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L'ancêtre a-protéobactérie endosymbiotique à l'origine des mitochondries avait son propre génome, codant pour de nombreuses fonctions redondantes, voire totalement inutiles dans la cellule hôte. Ces informations ont disparu avec le temps, alors que les autres gènes indispensables ont en grande partie été transférés au noyau de la cellule eucaryote. Aujourd'hui, plus de quatre vingt quinze pourcents des protéines mitochondriales sont codées par le génome nucléaire. La question se pose de savoir pourquoi ces gènes sont maintenus dans les organites. Une manière de répondre expérimentalement à cette question consiste à relocaliser artificiellement au noyau les gènes des organites. Nous avons testé cette relocalisation nucléaire chez la levure Saccharomyces cerevisiae. Une première étape de l'étude a consisté à déléter le gène mitochondrial ATP9 natif. La délétion du gène ATP9 mitochondrial chez S. cerevisiae conduit à de multiples effets délétères sur la stabilité du génome mitochondrial, son expression, le contenu en complexes de la chaîne respiratoire, mais aussi sur la morphologie des mitochondries. Des expériences antérieures, décrites dans la littérature, avaient échouées dans la relocalisation nucléaire du gène ATP9 de S. cerevisiae. J'ai réussi la relocalisation nucléaire de ce gène chez la levure par une approche différente, avec cette fois un gène ATP9 déjà nucléaire, celui de Podospora anserina. Malgré une différence de 30% dans la séquence primaire des protéines, la protéine Atp9p de P. anserina exprimée depuis le noyau chez S. cerevisiae peut complémenter la délétion mitochondriale du gène ATP9. La levure modifiée peut former des ATP synthases hybrides ayant une bonne activité in vitro. En parallèle de cela, le travail sur P. anserina a donné lieu à une collaboration qui nous a permis d'en savoir un peu plus sur l'expression des deux gènes ATP9 de ce champignon filamenteux. Notons que P. anserina a deux gènes ATP9, nativement nucléaires, chacun étant exprimés à des moments précis du cycle de vie de ce champignon filamenteux. Dans l'évolution, le transfert fonctionnel du gène ATP9 chez P. anserina, comme chez les mammifères, a permis l'acquisition d'un mécanisme de régulation de la quantité d'ATP synthase en fonction des conditions physiologiques de la cellule. / The endosymbiotic a-protéobacteria ancestor of mitochondria had its own genome, specifying rebounding functions, sometimes useless inside the host cell. This piece of information have been lost during the evolution, while other essential genes have in part been transferred to the nucleus of the eukaryotic cell. Nowadays, more than 95% of the mitochondrial proteins are encoded by the nucleus. We ask the question of why there is still genes remaining in the mitochondrial genome. One way to answer experimentally that question is to artificially relocalize those mitochondrial genes to the nucleus. We have tested the nuclear relocation in Saccharomyces cerevisiae. A first step consisted in deleting the mitochondrial ATP9 gene. This deletion led to multiple deleterious effects on the stability of the mitochondrial genome, its expression, on the content of the respiratory complexes, but also on the mitochondrial morphology. Previous studies, described in the literature, have failed in the nuclear relocation of ATP9 of S. cerevisiae. I succeeded in the nuclear relocation of ATP9 using an already nuclear version of the gene, that of Podospora anserina. Despite a 30% divergence of the proteic sequences, the Atp9p of P. anserina expressed from the nucleus in S. cerevisiae can complement the ATP9 deletion. The modified yeast can form hybrid ATP synthases with a rather good in vitro activity. In parallel to that work on P. anserina, this has led to a collaboration which gave us more information on the expression of ATP9 in P. anserina. It is to notify that P. anserina has two ATP9 genes, natively nuclear, each of them being expressed at different times during the life cycle of the fungus. During evolution, the functional transfer of ATP9 to the nucleus, like it is the case in mammals too, has allowed the acquisition of regulatory mechanisms to control the amount of ATP synthases depending on physiological constraints of the cell.

The production of reactive oxygen species by mitochondria is implicated in mitochondrial dysfunction associated with a range of diseases and ageing. In addition, reactive oxygen species produced by mitochondria are involved in redox signalling pathways that modulate a number of cell processes. Mitochondria targeted antioxidants comprised of an antioxidant moiety linked to a lipophilic triphenylphosphonium cation have recently been used to decrease oxidative damage to mitochondria and to investigate the involvement of mitochondrial reactive oxygen species in redox signalling. These lipophilic cations are selectively accumulated by mitochondria within cells due to the mitochondria membrane potential. This thesis presents the synthesis and characterization of mitochondria targeted membrane uncoupler, cyclic nitroxide and alkyl thionitrite derivatives, all of which had the potential to influence reactive oxygen species. The biological analysis of these compounds is also presented. A triphenylphosphonium derivative of the membrane uncoupler 2,4-dinitrophenol (DNP) was anticipated to act as a self regulating protonophore. The DNP moiety would influence the scale of the membrane potential while the triphenylphosphonium cation would respond to the membrane potential. These two factors would combine so that as the membrane potential was dissipated by the uncoupler, the phosphonium cation would be released from the mitochondria and the effect of the uncoupler would thereby be nullified until the membrane potential had increased again. The compound was prepared by nitration of 3-(4-hydroxyphenyl)propyl triphenylphosphonium bromide. An untargeted derivative was also prepared by nitration of 3-(4-hydroxyphenyl)-1-propanol. Unfortunately, while this compound had appropriate acidity and lipophilicity to act as a membrane uncoupler, and did enter mitochondria in response to the membrane potential, it did not act as an uncoupler. A chemically stable targeted cyclic nitroxide based on Tempol was prepared following literature procedure, although other synthetic routes were also trialled. This compound was shown to concentrate in mitochondria in response to the membrane potential, was reduced by ubiquinol of the coenzyme Q pool, acted as a superoxide dismutase mimetic, and protected membranes against lipid peroxidation. A mitochondria targeted thionitrite or nitric oxide (NO) donor was anticipated to exhibit an effect on respiration at low oxygen concentrations as the released NO interacted with aspects of the respiratory chain. The alkyl thionitrites were synthesised from appropriate thiol precursors, several of which were prepared. Two targeted alkyl thionitrites were prepared with primary or tertiary carbon arrays next to the thionitrite functionality. Another targeted thionitrite, based on S-nitroso-N-acetylpenicillamine (SNAP), was also prepared. These compounds were difficult to characterise because of issues surrounding their stability. However, modified high resolution positive ion electrospray mass spectrometry in combination with HPLC and NMR was used to identify the compounds and to gauge the purity of the samples. Initial biological investigations verified that the primary alkylthionitrite derivative accumulated in mitochondria, released NO, and had an effect on respiration at low oxygen concentrations.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007. / Includes bibliographical references (p. 129-140).

Graduation date: 1994

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.

Sequences of the cytochrome b gene and genotypes from 11 polymorphic microsatellite loci were used to assess phylogeographic variation in ferruginous pygmy-owls (Glaucidium brasilianum) from Arizona, Mexico, and Texas. Analysis of mtDNA indicated that pygmy-owl populations in Arizona and Texas are unique, with no shared haplotypes. Populations from Sonora and Sinaloa, Mexico, were distinct from remaining populations in Mexico and grouped closest to haplotypes in Arizona. Nested clade analysis of mtDNA sequence data indicated past fragmentation separated pygmy-owls into two major groups: 1) Arizona, Sonora and Sinaloa, Mexico, and 2) southwestern (Nayarit and Michoacan), south-central (Oaxaca and Chiapas), and eastern Mexico, along the eastern slope of the Sierra Madre Oriental from Texas to Central America. In addition, analysis of mtDNA variation in several species of Glaucidium support the recommendation that populations of G. brasilianum from Mexico, Texas, and Arizona represent a phylogenetically distinct group from populations occurring in South America. The level of separation between the North and South Americanpopulations justifies granting species status (G. ridgwayi) to the North American population. Analysis of distance matrices derived from genotypes of 11 polymorphic microsatellite loci supports restricted gene flow between pygmy-owl populations in Arizona-Sonora and Sinaloa, and Texas-Tamaulipas and the remainder of states in Mexico. The Arizona-Sonora population showed signs of a recent genetic bottleneck, an observation supported by low population estimates for Arizona (13-117 individuals). Heterozygosity in Arizona, however, was equal to levels recorded throughout Mexico and Texas. Congruent patterns revealed by both mtDNA and nuclear DNA (microsatellites) indicate Arizona and Texas populations are distinct subspecies that require the design and implementation of separate management plans for recovery and conservation efforts.

Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains ix, 36 p. : ill. Includes bibliographical references.

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