A seqüência completa do genoma mitocondrial de uma espécie pode ajudar no mapeamento de restrição e desenho de primers para PCR. Estes poderão servir para amplificação e posterior seqüenciamento de regiões específicas de outras espécies e populações relacionadas, para estudos filogenéticos e de dinâmica populacional. Até o momento, temos na literatura a seqüência completa do DNA mitocondrial (DNAmt) de um único himenóptero, Apis mellifera, espécie que é endêmica do Velho Mundo. Nenhum genoma mitocondrial de uma espécie de abelha nativa do Brasil foi até o momento descrito. Com a devastação crescente dos ecossistemas, há a perda de espécies de abelhas ainda pouco estudadas, e talvez até outras ainda não conhecidas. Entre os meliponíneos, há espécies-chave de diversos ecossistemas brasileiros, tendo portanto uma enorme importância ecológica. No decorrer deste projeto, foram amplificados via PCR e seqüenciados 77% do genoma mitocondrial da abelha sem ferrão Melipona bicolor (Apidae, Meliponini), contendo todos os 13 genes mitocondriais codificadores para proteínas, 18 dos 22 genes para RNAt e os dois genes para RNAr (sendo um integral e o outro parcialmente seqüenciado). Além do seqüenciamento, foram realizados neste trabalho: análise da organização do genoma (conteúdo e ordem gênica); análise da tradução dos genes para proteínas e código genético; análise de outras características moleculares (freqüência das bases, códons utilizados, iniciação e terminação de genes, freqüência de aminoácidos etc); e comparação das características acima mencionadas com o genoma mitocondrial de A. mellifera e também com outros insetos. O viés para o uso de bases A+T, bastante evidente em A. mellifera, mostrou-se ainda mais acentuado em M. bicolor. Foram encontradas diferenças no tamanho e composição dos genes. Pelo menos nove rearranjos na ordem gênica mitocondrial foram observados entre as duas espécies de abelhas, um fenômeno raro entre organismos tão próximos. Considerando que essas espécies compartilham um comportamento intrigante, a eussocialidade, esses rearranjos podem servir como um excelente marcador para estudar a origem e a evolução desse comportamento no grupo. / The complete sequence of the mitochondrial genome of a species may help on restriction mapping and to design PCR primers. These can be useful to amplify and sequence specific regions from other species and analyze populations, in phylogenetic and demographic studies. So far, there was reported on literature the mtDNA complete sequence for only one hymenopteran, Apis mellifera, endemic from the Old World. No mitocondrial genome of a Brazilian native bee was ever described. With the increasing devastation of natural environments, several bee species can be led to extinction, including those poorly studied and maybe some unknown species. The meliponines (stingless bees) include key species to several Brazilian ecosystems, so they play an important ecological role. In this project, we have PCR amplified and sequenced 77% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was only partially sequenced). Besides sequencing, this work consisted of: analysis of genome organization (gene content and order); analysis of gene translation and genetic code; analysis of other molecular features (base frequencies, codon usage, gene initiation and termination, amino acid frequencies etc.); and comparison of the characteristics mentioned above with A. mellifera mitocondrial genome and also other insects. The highly biased A+T content is a typical characteristic of A. mellifera mitochondrial genome, and it is even more extreme on M. bicolor mtDNA. There are length and compositional differences on genes between M. bicolor and A. mellifera. At least nine gene order rearrangements were observed by comparing the mtDNA of these species, what is a rare event on closely related organisms. Considering that both species share an intriguing behavior, eusociality, these gene rearrangements may be used as an excellent marker to study the origin and evolution of that behavior on bees.

Este trabalho teve como meta principal contribuir para elucidar algumas das questões em aberto pertinentes à história evolutiva e antropológica de populações nativas americanas. Para isso investigou-se marcadores uniparentais paternos, ligados à NRY, e materno, mtDNA. Para o cromossomo Y foram investigados 108 indivíduos (85 sulameríndios de 16 tribos, pertencentes a 5 grupos lingüísticos, além de 23 asiáticos (siberianos), compreendendo 6 grupos étnicos distintos). Para o mtDNA foram investigados 160 indivíduos (homens e mulheres), compreendendo 10 tribos sulamericanas, pertencentes a 5 grupos lingüísticos distintos. Para o cromossomo Y foram utilizados 26 marcadores (SNPs). Para o mtDNA a região controladora-RC (HVS-I: da posição 16.024 até 16.569, e HVS-II: da posição 001 até 576) e a região imediatamente 5’ à região controladora foram seqüenciadas. Foi possível determinar para o cromossomo Y que Q1a3a* (autóctone nativo-americano, de provável origem beringiana) está fixado em 63% das tribos; o haplogrupo Q1a3*, que por outro lado também é encontrado na Ásia, foi observado entre os Araweté (25%), Jamamadi (100%), Lengua (25%) e esquimós asiáticos (17%). Merece destaque que Q1a3* parece ser o que até agora era identificado como sendo apenas “haplogrupo Q*”, ou seja, cromossomo Y portador do alelo derivado no loco M242, mas com alelo ancestral para o M3. Nenhuma das novas mutações mencionadas na atual árvore filogenética do cromossomo Y (com exceção do M346, que define Q1a3*) foram encontradas. O seqüenciamento de regiões do cromossomo Y não revelou nenhuma nova mutação. No caso do mtDNA, os indígenas do tronco Ge mostram os haplogrupos B e A como sendo os mais freqüentes, enquanto que nos Tupi esses haplogrupos apresentam freqüências mais elevadas apenas em regiões bastante restritas, ficando o haplogrupo D como o mais freqüente. Cabe salientar que o haplogrupo C apresenta freqüência muito baixa tanto para os Ge quanto para os Tupi, sendo que freqüências um pouco mais elevadas estão quase que geograficamente opostas, ficando no sul do Brasil para os Ge e no norte para os Tupi. Avaliando o modelo de fissão-fusão pôde-se sugerir que: 1) As linhagens mitocondriais tribo-específicas dentro das tribos Kayapó aqui investigadas dificilmente representariam linhagens autóctones, já que o tempo de surgimento de cada tribo por processo de fissão é pequeno para comportar uma rede de novas mutações. As especificidades poderiam estar vinculadas ao modelo de fissão envolvendo grupos de pessoas aparentadas via materna. Nesse caso, grupos de parentes carregariam para fora do grupo parental todas as seqüências pertencentes a uma determinada linhagem. Assim a linhagem estaria presente somente no grupo derivado e não mais no parental; 2) Perda de linhagens parentais na dispersão e/ou por deriva na formação do novo grupo, o que resultaria na diferença encontrada entre os grupos derivados; 3) Embora não se possa excluir alguma fusão posterir a fissão, a quantidade de linhages exclusivas nas tribos Kayapó estaria indicando relativo isolamento dos grupos depois da fissão (ausência ou baixa freqüência de fluxo gênico entre os grupos fissionados levando à relativa baixa freqüência de linhagens compartilhadas), o que denota o fato do fenômeno ser recente (atritos ainda presentes na memória coletiva e/ou familiar dos grupos fissionados) como estabelecido pelos dados históricos (início do século XVII). Esse fato poderia sugerir que a fusão demanda mais tempo para ocorrer; 4) O compartilhamento das linhagens mais comuns, normalmente na raiz das networks, entre os Tupi e os Ge, parece denotar mais ancestralidade comum do que importante fluxo gênico depois da formação desses dois grandes estoques lingüísticos. / This work has as its main aim to elucidate some of the still open questions about the evolutive and anthropological history of the Native American populations. Paternal uniparental markers, in the NRY, and maternal, mtDNA, were investigated to do that. For the Y chromosome, 108 individuals were investigated (85 South-Amerindians from 16 tribes, belonging to 5 linguistic groups, and 23 Asians (Siberians), covering 6 distinct ethnical groups). For the mtDNA, 160 individuals (men and women) were evaluated, covering 10 South-American tribes, belonging to 5 distinct linguistic groups. For the Y chromosome 26 SNPs were tested and some regions sequenced. For the mtDNA the control region-CR (HVS-I: from position 16.024 to 16.569, and HVS-II: from position 001 to 576) and the region immediately 5’ of the control region were sequenced. It was possible to determine that Q1a3a* (a Native American autoctonous chromosome, probably of Beringian origin) is fixed in 63% of the tribes; the haplogroup Q1a3*, which, moreover, is also encountered in Asia, was observed in Araweté (25%), Jamamadi (100%), Lengua (25%) and Asian Eskimos (17%). It is worth mentioning that Q1a3* appears to be what until now has been identified as “haplogroup Q*” only, that is, Y chromosome carrier of the derived allele in the M242 locus, but with an ancestral allele for M3. Any of the new mutations mentioned in the current Y chromosome phylogenetic tree (except M346, which defines Q1a3*) were encountered. Sequencing of Y chromosome regions hasn’t revealed any new mutation. In the mtDNA’s case, the Ge indians show the haplogroups B and A as the most frequent ones, while in the Tupi indians these haplogroups show high frequencies only in very restrict regions, being haplogroup D the most frequent. It should be noted that haplogroup C shows very low frequency in both Ge and Tupi, the slightly higher frequencies occuping almost geographically opposite locations, at the South of Brazil for the Ge and on the North for the Tupi. On evaluating the fission-fusion model it could be suggested that: 1) Tribe-specific lineages in the Kayapó tribes investigated here would hardly represent autoctonous lineages, since the time of emergence of each tribe by fission process is small to bear a web of new mutations. The specificities could be related to the fission model involving maternally related groups of people. In this case, groups of relatives would carry out of the parental group all the sequences belonging to a determined lineage. Therefore the lineage would be present only in the derived group and not in the parental anymore; 2) Loss of parental lineages in the dispersion and/or by drift in the new group’s formation, which would result in the differences found between the derived groups; 3) Though some fusion posterior to the fission cannot be excluded, the amount of exclusive lineages in the Kayapó tribes would indicate a relative isolation of the groups after the fission (absence or low frequency of gene flow between the fissioned groups leading to relative low frequency of the shared lineages), which denotes the fact of the phenomenon being recent (struggles still present in the collective and/or familiar memories of the fissioned groups) as estabilished by historical data (beginning of the XVII century). This fact could suggest that the fusion demands more time to occur; 4) The sharing of the more common lineages, normally in the networks’ nodes, between Tupi and Ge, appears to denote more common ancestrality than important gene flow after the formation of these two great linguistic stocks.

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 DNA é um alvo constante de modificações químicas, as quais resultam na ativação dos programas de reparo de danos no DNA. O DNA mitocondrial (DNAmt), uma molécula circular contendo aproximadamente 16,6 kb de extensão, é constantemente exposto às espécies reativas de oxigênio (EROs) devido a sua proximidade da cadeia transportadora de elétrons, presente na membrana mitocondrial interna. Quase todas as vias de reparo de DNA presentes no núcleo atuam também na mitocôndria, entretanto, a regulação das vias mitocondriais não é bem compreendida. As proteínas p53, ATM, mTOR e PGC1α participam, dentre outros papéis, do controle do metabolismo energético e das respostas a lesões no DNA nuclear. Dessa forma, decidimos gerar linhagens celulares com níveis reduzidos dessas proteínas como uma ferramenta para o estudo dos seus papéis na manutenção do DNAmt. Para isso, foram geradas linhagens celulares de HEK293 expressando constitutivamente shRNAs alvo-específicos, cuja diminuição da expressão das proteínas alvo foi confirmada através de western blotting. Neste trabalho, também foi estudado o papel de p53 na resposta ao estresse oxidativo mitocondrial provocado por azul de metileno (AM). O AM é um corante fotoativo capaz de atravessar membranas biológicas e, em células de mamíferos, se acumula em organelas, tais como a mitocôndria. Uma vez que p53 participa de diversas funções celulares e transloca para a mitocôndria sob condições de estresse, onde pode induzir apoptose ou modular o reparo de DNAmt, nós investigamos se p53 está envolvido na indução de morte celular após tratamento com AM fotoativado. Para isso, foram utilizados 2 clones com níveis reduzidos de p53 obtidos na primeira etapa deste trabalho. Sob condições normais, foi demonstrado que o silenciamento de p53 induziu uma forte redução do número de cópias de DNAmt e estimulou a proliferação celular quando fornecemos glicose ou galactose como substratos energéticos. A depleção de p53, ou a sua inibição farmacológica, resultaram em uma ligeira proteção quando as células foram submetidas ao tratamento com AM. Também foi demonstrado que AM provoca morte celular apoptótica de uma maneira dependente de p53, uma vez que a depleção dessa proteína protegeu a população do acúmulo de células em sub-G1. Portanto, nossos resultados sugerem que AM induz morte celular apoptótica em células HEK293, de uma maneira dependente de p53. Esse efeito pode ser mediado diretamente por p53, ou ainda, pelo seu papel na manutenção do número de cópias do DNAmt. / DNA is constantly being chemically modified, which results in activation of the DNA damage response program. The mitochondrial DNA (mtDNA), a circular molecule of 16.6 kb in length, is primary target of reactive oxygen species (ROS) due its proximity to the electron transport chain, in the mitochondrial inner membrane. Almost all known DNA damage repair pathways operating in the nucleus were also found in the mitochondrion; however, their regulation remains not well understood. The proteins p53, ATM, mTOR e PGC1α have many cellular functions, including control of energy metabolism and cell fate after stress. Thus, we hypothesized that those proteins could participate in maintaining of mtDNA, through direct or indirect roles. To test this hypothesis, we generated isogenic knockdown cell lines to further use them to study their role in the mtDNA damage response. For that, were generated HEK293 knockdown cell lines that stably express target-specific shRNAs. Efficient knockdown was checked using western blotting. Here, we also studied the role of p53 in the cellular response to mitochondrial oxidative stress induced by methylene blue (MB). MB is a photoactive dye that crosses biological membranes due to its lypophylic character and, in mammalian cells, accumulates in organelles such as mitochondria; however, its cytotoxic mechanism is not well understood. As the p53 protein participates in several cellular functions and translocates to mitochondria under stress conditions, where it can induce apoptosis or modulate mtDNA repair, we investigated whether p53 was involved in MB + light-induced cell death using p53 knockdown clones selected from the cell lines generated in the first phase of this work. Under normal conditions, p53 knockdown caused a decrease in mtDNA copy number and stimulated cellular growth supported by either glucose or galactose. After MB treatment, p53-kd cells showed a slight decrease in cell death compared to scrambled shRNA controls. Evaluation of cell death after MB treatment, using flow cytometry analysis, indicated that MB was able to induce significant levels of apoptotic cell death, which was dependent on p53 levels. Taken together, our results suggest that MB induces cell death, probably via apoptosis, in a p53 dependent manner. This effect may be mediated by p53 directly or by its role in mtDNA copy number maintenance.

O DNA é um alvo constante de modificações químicas, as quais resultam na ativação dos programas de reparo de danos no DNA. O DNA mitocondrial (DNAmt), uma molécula circular contendo aproximadamente 16,6 kb de extensão, é constantemente exposto às espécies reativas de oxigênio (EROs) devido a sua proximidade da cadeia transportadora de elétrons, presente na membrana mitocondrial interna. Quase todas as vias de reparo de DNA presentes no núcleo atuam também na mitocôndria, entretanto, a regulação das vias mitocondriais não é bem compreendida. As proteínas p53, ATM, mTOR e PGC1α participam, dentre outros papéis, do controle do metabolismo energético e das respostas a lesões no DNA nuclear. Dessa forma, decidimos gerar linhagens celulares com níveis reduzidos dessas proteínas como uma ferramenta para o estudo dos seus papéis na manutenção do DNAmt. Para isso, foram geradas linhagens celulares de HEK293 expressando constitutivamente shRNAs alvo-específicos, cuja diminuição da expressão das proteínas alvo foi confirmada através de western blotting. Neste trabalho, também foi estudado o papel de p53 na resposta ao estresse oxidativo mitocondrial provocado por azul de metileno (AM). O AM é um corante fotoativo capaz de atravessar membranas biológicas e, em células de mamíferos, se acumula em organelas, tais como a mitocôndria. Uma vez que p53 participa de diversas funções celulares e transloca para a mitocôndria sob condições de estresse, onde pode induzir apoptose ou modular o reparo de DNAmt, nós investigamos se p53 está envolvido na indução de morte celular após tratamento com AM fotoativado. Para isso, foram utilizados 2 clones com níveis reduzidos de p53 obtidos na primeira etapa deste trabalho. Sob condições normais, foi demonstrado que o silenciamento de p53 induziu uma forte redução do número de cópias de DNAmt e estimulou a proliferação celular quando fornecemos glicose ou galactose como substratos energéticos. A depleção de p53, ou a sua inibição farmacológica, resultaram em uma ligeira proteção quando as células foram submetidas ao tratamento com AM. Também foi demonstrado que AM provoca morte celular apoptótica de uma maneira dependente de p53, uma vez que a depleção dessa proteína protegeu a população do acúmulo de células em sub-G1. Portanto, nossos resultados sugerem que AM induz morte celular apoptótica em células HEK293, de uma maneira dependente de p53. Esse efeito pode ser mediado diretamente por p53, ou ainda, pelo seu papel na manutenção do número de cópias do DNAmt. / DNA is constantly being chemically modified, which results in activation of the DNA damage response program. The mitochondrial DNA (mtDNA), a circular molecule of 16.6 kb in length, is primary target of reactive oxygen species (ROS) due its proximity to the electron transport chain, in the mitochondrial inner membrane. Almost all known DNA damage repair pathways operating in the nucleus were also found in the mitochondrion; however, their regulation remains not well understood. The proteins p53, ATM, mTOR e PGC1α have many cellular functions, including control of energy metabolism and cell fate after stress. Thus, we hypothesized that those proteins could participate in maintaining of mtDNA, through direct or indirect roles. To test this hypothesis, we generated isogenic knockdown cell lines to further use them to study their role in the mtDNA damage response. For that, were generated HEK293 knockdown cell lines that stably express target-specific shRNAs. Efficient knockdown was checked using western blotting. Here, we also studied the role of p53 in the cellular response to mitochondrial oxidative stress induced by methylene blue (MB). MB is a photoactive dye that crosses biological membranes due to its lypophylic character and, in mammalian cells, accumulates in organelles such as mitochondria; however, its cytotoxic mechanism is not well understood. As the p53 protein participates in several cellular functions and translocates to mitochondria under stress conditions, where it can induce apoptosis or modulate mtDNA repair, we investigated whether p53 was involved in MB + light-induced cell death using p53 knockdown clones selected from the cell lines generated in the first phase of this work. Under normal conditions, p53 knockdown caused a decrease in mtDNA copy number and stimulated cellular growth supported by either glucose or galactose. After MB treatment, p53-kd cells showed a slight decrease in cell death compared to scrambled shRNA controls. Evaluation of cell death after MB treatment, using flow cytometry analysis, indicated that MB was able to induce significant levels of apoptotic cell death, which was dependent on p53 levels. Taken together, our results suggest that MB induces cell death, probably via apoptosis, in a p53 dependent manner. This effect may be mediated by p53 directly or by its role in mtDNA copy number maintenance.

In this study, I sequenced the mitochondrial cytochrome b gene to elucidate the extent and pattern of genetic variations among and within populations of Rhinichthys osculus (Santa Ana Speckled Dace) found in the different watersheds in Southern California, Central California Coast and Eastern California Desert. I described and analyzed the structural characteristics and pattern of base sequence substitutions in the cytochrome b gene to understand the molecular evolution of the gene. The Phylogenetic analyses showed that the Southern California Santa Ana Speckled dace is a distinct population from the Central California Coast dace population and Eastern California Desert dace population, and is more closely related to the Colorado River speckled dace population. There is a high degree of genetic variation among all populations including a significant genetic structure associated with watersheds, mountain ranges, and geographic grouping based on locations. The SWISS-Model automated protein structure homology elucidated the conserved and invariant residues within the cytochrome b gene where the amino acid substitutions are located in the trans-membrane of the protein sequence. The implication for conservation and management of the Southern California Santa Ana Speckled dace is high because of habitat lost for this distinct dace population. The data from this study will contribute to preserving the genetic variability of the Santa Ana Speckled Dace as a separate taxa and species, as well as to help maintain intact the population in the different Southern California creeks.

La dynamique mitochondriale est impliquée dans la maintenance et la fonction des mitochondries. Dans les graines sèches tout les processus cellulaires sont arrêtés du fait de la faible teneur en eau des tissues, et la transition développementale que représente la germination requiert la réactivation de la dynamique cellulaire. Une approche de bio-imagerie sur la plante modèle Arabidopsis a été utilisée afin d’étudier la réactivation des mitochondries nécessaire à la germination. La réactivation bioénergétique des mitochondries, mesurée par la présence du potentiel membranaire, intervient dès le début de l’hydratation des tissus. Cependant les mitochondries restent statiques et la dynamique mitochondriale ne reprend que plus tardivement. La réactivation des mitochondries provoque une réorganisation du chondriome impliquant la biogenèse de membranes et une fusion massive menant à la formation de structures réticulaires et périnucléaires, qui permet le mélange des nucléoïdes d’ADNmt précédemment isolés en unités discrètes. La mitophagie, un indicateur de la qualité mitochondriale, est réactivée de manière concomitante à la dynamique, alors qu’elle est réprimée durant la biogenèse des mitochondries. La fin de la germination coïncide avec la fragmentation du chondriome tubulaire, menant au doublement du nombre de mitochondrie et à une redistribution hétérogène des nucléoïdes dans le chondriome, générant une population de mitochondrie adaptée à la croissance des plantules. Cette thèse met en évidence l’imbrication des processus de dynamique mitochondriale, de biogenèse et de contrôle qualité des mitochondries requis pour la germination et pour la transition vers l’autotrophie. / Mitochondrial dynamics underpin their function and maintenance. In dry seeds, all cellular processes are in stasis due to a low water content. Thus, the developmental switch leading to germination necessarily involves a reactivation of cellular dynamics. In order tobetter understand the role played by mitochondrial dynamics during germination we used Arabidopsis as a model for a bioimaging approach to investigate the rapid reactivation of mitochondria that is required in order to provide ATP to support germination. Bioenergetic reactivation, visualised as the presence of a mitochondrial membrane potential, is almost immediate upon rehydration. However, the reactivation of mitochondrial dynamics only occurs after several hours of rehydration. The reactivation of mitochondrialbioenergetics and dynamics lead to a dramatic reorganisation of the chondriome involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. Mitophagy, an indicator of mitochondrial quality, is reactivated concomitant with a reactivation of mitochondrial dynamics, but is repressed at time of mitochondrial biogenesis. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondrialnumber and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth. This thesis provides strong evidence for the tight interweaving of mitochondrial dynamics, mitochondrialbiogenesis and mitochondrial quality control that is required to ensure effective germination and the transition to autotrophy.

The influence of genotype variation on phenotype has been a longstanding question in biology but it is now one of the greatest challenges of the post-genomics era. Discovering the link between common gene variants that affect phenotypes within and between populations is likely to provide insight into the molecular physiology of phenotypic traits and the mechanisms by which they evolve. The overall goal of this thesis is to link naturally occurring genotypic variation with the organism??s phenotype. The specific goal of this thesis is to connect natural variation in the mitochondrial genotype with the organismal phenotype using the model organism Drosophila simulans. Mitochondria are intracellular organelles found in most eukaryotes and produce over 90% of the energy needed by cells. Determining the connection of mitochondrial genotype to whole organism phenotype is of particular interest because of the broad use of mitochondrial (mt) DNA as a molecular marker in evolutionary biology and population genetics, the organelle??s central role in cellular energy production, the potential for the mitochondria to influence organismal distribution particularly in the face of climate change and in human degenerative disease. I use the model organism D. simulans because it has high genetic variability, can be easily sampled from the wild and manipulated in the lab, and the energy producing reactions that take place in its mitochondria are highly conserved among metazoa. I studied naturally occurring mutations to understand the influence of these changes in natural populations. The four studies in this thesis have employed a Genotype-Biochemistry-Phenotype (GBP) model to link naturally occurring variation in the mitochondrial genotype with organism phenotype in D. simulans mitochondria. Three major conclusions can be drawn from the thesis that follow the genotype to biochemistry to phenotype model. Firstly, a subset of the mutations in genes that comprise the mitochondrial genotype is functionally significant. Secondly, the biochemical efficiency of OXPHOS is regulated by mitochondrial homeostasis. Thirdly, key organismal life history traits influenced by the mitochondrial genotype and this is mediated through the biochemistry of OXPHOS.

<p>The phylogeography of a wide ranging temperate species, the adder, <i>Vipera berus</i>, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders.</p><p>The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of <i>V. b. bosniensis</i>. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia. </p>

<p>The white-tailed eagle is a formerly threatened raptor that is commonly used as a flagship and indicator species in conservation work. This thesis uses molecular genetic methods to study sex determination of nestlings, genetic variability, population structure and phylogeography of the white-tailed eagle.</p><p>Fourteen microsatellite markers were developed and tested for the white-tailed eagle.</p><p>A method to sex white-tailed eagle nestlings in the field is presented. The method is based on just one tarsus measure, and is suitable for situations where a single person is handling the nestlings alone in a treetop.</p><p>Most European white-tailed eagle populations underwent extreme declines during the 20th century. The results presented here show that bottlenecked populations have maintained significant levels of genetic diversity. Gene flow between regions is not a main explanation for this, as indicated by both genetic and ringing data. Instead, the long generation time of white-tailed eagles has acted as an intrinsic buffer against rapid loss of genetic diversity. Additionally, local conservation led to protection of more genetic diversity than if conservation had focused on the large remnant population in Norway.</p><p>Mitochondrial DNA of white-tailed eagles is structured in two main clades with a predominantly eastern and western Eurasian distribution. The clades likely correspond to separate Ice Age refugia but do not grant classification as evolutionary significant units given their current extensive overlap across large parts of Eurasia.</p><p>Microsatellite variation was studied in populations across Eurasia. Variability was rather constant across the continent, but clearly lower on Iceland and Greenland. This is best explained by founder effects during their colonisation, but only weak bottlenecks during colonisation of and persistence on the continent. Current population differentiation between Europe and eastern Eurasia is not compatible with a zero gene flow model but requires some amount of gene flow over evolutionary time scales.</p>