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Liu, Oscar H. "RNAseq Analysis of Gastric Bacteria in Helicobacter pylori-Associated Carcinogenesis." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-9937.
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Helicobacter pylori infects more than half of the world's population, and is known to be involved in several diseases including gastric cancer. Its close interactions with the stomach and host immune system serves as a good model to study the co-adaptation and co-evolution of the organisms in the stomach micro-environment. In this project, we utilized RNA-seq and data analysis tools to investigate differentially expressed genes by H. pylori in patients at different stages of early gastric cancer development. We also investigated the abundance and diversity of bacterial genera other than H. pylori, and looked for correlations with H. pylori presence and number. For differential gene expression of H. pylori, one gene was differentially expressed between samples of corpus atrophy without metaplasia vs. samples of antrum gastritis, and eight genes were found to be differentially expressed between samples of corpus atrophy with metaplasia vs. samples with pan-gastritis. When samples were clustered into different groups based on the expression data, 52 genes (shared or unique to the specific comparison groups) were found to be differentially expressed, but no apparent patterns were observed that could be explained by medical or sample collection data. For bacterial diversity and abundances, we found several genera colonizing the stomach, of which some have been previously identified. While most of these bacteria colonize regardless of the presence of H. pylori, the abundance of three genera, Wolinella, Campylobacter, and Veillonella, seem to be correlated with the presence of H. pylori.
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Simon, Svenja [Verfasser]. "Visual Analysis of RNAseq Data : Discovering Genes in Bacteria / Svenja Simon." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1114886580/34.
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Aksamit, Matthew Stephen. "Bioinformatic analysis of pea aphid salivary gland transcripts." Thesis, Kansas State University, 2014. http://hdl.handle.net/2097/32836.
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Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental Program
Gerald Reeck
Pea aphids (Acyrthosiphon pisum) are sap-sucking insects that feed on the phloem sap of some plants of the family Fabaceae (legumes). Aphids feed on host plants by inserting their stylets between plant cells to feed from phloem sap in sieve elements. Their feeding is of major agronomical importance, as aphids cause hundreds of millions of dollars in crop damage worldwide, annually. Salivary gland transcripts from plant-fed and diet-fed pea aphids were studied by RNASeq to analyze their expression. Most transcripts had higher expression in plant-fed pea aphids, likely due to the need for saliva protein in the aphid/plant interaction. Numerous salivary gland transcripts and saliva proteins have been identified in aphids, including a glutathione peroxidase. Glutathione peroxidases are a group of enzymes with the purpose of protecting organisms from oxidative damage. Here, I present a bioinformatic analysis of pea aphid expressed sequence tag libraries that identified four unique glutathione peroxidases in pea aphids. One glutathione peroxidase, ApGPx1 has high expression in the pea aphid salivary gland. Two glutathione peroxidase genes are present in the current annotation of the pea aphid genome. My work indicates that the two genes need to be revised.
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Ahmed, Firdous. "Identification of potential biomarkers in lung cancer as possible diagnostic agents using bioinformatics and molecular approaches." University of the Western Cape, 2015. http://hdl.handle.net/11394/4862.
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>Magister Scientiae - MScLung cancer remains the leading cause of cancer deaths worldwide, with the majority of cases attributed to non-small cell lung carcinomas. At the time of diagnosis, a large percentage of patients present with advanced stage of disease, ultimately resulting in a poor prognosis. The identification circulatory markers, overexpressed by the tumour tissue, could facilitate the discovery of an early, specific, non-invasive diagnostic tool as well as improving prognosis and treatment protocols. The aim was to analyse gene expression data from both microarray and RNA sequencing platforms, using bioinformatics and statistical analysis tools. Enrichment analysis sought to identify genes, which were differentially expressed (p < 0.05, FC > 2) and had the potential to be secreted into the extracellular circulation, by using Gene Ontology terms of the Cellular Component. Results identified 1 657 statically significant genes between normal and early lung cancer tissue, with only 1 gene differentially expressed (DE) between the early and late stage disease. Following statistical analysis, 171 DE genes selected as potential early stage biomarkers. The overall sensitivity of RNAseq, in comparison to arrays enabled the identification of 57 potential serum markers. These genes of interest were all downregulated in the tumour tissue, and while they did not facilitate the discovery of an ideal diagnostic marker based on the set criteria in this study, their roles in disease initiation and progression require further analysis.
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Sadacca, Benjamin. "Pharmacogenomic and High-Throughput Data Analysis to Overcome Triple Negative Breast Cancers Drug Resistance." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS538/document.
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Devant le grand nombre de tumeurs du sein triple négatif résistant aux traitements, il est essentiel de comprendre les mécanismes de résistance et de trouver de nouvelles molécules efficaces. En premier lieu, nous analysons deux ensembles de données pharmacogénomiques à grande échelle. Nous proposons une nouvelle classification basée sur des profils transcriptomiques de lignées cellulaires, selon un processus de sélection de gènes basé sur des réseaux biologiques. Notre classification moléculaire montre une plus grande homogénéité dans la réponse aux médicaments que lorsque l’on regroupe les lignées cellulaires en fonction de leur tissu d'origine. Elle permet également d’identifier des profils similaires de réponse aux traitements. Dans un second travail, nous étudions une cohorte de patients atteints d’un cancer du sein triple négatif ayant résisté à la chimiothérapie néoadjuvante. Nous effectuons des analyses moléculaires complètes basées sur du RNAseq et WES. Nous constatons une forte hétérogénéité moléculaire des tumeurs avant et après traitement. Bien que nous observons une évolution clonale sous traitement, aucun mécanisme récurrent de résistance n’a pu être identifié. Nos résultats suggèrent fortement que chaque tumeur a un profil moléculaire unique et qu'il est important d'étudier de grandes séries de tumeurs. Enfin, nous améliorons une méthode pour tester la surreprésentation de motifs connus de protéines de liaison à l'ARN, dans un ensemble donné de séquences régulées. Cet outil utilise une approche innovante pour contrôler la proportion de faux positifs qui n'est pas réalisé par l'algorithme existant. Nous montrons l'efficacité de notre approche en utilisant deux séries de données différentesGiven the large number of treatment-resistant triple-negative breast cancers, it is essential to understand the mechanisms of resistance and to find new effective molecules. First, we analyze two large-scale pharmacogenomic datasets. We propose a novel classification based on transcriptomic profiles of cell lines, according to a biological network-driven gene selection process. Our molecular classification shows greater homogeneity in drug response than when cell lines are grouped according to their original tissue. It also helps identify similar patterns of treatment response. In a second analysis, we study a cohort of patients with triple-negative breast cancer who have resisted to neoadjuvant chemotherapy. We perform complete molecular analyzes based on RNAseq and WES. We observe a high molecular heterogeneity of tumors before and after treatment. Although we highlighted clonal evolution under treatment, no recurrent mechanism of resistance could be identified Our results strongly suggest that each tumor has a unique molecular profile and that that it is increasingly important to have large series of tumors. Finally, we are improving a method for testing the overrepresentation of known RNA binding protein motifs in a given set of regulated sequences. This tool uses an innovative approach to control the proportion of false positives that is not realized by the existing algorithm. We show the effectiveness of our approach using two different datasets
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Grosse-Holz, Friederike. "Proteases and inhibitors in the interaction between Nicotiana benthamiana and Agrobacterium tumefaciens : systematic analysis and emerging solutions for molecular farming." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6146918c-3749-4604-88fa-01d426e4a817.
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Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.
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DAS, VIVEK. "LEVERAGING TRANSCRIPTOMIC ANALYSIS TO IDENTIFY TRANSCRIPTION FACTORS ORCHESTRATING CANCER PROGRESSION." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/559711.
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Next generation sequencing (NGS) technology is currently employed to explore the molecular profiles associated to different biological contexts. The application of this technology provides at same time a high-resolution and global view of the genome and epigenome phenomena, enabling us to study the molecular events underlying many human diseases, including cancer. Our lab tries to exploit the utility of high throughput sequencing technologies generating genomic, transcriptomic and epigenomic data from patient's cohort to study the underlying molecular mechanisms that characterize the specific diseases and map the key regulators that can be critical targets for relevant therapeutic measures. I take the advantage of this technology to mainly understand two aggressive cancers: Ovarian Cancer (OC) and Glioblastoma multiforme (GBM).
OC is a leading cause of cancer-related death for which no significant therapeutic progress has been made in the last decades. Also, in this case, despite multimodal treatment its prognosis remains extremely poor. This is due to the fact that the molecular mechanisms underlying OC tumorigenesis and progression are still poorly understood (Vaughan et al., 2011). GBM is the most common and aggressive primary brain malignancy with very poor prognosis (Frattini et al., 2013). The median survival rate is of 12-15 months (Singh et al., 2012) with 5-year survival that is less than 5% despite the multimodal treatment which include surgery, radiotherapy and chemotherapy. To this end, I will be integrating various genomic and transcriptomic analysis to define the key regulatory actors that characterize the disease progression paving. This integrated analysis has been devised in form of a computational workflow that gives way for a discovery pipeline for physiopathologically meaningful epigenetic targets that can lead to therapies.
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Couto, Joana Manuel Gonçalves Teixeira. "Transcriptomic analysis of Anopheles Stephensi salivary glands during the infection with Plasmodium Berghei." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/14639.
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Mestrado em Biologia Molecular e CelularMalaria remains the leading cause of morbidity and mortality in tropical and subtropical regions, contributing to the emergence of 198 million clinical cases in 2013. The mosquito Anopheles stephensi is one of the most prevalent malaria vectors in the Asian region having recently been implicated in malaria resurgence in Djibouti. Using techniques as RNA sequencing, differentially expressed genes in the salivary glands of the mosquito in response to infection by Plasmodium berghei were identified. Some of these genes can be selected to evaluate their potential as targets for malaria transmission blocking. Among the genes with differentially expression resulting from the analysis of RNA-seq results and confirmation by qPCR, a gene related transport of Cl- and HCO3 2-, prestin, was upregulated after infection with P. berghei. This gene plays a crucial role in parasite invasion in the midgut and the optimization of the environment in which the parasite develops. For this reason, the silencing of this transcript was made to evaluate the function of prestin in salivary glands. The gene silencing, using RNA interference technique, allow inferring about the role or function of prestin gene in a particular metabolic or physiological process. After prestin gene silencing, the number of viable mosquitoes had a significant decrease in comparison with the control (β2M). There was also a significant decrease in the number of mosquitoes before injection and at the last day after injection. The number of sporozoites were not generally affected by silencing of prestin when compared with the control. To clarify other results obtained during the study, as the influence of the silencing of prestin in the survival of mosquitoes and the presence and number of sporozoites in the salivary glands, will be essential to perform qPCR to determine differential expression of this gene after silencing. Furthermore, it is also important to examine differential expression of off-target (ASTE006714) after silencing prestin, since the sequence of this gene have a high percentage of identity with prestin.
A malária continua a ser a principal causa de morbilidade e mortalidade nas regiões tropicais e subtropicais, contribuindo para o surgimento de 198 milhões de casos clínicos no ano de 2013. O mosquito Anopheles stephensi é um dos vectores de malária mais prevalentes na região asiática, tendo sido recentemente implicado no ressurgimento de malária em Djibouti. Através de técnicas como sequenciação de RNA, genes diferenciadamente expressos nas glândulas salivares deste mosquito em resposta à infecção por Plasmodium berghei foram identificados. Alguns destes genes podem ser selecionados para avaliar a sua potencialidade como alvos para bloqueio da transmissão da malária. Entre os genes com expressão diferencial resultante da análise dos resultados de RNA-seq e confirmação por qPCR, um gene relacionado com o transporte de Cl- e HCO3 2-, prestin, estava sobrexpresso após infeção com P. berghei. Este gene tem um papel crucial na invasão do parasita no intestino médio e na optimização do meio em que o parasita se desenvolve. Por esse motivo, o silenciamento deste transcrito foi efectuado para averiguar o papel funcional nas glândulas salivares. O silenciamento de genes utilizando a técnica de RNA de interferência permite inferir sobre o seu papel ou função num dado processo metabólico ou fisiológico. Após o silenciamento do gene prestin, o número de mosquitos viáveis apresentou um decréscimo significativo em comparação com o controlo (β2M). Também houve uma queda significativa entre o número de mosquitos antes da injeção e no último dia após injecção. O número de esporozoítos em geral não foi afectado pelo silenciamento da prestin quando comparado com o controlo. Para esclarecer resultados obtidos durante o estudo, tais como a influência do silenciamento da prestin na sobrevivência dos mosquitos e a presença e número de esporozoítos na glândulas salivares, seria fundamental realizar ensaios de qPCR para determinar a expressão diferencial deste gene após silenciamento. Além disso, será também importante analisar a expressão diferencial do off-target (ASTE006714) após silenciamento da prestin, uma vez que a semelhança da sequência entre este e a prestin é elevada.
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Ahmed, Fathima Zuba. "Unravelling genes responsible for successful anthocyanin production in Nicotiana benthamiana." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/230763/1/Fathima%20Zuba_Ahmed_Thesis.pdf.
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This thesis utilised two approaches, forward genetics and comparative transcriptomic analysis, to investigate the contrasting response to anthocyanin production observed in two distinct Nicotiana benthamiana ecotypes, LAB and QLD. The thesis is a step forward in utilising N. benthamiana as a candidate in forward genetics, currently limited due to its large complex genome and polyploid nature. The study utilised a cross-population between LAB and QLD to investigate the nature of inheritance of the contrasting parental phenotypes in its progeny. Additionally, expression profiles of anthocyanin biosynthesis genes were analysed via differential expression analysis and a novel method of mathematical manifold analysis
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Meunier, Léa. "Analyse de signatures transcriptomiques et épigénétiques des carcinomes hépatocellulaires." Thesis, Université de Paris (2019-....), 2020. http://www.theses.fr/2020UNIP7082.
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Élucider les processus transcriptionnels et épigénétiques dérégulés dans les cancers est fondamental pour mieux comprendre les voies biologiques impliquées et proposer une thérapie adaptée au phénotype moléculaire de chaque tumeur. Les approches classiques de classification non supervisée définissent des groupes moléculaires principaux pour chaque type tumoral. Cependant, ces méthodes, appliquées à des tumeurs complexes comme le carcinome hépatocellulaire (CHC), le 3ème cancer le plus mortel au monde, définissent des groupes qui restent relativement hétérogènes et ne reflètent qu’imparfaitement la diversité des mécanismes biologiques à l’œuvre dans ces tumeurs. Au cours de ma thèse, j’ai développé une stratégie d’analyses innovante, basée sur l’analyse en composantes indépendantes (ACI), pour extraire des signatures de processus biologiques précis à partir de grands jeux de données transcriptomiques et épigénétiques. Grace à cette nouvelle approche, j’ai identifié des groupes de gènes co-régulés, associés à des phénotypes ou altérations moléculaires précises. De même, l’analyse en composantes indépendantes du méthylome de 738 CHC m’a permis d’isoler 13 signatures épigénétiques stables, préférentiellement actives dans certaines tumeurs et certains sites CpG. Ces signatures incluent des signatures de méthylation précédemment associées au vieillissement et au cancer, mais aussi de nouvelles signatures d'hyper- et d'hypométhylation liées à des événements « drivers » et sous-groupes moléculaires spécifiques. Ces résultats nous éclairent sur la diversité des mécanismes moléculaires impliqués dans la carcinogenèse hépatique. Les outils d’analyse biostatistique innovants que j’ai développés ont été incorporés dans un package R librement utilisable par la communauté scientifiqueElucidating deregulated transcriptional and epigenetic processes in cancers is fundamental to better understand the biological pathways involved and to propose a therapy adapted to the molecular phenotype of each tumor. Classical unsupervised classification approaches define, for each tumor type, the main molecular groups. However, these methods, applied to complex tumors such as hepatocellular carcinoma (HCC), the 3rd cause of cancer-associated mortality worldwide, define groups that remain relatively heterogeneous and only imperfectly reflect the diversity of biological mechanisms at work in these tumors. During my PhD, I developed a, innovative strategy involving independent component analysis (ICA) to extract signatures of precise biological processes in large transcriptomic and epigenetic tumor data sets. This new approach allowed me to identify groups of co-regulated genes associated with specific phenotypes or molecular alterations. Similarly, independent component analysis of the methylomes of 738 HCC revealed 13 stable epigenetic signatures preferentially active in specific tumors and CpG sites. These signatures include signatures previously associated with ageing and cancer, but also new hyper- and hypomethylation signatures related to specific driver events and molecular subgroups. The work presented in this thesis sheds light on the diversity of molecular processes remodeling liver cancer transcriptomes and methylomes, improve the understanding of the molecular mechanisms involved in hepatic carcinogenesis and provides a statistical framework to unravel the signatures of these processes
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Lim, Chee Yee. "Understanding transcriptional regulation through computational analysis of single-cell transcriptomics." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267786.
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Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.
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Marchant, Axelle. "Le processus de domiciliation des punaises hématophages vectrices de la maladie de Chagas : apport de l’étude du transcriptome chimiosensoriel." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS008/document.
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En Amérique Latine, les punaises hématophages Triatominae transmettent à l’homme le parasite Trypanosoma cruzi, responsable de la maladie de Chagas touchant actuellement 5 millions de personnes. Même si les programmes d’éradication chimique des vecteurs sont efficaces, la maladie persiste du fait de la recolonisation des habitations humaines par des vecteurs provenant d’habitats naturels. Ainsi, certaines espèces présentent une capacité d’adaptation aux anthroposystèmes (processus de domiciliation), alors que d’autres espèces apparentées ne l’ont pas. Comprendre cette capacité d’adaptation est crucial d’un point de vue épidémiologique afin de cibler les espèces présentant un risque pour l’homme. La capacité à s’adapter à un nouvel habitat pourrait être liée à l’évolution du répertoire de gènes du système chimiosensoriel, important pour la perception du milieu. Cette étude a porté sur le système chimiosensoriel des Triatominae dans le but de documenter le processus d’adaptation et donc de domiciliation des vecteurs. Des données transcriptomiques obtenues en séquençage à haut débit ont été utilisées pour annoter et répertorier les gènes chimiosensoriels ainsi que pour comparer leur expression au sein de punaises hématophages d’habitats différents. L’existence d’une relation entre les variations de ces gènes chez différentes espèces de Triatominae et leur capacité d’adaptation à un habitat a par la suite été évaluée. L’espèce T. brasiliensis en voie de domiciliation au Brésil et présentant à la fois des populations sylvatiques, péri-domiciliaires et domiciliaires, et différentes espèces du genre Rhodnius d’habitats variés, ont été étudiées, notamment les deux espèces sœurs, R. robustus, sylvatique en Amazonie et R. prolixus majoritairement domiciliée dans toute son aire de répartition. En l’absence de génomes de références suffisamment proches de T. brasiliensis et des 10 espèces de Rhodnius étudiées, leurs transcriptomes ont été assemblés de novo. Les transcriptomes des deux espèces R. prolixus et R. robustus ont été assemblés par alignement sur le génome de R. prolixus. Chez ces différentes espèces de Triatominae étudiées, l’analyse du répertoire des gènes chimiosensoriels codant les OBPs et CSPs (familles multigéniques) comparé à celui d’autres Paranéoptères a montré des expansions géniques pouvant refléter des processus adaptatifs. Par ailleurs, chez les différentes espèces du genre Rhodnius, il existe une corrélation positive entre le nombre de gènes codant les OBPs et la capacité de domiciliation, suggérant l’implication de cette famille de gènes dans l’adaptation au milieu anthropique. Les analyses d’expression différentielle concernant les différentes populations de T. brasiliensis et les espèces R. prolixus/R. robustus ont montré qu’un certain nombre de transcrits sont différentiellement exprimés selon l’environnement dans lequel ont évolué les punaises notamment des gènes chimiosensoriels (OBPs, CSPs) ainsi que des gènes impliqués dans le rythme circadien et le comportement de recherche alimentaire (Takeout), dans la réponse à des stress environnementaux comme des gènes de détoxification (P450, glutathione S-transférase), dans la résistance à des changements climatiques (Heat-shock protéines) et dans la protection du milieu extérieur (protéines cuticulaires). Ce travail a permis de mettre à la disposition de la communauté scientifique des outils performants pour l’étude du processus de domiciliation des vecteurs de la maladie de Chagas (transcriptome, répertoire de gènes). Il a également permis de révéler des gènes qui pourraient être impliqués dans l’adaptation et/ou la plasticité phénotypique en réponse à un changement d’habitat. La compréhension des bases moléculaires de l’adaptation des vecteurs aux habitations humaines ouvre des potentialités de développer des méthodes alternatives de lutte contre les vecteurs qui pourraient être basées sur une perturbation de la communication chimiqueIn Latin America, the bloodsucking bugs (Triatominae, Hemiptera, Reduviidae) are vectors of the parasite Trypanosoma cruzi, which causes Chagas disease. More than five million people are infected. Even if chemical control campaigns are effective against vectors, the disease persists due to the recolonization of human habitations by vectors from natural habitats. Some species have the capacity to adapt to anthroposystems (domiciliation process), while other related species do not. Understanding this capacity to adapt is crucial from an epidemiological perspective to target species at risk to humans. The capacity to adapt to a new habitat could be linked to changes in the repertoire of chemosensory system genes, particularly for odorant binding proteins (OBP) and chemosensory proteins (CSP), which are important proteins to detect various odor stimuli. This study is based on the chemosensory system of Triatominae to document the adaptation process and then the domiciliation of the vectors. Transcriptomic data obtained by high-throughput sequencing were used to annotate and list the chemosensory genes and also to compare their expression in bloodsucking bugs from different habitats. The relationship between changes in these genes in different Triatominae species and their ability to adapt to a new habitat was evaluated. The species T. brasiliensis, which is in the process of domiciliation in Brazil with sylvatic, peridomiciliary and domiciliary populations, and various species of the genus Rhodnius from diverse habitats were studied, especially the two sibling species R. robustus, sylvatic in the Amazonia and R. prolixus mostly domiciliary throughout its geographical range. In the absence of a reference genome for T. brasiliensis, a reference transcriptome via de novo assembly (data 454 and Illumina) was achieved. The reference transcriptomes for 10 Rhodnius species were also established using the de novo assembly method. A genome reference based method on R. prolixus was also used to assemble the transcriptome of the two species R. prolixus and R. robustus. In the different species of the Triatominae studied, the chemosensory gene repertoire showed a high diversity and genic expansions compared to that of others Paraneoptera, which could reflect adaptive process. Furthermore, a positive correlation was shown between the number of OBP genes in Rhodnius species and their domiciliation ability, suggesting that this gene family is involved in the adaptation to anthropogenic environment. The differential expression analyses on the T. brasiliensis populations and the R. prolixus / R. robustus species showed that some transcripts are differentially expressed according to the environment in which the bugs have evolved, especially the chemosensory genes (OBP, CSP) and also genes involved in the circadian rhythm and foraging behavior (Takeout), in the response to environmental stress such as detoxification genes (P450, glutathione S-transferase), in resistance to climatic changes (heat-shock proteins) and in protection from the external environment (cuticular proteins).This work has helped make available to the scientific community powerful tools for studying the process of domiciliation of Chagas disease vectors (transcriptome, gene repertoire). It also revealed genes that could be involved in the adaptation and/or phenotypic plasticity in response to a change in habitat. Understanding the molecular basis of vector adaptation to human dwellings opens the potential to develop new tools to control the disease vectors, for example by disrupting chemical communication
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Gonzalez, Claudia. "Étude des mécanismes immunitaires impliqués dans le contrôle de l'infection par le virus Nipah." Electronic Thesis or Diss., Lyon, École normale supérieure, 2024. http://www.theses.fr/2024ENSL0035.
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Le virus Nipah (NiV) est un paramyxovirus hautement pathogène pour les humains faisant partie de la liste prioritaire pour la recherche et le développement de l’OMS. Les chauves-souris Pteropus sont le réservoir naturel asymptomatique du NiV et nous nous sommes intéressés aux mécanismes leur permettant de contrôler l’infection. Pour cela, nous avons réalisé une analyse transcriptomique comparative entre des cellules de chauves-souris et des cellules humaines. Nous avons tout d’abord observé des profils immunitaires innés distincts à l’état basal. Les cellules de chauves-souris présentent des niveaux élevés de récepteurs aux pathogènes comme TLR-3 et TLR-8, pouvant expliquer une détection rapide du virus. De plus, la cinétique d’activation des gènes a montré une différence entre les deux espèces, avec une activation précoce chez les chauves-souris alors que plus tardive et de plus grande amplitude chez les humains. L’activation précoce de la voie NF-kB a été observée chez les chauves-souris et parmi ces facteurs, c-Rel faisait partie des gènes les plus exprimés. L’analyse fonctionnelle a révélé que les protéines c-Rel humaine et de chauve-souris induisent l’activation de la voie NF-kB et sont inhibées par la protéine virale non structurale NiV-W. Nous avons aussi montré la capacité du c-Rel de chauve-souris, contrairement au c-Rel humain, de moduler l’activité du promoteur de réponse aux IFN (ISRE) après stimulation par l’IFN. Cette étude suggère que la réponse rapide et transitoire des Pteropus pourrait favoriser une meilleure régulation des réponses pro-inflammatoires et contribuer à leur capacité de contrôler l’infection NiV. Étant donné que nous ne disposons ni de traitement ni de vaccin pour ce virus, le travail a aussi porté sur l’évaluation d’un inhibiteur de fusion agissant sur l’entrée du virus, et un essai vaccinal chez un modèle primate. Ce dernier, combinant le récepteur CD40 à l’ectodomaine de la protéine NiV-G a été validé in vivo, démontrant une protection complète chez les singes immunisés. L’ensemble de ces résultats ouvre de nouvelles perspectives vers des approches antivirales innovantesNipah virus (NiV) is a highly pathogenic paramyxovirus for humans, listed as a priority for research and development by the WHO. Pteropus bats are the natural asymptomatic reservoir of NiV and we investigated on the mechanisms allowing them to control the infection. For this, we conducted a comparative transcriptomic analysis between bat and human cells. We observed distinct immune profiles at the basal state. Bat cells show high levels of receptors like TLR-3 and TLR-8, which may explain the rapid viral detection. Additionally, the kinetics of gene expression resulted to be different among the two species, as we detected early gene activation in bats, while the response in humans was delayed. Early activation of the NF-kB pathway was observed in bats, and among these factors, c-Rel was one of the most expressed genes. Functional analysis revealed that both human and bat c-Rel proteins induce NF-kB pathway activation and are inhibited by the non-structural protein NiV-W. We also demonstrated the ability of bat c-Rel, unlike human c-Rel, to modulate IFN response promoter (ISRE) activity after IFN stimulation. This study suggests that the rapid and transient response of Pteropus may promote better regulation of pro-inflammatory responses and contribute to their ability to control NiV infection. Since no treatment or vaccine is available for this virus, the work also focused on evaluating a fusion inhibitor acting on virus entry and a vaccine. The latter, combining the CD40 receptor with the ectodomain of the G protein, was validated in vivo, demonstrating complete protection Nipah virus (NiV) is a highly pathogenic paramyxovirus for humans, listed as a priority for research and development by the WHO. Pteropus bats are the natural asymptomatic reservoir of NiV and we investigated on the mechanisms allowing them to control the infection. For this, we conducted a comparative transcriptomic analysis between bat and human cells. We observed distinct immune profiles at the basal state. Bat cells show high levels of receptors like TLR-3 and TLR-8, which may explain the rapid viral detection. Additionally, the kinetics of gene expression resulted to be different among the two species, as we detected early gene activation in bats, while the response in humans was delayed. Early activation of the NF-kB pathway was observed in bats, and among these factors, c-Rel was one of the most expressed genes. Functional analysis revealed that both human and bat c-Rel proteins induce NF-kB pathway activation and are inhibited by the non-structural protein NiV-W. We also demonstrated the ability of bat c-Rel, unlike human c-Rel, to modulate IFN response promoter (ISRE) activity after IFN stimulation. This study suggests that the rapid and transient response of Pteropus may promote better regulation of pro-inflammatory responses and contribute to their ability to control NiV infection. Since no treatment or vaccine is available for this virus, the work also focused on evaluating a fusion inhibitor acting on virus entry and a vaccine. The latter, combining the CD40 receptor with the ectodomain of the G protein, was validated in vivo, demonstrating complete protection in immunized monkeys. These results open new perspectives for innovative antiviral approaches
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Dondi, Cristiana. "Molecular and functional analysis of cardiac diversification by cell specific translatomic approaches in Drosophila Melanogaster." Thesis, Université Clermont Auvergne (2017-2020), 2018. http://www.theses.fr/2018CLFAS003/document.
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Le cœur humain est un organe composé de différents types cellulaires tels que les cardiomyocytes, les fibroblastes, les muscles lisses et les cellules endothéliales. Ces cellules se diversifient grâce à des mécanismes moléculaires spécifiques en acquérant leurs propriétés fonctionnelles spécifiques. L’embryon de Drosophile est un modèle simple et adapté pour étudier la diversification des cellules cardiaques et leurs propriétés spécifiques. Le but du projet est d’améliorer notre connaissance sur les acteurs moléculaires qui contrôlent la diversification des cellules cardiaques. Pour atteindre cet objectif nous avons appliqué la méthode TRAP-rc ("rare cell Translation Ribosome Affinity Purification") suivie du séquençage ARN pour identifier les ARN messagers en cours de traduction spécifiques des cellules cardiaques Tin et Lb (Tin CBs et Lb CBs) à deux stades de développement corrélés avec la morphogenèse du cœur embryonnaire. Dans une première analyse focalisée sur l'analyse des données issues des TRAP-Seq sur cellules Tin nous avons mis en évidence que CAP et MSP-300 sont impliqués dans la migration des cardioblasts pendant la fermeture du cœur. En parallèle, nous avons également identifié deux autres gènes impliqués dans la morphogenèse, kon-tiki et dGrip qui semblent contrôler la cohésion des CBs au cours de la migration. En outre, nous avons trouvé qu'au stade 16, environ 60% des gènes enrichis sont communs entre les populations Tin et Lb. Parmi ces gènes, Src42, sqa et flr participent à la régulation du cytosquelette d'actine et nos analyses ont permis de démontrer qu'ils avaient également des fonctions dans la morphogenèse cardiaque. Nous avons également identifié des groupes de gènes plus spécifiques à chacune des populations ciblées. Une catégorie fonctionnelle fortement associée à la population Lb, comprend les gènes qui régulent l'épissage des ARN messagers et certains de ces gènes semblent être requis au cours de la morphogenèse cardiaque. Enfin, nous avons comparé nos données de TRAP-seq cardiaque avec des données de TRAP-Seq issues du muscle somatique (de l'équipe), et ainsi identifié près de 90 gènes qui présentent des isoformes protéiques spécifiques à chaque tissu notamment impliquées dans la formation de l'unité contractile sarcomérique. Ceci suggère que des mécanismes d'épissage spécifiques sont mis en place dans différents types cellulaires pour moduler les fonctions de certaines protéines musculaires. A travers ce projet, nous avons identifié de nouveaux acteurs généraux de la migration collective des cardioblastes au cours de la fermeture du cœur mais également de nouveaux gènes potentiellement impliqués dans l’acquisition des propriétés spécifiques dessous populations cardiaques Tin et Lb et de tissus musculaires distincts. Nous espérons que les données générées permettront dans le futur de mieux comprendre les mécanismes de la cardiogenèse des vertébrés ainsi que l’étiologie de maladies cardiaquesCardiac cells diversification is required for the formation of a functional heart. Human heart is a multi-lineage organ that develops through progressive diversification of progenitors derived from different heart fields. This process is underlined by numerous changes in the expression of a repertory of genes that allow cells to acquire their own identity and functions. The Drosophila embryo is a relatively simple model to study the diversification of cardiac cells and their properties. The goal of this project is to identify the repertories of genes that control the formation of different types of cardiac cells. To reach this objective we applied Translation Ribosome Affinity Purification (TRAP) method followed by RNA sequencing in order to identify mRNA engaged in translation specific to two cardiac cell types (Tinman (Tin) and Labybird (Lb) expressing cells), at two different time windows. We obtained a list of enriched genes for the different types of cardiac cells and time points. In a first part, we focused our attention on the Tindatasets and found that two genes, CAP and Msp300, are involved in cardioblasts migration during the heart closure. Then we identified two other candidate genes kontiki and dGrip that seem to contribute to maintain cohesion between CBs during heartmorphogenesis. Moreover by comparing our spatial datasets, we found that for the same time point, around 60% of Tin CBs enriched genes are common with Lb CBs enriched population and within this group we identified evolutionary conserved genes such as Src42, flr and sqa known to be involved in the cytoskeleton organization and in the actinpolymerization and depolymerisation. Our premiminary analyses show that they seem to be required for correct cardiac morphogenesis. We also identified sets of genes more specific for each cardiac cell population. Indeed, Lb CBs datasets show that in early stage there is the enrichment of genes mostly involved in transcriptional regulation and RNA splicing and some of these genes (prp8 and prp38) are involved in cardiac development. In parallel, we compared our TRAP-Seq dataset in the cardiac system with the TRAP-seqon muscle cells, and identified close to 90 genes that present cardiac or muscular specific isoforms. It is known that the alternative splicing, by increasing proteins diversity, contributes to the acquisition of specific cell properties. Furthermore, some cardiomyopathies are associated to defects in the alternative splicing of genes encoding sarcomeric proteins that we found in our dataset such as Tropomyosin and Zasp52. With this project, we have identified new actors of collective cardioblast migration and a set of genes with potential role in the acquisition of individual properties of Tin and Lbcardiac cells or of specific type of muscle tissue. We hope that our data could provide new insights into the genetic control of vertebrate cardiogenesis and into etiology of cardiac diseases
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Gantt, John Arthur. "Comparative Analysis of RNase P." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-09012003-153904/.
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This Master?s Thesis contains a description of the results of two research projects that were worked on over the course of two years. The first was an attempt to heterologously reconstitute the Bacillus subtilis RNase P RNA with the RNase P protein subunits of Methanothermobacter thermoautotrophicus, to identify a functional homology between one or more of the archaeal proteins with the Bacillus subtilis RNase P protein. Unfortunately, the reconstitution experiment could not be completed due to the poor quality of the pre-tRNAasp substrate synthesized. The second project dealt with comparative analysis of bacterial RNase P RNA. Eleven new RNase P RNA genes were amplified, sequenced, and their secondary structures derived by comparative analysis.
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Dichtl, Bernhard. "Genetic analysis of yeast RNase MRP." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/13645.
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RNase MRP is a ribonucleoprotein particle (RNP) with endoribonucleolytic activity which is involved in the processing of pre-rRNA at site A3. A genetic screen for mutants which are synthetic lethal (sl) with a temperature sensitive (ts) mutation in the RNA component of RNase MRP (rrp2-1) has been performed in order to identify new gene products which physically and/or functionally interact with RNase MRP. Analysis of the obtained sl mutant strains led to the identification of a new and essential gene, POP3. Depletion of Pop3p in vivo results in a phenotype characteristic of the loss of RNase MRP activity; A3 cleavage is inhibited, leading to under-accumulation of the short form of the 5.8S rRNA (5.8SS) and formation of an aberrant 5.8S rRNA precursor which is 5' extended to site A2. Pop3p depletion also inhibits pre-tRNA processing. Primary tRNA transcripts accumulate, as well as spliced but 5' and 3' unprocessed pre-tRNAs. The Pop3p depletion phenotypes resemble those previously described for mutations in components of RNase MRP and RNase P (rrp2-1, rpr1-1 and pop1-1). Immunoprecipitation of epitope tagged Pop3p efficiently co-precipitates the RNA components of both RNase MRP and RNase P. Thus, Pop3p is a common component of both RNPs and is required for the function of both enzymes in vivo. Strain SL158 carried a mutation in HAL2 which is sl in combination with rrp2-1. Hal2p is an enzyme that converts pAp (adenosine 3', 5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi.
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WILLIAMS, DANIEL. "ANALYSIS OF RNase P RNA STRUCTURE AND FUNCTION." NCSU, 2001. http://www.lib.ncsu.edu/theses/available/etd-20010821-140212.
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ABSTRACTWILLIAMS, DANIEL. Analysis of RNase P Structure and Function. (Under the direction of James W. Brown.) The diversity of Archaea in municipal wastewater sludge was investigated by amplification of rRNA sequences from sludge DNA using archaeal-specific primers. The most common sequences were extreme halophiles; also found were sequence members of environmental euryarchaeal and crenarchaeal groups. Only distant relatives of Methanosarcina among the Methanomicrobiales were found.A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P have largely disappeared, and even variation in the sequence and structure in the RNAs are similar in extent and type. The structure of the cruciform (P7-P11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences. Archaeal and bacterial RNase P RNAs are very similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions. In order to assess how readily the activity of the archaea RNA alone could be improved by point mutations in its sequence, in vitro selection was used to generate variants of the self-cleaving conjugant Methanobacterium formicicum: B. subtilus tRNAAsp (cpTP). Functional variants were generated with a broad spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection were only comparable to wildtype in catalytic activity; more performed significantly faster at lower ionic strength. These results suggest that the archaeal RNase P RNA is globally optimized and that the protein may play larger compensatory roles in catalysis than previously thought.
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Tufail, Muhammad Aammar. "Use of plant growth promoting endophytic bacteria to alleviate the effects of individual and combined abiotic stresses on plants as an innovative approach to discover new delivery strategies for bacterial bio-stimulants." Doctoral thesis, Università degli studi di Trento, 2021. http://hdl.handle.net/11572/305571.
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Bacterial endophytes are the organisms that live inside the plant for a full or a part of their life cycle. Endophytic bacteria have captured the interest of agriculture industry due to their plant beneficial properties, such as synthesis of phytohormones, solubilization of soil nutrients, and alleviation of biotic and abiotic stresses. Several studies have reported that stress tolerant endophytic bacteria can work with a similar performance as non-stressed conditions when inoculated to the plants under stressed conditions. Combination of abiotic stresses such as salinity, drought and low nitrogen stress can have additive or agonistic effects on bacterial and plant growth, and their interactions. However, very few studies have reported the impact of combined stress on endophytic bacterial assisted plant growth promotion. Therefore, understanding the underlying mechanisms of endophytic bacterial assisted plant’s tolerance abiotic stresses may provide the means of better exploiting the beneficial abilities of endophytic bacteria in agricultural production. Thus, the aim of this thesis was to study the stress tolerance mechanisms, beneficial characteristics, and plant growth promotion characteristics of endophytic bacteria under individual and combined abiotic stresses. Transcriptome analysis of endophytic bacteria revealed that tolerance mechanisms to deal with one kind of stress is different than concurrent stresses. Salinity and drought stress largely modulated the genes involved in flagellar assembly and membrane transport, showing reduced motility under stress conditions to preserve the energy. Additionally, bacterial endophyte that can fix nitrogen was studied with maize plant growth promotion under drought and low nitrogen stress conditions. The results suggested that diazotrophic bacterial endophyte can promote plant growth under moderate individual and combined stress conditions. Plant growth promoting endophytic bacteria can be utilized as an efficient tool to increase crop production under individual and concurrent abiotic stresses.
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Tufail, Muhammad Aammar. "Use of plant growth promoting endophytic bacteria to alleviate the effects of individual and combined abiotic stresses on plants as an innovative approach to discover new delivery strategies for bacterial bio-stimulants." Doctoral thesis, Università degli studi di Trento, 2021. http://hdl.handle.net/11572/305571.
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Bacterial endophytes are the organisms that live inside the plant for a full or a part of their life cycle. Endophytic bacteria have captured the interest of agriculture industry due to their plant beneficial properties, such as synthesis of phytohormones, solubilization of soil nutrients, and alleviation of biotic and abiotic stresses. Several studies have reported that stress tolerant endophytic bacteria can work with a similar performance as non-stressed conditions when inoculated to the plants under stressed conditions. Combination of abiotic stresses such as salinity, drought and low nitrogen stress can have additive or agonistic effects on bacterial and plant growth, and their interactions. However, very few studies have reported the impact of combined stress on endophytic bacterial assisted plant growth promotion. Therefore, understanding the underlying mechanisms of endophytic bacterial assisted plant’s tolerance abiotic stresses may provide the means of better exploiting the beneficial abilities of endophytic bacteria in agricultural production. Thus, the aim of this thesis was to study the stress tolerance mechanisms, beneficial characteristics, and plant growth promotion characteristics of endophytic bacteria under individual and combined abiotic stresses. Transcriptome analysis of endophytic bacteria revealed that tolerance mechanisms to deal with one kind of stress is different than concurrent stresses. Salinity and drought stress largely modulated the genes involved in flagellar assembly and membrane transport, showing reduced motility under stress conditions to preserve the energy. Additionally, bacterial endophyte that can fix nitrogen was studied with maize plant growth promotion under drought and low nitrogen stress conditions. The results suggested that diazotrophic bacterial endophyte can promote plant growth under moderate individual and combined stress conditions. Plant growth promoting endophytic bacteria can be utilized as an efficient tool to increase crop production under individual and concurrent abiotic stresses.
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Rios, Jonathan Joseph. "Genetic analysis of equine 2', 5'-oligoadenylate synthetase (OASI) and ribonculclease L (RNASEL) polymorphims and association to severe West Nile Virus disease." [College Station, Tex. : Texas A&M University, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-2749.
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Ghazal, Ghada. "Biochemical and genetic analysis of RNA processing and decay." Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/4287.
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Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.
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Xu, Jia. "Bioinformatics analysis of small silencing RNAs." Thesis, Boston University, 2011. https://hdl.handle.net/2144/38118.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
More than 180 genomes have been deciphered, however, much remains to be learned about how genes are regulated. Transcription factors harboring promoters and distal elements are known to activate or repress downstream gene expression, and DNA methylation and histone modifications add the complexity of epigenetic regulation. Furthermore, three classes of small RNA regulators have recently been discovered to repress the target gene and transposon expression. In flies, microRNAs (miRNAs) inhibit translation and expedite degradation of the target mRNAs. Small interfering RNAs (siRNAs) participate in a self defense mechanism called RNA interference (RNAi) to silence infected virus mRNAs or endogenous transposon elements. Piwi-interaction RNAs (piRNAs) efficiently silence the transposon elements in the gonad. The advent of next generation sequencing technologies has allowed us to sequence with sufficient coverage and accuracy and perform genome-wide bioinformatics analyses on small regulatory RNAs to enrich our knowledge on regulation. In this dissertation, I developed a suite of computational algorithms and programs to study small RNAs from next generation sequencing data. First I developed a de novo miRNA discovery pipeline to discover miRNAs in sea urchin and demonstrated one of the sources of endo-siRNAs in flies was overlapping complementary mRNAs. I further investigated the question of how miRNAs and siRNAs were sorted into their own pathways. First nucleotide composition and duplex structure were shown to significantly affect the sorting protein (R2D2) to decide small RNA's destiny. Next, I described collaboration work on piRNA pathway proteins, Ago3 and Rhino. Ago3 was found to catalyze the ping-pong amplification cycle in the piRNA pathway and Rhino, a HP1 homolog, was essential for dual strand piRNA clusters. Lastly, I demonstrated a sequencing-depth independent computational approach to quantify ping-pong efficiency and illustrated the function of each piRNA pathway protein after implementing. In addition, I developed a dynamic programming for detecting piRNA clusters to better annotate the piRNAs enriched segments in the genome and revealed the expression pattern for each cluster.
2031-01-01
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Gössringer, Markus. "In-vivo-Analysen zur Funktion bakterieller RNase-P-Proteine in Bacillus subtilis." [S.l. : s.n.], 2004. http://archiv.ub.uni-marburg.de/diss/z2004/0529/.
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Soltero, Stephanie Ruth. "Genetic analysis of the role of RNaseH2 in preventing genome instability." Diss., [La Jolla, Calif.] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3344708.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed April 1, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Nathania, Lilian. "Biochemical Analysis of Thermotoga maritima Ribonuclease III and its Ribosomal RNA Substrates." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/140013.
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ChemistryPh.D.
The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.
Temple University--Theses
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Smith, Alexandra Kimberly. "A MUTATIONAL-FUNCTIONAL ANALYSIS OF THE ESCHERICHIA COLI MACRODOMAIN PROTEIN, YMDB." Master's thesis, Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/539353.
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BiologyM.S.
Gene expression pathways exhibit many “twists and turns,” with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium Escherichia coli, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an E. coli protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. In E. coli, the model organism in this study, YmdB protein is encoded by the single ymdB gene, and has a predicted molecular mass of ~18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified Escherchia coli(Ec) YmdB and Aquifex aeolicus (Aa) YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of Ec-YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on Ec-YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated.
Temple University--Theses
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Snøve, Jr Ola. "Hardware-accelerated analysis of non-protein-coding RNAs." Doctoral thesis, Norwegian University of Science and Technology, Faculty of Information Technology, Mathematics and Electrical Engineering, 2005. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-713.
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A tremendous amount of genomic sequence data of relatively high quality has become publicly available due to the human genome sequencing projects that were completed a few years ago. Despite considerable efforts, we do not yet know everything that is to know about the various parts of the genome, what all the regions code for, and how their gene products contribute in the myriad of biological processes that are performed within the cells. New high-performance methods are needed to extract knowledge from this vast amount of information.
Furthermore, the traditional view that DNA codes for RNA that codes for protein, which is known as the central dogma of molecular biology, seems to be only part of the story. The discovery of many non-proteincoding gene families with housekeeping and regulatory functions brings an entirely new perspective to molecular biology. Also, sequence analysis of the new gene families require new methods, as there are significant differences between protein-coding and non-protein-coding genes.
This work describes a new search processor that can search for complex patterns in sequence data for which no efficient lookup-index is known. When several chips are mounted on search cards that are fitted into PCs in a small cluster configuration, the system’s performance is orders of magnitude higher than that of comparable solutions for selected applications. The applications treated in this work fall into two main categories, namely pattern screening and data mining, and both take advantage of the search capacity of the cluster to achieve adequate performance. Specifically, the thesis describes an interactive system for exploration of all types of genomic sequence data. Moreover, a genetic programming-based data mining system finds classifiers that consist of potentially complex patterns that are characteristic for groups of sequences. The screening and mining capacity has been used to develop an algorithm for identification of new non-protein-coding genes in bacteria; a system for rational design of effective and specific short interfering RNA for sequence-specific silencing of protein-coding genes; and an improved algorithmic step for identification of new regulatory targets for the microRNA family of non-protein-coding genes.
Paper V, VI, and VII are reprinted with kind permision of Elsevier, sciencedirect.com
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Miladi, Milad [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Computational analysis and annotation of structurally functional RNAs." Freiburg : Universität, 2020. http://d-nb.info/1230322159/34.
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Hughes, John Michael Xavier. "Molecular analysis of small RNAs of Saccharomyces cerevisiae." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35256.
Full textAbstract:
RNA has many diverse functions in living organisms, from serving as genome for many viruses, to regulating DNA replication, transcription, translation and other metabolic processes. Some RNA, like protein, has been shown to have catalytic activity. The great proportion of the mass of RNA in living cells, in the form of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), constitutes the machinery of protein synthesis, the remainder (approximately 2%) consists of many heterogeneous RNA species of relatively small size, loosely termed "small RNAs", the functions of many of which are completely unknown. In an attempt to understand some of these functions, three hitherto undescribed small RNAs of the budding yeast Saccharomyces cerevisiae were identified and their genes were cloned. These three small RNAs, which lack polyadenylation at their 3' ends, appear to represent the three most abundant RNA species in this organism after rRNA and tRNA. The most abundant of the three was found to be mainly cytoplasmic and was therefore called "small cytoplasmic RNA 1" (scR1). The other two RNAs, named snR17 and snR30, were found to be enriched in nuclear fractions and to possess trimethyl guanosine cap structures at their 5 ends, identifying them as belonging to the ubiquitous class of "U" small nuclear RNAs (U snRNAs), of which several are required for the endonucleolytic cleavage and splicing reactions in the maturation pathways of nuclear precursor mRNAs (pre-mRNA). Whereas scR1 and snR30 are both encoded by single genes, snR17 is the only yeast small RNA found so far to be encoded by two genes. SnR17 was found to be essential: haploid yeast strains lacking intact copies of one or other of the genes appeared to grow normally, but strains lacking both genes were inviable. The nucleotide sequences of the snR17 genes were determined, and the primary and predicted secondary structures of the RNA, 328 nucleotides in length, were found to show significant similarities to those of U3 snRNA, an abundant U snRNA, the function of which is not known. SnR17 belongs to a family of S. cerevisiae snRNAs which, unlike those involved in pre-mRNA splicing, are located in the nucleolus hydrogen-bonded to pre-rRNA, and are associated with antigenic protein that is recognized by human antibodies specific for a 36 kD polypeptide of the U3 small nuclear ribonucleoprotein (U3 snRNP) in mammals. U3 snRNA is also nucleolar and associated with pre-rRNA. Given their structural similarities, snR17 and U3 snRNA are presumably homologous. Yeast snRNAs associated with the anti-(U3)RNP antigen share with U3 snRNAs a conserved nucleotide sequence element. This sequence element alone, however, when injected into Xenopus oocytes, was not sufficient to direct binding of the antigen. The association of snRNAs with pre-rRNA suggests that they function in ribosomal biogenesis.
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O'Gorman, William Evert. "Analysis of cyclin H interaction with non-coding RNAs." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670092.
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Otto, Christina [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Optimizing algorithms for the comparative analysis of non-coding RNAs." Freiburg : Universität, 2015. http://d-nb.info/1115861808/34.
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Liu, Ding Xiang. "Translational analysis of the coronavirus infectious bronchitis virus messenger RNAs." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239182.
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Mead, Edward. "Discovery, Characterization, and Functional Analysis of micro RNAs in Culicidae." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/77433.
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MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs.
Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations.
Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages.
Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases.Ph. D.
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Kachouri-Lafond, Rym. "Computational identification of non-coding RNAs in hemiascomycete yeast genomes and structural evolution analysis of these RNAs." Université Louis Pasteur (Strasbourg) (1971-2008), 2008. http://www.theses.fr/2008STR13251.
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The central aim of the thesis is to contribute to the understanding of the relationships between the architecture and evolution of non-coding RNAs (ncRNAs), by comparative sequence analysis. The chosen model group is the one of the hemiascomycete yeasts, the group of Saccharomyces cerevisiae. The RNA annotation of these complete sequenced yeast genomes, led to the identification of an unconventional size of a RNase P RNA, which is an universal ribozyme that processes pre-tRNAs in all organisms of the three domains of life. This huge RNase P RNA, more than 1kb, was discovered in the human–pathogen yeast, Candida glabrata, which is very unusual, since in other eukaryotic and prokaryotic species the length is averaging 300 nucleotides (nt). Despite its length, the computational identified RNA is really the functional RNA in the cell. Another huge RNA was surprisingly discovered in the same yeast, namely the one of the telomerase ribonucleoprotein (RNP) complex, which protects chromosome by adding repeated sequences at their ends. It is the largest described to date, as it was also the case for the RNase P RNA. However, the telomerase RNA is much larger, with a size that reaches more than 2,5 kb and corresponds to more than 15 times the smallest telomerase RNA ever found in a eukaryote. Most of the time, the largest RNAs is found in C. Glabrata (the other example is U1 snRNA), but other hemiascomycete species express also ncRNAs with great sizes. [. . . ]
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Choudhury, Nila Roy. "Functional analysis of the non-coding RNAs of murine gammaherpesvirus 68." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4822.
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Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.
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Furió, Tarí Pedro. "Development of bioinformatic tools for massive sequencing analysis." Doctoral thesis, Universitat Politècnica de València, 2020. http://hdl.handle.net/10251/152485.
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[EN] Transcriptomics is one of the most important and relevant areas of bioinformatics. It allows detecting the genes that are expressed at a particular moment in time to explore the relation between genotype and phenotype. Transcriptomic analysis has been historically performed using microarrays until 2008 when high-throughput RNA sequencing (RNA-Seq) was launched on the market, replacing the old technique. However, despite the clear advantages over microarrays, it was necessary to understand factors such as the quality of the data, reproducibility and replicability of the analyses and potential biases.
The first section of the thesis covers these studies. First, an R package called NOISeq was developed and published in the public repository "Bioconductor", which includes a set of tools to better understand the quality of RNA-Seq data, minimise the impact of noise in any posterior analyses and implements two new methodologies (NOISeq and NOISeqBio) to overcome the difficulties of comparing two different groups of samples (differential expression). Second, I show our contribution to the Sequencing Quality Control (SEQC) project, a continuation of the Microarray Quality Control (MAQC) project led by the US Food and Drug Administration (FDA, United States) that aims to assess the reproducibility and replicability of any RNA-Seq analysis.
One of the most effective approaches to understand the different factors that influence the regulation of gene expression, such as the synergic effect of transcription factors, methylation events and chromatin accessibility, is the integration of transcriptomic with other omics data. To this aim, a file that contains the chromosomal position where the events take place is required. For this reason, in the second chapter, we present a new and easy to customise tool (RGmatch) to associate chromosomal positions to the exons, transcripts or genes that could regulate the events.
Another aspect of great interest is the study of non-coding genes, especially long non-coding RNAs (lncRNAs). Not long ago, these regions were thought not to play a relevant role and were only considered as transcriptional noise. However, they represent a high percentage of the human genes and it was recently shown that they actually play an important role in gene regulation. Due to these motivations, in the last chapter we focus, first, in trying to find a methodology to find out the generic functions of every lncRNA using publicly available data and, second, we develop a new tool (spongeScan) to predict the lncRNAs that could be involved in the sequestration of micro-RNAs (miRNAs) and therefore altering their regulation task.[ES] La transcriptómica es una de las áreas más importantes y destacadas en bioinformática, ya que permite ver qué genes están expresados en un momento dado para poder explorar la relación existente entre genotipo y fenotipo. El análisis transcriptómico se ha realizado históricamente mediante el uso de microarrays hasta que, en el año 2008, la secuenciación masiva de ARN (RNA-Seq) fue lanzada al mercado y comenzó a desplazar poco a poco su uso. Sin embargo, a pesar de las ventajas evidentes frente a los microarrays, resultaba necesario entender factores como la calidad de los datos, reproducibilidad y replicabilidad de los análisis así como los potenciales sesgos. La primera parte de la tesis aborda precisamente estos estudios. En primer lugar, se desarrolla un paquete de R llamado NOISeq, publicado en el repositorio público "Bioconductor", el cual incluye un conjunto de herramientas para entender la calidad de datos de RNA-Seq, herramientas de procesado para minimizar el impacto del ruido en posteriores análisis y dos nuevas metodologías (NOISeq y NOISeqBio) para abordar la problemática de la comparación entre dos grupos (expresión diferencial). Por otro lado, presento nuestra contribución al proyecto Sequencing Quality Control (SEQC), una continuación del proyecto Microarray Quality Control (MAQC) liderado por la US Food and Drug Administration (FDA) que pretende evaluar precisamente la reproducibilidad y replicabilidad de los análisis realizados sobre datos de RNA-Seq. Una de las estrategias más efectivas para entender los diferentes factores que influyen en la regulación de la expresión génica, como puede ser el efecto sinérgico de los factores de transcripción, eventos de metilación y accesibilidad de la cromatina, es la integración de la transcriptómica con otros datos ómicos. Para ello se necesita generar un fichero que indique las posiciones cromosómicas donde se producen estos eventos. Por este motivo, en el segundo capítulo de la tesis presentamos una nueva herramienta (RGmatch) altamente customizable que permite asociar estas posiciones cromosómicas a los posibles genes, transcritos o exones a los que podría estar regulando cada uno de estos eventos. Otro de los aspectos de gran interés en este campo es el estudio de los genes no codificantes, especialmente los ARN largos no codificantes (lncRNAs). Hasta no hace mucho, se pensaba que estos genes no jugaban ningún papel fundamental y se consideraban como simple ruido transcripcional. Sin embargo, suponen un alto porcentaje de los genes del ser humano y se ha demostrado que juegan un papel crucial en la regulación de otros genes. Por este motivo, en el último capítulo nos centramos, en un primer lugar, en intentar obtener una metodología que permita averiguar las funciones generales de cada lncRNA haciendo uso de datos ya publicados y, en segundo lugar, generamos una nueva herramienta (spongeScan) que permite predecir qué lncRNAs podrían estar secuestrando determinados micro-RNAs (miRNAs), alterando así la regulación llevada a cabo por estos últimos.
[CA] La transcriptòmica és una de les àrees més importants i destacades en bioinformàtica, ja que permet veure quins gens s'expressen en un moment donat per a poder explorar la relació existent entre genotip i fenotip. L'anàlisi transcriptòmic s'ha fet històricament per mitjà de l'ús de microarrays fins l'any 2008 quan la tècnica de seqüenciació massiva d'ARN (RNA-Seq) es va fer pública i va començar a desplaçar a poc a poc el seu ús. No obstant això, a pesar dels avantatges evidents enfront dels microarrays, resultava necessari entendre factors com la qualitat de les dades, reproducibilitat i replicabilitat dels anàlisis, així com els possibles caires introduïts. La primera part de la tesi aborda precisament estos estudis. En primer lloc, es va programar un paquet de R anomenat NOISeq publicat al repositori públic "Bioconductor", el qual inclou un conjunt d'eines per a entendre la qualitat de les dades de RNA-Seq, eines de processat per a minimitzar l'impact del soroll en anàlisis posteriors i dos noves metodologies (NOISeq i NOISeqBio) per a abordar la problemàtica de la comparació entre dos grups (expressió diferencial). D'altra banda, presente la nostra contribució al projecte Sequencing Quality Control (SEQC), una continuació del projecte Microarray Quality Control (MAQC) liderat per la US Food and Drug Administration (FDA) que pretén avaluar precisament la reproducibilitat i replicabilitat dels anàlisis realitzats sobre dades de RNA-Seq. Una de les estratègies més efectives per a entendre els diferents factors que influïxen a la regulació de l'expressió gènica, com pot ser l'efecte sinèrgic dels factors de transcripció, esdeveniments de metilació i accessibilitat de la cromatina, és la integració de la transcriptómica amb altres dades ómiques. Per això es necessita generar un fitxer que indique les posicions cromosòmiques on es produïxen aquests esdeveniments. Per aquest motiu, en el segon capítol de la tesi presentem una nova eina (RGmatch) altament customizable que permet associar aquestes posicions cromosòmiques als possibles gens, transcrits o exons als que podria estar regulant cada un d'aquests esdeveniments regulatoris. Altre dels aspectes de gran interés en aquest camp és l'estudi dels genes no codificants, especialment dels ARN llargs no codificants (lncRNAs). Fins no fa molt, encara es pensava que aquests gens no jugaven cap paper fonamental i es consideraven com a simple soroll transcripcional. No obstant això, suposen un alt percentatge dels gens de l'ésser humà i s'ha demostrat que juguen un paper crucial en la regulació d'altres gens. Per aquest motiu, en l'últim capítol ens centrem, en un primer lloc, en intentar obtenir una metodologia que permeta esbrinar les funcions generals de cada lncRNA fent ús de dades ja publicades i, en segon lloc, presentem una nova eina (spongeScan) que permet predeir quins lncRNAs podríen estar segrestant determinats micro-RNAs (miRNAs), alterant així la regulació duta a terme per aquests últims.
Furió Tarí, P. (2020). Development of bioinformatic tools for massive sequencing analysis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/152485
TESIS
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Findeiß, Sven. "Expanding the repertoire of bacterial (non-)coding RNAs." Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-67816.
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The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration.
Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene.
The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes.
The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes.
Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes.
Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions.
RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz.
During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins.
In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World.
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Thüring, Kathrin Luise [Verfasser]. "Analysis of translationally active RNAs : quantification by microscale thermophoresis and modification analysis by LC-MS/MS / Kathrin Luise Thüring." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/1123100837/34.
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Samols, Mark Atienza. "Identification and Functional Analysis of Micro-RNAs Encoded by Kaposi’s Sarcoma-Associated Herpesvirus." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1181143062.
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Burke, Caleb A. Burke. "Identification and Analysis of Novel, Biofilm-Specific small RNAs (sRNAs) in Staphylococcus aureus." Ohio University Honors Tutorial College / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1524837232775665.
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Li, Siwei. "High Throughput Automated Comparative Analysis of RNAs Using Isotope Labeling and LC-MS/MS." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1384427990.
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Bajak, Edyta Zofia. "Genotoxic stress: novel biomarkers and detection methods : uncovering RNAs role in epigenetics of carcinogenesis /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-415-5/.
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Kean, Joy. "Analysis of RNAs that interact with herpes simplex virus type 1 immediate early protein ICP27." Thesis, University of Glasgow, 2006. http://theses.gla.ac.uk/3964/.
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ICP27 is an HSV-1 immediate early protein required for the switch from early to late gene expression. ICP27 binds viral and cellular RNAs, and recently a yeast three-hybrid (Y3-H) analysis has identified an array of viral RNA sequences that interact with ICP27. Presented here are analyses of functional assays using a selection of the Y3-H identified RNA sequences inserted into the 5’ untranslated region (UTR) of a chloramphenicol acetyl transferase (CAT) reporter plasmid. A set of plasmids was transfected into baby hamster kidney (BHK) cells and CAT assays carried out to analyse the effects of the sequences on gene expression. Results indicated that expression was increased when ICP27-binding sequences were present even though no viral proteins were present. Comparison of sequences revealed that no common activation code or RNA structure was present that could be responsible for the increase in CAT gene expression. The levels of expression were further determined in the presence of wild type (wt), ICP27-null or ICP27 mutant HSV-1 infection to investigate whether ICP27 had any affect on CAT expression when ICP27-binding sequences were present. Interestingly, enhanced expression was observed during wt HSV-1-infection when ICP27-binding sequences were present, whereas little to no enhancement was observed during ICP27-null or mutant virus infections. However, a higher fold increase in CAT gene expression was observed during a HSV-1 infection when ICP27-binding sequences were not present. This indicated that an inhibitory effect on CAT expression observed during wt HSV-1 infection when ICP27-binding sequences were present was ICP27-depdnent. As a control, a noncoding, protein binding regulatory HPV RNA sequence was inserted into the CAT reporter plasmid, transfected into BHK cells and then infected with wt HSV-1, ICP27-null or ICP27 mutant viruses. Surprisingly, CAT expression was increased, albeit to only a limited extent, indicating that the previously observed increase in gene expression was not HSV-1 sequence specific. However, upon transfection of plasmids with the HPV control sequence inserted in the reverse orientation and a subsequent infection of cells no increase in CAT expression was observed. Analysis of the control sequence in the reverse orientation identified a shortage in G residues, which led to the construction of CAT reporter plasmids containing homopolymer sequences to inserted into the 5’UTR. A series of transfections and subsequent mock, wt HSV-1 or ICP27-null virus infections were carried out using this set of constructs. CAT assay analysis revealed an increase in CAT expression, to levels similar to those observed when the HSV-1 sequences were present, when poly(G) homopolymers were used as inserts during wt HSV-1 infection, whereas poly(A), (C) and (T) gave low levels of expression.
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Biryahwaho, B. "Analysis of RNAs and proteins of rotaviruses with rearranged genomes : A study of molecular variability." Thesis, University of Glasgow, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380397.
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Folkes, Leighton. "Computational analysis of small RNAs and the RNA degradome with application to plant water stress." Thesis, University of East Anglia, 2014. https://ueaeprints.uea.ac.uk/52038/.
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Water shortage is one of the most important environmental stress factors that affects plants, limiting crop yield in large areas worldwide. Plants can survive water stress by regulating gene expression at several levels. One of the recently discovered regulatory mechanisms involves small RNAs (sRNAs), which can regulate gene expression by targeting messenger RNAs (mRNAs) and directing endonucleolytic cleavage resulting in mRNA degradation. A snapshot of an mRNA degradation profile (degradome) can be captured through a new high-throughput technique called Parallel Analysis of RNA Ends (PARE) by using next generation sequencing technologies. In this thesis we describe a new user friendly degradome analysis software tool called PAREsnip that we have used for the rapid genome-wide discovery of sRNA/target interactions evidenced through the degradome. In addition to PAREsnip and based upon PAREsnip’s rapid capability, we also present a new software tool for the construction, analysis and visualisation of sRNA regulatory interaction networks. The two new tools were used to analyse PARE datasets obtained fromMedicago truncatula and Arabidopsis thaliana. In particular, we have used PAREsnip for the high-throughput analysis of PARE data obtained from Medicago when subjected to dehydration and found several sRNA/mRNA interactions that are potentially responsive to water stress. We also present how we used our new network visualisation and analysis tool with PARE datasets obtained from Arabidopsis and discovered several novel sRNA regulatory interaction networks. In building tools and using them for this kind of analysis, we gain a better understanding of the processes and mechanisms involved in sRNA mediated gene regulation and how plants respond to water stress which could lead to new strategies in improving stress tolerance.
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Videm, Pavankumar [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Analysis of high-throughput sequencing data related to small non-coding RNAs biogenesis and function." Freiburg : Universität, 2021. http://d-nb.info/1238518087/34.
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Christodoulou, Zoe. "An analysis of non-coding RNAs in Plasmodium falciparum and their potential role in antigenic variation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:60ea27e2-1129-4914-8abd-cfad018e0353.
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A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.
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MINOTTI, Linda. "Novel RNAs in cancer: large scale analysis of Affymetrix Human Exon chips and Next Generation Sequencing." Doctoral thesis, Università degli studi di Ferrara, 2019. http://hdl.handle.net/11392/2488087.
Full textAbstract:
Long non-coding RNAs (lncRNAs) are involved in numerous processes regulating locally protein-coding gene expression, acting at levels of chromatin, transcription and post- transcriptional mechanisms. LncRNAs control multiple cellular processes and are also involved in cancer pathway. Normally lncRNAs are detected with RNA-seq technology. Microarray technology is based on DNA fluorescent probes of specific sequence attached to a solid surface, that are used to hybridize a cDNA derived from RNA (target), under high stringent conditions. The probe-target hybridization produces fluorescence that is detected and quantified, determining the relative abundance of nucleic acid sequences. The limit of microarray technology is that probes are designed considering known genes, both protein or non-coding transcripts, but in a limited percentage compared to the extension of total genome. It follows that the microarray technology is less efficient than the RNA-seq (which is genome wide) in the search for new transcripts. However, Affymetrix has developed a chip, initially thought to study the splicing variants of genes, containing a huge number of probesets designed along the entire length of known transcripts, but also within intron region and based on predictive dataset. In our previous study, working on dataset analyzed with Affymetrix Human Exon chip, we were able to validate the lncRNA LOC107987281 situated in the first intron of the TGM2 gene. So we thought about extending the study to the whole genome, using datasets published in GEO, of cancer samples analyzed with Affymetrix Human Exon chip. Often this type of studies focuses on one tumor at a time. We have built a pipeline to work on a ‘pan-cancer’ model. For the bioinformatics analysis we designed and applied scripts in R and Python. We have constructed two datasets, one composed of tumor samples and normal tissues, coupled by patient, the other dataset formed by tumor tissue of several patients, cancer cell lines, considering leukocytes (PBMC) as normal samples. The pipeline allowed us to identify the probes that are differentially expressed in tumors compared to normal and which do not fall into already annotated transcripts. We have identified about 9000 probes with these characteristics. We have validated them with an independent dataset, both for samples and technology, analyzed in RNA-seq and derived from ENCODE. Finally, to confirm the specificity of the pipeline we re-annotated the probes identified with the latest version of Gencode, recently published, V29. In order to suggest the function of these new transcripts in cancer we carried out further bioinformatics analyses, including interspecific preservation, coding potentiality and transcriptional correlation with genes implicated in tumor processes (Cancer Census).I long non coding RNA (lncRNAs) sono coinvolti in numerosi processi cellulari durante la trascrizione genica o in meccanismi post-trascrizionali. I lncRNAs, controllando l’espressione genica, svolgono un ruolo chiave nello sviluppo del cancro. Allo stato attuale possono essere identificati e studiati attraverso la tecnologia del sequenziamento dell’RNA (RNA-seq), mentre nell’ultimo decennio molto utilizzati erano i microarrays. I microarray si basano su sonde specifiche attaccate a una superficie solida, che vengono ibridizzate al cDNA derivato dal RNA target. Il target ibridizzato con la sonda viene individuato e misurato tramite fluorescenza. Il limite fondamentale della tecnologia a microarray è che le sonde sono disegnate sulle sequenza geniche, relative sia a sequenze codificanti che non codificanti, ma in una percentuale limitata rispetto all’estensione del genoma totale. Ne deriva che i microarrays sono meno efficienti dell’ RNA-seq (che è genome wide) nella ricerca di nuovi trascritti. Tuttavia, Affymetrix ha sviluppato un chip (Human Exon) per studiare le varianti di splicing, in cui le sonde sono state disegnate non solo sui trascritti noti ma anche nelle regioni circostanti, negli introni e sui geni predetti. In uno studio precedente abbiamo utilizzato un dataset analizzato con Affymetrix Human Exon microarray, per studiare un lncRNA situato nel primo introne del gene TGM2. Quindi abbiamo pensato di estendere lo studio a tutto il genoma, utilizzando datasets pubblicati in GEO, di campioni di cancro analizzato con Affymetrix Human Exon chip. Spesso gli studi di questo tipo, si concentrano su un tumore alla volta. Noi abbiamo costruito una pipeline per lavorare su un modello di ‘pan-cancer’. Per l’analisi bioinformatica abbiamo disegnato e applicato scripts in R e Python. Abbiamo costruito due dataset, uno composto da campioni di tessuto tumorale e tessuto normale, accoppiati per paziente, l’altro formato da tessuti tumorali di diversi pazienti, linee cellulari di cancro, considerando leucociti (PBMC) come campioni normali. La pipeline ci ha permesso di individuare le sonde che sono differenzialmente espresse nei tumori rispetto ai normali e che non rientrano in trascritti già annotati. Abbiamo individuato circa 9000 sonde con queste caratteristiche. Le abbiamo validate con un dataset indipendente sia per campioni che per tecnologia, analizzato in RNA-seq e derivato da ENCODE. Infine per confermare la specificità della pipeline abbiamo ri-annotato le sonde cosi’ identificate con l’ultima versione di Gencode, recentemente pubblicata, V29. Al fine di suggerire la funzione di questi nuovi trascritti nel cancro abbiamo svolto ulteriori analisi bioinformatiche, incluso la conservazione interspecifica, la potenzialità codificante e la correlazione trascrizionale con geni implicati nei processi tumorali (Cancer Census).
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Waldmann, Barbara Ellen [Verfasser], and Petra [Akademischer Betreuer] Dersch. "Identification and expression analysis of small regulatory RNAs in Yersinia pseudotuberculosis / Barbara Ellen Waldmann ; Betreuer: Petra Dersch." Braunschweig : Technische Universität Braunschweig, 2014. http://d-nb.info/1175821578/34.
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Dalton, Kevin Paul. "Analysis of cis-acting signals required for the replication and packaging of infectious bronchitis virus defective-RNAs." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624435.
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