Les cancers MSI (Microsatellite Instable) sont caractérisés par un niveau élevé d'instabilité des séquences répétées de l'ADN, les microsatellites, suite à l'inactivation du système MMR (Mismatch Repair). L'étude de la carcinogenèse MSI a révélé des mutations somatiques (délétion/insertion) de nombreux gènes sur des microsatellites codants, qui décalent le cadre de lecture et engendrent la production de protéines tronquées dont la fonction est le plus souvent perdue. Ces altérations affectent des gènes suppresseurs de tumeurs ou apparentés agissant au niveau de voies de signalisation en rapport avec l'oncogenèse, et sont sélectionnées lorsqu'elles promeuvent le développement tumoral. Ces travaux font état de la découverte de la première mutation d'une chaperonne dans une pathologie tumorale, affectant l'oncogène codant pour HSP110 (Heat Shock Protein) sur un microsatellite non-codant intronique situé dans un site accepteur d'épissage. Cette mutation, conduisant à un saut d'exon, est inéluctable et bi-allélique dans l'ADN tumoral, et entraîne la perte des activités oncogéniques d'HSP110 dans les cellules cancéreuses (effet pro-apoptotique, chimio-sensibilisant, antiprolifératif et de ralentissement de la croissance tumorale). Ces résultats remettent en question les mécanismes de l'oncogenèse MSI et le caractère oncogénique de l'instabilité microsatellitaire. Au niveau physiopathologique et clinique, ils pointent HSP110 comme cible pronostique (facteur prédictif de réponse à la chimiothérapie) et thérapeutique. Je propose une approche de traitement pour les patients avec une tumeur MSI, visant à exacerber le caractère délétère de cette mutation dans les cellules tumorales. / MSI cancers (MicroSatellite Instability) are characterized by widespread instability of DNA repeated sequences, known as microsatellites, due to MMR system (Mismatch Repair) deficiency. Since the detection of this tumor phenotype, most of the oncogenic events reported in these tumors are somatic mutations (1-2 bp insertions or deletions) that affect coding DNA repeats, resulting in frameshifts and inactivation of the corresponding proteins. They accumulate in tumor cells due to positive selection during the MSI-driven tumorigenic process when they promote tumor development by inactivating genes with tumor suppressor-related functions. This work reports the first somatic mutation of a chaperone protein in a cancer so far, i.e. HSP110 (Heat Shock Protein) in MSI colorectal cancer. This mutational event consists in the somatic deletion of the intronic microsatellite, located in the splice acceptor site of HSP110. We demonstrate that it is almost systematic and bi-allelic in these cancers, leading to inactivation of the oncogenic functions of the HSP110 chaperone (pro-apoptotic and anti-proliferative impact leading to chemosensitization of tumor cells and tumor growth decrease). Our findings support an unexpected and paradoxical anticancer impact of the microsatellite instability-driven pathway in mismatch repair-deficient colon cancer. From a pathophysiological and clinical point of view, they highlight HSP110 as a putative relevant prognostic marker (improvement of patients’ response to chemotherapy) and therapeutic target. According to these findings, I propose a therapeutic strategy targeting HSP110 and its mutant for personalized medicine of MSI colon cancer patients.

Genetically structured populations arise when gene flow between groups of individuals is hindered by geographical, behavioural or temporal barriers. The identification of such groups is important for understanding evolution and has large implications for conservation concern. The field of population subdivision has received a lot of interest throughout the years and gained empirical support from a number of species. However, very little is known about population structure at small spatial scales, especially in a highly mobile species such as fish. The main object for my thesis was to further investigate population differentiation, explicitly at small spatial scales in the Eurasian perch. My results show that in this species, genetic differentiation occurs, even at very small spatial scales, both within lakes and in the Baltic Sea. Additionally, the differentiation can be stable over years and thus have a large impact in the evolution of adaptation to different environments. I also found barriers to gene flow that overlapped with the largest change in spring temperature, suggesting a temporal difference in spawning. Morphological differences were found at these small scales as well which indicates that a difference in food resources might be an underlying cause of change. My thesis work shows that the aquatic environment might not be as homogenous as widely thought and that there could be barriers or adaptations to different environments that hinder the fish from genetic panmixia. Slight patterns of isolation by distance (IBD) were found in the Baltic Sea, implying that the distance (i.e. currents) effect the level of differentiation via drifting of larvae and/or small fish. However, I have also theoretically investigated the IBD model of and seen that it is no longer correct when differences in population sizes are introduced. The pattern of IBD can mean high levels of gene flow or no gene flow at all, solely dependent on population size differences and fluctuations. My thesis has resulted in new and important findings regarding the existence and cause of genetic differentiation at very small spatial scales and thus added new knowledge into the field of evolution and speciation. In addition, my results also give insights into the contemporary state of the Eurasian perch and future evolutionary potential.

L'objectif principal de ce travail etait d'explorer les bactéries pathogènes que recèle le tube digestif des gorilles. Pour ce faire, un total de 48 échantillons de selles, provenant du Cameroun, appartenant à 21 gorilles ont été analysés. D'abord la culturomique et le pyroséquençage ont été utilisés pour évaluer exhaustivement la diversité bactérienne. En appliquant la culturomique, 86 conditions de culture dont des milieux fabriqués à base de plantes tropicales, sur un échantillon de selles de gorille, 12 800 colonies microbiennes ont été isolées et testées, et 147 espèces bactériennes identifiées. De nombreux pathogènes opportunistes ont été observés, dont 8 qui sont fréquemment associés à des maladies chez l'homme: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus et Clostridium botulinum. En utilisant la PCR en temps réel pour cribler des pathogènes bactériens dans les 48 échantillons de selles de gorilles, des bactéries fastidieuses telles que Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei ont été observées. Nous avons estimé la prévalence de ces agents pathogènes qui varie entre 4,76% et 85,7%. Ce travail a permis de savoir que l'homme et le gorille ont en commun plusieurs espèces bactériennes dont des pathogènes émergents. Par conséquent, les gorilles sauvages peuvent servir de réservoir et de source pour l'émergence et/ou la réémergence des bactéries pathogènes pour l'homme. / The main objective of this work is to explore the gorilla's potential role as a reservoir for pathogenic bacteria. We used both microbial culturomics and pyrosequencing to analyze the gorilla gut bacteria. By applying culturomics to one index gorilla, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic human pathogens were observed, including 8 frequently associated with human disease: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. Using specific real-time PCR on 48 gorilla fecal samples, we also observed the fastidious pathogens Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei. Using microsatellite analysis of the gorilla samples, we estimated that the prevalence of these pathogens was between 4.76% and 85.7%. Therefore, the gorilla shares many bacterial pathogens with humans, which suggests that wild gorillas might be a reservoir for the emergence and/or reemergence of these pathogens, especially in areas where human and gorilla habitats overlap and because of the increasing presence of humans in the African equatorial forests.

As alterações que a ação antrópica vem causando nos ambientes costeiros tem provocado impactos sobre as espécies a eles associadas. Larus dominicanus é uma espécie de ave marinha amplamente distribuída ao longo do Hemisfério Sul. Por possuir um hábito alimentar generalista, os resíduos da ação antrópica tem beneficiado a espécie, que, assim como outras gaivotas, vem apresentando um crescimento demográfico acelerado. O presente estudo, através do uso de marcadores de microssatélites, mostra que, apesar disso, a espécie possui uma baixa variabilidade genética, com fraca estruturação populacional, que provavelmente são o reflexo da origem recente da espécie e de uma diferenciação recente entre as populações. Múltiplas forças atuam para determinar a estruturação populacional, sendo elas o isolamento por distância, as barreiras físicas e a filopatria. Os sinais de gargalo populacional encontrados em algumas das colônias levantam a possibilidade de efeitos fundadores por colonização recente nas colônias mais ao norte da costa brasileira e redução populacional nas colônias da Argentina e da Antártica como conseqüência da última glaciação. Estes dados chamam a atenção para a necessidade de considerar as informações genéticas para a implantação de planos de manejo. Uma vez que a diferenciação entre as populações é recente, a variabilidade dentro de cada uma delas deve ser mantida. O controle populacional da espécie através de métodos diretos deve ser também acompanhado por planos de manejo ambiental, visando reduzir ou eliminar as condições que propiciam o crescimento desequilibrado dos gaivotões. / The changes in the coast that has been caused by human action has led to impacts on species associated with this environment. Larus dominicanus is a seabird species widely distributed throughout the Southern Hemisphere. The generalist feeding habits allow this species take advantage from human action, leading to population growth, which is also observed in other species of gulls. This study, through the use of microsatellite markers, shows that, despite of the population growth, Larus dominicanus has a low genetic variability, with low population structure, which probably reflects the recent origin of species and a recent differentiation among populations. Multiple forces act to determine the population structure, among them the isolation by distance, physical barriers and philopatry. Some colonies presents a bottleneck sign, raising the hypothesis of recent founder effects in the colonies to the north of Brazil and population reduction of colonies of Argentine and Antarctic as consequence of the last glaciation. These data show the need to consider genetic information for the implementation of management plans. The variability within populations must be maintained, since the differentiation between them is recent. Furthermore, the species population control by direct methods must also be accompanied by environmental management plans, to reduce or eliminate the conditions that favor the unbalanced growth of the gulls

Incidência de melanoma cutâneo maligno (MM) está aumentando em torno de 2,5 a 4% por ano no mundo. Os principais fatores de risco são história familiar de MM, múltiplos nevos benignos ou atípicos, e fatores adicionais como a imunossupressão, sensibilidade solar e exposição à radiação ultravioleta (UV). A instabilidade genômica é responsável pelo acúmulo de mutações que frequentemente estão envolvidas na transformação maligna. Podemos estudar a instabilidade genômica através de duas formas: microssatélites e RAPD (Random Amplified Polymorphic DNA). Na instabilidade genética o DNA repetitivo pode sofrer alterações. Através da Instabilidade de microssatélites (MSI) e da perda da heterozigosidade (LOH) podemos diferenciar tecidos normais de tumorais. A técnica de RAPD (baseada na PCR) produz fingerprints utilizados para detectar instabilidade genômica, polimorfismos, mutações e translocações quando comparados à fingerprints de amostras normais. No estudo de nove microssatélites encontramos um aumento de MSI (p=0.0132). D9S50 apresentou o maior número de alterações (28,5%) em nevos e MMs. D6S252, D9S52 e D9S180 são candidatos à marcador de prognóstico de MM porque apresentaram alterações (MSI + LOH) apenas em MMs. Na análise de 15 primers de RAPD em 12 amostras de MMs obtivemos 100 % de alteração com relação ao número ou posição das bandas. Os primers OPA-2 e OPA-14 são capazes de detectar alterações genéticas nos MMs. Dos padrões obtidos foram encontradas bandas que estavam ausentes nos tumores e estas foram clonadas e seqüenciadas. Estes procedimentos evidenciaram alterações em 9q33 e 12q15. O RAPD propicia o estudo do genoma humano sem a definição prévia de um lócus. Assim, podemos detectar alterações até então desconhecidas aumentando o conhecimento sobre a genômica tumoral. / The incidence of malignant skin melanoma (MM) increases around 2,5 a 4% each year in the world. The main risk factors are family history of MM, multiple benign or atypical nevi, and additional factors such as immunossuppression, sun sensibility and UV exposure. Genomic instability is responsible for a collection of mutations that are frequently involved in malignant transformation, and it can cause alterations in repetitive DNA sequences. There are two ways of studying genomic instability: microsatellites and RAPD (Random Amplified Polymorphic DNA). Through microsatellite instability (MSI) and loss of heterozygosis (LOH) we can separate normal from tumoral tissues. RAPD technique (which is based on PCR) generates fingerprints used for detection of genomic instability, polymorphisms, mutations and translocations that can be compared with fingerprint generated from normal tissue. Studying nine microsatellite, we found an increased MSI (p=0.0132). D9S50 showed the greater number of alterations (28,5%) in nevi and MM. D6S252, D9S52 e D9S180 are candidates for MM prognostic marker for showing alterations (MSI+LOH) in melanomas only. The analysis of 15 RAPD primers in 12 MM samples showed 100% of alteration related to the number or location of the bands. OPA-2 and OPA-14 primers are capable of detecting genetic alterations in MM. In the patterns obtained, two bands which were absent in tumors were found, and they were cloned and submitted to sequencing. These procedures highlighted alterations in loci 9q33 e 12q15. RAPD makes it possible to study the genome without a previous definition of a locus. So we are able to detect alterations so far unknown, increasing our knowledge on tumor genetics.

A Mata Atlântica representa um dos Biomas mais diversos do Planeta. No entanto, ainda pouco se sabe sobre os processos que levaram à alta diversidade de plantas nesta região. A maior parte dos estudos na Mata Atlântica trata este Bioma de forma ampla, sem considerar os diversos tipos vegetacionais encontrados na região. Apesar de ser dominado pela Floresta Pluvial Montana, este Bioma ainda inclui outros tipos vegetacionais periféricos, tais como: as Florestas Pluviais Baixo Montanas, Florestas Pluviais de Altitude, Florestas Pluviais Ripária, Florestas Pluviais em Manchas e as Florestas Paludosas Litorâneas, popularmente conhecidas como \"caxetais\". O nome \"caxetal\" foi dado devido à predominância da espécie Tabebuia Cassinoides (Lam.) DC., a qual é popularmente conhecida como \"caxeta\". Esta formação vegetal tem distribuição restrita a áreas com solos permanentemente encharcados do norte de Santa Catarina até o norte do Espírito Santo, onde ocorre de forma naturalmente fragmentada, formando ilhas ao longo de sua extensão de ocorrência. Populações com distribuição fragmentada são modelos em potencial para estudos de genética de populações, visto que a delimitação geográfica de populações naturais é um dos maiores problemas para estudos dessa natureza. Diante desse cenário, a presente tese teve como objetivo: (1) verificar como a distribuição geográfica da espécie arbórea T. cassinoides, agregada, bem delimitada e fragmentada na paisagem, pode influenciar na estruturação da diversidade genética em nível local; (2) caracterizar o grau de variabilidade genética inter e intrapopulacional em populações naturais de T. cassinoides ao longo da Mata Atlântica; e, (3) determinar a estrutura filogeográfica de T. cassinoides. Para tanto foram analisados dados de oito marcadores de microssatélites nucleares, além do sequenciamento da região trnC-ycf6 do DNA plastidial (cpDNA). Os dados genéticos obtidos foram primeiramente utilizados para o conhecimento da diversidade genética e estruturação da população em nível local, na região de Iguape (SP). Nossos resultados sugerem que a fragmentação dessas populações é somente geográfica, devido a suas necessidades edáficas, bem como que o modo de distribuição agregada e naturalmente fragmentada não indica a subdivisão genética das mesmas. A caracterização da variabilidade genética inter e intra-populacional em populações naturais de T. cassinoides ao longo de toda sua área de distribuição, com base nos marcadores de microssatélites nucleares indicou que a espécie possui baixos níveis de diversidade genética, e que 61% dessa diversidade é encontrada dentro das populações e 39% entre elas. Além disso, estes resultados evidenciaram significativos níveis de correlação entre distância genética e distância geográfica, sugerindo assim a presença de isolamento por distância. Nossos resultados indicam ainda a região geográfica do Rio de Janeiro como centro de diversidade para T. cassinoides. Uma forte estruturação genética foi encontrada para os dados de DNA nuclear e plastidial. Os resultados da análise de cpDNA, sugerem que a espécie T. cassinoides está dividida em três grupos filogeográficos: Norte, Central e Sul. Já os resultados de DNA nuclear apenas se diferenciam dos anteriores por considerar os grupos filogeográficos Norte e Central como um único grupo. Dados de modelagem de nicho ecológico foram gerados e associados com análises demográficas e de estruturação populacional visando um melhor entendimento dos padrões filogeográficos da espécie e inferências sobre a ocorrência de possíveis alterações populacionais ocorridas nos últimos 21 mil anos (Last Glacial Maximum - LGM) e 6 mil anos (Holocene Optimum - HO). Os resultados destas análises sugerem a estabilidade do tamanho populacional e das possíveis áreas de ocorrência de T. cassinoides no LGM e no HO. Assim, aventamos a hipótese de que a quebra de fluxo gênico entre as regiões filogeográficas propostas no presente estudo sejam anteriores ao LGM. Esse padrão converge com resultados encontrados para outras espécies de comunidades vegetais periféricas e diverge daqueles encontrados para espécies do núcleo principal da Floresta Ombrófila Densa, evidenciando a importância de estudos com espécies de diferentes comunidades vegetais da Floresta Atlântica para um melhor entendimento dos processos que moldaram a evolução das espécies nesse complexo bioma / The Atlantic Forest is one of the most biodiverse biomes on the planet. Nevertheless, little is known about the processes that have engendered such highly diverse plantlife in this region. Most studies on the Atlantic Forest approach the biome from an ample scope, without considering the various vegetational types that are found therein. Though broadly termed Montane Rain Forest, this biome also contains other peripheral forms of vegetation, such as Submontane Rain Forest, Cloud Forest, Riparian Rainforest, Rainforest Patches and Coastal Swamp Forest, commonly known as \"caxetais\" (plur). The term \"caxetal\" (sing) derives from the overwhelming predominance of Tabebuia cassinoides (Lam.) DC., the \"caxeta\". This naturally fragmented vegetational formation is found in patches on permanently waterlogged soils all the way from the north of Santa Catarina state up to northern most Espírito Santo. Populations with fragmented distribution are potential models for populational genetics studies, seen as the geographical delimitation of natural populations is one of the biggest problems facing research of this kind. In the light of this, the present thesis aims to: (1) verify how the aggregated, well-delimited and fragmented geographical distribution of the tree species T. cassinoides might influence the structuring of genetic diversity on a local level; (2) ascertain the degree of inter and intra-populational genetic variability in natural T. cassinoides populations throughout the Atlantic Forest, and (3) determine the phylogeographical structure of T. cassinoides. In order to achieve this we analyzed data for eight nuclear microsatellite markers and sequencing for the trnC-ycf6 region of plastidial DNA (cpDNA). The genetic data obtained was first used to generate knowledge concerning genetic diversity and populational structuring on a local level, in the Iguape (São Paulo state) region. Our results suggest that the fragmentation of these populations is geographical only, due to their edaphic needs, and that this locally aggregated and naturally fragmented mode of distribution does not indicate genetic subdivision. The characterization of inter and intra-populational genetic variability for T. cassinoides throughout its range of distribution, based on nuclear microsatellite markers, showed that the species has low levels of genetic diversity and that 61% of this diversity is encountered within, and 39% among, the populations. Furthermore, the results evince high and significant levels of correlation between genetic and geographic distance, thus suggesting that this isolation is distance-related. Our findings also indicate that the geographical region of Rio de Janeiro is the center of T. cassinoides diversity. Considerable genetic structuring was identified from the nuclear and plastidial DNA, though with partial distinctions. Results for cpDNA analysis suggest that T. cassinoides is divided into three phylogeographical groups: North, Central and South. Nuclear DNA analysis, however, took the Central and North phylogeographical groups to be one and the same. Ecological niche modeling data were generated and associated with demographic analyses and findings on populational structuring so that we could determine the species\' phylogeographical patterns and infer the occurrence of possible populational alterations during two distinct periods: 21 thousand years ago (the Last Glacial Minimum) and over the last 6 thousand years (Holocene Optimum - HO). The results of these analyses suggest that population sizes remained stable in possible areas of occurrence during the LGM and HO. Based on these findings we suggest the hypotheses that the stemming of gene flow among the phylogeographical regions proposed by this study predates the LGM. This pattern converges with results found for other species of peripheral vegetal communities but diverges from those for species from the Dense Coastal Hydrophilic Forest nucleus. As such, our research underscores the importance of further studies with different vegetal communities that comprise the Atlantic Forest in order to foster a better understanding of the processes that shaped the evolution of species in this complex biome

The Dinaric karst region in the Balkans is one of the hot spots of biodiversity with a high number of endemic freshwater fish. The major cause of the high degree of endemism are numerous paleoclimatic and geological events that have affected the formation of the entire area. Flows of karst poljes represent a specific ecosystem to which some fishes have adapted. The poljes have been periodically flooded and these floods may activate different underground channels, which lead to interconnection of poljes. These fish species spend a part of the year in surface water and a part in groundwater and they may use the underground flows between poljes for migration. The aim of this thesis is to evaluate the genetic variability of two endemic fish species of the genus Telestes living in karst poljes in the region of eastern Herzegovina, and to detect a possible current gene flow between populations of these species due to the connection of underground flows. For this purpose, the DNA from 102 individuals from five different localities was isolated. The mitochondrial marker cytochrom b and 16 microsatellite loci were used for the analyses. The results point to very low variability between species and populations. Based on mitochondrial marker analysis, no link between populations of the species was detected,...

Complex Campanula rotundifolia agg. is rich in endemic species, especially in the high- elevated mountain ranges. Extreme climatic conditions in the subalpine habitats can lead to a convergence in plant's morphology between plants isolated in different mountain ranges. Because of the lack of morphological differentiation, it is very difficult to reconstruct the evolutionary history of bellflowers C. scheuchzeri from the Alps, C. bohemica from the Krkonoše Mountains and C. tatrae from the Western Carpathians. A similarity in morphology and the same ploidy level can suggest vicariance from a large area of an ancestor species. However, regarding the continuous morphological variation, the high-altitudinal endemics could have originated by local adaptation from originally low-land species. DNA-ploidy level and genome size were detected by flow cytometry. Taxonomically important characters were found using multivariate morphometric analysis. The phenotypic plasticity of the taxon C. tatrae was tested by a cultivation experiment. Genetic structure of the studied species was revealed using molecular marker - microsatellites (7 variable primers). Studied taxa were tetraploids, but some possessed different genome size. The morphological differentiation was mainly due to characters like calyx lobes length...

Habitat fragmentation can adversely affect species populations due to reduced genetic diversity and increased population differentiation due to habitat isolation, reduced population size, and disruption of gene flow. Many species suffer from habitat fragmentation and isolation, and Campanula glomerata is a good example of a species that is noticeably declining in many places due to changes in landscape management. The question is how the model species responds to these changes and whether is the change are reflected in genetic diversity and population structure. The aim of this work is to determine the degree of genetic diversity and to reveal the genetic structure of populations of the model species C. glomerata on a small regional scale in the White Carpathians. This system will allow us to compare populations from two different regions that have been affected to varying degrees by agricultural intensification and fragmentation in the last century. It is a southwestern and northeastern part of the region, which differs from each other in the history of management in the landscape, but also in the continuity of meadows, species composition and environmental conditions. For this purpose, I developed and optimized a total of 16 usable microsatellite markers, 4 of which come from related species of...

Asexual modes of reproduction are usually based on the principle of copying (cloning) DNA from the female and passing it on to the offspring. For most asexually reproducing vertebrates the progeny develop from an unreduced and often unfertilised egg. This is driven by the mechanisms of parthenogenetic and gynogenetic reproduction. While in the former the clonal germ cell develops spontaneously and separately, in the latter a sexual partner is needed to activate the cleavage of the ovum, although without the fusion of the sperm and egg. Therefore in both cases there is no fertilization and the clonal progeny consist solely of daughters, hence the majority of previous studies have only focused on asexual female lineages. However, on rare occasions asexual clonal males can arise when the right fertilization occurs. Whilst these offspring are usually infertile, fertile diploid asexual males have been discovered in just three genera of hybrid origin in vertebrates. One of these unique cases is the European water frog complex of the genus Pelophylax, whose distribution includes the Czech Republic. In areas around the upper Odra River populations of hybrid males were recently discovered who form stable all-male lineages, similar to those formed by asexual females. The results of this study show that males produce...