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.

L’atrophie optique dominante (AOD, MIM#165500) est une pathologie héréditaire affectant un individu sur 30 000 environ. Elle touche principalement les cellules ganglionnaires de la rétine qui composent le nerf optique, entraînant une baisse d’acuité visuelle. Cette pathologie, génétiquement et cliniquement hétérogène, est majoritairement due aux mutations du gène OPA1. La protéine mitochondriale OPA1 est impliquée dans de multiples fonctions telles que la fusion des mitochondries, le métabolisme énergétique, l’apoptose et la maintenance de l’ADN mitochondrial. Afin d’appréhender les effets globaux des dysfonctions d’OPA1, nous avons développé des approches métabolomiques et lipidomiques non-targeted sur des plasmas et des fibroblastes de patients ainsi que sur des modèles murins. En dépit des spécificités de chaque modèle et matrice, nos études ont clairement révélé des altérations métaboliques communes dont une déficience en aspartate. Ce déficit est impliqué dans le métabolisme des nucléotides et est en relation directe avec le défaut énergétique révélé dans nos différents modèles. Avec l’approche lipidomique, nous avons montré dans les fibroblastes de souris invalidés pour le gène OPA1 une augmentation importante des triglycérides qui est en lien avec le défaut énergétique. De plus, nous avons mis en évidence un remaniement majeur des phospholipides qui témoigne d’un profond remodelage des structures membranaires mitochondriales. Ces approches nous ont ainsi permis d’avoir de nouvelles données sur les implications physiopathologiques d’OPA1. L’ensemble de ce travail ouvre de nouvelles perspectives pour une meilleure prise en charge de la pathologie. / Dominant Optic Atrophy (DOA, MIM #165500) is an inherited disease affecting one of 30,000 individuals. It mostly affects the retinal ganglion cells that make up the optic nerve, leading to the decrease invisual acuity. This genetically and clinically heterogeneous pathology is mainly related to the mutations on OPA1 gene. The mitochondrial protein OPA1 has been implicated in many functions including mitochondrial fusion, energy metabolism, apoptosis and maintenance of mitochondrial DNA. In order to investigate the overall effects of OPA1 dysfunctions, we developed non-targeted metabolomic and lipidomic approaches on patients' plasmas and fibroblasts as well as on OPA1 knock-out mouse fibroblast model. Despite the specificities of each model and matrix, we clearly revealed a common metabolic alteration including an aspartate deficiency due to the energy defect observed in all our models and responsible for the alteration of nucleotide metabolism. With a lipidomic approach, we revealed in the knock-out cell model a huge increase of triglycerides which is related to the energetic deficiency. Moreover, we highlighted a major alteration on phospholipids, testifying a deep remodeling of mitochondrial membrane structures. Taken together, our analysis revealed new pathophysiological roles of OPA1. Finally, our work opens new perspectives to improve the diagnosis and the patient care.