Contexte et objectif : Plusieurs facteurs de risque de cancer ont été identifiés et il a été estimé que plus de 40% des cas dans les pays développés pourraient être évités en modifiant les facteurs de risque connus. L'objectif général de cette thèse était de démontrer que l’intégration de données génomiques et épigénomiques aux données détaillées sur les expositions environnementales et le mode de vie peut être utile pour identifier des biomarqueurs de ces facteurs et contribuer à augmenter notre connaissance de l'étiologie du cancer. Résultats : Dans un premier temps, nous décrivons comment les signatures génomiques et épigénomiques peuvent être utilisées pour identifier des marqueurs d’exposition et déchiffrer l’étiologie du cancer. Ensuite, nous contribuons au débat relatif à l’hypothèse de la chance dans le développement du cancer et démontrons que les mutations induites par le tabagisme sont plus prédictives du risque de cancer que les mutations aléatoires. Nous introduisons un modèle probabiliste pour la simulation de données mutationnelles et comparons la performance des outils d’identification de ces signatures avec des données réelles et simulées. De plus, nous introduisons une nouvelle méthode pour l’identification des signatures mutationnelles. Enfin, nous utilisons les données de méthylation de la cohorte E3N pour étudier le lien entre l'exposition aux retardateurs de flamme bromés et aux composés perfluorés, deux substances classées parmi les perturbateurs endocriniens, et la méthylation de l’ADN sanguin. Globalement, notre étude ne fournit aucune preuve d'altérations globales du méthylome ou d'altérations à l’échelle des CpGs. Cependant, certains résultats suggèrent l’existence d'altérations de la méthylation de gènes impliqués dans des voies biologiques (ex., la réponse aux androgènes) et nécessitent des recherches supplémentaires.Conclusion : Ce travail contribue à la recherche méthodologique portant sur les signatures mutationnelles en introduisant un protocole de mesure de performance et d’identification des signatures mutationnelles pouvant servir de référence à de futures études méthodologiques ou appliquées. Nos recherches sur les signatures mutationnelles et le méthylome démontrent l'utilité de tels outils pour évaluer les expositions et élucider leur rôle dans l'étiologie du cancer. / Context and aim: Several risks factors have been identified for cancer, and it has been estimated that more than 40% of cases in developed countries are preventable through the modulation of known modifiable risk factors. The overall objective of this thesis was to demonstrate that the analysis of genomic and epigenomic data integrated with well-characterised exposure and lifestyle data may be used to identify markers of environmental exposures and lifestyle and may contribute to increase our understanding of cancer aetiology.Results: We first describe how genomic and epigenomic signatures can be used to identify markers of exposure and decipher the aetiology of cancer. Then, we adopt the mutational signatures framework to contribute to the debate about the “bad luck” hypothesis for cancer and demonstrate that tobacco-related mutations are more strongly correlated with cancer risk than random mutations. We introduce a probabilistic model for the simulation of mutational signature data and compare the performance of the available methods for the identification of mutational signatures using both simulated and real data. Additionally, we introduce a new method for the identification of such signatures. Finally, we use methylation array data in an epidemiological study within the E3N cohort to investigate the association between exposure to Brominated Flame Retardants and Per- and polyfluoroalkyl substances, two organic pollutants that are known endocrine disrupting chemicals, and methylation in DNA from blood. Overall, our study does not provide evidence of methylation alterations at the level of the whole genome, in regions or in single CpGs. Suggestive evidence of alterations in the methylation of genes within plausible biological pathways (e.g. androgen response) warrants further investigations. Conclusion: Our work on the methodological aspects of mutational signature research introduces an original framework for measuring the performance of tools for the identification of mutational signatures that may serve as reference for future methodological or applied research. Our applications of both mutational signature and methylome research demonstrate the usefulness of such tools to assess exposures and elucidate their role in cancer aetiology.

En bioinformatique structurale, la prédiction de la (des) structure(s) secondaire(s) des acides ribonucléiques (ARNs) constitue une direction de recherche majeure pour comprendre les mécanismes cellulaires. Une approche classique pour la prédiction de la structure postule qu'à l'équilibre thermodynamique, l'ARN adopte plusieurs conformations, caractérisées par leur énergie libre, dans l’ensemble de Boltzmann. Les approches modernes privilégient donc une considération des conformations dominantes. Ces approches voient leur précision limitées par l'imprécision des modèles d'énergie et les restrictions topologiques pesant sur les espaces de conformations.Les données expérimentales peuvent être utilisées pour pallier aux lacunes des méthodes de prédiction. Différents protocoles permettent ainsi la révélation d'informations structurales partielles via une exposition à un réactif chimique/enzymatique, dont l'effet dépend, et est donc révélateur, de la (les) structure(s) adoptée(s). Les données de sondage mono-réactif sont utilisées pour valider et complémenter les modèles d’énergie libre, permettant ainsi d’améliorer la précision des prédictions. En pratique, cependant, les praticiens basent leur modélisation sur des données de sondage produites dans diverses conditions expérimentales, utilisant différents réactifs ou associées à une collection de séquences mutées. Une telle approche intégrative est répandue mais reste manuelle, onéreuse et subjective. Au cours de cette thèse, nous avons développé des méthodes in silico pour une modélisation automatisée de la structure à partir de plusieurs sources de données de sondage.En premier lieu, nous avons établi des pipelines d’analyse automatisés pour l'acquisition de profils de réactivité à partir de données brutes produites à travers une série de protocoles. Nous avons ensuite conçu et implémenté une nouvelle méthode qui permet l'intégration simultanée de plusieurs profils de sondage. Basée sur une combinaison d'échantillonnage de l'ensemble de Boltzmann et de clustering structurel, notre méthode produit des conformations dominantes, stables et compatible avec les données de sondage. En favorisant les structures récurrentes, notre méthode permet d’exploiter la complémentarité entre plusieurs données de sondage. Ses performances dans le cas mono-sondage sont comparables ou meilleures que celles des méthodes prédictives de pointe.Cette méthode a permis de proposer des modèles pour les régions structurées des virus. En collaboration avec des expérimentalistes, nous avons suggéré une structure raffinée de l'IRES du VIH-1 Gag, compatible avec les données de sondage chimiques et enzymatiques, qui nous a permis d’identifier des sites d'interactions putatifs avec le ribosome. Nous avons également modélisé la structure des régions non traduites d'Ebola. Cohérents avec les données de sondage SHAPE et les données de covariation, nos modèles montrent l’existence d'une tige-boucle conservée et stable à l'extrémité 5', une structure typiquement présente dans les génomes viraux pour protéger l'ARN de la dégradation par les nucléases.L’extension de notre méthode pour l’analyse simultanée de variants, appliquée dans un premier temps sur des mutants produits par le protocole Mutate-and-Map et sondés par le DMS, a permis d'enregistrer une amélioration en précision de prédiction. Pour éviter la production systématique de mutants ponctuels et exploiter le protocole récent SHAPEMap, nous avons conçu un protocole expérimental basé sur une mutagenèse non dirigé et le séquençage, où plusieurs ARN mutés sont produits et simultanément sondés. Nous avons traité l’affectation des reads aux mutants de références à l'aide d'une instance de l'algorithme "Expectation-Maximization" dont les résultats préliminaires, sur un échantillon de reads réduit/simulé, ont montré un faible taux d’erreurs d'assignation par rapport à une affectation classique des reads aux séquences d'ARN de référence. / In structural bioinformatics, predicting the secondary structure(s) of ribonucleic acids (RNAs) represents a major direction of research to understand cellular mechanisms. A classic approach for structure postulates that, at the thermodynamic equilibrium, RNA adopts its various conformations according to a Boltzmann distribution based on its free energy. Modern approaches, therefore, favor the consideration of the dominant conformations. Such approaches are limited in accuracy due to the imprecision of the energy model and the structure topology restrictions.Experimental data can be used to circumvent the shortcomings of predictive computational methods. RNA probing encompasses a wide array of experimental protocols dedicated to revealing partial structural information through exposure to a chemical or enzymatic reagent, whose effect depends on, and thus reveals, features of its adopted structure(s). Accordingly, single-reagent probing data is used to supplement free-energy models within computational methods, leading to significant gains in prediction accuracy. In practice, however, structural biologists integrate probing data produced in various experimental conditions, using different reagents or over a collection of mutated sequences, to model RNA structure(s). This integrative approach remains manual, time-consuming and arguably subjective in its modeling principles. In this Ph.D., we contributed in silico methods for an automated modeling of RNA structure(s) from multiple sources of probing data.We have first established automated pipelines for the acquisition of reactivity profiles from primary data produced through a variety of protocols (SHAPE, DMS using Capillary Electrophoresis, SHAPE-Map/Ion Torrent). We have designed and implemented a new, versatile, method that simultaneously integrates multiple probing profiles. Based on a combination of Boltzmann sampling and structural clustering, it produces alternative stable conformations jointly supported by a set of probing experiments. As it favors recurrent structures, our method allows exploiting the complementarity of several probing assays. The quality of predictions produced using our method compared favorably against state-of-the-art computational predictive methods on single-probing assays.Our method was used to identify models for structured regions in RNA viruses. In collaboration with experimental partners, we suggested a refined structure of the HIV-1 Gag IRES, showing a good compatibility with chemical and enzymatic probing data. The predicted structure allowed us to build hypotheses on binding sites that are functionally relevant to the translation. We also proposed conserved structures in Ebola Untranslated regions, showing a high consistency with both SHAPE probing and evolutionary data. Our modeling allows us to detect conserved and stable stem-loop at the 5’end of each UTR, a typical structure found in viral genomes to protect the RNA from being degraded by nucleases.Our method was extended to the analysis of sequence variants. We analyzed a collection of DMS probed mutants, produced by the Mutate-and-Map protocol, leading to better structural models for the GIR1 lariat-capping ribozyme than from the sole wild-type sequence. To avoid systematic production of point-wise mutants, and exploit the recent SHAPEMap protocol, we designed an experimental protocol based on undirected mutagenesis and sequencing, where several mutated RNAs are produced and simultaneously probed. Produced reads must then be re-assigned to mutants to establish their reactivity profiles used later for structure modeling. The assignment problem was modeled as a likelihood maximization joint inference of mutational profiles and assignments, and solved using an instance of the "Expectation-Maximization" algorithm. Preliminary results on a reduced/simulated sample of reads showed a remarkable decrease of the reads assignment errors compared to a classic algorithm.

Background: Development of brain metastases in advanced melanoma patients is a frequent event that limits patients’ quality of life and survival. Despite recent insights into melanoma genetics, systematic analyses of genetic alterations in melanoma brain metastasis formation are lacking. Moreover, whether brain metastases harbor distinct genetic alterations beyond those observed at different anatomic sites of the same patient remains unknown. Experimental Design and Results: In our study, 54 intracranial and 18 corresponding extracranial melanoma metastases were analyzed for mutations using targeted next generation sequencing of 29 recurrently mutated driver genes in melanoma. In 11 of 16 paired samples, we detected nucleotide modifications in brain metastases that were absent in matched metastases at extracranial sites. Moreover, we identified novel genetic variants in ARID1A, ARID2, SMARCA4 and BAP1, genes that have not been linked to brain metastases before; albeit most frequent mutations were found in ARID1A, ARID2 and BRAF. Conclusion: Our data provide new insights into the genetic landscape of intracranial melanoma metastases supporting a branched evolution model of metastasis formation.

No description available.

INTRODUÇÃO: O melanoma primário da cavidade oral é uma neoplasia agressiva, rara e originada a partir da proliferação de melanócitos malignos da mucosa. Ele representa aproximadamente de 0,2 a 8% de todos os melanomas. Estudos recentes apontam algumas vias moleculares tem sido encontradas por estarem envolvidas na patogenia dos melanomas. Dentre essas vias destaca-se a via proliferativa da MAPK (mitogen activated protein kinase), esta cascata de sinalização está envolvida no controle do crescimento celular, proliferação e migração, e tem sido relacionada com um papel importante no desenvolvimento e progressão do melanoma cutâneo. OBJETIVOS: Analisar a expressão proteica e mutação pontual dos componentes da via MAPK e correlacionar com os dados clínicos-histológicos. MATERIAL E MÉTODOS: Através da imunoistoquímica avaliar a expressão proteica dos anticorpos RAS; BRAF; MEK1; MEK2; ERK1 e ERK2 em 35 casos de melanomas orais organizados em matriz (TMA: Tissue Microarray) e através de pirosequenciamento avaliar a mutação pontual dos genes BRAF; NRAS; KRAS em 14 casos de melanomas orais. RESULTADOS: Idade dos pacientes entre 9 e 91 anos, sem predileção por sexo, 75% caucasianos, 71,42% acometeram o palato, 80% com aspecto histológico grau III. A análise da expressão proteica foi: RAS (28,57%); BRAF (82,85%); MEK1 (0%); MEK2 (51,43%); ERK1 (20%)e ERK2 (74,28%). Na análise molecular observamos mutações para BRAF (9/14 casos) e NRAS (2/14 casos). CONCLUSÃO: Todos os aspectos da via MAPK necessita de outras elucidações em melanomas de áreas foto-protegidas e melanomas de mucosa e comparando diferentes populações. Entretanto, os resultados deste presente estudo apontam importante alterações na cascata RAS-RAF-MEK-ERK e estes são indicadores de prognóstico ruim em melanomas primários da mucosa oral, independente da exposição solar / BACKGROUND: Primary melanoma of the oral cavity is an aggressive and rare neoplasm and originated from the proliferation of malignant melanocytes of the mucosa. It represents approximately 0.2 to 8% of all melanomas. Recent studies indicate some molecular pathways have been found to be involved in the pathogenesis of melanomas. Among these means there is a proliferative MAPK pathway (\"mitogen activated protein kinase\"), this signaling pathway is involved in controlling cell growth, proliferation and migration, and it has been associated with a role in the development and progression of melanoma skin. OBJECTIVES: To analyze protein expression and mutation of components of the MAPK pathway and to correlate with the clinical, histological data. MATERIALS AND METHODS: Using immunohistochemistry to evaluate the protein expression of RAS, BRAF, MEK1, MEK2, ERK1 and ERK2 antibodies in 35 cases of oral melanomas organized array (TMA: Tissue Microarray) and using pyrosequencing to assess the mutation of the BRAF, NRAS, KRAS in 14 cases of oral melanomas. RESULTS: Age of patients between 9 and 91 years, regardless of gender, 75% Caucasian, 71.42% in palate, 80% with histologic grade III. Analysis of protein expression was: RAS (28.57%); BRAF (82.85%); MEK1 (0%), MEK2 (51.43%); ERK1 (20%) and ERK2 (74.28%). Molecular analysis we found BRAF mutations (9/14 cases) and NRAS (2/14 cases). CONCLUSION: All aspects of the MAPK pathway requires further elucidation in melanomas of photo-protected areas and mucosal melanomas and comparing different populations. However, the results of this study indicate important changes in the cascade RAS-RAF-MEK-ERK and these are indicators of poor prognosis in primary melanomas of the oral mucosa, regardless of sun exposure

The process of protein and RNA folding has been understood in general terms through the principle of minimal frustration, and is usually thought of as being guided by a folding funnel on the energy landscape, which is based around the native structure. However, more recently, various biomolecules have been associated with multifunnel energy landscapes, where each funnel exhibits a distinct structural ensemble and function. This work explores how the principle of minimal frustration may be extended to multifunnel energy landscapes that are associated with multifunctional biomolecules. To achieve this aim, the computational potential energy landscape framework is employed to analyse four example systems. Additionally, this study analyses mutants for all four systems, where the mutations are chosen to change properties of the systems without destabilising the native sequence ensemble entirely. The first system considered is a two-state coiled-coil. It is shown how mutations fundamentally change the energy landscape from the minimal frustrated organisation necessary to fulfil biological function. These changes can introduce alternative pathways for folding, as well as new structural ensembles. Similar effects are observed for ubiquitin. In addition, the landscape exploration allows us to calculate a number of experimentally determined properties for this protein, which exhibit excellent agreement, and we characterise folding at an atomistic level of detail. Next we consider the hormones oxytocin and vasopressin, which are themselves mutants of each other, along with a number of other mutants for both molecules. Again, the frustration in the landscape increases due to mutations, and a greater variety in the resulting structural ensembles is observed, leading to changes in binding affinities. Finally, the HP1 loop of RNA 7SK is analysed, revealing that the principles established for the energy landscapes of proteins extend to nucleic acids. Overall, the results indicate that sequences have evolved to exhibit the minimum number of funnels on the energy landscape to support multiple functions, extending the principle of minimal frustration to multifunnel energy landscapes.

Cystinuria is defined as an inherited disorder characterized by increased urinary excretion of cystine and the dibasic amino acids arginine, lysine and ornithine. The only clinical manifestation of cystinuria is renal cystine stone formation due to the low solubility of cystine in the urine. Cystinuria can be attributed to mutations in the SLC3A1 and SLC7A9 genes in the majority of all cases and it has been a common expectation that molecular genetic studies of cystinuria would aid in understanding of the varying clinical outcome seen in the disease. Besides human, the disease has been most extensively studied in the domestic dog. The present study was undertaken to investigate the molecular genetic basis of cystinuria in patients from Sweden and to correlate genetic findings with phenotypes produced regarding cystine and dibasic amino acid excretion. Further, attempts were made to elucidate the molecular genetics of cystinuria in the dog. The entire coding sequences of the SLC3A1 and SLC7A9 genes were analysed by means of SSCA and DNA sequencing in 53 cystinuria patients and genetic findings were related to urinary excretion of cystine and dibasic amino acids in a subset of the patient group. We detected a total number of 22 different mutations in the SLC3A1 and SLC7A9 genes, 18 of which were described for the first time. We have found a probable genetic cause of cystinuria in approximately 74 % of our patients and a possible contribution to the disease in another 19 %. Mutations in the SLC3A1 gene is the major cause of cystinuria in our group, with only a minor contribution of SLC7A9 mutations. The group of patients presenting SLC3A1 mutations in a heterozygous state or lacking mutations in both genes had higher values of total urinary cystine and dibasic amino acids compared to patients homozygous for SLC3A1 mutations. The reason for this discrepancy remains unclear, but the possible impact of medical treatment with sulfhydryl compounds on total cystine values was ruled out. Sequencing of the full-length canine SLC7A9 cDNA was accomplished using the RACE technology and results from mutation analyses of SLC7A9 and SLC3A1 in cystinuric dogs showed that only two out of 13 dogs have mutations with possible impact on protein function in these genes. DNA sequencing was used for all exons of both genes in the dog, and in human cystinuria patients, all samples lacking mutations or showing heterozygosity after SSCA screening were sequenced in both genes as well. This implies that all point mutations present have been detected, but the possibility of mutations escaping PCR based methods as well as mutations in regulatory parts of the SLC3A1 and SLC7A9 genes remains in cases lacking a full molecular genetic explanation of the disease. Finally, clinical and genetic data from our study of cystinuria both in man and dog exemplifies that manifestation and clinical severity of cystinuria is not determined by genetic alterations in the SLC3A1 and SLC7A9 alone. Environmental factors, congenital malformations and modulating genetic factors are all possible contributors to the clinical outcome of cystinuria.

The study of bacterial evolution is fundamental for addressing current problems of antibiotic resistance and emerging infectious diseases and lays a solid foundation for successful and rational design in biotechnology and synthetic biology. The main aim of this thesis is to test evolutionary hypotheses, largely based on theoretical considerations and sequence analysis, by designing scenarios in a laboratory setting to obtain experimental data. Paper I examines how genomic GC-content can be reduced following a change in mutation rate and spectrum. Transcription-related biases in mutation location were found, but no replicative bias was detected. Paper II explores the distribution of fitness effects of random substitutions in two ribosomal protein genes using a highly sensitive fitness assay. The substitutions had a weakly deleterious effect, with low frequencies of both neutral and inactivating mutations. The surprising finding that synonymous and non-synonymous substitutions have very similar distribution of fitness effects suggests that, at least for these genes, fitness constraints are present mainly on the level of mRNA instead of protein. Paper III examines selective barriers to inter-species gene transfer by constructing mutants with a native gene replaced by an orthologue from another species. Results suggest that the fitness costs of these gene replacements are large enough to provide a barrier to this kind of horizontal gene transfer in nature. The paper also examines possible compensatory mechanisms that can reduce the cost of the poorly functioning alien genes and found that gene amplification acts as a first step to improve the selective contribution after transfer. Paper IV investigates the fitness constraints on horizontal gene transfer by inserting DNA from other species into the Salmonella chromosome. Results suggest that insertion of foreign DNA often is neutral and the manuscript provides new experimental data for theoretical analysis of interspecies genome variation and horizontal gene transfer between species.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

INTRODUÇÃO: O melanoma primário da cavidade oral é uma neoplasia agressiva, rara e originada a partir da proliferação de melanócitos malignos da mucosa. Ele representa aproximadamente de 0,2 a 8% de todos os melanomas. Estudos recentes apontam algumas vias moleculares tem sido encontradas por estarem envolvidas na patogenia dos melanomas. Dentre essas vias destaca-se a via proliferativa da MAPK (mitogen activated protein kinase), esta cascata de sinalização está envolvida no controle do crescimento celular, proliferação e migração, e tem sido relacionada com um papel importante no desenvolvimento e progressão do melanoma cutâneo. OBJETIVOS: Analisar a expressão proteica e mutação pontual dos componentes da via MAPK e correlacionar com os dados clínicos-histológicos. MATERIAL E MÉTODOS: Através da imunoistoquímica avaliar a expressão proteica dos anticorpos RAS; BRAF; MEK1; MEK2; ERK1 e ERK2 em 35 casos de melanomas orais organizados em matriz (TMA: Tissue Microarray) e através de pirosequenciamento avaliar a mutação pontual dos genes BRAF; NRAS; KRAS em 14 casos de melanomas orais. RESULTADOS: Idade dos pacientes entre 9 e 91 anos, sem predileção por sexo, 75% caucasianos, 71,42% acometeram o palato, 80% com aspecto histológico grau III. A análise da expressão proteica foi: RAS (28,57%); BRAF (82,85%); MEK1 (0%); MEK2 (51,43%); ERK1 (20%)e ERK2 (74,28%). Na análise molecular observamos mutações para BRAF (9/14 casos) e NRAS (2/14 casos). CONCLUSÃO: Todos os aspectos da via MAPK necessita de outras elucidações em melanomas de áreas foto-protegidas e melanomas de mucosa e comparando diferentes populações. Entretanto, os resultados deste presente estudo apontam importante alterações na cascata RAS-RAF-MEK-ERK e estes são indicadores de prognóstico ruim em melanomas primários da mucosa oral, independente da exposição solar / BACKGROUND: Primary melanoma of the oral cavity is an aggressive and rare neoplasm and originated from the proliferation of malignant melanocytes of the mucosa. It represents approximately 0.2 to 8% of all melanomas. Recent studies indicate some molecular pathways have been found to be involved in the pathogenesis of melanomas. Among these means there is a proliferative MAPK pathway (\"mitogen activated protein kinase\"), this signaling pathway is involved in controlling cell growth, proliferation and migration, and it has been associated with a role in the development and progression of melanoma skin. OBJECTIVES: To analyze protein expression and mutation of components of the MAPK pathway and to correlate with the clinical, histological data. MATERIALS AND METHODS: Using immunohistochemistry to evaluate the protein expression of RAS, BRAF, MEK1, MEK2, ERK1 and ERK2 antibodies in 35 cases of oral melanomas organized array (TMA: Tissue Microarray) and using pyrosequencing to assess the mutation of the BRAF, NRAS, KRAS in 14 cases of oral melanomas. RESULTS: Age of patients between 9 and 91 years, regardless of gender, 75% Caucasian, 71.42% in palate, 80% with histologic grade III. Analysis of protein expression was: RAS (28.57%); BRAF (82.85%); MEK1 (0%), MEK2 (51.43%); ERK1 (20%) and ERK2 (74.28%). Molecular analysis we found BRAF mutations (9/14 cases) and NRAS (2/14 cases). CONCLUSION: All aspects of the MAPK pathway requires further elucidation in melanomas of photo-protected areas and mucosal melanomas and comparing different populations. However, the results of this study indicate important changes in the cascade RAS-RAF-MEK-ERK and these are indicators of poor prognosis in primary melanomas of the oral mucosa, regardless of sun exposure