A transmissão do VHC vem diminuindo após a implementação de diretrizes de triagem de doadores de sangue e adoção de políticas sociais para reduzir o risco de infecção em UDI, entretanto o VHC ainda constitui um grave problema de saúde pública mundial. Em torno de 10% dos pacientes infectados com VHC não referem exposição a nenhum fator de risco conhecido. Alguns estudos demonstraram a presença de RNA em diferentes secreções, sugerindo a existência de outras rotas de transmissão do VHC. Este estudo teve como objetivo analisar as relações filogenéticas de diferentes regiões genômicas do VHC de cônjuges portadores crônicos e correlacioná-las com seqüências de portadores crônicos não relacionados atendidos no mesmo ambulatório. Foram selecionados 18 pacientes (9 casais) com genótipo concordante entre eles e 42 controles (14 de cada genótipo encontrados nos casais). Foram amplificadas e seqüenciadas as regiões NS3 (~620nt) e NS5B (~360nt). As seqüências foram alinhadas usando o programa Clustal X e Bioedit 6.0.7. A presença do sinal filogenético, nas regiões estudadas, foi analisado através do mapeamento da verossimilhança pelo programa Tree-Puzzle. Os modelos evolucionários foram estimados pelo teste de razão de verossimilhança com o auxílio do programa Modeltest e utilizados para as análises das seqüências NS3, NS3+NS5B (TrN+I+G) e NS5B(TrNef+I+G). Foram empregados os métodos de distância com algoritmo de agrupamento de vizinhos e máxima verossimilhança com o algoritmo de rearranjo dos braços, seccionando a árvore em dois pedaços e ligando em outras partes, para a construção das árvores, pelo programa PAUP*4b10. Foram calculados os valores de bootstrap, com 1000 réplicas, para a verificação da sustentação de ramos nas topologias. Nas análises foram incluídas seqüências referencia do Genbank de diferentes genótipos. Todos os casais tiveram a região NS5B amplificada e seqüenciada, entretanto, não foi possível amplificar e seqüênciar a região NS3 de amostras de 2 casais. Considerando-se as três análises o sinal filogenético foi de 90.5% (NS5B - 199 nt), 92.9% (NS5B - 344 nt), 94.8% (NS3 - 619 nt ) e 96.1% (NS5B + NS3). Como esperado, o melhor sinal filogenético foi obtido com as seqüências das duas regiões concatenadas NS3+NS5B. As análises filogenéticas sugerem fortemente que os vírus dos casais 3, 4, 6, 7, e 8 têm a mesma origem. Na maioria das análises as seqüências dos vírus destes casais formaram um grupo monofilético com valores de bootstrap acima de 70. As seqüências dos outros casais, em algumas situações, apresentaram grupos monofiléticos, contudo os valores de bootstrap não foram significativos. A utilização de seqüências de duas regiões genômicas diferentes suportam a hipótese de que os vírus dos casais 3, 4, 6, 7 e 8 têm a mesma origem. A inclusão de seqüências controle, dos mesmos subtipos encontrados nas amostras dos casais, foram fundamentais para a confirmação dos resultados. Estes resultados indicam fortemente a possibilidade de transmissão entre casais. / HCV transmission has decreased with the adoption of universal blood donors screening and social policies to reduce risk of infection in IVDU, but HCV is still a worldwide health problem. The epidemiological route of infection cannot be identified in a significant proportion of patients. Some studies demonstrated the presence of viral RNA in different secretions, suggesting the existence of other routes for HCV transmission. The aim of this study was to evaluate the phylogenetical relationships among sequences from different HCV genomic regions from sexual partners of chronic infected patients when analyzed among themselves and when analyzed conjointly with sequences from virus found in non related chronic infected patients attended in the same clinic. Eighteen individuals (9 couples with stable relationship without other risk factors for HCV infection) and forty-two control patients (fourteen from each genotype found in the couples) were selected. NS3 (~620 nts) and NS5B (~360 nts) regions were amplified and sequenced. Sequences were aligned using clustal X 1.81 and Bioedit 6.0.7. Phylogenetic signal/noise ratio in the data set was investigated with a likelihood mapping analysis with the program TREE-PUZZLE. Evolutionary models were chosen by Hierarchical Likelihood Ratio Test (hLRTs) using Modeltest 3.06 and used for analyze NS3, NS3+NS5B (TrN+I+G) and NS5B (TrNef+I+G) sequences. Distance and maximum-likelihood (ML) phylogenetical analyses were performed with PAUP*4b10 and the trees were constructed with NJ and heuristic search. Tree bisection and reconnection (TBR) algorithm respectively.Robustness of trees was evaluated by analyzing 1000 bootstrap replicates. Genbank reference sequences from different genotypes were included in data analysis. Sequences from NS5B region were obtained for all samples while it was not possible to get NS3 sequences from only 2 couples. Considering the three analysis, phylogenetical signals were 90.5% (NS5B - 199 nt), 92.9% (NS5B - 344 nt), 94.8% (NS3 - 619 nt ) and 96.1% (NS5B + NS3). As expected, the best phylogenetical signal was obtained with concatened NS3+NS5B sequences. Phylogenetical analysis strongly suggested that virus from couples 3, 4, 6, 7 and 8 had a common origin. In the majority of the analysis, sequences inside these couples clustered in the same monophyletical group with bootstrap values higher than 70. For the other couples, monophyletical groups were observed but these results were not supported by the bootstrap analysis. In conclusion, using sequencing from two different viral genomic regions, we have strongly supported a common source of infection for the two members of five couples. Control sequences from the same subtypes than the couples were crucial to confirm the results. These data strongly support HCV transmission inside couples.

Submitted by ANGEL DE JESUS LAZARO VELASCO null (angelstrauss26@hotmail.com) on 2017-10-25T17:49:03Z No. of bitstreams: 1 DissertacaoAngel.pdf: 1392488 bytes, checksum: 30d16b1c40935e0572a92a6d9c6dc400 (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-10-31T17:00:42Z (GMT) No. of bitstreams: 1 velasco_ajl_me_jabo.pdf: 1392488 bytes, checksum: 30d16b1c40935e0572a92a6d9c6dc400 (MD5) / Made available in DSpace on 2017-10-31T17:00:42Z (GMT). No. of bitstreams: 1 velasco_ajl_me_jabo.pdf: 1392488 bytes, checksum: 30d16b1c40935e0572a92a6d9c6dc400 (MD5) Previous issue date: 2017-09-26 / Consejo Nacional de Ciencia y Tecnología (CONACYT) / Há evidências na literatura que a uniformidade das características fenotípicas dos peixes, entre outros animais, pode ser afetada por componentes genéticos, além de fatores ambientais. Maior uniformidade na despesca é desejada, a fim de reduzir a competição entre os animais e, assim, ter uma produção mais homogênea, visando maior retorno financeiro da atividade. O objetivo deste estudo foi estimar se as variâncias residuais do peso e comprimento à despesca de tilápias do Nilo (Oreochromis niloticus) são afetadas por componentes genéticos, para avaliar a possibilidade de seleção para uniformidade de produção. Empregou-se o modelo hierárquico linear generalizado duplo (DHGLM) para calcular os componentes de variância, os parâmetros de herdabilidade, coeficientes de variação e correlações genéticas para peso (não transformado e com transformação Box-Cox) e comprimento à despesca. Foi utilizado um total de 8.725 informações fenotípicas de tilápias provenientes de 271 famílias da linhagem GIFT (Malásia), criados na estação experimental de piscicultura da Universidade Estadual de Maringá. Os resultados apoiaram a hipótese de heterogeneidade genética da variância residual para peso e comprimento à despesca, e a oportunidade de seleção medida através do coeficiente genético de variação da variância residual. Além disso, observou-se a necessidade de um grande número de filhos para a obtenção de estimativas precisas do mérito genético para variação residual, conforme indicado pelas estimativas de baixa herdabilidade. A transformação de Box-Cox foi capaz de diminuir a dependência entre a variância e a média do peso à despesca. A transformação reduziu, mas não eliminou toda a heterogeneidade genética da variância residual, destacando sua presença além do efeito da escala. / There is evidence in the literature that the uniformity of phenotypic traits of fish, among other animals, can be affected by genetic components, as well as environmental factors. Greater uniformity is desired in order to reduce competition among animals and thus have a more uniform production, aiming a greater financial return of the activity. The objective of this study was to assess if the residual variance of the weight and harvest length of the Nile tilapia (Oreochromis niloticus) is affected by a genetic component, to evaluate the possibility of selection for uniformity of production. The double hierarchical generalized linear model was used to estimate the components of variance, heritability, coefficients of variation and genetic correlation for weight (untransformed and with Box-Cox transformation) and harvest length. Phenotypic records of 8,725 individuals from 271 families were analyzed. The results supported the existence of genetic heterogeneity of residual variance on harvest weight and length, and the opportunity to select for increasing uniformity. Moreover, the low heritability estimates showed the necessity of high number of progeny for an accurate estimate of the genetic merit for the residual variance. The Box-Cox transformation of harvest weight reduced the dependency among its mean and variance. The transformation reduced but did not eliminate the genetic heterogeneity of residual variance, highlighting its presence beyond the scale effect. / CONACYT: 579742/410471

A transmissão do VHC vem diminuindo após a implementação de diretrizes de triagem de doadores de sangue e adoção de políticas sociais para reduzir o risco de infecção em UDI, entretanto o VHC ainda constitui um grave problema de saúde pública mundial. Em torno de 10% dos pacientes infectados com VHC não referem exposição a nenhum fator de risco conhecido. Alguns estudos demonstraram a presença de RNA em diferentes secreções, sugerindo a existência de outras rotas de transmissão do VHC. Este estudo teve como objetivo analisar as relações filogenéticas de diferentes regiões genômicas do VHC de cônjuges portadores crônicos e correlacioná-las com seqüências de portadores crônicos não relacionados atendidos no mesmo ambulatório. Foram selecionados 18 pacientes (9 casais) com genótipo concordante entre eles e 42 controles (14 de cada genótipo encontrados nos casais). Foram amplificadas e seqüenciadas as regiões NS3 (~620nt) e NS5B (~360nt). As seqüências foram alinhadas usando o programa Clustal X e Bioedit 6.0.7. A presença do sinal filogenético, nas regiões estudadas, foi analisado através do mapeamento da verossimilhança pelo programa Tree-Puzzle. Os modelos evolucionários foram estimados pelo teste de razão de verossimilhança com o auxílio do programa Modeltest e utilizados para as análises das seqüências NS3, NS3+NS5B (TrN+I+G) e NS5B(TrNef+I+G). Foram empregados os métodos de distância com algoritmo de agrupamento de vizinhos e máxima verossimilhança com o algoritmo de rearranjo dos braços, seccionando a árvore em dois pedaços e ligando em outras partes, para a construção das árvores, pelo programa PAUP*4b10. Foram calculados os valores de bootstrap, com 1000 réplicas, para a verificação da sustentação de ramos nas topologias. Nas análises foram incluídas seqüências referencia do Genbank de diferentes genótipos. Todos os casais tiveram a região NS5B amplificada e seqüenciada, entretanto, não foi possível amplificar e seqüênciar a região NS3 de amostras de 2 casais. Considerando-se as três análises o sinal filogenético foi de 90.5% (NS5B - 199 nt), 92.9% (NS5B - 344 nt), 94.8% (NS3 - 619 nt ) e 96.1% (NS5B + NS3). Como esperado, o melhor sinal filogenético foi obtido com as seqüências das duas regiões concatenadas NS3+NS5B. As análises filogenéticas sugerem fortemente que os vírus dos casais 3, 4, 6, 7, e 8 têm a mesma origem. Na maioria das análises as seqüências dos vírus destes casais formaram um grupo monofilético com valores de bootstrap acima de 70. As seqüências dos outros casais, em algumas situações, apresentaram grupos monofiléticos, contudo os valores de bootstrap não foram significativos. A utilização de seqüências de duas regiões genômicas diferentes suportam a hipótese de que os vírus dos casais 3, 4, 6, 7 e 8 têm a mesma origem. A inclusão de seqüências controle, dos mesmos subtipos encontrados nas amostras dos casais, foram fundamentais para a confirmação dos resultados. Estes resultados indicam fortemente a possibilidade de transmissão entre casais. / HCV transmission has decreased with the adoption of universal blood donors screening and social policies to reduce risk of infection in IVDU, but HCV is still a worldwide health problem. The epidemiological route of infection cannot be identified in a significant proportion of patients. Some studies demonstrated the presence of viral RNA in different secretions, suggesting the existence of other routes for HCV transmission. The aim of this study was to evaluate the phylogenetical relationships among sequences from different HCV genomic regions from sexual partners of chronic infected patients when analyzed among themselves and when analyzed conjointly with sequences from virus found in non related chronic infected patients attended in the same clinic. Eighteen individuals (9 couples with stable relationship without other risk factors for HCV infection) and forty-two control patients (fourteen from each genotype found in the couples) were selected. NS3 (~620 nts) and NS5B (~360 nts) regions were amplified and sequenced. Sequences were aligned using clustal X 1.81 and Bioedit 6.0.7. Phylogenetic signal/noise ratio in the data set was investigated with a likelihood mapping analysis with the program TREE-PUZZLE. Evolutionary models were chosen by Hierarchical Likelihood Ratio Test (hLRTs) using Modeltest 3.06 and used for analyze NS3, NS3+NS5B (TrN+I+G) and NS5B (TrNef+I+G) sequences. Distance and maximum-likelihood (ML) phylogenetical analyses were performed with PAUP*4b10 and the trees were constructed with NJ and heuristic search. Tree bisection and reconnection (TBR) algorithm respectively.Robustness of trees was evaluated by analyzing 1000 bootstrap replicates. Genbank reference sequences from different genotypes were included in data analysis. Sequences from NS5B region were obtained for all samples while it was not possible to get NS3 sequences from only 2 couples. Considering the three analysis, phylogenetical signals were 90.5% (NS5B - 199 nt), 92.9% (NS5B - 344 nt), 94.8% (NS3 - 619 nt ) and 96.1% (NS5B + NS3). As expected, the best phylogenetical signal was obtained with concatened NS3+NS5B sequences. Phylogenetical analysis strongly suggested that virus from couples 3, 4, 6, 7 and 8 had a common origin. In the majority of the analysis, sequences inside these couples clustered in the same monophyletical group with bootstrap values higher than 70. For the other couples, monophyletical groups were observed but these results were not supported by the bootstrap analysis. In conclusion, using sequencing from two different viral genomic regions, we have strongly supported a common source of infection for the two members of five couples. Control sequences from the same subtypes than the couples were crucial to confirm the results. These data strongly support HCV transmission inside couples.

Orientador: Hercílio Martelli Júnior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T12:37:33Z (GMT). No. of bitstreams: 1 Aquino_SibeleNascimentode_D.pdf: 2038371 bytes, checksum: 4158cd336adb86cc754e37ade0322e0e (MD5) Previous issue date: 2013 / Resumo: Fissuras do lábio ou palato não sindrômicas (FL/PNS) são as anomalias congênitas craniofaciais mais comuns, com prevalência de 1:500-2.500 nascidos vivos. Possuem etiologia complexa, com participação de fatores ambientais e genéticos. Estudos de larga escala genômica (GWAS) descreveram várias regiões cromossômicas e genes candidatos à etiologia das FL/PNS, entretanto poucos foram confirmados em diversas populações, o que pode ser resultado da diferença na composição étnica das populações. Recentemente, GWAS realizados com populações da Europa e da Ásia identificaram polimorfismos de suscetibilidade para FL/PNS nos genes FGF12, VCL, CX43, e nos loci 1p36, 2p21, 3p11.1, 8q21.3, 10q25, 13q31.1, 15q22.2 e 17q22. Como o Brasil é composto por uma população miscigenada, torna-se importante confirmar se esses marcadores também mostram suscetibilidade à FL/PNS na população brasileira. O objetivo deste estudo foi avaliar o envolvimento de polimorfismos genéticos que foram descritos como marcadores de risco para o desenvolvimento de FL/PNS em pacientes brasileiros com FL/PNS. Este estudo caso-controle, com uma análise estruturada de acordo com as proporções de ancestralidade de cada individuo, avaliou 16 marcadores polimórficos de suscetibilidade as fissuras orais em 300 pacientes com FL/PNS e 395 indivíduos sem fissura provenientes de Minas Gerais, Brasil e 7 marcadores polimórficos em 505 pacientes com FL/PNS e 594 indivíduos sem fissura, provenientes de Minas Gerais e Bahia, Brasil. Os polimorfismos foram genotipados pelo método de discriminação alélica com sondas fluorescentes. A ancestralidade genômica de cada indivíduo foi determinada pela caracterização de 40 marcadores bialélicos de inserção/deleção (INDELs). A distribuição genotípica de todos os polimorfismos no grupo controle respeitou o equilíbrio de Hardy-Weinberg, exceto para o polimorfimo rs7632427 que foi excluído da análise. Foram observadas associações entre os polimorfismos rs227731, rs742071, rs1873147, rs8001641 e rs7590268 e de um haplótipo formado pelos polimorfismos rs10787760, rs6585429 e rs1871345 do gene VAX1 com FL/PNS. Após a correção de Bonferroni para múltiplos testes, foram observadas associações significativas com os polimorfismos rs742071, rs1873147 e rs227731. Entretanto, a frequência dos alelos de risco variou entre as regiões geográficas, de acordo com as proporções de ancestralidade européia e africana. O grupo com maior proporção de marcadores de origem européia mostrou associação com rs227731, enquanto que o grupo com proporção maior de marcadores de origem africana exibiu associação com o polimorfismo rs1873147. A associação significante com rs742071 foi detectada apenas com a combinação de amostras. Em síntese, os resultados demonstram a associação dos polimorfismos localizados na região 1p36 (rs742071), 15q22.2 (rs1873147) e 17q22 (rs227731), com a suscetibilidade genética ao desenvolvimento de FL/PNS na população brasileira, sendo observada uma influência da diversidade populacional nessas associações, uma discreta associação dos polimorfismos rs7590268 (2p21) e rs8001641 (13q31.1) com FL/PNS e uma modesta associação de um haplótipo no gene VAX1, sugerindo um efeito com baixa penetrância em FL/PNS / Abstract: Nonsyndromic cleft lip with or without palate (NSCL/P) is the most common orofacial birth defect with prevalence of 1:500-2,500 live births. NSCL/P has complex etiology, which is related to environmental and genetic risk factors. Recent genome-wide association (GWAS) studies have been identified a varied of chromosomal loci and candidate genes in association with NSCL/P development, however few of them has been replicated in different populations, which may be related to the differences in the ethnicity of the populations. Previous GWAS with populations from Europe and Asia have identified putative susceptibility markers for NSCL/P in FGF12, VCL, CX43 and polymorphism at the regions 1p36, 2p21, 3p11.1, 8q21.3, 10q25, 13q31.1, 15q22.2 and 17q22. As the Brazilian population is one of the most heterogeneous in the world, it is important to confirm the susceptibility of those markers identified in genetically homogenous populations in the Brazilian population. The purpose of this study was to verify the association of single nucleotide polymorphisms, which were identified as NSCL/P risk markers, with NSCL/P in a Brazilian population. We conducted a structured association study conditioned on the individual ancestry proportions to determine the role of 16 polymorphic markers in 300 patients with NSCL/P and 395 controls from Minas Gerais state, Brazil, and 7 polymorphic markers in 505 patients with NSCL/P and 594 controls from Minas Gerais state and Bahia state, Brazil. The polymorphisms were genotyping using the allelic discrimination method with fluorescence probes. The genomic ancestry of each individual was determined by genotyping 40 biallelic short insertion-deletion polymorphic markers (INDELs). The genotype frequencies observed for all studied polymorphisms in the control group did not reveal statistically significant differences compared to those expected under Hardy-Weinberg equilibrium, except for rs7632427 which was excluded from analysis. Associations with NSCL/P were observed with rs227731, rs742071, rs1873147, rs8001641 and rs7590268, and a haplotype formed by VAX1 rs10787760, rs6585429 and rs1871345 polymorphisms with NSCL/P. After Bonferroni correction for multiple tests, significant associations with NSCL/P were observed with rs742071, rs1873147 and rs227731. However, the frequency of the risk alleles varied between the geographical regions, according to the proportions of European and African genomic ancestry. The group enriched by European ancestry showed significant association with rs227731, whereas the group with high African ancestry was significantly associated with rs1873147 polymorphism. The significant association with rs742071 was only detected in the combined sample. In conclusion, our results demonstrated the association of the polymorphisms located at 1p36, 15q22.2 and 17q22 with NSCL/P Brazilian population, and that the diversity of the Brazilian population clearly influences the contribution of polymorphisms, a weekly association of the rs7590268 (2p21), rs8001641 (13q31.1) e rs1873147 (15q22.2) and a modest association of a haplotype of VAX1, suggesting a low penetrant gene for oral cleft / Doutorado / Patologia / Doutora em Estomatopatologia

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

Abstract Adaptation through polygenic selection is a prominent feature in nature. Still, the genetic backgrounds of polygenic adaptations are often unknown. The challenges of resolving adaptive processes are related to selection being distributed over several loci with often small effect sizes. Also, even a low level of population substructure can obstruct the inference. Further, demographic factors in the history of the species, such as population size changes and range expansions leave a confounding footprint in the background genomic variation. In this thesis, polygenic adaptation was studied with Scots pine (Pinus sylvestris L.), a widespread ecologically and economically important conifer. In this thesis, timing of bud set – an adaptive polygenic trait – was studied at the level of the phenotype in a common garden study, and at the genomic level by examining the sequence and allele frequency variation patterns in bud set timing related loci, with a sampling across a latitudinal transect in Europe. An association study, combining these two levels, was carried out with a new Bayesian multipopulation method. The congruence of allozyme and nucleotide level diversity was estimated, the level of neutral genetic population structure surveyed, and a demographic background model for statistical inference of selective signals redefined. Allozyme variation seemed to correlate well with the nucleotide level variation at the between species level, but within population, at the individual allozyme coding loci, does not describe the underlying level of nucleotide variation well. Indications of recent colonization history affecting the level of differentiation between populations were seen, and the need to control for the background effects of simultaneous range expansion and adaptation shown. Lower phenotypic and additive genetic variation in timing of bud set was found in northern compared to central European populations. Signs of heterogeneity in genetic basis of this trait were also found between these areas, which could indicate different timekeeping mechanisms due to different environmental cues in the two regions. The results in this thesis are of value to the study of adaptation, but also for breeding, conservation and prediction of responses of forest trees to future climate change. / Tiivistelmä Sopeutuminen perustuu usein polygeenisiin ominaisuuksiin. Näiden ominaisuuksien geneettiset taustat ovat silti vielä pitkälti selvittämättä. Sopeutumisominaisuuksien genetiikan selvittämistä vaikeuttaa valinnan vaikutusten jakautuminen usean, usein pienivaikutuksisen lokuksen kesken. Lisäksi vähäinenkin populaatiorakenne hankaloittaa geenien tunnistamista. Myös lajin historiassa tapahtuneet demografiset muutokset, kuten populaatiokoon vaihtelut ja kolonisaatio jättävät jälkensä genomiin. Väitöskirjassani tutkin polygeenistä sopeutumista ekologisesti ja taloudellisesti tärkeän havupuulajin, metsämännyn (Pinus sylvestris L.) avulla. Väitöskirjassani tutkin metsämännyn silmunmuodostuksen ajoitusta sekä fenotyypin että sekvenssimuuntelun tasolla. Ajoitusta mitattiin eri leveysasteilta peräisin olevista eurooppalaisista populaatioista yhteiskasvatuskokeessa, ja sekvenssimuuntelua sekä alleelifrekvenssien jakautumista tutkittiin vastaavasta näytteestä. Geenikartoituskokeessa yhdistettiin nämä kaksi muuntelun tasoa hyödyntäen uutta, usean populaation tutkimiseen soveltuvaa analyysimenetelmää. Lisäksi tutkin allotsyymimuuntelun ja nukleotidimuuntelun keskinäistä tarkkuutta geneettisen diversiteetin kuvaajina, neutraalin populaatiorakenteen tasoa, sekä demografian vaikutusta metsämännyn genomissa. Allotsyymimuuntelun todettiin kuvaavan hyvin lajien välisiä diversiteettieroja. Populaation sisällä yksittäisten allotsyymien heterosygotia ei korreloinut entsyymiä koodaavan geenin muuntelun määrän kanssa. Pohjoisten populaatioiden vähäisemmät keskinäiset erot verrattuna keskieurooppalaisiin antoivat viitteitä siitä, että viimeisimmän jääkauden jälkeiset kolonisaatiotapahtumat voivat edelleen vaikuttaa populaatioiden erilaistumisasteeseen. Assosiaatiotutkimuksessa osoitettiin, kuinka tärkeää yhtäaikaisen sopeutumisen ja kolonisaation huomioiminen on sopeutumisominaisuuksien tutkimisessa. Fenotyyppinen muuntelu silmunmuodostuksen ajoituksessa oli vähäisempää pohjoisissa populaatioissa. Lisäksi löysimme merkkejä geneettisestä heterogeenisuudesta silmunmuodostuksen taustalla pohjoisten ja keskieurooppalaisten metsämäntyjen välillä, mikä voi johtua vuodenajan vaihtelun mittaamiseen käytettävien ympäristösignaalien eriytymisestä näiden alueiden välillä. Väitöskirjassani saadut tulokset hyödyttävät paitsi sopeutumistutkimusta, myös jalostus- ja luonnonsuojelututkimusta sekä ilmastonmuutoksen vaikutusten arviointia.

Contexte: Les champignons mycorhiziens à arbuscules (AMF) établissent des relations symbiotiques avec la plupart des plantes grâce à leurs réseaux d’hyphes qui s’associent avec les racines de leurs hôtes. De précédentes études ont révélé des niveaux de variation génétique extrêmes pour des loci spécifiques permettant de supposer que les AMF peuvent contenir des milliers de noyaux génétiquement divergents dans un même cytoplasme. Si aucun processus de reproduction sexuée n’a jusqu’ici été observé chez ces mycorhizes, on constate cependant que des niveaux élevés de variation génétique peuvent être maintenus à la fois par l’échange de noyaux entre hyphes et par des processus fréquents de recombinaison entre noyaux. Les AMF se propagent par l’intermédiaire de spores qui contiennent chacune un échantillon d’une population initiale de noyaux hétérogènes, directement hérités du mycélium parent. À notre connaissance les AMF sont les seuls organismes qui ne passent jamais par un stade mononucléaire, ce qui permet aux noyaux de diverger génétiquement dans un même cytoplasme. Ces aspects singuliers de la biologie des AMF rendent l’estimation de leur diversité génétique problématique. Ceci constitue un défi majeur pour les écologistes sur le terrain mais également pour les biologistes moléculaires dans leur laboratoire. Au-delà même des problématiques de diversité spécifique, l’amplitude du polymorphisme entre noyaux mycorhiziens est mal connue. Le travail proposé dans ce manuscrit de thèse explore donc les différents aspects de l’architecture génomique singulière des AMF. Résultats L’ampleur du polymorphisme intra-isolat a été déjà observée pour la grande sous-unité d’ARN ribosomal de l’isolat Glomus irregulare DAOM-197198 (précédemment identifié comme G. intraradices) et pour le gène de la polymerase1-like (PLS) de Glomus etunicatum isolat NPI. Dans un premier temps, nous avons pu confirmer ces résultats et nous avons également pu constater que ces variations étaient transcrites. Nous avons ensuite pu mettre en évidence la présence d’un goulot d’étranglement génétique au moment de la sporulation pour le locus PLS chez l’espèce G. etunicatum illustrant les importants effets d’échantillonnage qui se produisaient entre chaque génération de spore. Enfin, nous avons estimé la différentiation génétique des AMF en utilisant à la fois les réseaux de gènes appliqués aux données de séquençage haut-débit ainsi que cinq nouveaux marqueurs génomiques en copie unique. Ces analyses révèlent que la différenciation génomique est présente de manière systématique dans deux espèces (G. irregulare et G. diaphanum). Conclusions Les résultats de cette thèse fournissent des preuves supplémentaires en faveur du scénario d’une différenciation génomique entre noyaux au sein du même isolat mycorhizien. Ainsi, au moins trois membres du genre Glomus, G. irregulare, G. diaphanum and G. etunicatum, apparaissent comme des organismes dont l’organisation des génomes ne peut pas être décrit d’après un modèle Mendélien strict, ce qui corrobore l’hypothèse que les noyaux mycorhiziens génétiquement différenciés forment un pangenome. / Background: Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi whose hyphal networks form symbioses with plants. Previous studies have revealed extremely high levels of genetic variation for some loci, which has lead to the proposition that AMF contain thousands of genetically divergent nuclei that share the same cytoplasm, i.e. they are heterokaryotic coenocytes. No reproductive stage has as yet been observed in AMF, yet evidence is accumulating that the observed high levels of diversity could be maintained by the exchange of nuclei between hyphal systems and (meiotic) recombination. AMF spores contain varying fractions of this heterogeneous population of nuclei, which migrate directly from the parent mycelium. To our knowledge, AMF are the only organisms that never pass through a single nucleus stage in their life cycle, which allows nuclei to diverge into genetically distinct nuclei within the same cytoplasm. Thus, estimating genetic diversity in arbuscular mycorrhizal fungi (AMF) is a major challenge, not only for ecologists in the field but also for molecular biologists in the lab. It is unclear what the extent of polymorphism is in AMF genomes. The present thesis investigates different aspects of this peculiar genome organization. Results The second chapter in this thesis confirms the extensive intra-isolate polymorphism that was previously observed for large subunit rDNA (in G. irregulare DAOM-197198) and the polymerase1-like gene, PLS (in G. etunicatum), and shows that this polymorphism is transcribed. In the third chapter I report the presence of a bottleneck of genetic variation at sporulation for the PLS locus, in G. etunicatum. Analyses in the fourth chapter, based on a conservative network-based clustering approach and five novel single copy genomic markers, reveal extensive genome-wide patterns of diversity in two different AMF species (G. irregulare and G. diaphanum). Conclusions The results from this thesis provide additional evidence in favor of genome differentiation between nuclei in the same isolate for AMF. Thus, at least three members of the Glomus genus, G. irregulare, G. diaphanum and G. etunicatum appear to be organisms whose genome organization cannot be described by a single genome sequence: genetically differentiated nuclei in AMF form a pangenome.

<p>HER2 Gene-Protein Assay (GPA) је посебно погодан за истовремено процењивање експресије HER2 протеина и статуса амплификације HER2 гена на нивоу појединачних ћелија и њихово повезивање са ћелијском морфологијом. Циљ истраживања био је испитати да ли су постојећи критеријуми препоручени од стране ASCO/CAP довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску хетерогеност, како у примарним туморима тако и у метастазама у регионалне лимфне чворове, учесталост HER2 хетерогености у макрометастазама лоцираним у лимфним чворовима, те да ли постоји јасна корелација између хетерогености нађене у примарном тумору дојке и припадајућим метастазама у лимфним чворовима. Испитивање је обухватило 41 од планиране 51 пацијенткиње које су испуниле све критеријуме укључивања. Репрезентативни парафински блокови метастатских лимфних чворова одабрани су из архивираног материјала, обојени GPA методом и процењени у складу са критеријумима ASCO/CAP 2013. Анализирано је 120 ћелија у хистолошком резу сваког метастатског лимфног чвора. Статус HER2 се разликовао између примарног тумора и његових метастаза у 13,2% (5/38) случајева. Један случај HER2 позитивног примарног тумора имао је HER2 негативне метастазе, два додатна случаја са HER2 позитивним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина и два случаја са HER2 негативним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина. У 17.4% (4/23) случајева са HER2 не-амплификованим примарним тумором метастазе су постале граничне у статусу генске амплификације. Једна од четири метастазе HER2 негативног примарног тумора показала је мали фокус HER2 позитивних туморских ћелија (&lt;3% тумора). Микрохетерогеност је анализирана у 108 лимфних чворова код 38 пацијенткиња и уочена у 22 лимфна чвора, тј. код четири пацијенткиње у свим анализираним лимфним чворовима, док је код једне пацијенткиње од 4 анализирана лимфна чвора микрохетерогеност потврђена у једном лимфном чвору. На основу добијених резултата може се закључити да постојећи критеријуми препоручени од стране ASCO/CAP применом прихваћених метода нису довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску и интертуморску хетерогеност како у примарним туморима тако и у метастазама, те да постоји статистички високо сигнификантан број макрометастаза лоцираних у лимфним чворовима које показују HER2 хетерогеност и позитивна корелација између хетерогености нађене у примарним туморима и припадајућим метастазама у лимфним чворовима.</p> / <p>HER2 Gene-Protein Assay (GPA) je posebno pogodan za istovremeno procenjivanje ekspresije HER2 proteina i statusa amplifikacije HER2 gena na nivou pojedinačnih ćelija i njihovo povezivanje sa ćelijskom morfologijom. Cilj istraživanja bio je ispitati da li su postojeći kriterijumi preporučeni od strane ASCO/CAP dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku heterogenost, kako u primarnim tumorima tako i u metastazama u regionalne limfne čvorove, učestalost HER2 heterogenosti u makrometastazama lociranim u limfnim čvorovima, te da li postoji jasna korelacija između heterogenosti nađene u primarnom tumoru dojke i pripadajućim metastazama u limfnim čvorovima. Ispitivanje je obuhvatilo 41 od planirane 51 pacijentkinje koje su ispunile sve kriterijume uključivanja. Reprezentativni parafinski blokovi metastatskih limfnih čvorova odabrani su iz arhiviranog materijala, obojeni GPA metodom i procenjeni u skladu sa kriterijumima ASCO/CAP 2013. Analizirano je 120 ćelija u histološkom rezu svakog metastatskog limfnog čvora. Status HER2 se razlikovao između primarnog tumora i njegovih metastaza u 13,2% (5/38) slučajeva. Jedan slučaj HER2 pozitivnog primarnog tumora imao je HER2 negativne metastaze, dva dodatna slučaja sa HER2 pozitivnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina i dva slučaja sa HER2 negativnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina. U 17.4% (4/23) slučajeva sa HER2 ne-amplifikovanim primarnim tumorom metastaze su postale granične u statusu genske amplifikacije. Jedna od četiri metastaze HER2 negativnog primarnog tumora pokazala je mali fokus HER2 pozitivnih tumorskih ćelija (&lt;3% tumora). Mikroheterogenost je analizirana u 108 limfnih čvorova kod 38 pacijentkinja i uočena u 22 limfna čvora, tj. kod četiri pacijentkinje u svim analiziranim limfnim čvorovima, dok je kod jedne pacijentkinje od 4 analizirana limfna čvora mikroheterogenost potvrđena u jednom limfnom čvoru. Na osnovu dobijenih rezultata može se zaključiti da postojeći kriterijumi preporučeni od strane ASCO/CAP primenom prihvaćenih metoda nisu dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku i intertumorsku heterogenost kako u primarnim tumorima tako i u metastazama, te da postoji statistički visoko signifikantan broj makrometastaza lociranih u limfnim čvorovima koje pokazuju HER2 heterogenost i pozitivna korelacija između heterogenosti nađene u primarnim tumorima i pripadajućim metastazama u limfnim čvorovima.</p> / <p><!--[if gte mso 9]><xml> <o:DocumentProperties> <o:Author>ilija vogel</o:Author> <o:Version>16.00</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings></xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> 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Contexte: Les champignons mycorhiziens à arbuscules (AMF) établissent des relations symbiotiques avec la plupart des plantes grâce à leurs réseaux d’hyphes qui s’associent avec les racines de leurs hôtes. De précédentes études ont révélé des niveaux de variation génétique extrêmes pour des loci spécifiques permettant de supposer que les AMF peuvent contenir des milliers de noyaux génétiquement divergents dans un même cytoplasme. Si aucun processus de reproduction sexuée n’a jusqu’ici été observé chez ces mycorhizes, on constate cependant que des niveaux élevés de variation génétique peuvent être maintenus à la fois par l’échange de noyaux entre hyphes et par des processus fréquents de recombinaison entre noyaux. Les AMF se propagent par l’intermédiaire de spores qui contiennent chacune un échantillon d’une population initiale de noyaux hétérogènes, directement hérités du mycélium parent. À notre connaissance les AMF sont les seuls organismes qui ne passent jamais par un stade mononucléaire, ce qui permet aux noyaux de diverger génétiquement dans un même cytoplasme. Ces aspects singuliers de la biologie des AMF rendent l’estimation de leur diversité génétique problématique. Ceci constitue un défi majeur pour les écologistes sur le terrain mais également pour les biologistes moléculaires dans leur laboratoire. Au-delà même des problématiques de diversité spécifique, l’amplitude du polymorphisme entre noyaux mycorhiziens est mal connue. Le travail proposé dans ce manuscrit de thèse explore donc les différents aspects de l’architecture génomique singulière des AMF. Résultats L’ampleur du polymorphisme intra-isolat a été déjà observée pour la grande sous-unité d’ARN ribosomal de l’isolat Glomus irregulare DAOM-197198 (précédemment identifié comme G. intraradices) et pour le gène de la polymerase1-like (PLS) de Glomus etunicatum isolat NPI. Dans un premier temps, nous avons pu confirmer ces résultats et nous avons également pu constater que ces variations étaient transcrites. Nous avons ensuite pu mettre en évidence la présence d’un goulot d’étranglement génétique au moment de la sporulation pour le locus PLS chez l’espèce G. etunicatum illustrant les importants effets d’échantillonnage qui se produisaient entre chaque génération de spore. Enfin, nous avons estimé la différentiation génétique des AMF en utilisant à la fois les réseaux de gènes appliqués aux données de séquençage haut-débit ainsi que cinq nouveaux marqueurs génomiques en copie unique. Ces analyses révèlent que la différenciation génomique est présente de manière systématique dans deux espèces (G. irregulare et G. diaphanum). Conclusions Les résultats de cette thèse fournissent des preuves supplémentaires en faveur du scénario d’une différenciation génomique entre noyaux au sein du même isolat mycorhizien. Ainsi, au moins trois membres du genre Glomus, G. irregulare, G. diaphanum and G. etunicatum, apparaissent comme des organismes dont l’organisation des génomes ne peut pas être décrit d’après un modèle Mendélien strict, ce qui corrobore l’hypothèse que les noyaux mycorhiziens génétiquement différenciés forment un pangenome. / Background: Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi whose hyphal networks form symbioses with plants. Previous studies have revealed extremely high levels of genetic variation for some loci, which has lead to the proposition that AMF contain thousands of genetically divergent nuclei that share the same cytoplasm, i.e. they are heterokaryotic coenocytes. No reproductive stage has as yet been observed in AMF, yet evidence is accumulating that the observed high levels of diversity could be maintained by the exchange of nuclei between hyphal systems and (meiotic) recombination. AMF spores contain varying fractions of this heterogeneous population of nuclei, which migrate directly from the parent mycelium. To our knowledge, AMF are the only organisms that never pass through a single nucleus stage in their life cycle, which allows nuclei to diverge into genetically distinct nuclei within the same cytoplasm. Thus, estimating genetic diversity in arbuscular mycorrhizal fungi (AMF) is a major challenge, not only for ecologists in the field but also for molecular biologists in the lab. It is unclear what the extent of polymorphism is in AMF genomes. The present thesis investigates different aspects of this peculiar genome organization. Results The second chapter in this thesis confirms the extensive intra-isolate polymorphism that was previously observed for large subunit rDNA (in G. irregulare DAOM-197198) and the polymerase1-like gene, PLS (in G. etunicatum), and shows that this polymorphism is transcribed. In the third chapter I report the presence of a bottleneck of genetic variation at sporulation for the PLS locus, in G. etunicatum. Analyses in the fourth chapter, based on a conservative network-based clustering approach and five novel single copy genomic markers, reveal extensive genome-wide patterns of diversity in two different AMF species (G. irregulare and G. diaphanum). Conclusions The results from this thesis provide additional evidence in favor of genome differentiation between nuclei in the same isolate for AMF. Thus, at least three members of the Glomus genus, G. irregulare, G. diaphanum and G. etunicatum appear to be organisms whose genome organization cannot be described by a single genome sequence: genetically differentiated nuclei in AMF form a pangenome.