A espécie Bos indicus, particularmente a raça Nelore, é grande maioria no rebanho bovino da região acima do trópico no Brasil. Embora a habilidade desses animais em resistir às doenças parasitárias, condições climáticas e pastagens de baixa qualidade enalteçam a utilização em larga escala desta raça, estes animais não são considerados bons conversores de alimento e, conseqüentemente, precoces em comparação aos seus homólogos Bos taurus. Durante a formação das raças zebuínas brasileiras, houve uma participação das linhas maternas de Bos taurus, que pode ser demonstrada pela contribuição majoritária do genoma mitocondrial desta subespécie. Embora em escala muito menor, estima-se que exista também uma participação destas linhas maternas no genoma nuclear. O objetivo deste trabalho foi iniciar os estudos para estimar esta participação. Para os estudos foram utilizados 104 animais da raça Nelore e 8 animais de diferentes raças européias. Cinco regiões do DNA que produzem fragmentos microssatélites taurus/indicus específicos (HEL1, HEL9, ETH225, ILSTS005 e INRA063) foram amplificadas com a utilização de primers marcados com sondas fluorescentes. Os fragmentos foram submetidos à eletroforese em gel de poliacrilamida desnaturante 6% e visualizados após excitação com laser. No total foram encontrados 23 alelos para os microssatélites analisados o que representa uma média de 4,6±1,82 alelos por locus. Amplificou-se também uma região do DNA satélite 1711b que posteriormente foi digerida com a enzima de restrição Msp I. Verificou-se a existência de três possíveis genótipos entre os animais Nelore mtDNA Bos taurus e mtDNA Bos indicus. Os animais europeus analisados apresentaram sempre o mesmo padrão de restrição. A comparação dos componentes de variância do tamanho dos alelos intra e inter população usando os fragmentos microssatélites permitem a separação dos animais Bos taurus dos animais Nelore, mas não dos Nelore de origem materna distinta. No entanto, a freqüência de alelos indicus específicos nos microssatélites e de padrões de digestão do DNA satélite também indicus específicos sugerem uma participação da ordem de aproximadamente 6% do genoma taurus na população de gado Nelore. / Bos indicus specie, especially Nellore breed is responsible for the majority of Brazilian tropical herd. These animals are notably capable to endure parasite infection as well as hot weather and low quality feed. In one hand this qualities suggest the large scale application of this breed, but in other hand this same breed is well characterized as bad food converter and consequently far from having good precocity status compared with its Bos taurus homologues. It has been reported a matrilineal European participation in Zebu cattle since its introduction in American lands. This hybridization is confirmed by the majority contribution of Bos taurus mtDNA in these animals. Although in a much lower frequency, we hypothesize a Bos taurus cow participation in nuclei genome. The main aim of this work was to give the firsts steps towards the estimation of this participation. A total of 104 Nellore and 8 animals of different European breeds were used for DNA analysis. Five microsatellites fragments (HEL1, HEL9, ETH225, ILSTS005 e INRA063) were amplified applying primers with fluorescent dye. Amplified fragments were used in 6% polyacrilamide electrophoresis and visualized after laser excitation. Overall 23 alleles were detected averaging 4.6±1,82/locus. Variance components of microsatellites allele size comparisons allowed the formation of two clusters separating both subspecies. No significant variation was observed between Nellore with different maternal origins. A satellite 1711b DNA was also amplified and digested with the restriction enzyme Msp I. Three possible genotypes were identified in Nellore animals harboring B. taurus and B. indicus mtDNA. European originated animals always showed the same restriction pattern. Finally B. indicus specific microsatellite allele and satellite 1711b digestion patterns frequency allowed the estimation of 6% of B. taurus contribution in purebred Nellore. These results are discussed in terms of application in cattle genetic improvement.

O grupo Characidium lauroi pertence ao gênero Characidium e é formado por seis espécies, C. lauroi, Characidium sp. piabanha, C. japuhybense, C. oiticicai, C. schubarti, e Characidium sp. iguaçu, que se distribuem nas bacias do Rio Paraíba do Sul, Drenagens Costeiras da Baía de Ilha Grande, Rio Tietê, Rio Paranapanema e Rio Iguaçu, respectivamente. Os estudos disponíveis sobre as relações evolutivas entre as espécies que compõem esse grupo baseiam-se em dados morfológicos que, entretanto, não foram suficientes para resolver as relações entre C. oiticicai, C. schubarti e Characidium sp. iguaçu. Estudos citogenéticos ou moleculares não foram aplicados até agora às espécies do gênero Characidium com o intuito de verificar a existência do grupo C. lauroi. Técnicas que envolvem o uso de DNAmt, como PCR-RFLP, DNA barcoding e sequenciamento podem auxiliar na resolução dessa questão, uma vez que as espécies do gênero Characidium formam um grupo interessante para a aplicação dessas técnicas, principalmente por apresentar algumas espécies com limites taxonômicos muito próximos, como as espécies que compõem o grupo C. lauroi. No presente estudo, técnicas de citogenética clássica e molecular, assim como a técnica de PCR-RFLP, o uso do DNA barcoding e sequenciamento foram utilizados na tentativa de analisar e elucidar as relações filogenéticas entre as espécies do gênero Characidium, principalmente com enfoque nas relações entre as espécies do grupo C. lauroi. Os resultados citogenéticos encontrados indicam que esse grupo de espécies pode ser definido pela presença de cromossomos sexuais diferenciados do tipo ZZ/ZW portadores de cístrons ribossomais e pela presença de um cariótipo relativamente estável. No entanto, essas características não estão restritas ao grupo C. lauroi. Os resultados de PCR-RFLP, assim como os resultados obtidos por meio do DNA barcoding e sequenciamento, indicam que as espécies C. lauroi e C. sp. piabanha podem formar uma única espécie, da mesma forma que as populações de C. schubarti. No entanto, as espécies do grupo C. lauroi não formam um grupo monofilético. A relação entre as espécies do grupo C. lauroi com C. pterostictum e C. lanei pode indicar que essas espécies tenham passado por uma separação recente e que, não necessariamente essas duas espécies façam parte do grupo C. lauroi, mas sim que fazem parte de um grupo maior de espécies do gênero Characidium, o que é corroborado até por dados citogenéticos. Comparações entre as topologias encontradas com base em dados morfológicos e moleculares e suas implicações na delimitação e nas relações encontradas entre as espécies do grupo C. lauroi são discutidas. / The group Characidium lauroi belongs to the genus Characidium and consists of six species, C. lauroi, Characidium sp. piabanha, C. japuhybense, C. oiticicai, C. schubarti and Characidium sp. iguaçu; which are distributed in the basins of the Paraiba do Sul River, Drainage Coastal Bay of Ilha Grande, Tietê River, Paranapanema River and Iguaçu River, respectively. Available studies on the evolutionary relationships between species that make up this group are based on morphological data, however, were not sufficient to address the relationship between C. oiticicai, C. schubarti and Characidium sp. iguaçu. Cytogenetic and molecular studies have not been applied so far to the species of the genus Characidium in order to verify the existence of group C. lauroi. Techniques that involve the use of mtDNA, such as PCRRFLP, DNA barcoding and sequencing may help in resolving this issue, since the genus Characidium form an interesting group for the application of these techniques, mainly for some species with taxonomic boundaries very close, as the species that make up group C. lauroi. In this study, techniques of classical and molecular cytogenetics, as well as PCR-RFLP, the use of DNA barcoding and sequencing were used in an attempt to analyze and elucidate the phylogenetic relationships among species of the genus Characidium, mainly focusing on relations between species of group C. lauroi. The cytogenetic results obtained indicate that this group of species can be defined by the presence of sex chromosomes differentiated ZZ / ZW carrying ribosomal cistrons and the presence of a relatively stable karyotype. However, these features are not restricted to the group C. lauroi. The results of PCR-RFLP and the results obtained by barcoding DNA and the sequencing, indicate that C. lauroi and Characidium sp. piabanha may form a single species, in the same way that populations of C. schubarti. However, the species of group C. lauroi not form a monophyletic group. The relationship between species of group C. lauroi with C. pterostictum and C. lanei may indicate that these species have experienced a recent separation and, not necessarily these two species are part of group C. lauroi, but they are part of a larger group of species of the genus Characidium, which is supported even by cytogenetic data. Comparisons between the topologies found based on morphological and molecular data and its implications for delimitation and relationships found between species of group C. lauroi are discussed.

O fungo dimórfico Paracoccidioides brasiliensis, é o agente etiológico da paracoccidioidomicose, uma das micoses sistêmicas humanas mais prevalentes na América Latina. Componentes da cadeia respiratória mitocondrial são potenciais alvos quimioterapêuticos. Nesse sentido, em nosso trabalho demonstramos diferenças entre a cadeia respiratória do fungo P. brasiliensis e do hospedeiro mamífero. A respiração, potencial de membrana e fosforilação oxidativa mitocondrial de esferoplastos de P. brasiliensis foram avaliados in situ, os quais demonstraram a presença de uma cadeia respiratória funcional. A adição de ADP induziu a transição da respiração do estado de repouso para o fosforilativo, em esferoplastos energizados com succinato, NADH, NAD+ e substratos ligados ao complex I. A presença de uma NADH-ubiquinona oxidoredutase foi demonstrada pela capacidade das células oxidarem NADH exógeno, assim como, pela respiração insensível a rotenona e sensível a flavona. Além disso, a respiração mantida por NAD+ foi sensível a rotenona e flavona, sugerindo que vias metabólicas citosolicas contribuem para a produção de NADH e substratos ligados ao complexo I. Foi demonstrado também que os níveis de expressão do complexo I e da NADH desidrogenase alternativa alternam-se durante a curva de crescimento de leveduras de P. brasiliensis. A respiração induzida por NADH ou succinato foi parcialmente inibida por KCN ou antimicina A e sensível à inibição por BHAM, indicando a presença de uma oxidase alternativa. A fim de se caracterizar esta enzima oxidase alternativa, o seu gene foi clonado e expressado em xii S. cerevisiae e E. coli, o qual conferiu uma respiração resistente a cianeto e sensível a BHAM. S. cerevisiae expressando oxidase alternativa mostraram uma menor taxa de multiplicação celular e diminuição na geração de EROs. Nós também observamos que inibidores da cadeia transportadora de elétrons retardaram a transição de micélio para levedura em culturas de P. brasiliensis. Além disso, estes inibidores e outras drogas geradoras de EROs aumentaram a expressão do gene da oxidase alternativa, assim como a produção de EROs em leveduras de P. brasiliensis. / Paracoccidioides brasiliensis, a thermically dimorphic fungus, is the etiological agent of endemic paracoccidioidomycosis, one of the most prevalent human systemic mycosis in Latin America. Components of the respiratory chain constitute potential pharmacological targets, and here are reported differences between the respiratory chain of the mammalian host and the fungus P. brasiliensis. Respiration, membrane potential and oxidative phosphorylation of mitochondria from P. brasiliensis spheroplasts were evaluated in situ, which demonstrated the existence of a functional respiratory chain. Adenosine 5\'-diphosphate (ADP) induced a transition from resting to phosphorylating respiration in mitochondria energized by succinate, NADH, NAD+ and complex I linked substrates. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: the ability of the fungus to oxidize exogenous NADH; the insensitivity substrate-supported respiration to rotenone and sensitivity of this respiration to flavone. In addition, the sensitivity of NAD+-supported respiration to rotenone and flavone suggest that citosolic pathways contribute to NADH and complex I linked substrates production. Moreover, it was demonstrated that expression levels of complex I and alternative NADH dehydrogenase change during P. brasiliensis growth curve. The partial sensitivity of NADH or succinate-supported respiration to antimycin A and cyanide, as well as the sensitivity to BHAM, indicates the presence of an alternative oxidase. Therefore, to characterize the alternative oxidase its gene was cloned and heterologously xii expressed in S. cerevisiae and E. coli, wich confered, a cyanide resistant respiration and BHAM sensitivity. Moreover, S. cerevisiae that expressed alternative oxidase showed slower growth rate and decreased ROS generation. We also observed that electron transport pathways inhibitors delayed the P. brasiliensis mycelium to yeast transition in cultures. Besides, these inhibitors and other ROS generated drugs increase the ROS production and altenative oxidase expression.

O gênero Noctilio pertence à superfamília Noctilionoidea, família Noctilionidae, e inclui atualmente duas espécies de distribuição Neotropical, N. leporinus e N. albiventris. Estas ocorrem em simpatria nas áreas de planície desde a costa pacífica do México até o Norte da Argentina e Uruguai. N. leporinus possui características morfológicas externas e funcionais que o tornam adaptado à piscivoria, apesar de trabalhos citarem para sua alimentação um consumo equivalente de insetos, crustáceos e aracnídeos. N. albiventris possui hábito insetívoro, apresentando características morfológicas externas que se assemelham muito a N. leporinus, sendo apenas de menor tamanho. As duas espécies apresentam variação morfológica ao longo de sua distribuição geográfica, com a descrição de subespécies para ambas. Dados moleculares foram utilizados recentemente para investigar as relações intragenéricas em Noctilio, indicando uma origem recente para N. leporinus e a parafilia de N. albiventris. O presente trabalho teve como objetivo a caracterização genética intraespecífica e a determinação do padrão filogeográfico das espécies do gênero Noctilio, verificando a possibilidade de existência de mais que duas linhagens evolutivas para o táxon. A amostragem incluiu 63 indivíduos de N. leporinus distribuídos por 35 localidades, e 43 indivíduos de N. albiventris de 19 localidades distintas, tendo sido utilizados como marcadores moleculares o gene mitocondrial citocromo b e a região controle do DNAmit. Os resultados corroboraram a parafilia descrita para N. albiventris, além de evidenciarem níveis de divergência filogenética mais significativos do que o alcançado pelo estudo anterior. Foram encontrados em N. albiventris três filogrupos com alto suporte, igualmente distantes do filogrupo de N. leporinus, as quais apresentam uma correlação geográfica com as subespécies propostas. Os testes de desvio da neutralidade em N. leporinus foram significativos, indicando uma rápida expansão populacional após o surgimento da espécie. Estimativas para o cálculo do ACMR das linhagens evolutivas remetem ao período pleistocênico, tanto para o surgimento de N. leporinus como para os três clados encontrados em N. albiventris. A divergência observada entre estas linhagens variou entre 4,3 e 6,1%. O presente estudo permitiu a confirmação do surgimento recente da espécie N. leporinus a partir de uma linhagem de N. albiventris. A hipótese proposta para a origem da linhagem piscívora do gênero foi a colonização das pequenas Antilhas por uma população de N. albiventris durante os ciclos glaciais do Pleistoceno. Ali a população teria se diferenciado, originando N. leporinus, e posteriormente sofrido uma rápida expansão populacional devido à ocupação de um nicho inexplorado por morcegos neotropicais. / The genus Noctilio belongs to the Superfamily Noctilionoidea, Family Noctilionidae, and currently it includes two species with Neotropical distribution, N. leporinus and N. albiventris. Both species occurs simpatrically in lowland areas from western and eastern Mexico, southward to northern Argentina and Uruguay. N. leporinus have functional and external morphologic characteristics that enables it to piscivory, although many works describe an equivalent consume of arachnids, insects and crustaceans in its diet. N. albiventris is insectivore and resembles N. leporinus in most external and cranial features, but is a smaller species. Both species show morphological variation along their geographic range, congruent with the subspecies distribution. A recent study on molecular phylogeny suggested a recent origin for N. leporinus and to the paraphyly of N. albiventris. The main purpose of this work was the intraspecific genetic characterization and the description of phylogeographic patterns of both species of genus Noctilio, in attempt to investigate how many evolutionary lineages are included in the taxon. Sampling included 63 individuals of N. leporinus from 35 distinct localities and 43 individuals of N. albiventris from 19 localities. The mitochondrial gene cytocrome b and the control region from mtDNA were used as molecular markers. The results corroborate the paraphyly described for N. albiventris, and reveals more significant levels of phylogenetic divergence than previously observed. Three highly supported phylogroups were found within N. albiventris, all of them presenting similar phylogenetic distances to the lineage of N. leporinus. These phylogroups showed geographic structure congruent with the N. albiventris subspecies. The tests against neutrality hypothesis in N. leporinus were significant, suggesting a rapid population growth after the origin of the species. Estimates of the MRCA of the evolutionary lineages dates to the Pleistocene, for both the origin of N. leporinus and all three clades found for N. albiventris. Observed divergence between lineages varied from 4.3 to 6.1%. This study corroborates the recent origin of N. leporinus and its closest phylogenetic relationship with a N. albiventris lineage. The hypothesis for the piscivory in the genus Noctilio was the colonization of the Lesser Antilles by a population of N. albiventris during glacial cycles in the Pleistocene. This population may have differentiated, giving rise to N. leporinus, and then getting through a rapid expansion due to a trophic niche previously unexplored by any neotropical bat lineage.

As tartarugas marinhas são répteis existentes ao longo da costa brasileira, principalmente em áreas de alimentação e anidação. Por possuírem comportamento migratório, estudos ecológicos sobre esses animais são muito difíceis de realizar e muitas questões sobre sua biologia permanecem obscuras. A biologia molecular é uma importante ferramenta, que auxilia os pesquisadores na obtenção de informações valiosas a respeito de estrutura populacional, filogeografia e genealogia de populações naturais. Assim, este trabalho teve como objetivo analisar populações de tartarugas verdes (Chelonia mydas) que freqüentam a região de Cananéia, SP, com base na variedade haplotípica das seqüências gênicas correspondentes ao tRNA-Pro+D-loop do DNA mitocondrial. A área de estudo foi descrita como uma área de alimentação para juvenis de tartarugas verdes, Chelonia mydas, sendo caracterizada pela presença de um estoque misto, composto por animais provenientes de pelo menos seis diferentes áreas de desova analisadas durante quatro anos. A variabilidade haplotípica e nucleotídica é intermediária em relação às demais áreas de alimentação estudadas no Oceano Atlântico, sendo a amostra composta principalmente por indivíduos dos haplótipos CM-08 (63%) e CM-05 (26%). Os demais haplótipos foram relativamente raros, apresentando-se em frequências menores que 5% (CM-01, CM-06, CM-09, CM-10, CM-24, CM-32, CM-36, CM-45). Foi detectada diferença significativa entre as amostras anuais (p < 0.05) e não houve diferença entre as amostras sazonais (p >0.05). Análises indicam que Cananéia encontra-se conectada com as demais áreas de desova e alimentação estudadas no Oceano Atlântico, dependendo destas para o equilíbrio de populações saudáveis e necessitando de atenção, visto o grau atual de ameaça que sofrem. A importância da incorporação de parâmetros ecológicos que contribuam para o entendimento da dinâmica populacional da região, às análises genéticas, é imprescindível para que esses dados sejam validados e tenham utilidade e aplicação direta na proteção das tartarugas marinhas. As informações obtidas através deste trabalho foram utilizadas na elaboração de um documento que será anexado ao plano de manejo do Parque Estadual Ilha do Cardoso e contribuirão para a elaboração do plano de manejo da Área de Proteção Ambiental de Ilha Comprida, de modo que a região de Cananéia seja considerada prioritária na preservação das tartarugas verdes. / The green turtle, Chelonia mydas, is an endangered marine reptile that nests and forages along the Brazilian coast and oceanic islands, among other tropical areas. Due to the highly migratory and oceanic nature of these animals, ecological studies are many times difficult to carry out, and many questions about their biology remain. Molecular genetic analysis is a powerful tool for bridging these gaps, providing valuable information about population structure, phylogeography, and genealogy. This study analyzes mitochondrial DNA sequences (tRNA-Pro + D-loop) from green sea turtles of the Cananéia regional juvenile feeding ground over a four-year period. Significant differences were found between annual samples (p<0.05), however no significant differences were found between seasons (p>0.05). The sample was composed primarily of individuals of haplotypes CM-08 (63%) and CM-05 (26%). All other haplotypes (n = 8) were relative rare, with frequencies lower than 5%. The analysis revealed the area to be a mixed stock, composed of animals drawn from at least six different nesting areas. Because Cananéia is connected to other nesting and feeding grounds throughout the Atlantic Ocean, the health of these populations is interdependent, and threats in one area will likely impact connected sites. This connectivity underscores the importance of understanding regional population dynamics using genetic analysis, with conservation applications. These data will be included in the management plans for the Ilha do Cardoso State Park and the Environmental Protection Area of Ilha Comprida, so that Cananéia will be considered a priority area for the preservation of green sea turtles in Brazil.

Patologias causadas por mutações no DNA mitocondrial (mtDNA) constituem um importante grupo de doenças genéticas em humanos. Todavia, devido ao desconhecimento dos mecanismos que governam a herança mitocondrial, não existem métodos eficientes que permitam prever ou intervir na herança destas patologias. Estudos recentes indicam que mutações no mtDNA são seletivamente eliminadas na linhagem germinativa. O presente projeto investigou se o embrião é capaz de eliminar mitocôndrias disfuncionais durante o desenvolvimento pré-implantação. Para tanto, zigotos de camundongo foram tratados com clorometil-X-rosamina (MitoTracker Red CMXRos) e fotossensibilizados por 0, 2,5, 5, 10, 20 e 60 s. Houve diminuição da taxa de blastocisto, com bloqueio total do desenvolvimento quando a fotossensibilização foi realizada por período igual ou superior a 20 s. A fotossensibilização também resultou em disfunção mitocondrial, como indicado por diminuição do potencial de membrana mitocondrial. No entanto, a transferência de citoplasma de zigotos NZB/BINJ (NZB) fotossensibilizados por 20 s não afetou o desenvolvimento de embriões C57BL/6 (B6). A quantidade de mtDNA NZB também não diferiu entre os zigotos B6, independente de terem recebido citoplasma exposto ou não à fotossensibilização (30,6% ± 1,73 vs. 30,8% ± 1,73). Porém, a quantidade de mtDNA NZB foi menor (P = 0,008) nos blastocistos que receberam citoplasma fotossensibilizado (31,4% ± 1,43 vs. 24,7% ± 1,43). Como a quantidade total de mtDNA não diferiu entre os grupos, estes resultados sugerem que as mitocôndrias disfuncionais introduzidas foram destruídas. A análise de autofagossomos indicou, no entanto, que as mitocôndrias NZB não foram eliminadas por mitofagia. Diferente do esperado, o cultivo na presença de rapamicina reverteu o efeito causado pela introdução de citoplasma fotossensibilizado, resultando em níveis semelhantes de mtDNA NZB em comparação com os blastocistos que receberam citoplasma não fotossensibilizado. Concluiu-se que o embrião de camundongo é capaz de destruir mitocôndrias disfuncionais durante o desenvolvimento à blastocisto. Novos estudos deverão fornecer evidências adicionais e esclarecer os mecanismos moleculares que fundamentam esses achados. / Pathologies caused by mutations in mitochondrial DNA (mtDNA) represent an important group of genetic diseases in humans. Nonetheless, due to our limited understanding of the molecular mechanisms of mitochondrial inheritance there are no efficient methods to predict or intervene in the inheritance of these diseases. Recent studies indicate that mutations in mtDNA are selectively eliminated in the germline. This project investigated the ability of the embryo to target and eliminate dysfunctional mitochondria during early development. To test that, mouse zygotes were treated with chloromethyl-X-rosamina (MitoTracker Red CMXRos) and photosensitized for 0, 2.5, 5, 10, 20 and 60 s. There was a decrease in the rate of blastocyst development and a developmental arrest when the photosensitization was performed for a period equal to or greater than 20 s. Photosensitization also resulted in mitochondrial dysfunction, as indicated by a decreased of mitochondrial membrane potential. However, cytoplasmic transfer from NZB/BINJ (NZB) zygotes photosensitized for 20 s resulted in no effect on development of C57BL/6 (B6) embryos. The amount of NZB mtDNA introduced also did not differ between B6 zygotes, regardless of whether they received or not photosensitized cytoplasm (30.6% ± 1.73 vs. 30.8 ± 1.73%). On the other hand, the amount of NZB mtDNA was lower (P = 0.008) in the blastocysts receiving photosensitized cytoplasm (31.4% ± 24.7% ± 1.43 vs. 1.43). Since the total amount of mtDNA was not different between the groups, these results suggest that dysfunctional mitochondria introduced by cytoplasmic transfer were destroyed. Analysis of autophagosomes indicated, however, that the NZB mitochondria were not eliminated by mitophagy. Different than expected, culture in the presence of rapamycin reversed the effect caused by introduction of photosensitized cytoplasm, resulting in similar levels of NZB mtDNA compared to blastocysts receiving cytoplasm not photosensitized. It was concluded that the mouse embryo may destroy dysfunctional mitochondria during development into blastocysts. Further studies should provide additional evidence and elucidate the molecular mechanisms underlying these findings.

Cheung Shiu-fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / 摘要 --- p.vi / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xviii / ABBREVIATIONS --- p.xix / PUBLICATION --- p.xxi / AWARD --- p.xix / Chapter SECTION 1. --- INTRODUCTION OF HEPATOCELLULAR CARCINOMA --- p.1 / Chapter 1.1 --- Epidemiology of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- Etiologies of HCC --- p.1 / Chapter 1.2.1 --- Hepatitis B Virus and Hepatitis C Virus --- p.2 / Chapter 1.2.2 --- Aflatoxins and Alcohol --- p.3 / Chapter 1.3 --- Major Diagnostic and Prognostic Markers of HCC --- p.4 / Chapter 1.3.1 --- Biochemical Tumor Markers --- p.5 / Chapter 1.3.2 --- Clinico-pathological Features of HCC --- p.6 / Chapter SECTION 2. --- THE MITOCHONDRION --- p.7 / Chapter 2.1 --- Structure of the Mitochondrial Genome --- p.9 / Chapter 2.1.1 --- Nicotinamide Adenine Dinucleotide Dehydrogenase --- p.10 / Chapter 2.1.2 --- Cytochrome b --- p.10 / Chapter 2.1.3 --- Cytochrome c Oxidase --- p.11 / Chapter 2.1.4 --- ATP Synthase --- p.11 / Chapter 2.1.5 --- Ribosomal RNA --- p.11 / Chapter 2.1.6 --- Transfer RNA --- p.12 / Chapter 2.1.7 --- Displacement Loop --- p.12 / Chapter 2.2 --- Replication of Mitochondrial DNA --- p.17 / Chapter 2.3 --- Transcription of Mitochondrial DNA --- p.17 / Chapter SECTION 3. --- PHYSIOLOGY OF MITOCHONDRIA --- p.19 / Chapter 3.1 --- Energy Production by Oxidative Phosphorylation (OXPHOS) --- p.19 / Chapter 3.2 --- Programmed Cell Death: Apoptosis --- p.22 / Chapter 3.3 --- Morphology of Mitochondria in Hepatocytes --- p.25 / Chapter SECTION 4. --- MUTATIONS OF MITOCHONDRIAL DNA --- p.26 / Chapter 4.1 --- Special Terms Used in This Study --- p.26 / Chapter 4.1.1 --- Somatic Mutations and Polymorphisms --- p.26 / Chapter 4.1.2 --- Homoplasmic and Heteroplasmic Mutations --- p.26 / Chapter 4.2 --- Factors Causing High Mutation Frequency in mtDNA --- p.27 / Chapter 4.2.1 --- Presence of Reactive Oxygen Species --- p.27 / Chapter 4.2.2 --- Lack of Protective Histories --- p.28 / Chapter 4.2.3 --- Limited DNA Repair Mechanism --- p.29 / Chapter 4.3 --- Theories of Homoplasmic Mutations --- p.31 / Chapter 4.3.1 --- Replicative Advantage on Mutated mtDNA Sequence Selection --- p.31 / Chapter 4.3.2 --- Random Mutagenesis and Segregation --- p.32 / Chapter 4.4 --- MtDNA Mutations in Mitochondrial Disease and Aging --- p.33 / Chapter 4.5 --- MtDNA Deletions in Cancer --- p.34 / Chapter 4.6 --- Somatic Mutations of MtDNA in Various Cancers --- p.35 / Chapter 4.6.1 --- Frequencies of Somatic Mutations --- p.35 / Chapter 4.6.2 --- Distribution of Somatic Mutations in mtDNA --- p.36 / Chapter 4.7 --- Somatic Mutations of Mitochondrial DNA in HCC --- p.37 / Chapter SECTION 5. --- OBJECTIVES OF THIS STUDY --- p.44 / Chapter SECTION 6. --- MATERIALS AND METHODS --- p.46 / Chapter 6.1 --- Patients and Samples Collection --- p.46 / Chapter 6.2 --- DNA Extraction from Liver Tissues --- p.46 / Chapter 6.3 --- Amplification of Mitochondrial DNA by Polymerase Chain Reaction --- p.51 / Chapter 6.3.1 --- Design of Primers --- p.51 / Chapter 6.3.2 --- PCR Conditions and Contents --- p.54 / Chapter 6.3.3 --- Assessment of PCR Products by Agarose Gel Electrophoresis --- p.54 / Chapter 6.4 --- Purification of PCR Products --- p.54 / Chapter 6.5 --- Cyclesequencing of Mitochondrial DNA --- p.55 / Chapter 6.5.1 --- Design of Primers --- p.55 / Chapter 6.5.2 --- PCR Contents and Cycle Sequencing Procedures --- p.56 / Chapter 6.6 --- Purification of Sequencing Products --- p.56 / Chapter 6.7 --- Sequence Analysis by Automated Sequencer --- p.57 / Chapter 6.7.1 --- Preparation of Polyacrylamide Gel --- p.57 / Chapter 6.7.2 --- Sequence Analysis by Automated Sequencer --- p.58 / Chapter 6.7.3 --- "Search for Sequence Variants, Polymorphisms and Somatic Mutations" --- p.58 / Chapter 6.8 --- Further Studies on mtDNA Mutations --- p.59 / Chapter 6.8.1 --- Sequence Analysis in Buffy Coat --- p.59 / Chapter 6.8.2 --- Detection of the Presence of Somatic mtDNA Mutations in Plasma --- p.59 / Chapter 6.8.3 --- Frequency of Mutations in Two Nucleotide Repeat Sequences --- p.60 / Chapter 6.9 --- Clinical Data and Statistical Analysis --- p.61 / Chapter 6.9.1 --- Clinical and Pathological Data --- p.61 / Chapter 6.9.2 --- Statistical Analysis --- p.61 / Chapter SECTION 7. --- RESULTS --- p.63 / Chapter 7.1 --- Sequence Analysis of the Entire Mitochondrial Genome --- p.63 / Chapter 7.1.1 --- Sequence Variants and Polymorphisms --- p.63 / Chapter 7.1.2 --- Somatic Mutations --- p.71 / Chapter 7.2 --- Study of Mitochondrial Sequence in Lymphocytes --- p.78 / Chapter 7.3 --- Detection of Tumor DNA in Serum --- p.78 / Chapter 7.4 --- Analysis of Nucleotide Repeat Sequences --- p.79 / Chapter 7.4.1 --- General Results --- p.79 / Chapter 7.4.2 --- Statistical Analysis --- p.84 / Chapter SECTION 8. --- DISCUSSION --- p.89 / Chapter 8.1 --- Comparative Analysis of mtDNA Mutations with Two Previous HCC Studies --- p.89 / Chapter 8.1.1 --- Number of Cases and Region Studied --- p.89 / Chapter 8.1.2 --- Number and Distribution of Mutations in Normal Controls --- p.89 / Chapter 8.1.3 --- Number of Somatic Mutations --- p.90 / Chapter 8.1.4 --- Distribution of Somatic Mutations --- p.91 / Chapter 8.2 --- Similarities of Somatic mtDNA Mutations in This Study with Other Cancer Types --- p.93 / Chapter 8.2.1 --- Frequency and Distribution of Somatic Mutations --- p.93 / Chapter 8.2.2 --- Number of Homoplasmic Mutations --- p.93 / Chapter 8.3 --- Evaluation of Somatic Mutations of mtDNA in This Study --- p.96 / Chapter 8.3.1 --- Specificity of Somatic Mutations in Tumor Proved by Sequence Analysis in Lymphocytes --- p.96 / Chapter 8.3.2 --- Importance of Conserved Amino Acid Sequences with Other Species to the Presence of Somatic Mutations in Tumor --- p.96 / Chapter 8.3.3 --- Four Somatic Mutation Sites Are Detected in More than One Cancer Type --- p.101 / Chapter 8.3.4 --- Presence of Homoplasmic and Heteroplasmic Mutations --- p.101 / Chapter 8.3.5 --- Absence of Large-scale Deletions in Tumor Tissues --- p.102 / Chapter 8.4 --- Mutation Hotspots Region: Hypervariable Displacement-loop --- p.103 / Chapter 8.5 --- D310 Mononucleotide Repeats --- p.106 / Chapter 8.5.1 --- Description of D310 Mononucleotide Repeats --- p.106 / Chapter 8.5.2 --- Possible Causes of Varied Sequences at D310 --- p.106 / Chapter 8.5.3 --- Appearance of Nucleotide Repeats at D310 in Tumors --- p.107 / Chapter 8.5.4 --- Possible Outcomes of D310 Aberrations in mtDNA Replication and Transcription --- p.108 / Chapter 8.5.5 --- Comparison of D310 Alternations in HCC with Other Cancers --- p.109 / Chapter 8.6 --- Other Nucleotide Repeat Sequences --- p.112 / Chapter 8.6.1 --- The CA Dinucleotide Repeats --- p.112 / Chapter 8.6.2 --- Other Nucleotide Repeat Sequences Showing Genome Instability --- p.112 / Chapter 8.7 --- Evaluation of Somatic mtDNA Mutations as a Cancer Diagnostic Marker --- p.114 / Chapter 8.7.1 --- Coding Region --- p.114 / Chapter 8.7.2 --- D-loop Region --- p.114 / Chapter 8.7.3 --- D310 Nucleotide Repeats --- p.115 / Chapter 8.7.4 --- Possibility of Detecting Somatic Mutations in Serum --- p.116 / Chapter 8.8 --- Somatic mtDNA Mutations May Be a Prognostic Marker in HCC --- p.117 / Chapter 8.8.1 --- Possible Problems in Current Prognostic Factors --- p.117 / Chapter 8.8.2 --- Interpretation of Results --- p.117 / Chapter 8.8.3 --- Prognostic Values of Somatic Mutations at D310 --- p.118 / Chapter 8.9 --- Hypothesis of Somatic MtDNA Mutations on Tumorigenesis and Tumor Progression --- p.119 / Chapter 8.9.1 --- Somatic mtDNA Mutations Decline OXPHOS and May Inactivate Apoptotic Pathways --- p.119 / Chapter 8.9.2 --- Moderate Reactive Oxygen Species Production May Promote Mitosis --- p.120 / Chapter 8.10 --- Possible Appearance of Somatic Mutations in HCC with Chronic HBV Infection --- p.123 / Chapter 8.11 --- Possibility of HBx Protein Integration to MtDNA Mutations --- p.123 / Chapter 8.12 --- Conclusions --- p.125 / Chapter SECTION 9. --- LIMITATIONS AND FURTHER STUDIES --- p.127 / Chapter 9.1 --- Limitations and Improvements of Study --- p.127 / Chapter 9.1.1 --- Small Sample Size --- p.127 / Chapter 9.1.2 --- Sequence Analysis Method --- p.127 / Chapter 9.1.3 --- Fidelity of PCR Reactions and Long-range PCR Fragments --- p.128 / Chapter 9.2 --- Further Studies --- p.129 / Chapter SECTION 10. --- REFERENCES --- p.131

Mitochondria are some of the most complex organelles of eukaryotic cell. They have their own genome and transcriptional apparatus and maintain several key cellular functions. A substantial part of cellular energetic metabolism happens in the mitochondria, as well as formation of iron-sulfur complexes, synthesis of several key molecules and they are also the essential organelles for the apoptotic pathway. In order to maintain the quality of proteins in their oxidative environment, mitochondria have developed a complex system of proteases that reaches all the mitochondrial compartments that degrade damaged proteins and thus promote mitochondrial turnover. The aim of this work was to characterise function of ClpP subunit of ClpXP matrix protease, which role was not yet extensively investigated in human cells. Therefore, we used RNA-interference to silence expression of ClpP in HEK 293 cells and then we performed rescue experiment during which we reintroduced ClpP in cells. Our results show that the ClpP subunit does not actively participate in apoptotic pathway, nevertheless it is essential for correct assembly of all the respiratory complexes as well as the quality of mitochondria itself. We have also shown that the system of mitochondrial proteases is highly functional and that a lack of ClpP...

Submitted by Francine Silva (francine.silva@unipampa.edu.br) on 2019-03-27T13:53:55Z No. of bitstreams: 1 Ancestralidade e demografia genética de uma amostra da população humana do Rio Grande do Sul.pdf: 850877 bytes, checksum: 04f5841bad6d5ec7f458676b481d270b (MD5) / Made available in DSpace on 2019-03-27T13:53:55Z (GMT). No. of bitstreams: 1 Ancestralidade e demografia genética de uma amostra da população humana do Rio Grande do Sul.pdf: 850877 bytes, checksum: 04f5841bad6d5ec7f458676b481d270b (MD5) Previous issue date: 2013-02-19 / Os marcadores mitocondriais (mtDNA) e cromossomo Y têm sido utilizados para avaliar o grau de miscigenação. No caso da América Latina, três estoques principais originaram a população atual: europeus, ameríndios e africanos. No Rio Grande do Sul além de portugueses e espanhóis, foi marcante a imigração de outros europeus, especialmente alemães e italianos. O presente trabalho busca avaliar a contribuição europeia, ameríndia e africana, para a formação da população gaúcha. Para isso foram utilizados os dois sistemas uniparentais e sobrenomes dos indivíduos. Foram analisados 190 voluntários, nascidos nas sete mesorregiões que compõem o Rio Grande do Sul, dos quais se coletou uma amostra de sangue. Observou-se uma elevada frequência de contribuição europeia (87% no cromossomo Y e 76% no mtDNA), condizente com a vinda de casais de imigrantes portugueses, alemães e italianos. Os dados de sobrenomes demonstram também serem estes os sobrenomes mais encontrados na população gaúcha. / Mitochondrial markers (mtDNA) and Y chromosome have been used to assess degree of miscigenation. In case of Latin America, main three stocks generated current population: Europeans, Amerindians and Africans. In Rio Grande do Sul as well as Portuguese and Spanish, was significant immigration from other European, especially Germans and Italians. This study aims to evaluate the European, Amerindian and African contribution to formation of gaucho population. For this we used the two uniparental systems and surnames of individuals. Has been analysed 190 volunteers, borned in the seven mesoregions has compound Rio Grande do Sul, those colected a blood sample. It was observed that a high frequency of European contribution (87% on the Y chromosome and 76% in mtDNA), consistent with the couple’s coming of Portuguese immigrants, Germans and Italians. Surnames’ data demonstrate also these surnames are most commonly found in gaucho population.

La cellule cardiaque requiert un apport énergétique conséquent qui exige une production et un transfert énergétiques efficaces. Si la production de l’énergie dépend essentiellement des propriétés intrinsèques des mitochondries, il semblerait que l’efficacité du transfert d’énergie du site de production vers les sites consommateurs (ATPases) pourrait être liée à l’architecture spécifique du cardiomyocyte qui conduit à une organisation spatiale singulière des structures internes (mitochondries, réticulum sarcoplasmique, myofilaments). Pour comprendre ce qui lie la cytoarchitecture, la compartimentation cellulaire et la fonction contractile, il a été entrepris d’étudier l’architecture cellulaire et la signalisation énergétique de cardiomyocytes au cours du processus de maturation de la cytoarchitecture et dans un modèle présentant une désorganisation des structures intracellulaires. La première partie de ce travail, réalisée durant le développement postnatal de la souris, a permis de démontré qu’il existe une synchronisation parfaite entre la mise en place de la cytoarchitecture et la maturation fonctionnelle du transfert d’énergie par canalisation directe des nucléotides adényliques entre les mitochondries et les ATPases. Si cette étude apporte un élément qui tendrait à démontrer l’implication de l’architecture cellulaire dans l’efficacité des transferts d’énergie, elle a également mis en avant la maturation très précoce de l’énergétique cellulaire. La mitochondrie faisant partie intégrante de cette architecture et étant modelée par des mécanismes de fusion et de fission, la deuxième étape de ce travail de thèse a consisté à étudier l’implication de la morphologie mitochondriale dans l’énergétique du cardiomyocyte. Il a ainsi été montré que, chez la souris, la diminution d’expression de la protéine OPA1, impliquée dans la fusion mitochondriale, conduit à des perturbations de la morphologie mitochondriale qui n’affectent pas la fonction intrinsèque mitochondriale mais qui altèrent le système de canalisation directe entre les mitochondries et les ATPases des myofilaments. De manière générale, ces résultats démontrent clairement une dépendance des transferts d’énergie à l’architecture cellulaire spécifique de la cellule musculaire cardiaque. / The cardiac cell function requires a large amount of energy and therefore needs a high efficiency of energetic production and energetic transfer. While the energy production depends on the intrinsic properties of the mitochondria, it appears that the efficiency of energetic transfers from the main producers (mitochondria) to consumers (ATPases) could be related to the specific architecture of the cardiomyocyte, which ensures a unique spatial organization of internal structures (mitochondria, sarcoplasmic reticulum, myofilaments). In order to reveal the role of mitochondrial network organization in cardiac energy metabolism, we studied the cellular architecture and the energetic signalling of cardiomyocytes in the process of maturation of the cytoarchitecture and in a model which exhibits a perturbation of the mitochondrial dynamics. The first part of this work, which was performed during postnatal development of the mouse, showed the perfect synchronisation between the establishment of the cytoarchitecture and the maturation of the transfer of energy by direct channelling of adenine nucleotides between mitochondria and ATPases. While this study provides an element which would demonstrate the involvement of cellular architecture in the efficiency of energy transfer, it also highlighted the very early maturation of the energetic system of the cell. Knowing that the mitochondria are an integral part of the cell architecture and that the mitochondrial network is controlled by fusion and fission mechanisms, the second step of this work consisted in investigating the involvement of mitochondrial dynamics in cardiomyocyte energetics. Our work has shown that a decrease in expression of OPA1, a protein responsible for mitochondrial fusion, leads to disruption of mitochondrial morphology which does not affect intrinsic mitochondrial function but affects the direct channelling of ATP and ADP between mitochondria and ATPases of the myofilaments. Overall, these results clearly demonstrate that energy transfer in cardiomyocytes strictly depends on specific cellular architecture.