Mitochondrial function is critical for the survival of eukaryotes. Hence, mitochondrial dysfunctions are involved in numerous human diseases. An essential process for a normal mitochondrial function is mitochondrial gene expression which is tightly regulated in response to various physiological changes. The accurate control of mitochondrial gene expression is essential in order to provide the appropriate oxidative phosphorylation capacity for diverse metabolic demands. Recent findings in the basic mitochondrial replication and transcription regulation helped advance our understanding of organelle function and basic pathogenetic mechanisms of mitochondrial DNA mutations associated with oxidative phosphorylation defects. Mitochondrial transcription is regulated by the mitochondrial transcription termination factor (mTERF) both at the initiation and termination levels. A protein family containing highly conserved mTERF motifs has been identified recently and its members named generically as "terfins." In this work, one of these factors, mTERFD3, has been characterized in vitro and in vivo. The mTERFD3 protein is highly conserved throughout evolution. It is a mitochondrial protein localized to the matrix and is abundantly expressed in high energy demand tissues. We found that it contains 4 putative leucine zippers and is able to form dimers in vitro. We showed that mTERFD3 binds mtDNA at the transcription initiation site in the mtDNA regulatory region. These findings suggest that mTERFD3 may be involved in regulating mitochondrial gene expression at the transcriptional initiation level. In order to study the functional significance of mTERFD3 in vivo we developed a mouse deficient in mTERFD3 using a gene trapping strategy. The KO mice had a normal lifespan but showed decreased weight gain and decreased fat content in females. Fibroblasts isolated from KO mice displayed decreased growth rate when compared with WT in respiratory media, and had decreased complex IV activity. Consistent with the above findings, we found that muscle, one of the tissues with high energy demands, showed abnormal mitochondrial function, displaying features characteristic of mitochondrial myopathy such as decreased muscle strength and endurance. Muscle mitochondria of the KO mice showed a significant decrease in the complex II +III and complex IV activity. The decrease in OXPHOS complexes activity was associated with increased citrate synthase activity, suggesting mitochondrial proliferation, a feature typical for mitochondrial disorders. Another important finding was a decrease in the muscle mitochondrial transcripts in the KO animals associated with decreased steady state levels of OXPHOS subunits. Together these data suggest that mTERFD3 is a mitochondrial protein involved in the regulation of mtDNA transcription. mTERFD3 KO is not embryonic lethal suggesting that it is involved in the fine tuning of mitochondrial transcription. We conclude that mTERFD3 is a mitochondrial protein that modulates oxidative phosphorylation function, probably by directed interactions with the mtDNA regulatory region. This work shows the importance of mTERFD3, an mTERF family member, in the mitochondrial gene expression regulation.

Le complexe III de la chaine respiratoire mitochondriale (OXPHOS III) chez S. cerevisiae est assemblé à partir de dix sous-unités structurales codées par le génome soit nucléaire, soit mitochondrial et fait intervenir une douzaine de protéines extrinsèques au complexe. Nous avons étudié l’une d’entre elle, Bcs1, une ATPase oligomérique conservée de la famille des protéines AAA (ATPases Associated with diverse cellular Activities), qui contrôle la dernière étape de l’assemblage du complexe III. Chez l’Homme, des mutations dans l’orthologue de BCS1, BCS1L, sont associées à différentes maladies. Nous avons montré que des mutations dans les résidus conservés du domaine AAA de Bcs1 peuvent être compensées par des mutations dans les sous-unités de l’ATP synthase mitochondriale (OXPHOS V). Ces mutations compensatrices diminuent toutes l’activité d’hydrolyse de l’ATP de l’enzyme et nous avons proposé que la biogenèse du complexe III puisse être modulée selon l’état énergétique mitochondrial par Bcs1 via sa dépendance à l’ATP. Nous avons aussi identifié des mutations compensatrices dans d’autres gènes et le cas particulier de la délétion du RRF1, facteur général du recyclage des ribosomes mitochondriaux, a été étudié. Nous avons montré que l’absence de Rrf1 a un effet différent sur la stabilité et la traduction des divers ARNm mitochondriaux. Nos résultats suggèrent une coopération entre les facteurs généraux et les facteurs spécifiques de la traduction mitochondriale dans le contrôle de l’expression des sous-unités des complexes OXPHOS traduites dans la mitochondrie. / OXPHOS complexes are multi-subunit complexes embedded in the inner mitochondrial membrane. We have studied the assembly factor Bcs1 that is a membrane-bound AAA-ATPase, required for the assembly of complex III. Mutations in the human gene BCS1L are responsible for various mild to lethal pathologies. Extragenic compensatory mutations able to restore the assembly of complex III in yeast bcs1 mutants were found in different genes not directly connected to the complex, revealing new networks of protein interactions. Mutations in catalytic subunits of ATP synthase were identified and thoroughly characterized. This work has allowed us to propose a novel regulatory loop via the ATP-dependent activity of Bcs1 protein, connecting the production of mitochondrial complex III and the activity of the ATP synthase. Moreover, these results hold promise for the development of therapies, targeting the mitochondrial adenine nucleotide pool, in treatment of BCS1-based disorders. We also show that the absence of RRF1, a mitochondrial ribosome recycling factor, is able to compensate defects of bcs1 mutants. Deletion of RRF1 has a differential impact on the stability and translation of mitochondrial mRNAs. Our results suggest cooperation between general and specific translation factors in controlling the expression of mtDNA-encoded subunits of the OXPHOS complexes.

El genoma mitocondrial humano es una molécula circular de doble cadena de 16,5 kb. En su secuencia existe información para 13 polipéptidos de diferentes subunidades de los complejos de la cadena de transporte electrónico (CTE), para 22 ARNt y para 2 ARNr. Una mutación en cualquiera de estos genes puede provocar que la CTE no funcione correctamente, dando lugar a una disfunción del sistema de fosforilación oxidativa. Todo ello puede provocar por un lado un déficit de energía en las células o tejidos, o por otro lado un incremento de la producción de especies reactivas de oxígeno (ROS). Según la demanda energética de cada tejido este déficit de producción de energía será más o menos importante, pudiendo incluso provocar graves trastornos fisiopatológicos.El incremento de la producción de ROS por parte de la cadena de transporte electrónico puede ser eliminado con ayuda de las defensas antioxidantes celulares. Si la producción de ROS es más importante que la acción de estas defensas, ello puede llegar a provocar lesiones en diferentes componentes celulares tales como lípidos, proteínas o al propio ADNmt. Para profundizar en este campo, en este trabajo en primer lugar se han diagnosticado a cuatro pacientes con enfermedad mitocondrial, portadores de una mutación en su genoma mitocondrial. A partir de plaquetas de estos pacientes se han generado cíbridos transmitocondriales, que se han utilizado como modelo de estudio. Se han estudiado las siguientes mutaciones en genes mitocondriales: T14487C en la subunidad ND6 del complejo I, A3243G en el ARN de transferencia Leu (UUR), A8344G en el ARN de transferencia Lys y G6930A en la subunidad COXI del complejo IV. Analizando la producción de peróxido de hidrógeno como medida de la producción de ROS en estas cuatro líneas, hemos observado que las líneas portadoras de una mutación que afectase al funcionamiento del complejo I y III (descritos ampliamente en la literatura como principales productores de ROS en la mitocondria) es decir A3243G, A8344G y T144874C, sí provocan un incremento de la producción, mientras que la mutación que no afectaba a estos complejos (G6930A) no provocaba incremento. Posteriormente se ha estudiado si este incremento producía daño oxidativo a diferentes componentes celulares, tales como lípidos, proteínas y el propio ADNmt. Previamente, debido a que en la literatura no existía un consenso claro sobre el mejor método de análisis de la peroxidación lipídica, se realizó un pequeño estudio sobre cuál era el mejor inhibidor de la peroxidación lipídica a utilizar y en que concentración, obteniendo que el mejor a utilizar era el BHT a una concentración de 3mM.En cuanto los resultados de daño oxidativo se observó que en los lípidos solo se observaba daño oxidativo en la línea portadora de la mutación T144874C, mientras que las otras no lo presentaban. En la oxidación de proteínas no se observó daño en ninguna de las cuatro líneas portadoras de la mutación y en cuanto a la oxidación del ADNmt, se observó daño oxidativo en las líneas portadoras de las mutaciones A8344G y T14487C. Con estos resultados se observa que en algunas mutaciones en el genoma mitocondrial la producción de ROS generada es superior a la capacidad detoxificadora de la célula, provocando daño oxidativo, mientras que en otras la producción de ROS no supera la acción de las enzimas antioxidantes. / Mitochondrial encephalomyopathies caused by mutations in mitochondrial DNA (mtDNA) are a heterogeneous group of disorders characterized by primary dysfunction of the oxidative phosphorylation system (OXPHOS) with a decrease in ATP production. Clinical and biochemical heterogeneity of mitochondrial disorders is due to the ubiquitous nature of mitochondria and the dual genetic (mitochondrial and nuclear DNA) control of OXPHOS. Some unique features of mitochondrial genetics, such as heteroplasmy and tissue segregation, contribute to this phenomenon. However, the precise mechanisms leading to this heterogeneity are still largely unclear.Mitochondria are the major source of reactive oxygen species (ROS), which are generated as toxic by-products of redox-coupled reactions in the electron transport chain (ETC). Inhibition of the ETC in vitro using some respiratory complex inhibitors results in a significant increase in the mitochondrial production of ROS. This increase suggests that when dysfunction of the respiratory chain complexes occurs, electrons can be transferred directly to the molecular oxygen. However, cells are well protected by antioxidant enzymes: the manganese superoxide dismutase (Mn-SOD) and copper-Zinc superoxide dismutase (CuZn-SOD) to eliminate superoxide anion (O2.-) and the glutahione peroxidase (GSH-Px) and catalase (CAT) to eliminate hydrogen peroxide. Oxidative stress results when the balance of prooxidants and antioxidants is altered in favour of the prooxidants. In turn, an excess of ROS may contribute to OXPHOS damage. Thus, to define the relationship between mtDNA mutations and production of ROS, several transmitochondrial cell lines (cybrids) carrying different mutations in their mtDNA were obtained from different mitochondrial patients. These included two common and well characterized mtDNA mutations in tRNA genes, the A3243G transition in the tRNALeu(UUR) derived from a patient with MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), and the A8344G mutation in the tRNALys, derived from a patient with the MERRF syndrome (myoclonus epilepsy with ragged-red fibers). In addition, another two cybrids cell lines were studied, harbouring the G6930A mutation in the gene encoding the subunit I (COI) of the cytochrome c oxidase (COX). This mutation changes the amino acid glycine into a premature termination codon, resulting in the loss of the last 170 amino acids (33%) of the polypeptide, thus causing a complete disruption in the COX assembly. The last cybrid cell line studied carried the mutation T144874C in the subunit 6 of the complex I of the ETC.Hydrogen peroxide production was increased in cybrids harbouring tRNA and complex I mutations, but no changes were observed in cybrids harbouring the mutation in complex IV. No oxidative damage to lipids, proteins or mtDNA was detected in cybrids harbouring A3243G and G6930A mutations. In the cybrid cell line harbouring A8344G mutation, only oxidative damage to mtDNA was observed and in the cybrids harbouring the mutation in complex I, mtDNA and lipid oxidative damage were detected.These results suggest that some mutations in mtDNA may increase the production of hydrogen peroxide (i.e., those mutations which affect complex I or III of the ETC) meanwhile other mutations do not. Furthermore this increase can sometimes override the antioxidant defences of the cells and produce oxidative damage to key cellular components.

Mitochondria are a major site of reactive oxygen species (ROS) production in cells. While ROS can cause oxidative damage, they are vital in many signaling processes. Recently, mitochondrial superoxide flashes (mSOF) were defined through sensitive measurements of temporal and spatial differences in superoxide production. mSOF are stochastic events of quantal bursts in superoxide production, which are temporally linked to transient mitochondrial inner membrane depolarizations. The aims of the present study were to characterize a hydrogen peroxide sensitive biosensor to monitor these events and elucidate a role for uncoupling protein 3 (UCP3) and the mechanistic details of mSOF. While pHyPer- dmito was sensitive enough to monitor these dynamic changes its kinetics were insufficient to detect these ~20s long flashes. Additionally, analyses showed a prolonged duration of flashes in the absence of UCP3. Furthermore, we unearthed a novel relationship between flash amplitude and mitochondrial depolarization. Finally, investigations of mSOF in muscles of various fiber type compositions showed no differences, though additional investigations are warranted.

Un des rôles de la mitochondrie, qui possède son propre ADN (ADNmt), est la production de l'énergie nécessaire à la cellule, qu'elle synthétise sous forme d'ATP grâce aux oxydations phosphorylantes (OXPHOS). Ainsi, une altération du métabolisme énergétique mitochondrial peut provoquer l'apparition de pathologies mitochondriales dont, généralement, la sévérité est inversement proportionnelle à l'âge de début. De nombreuses études s'intéressent aux mécanismes d'apparition et de développement de ces maladies afin de mieux les comprendre et de pouvoir proposer des thérapies. Cependant, à ce jour, il est encore difficile de transformer l'ADNmt de façon ciblée (remaniement ou mutation). De plus, il existe encore peu de modèles animaux de pathologies mitochondriales qui permettraient de réaliser des études intégratives et d'essayer d'éventuelles molécules thérapeutiques. Dans le cadre de cette thèse, nous avons étudié deux types de modèles impliquant la modification du métabolisme mitochondrial. Dans un premier temps, nous nous sommes intéressés à la réalisation d'un modèle murin exprimant un grand nombre de caractéristiques du syndrome de Leigh, une maladie neurologique progressive. Pour cela nous avons utilisé une neurotoxine (MPTP) qui est connue pour sa toxicité envers les neurones dopaminergiques et aussi comme inhibiteur de la chaine respiratoire. Nous avons analysé l'activité de chaque complexe OXPHOS de différents tissus cérébraux et de tissus périphériques (cœur, foie, muscle et rein), prélevés sur des souris traitées et non-traitées. Nous avons retrouvé une inhibition des complexes III et/ou IV de la chaîne respiratoire dans le foie, le cortex, le striatum et le cervelet. Ces résultats, ajoutés à une neuro- dégénérescence accrue retrouvée dans une étude précédente, sont tous caractéristiques du syndrome de Leigh. Ces souris traitées par le MPTP semblent donc être un bon modèle pour l'étude de cette pathologie mitochondriale. Dans un second projet, nous nous sommes intéressés à l'effet des haplogroupes de l'ADNmt sur le métabolisme mitochondrial. En effet, bien qu'ils soient définis par des mutations neutres de l'ADNmt (polymorphismes), plusieurs études ont démontré des associations entre les haplogroupes et les pathologies, suggérant que les haplogroupes sont capables d'avoir un effet protecteur ou aggravant dans l'apparition d'une pathologie. Récemment, notre laboratoire a montré que certains haplogroupes avaient la capacité d'influencer le fonctionnement du métabolisme énergétique mitochondrial. Mon projet de recherche a donc consisté à mettre en place un modèle afin d'étudier les mécanismes cellulaires et moléculaires impliqués dans ce phénomène. Pour cela, nous avons recherché des haplogroupes d'intérêt dans la population française afin d'élaborer une collection de " cybrides " où chaque lignée de cellules possède un haplogroupe particulier mais un fonds génétique nucléaire commun à toutes les lignées. Nous avons caractérisé ces cybrides de manière biochimique (analyse de l'activité et des paramètres cinétiques de chaque complexe) et phénotypique (courbes de croissance). L'ensemble de ces résultats a été intégré dans un modèle informatique spécifiquement développé dans notre laboratoire pour modéliser la physiologie de la mitochondrie. Ce projet nous a permis de mettre en évidence l'influence des haplogroupes de l'ADNmt sur le métabolisme mitochondrial et de proposer une vision modulée des pathologies mitochondriales tant pour leur étude que pour leur diagnostic, en faisant ressortir la notion de médecine personnalisée. A l'avenir, il sera nécessaire de tenir compte du contexte génétique de l'ADNmt pour trouver de nouvelles stratégies ou de nouvelles cibles pour les thérapies des maladies mitochondriales. / The mitochondrion is an intracellular organelle responsible for the cellular energy production, by synthesizing ATP through the oxidative phosphorylation (OXPHOS). One of the characteristics of this organelle is that it has its own DNA (mtDNA) encoding for subunits of OXPHOS complexes. Any alterations of mitochondrial energy metabolism cause mitochondrial pathologies whose severity is generally inversely proportional to the age of onset. Some scientific studies are looking at the mechanisms of occurrence and development of these diseases in order to better understand them and to be able to offer therapies. However, there is no tool that can transform mtDNA in a targeted way by mutation or DNA rearrangement. Moreover, there are still few animal models of mitochondrial pathology that would allow integrative studies on the one hand, and on the other hand, to try out possible therapeutic molecules. In this thesis, we studied two types of models involving the modification of mitochondrial metabolism either by chemical treatment or by the use of mutations found in individuals. In a first part, we were interested in the realization of mouse model with a large number of characteristics of the Leigh syndrome, a progressive neurological disease characterized by neuropathological lesions associating a damage of the brain stem and the basal ganglia. For this study, we have used the 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) neurotoxin, known for its toxicity to dopamine neurons and also as an inhibitor of mitochondrial respiratory chain. We analyzed the activity of the OXPHOS complexes I to IV on brain tissues (cerebelum, cortex, striatum and substancia nigra) and peripheral tissues (heart, liver, muscle and kidney) from treated and untreated mice. Inhibition of complexes III and/or IV in the liver, cortex, striatum and cerebellum was found. These results, combined with an increased neurodegeneration found in a previous study, are all characteristics of Leigh Syndrome. Mice treated with MPTP seem to be a good model for this mitochondrial pathology. In the second project, we looked at the effect of mtDNA haplogroups (haplotypes grouping) on mitochondrial metabolism. Although, haplogroups are defined by neutral mutations of mtDNA (polymorphism), several studies have shown associations between haplogroups and some pathologies suggesting that haplogroups are able to have a protective effect or being a risk factor in the pathology development. Recently, our laboratory has confirmed that some haplogroups may not be neutral and have the ability to influence the mitochondrial energy metabolism functioning. Therefore, my research project consisted of setting up a model to study these cellular and molecular mechanisms. We looked for haplogroups of interest in the population in order to elaborate a cellular collection where each cell line has a particular haplogroup but with a common nuclear genetic background in all the cell lines. This collection was obtained by cybride constructions. We characterized these cybrides biochemically by analyzing the activity of each complex, determining kinetic parameters (KM and Vmax) and titration specific respiratory chain inhibitors. Concomitantly, we defined cell parameters via growth curves. All these results were integrated into a computer model specifically developed in our laboratory to model mitochondrial processes. This project gives us some evidence of the mtDNA haplogroups' influence on mitochondrial metabolism and to propose a modulated vision of mitochondrial pathologies for their study and their diagnosis, highlighting the notion of personalized medicine. As each haplogroup modulates in the different way the mitochondrial metabolism, each individual could have a personal response to the same mutation or pathology. In future, the mtDNA genetics background should be taken into account to find new strategies or new targets for the therapies of mitochondrial diseases.

O câncer de mama ocupa o primeiro lugar em mortalidade dentre todos os tipos de câncer. O subtipo triplo-negativo (triple-negative breast cancer - TNBC) representa 15-20% de todos os tipos de câncer de mama com alta prevalência em mulheres pré-menopausa e destaca-se pelo seu grande tamanho tumoral e agressividade no estabelecimento de metástases, com impacto direto na redução da sobrevida dos pacientes. Apesar das evidências sobre os efeitos anti-tumorigênicos do exercício físico, tanto na prevenção como durante a carcinogênese, é comum que pacientes alterem sua rotina após o diagnóstico de câncer, frequentemente reduzindo as atividades físicas durante e após o tratamento. Em adição, os mecanismos pelos quais o exercício físico exerce papel anti-tumoral são pouco compreendidos. O objetivo deste estudo foi avaliar os efeitos do exercício físico aeróbico moderado em modelo experimental de câncer de mama de tipo triplo-negativo, com ênfase na modulação do metabolismo energético tumoral. Foram utilizados camundongos fêmeas BALB/c em desenho experimental de 12 semanas, cuja inoculação de 1x104 células 4T1 foi realizada após 8 semanas de treinamento. Após protocolo de exercício aeróbico moderado em esteira, foram realizadas análises do metabolismo mitocondrial tumoral, composição lipídica e expressão de genes relacionados à bioenergética e proliferação celular. Os resultados mostraram que o exercício aeróbico moderado reduziu 54,5% do volume e 42% da massa tumoral de animais que foram treinados antes e após a inoculação tumoral. Animais treinados apresentaram fosforilação oxidativa mais próxima ao seu limite máximo respiratório e menor respiração mitocondrial no tecido tumoral quando comparados ao grupo sedentário. O treinamento ocasionou redução no conteúdo de ácido fosfatídico e fosfatidilcolina. Enquanto a análise de expressão relativa de mRNA demonstrou aumento na expressão de genes relacionados à via metabólica glicolítica, como Hif1a, Glut-1, HKII, Ldha e Pdk, além dos supressores tumorais p53 e Lats2. Nossos resultados sugerem que a redução na velocidade de crescimento tumoral proporcionada pelo exercício físico aeróbico de carga moderada seja devida, pelo menos em parte, à modulação do metabolismo energético tumoral. Em conjunto, os dados do nosso estudo abrem novas perspectivas para a identificação de vias metabólicas sensíveis ao exercício físico, permitindo o melhor o entendimento de seus efeitos antitumorigênicos / Breast cancer ranks first in mortality among all types of cancer. The triple-negative breast cancer (TNBC) accounts for 15-20% of all types of breast cancer with a high prevalence in premenopausal women and is notable for its large tumor size and aggressiveness in the establishment of metastasis, with a direct impact on the reduction of patients\' survival. Altough evidence highlight the anti-tumorigenic effects of physical exercise both on the prevention as well as during carcinogenesis, patients commonly change their routine after cancer diagnostic, usually reducing physical activity during and after treatment. Moreover, the mechanisms underlying the anti-tumor role of physical exercise remain poorly understood. The objective of this study was to evaluate the effects of moderate aerobic physical exercise in an experimental model of triple-negative breast cancer, with emphasis on the modulation of tumor energy metabolism. Female BALB / c mice were used in a 12-week experimental design, whose inoculation of 1x104 4T1 cells was performed after 8 weeks of training. After the protocol of moderate aerobic exercise was carried out on the treadmill, analyzes of mitochondrial tumor metabolism, lipid content and qPCR of genes related to bioenergetics and tumorigenic process were performed. The results showed that moderate aerobic exercise reduced 54.5% of the volume and 42% the tumor mass of animals trained before and after tumor inoculation. Trained animals showed oxidative phosphorylation closest to the maximum respiratory limit and lower mitochondrial respiration in tumor tissue when compared to the sedentary group. The training resulted in a reduction in the content of phosphatidic acid and phosphatidylcholine. In the trained group, relative mRNA quantification analysis showed increased expression of genes related to the glycolytic metabolic pathway, such as Hif1a, Glut-1, HKII, Ldha, and Pdk, as well as of the tumor suppressors p53 and Lats2. Our results suggest that the reduction in tumor growth velocity provided by moderate-intensity aerobic physical exercise is due, at least in part, to the modulation of tumor energy metabolism. Together, data from our study open new perspectives for the identification of metabolic pathways sensitive to exercise, allowing better understanding of its anti-tumorigenic effects

Mitochondria are organelles present in eukaryotic cells that play a key role in regulating cells’ metabolic processes as well as cell death. The main function of mitochondria is to produce ATP, by oxidizing nutrients in a process called oxidative phosphorylation (OXPHOS). Besides this, mitochondria also play a critical role in calcium homeostasis, cell signaling, and apoptosis. Mitochondrial dysfunction is implicated in a plethora of diseases including neurodegenerative diseases, metabolic disorders as well as ageing and cancer. The triphenylphosphonium (TPP+) moiety has been used as a carrier to direct a wide variety of therapeutic and diagnostic cargo to mitochondria, in an effort to study and treat mitochondrial dysfunction. Studies in recent years show that TPP+ is not an inert carrier as previously thought. Many TPP+ conjugates have been shown to exert a negative effect on mitochondrial and cellular bioenergetics by decreasing the efficiency of OXPHOS. This phenomenon is called ‘mitochondrial uncoupling’. While mitochondrial uncoupling is undesirable for the TPP+ moiety to function as a carrier of cargo to mitochondria, controlled uncoupling has therapeutic applications in treatment of obesity and cancer. The extent of mitochondrial accumulation as well as potency of mitochondrial uncoupling caused by the TPP+ moiety increases with increasing length of the linker functionality in TPP+ conjugates. Most of the studies to date have focused on altering the linker length of the TPP+-linker-cargo conjugate to optimize the balance between safety and efficacy. However, very little is known about how structural modification of the TPP+ moiety itself affects mitochondrial uncoupling potency. Therefore, there is a need to understand the structure activity relationship (SAR) between modification of TPP+ structure and the effect of these structural changes on mitochondrial uncoupling and uptake. Towards this end, the first goal of this study was to understand the effect of modulating electron density on the phosphorus atom of TPP+ on the potency of uncoupling OXPHOS. Modifications to the TPP+ moiety included substitution of electron withdrawing and donating groups on the phenyl rings of TPP+, and replacing phenyl rings with bulkier napthyl rings. Modified TPP+ moieties were conjugated to five different linkers, which varied in length and lipophilicity, and the effect of these conjugates on mitochondrial bioenergetics was studied. The second goal of the study was to evaluate if the propensity of TPP+ to uncouple mitochondrial respiration can be modulated, independently of mitochondrial uptake. For this purpose, the uptake of modified TPP+-linker conjugates into isolated mitochondria and the uptake of fluorescently labeled modified TPP+-linker conjugates into mitochondria within whole cells was investigated. The ability of modified TPP+ to protect cells from oxidative stress by successfully delivering an anti-oxidant cargo to mitochondria within cells was also assessed. The results of these studies establish the first SAR for modulating TPP+ structure to either eliminate, optimize, or maximize uncoupling of mitochondrial OXPHOS, and led to identification of lead molecules for potential applications in the fields of mitochondrial delivery, anti-obesity therapy and anti-cancer therapy.

Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Mitochondrial complex I, a large respiratory enzyme located in the inner mitochondrial membrane, catalyses electron transfer from NADH to ubiquinone while concomitantly translocating protons across the membrane to sustain ATP synthesis. A crucial aspect of the pharmacology of complex I is drug-induced mitochondrial dysfunction, particularly its role in liver toxicity. Complex I inhibition causes an energy deficit and can lead to adverse changes in the status of the mitochondrial [NADH]/[NAD+] pool and increased reactive oxygen species production, causing widespread damage. A library of molecules that are known candidates for causing complex I-driven drug- induced mitochondrial dysfunction was compiled using database and literature searches and then tested with assays on isolated mammalian complex I, mitochondrial membranes and cultured mammalian cells. The results extend the knowledge of complex I-linked drug toxicity and define a proof-of-principle methodology for the investigation of further unknown candidate molecules. Using this methodology, the Screen-Well V2 library from Enzo Life Sciences, containing 786 FDA-approved drugs, was used to investigate the role of complex I-linked drug toxicity on a wider scale. The results show that complex I is targeted by many structurally unrelated pharmacological compounds, but whether catalysis is inhibited in vivo requires drug transport into the mitochondrion, limiting the adverse physiological consequences in most cases tested. Furthermore, three structure-activity relationship studies were carried out on specific classes of complex I inhibitors: rotenoid natural product compounds, a family of pyrazole-based compounds under investigation as anticancer drugs, and variants on the drug Mubritinib. These studies identified structural determinants of binding to complex I and improve our understanding of complex I inhibition.