The purpose of this study is to examine mitochondrial DNA variation in the Southwest Pacific and determine what factors contribute to the degree and patterning of the observed variation. Population variation is known to be influenced by factors including demographic history, natural selection, climate, isolation, island area/complexity, and population age, as older populations are generally more diverse. The groups compared are from three regions in the Southwest Pacific; (a) northeast New Guinea, (b) Manus in northern Island Melanesia and (c) Easter Island in eastern Polynesia. MtDNA surveys have revealed highly significant differences in molecular variance across these populations. According to traditional biogeographical theory, the likely determinants of these differences are (a) length of time since initial settlement, (b) the comparative isolation of particular islands or regions since settlement, and (c) the size and complexity of settlement areas. Evidence from archaeology and linguistics provides the necessary framework for the study. Detailed archaeological surveys for several of the study regions provides evidence for settlement dates as well as evidence for isolation and/or frequent contact with other areas, usually in the form of trade and translocation of animals and artifacts. Linguistics, though not as informative as archaeology for settlement dates, provides detailed evidence for isolation and/or contact in the form of language isolates, language families, borrowing and linguistic divergence. The mtDNA haplogroups found in this study belong to several documented haplogroups, some of Melanesian origin, and some of Southeast Asian origin. The distribution of mtDNA variants and the pattern and degree of variation was examined using Analysis of Molecular Variance, standard diversity measures and partial Mantel matrix correlations. There were strong positive correlations between insular area, isolation and degree of variation. There were also measurable differences between inland and coastal populations on the larger islands where diversity in the isolated inland populations was greater than diversity in the coastal population. While there was some confounding of the variables, the results of our analysis indicate that insular area/complexity and isolation influence the pattern of variance more than length of settlement time. / Anthropology

Cyprinodon variegatus is a pupfish that inhabits the Atlantic coast of North America, nearly continuously, from Massachusetts to Belize. This research attempts to resolve the phlylogeography of C. variegatus by investigating the genetic sequence structure of the mitochondrial control region (D-loop, non-coding origin of replication) and cytochrome-b gene for evidence of northern and southern subspecies within the Atlantic Coast of the eastern United States. Additionally, it will be may be possible to determine if secondary hybrid zones developed as a result of the last retreat of ice from North America during the Pleistocene, about 17,000 years ago. A definitive monophyletic northern clade was found using parsimony, distance, and maximum likelihood phylogenetic methods to analyze the control region data. The cytochrome-b sequence data supported this monophyletic northern clade, although their utility for this analysis is marginal. Little evidence was found for the existence of a hybrid zone. More thorough sampling will be needed to make more accurate determinations regarding the existence of such a zone. / Master of Science

No / The 15th century Inuit mummies excavated at Qilakitsoq in Greenland in 1978 were exceptionally well preserved and represent the largest find of naturally mummified specimens from the Arctic. The estimated ages of the individuals, their distribution between two adjacent graves, the results of tissue typing, and incomplete STR results led researchers to conclude that the eight mummies formed two distinct family groups: A grandmother (I/5), two daughters (I/3, I/4), and their two children (I/1, I/2) in one grave, and two sisters (II/6, II/8) and a daughter (II/7) of one of them in the other. Using mtDNA from hair and nail, we have reanalyzed the mummies. The results allowed the unambiguous assignment of each of the mummies to one of three mtDNA haplogroups: A2b (I/5); A2a (I/2, I/3, II/6, II/8); A2a-311 (I/1, I/4, II/7), excluded some of the previous relations, and pointed to new ones. I/5 is not the grandmother/mother of the individuals in Grave I, and she is not maternally related to any of the seven other mummies; I/3 and I/4 are not sisters and II/7 is neither the daughter of II/6 nor of II/8. However, I/1 may be the child of either I/4 or II/7 and these two may be sisters. I/2 may be the son of I/3, who may be the daughter of either II/6 or II/8, and these two may be sisters. The observation of haplogroups A2a and A2b amongst the 550-year-old Inuit puts a lower limit on the age of the two lineages in Greenland.

The thesis is focused on two mtDNA haplogroups of Eurasian origin that occur mostly among the Fulani, herders of the African Sahel. On the initial analysis 544 DNA samples from unrelated Fulani individuals were used. These samples were classified to haplogroups according to hypervariable segment I (HVS-I) of mtDNA and subsequently, specific Eurasian haplotypes were chosen for the whole mtDNA genome sequencing. Obtained mito-genomes were assigned to phylogenetic trees and dated. It turned out that they belonged to haplogroups U5b1b1b and H1ca1a with the probable origin in the Iberian Peninsula, presumably in Franco-Cantabrian refugium where their ancestors originated in the period between the Late Glacial and the first half of the Holocene. Afterwards they migrated through the Strait of Gibraltar to North Africa and the Sahara, where the proto-Fulani pastoral population was being formed. It may also be assumed that a part of this population came to Africa from the Near East, along with cattle. In the second half of the Holocene, this pastoral population migrated from the drying Sahara to the Sahel. Probably due to small number of female migrants the females from the local populations were integrated, which explains the major representation of West African mtDNA haplogroups in contemporary Fulani....

A superfamília Majoidea contém os caranguejos conhecido popularmente como aranha, encontrados em ambientes marinhos e costeiros, em áreas intermareais rochosas ou de recifes. A imensa diversidade do grupo gerou diferentes propostas de classificação, sendo atualmente aceitas as seguintes famílias: Epialtidae, Inachidae, Inachoididae, Majidae, Mithracidae e Oregoniidae. Epialtidae contém 87 gêneros, dentre eles Libinia com 10 espécies válidas, das quais três ocorrem no Pacífico Oriental (México e Peru) e sete ocorrem no Atlântico Ocidental, desde o Canadá até a Argentina. Em diferentes hipóteses filogenéticas, o gênero posiciona-se com distintos grupos-irmãos (Herbstia, Leucippa, Notolopas, Pisa, Rochinia, Taliepus) que pertencem a subfamílias diferentes, sugerindo o parafiletismo da família, mas sem resolver as relações de parentesco do gênero. No Brasil, embora a posição taxonômica de L. ferreirae e L spinosa não seja duvidosa, podem conviver em simpatria além de possuírem alguns hábitos similares. No entanto, não havia estudos que esclarecessem suas relações de parentesco. Libinia dubia, L. emarginata e L. erinacea coexistem no Golfo do México, em habitat e profundidade similares; apresentaram diferenças morfológicas bastante sutis que justificaram o estudo comparativo das mesmas. Libinia cavirostris foi descrita em 1942, seus caracteres morfológicos a separam das demais espécies do gênero; no entanto não houve mais registros de ocorrência e como consequência ausência de outras informações. Libinia rhomboidea tem distribuição limitada, poucos registros recentes e localidade-tipo duvidosa, o que instigou o exame detalhado de outros espécimes e localidades. Três espécies ocorrem no Pacífico Oriental, L. mexicana, L. setosa e L. peruana, sendo as duas primeiras com distribuição no México e a segunda apenas no Peru, os quais são distinguíveis entre sim, porém não se tem mais registro de L. peruana desde sua descrição em 1983. O presente estudo investigou as relações de parentesco entre as 10 espécies que compõe o gênero Libinia e avaliou a hipótese de monofilia; comparou a morfologia das mesmas e realizou a revisão taxonômica do gênero, levantando caracteres morfológicos informativos de adultos e de jovens de diferentes localidades. As árvores filogenéticas foram construídas a partir de sequências de DNA dos genes 16S e COI alinhadas individualmente e concatenadas, por meio dos métodos de máxima verossimilhança e inferência bayesiana. Todas as dez espécies foram consideradas válidas. As hipóteses filogenéticas suportaram a monofilia de Libinia, indicando pouca proximidade com Stratiolibinia e corroborando a criação do gênero. Gomes, A.F.T. - Tese de Doutorado 2 De forma geral, a morfologia das espécies que ocorrem no Pacífico Oriental é similar e as distinguem das espécies que ocorrem no Atlântico Ocidental. Ainda sim, a hipótese filogenética do gênero não indica clados monofileticos para estes grupos geográficos e ainda evidenciou pouca proximidade entre L. mexicana e L. setosa. Libinia spinosa posicionou-se externamente às demais espécies, L. mexicana é próxima à L. cavirostris, e L. ferreirae tem proximidade filogenética com as espécies que ocorrem no Golfo do México e Caribe. Assim, a hipótese filogenética do gênero Libinia que sugeriu sua monofilia foi confirmada, não apresentando indicativos de clados distintos para as espécies do Pacífico e do Atlântico, e esclareceu as relações internas dentro do gênero / The Majoidea superfamily contains the popularly known spider crabs, found in marine and coastal environments on rocky or reef intertidal areas. Its great diversity resulted on different proposals of classification, thus, combining more recent propositions, there the following families: Epialtidae, Inachidae, Inachoididae, Majidae, Mithracidae e Oregoniidae. Epialtidade consists of 87 genera among them Libinia with 10 valid species, three of them occur in the Eastern Pacific (Mexico and Peru) and seven in the Western Atlantic, from Canada to Argentina. According to distinct phylogenetic hypotheses, this genus can be related to more than one outgroup (Herbstia, Leucippa, Notolopas, Pisa, Rochinia, Taliepus) each belonging to different subfamilies, suggesting that the family is paraphyletic, but without solving the intern relationships. In Brazil, although the taxonomic position of L. ferreirae and L spinosa are not doubtful, they are found in sympatry besides having some similar habits. However, there were no studies that clarified their relationship. Libinia dubia, L. emarginata and L. erinacea occur together in the Gulf of Mexico, or similar habitat and depth; they show subtle morphological differences that justified a comparative study. Libinia cavirostris was described in 1942, its morphological characters differed it from the other species of the genus; however, there were no more recent records, and as a consequence no further information. Libinia rhomboidea has a restricted distribution, few recent records and its type-locality is doubtful, which instigated the detailed examination of other specimens and ocalities. Three species occur in the Eastern Pacific, L. mexicana, L. setosa and L. peruana, the former with distribution in Mexico and the latter only in Peru; they are distinguishable from each other, but L. peruana was not sample since its description in 1983. The main goal of this study was investigating the phylogenetic relationship among the 10 species of Libinia and evaluated the hypothesis of monophyly; compared their morphology and performed the taxonomic revision of the genus, proposing informative morphological characters for adults and juveniles. Phylogenetic trees were obtained from DNA sequences of 16S and COI genes separated and concatenated through the maximum likelihood and Bayesian inference approaches. All species were considered valid. The phylogenetic hypotheses supported the monophyly of Libinia and indicated low proximity with Stratiolibinia, confirming the description of the genus. In general, the morphology of the species that occur in the Eastern Pacific is similar and distinguish them from the ones in the Western Atlantic. Moreover, the phylogenetic hypothesis of the genus does not suggest monophyletic clades for these geographic groups and still suggests low proximity between L. mexicana and L. setosa. Libinia spinosa was positioned externally to the other species, L. mexicana was sister group of L. cavirostris, and L. ferreirae was close related to the species that occur in the Gulf of Mexico and the Caribbean. Finally, the phylogenetic hypothesis of the Libinia genus suggested its monophyly, it did not indicate distinct clades of the Pacific and Atlantic species and clarified the internal relationship among the species of the genus

Todos os organismos vivos estão constantemente expostos a uma variedade de agentes que podem causar modificações químicas e/ou estruturais no DNA, afetando processos essenciais como replicação e transcrição. Ao longo da evolução várias estratégias de reparo do DNA foram desenvolvidas para remover essas modificações, prevenindo o efeito citotóxico ou mutagênico dessas lesões. Mutações no mtDNA são frequentemente observadas em inúmeras patologias, o que reflete em alterações metabólicas ou até mesmo atenuação da resposta apoptótica a terapias antineoplásicas. Para manter a integridade genômica mitocondrial, alguns mecanismos de reparo são recrutados à organela, principalmente a via de reparo por excisão de bases, BER. No núcleo, a proteína supressora de tumor p53 colabora para a manutenção da estabilidade do DNA, em parte pela estimulação direta da via BER. Em resposta a certos estímulos celulares, p53 transloca-se para a mitocôndria, onde pode desencadear uma resposta apoptótica. Entretanto, foi demonstrado anteriormente que p53 pode estimular a atividade catalítica da DNA polimerase mitocondrial, DNA polimerase gama (pol γ), que participa da replicação e do reparo do mtDNA. Sendo assim, nesse trabalho buscou-se compreender, molecularmente, essa interação entre p53 e pol γ durante a modulação de BER em mitocôndrias de células humanas, investigando se: i) p53 se associa fisicamente a pol γ; ii) a proteína TFAM modula o papel de p53 no reparo do DNA em mitocôndrias; e iii) a translocação de p53 para a mitocôndria é mediada por processos redox durante o reparo do mtDNA. Para isso, métodos bioquímicos e moleculares foram empregados nos estudos da interação entre essas proteínas. Em conjunto, os resultados sugerem o envolvimento da proteína p53 no reparo por excisão de bases em mitocôndrias de células humanas, e a dependência de sua interação com TFAM e pol γ para o sustento dessa via. Isso reforça a importância dessas proteínas para a manutenção da estabilidade genômica mitocondrial e, provavelmente, para a função mitocondrial. / All living organisms are constantly exposed to a variety of agents that can cause chemical and/or structural changes in DNA, affecting essential processes like replication and transcription. Throughout the evolution several strategies of DNA repair have been developed to remove these modifications, preventing the cytotoxic or mutagenic effect of these lesions. Mutations in mtDNA are often observed in numerous pathologies, reflecting metabolic changes or even attenuation of the apoptotic response to antineoplastic therapies. To maintain mitochondrial genomic integrity, some repair mechanisms are recruited to the organelle, mainly the base excision repair route, BER. In the nucleus, p53 tumor suppressor protein contributes to the maintenance of DNA stability, in part by direct stimulation of the BER pathway. In response to certain cellular stimuli, p53 translocate to the mitochondria, where it can trigger an apoptotic response. However, it has been shown previously that p53 can stimulate the catalytic activity of mitochondrial DNA polymerase, gamma polymerase (pol γ), which participates in the replication and repair of mtDNA. Thus, in this work we sought to understand, molecularly, this interaction between p53 and pol γ during a modulation of BER in mitochondria human cells, investigating if: i) p53 is physically associated with pol γ; ii) the TFAM protein modulates the role of p53 in DNA repair in mitochondria; and iii) the translocation of p53 to mitochondria is mediated by redox processes in mtDNA repair. For this, biochemical and molecular methods were used in the studies of protein interaction. Taken together, the results suggest the involvement of p53 protein in repair by base excision in mitochondria of human cells, and dependence of its interaction with TFAM and pol γ to support this pathway. This reinforces the importance of these proteins for the maintenance of mitochondrial genomic stability and probably for mitochondrial function.

Os herpesvírus são normalmente adaptados a um único grupo de hospedeiros, e esta associação parasita-hospedeiro está ligada à sua seleção e coevolução. Estes agentes podem causar infecções latentes, onde normalmente o vírus não se replica. Durante o ciclo lítico, no entanto, outras células são infectadas e liberam partículas virais capazes de infectar outros indivíduos. O CFPHV (Chelonid fibropapilloma-associated herpesvirus) tem sido apontado como principal agente infeccioso ligado a fibropapilomatose em tartarugas-marinhas. A doença caracteriza-se por uma proliferação cutânea benigna mas que, dependendo da sua severidade, pode comprometer a sobrevivência do indivíduo afetado, sendo por isso apontada como importante ameaça a conservação de tartarugas-marinhas, particularmente de tartarugas verdes (Chelonia mydas), a principal espécie acometida pela doença. Alguns aspectos da biologia do CFPHV e sua relação com as tartarugas verdes foram estudados no presente trabalho. Primeiramente, a capacidade deste agente em se disseminar pelo ambiente e infectar outros indivíduos, e as possíveis vias envolvidas nesta dispersão. Em seguida, avaliou-se os possíveis tecidos em que o herpesvírus pode estabelecer a infecção latente. Por fim, determinou-se a prevalência de indivíduos de Chelonia mydas infectados pelo CFPHV em duas áreas de alimentação (Ubatuba-SP e Vitória-ES) e em uma áreas mista - de alimentação e reprodução (Fernando de Noronha-PE). No primeiro estudo, observou-se que a prevalência de CFPHV nas amostras de secreções de Chelonia mydas variou entre 0%, no Espírito Santo, a 25%, em São Paulo. Os haplótipos afetados foram CMA-3 e CMA-8, e a variante viral encontrada não havia sido detectada anteriormente no Brasil, mas possui elevada similaridade com vírus provenientes do Golfo da Guiné e de Porto Rico. Os resultados sugerem que estes vírus podem ser transmitidos por secreções e também circular entre diferentes regiões. No segundo estudo, detectou-se a presença de CFPHV no cérebro de 5 animais necropsiados e também na pele e em lesões fibropapilomatosas. Em um dos animais foi detectada a presença de uma única variante de CFPHV no cérebro, pele e tumores. Esta variante ainda não havia sido detectada no Brasil e apresentou 100% de identidade com a variante detectada nas secreções. Para avaliar a relação entre haplótipos e variantes virais, o terceiro estudo determinou a prevalência de CFPHV em pele e tumores de 136 indivíduos - 9,56% de indivíduos sadios apresentavam o agente em tecido epitelial e 45,58% dos animais foram positivos para CFPHV, quando considerados também animais com fibropapilomatose. Duas novas variantes de herpesvírus foram encontradas: Var. 7, em Ubatuba-SP e Vitória-ES e Var. 8, em Vitória-ES. Não houve associação entre uma variante viral e um haplótipo. Os resultados observados permitem apontar que o CFPHV pode estabelecer infecções latentes; o vírus pode \"migrar\" entre diferentes regiões, junto com seus hospedeiros; partículas virais podem ser liberadas por secreções; duas novas variantes foram identificadas. Altas taxas de substituição de nucleotídeos em CFPHV podem indicar o surgimento das variantes destas áreas, mas a alta similaridade entre as variantes detectadas e àquelas de Porto Rico e Golfo da Guiné sugerem também a entrada de novas variantes na costa brasileira. / Herpesviruses are usually adapted to a single group of hosts, and this host-parasite association is linked to its selection and co-evolution. These agents can cause latent infections, where the virus usually does not replicate. During the lytic cycle, however, other cells are infected and release viral particles capable of infecting other individuals.The CFPHV (Chelonid fibropapilloma-associated herpesviru) has been indicated as the main infectious agent linked to fibropapillomatosis on sea turtles. The disease is characterized by a benign skin proliferation, but, depending on its severity, can compromise the survival of the affected individual, therefore considered an important threat to the conservation of sea turtles, especially green turtles (Chelonia mydas), the main species affected by the disease. Some aspects of the CFPHV biology and its relation to green turtles were studied in this work. Firstly, the ability of this agent to spread in the environment and infect other individuals, and the possible pathways involved in this dispersion. Then, potential tissues wherein the herpesvirus can establish latent infection were assessed. Finally, we determined the prevalence of Chelonia mydas individuals infected by CFPHV in two feeding areas (Ubatuba-SP and Vitória-ES) and in a mixed area of feeding and reproduction (Fernando de Noronha-PE). In the first study, it was observed that the prevalence of CFPHV in samples of Chelonia mydas secretions ranged from 0% in Espírito Santo, to 25% in São Paulo. Affected haplotypes were CM-A3 and CM-A8, and viral variant found had not been previously detected in Brazil, but it is significantly similar to viruses found in the Gulf of Guinea and Puerto Rico. The results suggest that these viruses can be transmitted by secretions and can also circulate among different regions. Considering the low maintenance of the agent within the environment, they are probably brought by individuals with the latent virus, being capable of releasing viral particles during the herpesvirus replication cycle. In the second study, the presence of CFPHV was detected inside the brain of 5 necropsied animals, besides the detection of the virus on the skin and fibropapillomatosis lesions. In one of the animals, it was possible to characterize the CFPHV and the presence of a single viral variant inside the brain, tumors and on the skin of the same animal was detected. This variant had not yet been detected in Brazil and showed 100% identity with the variant detected in secretions. These results indicate that the virus may establish a latent infection in nerve tissue. To evaluate the relationship between haplotypes and viral variants, the third study determined the prevalence of CFPHV on skin and tumors of 136 individuals - 9.56% of healthy individuals showed the agent in epithelial tissue and 45.58% of the animals were positive for CFPHV, when also considered animals with fibropapillomatosis. Two new variants of the herpesvirus were found, Var. 7 in Ubatuba-SP and Vitória-ES and Var. 8 only in Vitória-ES. C. mydas individuals of different haplotypes were infected, and there was no association between a viral variant and a haplotype. The observed results permitted to point that CFPHV can establish latent infections in Chelonia mydas; the virus can \"migrate\" among different regions, along with its hosts; viral particles can be released by secretion; viral variants previously detected were not found in these areas, but two new variants were detected. The high nucleotide substitution rates observed in CFPHV may indicate the emergence of these variants in these areas, but the high similarity among the detected variants and those identified in Puerto Rico and Gulf of Guinea also suggest the entry of new variants into the Brazilian coast.

A utilização de sequências do DNA mitocondrial (mtDNA) como marcadores moleculares na investigação da diversidade genética e evolução é muito difundida, auxiliando na realização de inferências em inúmeros trabalhos. Apesar de sua inegável importância, a utilização dessas sequências como marcadores moleculares suscita algumas questões. A heteroplasmia, por exemplo, é reconhecida como um desafio na utilização de sequências do mtDNA. Este estado ocorre quando um organismo apresenta diferentes haplótipos mitocondriais. Em um trabalho anterior, foram encontrados indícios que sugeriam a presença de heteroplasmia na espécie de abelha Bombus morio. O trabalho atual investigou de forma mais detalhada a presença de heteroplasmia nessa espécie, assim como fatores que podem influenciar na identificação desse estado. Os resultados obtidos confirmaram a existência de heteroplasmia nessa espécie, e identificaram que alguns haplótipos heteroplásmicos foram compartilhados entre indivíduos de localidades distintas. Esses haplótipos heteroplásmicos compartilhados sugerem a existência de heteroplasmia estável em B. morio, o que pode influenciar inferências evolutivas, e em especial, os estudos populacionais. Também foi detectada a presença de NUMTs, pseudogenes nucleares resultantes da transferência de sequências do mtDNA para o genoma nuclear. Esses NUMTs apresentaram grande divergência de sequência em relação aos haplótipos mitocondriais, o que poderia afetar análises filogenéticas e populacionais, além da identificação de espécies por meio do DNA barcoding. Ainda, erros de amplificação podem ser falsamente interpretados como variação intraindividual do DNA mitocondrial (mtDNA), superestimando o número de haplótipos, principalmente quando polimerases de baixa fidelidade são utilizadas. Por fim, os resultados observados neste trabalho sugerem que a utilização de sequências do mtDNA deve ser utilizada de forma cautelosa, e indícios de heteroplasmia, como a presença de picos duplos, não devem ser ignorados. Quando essas evidências são observadas investigações mais detalhadas devem ser aplicadas, a fim de aferir qual a sua origem, e, no caso da heteroplasmia ser confirmada, quais as possíveis consequências produzidas pela presença desse estado / The mitochondrial DNA sequences (mtDNA) have been widely applied as molecular markers in the investigation of genetic diversity and evolution. Despite its undeniable importance, the use of these sequences as molecular markers may present some drawbacks. Heteroplasmy, for example, is recognized as a challenge. This state occurs when an individual has different mitochondrial haplotypes. In a previous work, evidences suggesting the presence of heteroplasmy in the bumblebee Bombus morio were verified. The present work investigated in more detail the presence of heteroplasmy in this species, as well as factors that may influence the identification of this state. The results confirmed the existence of heteroplasmy in this species, and identified that some heteroplasmic haplotypes were shared between individuals from different locations. These shared heteroplasmic haplotypes suggest the existence of stable heteroplasmy in B. morio, which may interfere in evolutionary inferences, especially in population studies. NUMTs, nuclear pseudogenes resulting from the transfer of mtDNA sequences to the nuclear genome, were also detected. These NUMTs showed great sequence divergence from mitochondrial haplotypes, which could affect phylogenetic and population analyzes, as well as species identification through DNA barcoding. In addition, it was observed that amplification errors might be misinterpreted as mtDNA intraindividual variation and overestimates the number of intraindividual haplotypes, especially when low fidelity polymerases are used. Finally, the results observed in this study suggest that the use of mtDNA sequences should be used carefully, and evidences of heteroplasmy, such as the presence of double peaks, should not be ignored. Additional investigations should be applied in case of heteroplasmy evidences to ascertain your source and the consequences of the presence of this state

Todos os organismos vivos estão constantemente expostos a uma variedade de agentes que podem causar modificações químicas e/ou estruturais no DNA, afetando processos essenciais como replicação e transcrição. Ao longo da evolução várias estratégias de reparo do DNA foram desenvolvidas para remover essas modificações, prevenindo o efeito citotóxico ou mutagênico dessas lesões. Mutações no mtDNA são frequentemente observadas em inúmeras patologias, o que reflete em alterações metabólicas ou até mesmo atenuação da resposta apoptótica a terapias antineoplásicas. Para manter a integridade genômica mitocondrial, alguns mecanismos de reparo são recrutados à organela, principalmente a via de reparo por excisão de bases, BER. No núcleo, a proteína supressora de tumor p53 colabora para a manutenção da estabilidade do DNA, em parte pela estimulação direta da via BER. Em resposta a certos estímulos celulares, p53 transloca-se para a mitocôndria, onde pode desencadear uma resposta apoptótica. Entretanto, foi demonstrado anteriormente que p53 pode estimular a atividade catalítica da DNA polimerase mitocondrial, DNA polimerase gama (pol γ), que participa da replicação e do reparo do mtDNA. Sendo assim, nesse trabalho buscou-se compreender, molecularmente, essa interação entre p53 e pol γ durante a modulação de BER em mitocôndrias de células humanas, investigando se: i) p53 se associa fisicamente a pol γ; ii) a proteína TFAM modula o papel de p53 no reparo do DNA em mitocôndrias; e iii) a translocação de p53 para a mitocôndria é mediada por processos redox durante o reparo do mtDNA. Para isso, métodos bioquímicos e moleculares foram empregados nos estudos da interação entre essas proteínas. Em conjunto, os resultados sugerem o envolvimento da proteína p53 no reparo por excisão de bases em mitocôndrias de células humanas, e a dependência de sua interação com TFAM e pol γ para o sustento dessa via. Isso reforça a importância dessas proteínas para a manutenção da estabilidade genômica mitocondrial e, provavelmente, para a função mitocondrial. / All living organisms are constantly exposed to a variety of agents that can cause chemical and/or structural changes in DNA, affecting essential processes like replication and transcription. Throughout the evolution several strategies of DNA repair have been developed to remove these modifications, preventing the cytotoxic or mutagenic effect of these lesions. Mutations in mtDNA are often observed in numerous pathologies, reflecting metabolic changes or even attenuation of the apoptotic response to antineoplastic therapies. To maintain mitochondrial genomic integrity, some repair mechanisms are recruited to the organelle, mainly the base excision repair route, BER. In the nucleus, p53 tumor suppressor protein contributes to the maintenance of DNA stability, in part by direct stimulation of the BER pathway. In response to certain cellular stimuli, p53 translocate to the mitochondria, where it can trigger an apoptotic response. However, it has been shown previously that p53 can stimulate the catalytic activity of mitochondrial DNA polymerase, gamma polymerase (pol γ), which participates in the replication and repair of mtDNA. Thus, in this work we sought to understand, molecularly, this interaction between p53 and pol γ during a modulation of BER in mitochondria human cells, investigating if: i) p53 is physically associated with pol γ; ii) the TFAM protein modulates the role of p53 in DNA repair in mitochondria; and iii) the translocation of p53 to mitochondria is mediated by redox processes in mtDNA repair. For this, biochemical and molecular methods were used in the studies of protein interaction. Taken together, the results suggest the involvement of p53 protein in repair by base excision in mitochondria of human cells, and dependence of its interaction with TFAM and pol γ to support this pathway. This reinforces the importance of these proteins for the maintenance of mitochondrial genomic stability and probably for mitochondrial function.

Mitochondria are organelles present in most eukaryotic cells. They generate most of the cells adenosine triphosphate (ATP) supply which make them essential for cell viability. It is assumed that they are derived from a proteobacterial ancestor as they retain their own, drastically small genome. The importance in studying mitochondrial genome evolution came from the discovery of a large number of human diseases that are caused by mitochondrial dysfunction (e.g., Parkinson and Alzheimer). Many of these diseases are a result of a mutation in one of the mitochondrial genes or a defective mitochondrial DNA (mtDNA) maintenance, mostly caused by genetic defects in proteins involved in mtDNA replication. In order to explore the diversity and understand the evolution of mitochondrial genomes (mitogenomes) in animals, multiple methods have been developed in this study to deal with two biological problems related to the mitochondrial genome evolution. A new method for identifying the mitochondrial origins of replication is presented. This method deals with the problem of determining the origins of replication, which despite many previous efforts has remained non-trivial even in the small genomes of animal mitochondria. The replication mechanism is of central interest to understand the evolution of mitochondrial genomes since it allows the duplication of the genetic information. The extensive work that has been done to study the replication of mitochondrial genomes has generated the assumption of the strand displacement model (SDM) also known as the standard model of replication that is known to leave the mitochondrial H-strand in a single stranded state exposing it to mutation and damage. Later on, other models of replication have been suggested such as the strand coupled bidirectional replication model, its refinement which assumes the bidirectional mode but with a unidirectional start, and the \"RNA incorporation throughout the lagging strand\" (RITOLS) model proposed as a refinement of the strand displacement model. Based on the observation that the GC-skew is correlated with the distance from the replication origins in the light of the strand displacement model of replication, a new computational method to infer the position of both the heavy strand and the light strand origins from nucleotide skew data has been developed. The method has been applied in a comprehensive survey of deuterostome mitochondria where conserved positions of the replication origins for the vast majority of vertebrates and cephalochordates have been inferred. Deviations from the consensus picture are presumably associated with genome rearrangements. Additionally, two methods for the identification of tRNA remolding events throughout Metazoa have been developed. Remolding changes the identity of a tRNA by a duplication and a point mutation(s) of the anticodon. This new tRNA takes the identity of another tRNA which is then lost. This can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. In this work, novel methods are developed to detect tRNA remolding in large-scale data sets. The first method represents an extension of the similarity-based approach to determine remolding candidates with high confidence. This approach uses an extended set of criteria based on both sequence and structural similarities of the tRNAs in conjunction with statistical tests. The second method is a novel phylogeny-based likelihood method which evaluates specific topologies of gene phylogenies of the two tRNA families relevant to a putative remolding event. Both methods have been applied to survey tRNA remolding throughout animal evolution. At least three novel remolding events are identified in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived ancestral events.