eine APLT-Aktivität in der CM von Hepatozyten vorhanden ist, welche das Fehlen der Aminophospholipide in der Gallenflüssigkeit erklärt. Ein zweiter Schwerpunkt dieser Arbeit war die Untersuchung der Rolle von MDR-Proteinen (wie MDR3) bei der Lipidsekretion in die Gallenflüssigkeit. Aufgrund bisheriger Arbeiten wird vermutet, dass MDR3 daran als spezifischer Membrantransporter für PC beteiligt ist. In der vorliegenden Arbeit konnte jedoch gezeigt werden, dass verschiedene MDR-Inhibitoren die Anreicherung fluoreszierender Phospholipidanaloga in den BC von HepG2 Zellen nur wenig reduzieren. Diese Beobachtung kann unter der Annahme erklärt werden, dass MDR3 eher für die Exposition von PC an der lumenalen Seite der CM verantwortlich ist, als für den Tranport von PC über die Membran. Solche "Liftase"-Aktivität von MDR3 könnte endogenes PC der Detergenzwirkung von Gallensalzen zugänglich machen, ein Prozess, der für die hydrophileren fluoreszierenden PC-Analoga nicht nötig ist. Im dritten Teil wird die Rolle von Sphingolipiden und die Bildung von "Rafts" in der CM behandelt. Solche Membrandomänen sollten die Solubilisierung von Spingolipiden in die Gallenflüssigkeit verhindern. Eine Anreicherung fluoreszierender Sphingolipidanaloga in den BC wurde jedoch nachgewiesen, was darauf hindeutet, dass die verwendeten Analoga das Verhalten endogener Sphingolipide in der CM nicht korrekt wiederspiegeln. Im abschließenden Teil dieser Arbeit wurden die Grundlagen für eine Methode zur Aufklärung der physikochemischen Prozesse der Lipidsekretion an der Canalicularmembran gelegt. Die starke Umgebungsabhängigkeit der Fluoreszenzlebensdauer für verschiedene fluoreszierende Lipidanaloga wurde in einer Reihe von Modellumgebungen analysiert und deren Nutzbarkeit für die Vorhersage der Lipidorganisation geprüft. Insbesondere wurde die Wechselwirkung verschiedener Gallensalze mit Lipidanaloga und der Fluoreszenzresonanzenergietransfer zwischen verschiedenen Lipidanaloga charakterisiert. Diese Daten sind Ausgangsbasis für die mikroskopische Charakterisierung der Organisation von Lipidanaloga in den BC in vivo. / This thesis addresses the molecular processes which are important in the formation of bile fluid. The polar liver cells (hepatocytes) secrete the bile fluid at their apical (canalicular) membrane into tubular bile canaliculi (BC) which are formed between adjacent cells. The basolateral membrane of hepatocytes faces the blood vessel. Bile fluid possesses a remarkable specificity regarding its lipid composition. Even though phosphatidylcholine (PC) contributes to only 35% of the phospholipids in the canalicular membrane, it constitutes 95% of biliary phospholipids. In this thesis possible mechanism that might lead to the specificity in biliary lipid secretion are analysed and discussed. Phospholipids are secreted from the outer leaflet of the canalicular membrane into bile by the effect of bile salts. The interaction of bile salts with phospholipids was shown to be independent of the phospholipid headgroup. Solubilisation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by bile salts could be prevented by the action of an aminophospholipid translocase (APLT) which actively pumps these lipids to the cytoplasmic leaflet of the membrane. Experiments to demonstrate a canalicular APLT activity were performed to proof this hypothesis. For this, the hepatoma cell line HepG2 which is able to polarise and to form a canalicular vacuole (BC) was utilised. A panel of fluorescent lipid analogues with different affinities to this transporter was used and first characterised at the basolateral membrane of HepG2 cells, where an APLT activity was already demonstrated. The rapid APLT mediated uptake of aminophospholipid analogues representing appropriate substrates of APLT was reduced by applying the inhibitor suramin. The affinity of a pair of PS analogues with diether NBD-PS as a poor APLT substrate and diacyl NBD-PS representing a suitable substrate was confirmed. In a next step the enrichment of the same phospholipid analogues in the BC was investigated. There was a striking correlation between APLT mediated uptake of phospholipid analogues at the basolateral membrane and absence of these analogues from the BC. In the case of phospholipid analogues that were no or poor substrates of APLT the BC appeared highly fluorescent, indicating that indeed a canalicular APLT is responsible and sufficient for biliary absence of aminophospholipids. Further experiments were aimed on the investigation of the role of MDR proteins (as MDR3) in biliary lipid secretion. It has been proposed that MDR3, which is crucial for biliary phospholipid secretion, acts as a specific flippase for PC. However, different MDR inhibitors did not completely abolish the enrichment of fluorescent phospholipid analogues in the BC in this study. This observation can be explained assuming that MDR3 is responsible for the exposure of PC at the lumenal side of the canalicular membrane rather than for its transport across the membrane. Such a "liftase" activity of MDR could make endogenous PC accessible to the detergent bile salts which is not necessary for its more hydrophilic fluorescent analogues. The third part of this thesis addressed the role of sphingolipids and the formation of detergent resistant rafts in the canalicular membrane. Rafts are thought to prevent sphingolipid solubilisation into bile. Fluorescent sphingolipid analogues were found to enrich in the BC even at low temperatures, however. These experiments suggest that the applied analogues might not suitably represent the majority of sphingolipids in the canalicular membrane. The final part of this study provides the basis for a method to investigate the physico-chemical processes occurring during lipid secretion at the canalicular membrane. The sensitivity of fluorescence life times on environmental changes was analysed using fluorescent lipid analogues in a set of model environments and its utility for predicting biliary lipid organisation is discussed. Especially the interaction of different bile salts with lipid analogues and fluorescence energy transfer between distinct lipid analogues was characterised. These data can be utilised for characterisation of the organisation of biliary enriched lipid analogues in vivo at a microscopic level in future.

A oxidação do resíduo de cisteína 56 (ANT-cys56) do transportador de nucleotídeos de adenina (ANT) é descrita como evento crítico da Transição de Permeabilidade Mitocondrial (TPM), fenômeno caracterizado pela sensibilidade ao fármaco imunossupressor ciclosporina A (CsA), responsável pela ligação e inibição do componente promotor da abertura do Poro de Transição de Permeabilidade (PTP), a enzima peptidil-prolil-cis trans isomerase (cyp D). Aspectos bioquímico-estruturais do ANT, das cardiolipinas (CDL) que envolvem o transportador e da cyp D na TPM foram avaliados por meio de ensaios turbidimétricos de inchamento mitocondrial e estado conformacional do ANT em mitocôndrias isoladas de fígado de rato, associados a abordagens de química computacional para análises de campos de interação molecular (MIF) e dinâmica molecular (MD), visando a predição de eventos envolvidos na abertura do PTP. As análises computacionais revelaram aumento da mobilidade relativa do ANT-cys56, como resultado da interação preferencial do Ca2+ com a molécula de CDL ligada à hélice 4 do transportador, enquanto que a inversão da configuração do resíduo de prolina do ANT (ANT-pro61) potencializou o efeito induzido por Ca2+. A presença de ADP no interior do ANT preveniu o aumento da mobilidade relativa do ANT-cys56 promovida pelo Ca2+, enquanto que a inversão da configuração do ANT-pro61, de trans para cis, potencializou o efeito promovido pelo Ca2+ na mobilidade relativa do ANT-cys56, de forma insensível ao nucleotídeo. Os ensaios com mitocôndrias isoladas demonstraram que o Ca2+ induz a conformação c do ANT e promove abertura do PTP, de forma sensível à CsA e ADP. A presença de cyp D estabilizou a conformação c do ANT induzida por Ca2+, sendo que Atractilosídeo (ATR) tornou o efeito parcialmente insensível aos inibidores da TPM. Os resultados sugerem que a abertura do PTP induzida por Ca2+ envolve a mudança conformacional do ANT para o estado c, cuja estabilização é obtida pela cyp D na função de inversão do ANT-pro61, com base na avaliação da mobilidade relativa do ANT-cys56 parcialmente sensível ao ADP. / Oxidation of the Adenine Nucleotide Translocase (ANT) cysteine residue 56 (ANT-cys56) is potentially involved in Ca2+-induced Mitochondrial Permeability Transition (MPT), a process which is prevented by cyclosporine A (CsA), due to its inhibition of Permeability Transition Pore (PTP) opener component, the peptidyl-prolyl cis-trans isomerase cyclophylin D (cyp D). The main aspects of ANT, cardiolipins (CDL) and cyp D on Ca2+-induced PTP opening were addressed by employing light scattering techniques in isolated rat liver mitochondria to assess both ANT conformational change and mitochondrial swelling in association with computational chemistry analysis of Molecular Interaction Fields (MIF) and Molecular Dynamics (MD) for PTP events predictions. Computational analysis revealed that Ca2+ interacts preferentially with the ANT surrounding CDL bound to the H4 helix of the carrier and weakens the CDL/ANT interactions accounting for the ADP-sensitive increase of ANT-cys56 relative mobility while ANT-pro61 cis to trans configuration inversion intensified the Ca2+ effect in a ADP-insensitive way. The ANT conformation and mitochondrial swelling analyses demonstrated that Ca2+ induces conformation c of ANT and opens PTP in a CsA- and ADP-sensitive way. Cyp D stabilizes Ca2+-induced ANT conformation c, whereas ATR renders a PTP opening less sensitive to the inhibition by CsA or ADP. The results suggest that Ca2+-induced PTP opening involves ANT conformation c change supported by a cyp D-induced trans to cys ANT-pro61 configuration inversion based on the relative mobility of ANT-cys56, in a ADP-sensitive manner.

EcoR124I is a Type I restrictionmodification (RM) enzyme and as such forms multifunctional pentameric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on the motor subunit HsdR. When non-methylated invading DNA is recognized by the complex, two HsdR endonuclease/motor subunits start to translocate dsDNA without strand separation activity up to thousands base pairs towards the stationary enzyme while consuming ~1 molecule of ATP per base pair advanced. Whenever translocation is stalled the HsdR subunits cleave the dsDNA nonspecifically far from recognition site. The X-ray crystal structure of HsdR of EcoR124I bound to ATP gave a first insight of structural/functional correlation in the HsdR subunit. The four domains within the subunit were found to be in a square planer arrangement. Computational modeling including molecular dynamics in combination with crystallography, point mutations, in vivo and in vitro assays reveals how interactions between these four domains contribute to ATP-dependent DNA translocation, DNA cleavage or inter-domain communication between the translocase and endonuclease activities.

A oxidação do resíduo de cisteína 56 (ANT-cys56) do transportador de nucleotídeos de adenina (ANT) é descrita como evento crítico da Transição de Permeabilidade Mitocondrial (TPM), fenômeno caracterizado pela sensibilidade ao fármaco imunossupressor ciclosporina A (CsA), responsável pela ligação e inibição do componente promotor da abertura do Poro de Transição de Permeabilidade (PTP), a enzima peptidil-prolil-cis trans isomerase (cyp D). Aspectos bioquímico-estruturais do ANT, das cardiolipinas (CDL) que envolvem o transportador e da cyp D na TPM foram avaliados por meio de ensaios turbidimétricos de inchamento mitocondrial e estado conformacional do ANT em mitocôndrias isoladas de fígado de rato, associados a abordagens de química computacional para análises de campos de interação molecular (MIF) e dinâmica molecular (MD), visando a predição de eventos envolvidos na abertura do PTP. As análises computacionais revelaram aumento da mobilidade relativa do ANT-cys56, como resultado da interação preferencial do Ca2+ com a molécula de CDL ligada à hélice 4 do transportador, enquanto que a inversão da configuração do resíduo de prolina do ANT (ANT-pro61) potencializou o efeito induzido por Ca2+. A presença de ADP no interior do ANT preveniu o aumento da mobilidade relativa do ANT-cys56 promovida pelo Ca2+, enquanto que a inversão da configuração do ANT-pro61, de trans para cis, potencializou o efeito promovido pelo Ca2+ na mobilidade relativa do ANT-cys56, de forma insensível ao nucleotídeo. Os ensaios com mitocôndrias isoladas demonstraram que o Ca2+ induz a conformação c do ANT e promove abertura do PTP, de forma sensível à CsA e ADP. A presença de cyp D estabilizou a conformação c do ANT induzida por Ca2+, sendo que Atractilosídeo (ATR) tornou o efeito parcialmente insensível aos inibidores da TPM. Os resultados sugerem que a abertura do PTP induzida por Ca2+ envolve a mudança conformacional do ANT para o estado c, cuja estabilização é obtida pela cyp D na função de inversão do ANT-pro61, com base na avaliação da mobilidade relativa do ANT-cys56 parcialmente sensível ao ADP. / Oxidation of the Adenine Nucleotide Translocase (ANT) cysteine residue 56 (ANT-cys56) is potentially involved in Ca2+-induced Mitochondrial Permeability Transition (MPT), a process which is prevented by cyclosporine A (CsA), due to its inhibition of Permeability Transition Pore (PTP) opener component, the peptidyl-prolyl cis-trans isomerase cyclophylin D (cyp D). The main aspects of ANT, cardiolipins (CDL) and cyp D on Ca2+-induced PTP opening were addressed by employing light scattering techniques in isolated rat liver mitochondria to assess both ANT conformational change and mitochondrial swelling in association with computational chemistry analysis of Molecular Interaction Fields (MIF) and Molecular Dynamics (MD) for PTP events predictions. Computational analysis revealed that Ca2+ interacts preferentially with the ANT surrounding CDL bound to the H4 helix of the carrier and weakens the CDL/ANT interactions accounting for the ADP-sensitive increase of ANT-cys56 relative mobility while ANT-pro61 cis to trans configuration inversion intensified the Ca2+ effect in a ADP-insensitive way. The ANT conformation and mitochondrial swelling analyses demonstrated that Ca2+ induces conformation c of ANT and opens PTP in a CsA- and ADP-sensitive way. Cyp D stabilizes Ca2+-induced ANT conformation c, whereas ATR renders a PTP opening less sensitive to the inhibition by CsA or ADP. The results suggest that Ca2+-induced PTP opening involves ANT conformation c change supported by a cyp D-induced trans to cys ANT-pro61 configuration inversion based on the relative mobility of ANT-cys56, in a ADP-sensitive manner.

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...