Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

The effect of a free-living stage in host-parasite coevolution: a skink mite phylogenetic study in New Zealand. During the last decade, phylogenetic trees have even been used to compare ecologically related taxa such as parasites and their hosts, and are used to determine their level of coevolution or reciprocal adaptation in time. Diverse coevolutionary events have been detected for this ecological association, where generally the parasite has been regarded as one that feeds exclusively on the host and is likely to cospeciate with it. A different coevolutionary pattern might occur when the parasite has a free-living stage in its life cycle, in which the parasite may have the opportunity to abandon its host and successfully colonise a new species (host-switching) making cospeciation less likely. Many New Zealand skinks are infested with a parasitic mite, Odontacarus sp. (Prostigmata: Leeuwenhoekiidae), which becomes free-living as an adult. The genetic variation of these mites found on four hosts was analyzed for host- parasite coevolutionary events. The hosts were the McCann’s skink and the common skink in coastal Birdling Flat, Canterbury, plus these species and the Grand and Otago skinks in Macraes Flat, Central Otago, South Island, New Zealand. The genetic variation of fast evolving nuclear Internal Transcribed Spacers 2 and mitochondrial Cytochrome c Oxidase I in Odontacarus mites found on these hosts was determined by PCR and DNA sequencing and phylogenetic trees were built using the computer programs PAUP*4 and MrBayes 3. The results show that mite haplotypes only had a significant geographical division and no host-related differences. In Birdling Flat, the COI haplotypes were represented in two groups that infested both regional hosts and had 5.7 % divergence. The same individual mites belonged to a single ITS 2 haplotype, thus indicating a historical geographical division between two populations that now interbreed successfully. The Macraes Flat mites were divided into two COI haplotypes with 2.4% divergence and internal nodes, which showed greater genetic variability than the Birdling Flat populations. The Macraes Flat mites formed two ITS 2 haplotypes with 6% divergence. This greater geographical structure of the Otago mites is probably due to the older age of the mainland area compared to the recently exposed coastal locality of Birdling Flat. The COI haplotypes from the two different regions had a mean distance of 15.5%, with an earlier divergence time than that known for the hosts. For both genes, the haplotypes from different regions had 100% bootstrap support and the parasite showed no host specificity. Mites of the different COI and ITS haplotypes were found on most of the host species that were sampled in Canterbury and Otago. The results of this study suggest that a free-living stage in a parasite’s life cycle can favour coevolutionary events such as inertia (failure to speciate) and host-switching, probably as a result of resource-tracking of the parasite. NB: Electronic files contained on CD to accompany print copy are not included with this version of the thesis.

This thesis investigates the molecular evolutionary and macroevolutionary consequences of flight loss in insects. Chapter 2 tests the hypothesis that flightless groups have smaller effective population sizes than related flighted groups, expected to result in a consistent pattern of increased non-synonymous to synonymous ratios in flightless lineages due to the greater effect of genetic drift in smaller populations. Chapter 3 tests the hypothesis that reduced dispersal and species-level traits such as range size associated with flightlessness increase extinction rates, which over the long term will counteract increased speciation rates in flightless lineages, leading to lower net diversification. The wide-spread loss of flight in insects has led to increased molecular evolutionary rates and is associated with decreased long-term net diversification. I demonstrate that the fundamental trait of dispersal ability has shaped two forms of diversity—molecular and species—in the largest group of animals, and that microevolutionary and macroevolutionary patterns do not necessarily mirror each other. / Generously funded by NSERC with a Canada Graduate Scholarship and the Government of Ontario with an Ontario Graduate Scholarship to T. Fatima Mitterboeck; NSERC with a Discovery Grant to Dr. Sarah J. Adamowicz

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

Des modifications dans le gène codant pour la sous-unité II du cytochrome c oxydase du génome mâle (Mcox2) ont été recensées chez des espèces de bivalves présentant un mode unique de transmission mitochondriale nommé double transmission uniparentale (DUI). Dans la DUI, les mitochondries paternelles (et leur ADNmt mâle) ainsi que les mitochondries maternelles (et leur ADNmt femelle) sont transmises aux descendants mâles. Scrobicularia plana, une espèce de bivalves présentant ce modèle d'hérédité, possède une insertion importante d'environ 4,8 kb dans son gène Mcox2 qui ne change pas le cadre de lecture et qui est traduite en un polypeptide de 1 892 acides aminés, ce qui en fait la plus grande protéine COX2 connue à ce jour chez les métazoaires. L’objectif de cette étude était de caractériser l'évolution et la fonction potentielle de l'insertion dans Mcox2 chez S. plana par RT-PCR, tests immunologiques et analyses bio-informatiques. L'insertion est présente parmi les individus de différentes populations, contient des variations dans la longueur de sa séquence, est riche en zones de désordre intrinsèque et évolue sous sélection purificatrice. La longue insertion pourrait modifier la structure 3D du complexe IV de la chaîne de transport d'électrons (CTE), affectant sa fonction dans la phosphorylation oxydative (OXPHOS) ce qui pourrait expliquer les faibles taux d'OXPHOS observés dans les mitochondries mâles des bivalves à DUI. L'insertion pourrait également modifier le métabolisme mitochondrial mâle en interagissant avec d'autres complexes de la CTE et avec l'ATP synthase. Comme pour les autres modifications de Mcox2 chez les bivalves à DUI, un rôle potentiel dans la détermination du sexe peut être prédit pour MCOX2 chez S. plana. / Modifications in the cytochrome c oxidase subunit II gene of male-transmitted genome (Mcox2) have been found in some bivalve species that exhibit a unique mode of mitochondrial transmission named doubly uniparental inheritance (DUI). In DUI, paternal mitochondria (and their male mtDNA) as well as maternal mitochondria (and their female mtDNA) are transmitted to male offspring. Scrobicularia plana, a bivalve specie exhibiting this inheritance model possesses an important in-frame insertion of approximately 4,8 kb in its Mcox2 gene that is translated into a polypeptide of 1 892 amino acids making it the largest metazoan COX2 protein known to date. The aim of this study was to characterize the evolution and possible function of the Mcox2 insertion in S. plana through RT-PCRs, immunoassays, and bioinformatic analysis. The insertion is present amongst individuals from different populations, contains some variations in its sequence length, is rich in intrinsically disordered regions and evolves under purifying selection. The long insertion could modify the 3D structure of complex IV in the electron transport chain (ETC), impacting its function in oxidative phosphorylation (OXPHOS) which could explain low OXPHOS rates that were found in male mitochondria of DUI bivalves. The insertion could also alter male mitochondrial metabolism by interacting with other complexes of the ETC and with ATP synthase. As for other modifications of Mcox2 in DUI bivalves, a role in sex determination can also be predicted for MCOX2 in S.plana.