Made available in DSpace on 2016-06-02T20:20:37Z (GMT). No. of bitstreams: 1 6268.pdf: 2312191 bytes, checksum: f16c107fdc91ed439efa845247c55dc4 (MD5) Previous issue date: 2014-06-30 / Financiadora de Estudos e Projetos / This thesis has been divided into three chapters for better understanding of the experiments. The first chapter refers to a brief Literature Review, with the main concepts and justifications for this work. The second chapter discusses the strategy of positional and functional candidate genes for traits of feed efficiency in Nelore cattle, and the third chapter presents a genome-wide association study for feed efficiency traits in the same population. Feed efficiency is an important production trait in beef cattle production systems. The aim of this study was to identify genes/QTLs associated with feed efficiency traits in Nelore cattle genotypes using the Illumina BeadChip BovineHD (770K) of 593 Nelore steers. The traits analyzed were: average daily weight gain (ADG), dry matter intake (DMI), feed conversion (FC), feed efficiency (FE), residual feed intake (RFI), efficiency of maintenance (EM), efficiency gain (EG), partial efficiency of growth (PEG) and relative growth rate (RGR). The first strategy discussed in this work, using candidate genes, investigated the association of Neurogenic differentiaton 1 (NEUROD1), Kv channel interacting protein 4 (KCNIP4), and Vascular endothelial growth factor C (VEGFC) genes, with production traits in the same population. Our results are in according to literature that indicate NEUROD1, KCNIP4 and VEGFC as candidate genes for feed efficiency; and this study is the first to suggest the NEUROD1 gene as a candidate for gene backfat thickness, body weigth and metabolic weight in Nelore cattle. In the genome-wide association study some QTL regions identified in this study minimally overlapped with QTL regions previously reported for feed efficiency in Bos Taurus cattle, and harbor genes with biological functions related to metabolic processes, lipid and protein metabolism, energy generation and growth. A comparison with published results indicates that different QTL and genes may be involved in the control of feed efficiency in this population of Nelore cattle. The validation of the results obtained in this work in independent populations may contribute to elucidation of the mechanisms will involved in the variation of feed consumption and specific improvement in Nelore programs. / Esta tese foi dividida em três capítulos para melhor compreensão dos experimentos realizados. O primeiro capítulo refere-se a uma breve Revisão de Literatura, com os principais conceitos e justificativas para a realização deste trabalho. O segundo capítulo aborda a estratégia de genes candidatos posicionais e funcionais para características de eficiência alimentar na raça Nelore e o terceiro capítulo apresenta um estudo de associação genômica ampla para características de eficiência alimentar na mesma população. A eficiência alimentar é uma característica produtiva importante em sistemas de produção de bovinos de corte. O objetivo deste estudo foi identificar genes/QTLs associados com características de eficiência alimentar em bovinos da raça Nelore utilizando genótipos do Illumina BovineHD BeadChip (770K). As características analisadas foram: ganho de peso médio diário (GMD), consumo de matéria seca (CMS), conversão alimentar (CA), eficiência alimentar (EA), consumo alimentar residual (CAR), eficiência da mantença (EM), eficiência de ganho (EG), eficiência parcial de crescimento (EPC) e taxa de crescimento relativo (TCR). A primeira estratégia abordada neste trabalho, a de genes candidatos, investigou a associação dos genes Neurogenic differentiaton 1 (NEUROD1), Kv channel interacting protein 4 (KCNIP4), e Vascular endothelial growth factor C (VEGFC), com características produtivas nesta mesma população. Nossos resultados corroboram com resultados da literatura que indicam os genes KCNIP4 e VEGFC como genes candidatos para eficiência alimentar; e este estudo é o primeiro a sugerir o gene NEUROD1 como gene candidato para espessura de gordura subcutânea, peso médio e peso metabólico em bovinos da raça Nelore. No estudo de associação genômica, algumas regiões de QTL identificadas foram sobrepostas minimamente com regiões de QTL relatadas anteriormente para eficiência alimentar em bovinos Bos taurus, e abrigam genes com funções biológicas relacionadas com processos metabólicos, metabolismo lipídico e proteíco, geração de energia e crescimento. A comparação com os resultados publicados indicam que diferentes QTL e genes podem estar envolvidos no controle da eficiência alimentar nesta população de bovinos da raça Nelore. A validação dos resultados obtidos neste trabalho em populações independentes pode contribuir para elucidação dos mecanimos envolvidos na variação do consumo alimentar e para programas de melhoramento específicos para a raça Nelore.

Le cancer du sein est une maladie complexe et hétérogène. La classification histo-clinique connait des limites et doit donc évoluer pour une meilleure prise en charge des patientes. Depuis près de 10 ans, une nouvelle taxonomie moléculaire basée sur la similitude d’expression génique permet de classer les cancers du sein en plusieurs sous-types moléculaires distinguant des classes tumorales de phénotypes et d’évolutions cliniques différents. L’amélioration de l’approche moléculaire en complément de la classification conventionnelle devrait avoir un impact considérable sur la prise en charge des Patientes. Il a déjà été montré que la caractérisation d’altérations génomiques telles que les amplifications d’oncogènes et les pertes de gènes suppresseurs de tumeurs (TSG) combinée aux données d’expression permettaient d’identifier des gènes candidats (oncogènes et TSG). Certains sont considérés comme marqueurs au diagnostique et/ou pronostique spécifiques de sous-types moléculaires ou d’entités cliniques et peuvent constituer de futures cibles thérapeutiques potentielles. De plus, l’identification de ces altérations récurrentes améliore notre compréhension des mécanismes biologiques impliqués. Ainsi, les analyses moléculaires à haut débit du cancer du sein ont déjà révélé une partie de leur potentiel. Cependant, malgré la promesse des signatures pronostiques déjà décrites, nous manquons de données moléculaires dans certaines entités cliniques et sous-types moléculaires à phénotype particulièrement agressif. Dans cette thèse, nous nous sommes intéressés aux cancers du sein de sous-type moléculaire luminal B dont l’évolution clinique est particulièrement péjorative et pour lesquels aucune thérapie ciblée n’existe. Mieux comprendre ces cancers permettrait peut-être de mieux les traiter. Ainsi dans cette thèse vous sont présentés (i) le gène candidat L3MBTL4, cible de multiples altérations génomiques (perte, mutation, point de cassure) suggérant son implication comme TSG potentiel dans les cancers du sein. (ii) l’existence de quatorze TSG potentiels du chromosome 18p qui pourraient expliquer le phénotype agressif associé à la perte du 18p dans les cancers du sein.(iii) l’analyse intégrée des données génomiques et d’expression des carcinomes mammaires et l’identification de gènes candidats spécifiques du sous-type moléculaire luminal B. / Breast cancer is a complex and heterogeneous disease. Histoclinical classification has limitations and must evolve to better care for patients. For nearly 10 years, a new molecular taxonomy based on similarity of gene expression used to classify breast cancers into several molecular subtypes distinguished tumor classes with different phenotypes and clinical courses. Improved molecular approach complementary to conventional classification should have a considerable impact on patients care. It has already been shown that the characterization of genomic alterations such as amplification of oncogenes and loss of tumor suppressor genes (TSG) combined with expression data allow to identify candidate genes (oncogenes and TSG). Some are considered as diagnostic and / or prognostic markers specific of molecular subtypes or clinical entities and may constitute future therapeutic targets. Furthermore, identification of these recurrent alterations improves our understanding of biological mechanisms involved.Thus, high throughput molecular analyses of breast cancer have revealed some of their potential. However, despite the promise of prognostic signatures already described, we lack molecular data in certain clinical entities and molecular subtypes with a particularly aggressive phenotype.In this thesis, we focused on luminal B breast cancer molecular subtype whose clinical course is particularly pejorative and for which no targeted therapy exists. Better understanding of these cancers might help them better deal.Thus in this thesis are presented: (i) the candidate gene L3MBTL4, target by multiple genomic alterations (loss, mutation, break-point), suggesting its involvement as a potential TSG in breast cancer.(ii) the existence of fourteen chromosome 18p potential TSG that could explain the aggressive phenotype associated with 18p loss in breast cancers.(iii) the integrated analysis of genomic and expression data of mammary carcinoma and the identification of specific luminal B molecular subtype candidate genes.

Depuis plusieurs années, les programmes de recherche chez le cacaoyer ont mis l'accent sur l'étude des bases génétiques des caractères agronomiques d'intérêt, notamment concernant la résistance aux maladies et la qualité des fèves de cacao, qui représentent deux attributs importants pour la cacaoculture et la production de chocolat. Ce travail présente, par l'exploration du transcriptome et du génome du cacaoyer, la constitution de ressources moléculaires et l'analyse des voies de biosynthèse impliquées dans plusieurs de ces caractères agronomiques d'intérêt. L'étude du transcriptome a permis l'identification de plusieurs dizaines de milliers de gènes exprimés dans divers organes et pour différentes conditions environnementales et ont fourni de nombreux marqueurs moléculaires qui ont été utilisés pour réaliser des cartes génétiques haute densité. Les informations apportées par ce travail ont permis d’engager le séquençage du génome de la variété Criollo du cacaoyer. Son analyse et son annotation ont apporté un ensemble d'informations biologiques cruciales, depuis le catalogue des gènes et éléments mobiles jusqu'aux aspects évolutifs qui a révélé une structure du génome peu remanié par rapport à l'ancêtre commun aux dicotylédones. Par la suite, les travaux que nous avons menés pour améliorer la séquence complète ont conduit à une réduction considérable de la fragmentation chromosomique observée dans la première version. Par ailleurs 97% de la séquence assemblée et 99% des gènes sont désormais ancrés sur les chromosomes du cacaoyer. Pour commencer à exploiter cette nouvelle séquence du génome, nous avons conduit une étude QTL à partir d'une descendance entre Trinitario implantée en Guyane, permettant de localiser les régions génomiques impliquées dans la variation de la couleur des fèves de cacao. En s'appuyant sur la version améliorée du génome du Criollo, nous avons identifié deux gènes potentiellement impliqués dans la voie de biosynthèse des anthocyanines et flavonoïdes dans la principale région génomique concernée. Un des deux gènes, situé proche du marqueur situé au pic du QTL et codant pour une chalcone synthase, semble être un gène candidat prometteur. L'étude comparative de sa structure dans le génome du Criollo (à fève blanche) et du génome de l'Amelonado (à fève violette) a mis en évidence des différences structurales pouvant être à l'origine d'une modification fonctionnelle. L'ensemble des résultats présentés dans ce travail de thèse apporte une connaissance et des outils variés qui peuvent être exploités par de multiples approches intégrées pour étudier la génétique du cacaoyer. / For several years, cocoa research programs have focused on studying the genetic basis of agronomic traits of interest, especially for disease resistance and quality of cocoa beans, two important attributes for cocoa farmers and chocolate production. This work presents, by exploring the transcriptome and cocoa genome, the constitution of molecular resources and the analysis of biosynthesis pathways involved in several of these agronomical traits. The transcriptome study allowed the identification of several tens of thousands of genes expressed in various organs and for different environmental conditions and provided numerous molecular markers, used to produce high-density genetic maps. The information provided by this work led to the genome sequencing of a cocoa Criollo variety. Its analysis and annotation have provided a set of crucial biological information, from the catalog of genes and transposable elements to evolutionary aspects, which revealed a close evolutionary relationship to the eudicot putative ancestor, showing a limited number of recombination between ancestral chromosomes. Subsequently, the work we carried out to improve the complete sequence led to a considerable reduction of the chromosomal fragmentation observed in the first version. In addition, 97% of the assembled sequence and 99% of the genes are now anchored on the cocoa chromosomes. To exploit this new sequence of the genome, we conducted a QTL study from a progeny between Trinitario clones established in French Guiana, allowing to identify genomic regions involved in color trait variation observed in cocoa beans. Based on the improved version of the Criollo genome, we identified two genes potentially involved in the biosynthesis pathway of anthocyanins and flavonoids in the main genomic region concerned. One of the two genes is located nearby the QTL peak and encoding a chalcone synthase, appears to be a promising candidate gene. The comparative study of its structure in the Criollo genome (white bean) and in the Amelonado genome (purple bean) revealed several variations that could be responsible for functional modifications. The results presented in this thesis provide a variety of knowledge and tools useful to conduct multiple integrated approaches to studying cocoa tree genetics.

Afin d’estimer la capacité de réponse du chêne sessile (Quercus petraea Matt. Liebl.) aux changements climatiques en cours, le potentiel d’adaptation de cette espèce pour le débourrement a été mesuré en populations naturelles. Ces populations sont situées le long d’un gradient altitudinal comprenant 12 populations entre 131 et 1630 m, et d’un gradient latitudinal comprenant 21 populations de l’ensemble de l’aire de répartition. Tout d’abord l’empreinte démographique sur les niveaux de diversité génétique a été estimée sur les populations du gradient altitudinal à partir de marqueurs neutre. Les résultats ont montré que la diversité est maintenue le long du gradient altitudinal grâce notamment à des forts flux de gènes entre populations. La variabilité génétique du débourrement à été mesurée en tests de provenances pour 10 populations du gradient altitudinal. Les résultats ont montré une forte différenciation ainsi qu’une héritabilité élevée du trait. Une variabilité génétique importante est maintenue à l’intérieur des populations et semble indiquer que de multiples pressions de sélection agissent de manière fluctuante et/ou opposée. La diversité de gènes candidats pour le débourrement a été étudiée sur les populations des deux gradients environnementaux. Un niveau de diversité nucléotidique relativement fort et un faible déséquilibre de liaison qui décroit rapidement avec la distance ont été observés. Des signatures de sélections ont été mises en évidence sur un ensemble de gènes candidats. Une étude d’association a été menée entre variabilité du caractère et polymorphisme au sein des gènes candidats sur les populations des deux gradients. Un total de 16 associations significatives a été observé impliquant 10 gènes candidats. Ces résultats suggèrent un potentiel d’adaptation important face aux changements climatiques et offrent des perspectives intéressantes pour la compréhension des processus évolutifs qui régissent l’adaptation du chêne sessile pour le débourrement. / In order to assess the capacity of sessile oak (Quercus petraea Matt. Liebl.) to withstand the ongoing climate changes, we estimated its adaptative potential for bud burst within natural populations. These populations are located along two steep temperature gradients: an altitudinal gradient comprising 12 populations located between 131 m 1630 m, and a latitudinal gradient including 21 populations from the species’ distribution range. First the demographic imprint on the overall genetic diversity was assessed on the altitudinal gradient populations using neutral markers. Results showed that genetic diversity was homogeneously distributed along the gradient and maintained at high altitudes. The genetic variability of bud burst was measured in provenance tests for 10 populations of the altitudinal gradient. We found a high level of genetic differentiation and a high heritability for the trait. A high variability was also observed within populations, indicating that selection pressures may fluctuate in natural conditions. Genetic diversity of candidate genes for bud burst was assessed on populations from both gradients. A high level of nucleotide diversity was observed, and linkage disequilibrium was low. Selective signatures were observed on few candidate genes. An association mapping study was performed between bud burst variability and polymorphism at the candidate genes on populations of both gradients separately. A total of 16 associations involving 10 genes were observed. These results suggest an important adaptive potential of sessile oak for bud burst in the face of climate change and provide interesting perspectives for the comprehension of evolutionary processes controlling bud burst adaptation of sessile oak.

A displasia frontonasal (DFN) compreende quadros de aparência facial variável, sendo clinicamente caracterizada por dois ou mais dos seguintes sinais: hipertelorismo ocular com consequente alargamento da base nasal; fissura facial mediana afetando o nariz ou o nariz e lábio superior e, por vezes, o palato; fissura alar (uni ou bilateral); ponta nasal ausente; crânio anterior bífido oculto, e implantação em 'V' dos cabelos na fronte. A DFN pode ser vista como um defeito de desenvolvimento que pode ocorrer por si só ou como parte do quadro clínico de várias síndromes. A maioria dos casos de DFN é esporádica, e em raras circunstâncias foram observadas alterações cromossômicas em alguns indivíduos. Até o momento, quatro genes foram relacionados à patogênese molecular de algumas das síndromes com DFN, EFNB1, associado a uma forma de DFN ligada ao X e os genes ALX1, ALX3 e ALX4, todos associados a formas de DFN com herança autossômica recessiva. Embora esteja claro haver heterogeneidade etiológica, na maioria dos casos de DFN a causa não é conhecida, dificultando o adequado aconselhamento genético aos pacientes e seus familiares. Sendo assim, realizamos estudos com diferentes estratégias metodológicas buscando melhor compreender as possíveis causas genéticas da DFN. Ao todo foram analisados 10 pacientes: um caso familial de DFN leve com herança aparentemente autossômica dominante, um caso clinicamente sugestivo de mutação em ALX1, e oito casos de DFN associada a atraso de desenvolvimento com ou sem outras anomalias, dos quais um apresentava um rearranjo de novo aparentemente balanceado entre os cromossomos 4 e 12. Optamos por realizar sequenciamento dos genes previamente relacionados a fenótipos com DFN em todos os casos; para aqueles em que não foram detectadas mutações patogênicas, realizamos análise de variações de número de cópias (CNV) por microarray de polimorfismos de base única e, para o paciente com rearranjo cromossômico, realizamos o mapeamento do ponto de quebra por hibridação in situ fluorescente. Constatamos uma mutação em heterozigose no gene ALX4 co-segregando com o fenótipo do caso familial, sendo esta a primeira descrição de alteração em tal gene causando uma forma de DFN com herança dominante, e sugerimos pela primeira vez um mecanismo de dominância negativa. No caso sugestivo de mutação em ALX1, o diagnóstico foi confirmado através da identificação de uma mutação em homozigose neste gene do paciente; este caso consiste no 3o da literatura mundial e evidencia pela primeira vez que mutações em ALX1 não necessariamente levam a atraso de desenvolvimento ou deficiência intelectual. Os estudos citogenéticos e moleculares dos pontos de quebra do paciente com rearranjo cromossômico sugeriram os genes ARAP2 e CAND1 como possíveis responsáveis por seu quadro clínico, enquanto o estudo de CNVs nos indivíduos com DFN associada a atraso de desenvolvimento apontou os genes DNAJB12 e ENOX2 como possíveis candidatos para explicar o fenótipo de dois dos pacientes. É preciso que novos estudos sejam realizados a fim de melhor compreender o significado de tais achados e a real contribuição de cada gene para o desenvolvimento craniofacial humano e para a etiologia da DFN. Para os casos em que não foram identificadas alterações conclusivas no presente estudo, embora causas ambientais não possam ser descartadas, é preciso que seja investigada também a existência de fatores genéticos e epigenéticos não detectáveis pelas metodologias utilizadas, bem como a hipótese de mosaicismo somático. Nossos resultados, além de corroborarem o envolvimento dos genes ALX1 e ALX4 em fenótipos com DFN, sugerem também novos genes candidatos: ARAP2, CAND1, DNAJB12 e ENOX2 / Frontonasal dysplasia (FND) is a rare group of disorders that comprises cases with a variety of facial appearances, and is clinically characterized by two or more of the following signs: ocular hypertelorism with consequent broadening of the nasal root; median facial cleft affecting the nose and/or upper lip and palate; clefting of the alae nasi (uni or bilateral); lack of formation of the nasal tip; anterior cranium bifidum occultum; and a V-shaped frontal hairline. FND is a developmental defect that can occur alone or as part of several syndromes. Most cases of FND are sporadic, and in rare circumstances chromosomal alterations were observed in affected individuals. To date, four genes have been related to the molecular pathogenesis of some syndromes with DFN, one (EFNB1) is associated with an X-linked form while the 3 others (ALX1, ALX3 and ALX4) are associated with autosomal recessive forms. Although it is clear that FND is etiologic heterogeneous, the causative mechanism is unknown in most cases which makes it hard to give proper genetic counseling to patients and their families. In order to get new insights into the genetic mechanisms leading to FND, we performed studies with different methodologies. Altogether, 10 patients were analyzed: a familial case of a mild form of FND with an apparently autosomal dominant inheritance pattern, a case clinically suggestive of mutation in ALX1, and eight cases of FND associated with developmental delay with or without other anomalies, one of which with an apparently balanced de novo rearrangement between chromosomes 4 and 12. We chose to sequence the genes previously associated with FND phenotypes in all cases; for those in which pathogenic mutations were not detected, we conducted an analysis of copy number variations (CNV) by single nucleotide polymorphisms microarrays; for the patient with chromosomal rearrangement, we also mapped the breakpoints by using fluorescence in situ hybridization. We found a heterozygous mutation in ALX4 co-segregating with the phenotype of the familial case; this is the first description of mutation in this gene causing a form of FND with dominant inheritance pattern, and we suggested for the first time a dominant negative mechanism. In the case suggestive of mutation in ALX1, the diagnosis was confirmed by the identification of a homozygous mutation in this gene; this is the third case of the literature and shows for the first time that mutations in ALX1 are not necessarily related to developmental delay or intellectual disability. Breakpoints cytogenetic and molecular studies done with the patient with chromosomal rearrangement suggested ARAP2 and CAND1 genes as causative candidates for his condition, while the study of CNVs in individuals with FND associated with developmental delay pointed DNAJB12 and ENOX2 genes as possible candidates to explain the phenotypes of two of the patients. Further studies are necessary to better understand the significance of such findings and the actual contribution of each of these genes to human craniofacial development and the etiology of FND. Although environmental causes cannot be ruled out, it should also be investigated the existence of genetic and epigenetic factors as well as the possibility of somatic mosaicism, among the cases negative for the molecular approaches used in our study. Our results corroborate the involvement of ALX1 and ALX4 in FND phenotypes, and suggest new candidate genes: ARAP2, CAND1, DNAJB12 and ENOX2.

Les patrons de variation au sein des espèces d'arbres résultent d'évolutions complexes dont certaines sont particulièrement liées à la condition d'arbre. Si les facteurs expliquant cette variabilité ont été abordés pour les espèces des zones tempérées, ils restent encore peu connus pour des espèces fruitières des zones tropicales sèches offrant des particularités sur le plan adaptatif et relevant d'un processus de domestication ancien. Pour répondre à cet enjeu, l'objectif scientifique de cette thèse est de mieux comprendre l'impact des facteurs évolutifs sur la variabilité au sein d'une espèce d'arbre en zone soudano-sahélienne. Nous nous sommes intéressés à Vitellaria paradoxa (karité), une espèce jouant un rôle économique majeur par la production de beurre à partir des graines, dont l'aire de distribution s'étend du Sénégal à l'Ouganda sous la forme de peuplements naturels et agroforestiers. Dans une approche phylogéographique basée sur l'analyse du polymorphisme de marqueurs moléculaires supposés neutres (microsatellites chloroplastiques et nucléaires, et régions intergéniques du chloroplaste) et sur la modélisation des niches écologiques du Karité à différentes périodes du Quaternaire, nous mettons en évidence l'impact des perturbations liées aux dernières glaciations (il y a 20000 ans) sur la diversité génétique de l'espèce. Dans une seconde approche méthodologique, nous étudions l'impact des déterminants environnementaux sur la variabilité des constituants chimiques des graines de Karité, permettant d'énoncer de nouvelles hypothèses. Enfin, nous nous intéressons à l'expression du polymorphisme de gènes candidats, codant les enzymes stéaroyl-ACP-désaturase (SAD) et homogentisate phytyltransférase (HPT), en relation avec la variabilité de la composition chimique en acides gras et en tocophérols des graines. Les résultats obtenus permettent notamment de discuter de l'impact de la sélection naturelle, de la domestication et de la dérive génétique, et d'apporter des hypothèses évolutives et fonctionnelles potentielles expliquant la variabilité observée. / Patterns of variation within tree species are the result of complex evolutionary changes, some of which are particularly related to the condition of trees. If the factors explaining this variability were discussed for species in temperate zones, they remain little known for fruit tree species of dry tropics, which show special adaptive features and result from an old domestication process. To meet this challenge, the scientific purpose of this thesis is to improve our understanding of evolutionary factors affecting the variability within a tree species in the Sudano-Sahelian zone. We got interested in Vitellaria paradoxa (Shea tree), a species which plays a major economic role for the production of butter from seeds and whose distribution range extends from Senegal to Uganda. In a phylogeographic approach based on the analysis of polymorphism supposedly neutral molecular markers (chloroplast and nuclear microsatellites and chloroplast intergenic regions) and based on ecological niche modelling of Shea tree at different periods of the Quaternary, we shape the role of last glacial maximum (20.000 YBP) on the genetic diversity of the species. In a second methodological approach, we study the impact of environmental determinants on the variability of Shea nuts chemical constituents, stating novel hypotheses. Finally, we investigate the polymorphism of candidate genes, encoding enzymes stearoyl-ACP-desaturase (SAD) and homogentisate phytyltransférase (HPT), in connection with the variability of relative fatty acids compositions and tocopherols contents in seeds. Through the results obtained, we discuss the impact of natural selection, domestication and genetic drift, and provide evolutionary and functional hypotheses that potentially explain the varia bility observed.

Speciation, the formation of new species, is an essential evolutionary process that causes species diversity on the Earth. At the beginning of this process is the separation of two populations by a reproductive barrier that prevents gene flow between these populations. One of the mechanisms, which enable reproductive isolation, is hybrid sterility (HS). It is a mechanism of postzygotic isolation that is described in a number of eukaryotes. The first discovered gene of hybrid sterility in vertebrates is the mice gene Hst1, later identified as gene Prdm9. By genetic and molecular analysis the locus on the X chromosome was determined, whose interaction with Prdm9 causes sterility or reduced fitness in male hybrids. This locus contains two genetic factors: Hstx1, causing an abnormal morphology of spermatozoa, and Hstx2, causing an arrest in spermatogenesis in pachytene spermatocytes and sterility. In my thesis I focus on the effect of deletion of a candidate hybrid sterility gene Fmr1nb on the X chromosome. The analysis of males B6N.Fmr1nbmut with deletion variants of the Fmr1nb gene showed that Fmr1nb is one of the factors influencing spermatogenesis. An increase in morphologic abnormalities in spermatozoa occurred in males with Fmr1nb gene deletion. This phenotype is identical with Hstx1. The effect...

Anomalies de structure du génome et Déficience Intellectuelle : Recherche des gènes candidats de Déficience intellectuelle en utilisant l'analyse chromosomique sur microréseau d'ADN pangénomique 180K et l'analyse chromosomique sur microréseau d'ADN de haute résolution 1M ciblée des gènes candidats de Déficience intellectuelle. L'analyse chromosomique sur microréseau d'ADN (ACM) de haute résolution est une innovation technologique puissante afin de détecter les aberrations chromosomiques concernant les variations du nombre de copies. En utilisant l'ACM 180K, l'ACM 1M et la PCR quantitative, nous avons identifié les 5 variations du nombre de copies (CNV) intragéniques pathogènes de novo impliquant les gènes : RUNX1T1, KIAA1468, FABP7, ZEB2 (syndrome de Mowat-Wilson) et ANKRD11 (syndrome de KBG). Les 5 patients ayant une DI et une dysmorphie faciale. L'ACM 180K a révélé une délétion d'une taille de 92 kb emportant le gène KIAA1468 candidat pour le syndrome de West chez un enfant de 3 ans présentant une DI sévère et une encéphalopathie épileptique infantile précoce. Le criblage des mutations du gène KIAA1468 a été réalisé chez 35 patients atteints de syndrome de West. Un variant intronique c.2761-7 T>C et un variant faux-sens hérité de la mère c.3547 G>A avec signification clinque inconnue ont été identifiés. En utilisant des approches par l'ACM 1M de haute résolution chez 45 patients atteints de DI idiopathique modérée à sévère, un seul CNV causal a été identifié, une délétion intragénique d'une taille de 28.37 kb du gène ZEB2. Notre étude confirme une fréquence très faible des délétions/duplications intragéniques avec la détection d'une seule aberration chromosomique (1/45). En conclusion, si la fréquence des mutations ponctuelles est élevée, nous avons également souligné l'application de la technique de séquençage à haut débit avec un rendement diagnostique jusqu'à 45%-55% des cas de DI sévère idiopathique chez lesquels aucun CNV n'a été détecté sur ACM / Chromosomal structural abnormalities and Intellectual Disability : In search of intellectual disability candidate genes by using pangenomic comparative genomic hybridization 180 K and high resolution comparative genomic hybridization 1M targeting intellectual disability candidate gene.High resolution microarray-based comparative genomic hybridization (a-CGH) has been a powerful technical innovation in order to detect submicroscopic chromosomal aberrations related to copy number variations. By using a-CGH 180K, 1M high resolution a-CGH and quantitative PCR, we have identified 5 pathogenic intragenic copy number variations (CNVs) de novo : RUNX1T1, KIAA1468, FABP7, ZEB2 (Mowat-Wilson syndrome) and ANKRD11 (KBG syndrome). All five patients had intellectual disability (ID) and facial dysmorphism. Interestingly, a-CGH 180K has revealed a 92 kb deletion of a candidate gene KIAA1468 for West syndrome in a 3 year-old boy with severe ID and early infantile epileptic encephalopathy. Mutational screening for candidate gene KIAA1468 was performed in 35 patients with West syndrome. An intronic variant c.2761-7 T>C and a non synonymous maternally inherited variant c.3547 G>A with unknown clinical significance were identified. By using 1M high-resolution a-CGH approach in 45 patients presenting moderate to severe idiopathic ID, only one causal CNV was identified, a 28.37 kb intragenic ZEB2 deletion. Our study has confirmed the low frequency of intragenic deletion/duplication with the detection of only one chromosomal aberration (1/45). In conclusion, providing that the high frequency of intragenic point mutation, we also stressed the application of next-generation sequencing technology with 45-55% diagnostic yield in patients with idiopathic severe ID in case of no apparent CNV(s) on high-resolution a-CGH

Ziel unserer molekulargenetischen Studien ist es, Gene der genetisch komplexen idiopathisch generalisierten Epilepsien (IGE) im Genom des Menschen zu lokalisieren und die verantwortlichen Genstörungen durch die Mutationsanalyse von positionell und funktionell plausiblen Kandidatengenen zu identifizieren. Unsere Kopplungsanalysen konnten einen IGE-Locus (Locus-Symbol: EJM1) in der chromosomalen Region 6p21.3 bestätigen und die Kandidatengenregion auf ein chromosomales Segment von 10 centiMorgan (cM) eingrenzen. Ein positionell und funktionell plausibles Kandidatengen ist das Gen einer Untereinheit des heterodimeren GABAB Rezeptors (Gen-Symbol: GABA-BR1). Die systematische Mutationsanalyse des GABA-BR1 Gens und eine Assoziationsstudie mit drei Sequenzpolymorphismen in den Exonen 1a1, 7 und 11 ergaben keinen Anhalt für eine Beteiligung des GABA-BR1 Gens bei der Epileptogenese der IGE. Kopplungshinweise in den chromosomalen Regionen 20q13, 8q24 und 15q14 konnten wir in unserem Familienkollektiv nicht bestätigen. Die Mutationsanalyse der Kandidatengene CHRNA4 und KCNQ2 in der Kandidatengenregion 20q13 und von zwei Kalziumkanal-Genen (CACNA1A, CACNB4) ergaben keinen Hinweis auf disponierende Sequenzvarianten bei IGE-Patienten. Unsere systematische Genomanalyse bei 130 Familien mit mehreren IGE-Angehörigen zielte auf die positionelle Eingrenzung von Genstörungen, die an der Disposition eines breiten IGE-Spektrums beteiligt sind. Bei 360 der 694 Familienangehörigen lag ein IGE-Phänotyp vor. Bei 617 Familienangehörigen wurden für die systematische Genomanalyse insgesamt 416 Mikrosatelliten-Polymorphismen mit einem durchschnittlichen Abstand von 10 cM genotypisiert. Die parameter-freien Kopplungsanalysen ergaben einen signifikanten Kopplungsbefund in der chromosomalen Region 3q26 (P = 1,7 x 10-5 bei D3S3725) sowie zwei Kopplungshinweise in den chromosomalen Regionen 2q36.1 (P = 5,4 x 10-4 bei D2S1371) und 14q23 (P = 5,6 x 10-4 bei D14S63). Positionell und funktionell plausible Kandidatengene sind die Gene des Kalium-Kanals KCNA1B und des Chlorid-Kanals CLCN2 in der Region 3q26, das Gen des Chlorid-Bikarbonat Austauschers SLC4A3 in der Region 2q36, und das Gen des Natrium-Kalzium Austauschers SLC8A3 in der Region 14q23. Der molekulargenetische Nachweis von Genmutationen für die IGE wird konkrete Einblicke in die molekularen Mechanismen der Epileptogenese eröffnen und die Voraussetzungen dafür schaffen, rational begründete Therapieansätze zu entwickeln. / The aim of our molecular genetic studies is to map genes of the genetically complex idiopathic generalized epilepsies on the human genome and to identify the causative gene variants by mutation analyses of positional and functional plausible candidate genes. Our linkage studies confirmed an IGE-locus (locus symbol: EJM1) in the chromosomal region 6p21.3 and to refine the candidate region to a chromosomal segment of 10 centiMorgan (cM). A positional and functional candidate gene is the gene encoding a subunit of the heterodimeric GABAB receptor (gene symbol: GABA-BR1). The systematic mutation screening of the GABA-BR1 gene and an association analysis with three sequence polymorphisms in exons 1a1, 7 and 11 provided no evidence that the GABA-BR1 gene confers susceptibility to the epileptogenesis of IGE. We failed to replicate previous linkage findings in the chromosomal regions 20q13, 8q24 and 15q14 in our family sample. Mutation analysis of the candidate genes CHRNA4 and KCNQ2 and two genes encoding calcium channel subunits (CACNA1A, CACNB4) did not detect common susceptibility alleles in IGE patients. Our systematic genome scan was designed to identify susceptibility loci that predispose to a broad spectrum of common IGE syndromes. Our study included 130 families with two or more siblings affected by an IGE. In total, 360 out of 694 family members were affected by an IGE-trait. 617 family members were genotyped for 416 microsatellite polymorphisms with an average distance of 10 cM. Non-parametric linkage analysis provided significant evidence for a novel IGE susceptibility locus on chromosome 3q26 (ZNPL = 4.19 at D3S3725; P = 0.000017) and suggestive evidence for two IGE loci on chromosome 14q23 (ZNPL = 3.28 at D14S63; P = 0.000566), and chromosome 2q36 (ZNPL = 2.98 at D2S1371; P = 0.000535). Positional and functional candidate genes include the potassium channel gene KCNA1B and the chloride channel gene CLCN2 in the region 3q26, the chloride-bicarbonate anion exchanger gene SLC4A3 in the region 2q36, and the sodium-calcium exchanger gene SLC8A3 in the region 14q23. The molecular genetic detection of susceptibility genes for IGE will provide clues to elucidate the complex molecular pathways of epileptogenesis, and, finally, will help to develop rational treatment strategies.

Escherichia coli diarrhoea is the most important source of mortality in piglets. The most frequently isolated strain in enterotoxigenic E. coli diarrhoea is F4ab/ac. Recent studies in South Africa reported non-fimbrial strains such as PAA and EAST-1 to be prevalent. The objective of the study was to determine whether there are breed differences among pigs with respect to E. coli adhesion phenotypes and correlate them to polymorphisms at selected candidate genes in the South African population. A total of 225 pigs aged 3-12 weeks of the imported (Large White, Landrace and Duroc), local and crossbreds, were sampled from the Eastern Cape and Limpopo provinces of South Africa and genotyped for PCR-RFLP polymorphisms at four candidate genes associated with E. coli F4ab/ac resistance/susceptibility. These genes were Mucin 4 (MUC4), Mucin 13, (MUC13), Mucin 20 (MUC20) and Transferrin Receptor (TFRC). The TFRC and MUC13 genes were less polymorphic, the C allele was close to fixation and the homozygous CC genotype was the most frequent in all three pig populations. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for the TFRC locus. The g.8227G>C polymorphism in MUC4 segregated in all three breeds and the marker was moderately polymorphic. There was a significant difference (P <0.05) in genotypic distribution amongst breeds for MUC4.The g.191C>T polymorphism in MUC20 segregated in the local and crossbred pigs and was close to fixation in the imported pigs. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for MUC20, which was moderately polymorphic. There was a reduction in heterozygosity in both the TFRC and MUC13 loci, although MUC4 and MUC20 genes had higher heterozygosity levels. The MUC4 gene had a negative FIS value, indicating outbreeding at this locus. The MUC20, MUC13 and TFRC genes had a positive FIS value, indicating inbreeding at these loci. Overall, the studied population was outbred. Imported pigs in TFRC and MUC20 deviated from Hardy-Weinberg equilibrium (HWE). All breeds were in HWE at the MUC4 and MUC13 genes. There was no linkage disequilibrium observed amongst the analysed loci. iv A total of 109 piglets of three breeds (Large White, indigenous and crossbred) aged 3-5 weeks, were investigated for the susceptibility to E. coli F4, PAA strains and EAST-1 toxin. Adhesion tests were conducted on pig intestinal cells, which were viewed under a phase contrast microscope. Three phenotypes were identified as, adhesive, weakly adhesive and non-adhesive. There was a significant association (P <0.05) between breed and level of adherence of the F4 and PAA strains. Highest frequencies of adhesion phenotypes were observed in the indigenous pigs for both F4 and PAA E. coli strains. Large White pigs had the lowest frequency of non-adhesion in F4 and PAA E. coli strains. The F4 strain had a higher (P <0.05) level of adherence compared to PAA and EAST-1 in Large White pigs. Age of pigs had a significant effect on the level of E. coli adherence in indigenous and crossbred pigs (P <0.05). Adhesion of F4 and EAST-1 was higher in weaned indigenous and crossbred pigs, respectively, than in suckling piglets. There was no significant difference between F4 adhesion and the genotypes at all four candidate genes genotypes. The study showed that both imported and local pig populations carry receptors and are susceptible to F4, PAA and EAST-1 E. coli infections. Indigenous pigs were less susceptible than Large White to E. coli infection. Although polymorphic and segregating in the populations, the MUC4 g.8227G>C and MUC20 g.191C>T mutations were not associated with the adhesion phenotypes and cannot be used in the selection of susceptible animals. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.