Important candidate genes to human bone phenotypes are those involved in the regulation of hormonal action, such as the vitamin D receptor (VDR) and the estrogen receptor-α (ERα) genes and their cofactors. RIZ1 is a specific ERα cofactor proved to strongly enhance the function of the ERα. The main focus of this thesis has been to study genetic variants in the VDR and RIZ genes and their associations to human bone phenotypes using candidate gene and functional approaches. Specifically, polymorphisms in the VDR 3’ untranslated region (UTR) and a deletion/insertion polymorphism of a proline in the RIZ gene were investigated. The candidate gene approach was applied to large-scale population-based cohorts of pre-and post-menopausal women from Sweden and of elderly men from Sweden and Hong Kong. VDR 3’ UTR polymorphisms were associated with peak bone mass and body composition in young women. Further analysis of common VDR 3’ UTR haplotypes confirmed the association with BMD and risk of fractures in elderly men from Sweden and Hong Kong. The VDR polymorphisms were investigated for cis-acting effects, affecting allelic expression in the normal chromosomal context of human bone cells. The VDR allelic transcripts in the bone samples were unequally expressed, suggesting presence of regulatory variants in the 3’ UTR. The polymorphism in the RIZ gene was strongly associated to BMD in pre- and postmenopausal women and in elderly men. The functional analyses included reporter constructs containing the RIZ polymorphic variants transfected in a cell line and its abilities in coactivating the ERα were examined. The variants were functionally different in coactivating the ERα-receptor complex. To summarize, the results of this thesis show novel evidence for functional relevant polymorphisms in candidate genes to human bone phenotypes. These polymorphisms may contribute to the variation seen in BMD and risk of fractures in the population.

Currently infertility affects 10 to 15 percent of couples. In nearly 50% of all cases male factor contributes to infertility of the couple. Majority of causes of male infertility remains unexplained. For this reason, finding simple and clinically usefµl tools for improving male infertility diagnostics can be important at present. Based on the analysis of sperm transcriptome, in this diploma thesis we aimed to find genes that show differential expression between normal and pathological sperm, this could provide information about molecular basis of male infertility, moreover, expression profile of such genes in sperm could be employed for noninvasive diagnosis of male infertility. This study was conducted by using 67 sperm samples, including 16 control sperm samples from fertile men. The samples of infertile patients were divided into 3 groups according to morphology and motility using results of standard sperm evaluation according to WHO. First group included 20 patients diagnosed with astenoteratozoospermia (low sperm motility and abnormal morphology), second group was 15 patients diagnosed with asthenozoospermia (normal morphology and low sperm motility), the third group comprised 16 samples from infertile patients with normal spermiogram. 16 control samples were from fertile men (conception of...

Dentre o setor agropecuário, a avicultura é a que mais tem demonstrado índices de evolução nos últimos anos. Esses avanços são obtidos principalmente por meio da nutrição, manejo dos animais e seleção genética. A biotecnologia tem ganhado papel de destaque com o uso de marcadores moleculares como ferramenta para acrescentar informações genômicas aos processos de melhoramento convencional. Estudos anteriores em uma população F2 originada do cruzamento de frangos de corte e postura permitiram a identificação de um SNP no gene FGFBP1 (Proteína de ligação do fator de crescimento do fibroblasto 1) (g. 2014 G> A) no cromossomo 4 de Gallus gallus (GGA4). Este gene está em uma região de QTLs associado com rendimentos de coxa e sobrecoxa, peso vivo aos 35 e 41 dias de idade. O objetivo deste trabalho foi investigar um QTL previamente descrito para identificação de polimorfismos adicionais e suas associações com características de importância econômica utilizando testes de associação de um ou mais marcadores. Três genes candidatos posicionais foram identificados nesta região de QTL: KLF3(Krüeppel-like factor 3), SLIT2 (Slit homolog 2) e PPARG (Peroxisome proliferator-activated receptor gamma, coactivator 1alpha). O sequenciamento destes genes em onze (n=11) animais F1 permitiu a identificação de um polimorfismo em cada gene: g.6763 C> T (KLF3), g.187737 C> A (SLIT2) e g.76638826 -/TTTCT (PPARGC1A). Essas mutações foram genotipadas em uma população segregante F2 (n=276) e em uma linhagem pura de corte TT (n=840) da Embrapa Suínos e Aves. A frequência dos alelos para o gene KLF3 na população F2 foi de C=50% T=50% e na pura TT de C=98% T=2%, para o gene SLIT2 na população F2 foi de A=25% C=75% e na pura TT de A=30% C=70%, para o gene PPARGC1A na população F2 foi de Del=43% In=57% e na pura TT Del=33% C=67%, representando que estes polimorfismos estão segregando nas duas populações. Associações destes polimorfismos foram observadas (P<0,05) com várias características de desenvolvimento e carcaça na população F2 e na linhagem pura TT, sendo que algumas foram confirmadas nesta população como: pesos de fígado, coxas, ganhos de peso dos 35 aos 41 dias com g.6763 C> T (KLF3) e pesos das asas, cabeça, carcaça, dorso, coxas, peito, fígado e gordura abdominal com g.76638826 -/TTTCT (PPARGC1A) indicando que esta região de QTL é importante para características de produção e desempenho em frangos de corte. / Within the livestock sector, the broiler industry has showed fastest growing rates in past decades. Those advances were achieved mainly because a better understanding of the nutrition requirements, animal management and animal genetics. Biotechnology has gained a prominent role with the use of molecular markers as a tool for adding genomic information to conventional breeding processes. Previous studies using an F2 population developed from a broiler x layer cross led to the identification of a SNP on the Fibroblast growth factor binding protein 1 (FGFBP1) (g. 2014G> A) on Gallus gallus chromosome 4 (GGA4). This gene is part of a QTL associated with thigh & drumstick yields, weight gain at 35 and 41 days. This paper investigates the previously identified QTL for the identification of additional polymorphisms and their associations with important economic traits using a single and multiple markers tests. Three positional candidate genes were identified on the QTL region: KLF3 (Krüeppel-like factor 3), SLIT2 (Slit homolog 2) and PPARG (Peroxisome proliferator-activated receptor gamma, coactivator 1alpha). Sequencing of those genes was conducted in eleven (n=11) F1 animals and one polymorphism was identified in each gene g.6763 C> T (KLF3), g.187737 C> A (SLIT2) and g.76638826 -/TTTCT (PPARGC1A). These mutations were genotyped in an F2 (n=276) and a pure broiler line (n=840) from Embrapa. The frequency of the genes alleles were: KLF3 gene in F2 population C = 50% T = 50% and pure TT population C = 98% T = 2%; SLIT2 gene in F2 population A = 25% C = 75% and pure TT population A = 30% C = 70%; PPARGC1A gene in F2 population Del =43% and In = 57% and pure TT population Del = 33% C = 67% indicating that those polymorphisms are still segregating in both populations. Association was identified (P < 0,05) with several carcass and development traits in the F2 and Pure TT population, some which were confirmed like liver, drumstick weights, weight gain from 35 to 41 days with g.6763 C> T (KLF3) and wings, had, carcass, back, drumstick, breast and liver and abdominal fat weights with g.76638826 -/TTTCT (PPARGC1A) indicating that this QTL region is important for production and performance traits of broiler.

Submitted by Camila Urbano Braz null (camila_urbano@yahoo.com.br) on 2016-08-29T20:11:34Z No. of bitstreams: 1 Tese_Camila_Urbano_Braz.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-08-30T18:25:24Z (GMT) No. of bitstreams: 1 braz_cu_dr_jabo.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) / Made available in DSpace on 2016-08-30T18:25:24Z (GMT). No. of bitstreams: 1 braz_cu_dr_jabo.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) Previous issue date: 2016-07-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O mercado consumidor tem sido cada vez mais exigente quanto à qualidade da carne e, portanto, a pecuária precisa melhorar sua eficiência e fornecer produtos diferenciados, padronizados e com qualidade. Entre as características de qualidade de carne, a maciez é a que mais influencia na satisfação dos consumidores. Considerando a importância dada à maciez da carne, pesquisas têm sido desenvolvidas para melhor compreender os mecanismos relacionados à expressão fenotípica desta característica. Os estudos de genes candidatos têm possibilitado a identificação de polimorfismos que modificam as estruturas das proteínas ou ainda, que estejam em desequilíbrio de ligação com alterações funcionais no DNA. Com a automatização dos polimorfismos de nucleotídeo único (SNPs) muitas regiões ao longo do genoma foram identificadas como responsáveis pela variação fenotípica da maciez da carne. No entanto, os marcadores SNPs podem ter baixa capacidade de identificar locos que atuam nas características quantitativas (QTL) por serem, na grande maioria, bi-alélicos e, mesmo que aconteçam mutações, as mudanças nas frequências alélicas dos SNPs podem permanecer quase inalteradas. Por outro lado, com a utilização de haplótipos, mutações tendem a causar mudanças nas frequências dos haplótipos, aumentando as chances de identificação dos QTL. Sendo assim, este estudo teve como objetivos detectar QTL e mutações causais em genes candidatos e identificar QTL por meio de uma análise de associação genômica ampla, para a característica maciez da carne em bovinos da raça Nelore, utilizando haplótipos como unidades fundamentais dos testes de associação. Foram utilizadas informações fenotípicas, genotípicas e de pedigree de animais provenientes de fazendas que integram os programas de melhoramento genético DeltaGen e PAINT. Cinquenta e dois genes candidatos foram escolhidos para serem analisados utilizando haplótipos construídos com base no desequilíbrio de ligação, utilizando 1.616 animais. Do total de haplótipos, dois foram significativos, sendo que os éxons próximos e dentro destes foram sequenciados visando buscar a mutação causal. O sequenciamento foi realizado com 298 animais e os SNPs identificados foram imputados para os 1.318 animais remanescentes. Foram realizadas análises de associação utilizando haplótipos construídos com base na metodologia de janelas sobrepostas, sendo que seus efeitos foram estimados pelo método Genomic Best Linear Unbiased Predictor (GBLUP). Os valores genéticos dos animais foram estimados para cada haplótipo e SNP e, após, as variâncias genéticas aditivas foram calculadas. Utilizando haplótipos construídos com base em janelas sobrepostas, verificou-se que o aumento do número de SNPs no haplótipo permitiu capturar maior proporção da variância genética aditiva da característica maciez da carne. Seis possíveis QTL foram identificados explicando as maiores proporções de variância genética aditiva para maciez da carne, dos quais um está no gene CAPN1 e cinco no gene ASAP1. Não houve evidências de que a mutação causal para a maciez da carne tenha sido identificada nos dois genes. Uma análise de associação genômica ampla foi realizada utilizando haplótipos construídos com base na metodologia de janelas sobrepostas de tamanhos variados. Foram utilizados nesta análise 1.405 animais genotipados com o painel Illumina Bovine HD e 1.756 animais genotipados com painel de menor densidade (70 K Neogen) e, posteriormente, imputados para o painel HD, em um total de 3.161 animais analisados. Os efeitos dos haplótipos e SNPs foram estimados pelo método GBLUP e as variâncias genética aditivas de cada haplótipo e SNP foram calculadas. Os genes NOS1AP, SUCLG1, PHLDB2 e LOC107132946 foram associados com a característica maciez da carne em bovinos da raça Nelore, por meio de análises de associação genômica ampla utilizando SNPs individuais e haplótipos. A análise utilizando SNPs identificou QTL diferentes das análises com haplótipos e, em alguns casos, SNPs apresentaram variâncias genéticas aditivas maiores do que as apresentadas pelos haplótipos. A análise que utilizou haplótipos construídos com cinco SNPs identificou mais QTL do que as análises de haplótipos construídos com sete e nove SNPs. Sugere-se que análises utilizando haplótipos, baseados em janelas sobrepostas, sejam realizadas para complementar análises de SNPs individuais em estudos de associação genômica ampla. / The consumer market has been increasingly demanding about the meat quality and therefore livestock needs to improve its efficiency and provide differentiated products, standardized and with quality. Considering the importance given to the meat tenderness, research has been undertaken to better understand the mechanisms related to the phenotypic expression of this trait. The candidate gene studies have allowed the identification of polymorphisms that change the structures of the proteins or that are in linkage disequilibrium with functional alterations in the DNA. With the advent of single nucleotide polymorphisms (SNPs) throughout many regions of the genome have been identified as responsible for phenotypic variation in meat tenderness. However, SNPs markers may have low ability to identify mutations, because SNPs are commonly bi-allelic and even when mutations have occurred, allelic frequencies can remain unaltered. On the other hand, using haplotype, mutations tend to cause major changes in haplotype frequencies, increasing the chances of identification of QTL. Thus, this study aimed to detect QTL and causal mutations by the approach of candidate genes and identify possible QTL through a genome-wide association analysis, for the trait meat tenderness in Nelore cattle using haplotypes as fundamental units of association tests. Information of the phenotypic, genotypic and pedigree were used from farms that belong to the breeding programs DeltaGen and PAINT. Fifty-two candidate genes were chosen for analysis using haplotypes constructed based on linkage disequilibrium using 1,616 animals. Two haplotypes were significant, and the exons near and within these haplotypes were sequenced to search for the causal mutation. The sequencing was performed using 298 animals and the identified SNPs were imputed for 1,318 remaining animals. Association analysis using haplotypes constructed based on the method of overlapping sliding windows were carried out and the SNPs and haplotypes effects were estimated using Genomic Best Linear Unbiased Predictor (GBLUP) method. The breeding values were estimated for each haplotype and SNPs and the additive genetic variances were calculated. Using haplotypes constructed based on overlapping sliding windows, we found that increasing the number of SNPs in the haplotype allowed to capture a greater proportion of additive genetic variance of meat tenderness. Six putative QTL were identified with the greatest additive genetic variances for meat tenderness, which one was in CAPN1 gene and five in ASAP1 gene. There was no evidence that the causal mutation for meat tenderness trait has been identified in these genes. A genome-wide association analysis was performed using haplotypes constructed based on the methodology of overlapping sliding windows of varying sizes. In this analysis, 1,405 animals genotyped with the Illumina Bovine HD panel and 1,756 genotyped animals with lower density panel (Neogen 70 K) were used and then, the genotypes of the 1,756 were imputed to the HD panel, in a total of 3,161 animals analyzed. The haplotypes and SNPs effects were estimated by the method GBLUP and the additive genetic variances were calculated for each haplotype and SNP. The NOS1AP, SUCLG1, PHLDB2 and LOC107132946 genes were associated with the meat tenderness trait in Nelore cattle through genome-wide association analysis using individual SNPs and haplotypes. The analysis using SNPs identified different QTL of the haplotype analyzes, and in some cases, the SNPs showed additive genetic variance greater than those presented by the haplotypes. The analysis used haplotypes constructed with five SNPs identified more QTL than analysis of haplotypes constructed with seven and nine SNPs. Analyzes using haplotypes based on overlapping sliding windows, should be conducted as additional analyzes for individual SNPs in genome-wide association studies. / FAPESP: 2013/00035-9

Orientadores: Luciana Lasry Benchimol Reis, Luis Eduardo Aranha Camargo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T04:26:18Z (GMT). No. of bitstreams: 1 Oblessuc_PaulaRodrigues_D.pdf: 15432495 bytes, checksum: 30c1cd638125be03e494a50fae4fe12b (MD5) Previous issue date: 2013 / Resumo: O feijão comum (Phaseolus vulgaris L.) é uma importante fonte de proteínas na dieta humana. A mancha angular (ALS), causada pelo fungo Pseudocercospora griseola (Sacc.) Crous & Braun, acarreta grandes prejuízos na produção do feijão. O melhoramento do feijoeiro busca ferramentas que agilizem a transferência de genes de resistência às doenças para cultivares em desenvolvimento. Assim, foi objetivo deste trabalho estudar os mecanismos genéticos e moleculares envolvidos na resposta do feijão à ALS, e com isso contribuir para o melhoramento dessa cultura. Primeiramente, QTLs (Quantitative Trait Locus) de resistência à ALS foram identificados utilizando a população de mapeamento UC (IAC-UNA x CAL 143) a partir do mapa genético previamente desenvolvido com marcadores microssatélites. O estudo quantitativo da severidade da ALS revelou distribuição normal e transgressiva na população UC, com resistência quantitativa observada em CAL 143. Ao todo foram mapeados sete QTLs em cinco diferentes grupos de ligação de feijão. Dentre estes, o loco ALS10.1 mostrou maior efeito (16% - 22%) e estabilidade nos três ambientes analisados: (1) condições naturais de infecção em época chuvosa de plantio; (2) condições naturais de infecção em época seca de plantio; e (3) condições controladas de infecção raça-específica em casa de vegetação. A região do loco ALS10.1 foi saturada com marcadores microssatélites, SCARs e Sequence-Tagged Site-DArTs (STS-DArTs); este último através da técnica de bulk segregant analysis (BSA). O intervalo de confiança foi reduzido de 13.4 cM para 3.0 cM após a saturação do loco, que teve seu número de marcadores aumentado de quatro para 10. O estudo do contexto genômico do ALS10.1 através do alinhamento dos marcadores com o rascunho do genoma do feijão possibilitou definir uma região core para o QTL na extremidade do cromossomo Pv10, com aproximadamente 5,3 Mb. Análise de Gene Onthology (GO) dos 323 genes preditos nesta região do genoma demonstrou que 61,6% destes genes estão envolvidos na resposta a estresse. Cluster de genes TIR-NB-ARC (domínios altamente conservado em genes (R) de Resistência) foi identificado cobrindo aproximadamente 849 Kb na região core de ALS10.1; além de esta região conter outros genes sabidamente relacionados à imunidade de plantas. Sete genes presentes na região core de ALS10.1 foram selecionados com base na função na resistência à patógenos dos respectivos ortólogos em Arabidopsis thaliana, e tiveram sua expressão gênica avaliada na resposta à P. griseola. Gene R TIR-NB-ARC (Phvul.010G025700) foi induzido em resposta compatível no genótipo IAC-UNA, com isso deve permitir a proliferação do patógeno, possivelmente através do reconhecimento do Avr (avirulência) do fungo, bloqueando a resposta de defesa. Além disso, genes putativos de regulação negativa da resposta imune pela inativação da via do ácido salicílico (SA) foram reprimidos durante resposta incompatível no genótipo CAL 143. O AS é um hormônio chave para resposta de defesa induzida por patógenos em plantas. Com isso, o reconhecimento do patógeno pelo feijão deve ocorrer através de genes R para sinalização downstream da resposta de defesa mediada por AS. Os resultados deste trabalho permitirão que o melhorista manipule a diversidade genética do feijão, seja pela introgressão e piramidação dos genes de resistência através de seleção assistida por marcadores ou transgenia; seja pela identificação de cultivares geneticamente resistentes pela análise de expressão gênica / Resumo: O feijão comum (Phaseolus vulgaris L.) é uma importante fonte de proteínas na dieta humana. A mancha angular (ALS), causada pelo fungo Pseudocercospora griseola (Sacc.) Crous & Braun, acarreta grandes prejuízos na produção do feijão. O melhoramento do feijoeiro busca ferramentas que agilizem a transferência de genes de resistência às doenças para cultivares em desenvolvimento. Assim, foi objetivo deste trabalho estudar os mecanismos genéticos e moleculares envolvidos na resposta do feijão à ALS, e com isso contribuir para o melhoramento dessa cultura. Primeiramente, QTLs (Quantitative Trait Locus) de resistência à ALS foram identificados utilizando a população de mapeamento UC (IAC-UNA x CAL 143) a partir do mapa genético previamente desenvolvido com marcadores microssatélites. O estudo quantitativo da severidade da ALS revelou distribuição normal e transgressiva na população UC, com resistência quantitativa observada em CAL 143. Ao todo foram mapeados sete QTLs em cinco diferentes grupos de ligação de feijão. Dentre estes, o loco ALS10.1 mostrou maior efeito (16% - 22%) e estabilidade nos três ambientes analisados: (1) condições naturais de infecção em época chuvosa de plantio; (2) condições naturais de infecção em época seca de plantio; e (3) condições controladas de infecção raça-específica em casa de vegetação. A região do loco ALS10.1 foi saturada com marcadores microssatélites, SCARs e Sequence-Tagged Site-DArTs (STS-DArTs); este último através da técnica de bulk segregant analysis (BSA). O intervalo de confiança foi reduzido de 13.4 cM para 3.0 cM após a saturação do loco, que teve seu número de marcadores aumentado de quatro para 10. O estudo do contexto genômico do ALS10.1 através do alinhamento dos marcadores com o rascunho do genoma do feijão possibilitou definir uma região core para o QTL na extremidade do cromossomo Pv10, com aproximadamente 5,3 Mb. Análise de Gene Onthology (GO) dos 323 genes preditos nesta região do genoma demonstrou que 61,6% destes genes estão envolvidos na resposta a estresse. Cluster de genes TIR-NB-ARC (domínios altamente conservado em genes (R) de Resistência) foi identificado cobrindo aproximadamente 849 Kb na região core de ALS10.1; além de esta região conter outros genes sabidamente relacionados à imunidade de plantas. Sete genes presentes na região core de ALS10.1 foram selecionados com base na função na resistência à patógenos dos respectivos ortólogos em Arabidopsis thaliana, e tiveram sua expressão gênica avaliada na resposta à P. griseola. Gene R TIR-NB-ARC (Phvul.010G025700) foi induzido em resposta compatível no genótipo IAC-UNA, com isso deve permitir a proliferação do patógeno, possivelmente através do reconhecimento do Avr (avirulência) do fungo, bloqueando a resposta de defesa. Além disso, genes putativos de regulação negativa da resposta imune pela inativação da via do ácido salicílico (SA) foram reprimidos durante resposta incompatível no genótipo CAL 143. O AS é um hormônio chave para resposta de defesa induzida por patógenos em plantas. Com isso, o reconhecimento do patógeno pelo feijão deve ocorrer através de genes R para sinalização downstream da resposta de defesa mediada por AS. Os resultados deste trabalho permitirão que o melhorista manipule a diversidade genética do feijão, seja pela introgressão e piramidação dos genes de resistência através de seleção assistida por marcadores ou transgenia; seja pela identificação de cultivares geneticamente resistentes pela análise de expressão gênica / Abstract: The common bean (Phaseolus vulgaris L.) is an important protein source in human diet. The angular leaf spot (ALS), caused by the fungus Pseudocercospora griseola (Sacc.) Crous & Braun, leads to great common bean yield losses. The common bean breeding search for tools that improve the transferring of disease resistance genes to developing cultivars. Therefore, the objective of the present work was study the genetic and molecular mechanisms enrolled in the common bean responses to ALS, and with this contribute to this crop breeding. Initially, ALS resistance QTLs were identified using the UC (IAC-UNA x CAL 143) mapping population based on the genetic map previously developed with microsatellites markers. The quantitative study of the ALS disease severity reveals normal and transgressive distribution on the UC population, with quantitative resistance observed in CAL 143. Seven QTLs were mapped in five different common bean linkage groups. Of these, the ALS10.1 locus showed major effect (16% - 22%) and stability in all three environments analyzed: (1) natural infection conditions in the dry season; (2) natural infection conditions in wet season; and (3) race-specific controlled infection conditions in greenhouse. The ALS10.1 locos region was saturated with microsatellites, SCARs and Sequence-Tagged Site-DArTs (STS-DArTs) markers; the latter using the bulk segregant analysis (BSA). The confidence interval was reduced from 13.4 cM to 3.0 cM after the locus saturation, which had the markers number increased from four to 10. The study of the ALS10.1 genomic context through the alignment of the markers to the draft of the common bean genome enabled the identification of the QTL core at the end of the chromosome Pv10, with approximately 3.5 Mb. The Gene Ontology (GO) analyses of the 323 predicted genes for this genomic region demonstrated that 61.6% of the genes are involved in stress responses. A TIR-NB-ARC gene cluster (domains highly conserved in Resistance (R) genes), was observed covering approximately 849 Kb on the ALS10.1 core region; besides this region also presents other genes known to be related to plant immunity. Seven genes on ALS10.1 core region were selected based on the role in pathogen resistance of the respective Arabidopsis thaliana orthologs, and had their gene expression pattern evaluated in response to P. griseola. The R gene TIR-NB-ARC was induced during the compatible response of the genotype IAC-UNA; therewith it should enable the pathogen proliferation, probably through the fungus Avr recognition, blocking the defense response. In addition, putative negative regulator genes of immune response through the inactivation of salicylic acid (SA) via were repressed during the incompatible response of the CAL 143. The SA is a key hormone to pathogen induced plant defense response. Therefore, the common bean pathogen recognition should take place through the R genes to the downstream signaling of the SA-mediated defense response. The results of the present work will enable the manipulation of the bean genetic diversity by the breeder either by introgression and pyramiding of resistance genes through marker assisted selection or transgenesis; or by the identification of genetically resistant cultivars through gene expression analysis / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

Answers to the most complex biological questions are rarely determined solely from the experimental evidence. It requires subsequent analysis of many data sources that are often heterogeneous. Most biological data repositories focus on providing only one particular type of data, such as sequences, molecular interactions, protein structure, or gene expression. In many cases, it is required for researchers to visit several different databases to answer one scientific question. It is essential to develop strategies to integrate disparate biological data sources that are efficient and seamless to facilitate the discovery of novel associations and validate existing hypotheses. This thesis presents the design and development of different integration strategies of biological and clinical systems. The BioSPIDA system is a data warehousing solution that integrates many NCBI databases and other biological sources on protein sequences, protein domains, and biological pathways. It utilizes a universal parser facilitating integration without developing separate source code for each data site. This enables users to execute fine-grained queries that can filter genes by their protein interactions, gene expressions, functional annotation, and protein domain representation. Relational databases can powerfully return and generate quickly filtered results to research questions, but they are not the most suitable solution in all cases. Clinical patients and genes are typically annotated by concepts in hierarchical ontologies and performance of relational databases are weakened considerably when traversing and representing graph structures. This thesis illustrates when relational databases are most suitable as well as comparing the performance benchmarks of semantic web technologies and graph databases when comparing ontological concepts. Several approaches of analyzing integrated data will be discussed to demonstrate the advantages over dependencies on remote data centers. Intensive Care Patients are prioritized by their length of stay and their severity class is estimated by their diagnosis to help minimize wait time and preferentially treat patients by their condition. In a separate study, semantic clustering of patients is conducted by integrating a clinical database and a medical ontology to help identify multi-morbidity patterns. In the biological area, gene pathways, protein interaction networks, and functional annotation are integrated to help predict and prioritize candidate disease genes. This thesis will present the results that were able to be generated from each project through utilizing a local repository of genes, functional annotations, protein interactions, clinical patients, and medical ontologies.

La sténose valvulaire aortique (SVA) est une valvulopathie résultant en l'ouverture incomplète de la valve aortique. La calcification des feuillets associée au vieillissement est la cause la plus importante de la SVA. Sa pathogénèse implique des dépôts de lipoprotéines, de l'inflammation et de la calcification des feuillets. Notre étude vise à identifier les gènes associés à une prédisposition à la SVA afin de mieux comprendre les mécanismes sous-jacents à cette maladie et potentiellement identifier de nouvelles cibles thérapeutiques. Pour ce faire, nous avons recruté 190 patients avec SVA dégénérative et 192 témoins, appariés pour l'âge et le sexe, puis effectué une étude d’association par gènes candidats en utilisant des marqueurs génétiques polymorphiques (SNP). Les gènes candidats choisis incluent (1) ceux dont les polymorphismes ont été présumés associés à la SVA dans des études antérieures (APOB, APOE, ESR1, PTH et VDR) (2) des gènes dont les polymorphismes ont été significativement associés et validés pour quelques maladies inflammatoires (IL-10, TNFAIP3) ou pour le métabolisme lipidique (PCSK9, LDLR) dans des études d’association pangénomiques, et (3) des gènes impliqués dans la pathogénie de la SVA à partir d’études faites sur des modèles animaux en lien avec la calcification (BMP2, CCR5, CTGF, LRP5, MSX2, WNT3), le remodelage tissulaire (CTSS, MMP9) ou le métabolisme lipidique (SMPD1). Pour les gènes des groupes (1) et (2), nous avons utilisé les SNPs rapportés dans la littérature comme étant significativement associés. Pour le groupe (3), nous avons effectué une approche par «tagSNP» qui consiste à sélectionner un groupe de SNP capturant la variabilité génétique dans la région ciblée. Au total, 81 SNPs dans 18 gènes ont été testés. Nous avons trouvé une association nominale avec les gènes BMP2 (OR = 1.55, IC95%: 1.14-2.10, p = 0.004) et LRP5 (OR = 1.47, IC95%: 1.06-2.03, p = 0.023) après ajustement pour la maladie coronarienne. Les gènes BMP2 et LRP5, impliqués dans la calcification selon certains modèles expérimentaux, sont donc associés à la SVA. Ce travail devrait être validé dans une cohorte indépendante plus large dans un avenir rapproché et il pourrait être étendu à d’autres gènes. / Aortic valve stenosis (AVS) is a valvular heart disease caused by calcification leading to incomplete opening of the aortic valve. Calcification of valve leaflets associated with aging is the most common cause of AVS. AVS pathogenesis involves lipoprotein deposits, chronic inflammation and calcification of the aortic valve leaflets. Our study aims to identify genes associated with AVS in order to better understand its mechanisms and potentially identify new therapeutic targets. We recruited 190 cases with AVS of different severity and 192 controls matched for age and sex. Then we conducted a candidate gene association study using single nucleotide polymorphisms (SNPs). The candidate genes selected include: (1) those with polymorphisms putatively implicated in previous genetic association studies of AVS (APOB, APOE, ESR1, PTH and VDR); (2) those with validated associations to inflammatory diseases (IL-10, TNFAIP3) or lipid metabolism (LDLR ,PCSK9) in genome-wide association studies and, (3) genes impliated in AVS pathogenesis from studies with animal models and thought to be involved in calcification (BMP2, CCR5, CTGF, LRP5, MXS2, WNT3); tissue remodeling (CTSS, MMP9) or lipid metabolism (SMPD1). For the first two categories of genes, we tested the SNPs reported to be associated in the literature and, in the third category we used a tag-SNP approach which consists of selecting a subset of SNPs to capture variability in the target region. Finally, 81 SNPs in 18 genes were tested. We found a nominal association of BMP2 (OR=1.55, CI: 1.14 – 2.10, p=0.004) and LRP5 (OR=1.47, CI: 1.06 – 2.03, p=0.023) with presence of AVS after adjustment for coronary heart disease. The genes BMP2 and LRP5, which are known to be involved in calcification based on animal models, are associated with AVS. The result of the current study should be validated in a larger independent cohort in the near future and then, it could also be extended to the study of other genes.

Ce travail a porté sur l'identification et la caractérisation de nouveaux facteurs de l'hôte nécessaires au cycle infectieux des virus à ARN (genre Potyvirus) chez Arabidopsis thaliana, ces facteurs pouvant constituer des cibles pour la lutte génétique contre les phytovirus. L'exploration de la diversité génétique naturelle d'Arabidopsis a conduit à l'identification d'un gène récessif de résistance au Watermelon mosaic virus (WMV) chez l'accession Cvi-0. Il s'agit du gène rwm1 qui agit à un stade précoce de l'interaction en empêchant l'accumulation du virus au niveau des premières cellules infectées. Les résultats de clonage positionnel de rwm1, de séquençage d'allèles de résistance et de sensibilité et de validation fonctionnelle argumentent en faveur d'un rôle de la phosphoglycérate kinase chloroplastique (cPGK), une enzyme clé de la photosynthèse, dans la résistance conférée par rwm1. En parallèle, une stratégie gène candidat a été mise en œuvre pour étudier le rôle de la protéine kinase Target Of Rapamycin (TOR) dans l'interaction plante-potyvirus. La caractérisation de lignées RNAi pour le gène TOR d'Arabidopsis montre une diminution voire une suppression de l'accumulation du WMV, ainsi qu'une réduction de l'accumulation du Turnip mosaic virus. L'étude comparative de la composition des complexes de liaison à la coiffe entre une lignée sauvage et une lignée RNAi TOR en conditions saines et infectées par le WMV suggère un rôle de TOR dans l'interaction avec les potyvirus par le biais d'une régulation de l'activité du facteur d'initiation de la traduction eIF3. / This work describes the use of forward and reverse genetics in Arabidopsis thaliana to identify and characterize new host factors required for the infectious cycle of plant RNA viruses (genus Potyvirus). The exploration of the Arabidopsis natural genetic diversity led to the identification of the recessive rwm1 gene for resistance to Watermelon mosaic virus (WMV). Rwm1-mediated resistance was identified in the Cvi-0 accession where it acts at an early stage of the infection process by impairing virus accumulation in initially infected tissues. Map-based cloning results, allelic sequence analysis and functional validation experiments, strongly suggest the involvement of chloroplast phosphoglycerate kinase (cPGK), a key photosynthetic enzyme, in rwm1-mediated resistance. In parallel, a candidate gene approach was developed to investigate the potential role of the TOR kinase (Target Of Rapamycin) in the control of susceptibility to potyviruses. Arabidopsis RNAi lines displaying partial TOR inactivation showed a significant decrease of susceptibility to the infection by two potyviruses, namely WMV and Turnip mosaic virus. The results from a comparative proteomic analysis of cap-binding complexes between wild type and TOR RNAi infected and healthy lines suggest that TOR could play a role in plant-potyvirus interactions by regulating the activity of the translation initiation factor eIF3. Overall, this work opens a challenging research area to provide novel insights on the molecular principles underlying viral infection processes and opportunities to improve and optimize the way we tackle plant resistance to viruses through a diversification of genetic targets.

Notre travail s’est intéressé à l’albinisme oculocutané en étudiant ses aspects clinico- moléculaires. Malgré l’analyse approfondie des gènes connus d’albinisme oculocutané, 15 % des patients restent sans mutation identifiée indiquant que les mutations sont situées dans des régions géniques non analysées par les techniques classiques de diagnostic moléculaire, ou qu’il existe d’autres gènes d’albinisme oculocutané. Nous avons établi une base de données clinico- biologiques décrivant les caractéristiques de plus de 400 patients analysés. Des outils de diagnostic moléculaire ont été développés à la recherche de mutations situées dans les introns et les régions régulatrices et de réarrangements géniques. Différentes stratégies ont également été utilisées pour rechercher des gènes candidats. La puce à façon a permis l’identification de grands réarrangements dans les gènes TYR, OCA2 et SLC45A2 et un réarrangement complexe du gène OCA2 chez 2 patients non apparentés. L'analyse de gènes candidats nous a permis d'identifier, chez 5 patients non apparentés présentant un albinisme oculocutané non syndromique, des mutations dans le gène SLC24A5, très récemment associé à l’AOC6. Le séquençage d’exome de 6 patients a mis en évidence des gènes candidats pour lesquels des analyses complémentaires sont poursuivies afin de confirmer leur implication dans la pathogenèse de l’AOC.Les résultats de ce travail permettent de redéfinir les aspects cliniques et moléculaires de l’AOC, d’identifier de nouveaux mécanismes moléculaires à l’origine de l’AOC ainsi que des gènes candidats dont la fonction dans le développement pigmentaire reste à élucider. L’identification de nouveaux gènes impliqués dans l’AOC pourrait permettre de mieux comprendre et de mieux prendre en charge les patients avec un AOC. / Our work focused on oculocutaneous albinism (OCA) by studying its clinical and molecular aspects. Despite a thorough analysis of the known genes involved in oculocutaneous albinism, 15% of patients remain without diagnostic at the molecular level indicating that mutations are located in unexplored regions and are undetected by standard techniques or that other genes are involved in albinism. We established a clinicomolecular database describing more than 400 patients and developped molecular tools in order to improve molecular diagnostic including a custom high resolution array-CGH dedicated to the four OCA genes (TYR, OCA2, TYRP1 and SLC45A2). We also used different strategies to identify new genes. Array-CGH allows us to detect large deletion in TYR, OCA2 and SLC45A2 and a complexe rearrangement in OCA2 in 2 unrelated patients. We identified, in 5 patients presenting with a non syndromic OCA, mutations in SLC24A5, recently associated with OCA6. Exome sequencing of 6 different patients allows us to identify candidate genes, for which further studies are required to confirm their involvement in OCA pathogenesis. The results of this work allowed us to delineate clinical and genetics aspects of more than 400 OCA patients and to identify new molecular mechanisms leading to OCA and candidates genes for which exact nature of their functions has to be understood. Giving the complexity of pigmentary system development and its regulation, identification of new genes leading to OCA could help to better understand OCA and take care of patients

Les mécanismes physiopathologiques des hypersensibilités (HS) médicamenteuses ne sont que partiellement connus. Un terrain génétique favorisant est connu de longue date, mais peu de facteurs de risques sont formellement identifiés. A l’aide d’une approche par gènes candidats, nous avons évalué l’association entre des polymorphismes des gènes NOD1 et 2 et l’HS aux bétalactamines ; l’association entre plusieurs polymorphismes cytokiniques et différentes HS médicamenteuses. Enfin, nous avons utilisé une approche pangénomique à la recherche de gènes candidats au cours d’une HS spécifique, le DRESS. Parmi 368 cas et autant de contrôles italiens ainsi que 387 cas et 326 contrôles espagnols, nous avons mis en évidence une association entre l’un des polymorphismes de NOD2 et un faible risque d’HS immédiate aux bétalactamines chez les patients italiens, tandis qu’un autre polymorphisme de NOD2 était associé à un risque augmenté d’HS immédiate aux bétalactamines chez les patients espagnols. Aucune association avec le polymorphisme de NOD1 n’était identifiée. Parmi 118 patients et 236 contrôles, nous avons identifiés l’association entre les polymorphismes de l’IL1 (IL1-RN-A2 et IL1-[bêta] -511) et de l’IL10 (-592A) avec le risque de DRESS. Enfin, parmi 18 DRESS, l’analyse de puces CNV pangénomique nous a permis d’identifier des variations comportant les gènes KLRC2 et CESP1.Au total, nous avons pu démontrer l’implication de gènes modulant l’inflammation, la réponse antivirale ou le métabolisme des médicaments dans différentes HS médicamenteuses. Une confirmation à l’étage fonctionnelle de ces résultats est nécessaire / The physiopathology of drug hypersensitivity (HS) are only partially known. A genetic background for such drug allergy is still demonstrated but only few genes are identified. Using a candidate gene approach, we tested the association of NOD1 and 2 genes with betalactam HS and the association of several cytokines genes with some drug HS. Using a whole genome approach, we tried to discover new candidate gene for DRESS. Among 368 italian cases and controls and 387 spanish cases and 326 controls, we identified one polymorphism of NOD2 gene associated with a protective effect for italians and another polymorphism associated with higher risk of druh HS for spanians. No association with NOD1 polymorphims was identified. Among 118 cases and 236 controls, we noticed that IL1 polymorphisms (IL1-RN-A2 and IL1-? -511) and IL10 polymorphism (-592A) were associated with DRESS.Ending, among 18 DRESS, a whole-genome array let us identify variations containing KLRC2 and CESP1 genes. These studies demonstrate the implication of several genes involved in inflammation, antivirus response or drug metabolism in different drug HS.Fonctionnal studies are needed to confirm these results