Cerca de 10% dos casos de câncer de mama e/ou ovário são caracterizados como hereditários, onde a presença de mutações germinativas no gene de suscetibilidade BRCA1 aumenta o risco de desenvolver esses cânceres durante a vida da mulher. O BRCA1 é um gene supressor tumoral envolvido na resposta de danos ao DNA, controle do ciclo celular, na remodelação da cromatina, ubiquitinação e regulação da transcrição. O presente estudo tem como objetivo central caracterizar as mutações do gene BRCA1 associadas a Síndrome Hereditária do Câncer de Mama e/ou Ovário (HBOC) em pacientes atendidos no Serviço de Aconselhamento Genético do Câncer do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP/USP). Os vinte e dois éxons codificantes do BRCA1 foram analisados utilizando o método de High Resolution Melting (HRM) para triagem de mutações pontuais, seguido pelo sequenciamento de DNA dos casos selecionados para validação. A técnica de MLPA (Multiplex Ligation-dependent Probe Amplification) também foi usada para detectar grandes deleções e duplicações. Uma vez confirmada a mutação, membros da família considerados de alto risco, serão investigados para a mutação específica, a fim de proporcionar-lhes um aconselhamento genético apropriado para a detecção precoce do câncer. No presente estudo, foram investigados 41 pacientes que preencheram os critérios para o teste genético de acordo com NCCN Clinical Practice Guidelines in Oncology v.1.2010. Um total de 21 mutações foram identificadas, duas das quais são patogênicas: a deleção dos éxons 17-18 e a deleção dos éxon 19. Ambas estão localizadas no domínio BRCT do gene BRCA1, essencial para a ligação de fosfoproteínas críticas para a ativação do complexo de reparo do DNA. Outra mutação, a S616del, foi tratada como patogênica, mas apresenta informações controversas em diferentes estudos. O trabalho também identificou uma nova mutação, Val1117Ile. Um estudo de haplótipos das mutações identificadas nos pacientes foi realizado e revelou que um dos haplótipos, denominado de 6, contendo quatro resíduos mutados (871Leu, 1038Gly, 1183Arg e 1613Gly) estava presente em 50% das pacientes. O estudo de associação com 82 indivíduos saudáveis, mostrou diferença significativa (p=0,026) nos pacientes, sugerindo assim um risco aumentado de HBOC. Adicionalmente, foi analisada a mutação germinativa R337H no gene p53 para os casos suspeitos de Síndrome de Li-Fraumeni. Em síntese, o presente estudo contribui com a identificação de uma nova mutação não-sinônina no gene BRCA1 e sugere que o haplótipo 871Leu-1038Gly-1183Arg-1613Gly possa conferir risco aumentado do câncer de mama e/ou ovário em pacientes diagnosticados com HBOC. / About 10% of cases of breast and/or ovary cancer are characterized as hereditary, where the presence of germline mutations in susceptibility BRCA1 gene increases the risk of developing these cancers during womans lifetime. BRCA1 is a tumor suppressor gene involved in DNA damage response, cell cycle control, chromatin remodeling, ubiquitination and transcriptional regulation. The present study aims to characterize BRCA1 gene mutations associated with Hereditary Breast/Ovary Cancer Syndrome (HBOC) in patients from the Cancer Genetic Counseling Service of the General Hospital of the Medical School of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The twenty two coding exons of BRCA1 were analyzed using High Resolution Melting (HRM) method for the screening of point mutations, followed by DNA sequencing of the cases selected to validation. MLPA (Multiplex Ligation-dependent Probe Amplification) technique was also used to detect gross deletions and duplications. Once confirmed the mutation, family members most at risk will be analyzed for the specific mutation in order to provide them with an appropriate genetic counseling for early detection of cancer. In the present study, we investigated 41 patients that fulfilled the criteria for genetic testing according to NCCN Clinical Practice Guidelines in Oncology v.1.2010. A total of 21 mutations were identified, two of them are pathogenic: a deletion of exons 17-18 and a deletion of exon 19. Both of them are located in the BRCT domain of BRCA1 gene, impairing the binding of essential phosphoproteins critical to the activation of DNA repair complex. Another mutation, S616del, shows controversial information about its pathogenesis in different studies.The present study also describes a new mutation, Val1117Ile. A study of haplotypes of the mutations identified in patients was performed and revealed that one of the haplotypes, called 6, containing four mutated residues (871Leu, 1038Gly, and 1183Arg 1613Gly) was present in 50% of patients. The association study with 82 healthy subjects showed a significant difference (p = 0.026) in patients, thus suggesting an increased risk for HBOC. Additionally, the germline mutation R337H on p53 gene was also analyzed in the present study for suspected cases of Li-Fraumeni Syndrome. In summary, this study contributes to the identification of a new missense mutation in the BRCA1 gene and suggests that the haplotype-871Leu-1038Gly 1183Arg-1613Gly may confer increased risk of breast cancer and / or ovarian cancer in patients diagnosed with HBOC.

A mitocôndria é a organela citoplasmática responsável pelo maior sistema produtor de energia, a fosforilação oxidativa (OXPHOS). Foi proposto que em células tumorais a hiper-regulação da glicólise em condições normais de oxigênio (Efeito Warburg), está associada a defeitos na OXPHOS e pode regular o fenótipo tumoral, por exemplo, o potencial metastático da célula por meio da indução de vias pseudohipóxicas durante a normóxia. Estudos recentes mostraram que vários tipos de tumores possuem mutações somáticas em seu genoma mitocondrial, o que pode alterar as funções da OXPHOS levando a troca de metabolismo energético nas células tumorais e induzindo a tumorigênese. Diante disto, o presente trabalho avaliou a instabilidade do genoma mitocondrial em etapas bem definidas da progressão do câncer colorretal. O DNA genômico foi extraído de amostras de adenoma, adenocarcinoma, tecido adjacente e sangue periférico de nove pacientes diagnosticados com Câncer colorretal. O genoma mitocondrial foi amplificado e sequenciado para que fossem feitas as buscas por mutações nas amostras de sangue periférico, adenomas e adenocarcinoma. Foi também medido o número de cópias relativas do mtDNA. Foram encontradas um total de 233 mutações, das quais 162 foram em comum entre os três tecidos avaliados. As amostras de adenocarcinoma foram as que apresentaram uma maior média de mutações por amostra (44,6), seguidas dos adenoma (40,2) e do sangue periférico (34). As amostras de adenocarcinoma apresentaram uma maior instabilidade do mtDNA refletidas a partir de um maior número de mutações somáticas (tanto do tipo InDel como mutações de uma única base), mutações não sinônimas com maior patogenicidade, maior número de mutações em heteroplasmia e com taxa de heteroplasmia elevada. Já as amostras de adenoma apresentaram instabilidade dos seus mtDNA intermediários entre o tecido não tumoral e tumoral, refletindo bem a etapa de modificação celular no qual esses tecidos se encontram. Na análise do número de cópias relativas, as amostras de adenocarcinoma tiveram diminuição no número de cópias relativas quando comparadas com tecido adjacente (p= 0,01) e com adenomas (p= 0,04). Em síntese, o presente trabalho sugere que a instabilidade do genoma mitocondrial parece ter um papel importante no desenvolvimento de tumores colorretais. / The mitochondrion is a cytoplasmic organelle responsible for the major energy producing system, which is the oxidative phosphorylation enzyme pathway (OXPHOS). It was proposed that glycolysis up-regulation during normal oxygen conditions (Warburg effect) may induce defects in the mitochondrial respiration and regulate tumoral phenotypes, for example, metastatic potential through the induction of pseudohipoxic pathways during normoxia. Recent studies have shown that many kinds of tumors have mtDNA somatic mutations, which could alter the OXPHOS functions, leading to changes in glucose metabolismo and improvind tumorigenesis. This study analyzed the mitochondrial genome instability of well defined stages of colorectal cancer. Genomic DNA was extracted from adenoma, adenocarcinoma, adjacente tissue and peripheral blood of patients diagnosed with Colorectal cancer. The mitochondrial genome was amplified and sequenced for mutations screening in adenoma, adenocarcinoma e blood samples. It was also analyzed the relative mtDNA copy number. It was find a total of 233 mutations, which 162 were in common among the three analyzed tissues. The adenocarcinoma samples presented a greater mutation mean per sample (44.6) followed by adenomas samples (40.2) and blood samples (34). The adenocarcinoma samples also shown a greater mitochondrial genome instability refleted by increased of somatic mutations (InDels and single nucleotide variation), non sinonimous mutations with higher patogenicity, increased number of heteroplasmatic mutations and higher heteroplasmatic levels. The adenoma samples showed intermadiate instability of its mtDNA, which well reflects the intermediate stage of cellular modifications of this tissue. The mtDN copy number analysis shown that the adenocarcinoma samples presented decreased number of mtDNA content when compared with adjacente tissue (p= 0.01) and adenoma samples (p= 0.04). In summary the presente study suggests that the mitochondrial genomic instability seems to play an importante role in colorectal tumorigenesis.

Hipercolesterolemia familiar (HF) é uma doença autossômica dominante, caracterizada por elevados níveis plasmáticos da lipoproteína de baixa densidade (LDL), desenvolvimento de xantoma tendíneo e arco corneal, além do aumento do risco de doença coronariana e acidente vascular cerebral prematuros. Frequentemente subdiagnosticada, estima-se que apenas 10% dos 400.000 indivíduos com HF no Brasil têm conhecimento da própria doença; afetando, desta forma, a qualidade e a expetativa de vida dos pacientes. Mutações no gene do receptor da LDL (LDLR) são consideradas as alterações genéticas mais frequentes para o desenvolvimento da hipercolesterolemia familiar, pois comprometem a capacidade de remoção das partículas de LDL circulantes, promovendo seu aumento em níveis plasmáticos. Já foram descritas mais de 1600 mutações diferentes no gene LDLR associadas ao fenótipo da HF; entretanto, ainda é difícil determinar em muitas delas o efeito deletério na atividade do receptor. O objetivo desse estudo foi identificar e caracterizar funcionalmente mutações no gene LDLR não descritas na literatura para determinar sua patogenicidade na hipercolesterolemia familiar. Foi avaliada a atividade residual de LDLR através da captação de LDL marcado com fluoróforo lipofílico em cultura de linfócitos T dos pacientes portadores das mutações analisadas após estimulação dos linfócitos T por mitógenos específicos. As mutações Cys82Ser, Thr404Ser, Gly529Arg e His285Tyr foram consideradas patogênicas por acarretarem diminuição da atividade residual do receptor de LDL. As mutações Glu 602X e His388ProfsX53 confirmaram sua patogenicidade e podem ser considerados como controle positivo para futuros ensaios funcionais. Estudos que esclareçam os mecanismos moleculares da HF e da relação genótipo/fenótipo abrem perspectivas para o desenvolvimento de terapias mais específicas na redução dos níveis de colesterol e, consequentemente, da morbidade e mortalidade associadas às doenças cardiovasculares. / Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevated plasma levels of low-density lipoprotein (LDL), and development of corneal arcus tendinous xanthoma, and increased risk of coronary heart disease and premature stroke. Often misdiagnosed, it is estimated that only 10% of the 400.000 patients with FH in Brazil has knowledge of the disease itself, affecting in this way the quality and life expectancy of patients. Mutations in the LDL receptor (LDLR) are considered the most frequent genetic alterations for the development of familial hypercholesterolemia because compromise the ability of removal of circulating LDL particles, promoting its increase in plasma levels. Have been described over 1600 different mutations in the LDLR gene associated with the phenotype of FH, however, it is still difficult to determine in many of the deleterious effects on receptor activity. The aim of this study was to identify mutations in the LDLR gene and functionally characterize mutations not described in the literature to determine its pathogenicity in familial hypercholesterolemia. The residual activity of LDLR was evaluated by raising LDL labeled with lipophilic fluorophore in cultured T lymphocytes of patients with the analyzed mutations after stimulation of T lymphocytes by specific mitogen. The substitution mutations Cys82Ser, Thr404Ser, Gly529Arg e His285Tyr were considered pathogenic because it causes decrease of the residual activity of the LDL receptor in T lymphocytes. The His388ProfsX53 and Glu602X mutations confirmed their pathogenicity and can be considered as positive control for future functional assays. Studies to clarify the molecular mechanisms of HF and genotype/ phenotype open perspectives for the development of more specific therapies for reducing cholesterol levels, and therefore the morbidity and mortality associated with cardiovascular diseases.

In high-diversity populations, a complete accounting of de novo mutations can be difficult to obtain. Most analyses involve identifying such mutations by sequencing pedigrees on second-generation sequencing platforms and aligning the short reads to a reference assembly, the genomic sequence of a canonical member (or members) of a species. Often, large regions of the genomes under study may be greatly diverged from the reference sequence, or not represented at all (e.g. the HLA, antigenic genes, or other regions under balancing selective pressure). If the haplotypic background upon which a mutation occurs is absent, events can easily be missed (as reads have nowhere to align) and false-positives may abound (as the software forces the reads to align elsewhere). This thesis presents a novel method for de novo mutation discovery and allele identification. Rather than relying on alignment, our method is based on the de novo assembly of short-read sequence data using a multi-color de Bruijn graph. In this data structure, each sample is assigned a unique index (or "color"), reads from each sample are decomposed into smaller subsequences of length k (or "kmers"), and color-specific adjacency information between kmers is recorded. Mutations can be discovered in the graph itself by searching for characteristic motifs (e.g. a "bubble motifs", indicative of a SNP or indel, and "linear motifs" indicative of allelic and non-allelic recombination). De novo mutations differ from inherited mutations in that the kmers spanning the variant allele are absent in the parents; in a sense, they facilitate their own discovery by generating "novel" sequence. We exploit this fact to limit processing of the graph to only those regions containing these novel kmers. We verified our approach using simulations, validation, and visualization. On the simulations, we developed genome and read generation software driven by empirical distributions computed from real data to emit genomes with realistic features: recombinations, de novo variants, read fragment sizes, sequencing errors, and coverage profiles. In 20 artifical samples, we determined our sensitivity and specificity for novel kmer recovery to be approximately 98% and 100% at worst, respectively. Not every novel stretch can be reconstituted as a variant, owing to errors and homology in the graph. In simulations, our false discovery rate was 10% for "bubble" events and 12% for "linear" events. On validation, we obtained a high-quality draft assembly for a single P. falciparum child using a third-generation sequencing platform. We discovered three de novo events in the draft assembly, all three of which are recapitulated in our calls on the second-generation sequencing data for the same sample; no false-positives are present. On visualization, we developed an interactive web application capable of rendering a multi-color subgraph that assists in visually distinguishing between true variation and sequencing artifacts. We applied our caller to real datasets: 115 progeny across four previously analyzed experimental crosses of Plasmodium falciparum. We demonstrate our ability to access subtelomeric compartments of the genome, regions harboring antigenic genes under tremendous selective pressure, thus highly divergent between geographically distinct isolates and routinely masked and ignored in reference-based analyses. We also show our caller's ability to recover an important form of structural de novo variation: non-allelic homologous recombination (NAHR) events, an important mechanism for the pathogen to diversify its own antigenic repertoire. We demonstrate our ability to recover the few events in these samples known to exist, and overturn some previous findings indicating exchanges between "core" (non-subtelomeric) genes. We compute the SNP mutation rate to be approximately 2.91 per sample, insertion and deletion mutation rates to be 0.55 and 1.04 per sample, respectively, multi-nucleotide polymorphisms to be 0.72 per sample, and NAHR events to be 0.33 per sample. These findings are consistent across crosses. Finally, we investigated our method's scaling capabilities by processing a quintet of previously analyzed Pan troglodytes verus (western chimpanzee) samples. The genome of the chimpanzee is two orders of magnitude larger than the malaria parasite's (3, 300 Mbp versus 23 Mbp), diploid rather than haploid, poorly assembled, and the read dataset is lower coverage (20x versus 120x). Comparing to Sequenom validation data as well as visual validation, our sensitivity is expectedly low. However, this can be attributed to overaggressiveness in data cleaning applied by the de novo assembler atop which our software is built. We discuss the precise changes that would likely need to be made in future work to adapt our method to low-coverage samples.

The gene regulatory network (GRN) controls the expression of genes providing phenotypic traits in living organisms. In particular, transcriptional regulation is essential to life, as it governs all levels of gene products that enable cell survival and numerous cellular functions. However, there is still poor understanding of how shifts in gene regulation alter the underlying evolutionary dynamics and consequently generate evolutionary innovations. By employing Wagner's GRN model, this dissertation investigates how the interplay of simple evolutionary forces (mutation and recombination) with natural selection acting on gene regulatory dynamics can generate major evolutionary innovations. In this dissertation, firstly, I review all currently available research papers using Wagner's GRN model, which is also employed as the computational model used extensively in the remaining chapters. I then describe how Wagner's GRN model and its variants are implemented. Finally, network properties such as stability, robustness and path length in initial populations are investigated. In the first study, I explore the characteristics of compensatory mutation in the context of genetic networks. Specifically, I find that 1) compensatory mutations are relatively insensitive to the size and connectivity of the network, 2) compensatory mutations are more likely to occur in genes at or adjacent to the site of a previous deleterious mutation and 3) compensatory mutations are more likely to be driven by mutations with a relatively large regulatory impact. In the second study, I further investigate the evolutionary consequences of the properties of compensatory mutation discovered previously. Specifically, I find that 1) compensatory mutations can occur regardless of patterns of selection, 2) networks with compensatory mutations exhibit proportionately higher robustness when compensatory mutations interact closely with deleterious mutations or have large effects on gene regulation, and 3) regulatory complexity can arise as a consequence of the propensity for co-localised and large-effect compensatory mutations. In the third study, I provide a mechanistic understanding of how recombination benefits sexual lineages. Specifically, I find that 1) recombination together with selection for developmental stability can drive populations towards the optimum, 2) recombination does not frequently disrupt well-adapted lineages as conventionally expected, and 3) recombination facilitates finding good genetic combinations which are robust to disruption, although it also rapidly purges weaker configurations. In the final study, I show that the selection pressure acting on rewiring gene regulation is critical to increasing benefits for sexual lineages whilst mitigating costs of sex and recombination. Specifically, I find that 1) strong selection strength can greatly benefit low-fitness sexual lineages, especially at the early stage, 2) recombination is initially costly, but it can rapidly evolve to compensate for costs of sex and recombination, and 3) sexual lineages with low levels of sex and recombination can outcompete strictly asexual populations under higher selection pressure and lower mutation rates. The results presented for all of the studies are important for mechanistically understanding evolutionary innovations through altering transcriptional regulatory dynamics. These innovations include 1) facilitating alternative pathway evolution, 2) driving regulatory complexity, 3) benefiting sexual reproduction, and 4) resisting invasion against asexual lineages.

A hipercolesterolemia familiar (HF) é uma doença autossômica dominante causada por mutações no gene do receptor de LDL (LDLR) (cromossomo 19p13.1 - p13.3), que alteram parcialmente ou totalmente a função do LDLR. A HF é também uma das doenças genéticas mais comuns com freqüências estimadas de heterozigotos e homozigotos de 1/500 e 1/1.000.000, respectivamente. Manifesta-se com altos níveis de LDL colesterol, arco corneal, xantomas tendíneos e sintomas prematuros de doença coronariana.. A grande heterogeneidade observada na manifestação clínica desta doença pode ser explicada, ao menos parcialmente, pelo amplo espectro de mutações no gene do LDLR. O presente estudo teve por objetivo a caracterização molecular do gene LDLR em pacientes com HF do Rio Grande do Sul (RS), Brasil. Para isso, foram obtidas amostras de DNA de 40 indivíduos provenientes de cinco macrorregiões do Estado, representando seis diferentes populações de ascendência européia, para a realização do seqüenciamento direto do gene do LDLR, com posterior análise por meio das ferramentas de bioinformática. Quinze mutações pontuais foram identificadas no gene do LDLR, a saber: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) e c.2243A>G (D727G) na região codificadora, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11- 69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T e IVS17-42A>G na região intrônica, e *52G>A, *105T>G e *141G>A na região 3\'-UTR. Destas, oito ainda não foram descritas na literatura (três situadas nos exons, quatro nos introns e uma na região 3\'-UTR). A mutação*52G>A foi previamente identificada em pacientes com HF da região Sudeste do Brasil, sugerindo que possa exercer um importante efeito na patogênese da HF em pacientes brasileiros. Em relação às macrorregiões do RS, os portugueses, italianos e espanhóis apresentaram o maior número de mutações dentre os grupos étnicos analisados. Assim, os resultados obtidos confirmam que existe um amplo de espectro de mutações no gene do LDLR. As mutações nas regiões intrônicas precisam ser investigadas sobre seu efeito potencial no desenvolvimento de HF. Considerando que este é o primeiro estudo que teve por objetivo a caracterização molecular de pacientes com HF no RS, novos estudos que visem a elucidação das bases moleculares da HF devem ser realizados, a fim de obter uma melhor caracterização genética desta doença no Brasil. / Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene (chromosome 19p13.1 - p13.3), which alter partially or totally the LDLR function. FH is also one of the most common inherited disorders with frequencies of heterozygotes and homozygotes estimated to be 1/500 and 1/1.000.000, respectively. Affected individuals display high levels of LDL cholesterol, arcus corneae, tendon xanthomas and premature symptomatic coronary heart disease. The extensive heterogeneity observed in the clinical manifestation of this disorder may be explained, at least partially, by the broad spectrum of mutations identified in the LDLR gene. The present study had as the main goal the molecular characterization of the LDLR gene in patients with FH from Rio Grande do Sul (RS) State, Brazil. For this, DNA samples were obtained from 40 individuals living in five macroregions of RS, representing six different isolated populations of European ascendancy. The LDLR gene was subjected to the direct sequencing with further analysis through bioinformatics tools. Fifteen punctual mutations were identified in the LDLR gene, namely: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) and c.2243A>G (D727G) in the coding region, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11-69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T and IVS17-42A>G in the intronic region, and *52G>A, *105T>G and *141G>A in the 3\'-UTR region. Of these, eight were not yet described in the literature (three situated in exons, four in introns and one in 3\'- UTR region). The *52G>A mutation was previously identified in FH patients from Southeast Brazil, suggesting that it can exert an important effect in the pathogenesis of FH in Brazilian patients. In relation to the macroregions of Rio Grande do Sul, Portuguese, Italian and Spanish subjects carried the highest number of mutations among the ethnic groups analyzed. Thus, the results obtained confirm the existence of a broad spectrum of mutations in the LDLR gene. The mutations in intronic regions need to be investigated in relation to its potential effect in the development of FH. Taking into account that this is the first study that had as the goal the molecular characterization of FH patients in RS, further studies aimed at elucidating the molecular bases of FH should be performed, in order to obtain the better characterization of this disease in Brazil.

Atrial fibrillation (AF) is the most common cardiac arrhythmia causing morbidity and mortality. Despite recent advances, developing effective and safe anti-AF pharmaceutical therapies remains challenging and is prone to adverse effects in the ventricles. Atrial-selective therapies are promising in managing AF. A better understanding of the role of the atrial-specific ion channels in the atrial arrhythmogenesis and contractility, as well as the anti-AF effects of blocking these channels is of interests. Also, a 3D ventricle-torso model capable of modelling ventricular electrical activities and the resulting electrocardiogram (ECG) is a valuable tool in evaluating the selectiveness and safety of an anti-AF pharmaceutical therapy. In part I, the role of an atrial-specific ion channel, IKur, in atrial electrical and mechanical activities and the potential of the current as a pharmaceutical target for anti-AF therapies were investigated in silico. The role of IKur in atrial arrhythmogenesis and mechanical contraction was revealed by elucidating the functional impacts of the KCNA5 mutations exerting either gain- or loss-in-function, on the atria. First, novel IKur models were developed and incorporated into multiscale biophysical models of human atrial electrophysiology to assess the effects of mutated IKur on atrial electrical dynamics. Then, a family of single cell human atrial electromechanical models was developed and incorporated into an updated 3D anatomical electromechanical model of human atria to clarify the effects of mutated IKur on the atrial contractile function. Finally, the antiarrhythmic effect of IKur block was assessed together with INa and other K+-current block. It was shown that the gain-of-function in IKur impaired atrial contractility and promoted atrial arrhythmogenesis by shortening the APD, whereas the down-regulated IKur exerted positive inotropic effects and increased the susceptibility of the atria to the genesis of early-afterdepolarisations. Both simulated IKur and INa block in human-AF demonstrated antiarrhythmic effects; the multi-channel block exerted synergistic anti-AF effects and enhanced the AF-selectivity of INa inhibitions. In Part II, a human ventricle-torso model was developed through proposing a new family of single cell ventricular models accounting for transmural, apicobasal and interventricular electrical heterogeneities and integrating an updated 3D biophysical and anatomical model of human ventricles with a heterogeneous anatomical model of a human torso. First, using the model, the role of heterogeneities in the genesis of T-wave was revealed. Then, ECG manifestations of bundle branch block and ventricular ischaemia were simulated. Finally, the platform was applied to investigate the impact of a long-QT-linked mutation (KCNQ1-G269S) on the ventricles and ECG. Good agreement between simulated and experimental/clinical ECG was reached under both normal and diseased conditions. It was shown that the apicobasal heterogeneity had a more pronounced effect on the T-wave than other heterogeneities. Simulations of the KCNQ1-G269S elucidated the causal link between the mutation and ECG manifestations of the patients.

TLE proteins are a closely related family of vertebrate corepressors. They have no intrinsic DNA binding ability, but are recruited as transcriptional repressors by other sequence specific proteins. TLE proteins and their homologues in other species have been implicated in many developmental processes including neurogenesis, haematopoiesis and the formation of major organs. They have also been implicated in early lineage specification in vertebrates but a direct role in this has not been found in mammals. The aim of my PhD is therefore to analyse the function of TLE proteins in early lineage specification and cell fate decisions using mouse embryonic stem cells (ESCs) as a model. The investigation of this has previously been complicated, firstly by the large array of transcription factors that TLEs interact with and secondly by redundancy between similar TLE proteins hindering loss of function approaches. To circumvent these problems, I have used two complementary experimental strategies. The first was identification of point mutations in TLE1 that affect specific classes of DNA binding. Two of these mutations L743F and R534A were of particular interest and were reversibly overexpressed in ES cells to correlate phenotypes to biochemical activity. The second strategy was the mutation of the two primary TLC genes in ES cells and early mouse embryos, TLE3 and TLE4. Complementary evidence from these approaches revealed a role for TLEs in the promotion of ES cell differentiation by repression of pluripotency/self-renewal associated genes. Additionally, neural specification was increased by TLE1 expression especially by the TLE1 point mutations, highlighting opposing roles for negative effects on mesendodermal differentiation. Early mesoderm/primitive streak was increased by loss of TLE, probably through Wnt antagonism. Anterior endoderm was increased by reduced TLE, but a critical level of TLE was still necessary and TLE1 overexpression also upregulated some anterior endoderm markers suggesting both negative and positive roles for TLE proteins in this process.

Le cinéma chinois indépendant se développe dans les années 1980, alors que la Chine vient de commencer une politique d’ouverture et de modernisation. Le pays, dont la croissance est exceptionnelle et continue, devient rapidement une puissance économique émergente. Il connaît ainsi un processus de mutations qui bouleverse ses territoires et les conditions de vie de ses populations. Jia Zhang-ke, qui est l’un des principaux cinéastes indépendants chinois, s’attache tout au long de son œuvre à filmer et à interroger ce processus de mutation. Son projet est de garder une trace de ce que les mutations détruisent, font disparaître, de ce qui le remplace, et également de ce moment même de la mutation. Pour tenter de filmer ce passage, Jia Zhang-ke met en œuvre des dispositifs filmiques d’entre-deux et d’alternance, s’enracinant dans les héritages de l’art pictural chinois et des modernités cinématographiques européennes. Son cinéma, en cela, questionne le processus de mondialisation, ou de globalisation, et participe à l’élaboration de la connaissance, en même temps qu’il peut être considéré comme une forme de résistance. / The independent Chinese cinema develops in the 1980s, while China has just begun a policy of openness and modernization. The country, the growth of which is exceptional and continuous, quickly becomes an emergent economic power. It so knows a process of transformations which upsets its territories and the living conditions of its populations. Jia Zhang-ke, who is one of the main Chinese independent film-makers, works throughout his work to film and to question this process of transformation. His project is to keep a track of what the transformations destroy, remove, of what replaces it, and also of this same moment of the transformation. To try to film this passage, Jia Zhang-ke operates cinematic devices of jump ball and alternation, taking root in the inheritances of the Chinese pictorial art and the European film modernities. His cinema, in that respect, questions the process of globalization, and participates in the elaboration of the knowledge, at the same time as he can be considered as a shape of resistance.

As manifestações bucais da AIDS têm sido relacionadas a diversas características clínicas da infecção pelo HIV como decréscimo de células T CD4+, aumento de carga viral e falha terapêutica, entre outras. Os avanços recentes da medicina mostram que a falha terapêutica, nesses pacientes, está diretamente vinculada a mutações na transcriptase reversa (TR) e na protease (PR). O objetivo deste estudo foi descrever, em pacientes HIV+ apresentando falha terapêutica, o perfil de mutações do vírus e o perfil de resistência a antirretrovirais, e correlacioná-los as manifestações bucais da imunodeficiência. Foram acessados prontuários, laudos de genotipagem e informações de bancos de dados digitais de pacientes com AIDS, que se submeteram a genotipagem no Centro de Referência e Treinamento em Doenças Sexualmente Transmissíveis e AIDS (CRT-DST/AIDS), entre 2003 e 2010. Os dados foram transferidos para o Epiinfo, onde foi construído um banco de dados informatizado para posterior análise estatística. O evento lesões orais foi escolhido como variável dependente. Calculou-se o odds ratio para cada variável independente, utilizando intervalo de confiança de 95%. Foram cruzados dados sobre mutações encontradas no vírus e resistência às medicações com a presença e tipo de manifestações bucais. O teste de Bartlett foi utilizado para testar a normalidade dos dados. Para variáveis sem distribuição normal foram aplicados os testes de Mann-Whitney ou Kruskal-Wallis. Para comparação entre frequências e proporções, foi utilizado o Teste de Exato de Fisher ou o Qui quadrado. O nível de significância foi estabelecido como 0,05 ou 5%. A análise de características sociocomportamentais e clínico laboratoriais permitiu verificar que a presença de lesões orais pode ser relacionada estatisticamente a baixas taxas de CD4 (p<0,05), faixa de carga viral (p=0,048) e ao uso prévio de mais de cinco esquemas antirretrovirais diferentes (p=0,021). Verificou-se maior prevalência de lesões virais (75%) e bacterianas (66,7%) do que de lesões fúngicas (37,3%) apenas em pacientes que apresentavam resistência a inibidores de protease (IP) (p=0,02). Foram encontradas 146 mutações diferentes nos pacientes que apresentavam lesões orais, dentre essas, quatro (101E, 20T, 188L, 93L) apresentaram correlação negativa com a presença de lesões orais (respectivamente, p=0,01, p=0,01, p=0,03, p=0,03) e oito (215Y, 118I, 20R, 44D, 71I, 82I E 84V) apresentaram correlação positiva (respectivamente p=0,04, p=0,05, p=0,03, p=0,01, p=0,01, p=0,04, p=0,0004). Subsequentemente, as mutações que apresentaram correlação positiva com a presença de lesões orais foram avaliadas para verificar se sua presença estaria realmente associada a resistência aos ARVs (aos quais seriam supostamente resistentes). Foram excluídas dessa avaliação as mutações 71I e 82I, por apresentarem uma quantidade extremamente pequena de ocorrências. Todas as mutações apresentaram correlação estatística positiva para a resistência aos respectivos antirretrovirais (p<0,05). Em pacientes HIV+, que apresentavam falha terapêutica e manifestações bucais, foram identificadas as mutações 84V e 20R na PR e as mutações 215Y, 44D e 118I na TR e a presença dessas mutações foi associada a resistência a inibidores de protease e inibidores de transcriptase reversa nucleosídeos, respectivamente. / Oral manifestation of AIDS have been associated with several clinical characteristics of HIV infection such as reduction in T CD4+ cells, increase in viral load and treatment failure, among others. Recent advances have shown that treatment failure in these patients is directly linked to mutations in reverse transcriptases (RT) and in proteases (PR). The objective of the present study was to describe the profile of virus mutations and of resistance to antiretroviral drugs in HIV+ patients in treatment failure, and to correlate mutations to the oral manifestations of the immunodeficiency. Patient charts, genotyping results and information from digital databases of AIDS patients, who underwent genotyping at the Sexually Transmissible Diseases and AIDS Training and Reference Center (CRT-DST/AIDS) between 2003 and 2010, were accessed. Data were transferred to the Epiinfo program, in which a computerized database was built for statistical analysis. The event oral lesions was chosen as a dependent variable. Odds ratio for each independent variable was calculated, using a 95% confidence interval. Data found on virus mutations and drug resistance was analyzed to check for correlation with presence and type of oral manifestations. The Bartlett test was used to test normality of data. Mann-Whitney or Kruskal-Wallis tests were used for variables without a normal distribution. The Fisher Exact or Chi-square Tests were used to compare frequencies and proportions. A 0.05 or 5% significance level was established. The analysis of socio-behavioral and clinical-laboratorial characteristics allowed concluding that the presence of oral lesions may be related to statistically low CD4 rates (p<0.05), viral load range (p=0.048) and previous use of more than five different antiretroviral regimens (p=0.021). A higher prevalence of viral (75%) and bacterial (66.7%) lesions in relation to fungal lesions (37.3%) was observed only in patients who were resistant to protease inhibitors (PI) (p=0.02). We found 146 different mutations in patients with oral lesions, among which, four (101E, 20T, 188L, 93L) with a negative correlation with the presence of oral lesions (p=0.01, p=0.01, p=0.03, p=0.03, respectively) and eight (215Y, 118I, 20R, 44D, 71I, 82I E 84V) with a positive correlation (p=0.04, p=0.05, p=0.03, p=0.01, p=0.01, p=0.04, p=0.0004, respectively). Subsequently, mutations with a positive correlation with the presence of oral lesions were assessed to check if their presence would really be associated with resistance to ARVs (to which they supposedly would be resistant to). Mutations 71I and 82I were excluded from this assessment because they had an extremely low frequency. All mutations had a statistically positive correlation for resistance to their respective antiretroviral drugs (p<0.05). Mutations 84V and 20R were identified in PR, and mutations 215Y, 44D and 118I in TR of HIV+ in patients undergoing treatment failure and presenting oral manifestations. Moreover, the presence of these mutations was associated with resistance to protease inhibitors and to nucleoside reverse transcriptase inhibitors, respectively.