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Mammoth phylogeography south of the ice: large-scale sequencing of degraded DNA from temperate depositsEnk, Jacob M. 04 1900 (has links)
<p>Mammoths (<em>Mammuthus</em>) have been studied extensively at the genetic level. However due to both taphonomic and technological limitations, only one of several late Pleistocene mammoth species, the woolly mammoth (<em>M. primigenius</em>), has been investigated. This limits our impression of mammoth population history to the the northern latitudes, just one of several environments in which mammoths lived and went extinct. It also obscures their evolutionary chronology, which prevents proper climatic and biogeographic contextualization of their history. Fortunately recent technological advances in high-throughput sequencing and targeted enrichment promise to expand Pleistocene faunal population phylogeography to non-permafrost, non-cave burial contexts. However the capacity and behavior of these combined technologies for characterizing ancient DNA is largely unexplored, preventing efficient and routine use for population-level studies. In this thesis I test and apply these technologies to remains of mammoth species from throughout North America. I first demonstrate their potential for poorly-preserved DNA, and then I evaluate their efficient application to large sample sets, as well as for capturing complete nuclear genomes. I then use these technologies to sequence dozens of mitochondrial genomes from Columbian (<em>M. columbi</em>)<em> </em>and other non-woolly mammoths, reconstructing their matrilineal phylogeography south of the ice. The revealed patterns not only imply a deep chronology for mammoth matrilineal diversity, but also that North American mammoth evolution was occurred via separate episodes of interbreeding between resident and invading populations, and between ecotypes. Overall the biological and methodological discoveries afforded by this body of work outline future research avenues on mammoth evolution, behavior, and extinction.</p> / Doctor of Philosophy (PhD)
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Caratterizzazione della diversità microbica in fave di cacao fermentate / CHARACTERIZATION OF MICROBIAL BIODIVERSITY IN FERMENTED COCOA BEANSBORTOLINI, CRISTIAN 31 May 2017 (has links)
La qualità delle fave di cacao disponibili in commercio, che rappresentano la principale materia prima per la produzione di cioccolato, dipende da diversi fattori inclusi: il tipo di piantagione, le pratiche agricole ed il processo di post raccolta. Tra queste; fermentazione ed essicazzione sono generalmente considerate le più rilevanti, dal momento in cui, durante queste fasi, vengono formati e fissati i precursori degli aromi del cacao. Inoltre, esse rappresentano un step cruciale durante il quale possono verificarsi contaminazioni da parte dei funghi filamentosi. La fermentazione è caratterizzata da una successione ben definita di lieviti, batteri lattici e batteri acetici, a tal fine, lo scopo del presente lavoro di tesi è stato quello di esplorare e descrivere in modo completo le comunità batteriche e fungine coinvolte nella fermentazione delle fave di cacao e valutare se l’origine geografica ed il metodo di fermentazione potessero influenzare la loro composizione. Per ottenere tali risultati il gene 16s rRNA è stato usato come marker per descrivere la comunità batterica totale mediante High Throughput Sequencing (HTS), dimostrando come tale approccio abbia la capacità di evidenziare la totalità delle comunità batteriche a livello di specie. In un secondo approccio l’Internal Transcribed Spacer 1 (ITS1) ed il dominio D1/D2 della sub unità maggiore dell’RNA ribosomiale (26s rRNA) sono stati selezionati per descrivere la popolazione fungina. I risultati hanno evidenziato come le due regioni abbiano la capacità di descrivere la composizione generale delle popolazioni, sebbene il dominio D1/D2 sia stato in grado di analizzare più nel dettaglio la composizione. Infine gli stessi campioni sottoposti all’analisi mediante HTS sono stati analizzati mediante SPME-GC-MS per evidenziare i principali composti aromatici formatisi durante il processo di post raccolta.
Complessivamente i risultati indicano chiaramente che l’approccio mediante HTS ha le potenzialità per fornire una dettagliata visione d’insieme delle comunità batteriche e fungine presenti durante le fasi di post raccolta delle fave di cacao, inoltre le analisi statistiche hanno evidenziato come l’ITS1 ed i composti volatili possano essere utilizzati per la tracciabilità geografica. / The quality of commercial cocoa beans, the principal raw material for chocolate production, depends on several factors including type of plantations, the agricultural practices and the post-harvest processing. Among these, fermentation and drying are generally considered the most relevant, since during these phases cocoa flavors precursors are formed and fixed. Furthermore, they represent crucial steps during which filamentous fungi contamination might occur. Fermentation is characterized by a well-defined succession of yeasts, lactic acid bacteria and acetic acid bacteria, so that, the aim of the described studies was to explore total bacterial and fungal communities involved in cocoa bean fermentation and to evaluate if geographical origin and fermentation method might affect their composition. To achieve these results, 16s rRNA gene was used as marker to assess the total bacterial community by using High Throughput Sequencing (HTS), indicating that this approach has the ability to provide a comprehensive view of the cocoa bean microbiota at the species level. In a second approach, Internal Transcribed Spacer 1 (ITS1) and the D1/D2 domain of the Large subunit (LSU) of the nuclear ribosomal RNA (26S rRNA) were screened to assess the total fungal community. Results revealed the ability of these two genomic regions to describe reliably the general composition, even if D1/D2domain was able to go deeper into the fungal composition resulting in a higher resolution. In the last approach the same samples subjected to HTS investigation were analyzed through SPME-GC-MS in order to underline the principal key-aroma compounds formed during the post-harvest processing.
Overall, results point out clearly that HTS approach has the ability to provide a comprehensive view of the total bacterial and fungal communities, and statistical analyses have shown how analyses of ITS1 sequences and volatile compounds might be useful for the geographical traceability of the processed cocoa beans samples.
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Etude de petits ARN régulateurs chez Helicobacter pylori / Search for small regulatory RNA in Helicobacter pyloriReignier, Jérémy 14 December 2010 (has links)
Ces dernières années de nombreuses recherches ont montré l’importance des petits ARN dans la régulation de l’expression des gènes, chez tous les organismes vivants, des bactéries aux mammifères. Le projet de cette thèse était de recherche et d’identifier des petits ARN chez une bactérie pathogène pour l’homme, Helicobacter pylori (Hp). Cette bactérie colonise exclusivement l’estomac humain, un organe qui pendant longtemps a été considéré comme étant stérile, en raison du pH parfois très acide qui y règne. L’infection persistante de l’estomac humain causée par Hp est associée avec plusieurs pathologies gastriques tels que les gastrites, les ulcères peptiques, les cancers gastriques et les lymphomes du MALT. La moitié de la population est infectée par Hp, qui est responsable d’environ 1 million de décès par an à travers le monde, et de 6000 nouveaux cas de cancers gastrique par an en France. Au cours de ma thèse, j’ai travaillé en étroite collaboration avec le groupe du Pr. Jörg Vogel (RNA Biology, MPI, Berlin, Allemagne) pour développer une méthode rapide et efficace d’analyse du transcriptome complet d’Hp, en s’appuyant sur une nouvelle sur une technologie émergente de pyroséquençage haut-débit (HTPS 454 technology, Life Science, USA). Notre méthode de séquençage du transcriptome d’Hp à partir de banques enrichies en transcrits primaires (dRNA-seq), nous a permis d’identifier les sites d’initiation de la transcription (TSS) de milliers de d’ARN. Plus de la moitié de ces TSS ont été associés à des petits ARN non codants, de courte taille (de 50 à 250 nucléotides en moyenne), qui n’avaient jamais été découverts jusqu’alors, et dont les gènes sont localisés dans des régions intergéniques (sRNA) ou en antisens (asRNA) par rapport aux ORF précédemment annotées dans le génome d’Hp. Nos travaux ont également permis de mettre en évidence une forte activité de transcription antisens sur l’ensemble du génome de la bactérie, un phénomène déjà observé chez E. coli et les eucaryotes. Ainsi, au moins un TSS est localisé sur le brin opposé à 46 % des ORF et à 28% des régions « leaders » des précurseurs des ARNr 23S et 16S, et des ARNt. Enfin, l’approche dRNA-seq a permis l’identification de la première famille de toxines de type I (AapA) identifiée à ce jour chez Hp. Dans ces conditions normales de culture, la traduction de ces toxines est constitutivement réprimée par des petits ARN antisens (IsoA) qui ciblent les ARNm aapA par complémentarité de base. Malgré leur homologie avec des modules toxine-antitoxine identifiés chez d’autres bactéries, pour certaines impliquées dans la réponse aux stress, nous n’avons pas encore découvert les conditions dans lesquelles ces peptides aapA seraient exprimées chez Hp, et leur rôle biologique reste à élucider. / In the past few years, small regulatory RNAs have emerged as an important class of post-transcriptional regulators of gene expression. Indeed they have been identified and/or predicted to exist in all species ranging from bacteria to mammals. The project of this thesis was to search for small non coding RNAs in a human pathogen: Helicobacter pylori (Hp). This bacterium exclusively colonizes the human stomach, an organ that until recently was thought to be sterile due to its extreme acidity. It is now established that persistent colonization by Hp is associated with various gastric pathologies including gastritis, peptic ulcer, gastric cancer and MALT lymphoma. Half of the human population is infected by Hp that is responsible for about 1 million deaths per year and around 6000 cases of gastric cancer in France. During my thesis we , in a close collaboration with the group of Joerg Vogel (RNA biology, MPI, Berlin, Germany) developed a rapid and efficient method to reveal the whole transcriptome of Hp based on recent advances in high-throughput pyrosequencing technologies (HTPS 454 technology, Life Science, USA). By using specifically enriched libraries in primary transcripts, our strategy allowed us to map thousand (1907) of transcription start sites (TSS) on the Hp genome. More than half of these TSS correspond to new short transcripts (non coding RNAs, between 50 and 250 nucleotides in length) that have never been annotated in this genome and that are localized both in intergenic regions (sRNA) and in regions antisense to annotated ORFs (asRNA). Analysis of associations between primary transcription start sites (pTSS) revealed more complexity in the Hp transcriptome than previously anticipated: around one third (27%) of pTSS belong to antisense transcripts (aTSS). The strikingly high degree of antisense transcription occurs, similar to E. coli and higher eukaryotes, across the entire Hp genome. Overall, at least one aTSS is linked to ~46% of all ORFs, ~28% of tRNAs, and the 5’ leaders of 23S and 16S rRNA precursors. Finally our dRNA-seq approach led us to identify the first family of putative type I toxins (AapA) in the Hp genome. Under normal growth conditions these toxins are constitutively repressed by a sophisticated antisense RNA-mediated (IsoA) mechanism. Despite their homology to other toxin-antitoxin modules previously described in other bacteria, we have not found physiological conditions under which these peptides are expressed and have yet to determine the biological significance (if any ?) of these suicide genes.
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High-Throughput Data Analysis: Application to Micronuclei Frequency and T-cell Receptor SequencingMakowski, Mateusz 01 January 2015 (has links)
The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered.
ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest.
The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.
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Analyse bioinformatique du contrôle des éléments transposables par les siARN chez Arabidopsis thaliana / Bioinformatic analysis of siRNA control on transposable elements in Arabidopsis thalianaSarazin, Alexis 23 October 2012 (has links)
De nombreux mécanismes contrôlent et limitent la prolifération des éléments transposables (ET) dans les génomes dont ils menacent l'intégrité structurale et fonctionnelle. Chez les plantes l'interférence ARN (ARNi) joue un rôle important dans ces contrôles via des petits ARN d'environ 20nt qui guident la régulation de l'expression de séquences endogènes ou exogènes par deux types de mécanismes. Un premier mécanisme, partagé par de nombreux organismes eucaryotes, inhibe l'activité d'ARNm par un contrôle post-transcriptionnel. Un deuxième type de régulation, permet un contrôle transcriptionnel de l'activité des ET via un mécanisme appelé RNA directed DNA Methylation (RdDM) qui implique des siARN (« short-interfering RNA ») de 24nt qui guident la méthylation de l'ADN spécifiquement au niveau des séquences d'ET. Les siARN sont impliqués également dans la restauration progressive de la méthylation de l'ADN après une perte induite par la mutation du gène DDM1 (Decrease in DNA Methylation 1). L'objectif de cette thèse est de tirer avantage des technologies de séquençage à haut débit pour caractériser le contrôle des ET par les siARN chez la plante modèle Arabidopsis thaliana.Dans un premier temps, j'ai développé des méthodes et outils bioinformatiques afin de gérer efficacement les données de séquençage à haut débit de banques de petit ARN. Ces outils, regroupés en pipeline, visent à permettre l'étude de l'accumulation des siARN correspondant aux séquences d'ET ou de familles d'ET ainsi que leur visualisation de manière globale ou détaillée.Ces outils ont ensuite été appliqués pour caractériser, dans un contexte sauvage, l'association entre les siARN et les ET afin de déterminer des facteurs pouvant expliquer les différences d'abondance en siARN observées. Ces analyses, réalisées en tenant compte de l'état de méthylation de l'ADN et du contexte génomique des ET apportent une vue statique du contrôle des ET par les siARN et de leur impact sur les gènes situés à proximité.L'analyse de banques de petits ARN de mutants de la voie de l'ARNi a ensuite été réalisée afin mieux caractériser l'impact de la perte de méthylation de l'ADN sur les populations de siARN et notamment définir les mécanismes impliqués dans la production des siARN de 21nt induite dans le mutant ddm1. Ces analyses comparatives du contrôle des ET lors d'une perte de la méthylation de l'ADN ont permis de mettre en évidence une production de siARN de 24nt indépendante de la voie classique du RdDM et de proposer un modèle permettant d'expliquer la production de siARN de 21nt dans le mutant ddm1.Dans un dernier temps, j'ai cherché à mieux définir l'implication des siARN dans la restauration des états de méthylation de l'ADN. Les variations de méthylation de l'ADN induites par la mutation ddm1 ont été caractérisées ainsi que leur stabilité transgénérationnelle au sein d'une population d'epiRIL. La stabilité de l'hypométhylation de l'ADN a été étudiée, au regard de données de séquençage à haut débit de banques de petits ARN de lignées WT, ddm1 ainsi que pour 4 lignées epiRIL, afin d'apporter une notion temporelle à l'étude du contrôle des ET par les siARN.Les résultats soulignent le rôle majeur des petits ARN pour le contrôle des éléments transposables afin de préserver l'intégrité structurale et fonctionnelle du génome et ce, via des mécanismes variés en fonction des ET. Ce travail ouvre la voie vers une analyse du contrôle des ET par les siARN basée sur une approche regroupant les ET en réseaux en fonction des séquences de siARN qu'ils partagent. Cela permettrait d'étudier les « connections-siARN » entre ET afin de, par exemple, explorer l'action en trans des siARN pour la restauration de la méthylation de l'ADN. / Many mechanisms control and limit the proliferation of transposable elements (TEs) which could otherwise threaten the structural and functional integrity of the genome. In plants RNA interference (RNAi) plays an important role in this control through small RNAs that guide the expression regulation of endogenous or exogenous sequences by two types of mechanisms. The first such mechanism, shared by many eukaryotic organisms, acts at the post-transcriptionnal level to inhibit the activity of mRNA. A second type of regulation allows the transcriptional control of TEs activity through a mechanism called RNA directed DNA methylation (RdDM) which involves 24nt long siRNA ("short-interfering RNA") that guide DNA methylation specifically on TEs sequences. Furthermore, siRNAs are also involved in the progressive restoration of DNA methylation after a loss induced by mutation of the DDM1 gene (Decrease in DNA Methylation 1). The aim of this thesis is to take advantage of high-throughput sequencing technologies to characterize these TEs controls mechanisms by siRNA in the model plant Arabidopsis thaliana .At first, I developed methods and bioinformatics tools to effectively manage data produced by high-throughput sequencing of small RNA libraries. These tools, combined in a pipeline, are designed to allow the study the accumulation of siRNA corresponding to TE sequences or TE families as well as their global or detailed visualization.These tools were applied to characterize, in a wild type background, the association between siRNA and TEs in order to define factors that may explain the observed differences in siRNA abundance . These analyses were performed by taking into account both DNA methylation states and genomic context. It provides a static view of siRNA control of TEs and their impact on nearby genes. Then, analysis of small RNA libraries from mutants of the RNAi pathway was performed to better characterize the impact of DNA methylation loss on siRNA populations and to define the mechanisms involved in the production of 21nt siRNA induced in the ddm1 mutant. These comparative analyses of the TE control after loss of DNA methylation allow us to highlight the production of 24nt siRNA independently of the classical RdDM pathway and to propose a model explaining the production of 21nt siRNA in the ddm1 mutant. At last, I tried to clarify the involvement of siRNA in the restoration of DNA methylation. Changes in DNA methylation induced by ddm1 mutation were characterized as well as their transgenerational stability in an epiRIL population. The stability of DNA hypomethylation has been studied in relation to high-throughput sequencing of small RNAs data from WT, ddm1 and 4 epiRIL lines. It provides a temporal view of the TE control by siRNA. The results highlight the important role of small RNAs in the control of transposable elements in order to preserve structural and functional integrity of the genome through a variety of mechanisms depending on TE sequences. This work opens the way to the analysis of the siRNA control on TEs based on approaches that combine TEs in networks based on their shared siRNA sequences. It would allow to study "siRNA-connections" between TEs in order to explore, for example, the action in trans of siRNA in the restoration of DNA methylation defect.
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Ecologie des micro-organismes producteurs d'hydrogène des sources hydrothermales alcalines associées à la serpentinisation en Baie de Prony, Nouvelle-Calédonie / Ecology of hydrogen-producing microorganisms in alkaline hydrothermal springs from the serpentinization-hosted system of the Prony Bay, New CaledoniaMei, Nan 30 September 2016 (has links)
Le système hydrothermal de la baie de Prony en Nouvelle-Calédonie est composée de plusieurs sources émettant à faible profondeur des fluides hyperalcalins, mesothermiques, et anoxiques riches en hydrogène (H2) et méthane, produits par la réaction de serpentinisation. Dans le cadre de ces travaux de thèse, la capacité potentielle des microorganismes à produire de l’H2 dans ces écosystèmes a été évaluée par des analyses moléculaires et culturales. Nos premières analyses moléculaires ont mis en évidence une grande diversité de bactéries et une faible diversité d’archées. La diversité et la distribution des populations productrices d’H2 a ensuite été spécifiquement évaluée par des analyses métagénomiques et basées sur la PCR. Les séquences de gènes hydA, utilisés comme marqueur moléculaire des bactéries productrices d’H2, étaient principalement associées à celles du phylum des Firmicutes. Deux groupes de séquences hydA se sont distinguées en fonction de l’origine des échantillons. De plus, plusieurs nouvelles bactéries alcalophiles ont été isolées de différents sites. Parmi elles, la souche alcalophile et anaérobie, PROH2 est capable de produire efficacement de l’H2 par voie fermentaire en condition alcaline (pH 9,5), de façon comparable aux espèces de Clostridium neutrophiles. Ces travaux présentent également la caractérisation d’une nouvelle espèce bactérienne anaérobie, mésophile et alcalophile (pH optimum de 9,5) d’un nouveau genre, nommée Serpentinicella alkaliphila, isolée d’un site intertidal de la baie de Prony. L’ensemble des données démontre la capacité des Firmicutes des environnements associés à la serpentinisation à produire de l’H2 par voie fermentaire. / The Prony hydrothermal field (PHF, New Caledonia) is composed of several shallow-submarine springs discharging into the lagoon seawater high pH, moderate temperature, low-salinity fluids, enriched in hydrogen (H2) and methane produced by serpentinization. In this work, we evaluated the potential ability of microorganisms to produce H2 in this alkaline ecosystem by using both molecular and cultural approaches. Our first molecular analyses provided evidence of high bacterial abundance and diversity contrasting with low archaeal diversity in the PHF chimneys. The diversity and distribution of potential H2-producing bacteria were specifically investigated by using metagenomic analyses and different PCR-sequencing methods. The sequences of hydA genes encoding the catalytic subunit of [FeFe]-hydrogenases, used as molecular marker of H2-producing bacteria, were mainly related to those of Firmicutes. Two groups of hydA sequences were distinguished according to the origin of the samples. Moreover, novel alkaliphilic H2-producing Firmicutes were successfully cultivated from PHF chimneys. Among them, an alkaliphilic and anaerobic strain, Clostridium sp. PROH2, belonging to the genus Clostridium, demonstrated efficient H2 production at a high pH, comparable to neutrophilic clostridial species. This manuscript also present the characterization of a novel anaerobic, mesophilic and alkaliphilic species belonging to a new genus, named Serpentiniticella alkaliphila 3bT, isolated from an intertidal PHF site. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce H2 by fermentation.
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Genotipagem de bactérias anaeróbias associadas às lesões da periodontite bovina / Genotyping of anaerobic bacteria isolated from bovine periodontitis lesionsBorsanelli, Ana Carolina [UNESP] 07 February 2017 (has links)
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Previous issue date: 2017-02-07 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A periodontite bovina é um processo infeccioso purulento e progressivo associado com a presença de biofilme subgengival anaeróbio. De caráter sazonal e associada ao manejo do solo e à dieta, a doença tem variações na sua apresentação clínica, que inclui desde uma forma agressiva até manifestações crônicas. Os prejuízos econômicos são significativos e decorrem das dificuldades na preensão, mastigação e ruminação. O presente estudo teve como objetivo geral caracterizar a periodontite bovina e identificar por métodos moleculares os micro-organismos associados às lesões periodontais. Na avaliação da microbiota da bolsa periodontal (n=26) e do sulco subgengival (n=25) de bovinos, pela reação em cadeia da polimerase (PCR) e com o emprego de 35 iniciadores de espécies de patógenos potenciais, pôde-se associar a ocorrência de Actinobacillus naeslundii, Enterococcus faecium, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Treponema denticola e Treponema pectinovorum com a periodontite bovina. Em estudo complementar realizado na Escócia para verificar a ocorrência de lesões periodontais em animais abatidos, foram examinadas 200 arcadas dentárias, das quais 24 (12%) apresentaram lesões periodontais nos dentes incisivos ou mastigatórios. Este estudo inédito revela que a periodontite não é incomum em bovinos abatidos no oeste da Escócia e é claramente um problema sanitário negligenciado na produção e bem-estar animal. Na oportunidade, os fatores de risco associados à doença foram avaliados em um universo de 250 animais abatidos, dos quais 35 apresentavam lesões periodontais e 40 eram periodontalmente sadios. Pela análise de regressão logística foi avaliada a associação entre as variáveis independentes, sexo, idade e raça com periodontite. A idade dos animais foi significativamente associada à presença de lesões periodontais. Para cada ano de idade, um bovino tem 1,53 vezes chances de desenvolver periodontite (p<0,001). A variável sexo não se mostrou significativamente associada à periodontite (p=0,887), enquanto os animais de corte têm 0,36 a chance de desenvolver a doença quando comparados com os de aptidão leiteira. Na mesma ocasião, amostras de biofilme subgengival de 40 bovinos com periodontite e de 38 periodontalmente sadios foram coletadas e realizou-se o sequenciamento do gene 16S rRNA. No microbioma de animais sadios os gêneros mais prevalentes foram Gastranaerophilus, Planifilus, Burkholderia e Arcobacter. Já nos animais com periodontite, os filos mais prevalentes foram Elusimicrobia, Synergista e Fusobacteria e os gêneros Propionivibrio, Wolinella, Porphyromonas, Candidatus, Prevotella, Firmicutes (não cultivável), Bacteroides e Treponema. Em conclusão, os dois grupos de bovinos avaliados abrigaram perfis microbianos distintos, sendo que as amostras de bovinos com periodontite foram mais diversas em micro-organismos do que as de bovinos sadios. Nesse contexto inédito na microbiologia oral de bovinos, pode-se constatar os componentes principais na homeostase bacteriana do biofilme de sítios sadios e a disbiose nas lesões periodontais, fornecendo indicadores para e evolução do conhecimento sobre a periodontite bovina. / Bovine periodontitis is a progressive purulent infectious process associated with the presence of strict anaerobic subgingival biofilm. Seasonal and associated to soil and dietary management, the disease has variations in its clinical presentation, which includes since an aggressive form until chronic manifestations. The economic losses are significant and result from difficulties in gripping, chewing and rumination. The present study aimed to identify and characterize bovine periodontitis and identify the microorganisms associated with periodontal lesions by molecular methods. In the evaluation of the microbiota of the periodontal pocket (n=26) and gingival sulcus (n=25) of cattle, by polymerase chain reaction (PCR) and with the use of 35 primers of species of potential pathogens, it can be associate the occurrence of Actinobacillus naeslundii, Enterococcus faecium, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Treponema denticola and Treponema pectinovorum with bovine periodontitis. In a study carried out in Scotland to verify the occurrence of periodontal lesions in slaughtered animals, 200 dental arches were examined, of which 24 (12%) presented periodontal lesions in the incisors or masticatory teeth. This unpublished study reveals that periodontitis is not uncommon in cattle slaughtered in West of Scotland and is clearly a neglected health problem in animal production and welfare. At the opportunity, the risk factors associated with the disease were evaluated in a universe of 250 slaughtered animals, of which 35 had periodontal lesions and 40 were periodontally healthy. By the logistic regression analysis was evaluated the association between the independent variables, sex, age and race with periodontitis. The age of the animals was significantly associated with the presence of periodontal lesions. For each extra year in age, a cow is 1.53 times more likely to develop periodontitis (p<0.001). Gender was not significantly associated with periodontitis (p=0.887). Regarding the variable breed type, beef cattle were 0.36 times as likely to have periodontitis compared to dairy cattle. At the same occasion, samples of subgengival biofilm of 40 bovines with periodontitis and 38 periodontally healthy were submitted to high- throughput sequencing. In the bovine microbiome the most discriminative taxa in the samples of healthy animals were Gastranaerophilus, Planifilus, Burkholderia and Arcobacter. In animals with periodontitis, the most prevalent microorganisms were Elusimicrobia, Synergista, Propionivibrio, Fusobacteria, Wolinella, Porphyromonas, Candidatus, Prevotella, Firmicutes (uncultivable), Bacteroides and Treponema. In conclusion, the two groups of bovines evaluated harboured distinct microbial profiles, and the samples of bovines with periodontitis were more diverse in microorganisms than those of healthy cattle. In this unprecedented context, in the oral microbiology of bovines we can verify the main components in the bacterial homeostasis of the biofilm of healthy sites and the dysbiosis in the periodontal lesions, providing indicators for and evolution of the knowledge on bovine periodontitis. / FAPESP: 2015/06917-9 / FAPESP: 2013/13701-7
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Dynamique de la circulation des Entérovirus de l'homme à l'environnement : Etude par séquençage haut débit / Dynamic of enterovirus circulation from humans to environment : A study by high throughput sequencingBisseux, Maxime 21 November 2017 (has links)
Les entérovirus (EV) sont des Picornavirus (virus nus à génome ARN positif), caractérisés par une grande diversité génétique et antigénique (116 types classés en 4 espèces taxonomiques EV-A à D) et une évolution rapide. Les infections humaines sont très fréquentes, hautement contagieuses à partir des selles et épidémiques. La plupart des infections sont asymptomatiques ou bénignes ; elles peuvent être graves voire mortelles, en particulier chez les jeunes enfants. La poliomyélite, modèle d’infection à EV, est en voie d’éradication grâce aux programmes de vaccination et de surveillance sous l’égide de l’OMS. La détection de poliovirus sauvages dans des pays déclarés exempts de polio depuis plusieurs années et l’émergence récente de plusieurs EV non poliomyélitiques (EV-A71, EV-D68) associés à des manifestations cliniques sévères dans plusieurs régions du monde montrent l’importance de surveiller la circulation des EV dans la population humaine. Le but de la thèse était de rechercher et caractériser les EV dans les eaux usées de l’agglomération de Clermont-Ferrand et de comparer les données à celles de la surveillance clinique pour avoir une image plus complète de la circulation virale dans la population générale. Une méthode de concentration virale à partir des eaux usées prélevées en entrée (eaux usées brutes) et sortie (eaux usées traitées) de station d’épuration a été mise au point, permettant la détection moléculaire des EV et de 6 autres virus entériques humains. La présence de génomes viraux a été détectée dans tous les échantillons d’octobre 2014 à octobre 2015, avec une médiane de 6 virus différents en entrée de station et de 4 virus en sortie. L’analyse phylogénétique des séquences d’EV et des virus des hépatites A et E présents dans les eaux usées et les prélèvements cliniques des patients hospitalisés au CHU de Clermont-Ferrand pendant la même période, a validé l’approche mise en place pour surveiller la circulation communautaire d’un virus entérique. La diversité des EV présents dans les eaux usées brutes a été analysée par séquençage d’amplicons avec une technique haut débit Illumina (metabarcoding). Les résultats montrent la présence d’une grande diversité d’EV et la circulation silencieuse de 25 types (notamment 9 EV-C, dont des séquences de poliovirus 1 vaccinal) dans la population générale. L’analyse phylogénétique des variants intra-typiques a mis en évidence plusieurs profils épidémiques parmi les principaux types ayant circulé pendant la période d’étude. Les données obtenues montrent la faisabilité et la sensibilité de la stratégie développée pour détecter et caractériser les EV présents dans les eaux usées. Ils permettent de discuter la place de la surveillance environnementale dans la surveillance des infections à EV non polio (études épidémiologiques, prévention des épidémies, alertes sanitaires). Surveiller conjointement les virus entériques dans l’environnement et chez les patients permet une meilleure compréhension de leur prévalence. Cette approche globale de la circulation virale et de l’écologie de la santé représente un engagement important de la part des laboratoires et nécessitera une intégration dans des réseaux structurés de collaboration nationales et internationales dépassant la seule surveillance des EV. / Enterovirus (EV) are Picornaviruses (non-enveloped, positive-sense RNA viruses), characterized by a large genetic and antigenic diversity (116 types classified within 4 taxonomic species EV-A to D) and rapid evolution. Human infections are frequent, highly contagious from stools and occur as outbreaks. The infections are mainly asymptomatic or benign but severe or fatal cases can be reported in young children. Poliomyelitis is the model EV infection. Combined with clinical and virological surveillance, mass vaccination is closer than ever to achieve the WHO program of the Global Polio Eradication Initiative. However, the detection of wild type polioviruses in polio-free countries and the recent worldwide emergence of non-polio enteroviruses (EV-A71, EV-D68) associated with severe clinical manifestations underscore the importance of surveilling EV circulation in the general population. The aim of the PhD thesis was the detection and identification of EV strains in wastewater treated in the sewage treatment plant at Clermont-Ferrand (France). The viral data were compared with those reported through clinical surveillance to obtain a comprehensive picture of the viral circulation in the local population. A method was developed to concentrate viruses from raw and treated wastewater and molecular assays were used to detect EVs and 6 other human enteric viruses. The viral genomes were detected in all samples from October 2014 to October 2015, with a median of 6 and 4 different viruses in raw and treated wastewater respectively. Phylogenetic analysis of viral sequences (EV, hepatitis A and E viruses) determined in wastewater and reported in patients during the sampling period, showed the efficiency of the method for surveilling enteric viruses in the community. The EV diversity in raw wastewater was analyzed by sequencing of amplicons with the Illumina high throughput technology (metabarcoding). The analysis revealed a large viral diversity and the silent circulation of 25 types not detected from hospital data (in particular 9 EV-C, of which sequences of vaccine poliovirus 1). The phylogenetic analyses of intra-typic variants showed different epidemic patterns in the predominant EV types circulating over the study period. The data demonstrate the feasibility and sensitivity of the strategy developed for the detection and characterization of EV in wastewater and provide a future prospect for the implementation of environmental surveillance of non-polio EV infections in epidemiological studies, epidemic prevention, and for health alert. Combining the surveillance of enteric viruses in the environment and in the clinical setting allows a better understanding of their prevalence. This global approach of virus circulation and ecological health represents an important investment for laboratories, which will require integration in national and international collaboration networks beyond the scope of enterovirus surveillance.
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Genome-wide Analysis of Ctcf-RNA InteractionsKung, Johnny Tsun-Yi January 2014 (has links)
Ctcf is a "master regulator" of the genome that plays a role in a variety of gene regulatory functions as well as in genome architecture. Evidence from studying the epigenetic process of X-chromosome inactivation suggests that, in certain cases, Ctcf might carry out its functions through interacting with RNA. Using mouse embryonic stem (ES) cells and a modified protocol for UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), Ctcf is found to interact with a multitude of transcripts genome-wide, both protein-coding mRNA (or noncoding transcripts therein) as well as many long-noncoding RNA (lncRNA). Examples of the latter include both well-characterized species from imprinted loci and previously unannotated transcripts from intergenic space. RNA binding targets of Ctcf are validated by a variety of biochemical methods, and Ctcf is found to interact with RNA through its C-terminal domain, distinct from its DNA-binding zinc-finger domain. Ctcf chromatin immunoprecipitation (ChIP)-seq done in parallel reveals distinct but correlated binding of Ctcf to DNA and RNA. In addition, allelic analysis of Ctcf ChIP pattern reveals significant differences between Ctcf binding to the presumptive inactive and active X chromosomes. Together, the current work reveals a further layer of complexity to Ctcf biology by implicating a role for Ctcf-RNA interactions in its recruitment to genomic binding sites.
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Detection and characterization of gene-fusions in breast and ovarian cancer using high-throughput sequencingMittal, Vinay K. 21 September 2015 (has links)
Gene-fusions are a prevalent class of genetic variants that are often employed as cancer biomarkers and therapeutic targets. In recent years, high-throughput sequencing of the cellular genome and transcriptome have emerged as a promising approach for the investigation of gene-fusions at the DNA and RNA level. Although, large volumes of sequencing data and complexity of gene-fusion structures presents unique computational challenges. This dissertation describes research that first addresses the bioinformatics challenges associated with the analysis of the massive volumes of sequencing data by developing bioinformatics pipeline and more applied integrated computational workflows. Application of high-throughput sequencing and the proposed bioinformatics approaches for the breast and ovarian cancer study reveals unexpected complex structures of gene-fusions and their functional significance in the onset and progression of cancer. Integrative analysis of gene-fusions at DNA and RNA level shows the key importance of the regulation of gene-fusion at the transcription level in cancer.
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