Rozprawy doktorskie na temat „Expression QTL”

Expression quantitative trait loci (eQTLs) are genetic markers associated with transcription of Ribonucleic Acid (RNA). eQTLs are detected using association analysis to detect correlations between RNA expression data (microarray or RNA-SEQ) and the genotypes of individuals within a study. Trans-ethnic meta-analysis can increase power to detect genetic variants for eQTLs and improve fine-mapping resolution because of differential patterns of linkage disequilibrium (LD) between diverse populations. Lymphoblastoid cell lines (LCLs) from samples in the Phase II and III HapMap populations have been used to detect cis eQTLs using association analysis followed by meta-analysis. Phase III HapMap samples have also been imputed using the 1000 Genomes March 2012 "all ancestries" panel. The goals of this thesis are to perform meta-analysis on multi-ethnic association summary statistics in order to: Increase the power to detect eQTLs, leverage differences in LD between ancestry groups to fine map eQTL variants and investigate and characterize heterogeneity in allelic effect sizes on expression between diverse populations. In addition to this, eQTLs identified are used to perform integration with signals from genome-wide association studies (GWAS) of complex human traits. A pipeline has been developed where eSNPs from the eQTL datasets are integrated with disease SNPs (dSNPs) from the NHGRI GWAS catalog using reciprocal conditional analysis to determine whether eSNP and dSNP tag or are the same causal variant. Also, eQTLs which are also "absorption, distribution, metabolism, and excretion" (ADME) genes are studied in more detail, specifically looking for heterogeneity and enrichment in this dataset. The analysis shows that combining association analysis summary statistics using meta-analysis leads to an increase in power to detect eQTLs. Differences in LD between ancestry groups can be used to improve fine mapping resolution, as measured by "credible sets" of variants most likely to drive the eQTL signal, when all ancestry groups are combined. Considerable heterogeneity between ancestry groups has been detected, much of which is due to differing LD between tag SNP and causal variants across ancestry groups. Furthermore, the GWAS integration has led to the identification of several dSNP – eSNP pairs for disease such as Ulcerative Colitis, Inflammatory Bowel Disease, Bechet's Disease, Sarcoidosis, Crohn's Disease, Grave’s Disease and Primary Biliary Cirrhosis, and have provided potential novel insights of genes through which these disease association signals are mediated. Several eQTLs for genes within the ADME dataset have also been identified some of which have significant heterogeneity.

QTL mapping with segregating populations results in poor map resolution which limits the applicability of mapped QTL in further research such as gene cloning. The current research project aimed mainly at developing STepped Aligned Recombinant Inbred Strains (STAIRS) covering the top region of chromosome 3 and demonstrating the feasibility of using STAIRS in high resolution mapping of QTL in Arabidopsis. The top region of chromosome 3 of Arabidopsis had been reported to house QTL related to flowering time. This region was first saturated with 24 polymorphic microsatellite markers and 23 narrow STAIRS were produced within the region via a marker-assisted backcross breeding programme using whole chromosome substitution lines. The analysis of QTL with the narrow STAIRS revealed a major pleiotropic QTL within 2-3 cM affecting flowering time, leaf number at day 20 and rosette and cauline leaf numbers at flowering. A second QTL with less but opposite effect on the same traits were located within 15-20 cM. The search for candidate genes within 2-3 cM of chromosome 3, to locate possible candidate genes revealed COL-2, CONSTANS-Like gene which affects flowering time. Microarray gene expression profiling was performed using the two genotypically closest lines which differ for flowering time to compare the two lines at the same chronological and physiological ages in two experiments respectively. The lists of differentially expressed genes were obtained from the two experiments. Differential expression was observed for the possible candidate gene in the latter experiment. The results emphasized the power of STAIRS in fine mapping of QTL and the possibility of using them in transcriptional profiling to study the expression of genes.

L'alternance de production est définie comme la charge en fruit d'un arbre irrégulière sur plusieurs années consécutives. La principale hypothèse en soulignant l'alternance est que la charge en fruits d'une année en cours inhibe la formation de fleurs pour l'année suivante. Ce phénomène génère d'importants problèmes agronomiques pour les espèces fruitières comme le pommier, en réduisant la production de fruits au cours des années 'OFF' et la qualité des fruits au cours des années 'ON', tout en augmentant les coûts de gestion des vergers, en particulier pour l'éclaircissage des fruits. Une stratégie pour atténuer l'alternance est de développer de nouvelles variétés avec une production régulière. Les principaux objectifs de ma thèse étaient: (i) d'améliorer les stratégies de phénotypage et les méthodes pour caractériser l'alternance de production, (ii) de disséquer le contrôle génétique de l'alternance de production en utilisant une descendance de pommier en ségrégation et d'identifier les principales régions génétiques responsables de la variation du caractère, et (iii) d'étudier les processus physiologiques sous-jacents à l'alternance de production. J'ai combiné des méthodes comme la modélisation, la génétique quantitative, la détection de Quantitative Trait Loci (QTL) et de gènes candidats ainsi que la cartographie et l'expression de gènes.Mon étude a utilisé une population de pommier ségrégation obtenue à partir d'un croisement entre des parents contrastés pour les caractéristiques architecturales et de floraison (‘Starkrimson' x 'Granny Smith'). Le phénotypage de la population pour l'alternance de production a été réalisée à l'échelle d'arbres entiers, en observant les occurrences de floraison pendant six années consécutives, et à l'échelle locale, en observant la succession de méristèmes floraux vs végétative en position terminale de rameaux. De ces données, de nouveaux modèles ont été développés afin de quantifier l'alternance de la production, en tenant compte de la croissance ontogénétique de la production et la présence / absence de floraison entre les années successives le long des pousses courtes. Ceci nous a conduits à proposer de nouveaux descripteurs de la tendance d'un génotype à l'alternance de production dans les premiers stades de développement des arbres et ouvre des possibilités pour une évaluation plus rapide et plus précoce de ce caractère dans les programmes de sélection de fruits à pépins.Pour identifier les régions génomiques impliquées dans l'alternance, une détection de QTL a été réalisée sur la base des données phénotypiques et des valeurs de BLUP obtenues à partir des modèles. J'ai démontré que la régularité de la production est sous contrôle polygénique. J'ai extrait une liste de gènes qui sont présents au sein de ces QTL en utilisant la séquence du génome du pommier. Les principaux gènes candidats identifiés sont liés aux gibbérellines, aux auxines, et à la floraison. J'ai étudié l'expression des gènes candidats co-localisant avec des QTLs par PCR quantitative en utilisant les méristèmes prélevés sur les arbres portant une forte charge en fruits vs une faible charge. Une analyse microarray m'a permis d'obtenir un aperçu global des processus biologiques et de l'expression des gènes qui sont modulés dans le méristème lorsque des fruits sont présents. Certains gènes liés à la floraison, au développement du méristème, aux gibbérellines et aux auxines ont montré un profil d'expression affectée par la présence de fruits.Mes résultats fournissent des éclaircissements sur le contrôle physiologique et génétique de ce caractère complexe qui est l'alternance et ouvrent la perspective d'inclure la régularité de production dans les schémas de sélection de pommier et d'autres espèces fruitières
Biennial bearing is defined as the irregular crop load of a tree over consecutive years. The main assumption underlining biennial bearing is that the fruit load of a given year inhibits flower formation for the following year. This phenomenon generates major agronomic problems for fruit species such as apple, by reducing fruit production during ‘OFF' years and fruit quality during ‘ON' years, while increasing orchard management costs, especially for fruit thinning. A strategy to attenuate biennial bearing is to develop new varieties with regular production. The main objectives of my project were (i) to improve phenotyping strategy and methodology for biennial bearing characterisation, (ii) to dissect the genetic control of biennial bearing using an apple segregating progeny and to identify key genetic regions responsible for the trait variation, and (iii) to investigate physiological processes underlying biennial bearing. I combined methodologies such as modelling, quantitative genetics, candidate gene and Quantitative Trait Loci (QTL) mapping and gene expression.My study used an apple segregating population issued from a cross between contrasting parents for architectural and flowering features (‘Starkrimson' x ‘Granny Smith'). Phenotyping of the population for biennial bearing was achieved at whole tree scale by observing flowering occurrence for six consecutive years, and at local scale, by observing the succession of floral vs. vegetative meristems in terminal position of shoots. From this data, new models were constructed to quantify the alternation of production, taking into account the ontogenetic increasing trend of production and the presence/absence of flowering between successive years along short shoots. This led us to propose new descriptors of the tendency of a genotype to biennial bearing in the early stages of tree development and opens possibilities for a faster and earlier evaluation of this character in pipfruit breeding programmes and for orchard management.To identify genomic regions involved in biennial bearing, a QTL detection was performed on the basis of phenotypic data and BLUP values obtained from the models. I demonstrated that the regularity of production is under polygenic control. I mined a list of genes that are present within these QTLs using the apple genome sequence. The main candidate genes identified are related to gibberellins, auxins, and flowering.I investigated the expression of candidate genes co-locating with QTLs by quantitative PCR using meristems collected on trees bearing heavy fruit load vs. light crop. A microarray analysis enabled me to obtain a global overview of biological processes and gene expression that are modulated in the meristem when fruits are present. Some genes related to flowering, meristem development, gibberellins and auxins showed an expression profile affected by the presence of fruit.My results provide elucidation on the physiological and genetic control of the complex trait that is biennial bearing and open up the perspective of including regular bearing in breeding schemes for apple and other fruit species

O grupo de percevejos que mais frequentemente causa perdas econômicas à soja no Brasil é composto pelas espécies: Nezara viridula, Piezodorus guildinii e Euchistus heros. Assim, os objetivos desta pesquisa foram avaliar parâmetros genéticos e correlações entre as diferentes características de desenvolvimento e produção, mapear QTL associados à resistência da soja aos percevejos e determinar respostas de expressão gênica associadas à alimentação do inseto. Uma população F2:3 foi desenvolvida através do cruzamento de IAC- 100 (resistente) e CD-215 (suscetível) e avaliada em campo experimental. As características agronômicas avaliadas foram: número de dias para o florescimento (NDF), altura da planta no florescimento (APF), número de dias para a maturidade (NDM), altura da planta na maturidade (APM), acamamento (AC), valor agronômico (VA) e produtividade de grãos (PG). As características de resistência a percevejos avaliadas foram: período de enchimento de grãos (PEG), retenção foliar (RF), índice percentual de danos nas vagens (IPDV), número de vagens por planta (NVP), número de sementes (NS), peso de sementes manchadas (PSM), peso de sementes boas (PSB), e peso de cem sementes (PCS). Para se ter um total de 96 amostras, os dois pais foram genotipados juntamente com as 12 mais resistentes e 12 mais suscetíveis plantas F2 para as características PEG, PCS e PSB, e as 11 mais resistentes e 11 mais suscetíveis para a característica RF. Para determinar o tempo de resposta de expressão gênica nas vagens à alimentação de percevejos, um estudo de microarranjo foi realizado com CD-215 avaliando a expressão relativa 5.5, 21, 24 e 41 horas após infestação em condições de casa-de-vegetação. Dentre as características de resistência, os maiores valores de herdabilidade foram observados para PCS e PEG. O caráter PCS apresentou correlação genética positiva e significativa com PSM e PEG. Neste estudo, 337 SNP, 28 SSR, 13 TRAP e 41 AFLP foram mapeados em 20 grupos de ligação. Quatorze QTL foram encontrados usando o modelo restrito de múltiplos QTL e análise de Kruskal-Wallis. A maioria dos QTL foi detectada para mais de uma característica e composta por genes com efeito menor. Na análise de microarranjo foi observada uma expressão diferencial clara para as amostras de 21, 24 e 41 horas com P. guildinii. Assim, para o experimento de campo as vagens foram infestadas com esta espécie e amostras de vagens foram coletadas 0, 8, 24 e 46 horas após infestação. Nesta etapa foi sequenciado somente o RNA mensageiro da amostra 24 horas. Na análise de RNA-seq realizada nas vagens sem nenhum tratamento, a cultivar resistente (IAC- 100) apresentou 39,4% dos genes com maior expressão e 11,68% dos genes com menor expressão do que a cultivar suscetível (CD-215). Baseado nos resultados, a seleção indireta para PCS associado com PSB pode ser realizada com sucesso para a obtenção de genótipos resistentes a percevejo. Além disso, os resultados de mapeamento de QTL foram parcialmente consistentes com estudos anteriores para característica agronômicas, sugerindo que QTL reais foram mapeados.
The group of stink bugs most frequently causing economic losses in soybean in Brazil consists of the species: Nezara viridula, Piezodorus guildinii, and Euchistus heros. Therefore, the objectives of the current research were to evaluate genetic parameters and correlations among distinct development and yield traits, map QTL associated with soybean resistance to stink bugs, and determine plant gene expression profiles associated to insect feeding. An F2:3 population was developed by crossing IAC-100 (resistant) and CD-215 (susceptible) and it was evaluated at an experimental field. The agronomic traits evaluated were number of days to flowering (NDF), plant height at flowering (PHF), number of days to maturity (NDM), plant height at maturity (PHM), lodging (L), agronomic value (AV), and grain yield (GY). The characteristics of stink bug resistance evaluated were grain filling period (GFP), leaf retention (LR), percentage index of pod damage (PIPD), number of pods per plant (NPP), number of seeds (NS), weight of spotted seeds (WSS), healthy seed weight (HSW), and weight of a hundred seeds (WHS). To have a total of 96 samples, the two parent lines were genotyped along with the 12 most resistant and 12 most susceptible F2 plants for the traits GFP, WHS, and HSW, and the 11 most resistant and 11 most susceptible for the trait LR. In order to determine the timing of gene expression response in pods under stink bug feeding, a microarray study was carried out with the cultivar CD-215, evaluating relative transcription levels at 5.5, 21, 24, and 41 hours post-infestation under greenhouse conditions. Among the characteristics of resistance, the highest values of heritability were observed for WHS and GFP. The trait WHS exhibited positive and significant genotypic correlation with WSS and GFP. In this study, 337 SNP, 28 SSR, 13 TRAP, and 41 AFLP markers were mapped to 20 linkage groups. Fourteen QTL were found using the restricted multiple QTL model and Kruskal-Wallis analyses. The majority of the QTL was detected for more than one trait and consisted of genes with minor effects. A clear differential gene expression was observed in the microarray analysis for the samples at time points 21, 24, and 41 hours infested with P. guildinii. Thus, in field trials the pods were infested with this species and samples of pods were taken at 0, 8, 24, and 46 hours. In this study, only RNA from the 24 hour sample was sequenced. From RNA-seq analysis performed on pods without treatment, the resistant cultivar (IAC-100) showed 39.4% of genes with induced expression and 11.68% of genes with repressed expression in comparison to the susceptible cultivar (CD-215). Based on the results, indirect selection for WHS associated with HSW can be successfully employed for obtaining stink bug resistant genotypes. Moreover, mapping QTL results were partially consistent with previous studies for agronomic traits, suggesting that real QTL were mapped.

Doctor of Philosophy
Department of Plant Pathology
Bikram S. Gill
Wheat is the most widely grown and consumed grain crop in the world. In order to meet future agricultural production requirements of a growing population, it is essential that we achieve an increased understanding of the basic components and mechanisms shaping growth and productivity of the polyploid wheat plant. Fusarium head blight (FHB) (syn. "scab") poses a serious threat to the quantity and safety of the world's food supply. The resistance locus Fhb1 has provided partial resistance to FHB of wheat for nearly four decades. Map-based cloning of Fhb1 is justified by its significant and consistent effects on reducing disease levels, the importance of FHB in global wheat production and food safety, and because this gene confers partial resistance to this disease and does not appear to behave in a gene-for-gene manner. A bacterial artificial chromosome (BAC) contig spanning the Fhb1 region was developed from the cultivar 'Chinese Spring', sequenced and seven candidate genes were identified in an ~250 kb region. Cosmid clones for each of the seven candidate genes were isolated from a line containing Fhb1 and used for genetic transformation by biolistic bombardment. Transgenic lines were recovered for five candidate genes and evaluated for FHB resistance. All failed to complement the Fhb1 phenotype. Fhb1 is possibly one of the two remaining candidate genes, an unknown regulatory element in this region, or is not present in Chinese Spring. Traditional views on the effects of polyploidy in allohexaploid wheat have primarily emphasized aspects of coding sequence variation and the enhanced potential to acquire new gene functions through mutation of redundant loci. At the same time, the extent and significance of regulatory variation has been relatively unexplored. Recent investigations have suggested that differential expression of homoeologous transcripts, or subfunctionalization, is common in natural bread wheat. In order to establish a timeline for such regulatory changes and estimate the frequency of non-additive expression of homoeologous transcripts in newly formed T. aestivum, gene expression was characterized in a synthetic T. aestivum line and its T. turgidum and Aegilops tauschii parents by cDNA-SSCP and microarray expression experiments. The cDNA-SSCP analysis of 30 arbitrarily selected homoeologous transcripts revealed that four (~13%) showed differential expression of homoeoalleles in seedling leaf tissue of synthetic T. aestivum. In microarray expression experiments, synthetic T. aestivum gene expression was compared to mid-parent expression level estimates calculated from parental expression levels. Approximately 16% of genes were inferred to display non-additive expression in synthetic T. aestivum. Six homoeologous transcripts classified as non-additively expressed in microarray experiments were characterized by cDNA-SSCP. Expression patterns of these six transcripts suggest that cis-acting regulatory variation is often responsible for non-additive gene expression levels. These results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and non-additive gene expression in synthetic T. aestivum.

Le riz est l'une des céréales les plus importantes au monde. Au Vietnam, le riz est également considéré comme un produit agronomique clé pour l'exportation. Cependant, le stress dû à la sécheresse menace la production de riz de plus en plus fréquemment et sur de plus longues périodes. Les racines coronaires constituent une partie importante du système racinaire du riz et jouent un rôle crucial dans le maintien du rendement en cas de sécheresse. Le nombre de racines coronaires affecte la biomasse de racines et détermine la capacité de la plante à extraire les ressources du sol. Un QTL de NRC, qNCR11, localisé sur le chromosome 11, avait été détecté dans une étude d’association précédente utilisant un panel de riz vietnamiens. Dans notre étude, son effet sur le NCR, léger, a été validé par cartographie de QTL en utilisant une population biparentale . Afin de déterminer les gènes sous-jacents à qNCR11 et régissant l'initiation et le développement des racines coronaires, une étude de séquençage du génome entier et une étude d'expression ont été réalisées. Deux gènes candidats, NCR2 (NBS-LRR) et NCR3 (OsbHLH014) ont été identifiés. NCR2 portait un SNP non synonyme dans son ORF, provoquant un codon stop prématuré corrélé avec un NCR élevé; NCR3 était moins exprimé dans les bases de la tige des plantes à haplotype NCR élevé que chez les plantes à haplotype NCR faible. Des mutations dans ces gènes ont été obtenues à l'aide du système CRISPR / Cas9 et le phénotypage des lignées obtenues est en cours. Le QTL qNCR11 à effet mineur pourrait être utile aux sélectionneurs pour produire des variétés de riz avec un NCR augmenté ou diminué pour les différents écosystèmes-cibles, afin de favoriser l'extraction de l'eau dans des conditions de sécheresse
Rice is one of the most important cereals worldwide. In Vietnam, rice is also known as a key agronomic product for exportation. However, drought stresses threaten rice production with an increasing frequency and for longer periods. Crown roots are a major component of rice root system and play a crucial role in maintaining yield under drought. The number of crown roots (NCR) impacts on root biomass and determines the ability of a plant to acquire soil resources. qNCR11, a QTL for NCR located on chromosome 11, was detected in a previous genome-wide association study using a Vietnamese rice panel. qNCR11 was validated to have a slight effect on NCR by QTL mapping using a biparental population in this study. To determine the genes underlying qNCR11 and governing crown root initiation and development, whole genome sequencing and expression study were performed. Two candidate genes, NCR2 (NBS-LRR) and NCR3 (OsbHLH014) were identified. NCR2 carried a non-synonymous SNP inside its ORF, causing a premature stop-codon that correlates with the high NCR trait; NCR3 was less expressed in stem bases of the high NCR haplotype plants relative to the low NCR haplotype plants. Mutations in these genes were obtained using the CRISPR/Cas9 system and the phenotyping of the obtained lines is on-going. The minor-effect qNCR11 could be useful for breeders to generate rice varieties with increased or decreased NCR for different target agro-systems, in order to enhance water extraction under drought stress

Osteoporosis is a disease that leads to decreased bone mineral density (BMD), an altered bone micro-architecture and fragile bones. The disease is highly heritable and numerous genes are thought to be involved, making it difficult to identify the causative genetic elements.

Animal models, mainly intercrosses between laboratory strains of mice, have been succesfully used to map genes affecting these traits, but may not mirror the multifactorial genetic etiology of highly complex traits such as osteoporosis.

Over the course of tens of thousand years humans have kept domestic animals whose phenotypic repertoires have been tailored to meet our needs. Wild-type red junglefowl (RJ) and domestic White Leghorn (WL) chicken differ for several bone traits.

In this thesis Quantitative Trait Loci (QTL) mapping was used to trace the inheritance of bone traits in two separate intercrosses between RJ and WL. In these studies we identified several QTL that contributed to differences in BMD, bone size and biomechanical strength of bone. In a comparison of QTL identified in the two intercrosses it was observed that nine QTL had overlapping genomic positions, implicating these loci as important to bone phenotypic variation in chicken.

In two separate studies, microarray technology was used to compare global gene expression in bone tissue from RJ and WL. In these studies, differential expression was observed for 779 and 560 genes, respectively. Many differentially expressed genes were co-localized with QTL, which implicates them as QTL-candidates.

Results presented in this thesis link several genomic regions and genes to variation in bone traits. Increased knowledge about these identified genes and regions will contribute to a better understanding of the mechanisms underlying inter-individual differences in bone metabolism, both in chicken and man.

Chez les espèces fruitières, la floraison est un évènement majeur qui influencera fortement la fructification. Ce processus, finement régulé par de nombreux facteurs génétiques et environnementaux, est encore peu connu. Chez le cerisier doux (Prunus avium), les fleurs ne s’épanouissent qu’après avoir satisfait des besoins en froid et en chaud. Les effets du changement climatique sur la floraison sont déjà notables et pourraient induire d’importantes pertes économiques. La compréhension des déterminants génétiques et moléculaires impliqués dans la floraison permettra l’amélioration des programmes de sélection variétale visant l’obtention d’arbres adaptés aux futures conditions climatiques. L’objectif de cette thèse est d’accroître les connaissances sur ces déterminants et d’identifier les gènes contrôlant la floraison chez le cerisier. En étudiant les deux familles intra spécifiques ‘Regina’ × ‘Lapins’ et ‘Regina’ × ‘Garnet’, la détection de nombreux quantitative trait loci (QTL) sur l’ensemble des groupes de liaisons (GL) a permis de confirmer la forte implication des besoins en froid dans la floraison ainsi que la complexité de ces caractères. Un QTL à effet majeur a été localisé sur le GL4. Dans les régions couvertes par les QTL contrôlant la date de floraison, une centaine de gènes candidats (GC) pour ce caractère a été identifiée. Un sous ensemble de ces GC a ensuite été étudié pour leur expression au cours du développement des bourgeons par PCR quantitative (qPCR). A terme, ces travaux serviront de bases pour l’identification et la sélection de gènes qui permettront l’obtention de génotypes adaptés aux futures conditions climatiques
In fruit species, the flowering is a major event which strongly influences fructification. This process tiny controlled by many genetic and environmental factors is still little known. In sweet cherry (Prunus avium), flowers open out only after having satisfied chill and heat requirements. The effects of climate change on the flowering are already notable and could induce important economic losses. Identification of genetic and molecular determinants involved in the flowering will allow the improvement of varietal selection programs to obtain trees adapted to future climate conditions. Objective of this thesis is to increase the knowledge of these determinants and identify genes involved in flowering in sweet cherry. By studying two intraspecific progenies ‘Regina’ × ‘Lapins’ and ‘Regina’ × ‘Garnet’, detection of many quantitative trait loci (QTL) on all linkage groups (LG) has enabled us to confirm the strong involvement of chill requirements in the flowering as well as the complexity of these traits. QTL with major effect was localized on the LG4. In regions covered by all the QTLs controlling flowering date, a hundred candidate genes (CG) for this trait was identified. A subset of these GC was then studied for their expression during development of buds by quantitative PCR (qPCR). In the long term, this work will serve as a basis for the identification and selection of genes that allow obtaining genotypes adapted to future climate conditions

Les strongles gastro-intestinaux, dont Haemonchus contortus constituent un problème majeur pour l'élevage des ovins allaitants. Ils entrainent des pertes de production et le recours aux anthelminthiques est remis en question par l'apparition de souches de vers résistantes. La sélection d'ovins plus résistants fait partie des stratégies complémentaires de lutte les plus sérieuses. Cependant sa mise en oeuvre requiert une meilleure compréhension des mécanismes sous-jacents. Cette thèse vise à identifier les régions du génome ovin impliquées dans la résistance aux strongles gastro-intestinaux. Une analyse statistique d'association entre des marqueurs génétiques et des mesures de résistance d'un troupeau d'ovins croisés Martinik Black-belly x Romane a mis en évidence un nombre limité de régions d'intérêt. Parmi celles-ci, un segment du chromosome 12 a été choisi pour effectuer des accouplements raisonnés et valider son rôle dans la résistance à H. contortus. L'effet de cette région a été validé chez les descendants issus d'accouplements assistés par marqueurs génétiques. Cette région semble limiter fertilité des vers femelles tout en contribuant à une réponse immunitaire plus forte. Le rôle d'une région du chromosome 21 dans la variation de concentration plasmatique en pepsinogène, un marqueur de lésions abomasales, a également été confirmé. Un gène candidat sous-jacent est en cours de séquençage et l'analyse des polymorphismes devrait contribuer à la validation de son rôle. Deux autres gènes très proches pourraient également être impliqués et mériteraient une considération future. Ces travaux illustrent à la fois la variation génétique disponible pour les caractères de résistance à H. contortus et la complexité des mécanismes mis en jeu. Des études complémentaires de séquençage et d'étude d'expression par séquençage devrait contribuer à une meilleure compréhension des fonctions des gènes impliqués et de leurs interactions
Gastro-intestinal nematodes, among which Haemonchus contortus are a major threat to the meat sheep industry. They are responsible for production losses and the apparition of worm populations resistant to drugs limits their use as worm control strategy. Breeding more resistant sheep is among the most practicable alternative strategy. However its implementation requires a deeper understanding of underlying mechanisms. This PhD aims at identifying regions of the ovine genome affecting resistance to gastro-intestinal nematodes. A statistical analysis of existing associations between genetic markers and resistance traits of a Martinik Black-belly x Romane cross-bred sheep flock unraveled a limited number of key players. Among these, a fragment of the chromosome 12 was chosen to perform marker-assisted matings and to validate its role in resistance to H. contortus. The effect of this region was validated in the progenies born from matings. It seems this chromosomic fragment limits female worms fertility and is associated to a stronger immune response. The putative role played by a fragment of the chromosome 21 in plasmatic pepsinogen concentration (a biomarker of abomasal lesions) was also confirmed in this work. A candidate gene underlying this region has been sequenced and the analysis of the detected polymorphisms should confirm its role. Further, two other genes in its vicinity could also play a role in this biological phenomenon and they should also deserve future considerations. This work illustrated both the existing genetic variation for resistance to H. contortus and the associated complexity of underlying mechanisms. Additional sequencing and gene expression sequencing studies should help understanding gene functions and interactions

Les nématodes à kystes sont l'un des bioagresseurs causant le plus de dégâts sur les cultures de pommes de terre. La résistance trouvée chez l'accession spl88S.329.18, issue de l'espèce Solanum sparsipilum est caractérisée par un déterminisme oligogénique avec un QTL à localisé sur le chromosome V (GpaVspl) et un QTL mineur localisé sur le chromosome XI(GpaXIspl). Pour obtenir une résistance de haut niveau, l'effet du QTL GpaVspl, doit êtrecomplémenté par celui du QTL à effet faible GpaXIspl. Par génomique comparative, le locusGpaV a été localisé dans un intervalle compris entre 16 et 60 kb sur les génomes de la tomateet des espèces apparentées à la pomme de terre, Solanum demissum et Solanum phureja. Deuxgènes ont été annotés dans cet intervalle sur les génomes de la tomate et de S. demissum : lepremier appartient à la famille des TIR-NBS-LRR (TNL), famille de gènes de résistanceclassiques, et le second appartient à la famille des " mitochondrial, transcription terminationfactor " (mTERF), dont l'implication dans des mécanismes de résistance n'a jamais étédémontrée.Les objectifs de ma thèse étaient d'identifier le(s) gène(s) responsable(s) de la résistance àG. pallida, conférée par le locus GpaVspl, et d'étudier sa régulation. Suite à la publication de laséquence du génome de S. phureja, en 2011, nous avons mis en évidence que le locus GpaVétait dupliqué chez S. phureja et que cette duplication était également présente chezS. sparsipilum. Les quatre gènes annotés au locus GpaVspl ont été nommésSpl_mTERF18430, Spl_TNL18429, Spl_mTERF18453 et Spl_TNL18428.L'effet des deux gènes Spl_mTERF18430 et Spl_TNL18428 sur la résistance à G. pallida aété analysé via des expériences de transformation génétique suivies par des tests de résistancesur les plantes transformées. Un effet partiel du gène Spl_TNL18428 sur la résistance àG. pallida a été mis en évidence par complémentation de plantes sensibles. Aucun effetsignificatif n'a été détecté pour le gène Spl_mTERF18430. Des expériences d'extinctiongénique suggèrent que le deuxième gène TIR-NBS-LRR, Spl_TNL18429, qui est égalementexprimé dans les racines et qui présente un fort pourcentage d'identité de séquence avec legène Spl_TNL18428, pourrait également être impliqué dans la résistance à G. pallida.L'expression du gène rapporteur GFP, placé sous le contrôle du promoteur du gèneSpl_TNL18428, est fortement induite dans les cellules situées autour du syncytium. Cecirenforce l'hypothèse d'une implication du gène Spl_TNL18428 dans la résistance à G. pallida,car la localisation de l'expression de la GFP est similaire à celle de la nécrose, qui estcaractéristique de la réaction développée par les plantes résistantes autour du syncytium induitpar les nématodes.En tenant compte des données bibliographiques récentes, montrant que plusieurs gènes NBSLRRpeuvent être indispensables à l'expression d'une résistance, nos résultats suggèrent queles deux gènes Spl_TNL18428 et Spl_TNL18429 sont nécessaires à l'expression de larésistance à G. pallida

L'huître plate européenne, espèce endémique des côtes européennes, est classée dans la catégorie des " espèces menacées et/ou en déclin ". En effet, les gisements naturels de cette huître ont été progressivement décimés par la sur-exploitation et par l'émergence successive de maladies parasitaires. Le parasite responsable de la bonamiose a notamment contribué à réduire de façon drastique l'exploitation de cette huître en France, et en Europe. Les mollusques bivalves marins présentent deux caractéristiques qui restreignent de fait le potentiel d'action pour lutter contre les maladies : ils sont cultivés en milieu ouvert, et possèdent un système immunitaire inné dépourvu de la capacité de réponse adaptative. Dans ce contexte, la sélection d'animaux naturellement résistants à la bonamiose est une voie prometteuse pour relancer la culture de l'huître plate européenne. Afin de mieux comprendre le phénomène de résistance à la bonamiose, plusieurs études ont porté sur les mécanismes de réponse de l'huître plate et sur l'identification de régions génomiques potentiellement impliquées dans les mécanismes de résistance à la maladie.Le présent travail de thèse consistait à améliorer la compréhension de la résistance de l'huître plate européenne vis-à-vis de la bonamiose, mais également à mieux caractériser la ressource génétique et la structuration de ses populations naturelles. L'huître plate n'étant pas un organisme modèle, seule une carte génétique préliminaire était disponible chez cette espèce. Il a donc été nécessaire de développer de nouveaux outils moléculaires afin d'optimiser la couverture de son génome. Des marqueurs de type SNP (polymorphisme d'une seule base) ont ainsi été développés par séquençage de produits PCR et par séquençage à haut débit. Afin d'améliorer la compréhension de la résistance à la bonamiose, trois expériences d'infection avec le parasite responsable de cette maladie ont été réalisées et ont permis de caractériser les phénotypes de réponse de l'huître plate à plusieurs échelles d'études.1- À l'échelle inter-familiale, il s'agissait de détecter des régions du génome (QTL) associées aux mécanismes de réponse (survie / mortalité) à la bonamiose chez plusieurs familles d'huîtres. Cette approche a permis d'identifier plusieurs régions génomiques d'intérêt communes entre les familles, et de nouvelles régions d'intérêt qui n'avaient pas encore été détectées.2- À l'échelle intra-familiale, il s'agissait de détecter des régions génomiques associées à la régulation d'activités hémocytaires (QTL) ou à l'expression de gènes (eQTL) préalablement identifiés comme potentiellement impliqués dans la réponse à la bonamiose. Cette approche, nouvelle chez un mollusque bivalve, a notamment permis de mettre en évidence une concordance positionnelle entre les régions génomiques impliquées dans la survie ou la mortalité à la bonamiose et celles impliquées dans la régulation des réponses cellulaires et/ou moléculaires.3- À l'échelle des populations, il s'agissait d'étudier un éventuel différentiel de réponse à la bonamiose chez des huîtres provenant de trois populations naturelles géographiquement et écologiquement distinctes. Cette étude a notamment permis d'identifier une possible adaptation à la parasitose des huîtres provenant de la baie de Quiberon. Afin de mieux caractériser les ressources naturelles de l'huître plate européenne, plusieurs populations couvrant l'ensemble de l'aire de distribution de l'espèce ont également été étudiées. Cette étude a permis de confirmer la forte diversité nucléotidique de l'huître plate, en évaluant pour la première fois la diversité génétique globale des populations naturelles d'un mollusque bivalve marin. Cette étude a également permis d'identifier une structuration génétique des populations, avec coïncidence entre les discontinuités dans la distribution des fréquences alléliques des marqueurs moléculaires sous sélection positive ou divergente et les barrières biogéographiques.

Animal domestication, the process where animals become adapted to living in proximity to humans, is associated with the alteration of multiple traits, including decreased fearfulness and stress response. With an estimated population of 50 billion, the domesticated chicken is the most populous avian species in the world. Hundreds of chicken breeds have been developed for meat and egg production, hobby or research purposes. Multidirectional selection and the relaxation of natural selection in captivity have created immense phenotypic diversity amongst domesticates in a relatively short evolutionary time. The extensive phenotypic diversity, existence of the wild ancestor, and feasibility of intercrossing various breeds makes the chicken a suitable model animal for deciphering genetic determinants of complex traits such as stress response. We used chicken domestication as a model to gain insights about the mechanisms that regulate stress response in an avian species. We studied behavioural and physiological stress response in the ancestral Red Junglefowl and one of its domesticated progenies, White Leghorn. An advanced intercross between the aforementioned breeds was later used to map genetic loci underlying modification of stress response. The general pattern of the stress response in chickens was comparable with that reported in mammals, however we identified distinctive differences in the stress modulatory pathways in chickens. We showed that changes in the expression levels of several stress modulatory genes in the brain, the pituitary and the adrenal glands underlie the observed modified stress response in domesticated chickens. Using quantitative trait loci (QTL) mapping, several QTL underlying stress induced corticosterone, aldosterone and baseline dehydroepiandrosterone (DHEA) levels were detected. As a next step, we combined QTL mapping with gene expression (eQTL) mapping and narrowed two QTL down to the putative causal genes, SERPINA10 and PDE1C. Both of these genes were differentially expressed in the adrenal glands of White Leghorn and the Red Junglefowl, had overlapping eQTL with hormonal QTL, and their expression levels in the adrenal glands were correlated with plasma levels of corticosterone and al-dosterone. These two genes thus serve as strong candidates for further functional investigation concerning modification of the stress response during domestication. This dissertation increase the knowledge about genetics and physiology of the stress response in an avian species and its modification during domestication. Our findings expand the basic knowledge about the stress response in chicken, which can potentially be used to improve welfare through appropriate genetic selection.

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The Centro APTA Citros Sylvio Moreira/IAC has been conducting an extensive breeding program of citrus via directed crosses. In a previous study with Citrushuanglongbing pathosystem (HLB) held in our group, using a population obtained by hybridization between Citrus sunki and Poncirus trifoliata, differences were found in the multiplication of the bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB, in the parents and in the progeny. It was observed that the rate of infection and bacterial concentration was higher in C. sunki than in P. trifoliata. Thus, it is important to deepen the studies with this genus and hybrids to increase knowledge of which mechanisms could be involved in the tolerance to HLB, considered the most important disease of citrus currently. In this sense, the objective of this study was to establish sinteny between the linkage groups of the C. sunki and P. trifoliata maps with the genome of Citrus sinensis and to map genomic regions associated to tolerance CLas bacterium through phenotypic analysis (QTLs) and gene expression (eQTLs). With the comparative analysis between maps and genome, it was observed that all the linkage groups showed synteny with reference genome chromosomes used, with the exception of the linkage group 10 of the C. sunki map. For the phenotypic data, a population of 79 F1 hybrids between C. sunki and P. trifoliata was used. The quantification of the bacterium and accumulation of starch in the leaves were evaluated after two years of inoculation with the pathogen. Through the statistical analysis of the mixed model it was possible to group the hybrids into resistant, tolerant and susceptible, being important the validation of these data in the field. The expression of 14 candidate genes related to HLB was performed in 72 hybrids of the population and used as expression data for the mapping of eQTLs. It was possible to locate nine QTLs and 52 eQTLs on the C. sunki genitor map and 17 QTLs and 40 eQTLs were found on the P. trifoliata genitor map. The overlapping eQTLs of the majority genes of QTL (phenotypic data) indicates that the genes are related to the phenotype and are probably responsive to the pathogen infection.
O Centro APTA Citros Sylvio Moreira/IAC vem realizando um amplo programa de melhoramento genético de citros via cruzamentos dirigidos. Em um estudo prévio com o patossistema Citros-huanglongbing (HLB) realizado pelo nosso grupo, utilizando uma população obtida por hibridação controlada entre Citrus sunki e Poncirus trifoliata, foram verificadas diferenças na multiplicação da bactéria Candidatus Liberibacter asiaticus (CLas), agente causal do HLB, tanto nos genitores quanto na progênie. A taxa de infecção e a concentração de bactéria foi maior em Citrus sunki em relação ao P. trifoliata. Assim, é importante aprofundar os estudos com esses gêneros e seus híbridos para ampliar o conhecimento de quais mecanismos poderiam estar envolvidos na tolerância ao HLB, considerada a mais importante doença dos citros atualmente. O objetivo do trabalho foi estabelecer sintenia entre os grupos de ligação dos mapas de C. sunki e P. trifoliata com o genoma de Citrus sinensis e mapear regiões genômicas associadas à tolerância a CLas por meio de análise fenotípica (QTLs) e de expressão gênica (eQTLs). Com a análise comparativa entre mapas e genoma, foi observado que todos os grupos de ligação apresentaram sintenia com pseudocromossomos do genoma de referência utilizado, com exceção do grupo de ligação 10 do mapa da C. sunki. Para os dados fenotípicos foi utilizada uma população de 79 híbridos F1 entre C. sunki e P. trifoliata, sendo avaliada a quantificação da bactéria e acúmulo de amido nas folhas após dois anos da inoculação com o patógeno. Com a análise estatística utilizando modelo misto foi possível agrupar os híbridos em resistentes, tolerantes e suscetíveis, sendo importante a validação desses dados em campo. A análise de expressão de 14 genes candidatos relacionados ao HLB foi realizada em 72 híbridos da população e utilizados como dados de expressão para o mapeamento de eQTLs. Foram encontrados nove QTLs e 52 eQTLs no mapa do genitor C. sunki enquanto no mapa do genitor P. trifoliata foram encontrados 17 QTLs e 40 eQTLs. A sobreposição de eQTLs da grande maioria dos genes com QTLs dos dados fenotípicos, indicam que os genes têm relação com o fenótipo e que provavelmente são responsivos à infecção do patógeno.

In the first year, two upland rice varieties (Azucena and Bala), were screened for root response to drought at the West Africa Rice Development Association (WARDA), Cote d’Ivoire, in two fields of slightly different soil penetration resistance (PR) characteristics.  Changes in soil PR and soil water content were monitored during the drought period.  Root density and depth were significantly greater for Azucena than Bala, and on the irrigated plots compared to the droughted plots, although no consistent site differences in root density were observed.  At each site, on the droughted subplot, soil PR quickly increased near the surface (0-30 cm) in response to reduction in soil water content and soil matrix potential caused by root water extraction.  It is likely that this increase in PR would have either prevented or reduced the rate of downward growth of new roots entering or growing within this layer.  Under these conditions, varietal differences in root response to impedance would be important for drought avoidance. In the second year, also at WARDA, a mapping population based on a cross between Azucena and Bala were tested in two fields of contrasting soil physical properties and QTL for root density at 35 cm were identified.  There was no agreement between sites.  Site characterisation prior to field screening revealed the two sites to be very different in terms of soil texture and water relations.  These site differences would have restricted root growth in different ways and are likely to be reasons behind the lack of agreement in root density QTL between sites. In the third year, near-isogenic lines (NILs) differing only in single or multiple root growth QTL were screened in fields at the International Rice Research Institute (IRRI), Philippines, in soils of lower mechanical impedance than sites used at WARDA.  No major differences were observed for root density indicating the importance of interaction between root traits and the environment when considering contribution to drought resistance.

The main aim of this work was to identify the important cis-acting regulatory sequences, and the trans-acting factors with which they interact, which are required for the tissue-specific expression of the Xenopus borealis skeletal actin gene. All sequences necessary and sufficient for the correct spatial and temporal expression of the Xenopus borealis skeletal actin gene are located in a 156 bp fragment of the gene that spans from nucleotide residues -197 to -42 in its promoter. This region of the skeletal actin promoter contains three imperfect repeats of a CArG sequence motif that has been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion analysis of the promoter of the Xenopus borealis skeletal actin gene, using Xenopus micro-injection techniques as a transient assay system for promoter activity, have identified that CArG box3 is essential for skeletal actin gene expression. By using band shift assays I have demonstrated that, under my assay conditions, CArG box2 is unable to bind any proteins in vitro . Conversely, the CArG boxl sequence exhibits two binding activities on band shift analysis. One of these is antigenically related to the transcription factor SRF, whilst the second appears to be distinct from this protein. CArG box3 also interacts with a protein in vitro. Although this sequence exhibits a similar shift to that of the CArG boxl/SRF complex on band shift analysis, my experiments suggest that this protein is distinct from SRF. A combination of the CArG boxl and CArG box3 motifs is unable to confer muscle-specific gene expression on a heterologous promoter. Furthermore, I have identified an upstream regulatory element (URE) in the Xenopus borealis skeletal actin gene promoter that spans from nucleotides -197 to -167 that is required for the expression of the gene, at least when sequences between nucleotide -42 and +28 are absent. The URE of the Xenopus borealis skeletal actin gene is capable of interacting with a trans-acting factor(s) in vitro. In addition to this a further region of the gene which spans from nucleotide residues -83 to -42 is also capable of interacting with a factor(s) in vitro. The mechanisms by which these multiple regulatory elements control the tissue-specific expression of the Xenopus borealis skeletal actin gene will be discussed.

Genetic associations have been discovered for many human complex traits, and yet for most associated loci the causal variants and molecular mechanisms remain unknown. Studies mapping quantitative trait loci (QTLs) for molecular phenotypes, such as gene expression, RNA splicing, and chromatin accessibility, provide rich data that can link variant effects in specific cell types with complex traits. These genetic effects can also now be modeled in vitro by differentiating human induced pluripotent stem cells (iPSCs) into specific cell types, including inaccessible cell types such as those of the brain. In this thesis, I explore a range of approaches for using QTLs to identify causal variants and to link these with molecular functions and complex traits. In Chapter 2, I describe QTL mapping in 123 sensory neuronal cell lines differentiated from human iPSCs. I observed that gene expression was highly variable across iPSC-derived neuronal cultures in specific gene categories, and that a portion of this variability was explained by commonly used iPSC culture conditions, which influenced differentiation efficiency. A number of QTLs overlapped with common disease associations; however, using simulations I showed that identifying causal regulatory variants with a recall-by- genotype approach in iPSC-derived neurons is likely to require large sample sizes, even for variants with moderately large effect sizes. In Chapter 3, I developed a computational model that uses publicly available gene expression QTL data, along with molecular annotations, to generate cell type-specific probability of regulatory function (PRF) scores for each variant. I found that predictive power was improved when the model was modified to use the quantitative value of annotations. PRF scores outperformed other genome-wide scores, including CADD and GWAVA, in identifying likely causal eQTL variants. In Chapter 4, I used PRF scores to identify relevant cell types and to fine map potential causal variants using summary association statistics in six complex traits. By examining individual loci in detail, I showed how the enrichments contributing to a high PRF score are transparent, which can help to distinguish plausible causal variant predictions from model misspecification.

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The mechanisms whereby parasitic nematodes become resistant to anthelmintics, particularly ivermectin, remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of gene expression changes. The present study focused on Teladorsagia circumcincta; the predominant parasitic gastrointestinal nematode species in the UK and the predominant resistant species. The role of changes in gene expression were investigated in an ivermectin-susceptible isolate (CVL) and a multidrug resistant isolate (MOTRI), utilising a range of molecular biological techniques. In the first experiment, a panel of novel putative ivermectin resistance genes were identified from T. circumcincta, comprising 11 partial P-glycoprotein (Pgp) and 3 partial Cytochrome P450 (CYP) sequences. Both Pgps and CYPs have been implicated in the handling and metabolism of xenobiotics in other biological systems, but have not been investigated in T. circumcincta to date. Initial results, using semi-quantitative PCR identified changes in expression of this panel of genes between the CVL and MOTRI isolates. Constitutive differences in expression of the Pgps and CYPs between CVL and MOTRI were determined using the ΔΔCt TaqMan® real-time PCR method. A statistically significant increase in expression was observed for TeciPgp-9 NBD2 across all life-cycle stages but most notably in eggs (55-fold increase). A statistically significant reduction in expression of TeciPgp-2 NBD2 was observed in all but the adult stages of MOTRI compared to CVL. Analysis of a 208 base pair sequence of TeciPgp-9 NBD2 identified high levels of polymorphism, with at least four non-coding SNPs evident in the MOTRI isolate. These results merit further investigation. Inducible changes in the expression of the Pgps and CYPs were investigated in MOTRI before and after ivermectin treatment, using real-time PCR. Statistically significant fold changes in expression in most of the genes occurred in at least one life-cycle stage. Inducible expression of TeciPgp-2 NBD2 and TeciPgp-9 NBD2 was investigated further by comparing adult MOTRI parasites with those recovered three days after in vivo ivermectin exposure, and by exposing pools of MOTRI xL3 to ivermectin in the larval migration inhibition test. The survivors of ivermectin exposure exhibited a statistically significant reduced 13.68-fold expression of TeciPgp-2 NBD2 compared to MOTRI. Similarly, the MOTRI xL3 able to migrate in the presence of ivermectin in the LMIT had a 1.88-fold reduction in TeciPgp-2 NBD2 expression compared to MOTRI xL3 unexposed to ivermectin. These results indicate that inducible changes in TeciPgp-2 NBD2 and TeciPgp-9 NBD2 expression can occur, but the experimental design is critical to being able to identify the changes. In a more global approach, the transcriptomic response of MOTRI adults to in vitro ivermectin exposure was investigated using Roche 454 sequencing, generating 98,685 novel EST sequences, providing an important resource for a genome resource-poor organism. Objective bioinformatic analysis of the two datasets revealed statistically significant differences in the mean expression levels of the KEGG orthologous groups for ‘translation’, ‘amino acid metabolism’ ‘carbohydrate metabolism’ and ‘xenobiotic degradation and metabolism’. On combining the two datasets, and through application of a novel statistical method, 16 clusters of ESTs were identified as containing statistically significant differences in the mean proportion of exposed reads compared to unexposed reads under the conservative model, whilst a further 355 clusters were found to have statistically significant differences under the liberal model. One-way suppression subtractive hybridisation (SSH) was used to identify genes exhibiting increased expression in MOTRI adults compared to CVL adults. 28 contiguous sequences were identified from the SSH experiment; 6 contiguous sequences were selected for validation; 5 of these results were confirmed using semi-quantitative PCR. Each contig was BLAST searched against the Roche 454 dataset; contig SSH14 aligned most closely to one of the statistically significant clusters in the conservative model, SSHs 5, 6, 10 and 23 aligned most closely to statistically significant clusters in the liberal model. This suggests that changes in expression in these sequences occur both constitutively, between CVL and MOTRI isolates, and inducibly, following ivermectin exposure. This work has shown that changes in gene expression, particularly the constitutively reduced expression in TeciPgp-2 NBD2 and the constitutively increased expression in TeciPgp-9 NBD2 (coupled with the presence of SNPs) could play a role in allowing multidrug resistant T. circumcincta to survive ivermectin exposure. Roche 454 sequencing and SSH approaches identified gene expression changes associated with in vitro ivermectin exposure and ivermectin resistance. These could form the basis of a novel panel of candidate resistance genes whose altered expression profiles may allow multidrug resistant T. circumcincta to survive ivermectin exposure by some, as yet identified, mechanism. Finally, we have also shown that a multidrug T. circumcincta isolate is affected by ivermectin exposure and that changes in gene expression could have a role to play in the ivermectin resistance phenotype in T. circumcincta. The genetic changes underpinning these changes in gene expression remain to be elucidated, and need to be investigated in other isolates. These changes could form the basis of an ivermectin resistance molecular marker, to monitor the spread of resistance, and to evaluate management practices aimed at delaying its spread.

Homologues of developmentally important regulatory genes from other species have been found to be expressed in early amphibian development. The Drosophila dorso-ventral polarity .gene, dorsal, is involved in the formation of ventral structures and dorsal- mutants arise from a default state where only dorsal structures are formed. A Xenopus homologue of dorsal may play a crucial role in interpreting some of the earliest embryonic signals.

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The objectives of this work were to determine the genetic inheritance, map QTL Al tolerance and quantify the differential gene expression of Al tolerance genes in tropical origin maize. To determine the genetic control of Al tolerance were evaluated diallel crosses between contrasting genotypes in the hybrid maize germplasm and maize landrace germplasm. Affiliates generations of diallel crosses and parental of respective crosses were evaluated for Al tolerance through minimal solution methodology. The DIF data (root growth difference) were analyzed by diallel Griffing model (1956) Method 2 (parental, hybrid and reciprocal). Later was determined the tolerance inheritance from average analysis of segregating generations. The landrace genotypes showed overall higher DIF average to hybrid germplasm, confirming the greater tolerance in this group. The diallel crosses involving the V 06 landrace stood out by the high specific and general combining ability for Al tolerance. The results of the generation average analysis indicated, for all families studied, quantitative inheritance of Al tolerance with predominance of genetic variance explained by additive effects. The heritability in the narrow sense was of intermediate magnitude, indicating the possibility of genetic gains to artificial selection of Al tolerant genotypes in F2 generation. The QTL mapping was performed based on phenotypic analysis of 114 F2:3 progenies, evaluated at minimum solution in presence of 4 mg L-1 Al. Microsatellite and AFLPs polymorphic loci between the parental lines of the mapping population were used for construction of the linkage map with the help of MAPMAKER program version 3.0 and QTL detection performed by the composite interval mapping. Nine tolerance QTLs were mapped in eight linkage groups (chromosomes 2, 4, 5, 6, 7, 8, 9 and 10), which explained 70.3% of phenotypic variation in Al tolerance. The results confirmed the three major QTL (bins 6.00, 8.05 and 10.01) described in the literature for Al tolerance, which were responsible for the most of phenotypic variation (40.3%). The high number of mapped QTLs in F2:3 segregating population confirms the quantitative inheritance of Al tolerance in the tropical maize germplasm. The molecular screening of ZmMATE1 and ZmMATE2 genes in hybrid and landraces maize germplasm showed no association of the amplified fragments with Al tolerance index. For studies of gene expression, the tolerant (H 44 and V 18) and sensitive genotypes (V H 22 and 25) were subjected to minimal solution for different exposure periods (0, 1, 3, 6, 9, 12, 24 and 48 h). After total RNA extraction from each genotype and the synthesis of cDNAs from each sample, were performed qRT-PCR assays to quantitate the expression of ZmMATE1, ZmMATE2 and ZmNrat1 genes. The results showed the highest differential expression of tolerant landrace V 18 for ZmMATE1 gene, indicating that, possibly, the exudation of citrate should be the main mechanism of Al tolerance in this genotype. The results also demonstrated the possibility of another type Al tolerance mechanism for the tolerant hybrid H 44, since showed differential expression only for ZmNrat1 gene in initial phase of exposure (1 to 3 h). The same genotypes used in the gene expression quantification were evaluated for roots differential staining with hematoxylin. The genotypes were submitted to minimal solution with Al for 6, 12, 24 and 48 h exposure times, evaluating the differential staining of roots in the respective periods. It was not possible to observe differential staining with hematoxylin among maize tolerant and sensitive genotypes to Al.
Os objetivos do trabalho foram determinar a herança genética, mapear os QTLs de tolerância ao Al e quantificar a expressão gênica diferencial de genes de tolerância ao Al em milho de origem tropical. Para determinar o controle genético da tolerância ao Al foram avaliados cruzamentos dialélicos entre genótipos contrastantes dentro do germoplasma de milho híbrido e dentro do germoplasma de milho crioulo. As gerações filiais dos cruzamentos dialélicos e os respectivos parentais dos cruzamentos foram avaliados quanto à tolerância ao Al através da metodologia de solução mínima. Os dados do DIF (diferença de crescimento radicular) foram submetidos à análise dialélica pelo modelo de Griffing (1956) método 2 (parentais, híbridos e recíprocos). Posteriormente, foi determinada a herança da tolerância a partir da análise de médias de gerações segregantes. Os genótipos de milho crioulo demonstraram média geral de DIF superior ao germoplasma híbrido, confirmando a maior tolerância neste grupo. Os cruzamentos dialélicos que envolveram a variedade crioula V 06 destacaram-se pela alta capacidade específica e geral de combinação para a tolerância ao Al. Os resultados da análise de média de gerações indicaram, para o conjunto de famílias estudadas, herança quantitativa da tolerância ao Al, com predomínio da variância genética explicada pelos efeitos aditivos. A herdabilidade no sentido restrito foi de magnitude intermediária, indicando a possibilidade de ganhos genéticos com a seleção artificial de genótipos tolerantes ao Al na geração F2. O mapeamento de QTLs foi realizado com base na análise fenotípica de 114 progênies F2:3, avaliadas em solução mínima na presença de 4 mgL-1 de Al. Os locos microssatélites e AFLPs polimórficos entre as linhagens parentais da população de mapeamento foram utilizados para a construção do mapa de ligação com o auxílio do programa MAPMAKER versão 3.0 e a detecção dos QTLs realizada pelo mapeamento por intervalo composto. Foram mapeados nove QTLs de tolerância em oito grupos de ligação (cromossomos 2, 4, 5, 6, 7, 8, 9 e 10), os quais explicaram 70,3% da variação fenotípica em tolerância ao Al. Os resultados confirmaram os três principais QTLs (bins 6.00, 8.05 e 10.01) descritos na literatura para a tolerância ao Al, os quais foram responsáveis pela maior parte da variação fenotípica (40,3%). O elevado número de QTLs mapeados na população segregante F2:3 confirma a herança quantitativa da tolerância ao Al neste germoplasma de milho tropical. O screening molecular dos genes ZmMATE1 e ZmMATE2 nos germoplasmas de milho híbrido e de variedades crioulas não evidenciou associação dos fragmentos amplificados com os índices de tolerância ao Al. Para os estudos de expressão gênica, os genótipos tolerantes (H 44 e V 18) e os sensíveis (H 22 e V 25) foram submetidos à solução mínima por diferentes períodos de exposição (0, 1, 3, 6, 9, 12, 24 e 48 h). Após a extração de RNA total de cada um dos genótipos e a síntese dos cDNAs de cada amostra foram realizados ensaios de qRT-PCR para quantificar a expressão dos genes ZmMATE1, ZmMATE2 e ZmNrat1. Os resultados demonstraram a maior expressão diferencial da variedade crioula tolerante V 18 para o gene ZmMATE1, indicando que, possivelmente, a exsudação de citrato deva ser o principal mecanismo de tolerância ao Al neste genótipo. Os resultados também evidenciaram a possibilidade de outro tipo de mecanismo de tolerância ao Al para o híbrido tolerante H 44, pois apresentou expressão diferencial apenas para o gene ZmNrat1 na fase inicial da exposição (1 a 3 h). Os mesmos genótipos utilizados na quantificação da expressão gênica foram avaliados para a coloração diferencial de raízes com hematoxilina. Os genótipos foram submetidos à solução mínima com Al por períodos de exposição de 6, 12, 24 e 48 h, avaliando-se a coloração diferencial das raízes nos respectivos períodos. Não foi possível observar coloração diferencial com hematoxilina entre genótipos de milho tolerantes e sensíveis ao Al.

Orientador: Marcos Antonio Machado
Resumo: Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) e Phytophthora citrophthora (Smith & Smith), agentes causadores da gomose e podridão radicular, têm trazido graves danos em viveiros e pomares de citros no mundo inteiro. O Centro de Citricultura Sylvio Moreira/IAC vem realizando um amplo programa de melhoramento genético de citros via cruzamentos dirigidos com relação ao patossistema Phytophthora-citros, em que já foram verificadas diferenças no nível de resistência na progênie do cruzamento entre tangerina Sunki (Citrus sunki) e trifoliata Rubidoux (Poncirus trifoliata), a partir da detecção de genes diferencialmente expressos utilizando microarranjos, identificando transcritos envolvidos na resistência à Phytophthora, e do mapeamento genético. Este trabalho teve como objetivo principal mapear nos grupos de ligação dos mapas de P. trifoliata e C. sunki as regiões genômicas associadas à resistência à Phytophthora parasitica por meio de análise fenotípica (QTLs) e de expressão (eQTLs). Uma população de 110 híbridos, seus genitores e dois porta-enxertos de referência para a citricultura (limão Cravo e citrumelo Swingle), foi enxertada em limão Cravo e estabelecida em casa de vegetação. Cada híbrido foi conduzido com uma única haste e a inoculação foi realizada pelo método do disco a partir do meio de cultura contendo o micélio de P. parasitica, a 10 cm e 15 cm acima da região da enxertia, totalizando duas inoculações por genótipo. As plantas foram mantidas ... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Adult hippocampal neurogenesis is regulated at various levels and by various factors. Genetic influence is an important key determinant of adult neurogenesis and exerts its effects at all levels. In vivo studies have suggested that adult hippocampal neurogenesis is highly variable and heritable among different laboratory strains of mice. To dissect the genetic effect from other contributing factors, it is necessary to study adult neurogenesis under highly controlled environment conditions. We extracted adult hippocampal precursor cells (AHPCs) from 20 strains of the BXD set of recombinant inbred mice, cultured them and studied the effect of genetic background on neurogenesis. The BXD panel consists of mouse lines derived from an intercross between inbred parentals C57BL/6J and DBA/2J. Both of the parentals are fully sequenced and all the strains are well characterized in terms of genotypic and phenotypic characteristics. This allows us to use advanced genetic techniques to identify novel genomic loci and gene-gene interactions important in adult neurogenesis. Comparison of the AHPCs from 20 BXD strains, with respect to cell proliferation and neuronal and astrocytic differentiation in vitro, revealed a large variation for these traits across the strains. Proliferation, as measured by BrdU incorporation, showed over two- fold differences between the extremes. Similar differences were observed for neurogenic (4-fold) and astrogenic differentiation (2-fold). These three traits all showed strong heritability values indicating that the differences were mainly attributed to the genetic component. QTL mapping, with these phenotypic data, revealed that there was no major contribution from single loci controlling these traits. Instead, we found many loci with smaller effects associated with these traits. Gene expression profiling using RNA samples from proliferating cultures of the 20 BXD mice strains yielded two cis eQTL candidates that directly regulated proliferation, LRP6 and Chchd8. LRP6 is well known as a co-receptor of Wnt signaling, but the function of Chchd8 is not known. Further experimentation, using over expression and gene silencing demonstrated that LRP6 negatively regulates AHPCs proliferation. Thus, from this study using a system genetics approach, we were able to identify, LRP6 as a novel regulator of adult hippocampal neurogenesis.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...)
At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role

Variations in gene expression have long been hypothesised to be the major cause of individual differences. An initial focus of this research thesis is to elucidate the genetic regulatory architecture of gene expression. Expression quantitative trait locus (eQTL) mapping analyses have been performed on expression levels of over 22,000 mRNAs from three tissues of a panel of recombinant inbred mice. These analyses are "single-locus" where "linkage" (i.e. significant correlation) between an expression trait and a putative eQTL is considered independently of other loci. Major conclusions from these analyses are: 1. Gene expression is mainly influenced by genetic (sequence) variations that act in trans rather than in cis; 2. Subsets of genes are controlled by master regulators that influence multiple genes; 3. Gene expression is a polygenic trait with multiple regulators. Single-locus mapping analyses are not designed for detecting multiple regulators of gene expression, and so observation of multiple-linkages (i.e. one expression trait mapped to multiple eQTLs) formed the basis of the second objective of this research project: to investigate the relationship between multiple-linkages and genotype pattern-association. A locus-pair is said to have associated genotype patterns if they have similar inheritance pattern across a panel of individuals, and these are attributed to one of fours sources: 1. linkage disequilibrium between loci located on the same chromosome; 2. non-syntenic association; 3. random association; 4. un-associated. To understand the validity of multiple-linkages observed in single-locus mapping studies, a newly developed method, bqtl.twolocus, is applied to confirm two-locus effects for a total of 898 out of 1,233 multiple-linkages identified from the three studies mentioned above as well as from seven publicly available eQTL-mapping studies. Combining these results with information of genotype pattern-association, a subset of 478 multiple-linkages has been deduced for which there is high confidence to be real.

碩士
國立臺灣大學
農藝學研究所
106
High temperature reduces the fruit set rate of tomato, so does the yield. The total pollen number and the pollen viability were recognized as two major factors involving in the reduction of the fruit set rate. In this study, we measured these two traits of heat-sensitive variety CA4, heat-tolerant variety CLN1621N and their offspring population, 78 F7 recombinant inbred lines (RIL), under 30/25 °C day/night temperature. The results showed these two traits were highly correlated to the light intensity and their broad-sense heritability were from 0.3 to 0.6. In order to understand the genetic architectures of total pollen number and pollen viability under heat stress, we deployed two strategies, genetic mapping and gene expression profiling (ie. transcriptome), using next-generation sequencing technologies. The RNAseq gene profiling data of the RIL population were used to discover single nucleotide polymorphism (SNP) markers. A total of 7,550 SNP markers were identified in this RIL population to construct the high-density genetic map which was consisted of 330 SNP markers with 693.8 cM length. The genetic mapping analyses revealed five quantitative trait loci (QTLs) on chromosome 3, 4, 10 and 11. Each QTL can explain 8.74 % to 34.63% of total variance. In addition, the variation of gene expression of 14 genes in the QTL of the total pollen number on chromosome 4 showed statistically significant association with distinct marker genotypes of this QTL. These 14 genes are the candidate genes to maintain total pollen number under heat stress.

Le syndrome du QT long (SQTL) est une anomalie du coeur caractérisée par une prolongation de l'intervalle de repolarisation entre les ondes Q et T sur l'électrocardiogramme. Ce syndrome pouvant provoquer de l'arythmie et même la mort peut être causé par des mutations génétiques, ou par la prise de certains médicaments. Plusieurs médicaments vendus sous ordonnance qui induisaient le syndrome du QT long ont été retirés du marché au cours de la dernière décennie et plusieurs autres ont échoué les tests de contrôle avant même leur sortie en pharmacie. La plupart de ces médicaments provoquent le SQTL à des doses thérapeutiques et dans pratiquement tous les cas, le syndrome est causé par une interaction du médicament avec le canal potassique transmembranaire du human ether-à-go-go-related-gene (hERG). Selon plusieurs études, la majorité des molécules qui bloquent le canal hERG se lieraient à des sites localisés dans la partie intracellulaire de la région du pore (hélice transmembranaire S5 et/ou S6) ou dans la région extracellulaire qui connecte S5 et S6. Dans la présente recherche, le rôle du segment extracellulaire (lIe583 -Tyr597 ) dans le fonctionnement du canal hERG et dans le mécanisme du SQTL fut étudié. Pour ce faire, l'interaction de ce segment avec quatre médicaments cardiotoxiques (bépridil, cétirizine, diphenhydramine, pentamidine) fut analysée en plus de vérifier l'interaction de ce segment et des médicaments avec des membranes modèles. Une approche combinant la RMN de l'état liquide et de l'état solide jumelée avec des analyses de dichroïsme circulaire a permis d'obtenir les conclusions suivantes. Tout d'abord, les résultats des interactions peptide-médicaments étudiées par RMN de l'état liquide du ¹H par des mesures de diffusion à l'aide de gradients de champ pulsés suggèrent une faible interaction du peptide avec chacun des médicaments dans un environnement aqueux. Cependant, une très forte interaction des médicaments et du peptide avec la membrane a été observée, suggérant un rôle potentiel de cette dernière dans la cardiotoxicité des médicaments provoquant le SQTL. Ensuite, les résultats des interactions médicaments-membranes et peptide-membrane étudiées par RMN de l'état solide du ²H et du ³¹P suggèrent une importante perturbation de la membrane (tant au niveau des têtes polaires que des chaînes acyle) par le segment extracellulaire lIe583 -Tyr597 . Finalement, les analyses de dichroïsme circulaire ont permis de démontrer que ce segment n'adopte pas une structure secondaire bien définie malgré sa forte interaction avec la membrane. Cependant, la conformation de ce segment varie en fonction de la nature et de la charge de la membrane modèle, ce qui prouve sa grande flexibilité structurale. Ces résultats suggèrent que la membrane joue un rôle important dans le fonctionnement du canal hERG, probablement en stabilisant des conformations transitoires durant les processus d'ouverture et de fermeture contrôlés par voltage. En terminant, les expériences biochimiques réalisées ont permis d'exprimer avec succès le segment S5. Des expériences sont toujours en cours pour purifier ce segment. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Syndrome du QT long, hERG, Médicaments cardiotoxiques, Membrane modèle, RMN de l'état liquide, RMN de l'état solide, Interaction médicaments-membranes, Interaction peptide-membranes.