Estudos com farinha de banana verde (FBV), rica em amido resistente, mostram efeitos positivos sobre a saciedade, resposta glicêmica e melhora do funcionamento intestinal. Entretanto, pouco se sabe sobre a capacidade da FBV em estimular seletivamente o crescimento e/ou atividades de microbiota intestinal benéfica e os efeitos fisiológicos do consumo habitual. No presente trabalho foi investigado o efeito da ingestão regular e descontinuada de FBV sobre a microbiota intestinal em voluntários saudáveis, bem como as interações com hormônios relacionados à fome e saciedade, funcionamento intestinal e homeostase da glicose. Para tanto foi realizado estudo de intervenção, duplo cego paralelo controlado com placebo, no qual voluntários saudáveis consumiram FBV ou maltodextrina, veiculadas através de sopa prontas congeladas, três vezes por semana e durante seis semanas. Os resultados evidenciaram que a FBV pode aumentar a saciedade, promover redução no aporte energético de refeições subsequentes (14%) e melhorar o funcionamento intestinal. Ao mesmo tempo reduz a secreção plasmática de insulina no jejum e o Índice HOMA2-RI em 20%, sinalizando aumento na sensibilidade à insulina. A análise da microbiota intestinal utilizando o rDNA 16S mostrou que existem dois grupos distintos de indivíduos, os quais respondem diferentemente ao consumo de FBV. O consumo de FBV por voluntários, cujo microbioma era mais abundante no gênero Prevotella, apresentou aumento de genes envolvidos em vias metabólicas relacionadas à degradação anaeróbia de carboidratos (794 Kegg orthologs, FDR=0,05), como as vias do metabolismo de amido e glicose, do butirato, propionato. Paralelamente outros genes indicaram redução de algumas vias metabólicas, incluindo a biossíntese de lipopolissacarídeos. Este mesmo grupo de voluntários apresentou gêneros microbianos positivamente relacionados com conteúdo de ácidos graxos de cadeia curta (AGCC), em padrão distinto do outro grupo de voluntários que consumiu FBV e do grupo Controle. Foi possível demonstrar que o consumo de FBV pode promover a modulação do microbioma em indivíduos saudáveis com enterótipos distintos, trazendo efeitos benéficos para a saúde humana. / Studies with Unripe Banana Flour, rich in resistant starch, shave shown positive effects on satiety, glycemic response and improved intestinal function. Nevertheless, little is known about its capacity to selectively stimulate the intestinal microbiota\'s activity, or the physiological effects of its habitual consumption. This study investigated the effects of the regular, discontinued ingestion of UBF on the intestinal microbiome in healthy volunteers, as well as effects on hormones related to satiety, intestinal function and glucose homeostasis. To achieve these goals, a double blind, parallel, placebo controlled study was designed, in which healthy volunteers ingested UBF or maltodextrin added to a standardized frozen soup meal, 3 times a week for 6 weeks. The results showed that UBF can improve satiety, promote a reduction in energy intake at subsequent meals (14%) and improve intestinal function. At the same time, it reduces plasmatic secretion of fasting insulin and e the HOMA2-RI index by 20%, signaling an increase in insulin sensitivity. The analysis of the microbiome using the 16S rDNA gene showed that there are two clusters of individuals, which respond differently to the dietary intervention. The UBF consumption by volunteers with a Prevotella dominant microbiome showed an increase in genes related to anaerobic carbohydrate degradation (794 Kegg orthologs, FDR=0,05), such as members of the starch and glucose metabolism, propanoate metabolism and butyrate metabolism. At the same time, other genes were reduced, including the biosynthesis of lipopolysaccharides. The same volunteers presented several microbial groups positively correlated with the short chain fatty acids (SCFA) present in the fecal samples analyzed. This was a distinct pattern to that observed for the remaining volunteers. We demonstrated that the consumption of UBF can promote the overall health of the human host as well as the modulation of the intestinal microbiome in healthy individuals and that this effect is dependent on the enterotype present.

Le campagnol terrestre, Arvicola terrestris, occasionne en France, lors de ses pullulations cycliques interannuelles, d’importants dégâts aux prairies de montagne. Un groupe de travail constitué des équipes de recherche de l’Université de Franche-Comté (UFC), de l’INRA (Centre de Biologie et de Gestion des Populations) et d’organismes agricoles (Fédération Régionale de Défense contre les Organismes Nuisibles de Franche-Comté, FREDON), ont privilégié une approche « systémique » dans laquelle les interactions entre les campagnols, leur habitat (paysage, prédateurs) et les pratiques agricoles sont analysées de façon hiérarchisées (spatialement et temporellement). Un des objectifs est de mettre en évidence le plus grand nombre possible de facteurs de contrôle sur lesquels il est possible d’agir, et l'échelle à laquelle ces actions sont pertinentes. Ces études ont permis d’initier une stratégie, expérimentée avec succès, notamment en Franche-Comté et en Auvergne, et qui privilégie la lutte raisonnée. Il subsiste néanmoins des zones d’ombre relatives à la compréhension du cycle, notamment concernant les déterminants de la phase de déclin. Le rôle du cortège de pathogènes (parmi lesquels certains peuvent être transmis à l’homme) reste pour l’instant sujet de débat dans la littérature scientifique. La compréhension des facteurs clés déterminant cette phase devrait permettre aux éleveurs de mieux anticiper les impacts économiques et adopter les stratégies de contrôles des population les plus adéquates. Objectifs de la thèse (1) Tester les hypothèses des pathogènes et de la sénescence pour expliquer le déclin démographique. (2) Rechercher des indicateurs biologiques (diversité des pathogènes et/ou indicateurs immunitaires) qui permettent de prédire les phases de déclin et d’anticiper des mesures agricoles appropriées pour restaurer les prairies. (3) Evaluer le rôle de la transition entre la phase de forte densité et de déclin démographique pour l’émergence de pathogènes circulants par les populations de campagnols et responsables de maladies humaines. Méthodologie générale Des suivis de populations avec des prélèvements réguliers (mensuels) seront réalisés sur plusieurs populations (répliquats) dans la période qui encadre le déclin démographique. Des méthodes fondées sur le séquençage à haut débit (NGS : Next Generation Sequencing) pour l’épidémiologie permettent d’établir des catalogues complets des pathogènes (virus, bactéries, parasites) hébergées par les populations, et d’en mesurer les prévalences. / Context In France, during cyclic population surges, water voles, Arvicola terrestri, cause extensive damage to mountain grassland. A working group consisting of researchers from the University of Franche-Comté (UFC), INRA (Centre de Biologie et de Gestion des Populations) agricultural organizations (Fédération Régionale de Défense contre les Organismes Nuisibles de Franche-Comté, FREDON) are working on systems approach in which interactions between voles, their habitat (landscape, predators) and agricultural practices are analysed hierarchically (in space and time). One of the objectives is to highlight the largest possible number of control factors on which it is possible to act, and the scale at which these actions are relevant. These studies have helped initiate a strategy, successfully tested in Franche-Comté and in Auvergne, which promotes the integrated control of water vole populations. Nevertheless, there are still grey areas in the understanding of the cycle, particularly on the determinants of the decline phase. The role of pathogen communities (some species may even be transmitted to humans) so far remains the subject of debate in the scientific literature. The understanding of the key factors determining this phase should allow farmers to better anticipate economic impacts and to adopt optimal strategies for vole population control Objectives: (1) To test the pathogens and senescence hypotheses in order to explain the population decline. (2) To look for biological indicators (diversity of pathogens and / or immune indicators) that may predict the decline phase in order to anticipate appropriate measures to restore grasslands. (3) To assess the role of the transition between high population density phase and the decline phase for the emergence of pathogens in vole populations that may cause human diseases.General Methodology Population monitoring with regular (monthly) sampling will be made on several populations (replicates) in the period that brackets the vole population declines. Methods based on Next Generation Sequencing (NGS) makes it possible to establish extensive catalogues of pathogens (viruses, bacteria, other parasites) hosted by vole populations and to measure the prevalence.

Le secteur du tri des déchets est en pleine expansion. De par la nature de leurs activités, les centres de tri des déchets sont une source d’émission de bioaérosols dont l’exposition peut entrainer diverses troubles sur la santé des travailleurs. La composition des bioaérosols dans les centres de tri est peu documentée. Dans ce contexte, les objectifs de la thèse étaient de caractériser et de déployer dans un centre de tri (pour un suivi d’un an) une méthode d’analyse de la biodiversité microbienne dans les bioaérosols par séquençage à haut débit. Les travaux ont permis de connaitre les avantages et les limites de plusieurs méthodes d’analyse de la biodiversité et de mettre en lumière la nécessité de standardiser un processus de mesure dans le cadre d’études de comparaison. Le suivi de la biodiversité microbienne dans les bioaérosols émis en centre de tri a permis de mettre en évidence la grande complexité et la variabilité des taxons bactériens et fongiques à différents postes de l’entreprise. L’analyse statistique a mis en évidence le caractère multifactoriel de la variation et de la composition des bioaérosols émis en environnement professionnel. La quantité importante de données a permis d’améliorer les connaissances sur la composition des bioaérosols émis en centre de tri des déchets et sur la stratégie de prélèvement à adopter pour une évaluation du risque biologique représentative de l’entreprise / The waste sorting activities is constantly increasing. Due to the nature of their activities, waste sorting plants are a source of bioaerosol emissions whose exposure can lead to various health problems for workers. The composition of bioaerosols in sorting centers is poorly documented. In this context, the aims of the thesis were to characterize and deploy in a sorting center (for a follow- up of one year) a method of analysis of microbial biodiversity in bioaerosols using high throughput sequencing. The work provided information about the advantages and the limits of several methods of analysis of the biodiversity and highlighted the need to standardize a process of measurements for comparative studies. The monitoring of microbial biodiversity in bioaerosols emitted in a sorting plant revealed the high complexity and the variability of bacterial and fungal taxa at different places in the company. Statistical analysis highlighted the multifactorial nature of the variation and composition of bioaerosols emitted in this occupational environment. The large amount of collected data improved the knowledge on the composition of bioaerosols emitted in the waste sorting plant and the sampling strategy to be conducted for a representative biological risk assessment in the company

Anticiper l’impact de fluctuations environnementales de nature climatique ou alimentaire est un enjeu crucial dans les systèmes de productions animales, et plus particulièrement sur la volaille. Cette influence de l’environnement sur les phénotypes passe en partie par des phénomènes épigénétiques, notamment la méthylation de l’ADN, et qui peuvent intervenir dans la régulation de l'expression des gènes. Ce sont des mécanismes qui n'affectent pas la séquence d'ADN mais qui peuvent être transmis par la mitose ou la méiose. Ces interactions entre épigénomes et expression des gènes sont de plus en plus étudiées dans les modèles animaux et chez les plantes. Cependant, les mécanismes de régulation de l'expression du génome par la méthylation de l’ADN sont assez peu connus chez les oiseaux. Ce travail de thèse repose sur deux dispositifs expérimentaux réalisés chez la poule, le but étant de caractériser le méthylome par séquençage haut-débit. Les profils de méthylation le long du génome, et le lien avec l’expression, sont établis d’abord par un séquençage tout-génome (WGBS) au sein d’embryons entiers, puis par un séquençage d'une sous-représentation du génome (RRBS) au sein d’hypothalamus d’individus adultes. À ce jour, aucune étude d'analyses de méthylome par RRBS chez la poule n'a été publiée. Ces deux analyses sont réalisées grâce au développement d'un pipeline bioinformatique, optimisé, disponible à la communauté scientifique. Globalement, le profil de méthylation chez la poule est similaire à ce qui est connu chez les mammifères : les îlots CpG - régions riches en dinucléotides CG, souvent peu méthylées, qui ponctuent le génome principalement dans les régions promotrices des gènes - sont globalement peu méthylés dans les promoteurs sur les données WGBS et RRBS. Les analyses du méthylome des embryons ont confirmé l'absence d'un phénomène de compensation de dose sur les chromosomes sexuels, ou la présence sur le chromosome Z d'une région hyperméthylée. Les analyses des données RRBS révèlent une hyperméthylation globale des CG sur le génome, suggérant une réponse de la méthylation à un stress environnemental. Sur les données WGBS, le niveau de méthylation dans le promoteur est négativement corrélé à l'expression du gène associé. Une méthylation allèle spécifique est également détectée entre les lignées, phénomène mis en évidence pour la première fois chez la poule et dont la fréquence est comparable à ce qui a été observé chez l'Homme. Sur les données RRBS, des résultats préliminaires de la réponse du méthylome aux stress environnementaux montrent le caractère complexe de cette relation. L’utilisation d’aliments moins énergétiques entraînerait une plus grande mobilisation des réserves lipidiques, tandis que les individus soumis à un stress à la chaleur ont un poids corporel plus léger. Une intégration de ces données à des mesures phénotypiques permettrait de faire le lien entre méthylation et environnement. Au-delà de l'aspect fondamental de cette thèse, l'application plus concrète de ces connaissances peut s'appliquer aux systèmes d'élevage pour obtenir des animaux mieux adaptés à l’environnement, en améliorant les caractères de production / Anticipating the impact of environmental changes (on climate and feed) is a crucial issue for livestock production systems, including poultry. The influence of the environment on phenotypes is partly mediated by epigenetic phenomena, including DNA methylation, which may be involved in the regulation of gene expression. These mechanisms do not affect the DNA sequence but can be inherited by mitosis or meiosis. The interactions between epigenomes and gene expression are increasingly being studied in animal models and in plants. However, the mechanisms of regulation of genome expression through DNA methylation are relatively unknown in birds. This thesis work is based on two experimental devices realized in chicken aiming to characterize the methylome by high-throughput sequencing. The methylation patterns across the genome, and their link with expression, were first established by whole-genome bisulfite sequencing (WGBS) in whole embryos, following a reduced representation bisulfite sequencing (RRBS) from hypothalamus of adults. To date, no specific chicken RRBS study has been published. These two analyses were carried out by developing an optimized bioinformatics pipeline, available for scientific community. Overall, the pattern of methylation in chicken is like those in mammals: CpG islands - dinucleotides CG-rich regions which are often poorly methylated, and which are found mainly in the promoter regions of the genome - are generally poorly methylated in promoters on WGBS and RRBS data. Embryo methylome analyses confirmed the absence of a dose-compensation phenomenon on sex chromosomes, or the presence of a hypermethylated region on the Z chromosome. The analyses of RRBS data revealed an overall hypermethylation of CGs across the genome, suggesting a methylation response to environmental stress. From the analysis of WGBS data, we found that the level of methylation in promoters was negatively correlated with the expression of the associated gene. For the first time, a specific allele methylation was also detected between chicken lines whose frequency is comparable to that observed in humans. On the RRBS data, preliminary results of the methylome response to environmental stresses showed the complex nature of this relationship. The use of a low-energy diet would led to greater mobilization of body fat, while individuals with heat stress had a lighter body weight. Integrating these data with phenotypic measurements would allow to link methylation and environment. Beyond the fundamental aspect of this thesis, the method developed in this work could be applied to livestock systems to breed animals better adapted to a changing environment, by improving production traits.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-01-24T17:49:23Z No. of bitstreams: 1 2017 - Leandro Martins Ferreira.pdf: 2738374 bytes, checksum: a6ccde64b30f6cd52122b9030ab6c92e (MD5) / Made available in DSpace on 2018-01-24T17:49:25Z (GMT). No. of bitstreams: 1 2017 - Leandro Martins Ferreira.pdf: 2738374 bytes, checksum: a6ccde64b30f6cd52122b9030ab6c92e (MD5) Previous issue date: 2017-02-22 / CAPES / Faperj / CNPq / Among the abiotic stress, drought is a major environmental stress seriously limiting plant growth and crop productivity. Rice is one of the most important staple food crops in the world and requires a larger quantity of water to produce, once it is a crop extremely sensitive to drought stress. For this reason, to obtain rice plants that cope with drought stress without major reduction in productivity is the challenge for breeding programs nowadays. This work aimed: (i) identify upland rice varieties with contrasting drought tolerance through the evaluation of morphological and physiological traits, (ii) analyse root parameters which could explain the differences between tolerant and sensitive varieties to the drought stress and, (iii) identify new biotechnological targets related with the tolerance through transcript profile analysis in the contrasting varieties. Six experiments were performed, two in greenhouse and four in growth chamber conditions. The experimental design adopted was completely randomized. The first experiment started with ten rice varieties submitted to control and stress conditions during the reproductive stage. The contrasting varieties were selected based on morphological and physiological traits. Experiments from II to IV aimed to correlate the tolerance to the drought stress with the root development and morphology. Experiment V aimed to evaluate the regulation of genes related to the drought tolerance and the experiment VI aimed to analyse the differential expression of genes through the RNAseq analysis in rice roots. Data obtained from the productivity components, tolerance index and multivariate analysis through the evaluation of morphological and physiological traits allowed to identify Catet?o and Piau? variety as the most tolerant and Quebra Cacho and Mira as the most sensitive. Drought tolerance was correlated with a lower root angle and increase in the root density and emission of lateral roots by Catet?o variety during drought stress. Moreover, Catet?o variety has showed higher expression levels and early induction of genes and transcription factors related with drought tolerance. The RNAseq analysis allowed to identify several potential genes which can be used in future breeding programs aimimg the improvement of drought tolerance in rice. / Dentre os estresses abi?ticos que podem limitar o crescimento das culturas agr?colas, a seca ? considerada um dos principais, sendo capaz de reduzir consideravelmente a produ??o global de alimentos. O arroz ? uma das mais importantes culturas agr?colas do mundo e sua produ??o demanda grande quantidade de ?gua, pois ? uma esp?cie extremamente sens?vel ao d?ficit h?drico. Portanto, a obten??o de plantas de arroz que lidam com o estresse h?drico, sem redu??o significativa de produtividade ? um desafio para os programas de melhoramento atuais. Este trabalho teve como objetivos: (i) identificar variedades de arroz de sequeiro contrastantes quanto ? toler?ncia ao estresse h?drico por meio da avalia??o de caracter?sticas morfol?gicas e fisiol?gicas, (ii) analisar par?metros radiculares que possam explicar a diferen?a entre variedades tolerantes e sens?veis ao estresse h?drico e, (iii) identificar novos alvos biotecnol?gicos envolvidos com essa toler?ncia por meio da an?lise do perfil de transcritos nas variedades de arroz contrastantes. Foram realizados seis experimentos, sendo dois em casa de vegeta??o e quatro em c?mara de crescimento. O delineamento experimental utilizado foi o inteiramente casualizado. O primeiro experimento iniciou com dez variedades de arroz submetidas a condi??o controle e estresse h?drico durante o per?odo reprodutivo. As variedades contrastantes foram selecionadas com base em caracter?sticas morfol?gicas e fisiol?gicas analisadas. Os experimentos II a IV foram realizados a fim de correlacionar a toler?ncia ao estresse com o desenvolvimento e morfologia do sistema radicular. O experimento V foi realizado para avaliar a regula??o de genes relacionados a toler?ncia ao estresse h?drico e o experimento VI teve como objetivo analisar a express?o diferencial de genes por meio da t?cnica de RNA-seq em ra?zes de arroz. Os dados obtidos dos componentes de produtividade, ?ndices de toler?ncia ao estresse e an?lise multivariada das caracter?sticas morfol?gicas e fisiol?gicas permitiram identificar as variedades Catet?o e Piau? como as mais tolerantes ao estresse h?drico, e Quebra Cacho e Mira como as mais sens?veis. Foi observado que a toler?ncia ao estresse h?drico est? correlacionada com o menor ?ngulo radicular, aumento da densidade e emiss?o de ra?zes laterais em condi??es de d?ficit h?drico na variedade Catet?o. Al?m disso, essa variedade mostra indu??o r?pida e elevados n?veis de express?o de genes e fatores de transcri??o relacionados ? toler?ncia ao estresse h?drico em arroz. Por meio do sequenciamento do RNA foi poss?vel identificar diversos genes com potencial para serem utilizados em programas de melhoramento visando o aumento da toler?ncia ao estresse h?drico em arroz.

Le séquençage à haut débit est une technique très utile pour l’étude des ARN. Pendant mon doctorat, nous l’avons utilisé pour la caractérisation des ARN extracellulaire (ARNex) du plasma humain. Les ARNex du plasma sont retrouvés soit à l’état soluble sous forme de complexes ribonucléoprotéiques (RNP) ou encapsulés au sein de vésicules extracellulaires (VE) de diverses origines (exosomes, microvesicles, …). Dans ce projet, j’ai démontré que le plasma contenait principalement des micro ARN, le fragment hY4 et des ARN ribosomiques dégradés. Par ailleurs, après chromatographie à exclusion de tailles ou par traitement consécutif protéinase K/RNase A, des VE hautement purifiées peuvent être obtenus. Nous ne retrouvons plus en majorité les micro ARN et l’ARN hY4 dans ces échantillons mais plutôt des ARN du microbiote humain, montrant une composition différente entre les ARNex solubles et ceux des vésicules purifiées. Par ailleurs, j’ai également effectué une étude comparative de kits commerciaux qui sont supposés purifier les exosomes par précipitation. La composition en ARN de ces fractions est très similaire au plasma humain total, montrant une forte contamination par les RNP solubles. Ainsi, nous sommes en mesure de proposer un protocole pour l’étude des ARNex dans le cadre de biopsies liquides avec des échantillons cliniques afin de découvrir de potentiels biomarqueurs de diagnostic. Au-delà de la caractérisation d’ARN, le séquençage à haut-débit peut être utilisé pour la détection et quantification des modifications post-transcriptionnelles. Pendant ma thèse, j’ai utilisé le séquençage pour l’analyse des 2’O-méthylation des ARN de transfert chez E. coli par RiboMethSeq. Sous plusieurs conditions de stress (manque de nutriments ou des concentrations non létales d’antibiotiques), certaines 2’O-méthylations montrent une réponse adaptative. Alors que plus de la moitié des 2’O-méthylations en position 18 (Gm18) sont augmentées dans toutes les conditions de stress étudiées, les positions Nm34 montrent un effet opposé avec une diminution dans certains stress (chloramphénicol et streptomycine). Chacun de ces deux comportements peut être relié à un phénomène de régulation cellulaire en réponse au stress : un changement au niveau de la wobble base pourrait être un moyen de réguler la traduction en modifiant l’usage des codons. En ce qui concerne Gm18, son rôle dans l’évasion du système immunitaire inné lors de l’invasion d’un hôte est en cours d’élucidation. / For less than a decade, high-throughput sequencing became a very powerful, sensitive and precise technique for the study of ribonucleic acids. During my PhD thesis, I used this technology for in-depth characterization of the extracellular RNA (exRNA) content of human plasma. exRNA in plasma exists either in a “soluble state” as a component of ribonucleoprotein (RNP) complexes or encapsulated into extracellular vesicles (EV) of diverse origins (exosomes, microvesicles, …). In this project, I demonstrated that whole human plasma contains mostly micro RNA and the fragment of RNA hY4, as well as degraded ribosomal RNA. Moreover, using a rigorous strategy via size exclusion chromatography or consecutive proteinase K/RNase A treatments, highly purified EVs can be obtained. miRNAs and RNA hY4 fragments were not present in majority of samples, demonstrating a huge difference between soluble exRNA and exRNA from purified EVs. The RNA content of these EVs mainly reflects RNA composition of human microbiota. In addition, I also performed a comparative analysis of commercially available “exosome-enrichment” kits which are supposed to purify human exosomes by precipitation. Their RNA composition was found to be almost identical to human plasma, showing strong uncontrolled contamination by soluble RNPs. Based on this study, we were able to propose a protocol for studies in exRNA in the field of liquid biopsies with clinical sample in order to discover new diagnostic biomarkers. Apart from the characterization of RNA, high-throughput sequencing can be used for detection and quantification of RNA post-transcriptional modifications. During my PhD thesis I applied deep sequencing for analysis of transfer RNA (tRNA) 2’-O-methylations in model bacteria (E. coli) using RiboMethSeq. Under several stress conditions, such as starvation and non-lethal antibiotics concentrations, some 2’-O-methylated nucleotides show an adaptive response. While over than half of Gm18 show a global increase under all investigated stress conditions, ribomethylated residues at position 34 show an opposite effect for some antibiotic treatments (chloramphenicol and streptomycin). Each of these dynamic profiles can be linked to cell regulation in response to stress. Change at the tRNA wobble base (position 34) could be a way to regulate translation by modifying the codon usage. Concerning Gm18, its role in the escape from the human innate immune system during host invasion is currently elucidated.

Les impacts de la construction sur l'environnement et sur la santé des habitants sont aujourd'hui des enjeux prioritaires. Les matériaux en terre crue connaissent un essor important pour de nombreuses raisons (écologique, économique, etc.), mais des questions se posent sur leur sensibilité à la prolifération des moisissures vis-à-vis de la qualité de l'air intérieur. Au cours de ces travaux, les flores bactériennes et fongiques présentes sur des supports en terre crue, biosourcés ou non, au sein d'habitations, ainsi que dans les matières premières, ont été caractérisées selon des méthodes par culture et par métabarcoding. Les champignons détectés sont similaires à ceux présents dans les habitations conventionnelles. L'ajout de fibres végétales ne modifie pas la structure des communautés fongiques mais rend le matériau plus sensible à la prolifération. Celle-ci n'intervient qu'en condition d'accident hydrique. Des approches de lutte biologique à partir de bactéries ont été initiées pour inhiber la prolifération fongique sur ces matériaux. / Impacts of building on environment and on health of inhabitants are nowadays priority issues. The interest for earthen materials is increasing for many reasons (ecological, economical, etc.), but some questions are raised about their fungal proliferation sensitivity considering the quality of indoor air. This work consists in characterizing by cultural and metabarcoding methods the fungal and bacterial communities on the surface of earthen building materials, biobased or not, and raw materials. Detected fungi are related to those which are identified in conventional buildings. The addition of vegetal fibers don't modify the fungal communities structure, but make the material more favorable for proliferation. The fungal development appears only under water damage condition. Biocontrol methods with bacteria have been initiated in order to inhibit the fungal proliferation on these materials.

The recent advent of High-Throughput Sequencing (HTS) methods has triggered a revolution in gene regulation studies. Demand has never been higher to process the immense amount of emerging data to gain insight into the regulatory mechanisms of the cell. We address this issue by describing methods to analyze, integrate and interpret HTS data from different sources. In particular, we developed and benchmarked Pyicos, a powerful toolkit that offers flexibility, versatility and efficient memory usage. We applied it to data from ChIP-Seq on progesterone receptor in breast cancer cells to gain insight into regulatory mechanisms of hormones. Moreover, we embedded Pyicos into a pipeline to integrate HTS data from different sources. In order to do so, we used data sets from ENCODE to systematically calculate signal changes between two cell lines. We thus created a model that accurately predicts the regulatory outcome of gene expression, based on epigenetic changes in a gene locus. Finally, we provide the processed data in a Biomart database to the scientific community. / La llegada reciente de nuevos métodos de High-Throughput Sequencing (HTS) ha provocado una revolución en el estudio de la regulación génica. La necesidad de procesar la inmensa cantidad de datos generados, con el objectivo de estudiar los mecanismos regulatorios en la celula, nunca ha sido mayor. En esta tesis abordamos este tema presentando métodos para analizar, integrar e interpretar datos HTS de diferentes fuentes. En particular, hemos desarollado Pyicos, un potente conjunto de herramientas que ofrece flexibilidad, versatilidad y un uso eficiente de la memoria. Lo hemos aplicado a datos de ChIP-Seq del receptor de progesterona en células de cáncer de mama con el fin de investigar los mecanismos de la regulación por hormonas. Además, hemos incorporado Pyicos en una pipeline para integrar los datos HTS de diferentes fuentes. Hemos usado los conjuntos de datos de ENCODE para calcular de forma sistemática los cambios de señal entre dos líneas celulares. De esta manera hemos logrado crear un modelo que predice con bastante precisión los cambios de la expresión génica, basándose en los cambios epigenéticos en el locus de un gen. Por último, hemos puesto los datos procesados a disposición de la comunidad científica en una base de datos Biomart.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose. / AFRIKAANSE OPSOMMING: Volgende-generasie tegnologie om basis volgordes van nukleiensure te bepaal, word al meer gebruik in metagenomiese studies. Dit is veral weens die hoe data-omset kapasiteit en onbevooroordeelde aanslag in die bepaling van die genetiese samestelling van onbekende omgewingsmonsters. Hierdie studie het die aanwending van die Illumina volgende-generasie volgorde-bepalingsplatform in 'n metagenomiese studie van wingerdvirusse, ondersoek. Dit het ten doel gehad om die eerste volledige virus profiel, of viroom, van 'n geinfekteerde wingerd saam te stel. Blaarmateriaal is verkry vanaf 44 lukraak-gekose wingerdstokke in 'n rolblad-geinfekteerde wingerd in Suid-Afrika. Monster materiaal is saamgevoeg en dubbelstring-RNS geekstraheer. Die dubbelstring-RNS is onderwerp aan gepaarde-ent volgorde-bepaling deur gebruik te maak van die Illumina volgorde-bepaling-deur-sintese tegniek. Meer as 19 miljoen volgorde reekse, ekwivalent aan ongeveer 837 megabasisse volgorde data, is verkry. Van hierdie data kon ongeveer 400 megabasisse saamgevoeg word in 449 konstrukte ("scaffolds"), deur gebruik te maak van die de novo samesteller Velvet. Hierdie konstrukte is onderwerp aan BLAST soektogte teen die NCBI databasisse en die hoogste trefslag-telling is gebruik vir virus identifikasie. Op grond van die "BLAST" resultate is geskikte volgordes geselekteer vanaf die NCBI databasis en gebruik as verwysingvolgordes in MAQ kartering-analises. Met die bioinfomatika analises kon die virus spesies teenwoordig, asook die mees prominente variante en relatiewe voorkoms van elk, bepaal word. Vier bekende virus wingerdpatogene is geidentifiseer. Grapevine leafroll-associated virus 3, verteenwoordig deur 59% van die geanaliseerde kort-reeks volgorde data, is identifiseer as die mees prominente virus spesie. Drie variante van die virus is in die wingerdmonster opgespoor: GP18 kom die mees algemeen voor, gevolg deur 'n CL-766/NY1 variant en 'n potensiele nuwe wingerd rolblad-geassosieerde ampelovirus. 'n Enkele Grapevine rupestris stem pitting-associated virus variant, soortgelyk aan SG1, en 'n Grapevine virus A variant, 'n lid van molekulere groep III, is geidentifiseer. Hierdie studie is ook die eerste om die teenwoordigheid van Grapevine virus E (GVE) in Suid-Afrikaanse wingerde te rapporteer. Grapevine virus E is verder geneties gekarakteriseer en die genoomvolgorde van GVE isolaat SA94 is bepaal. Die GVE SA94 genoomvolgorde, 7568 nukleotiede lank, is die eerste volledige genoomvolgorde vir hierdie virus spesie. Die genoomorganisasie is tipies van vitivirusse, maar in kontras met ander RNA virusse is die AlkB domein binne-in die helikase domein van oopleesraam 1 (ORF 1) geleë. Grapevine virus E SA94 deel byna 100% nukleotied identiteit met die Japannese TvP15 isolaat en GVE 3404, 'n de novo konstruk gegenereer vanaf die metagenomiese volgorde data. Bioinformatika analises van die metagenomiese volgorde data het verder die teenwoordigheid van drie swam-infekterende virus families, die Chrysoviridae, Totiviridae en ongeklassifiseerde dubbelstring-RNS virus, Fusarium graminearum dsRNA mycovirus 4, aangetoon. 'n Virus van die Chrysoviridae familie, soortgelyk aan Penicillium chrysogenum virus, het die tweede meeste voorgekom in die wingerd monster. Hierdie studie demonstreer die suksesvolle toepassing van 'n kort reeks volgorde-bepalingstegnologie soos die Illumina platform, vir die opstel van 'n virusprofiel van 'n geinfekteerde wingerd. Sover ons kennis strek is hierdie die eerste aanwending van die Illumina tegnologie vir hierdie doel.

Isogenic clonal fish lines are a powerful resource for aquaculture-related research. Fully inbred individuals, clone founders, can be produced either through mitotic gynogenesis or androgenesis and a further generation from those propagates fully inbred clonal lines. Despite rapid generation, as opposed to successive generation of sibling mating as in mice, the production of such lines may be hampered due to (i) potential residual contribution from irradiated gametes associated with poorly optimised protocols, (ii) reduced survival of clone founders and (iii) spontaneous arisal of meiotic gynogenetics with varying degree of heterozygosity, contaminating fully homozygous progenies. This research set out to address challenges and gain insights into isogenic clonal fish lines development by using double-digest RADseq (ddRADseq) to generate large numbers of genetic markers covering the genome of interest. Analysis of potential contribution from irradiated sperm indicated successful uniparental inheritance in meiotic and mitotic gynogenetics European seabass. Exclusive transmission of maternal alleles was detected in G1 progeny of Atlantic salmon (with a duplicated genome), while G2 progenies presented varying levels of sire contribution suggesting sub-optimal UV irradiation which was undetected previously with 27 microsatellite markers. Identification of telomeric markers in European seabass, with higher recombination frequencies for efficient differentiation of meiotic and mitotic gynogenetics was successful, and a genetic linkage map was generated from this data. One clear case of a spontaneous meiotic gynogenetic fish was detected among 18 putative DH fish in European seabass, despite earlier screening for isogenicity using 11 microsatellite markers. An unidentified larval DNA restriction digestion inhibition mechanism observed in Nile tilapia prevented the construction of SNP-based genetic linkage map. In summary, this study provides strong evidence on efficacy of NGS technologies for the development and verification of isogenic clonal fish lines. Reliable establishment of isogenic clonal fish lines is critical for their utility as a research tool.