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1
Mayhew, Michael. "Coding regions under non-coding selection: implications for transcriptional and post-transcriptional gene regulation." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21995.
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The signals that facilitate transcriptional and post-transcriptional gene expression regulation are still being understood. Comparative genomics approaches based on the premise that sequence or structure conservation implies functionality have successfully distinguished true regulatory sequences and structures from prediction noise. Protein-coding regions of genes have often been overlooked as potential regulatory sequence regions. A set of 8785 coding region sequences were previously found to be conserved significantly above a baseline protein-coding conservation level. We call these sequences coding regions under non-coding selection or CRUNCS. Analysis of the sequences, the primary contribution of this work, revealed that CRUNCS bases are more often found in coding exon edges and in middle coding exons. CRUNCS-containing genes are more significantly enriched for regulation of transcription and translation, protein ubiquitination, and mRNA processing. CRUNCS are significantly enriched for RNA secondary structure elements. We also uncovered statistical evidence linking CRUNCS to gene splicing regulation.<br>Les méthodes de génomique comparatives qui sont tirées de la prémisse que la conservation de la séquence ou de la structure implique la conservation de la fonctionnalité, sont parvenues à identifier de vrais signaux régulateurs. Les régions codantes ont souvent été négligées comme des régions potentiellement régulatrices. Un ensemble de 8785 séquences de ces régions plus conservées que prévues a été précédement identifié. L'analyse de ces séquences appelées CRUNCS a révélé que les acides nucléiques des CRUNCS sont plus nombreux aux extrémités des exons et dans les exons centraux. Les gènes contenants des CRUNCS sont enrichis des catégories fonctionnelles comprenant : la régulation de la transcription et la traduction, l'ubiquitination des protéines et le traitement des ARNm. Les CRUNCS sont enrichis d'éléments de structure secondaire de l'ARN. Nous avons aussi découvert des preuves statistiques démontrant que les CRUNCS jouent un rôle dans la régulation de l'épissage des gènes.
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Serra, Barros Ana Cristina. "Transcriptional regulation by non-coding RNAs in Saccharomyces cerevisiae." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:e523d0ee-bb3a-4217-aeba-9e6e398fc86a.
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Genome-wide studies in Saccharomyces cerevisiae have revealed that the majority of the genome is transcribed on both strands, producing both coding and non-coding RNAs (ncRNAs). Initially, these ncRNAs were regarded as spurious transcripts but some have since been shown to have important roles as transcriptional regulators. Very little is understood about how ncRNAs are initiated, terminated and processed or how this influences their function. To address these questions, the expression, stability, and subcellular localization of the ncRNAs at the endogenous GAL locus was analysed. This revealed a complex interleaved transcript map, challenging the conventional view of a transcription unit (TU) flanked by 5’ sequences or promoters (P) that initiate transcription and 3’ regions, known as terminators (T), which control events such as transcript cleavage, polyadenylation, export and transcription termination. By creating conventional (PGAL-T) or unconventional (PGAL-P) hybrid TUs at the GAL locus, in which a promoter or terminator is positioned downstream of a galactose-inducible promoter, this work shows that both promoters and terminators are able to initiate antisense transcription to yield stable antisense transcripts. The data suggest that terminators contribute to efficient but variable expression from the promoter. An unconventional P-P TU, lacking a terminator, is transcribed on both strands but the sense transcript remains at low levels, through the repressive action of antisense transcription, and is retained in the nucleus. In contrast, the conventional P-T bi-directional TUs are plastic, with the Rrp6 component of the nuclear exosome and TATA-like sequences in the 3’ UTR determining whether the predominant transcript is antisense or sense. By relieving the repressive action of antisense transcription, this allows high levels of sense transcript to accumulate in the cytoplasm, contributing to gene expression, supporting a novel mode of gene regulation involving components of RNA quality control pathways acting through the 3’ region of genes.
3
Snead, Aaron Nathan. "Exploring the non-transcriptional activity of thyroid hormone derivatives." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3339205.
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Camilleri, Emily Therese. "The Transcriptional Role of FOXP1 in Non-Hodgkin's Lymphoma." Thesis, Griffith University, 2013. http://hdl.handle.net/10072/367974.
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Non-Hodgkin's lymphoma (NHL) is a family of lymphoid malignances that are the 6th leading cause of death in Australia. B cell lymhomas account for approximately 90% of all NHL cases, and the common subtypes Diffuse Large B cell Lymphoma (DLBCL) and Follicular Lymphoma (FL) together make uo 60% of all cases. DLBCL and FL are both germinal centre derived B cell lymphomas, however these malignancies represent opposite ends of the clinical scale, being aggressive compared to indolent disease respectively. Early detection of lymphoma is highly advantageous for aggressive subtypes like DLBCL, where without treatment patient survival can be less than 2 years. Currently, there are very few genes that are found to be associated with the risk of NHL. One gene that has been implicated in NHL development and progression is , but as yet few other genes have been identified. This thesis is aimed at characterising this gene but also identifying other genes involved in NHL. The identification of genes that predispose individuals to the development of NHL may firstly provide a diagnostic tool, and also provide insight into the molecular pathogenesis of this disease. Additionally, molecular characterisation of these genes may also aid in the development of pharmacogenomic therapies.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Schoolo of Medical Science<br>Griffith Health<br>Full Text
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Bogu, Gireesh K. 1984. "Understanding the transcriptional landscape of non-coding genome in mammals." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/572043.
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Widespread transcription in mammals revealed unexpected discovery of non-coding
elements like long noncoding RNAs (lncRNAs) and repetitive elements. First,
lncRNAs were previously identified in limited number of tissues or cell lines in mouse
and the discovery of lncRNAs was still pending in many other tissues in mouse. To
address this, we applied a computational pipeline that discovered 2,803 highconfidence
novel lncRNAs by mapping and de novo assembling billions of RNA-Seq
reads in eight tissues and a primary cell line in mouse. Further, we integrated this
catalog of lncRNAs with chromatin state maps and found many regulatory lncRNAs
(promoter-associated and enhancer-associated lncRNAs). Second, more than half of
the human genome contains repetitive elements. However, it is not clear how they
are expressed across all mammalian tissues. To address this, as a part of Genotype-
Tissue Expression (GTEx) project, we profiled repetitive elements using 8,551 poly-A
RNA-Seq datasets from 53 tissues across 550 individuals and found various repeat
families transcribed across multiple human tissues in a tissue-specific manner. In
summary, to understand the transcriptional landscape of non-coding genome, we
mainly analyzed RNA-Seq datasets across many tissues in mammals and show that
the non-coding elements like lncRNA and repetitive elements are not only
transcribed but also tissue-specific. Together, this thesis work defines a unique
collection of non-coding elements that are transcribed and tissue-specific in
mammalian tissues.<br>Una gran parte del genoma de mamiefores se expresa en forma de ARNs y se
conoce hoy en dia que una gran parte de estos transcritos son no codificantes
llamados lncRNAs y que contienen elementos repetitivos. En ratones, estos han
sido identificados recientemente en un número limitado de tejidos y líneas celulares.
Esta tesis presenta un trabajo exhaustivo de estudio de lnRNAs en ratón en ocho
tejidos y una línea celular. En este trabajo se descubrieron 2803 nuevos lncRNAs a
los cuáles se les asignó una función reguladora (asociados a promotores o
activadores “enhancers”) en el genoma usando datos del estado de la cromatina.
Asimismo, más de la mitad del genoma humano contiene elementos repetitivos.
Desafortunadamente no se conoce el patrón de expresión de estos elementos
repetitivos en los tejidos mamíferos. Como miembros del proyecto GTEx (GenotypeviTissue Expression), analizamos la expresión de estos elementos repetitivos en
8,551 muestras de polyA RNA-Seq en 53 tejidos de 550 individuos. Encontramos
que muchas familias de elementos repetitivos son expresadas en tejidos específicos
en varios individuos, y representan una característica peculiar de la identidad de
cada tejido en humanos.
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POLI, VALENTINA. "A non-transcriptional role of NFAT in regulating platelets functions." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241337.
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Le piastrine ricoprono un ruolo fondamentale nei processi di emostasi, ma, recentemente, è stato dimostrato che hanno anche un ruolo nel modulare la risposta infiammatoria, interagendo con le cellule del sistema immunitario in modo diretto e indiretto. Nonostante le piastrine siano delle cellule anucleate, esse contengono fattori trascrizionali (TF) che modulano la loro attivatione attraverso attività non-trascrizionali. In particolare, è stato dimostrato che le piastrine contengono NF-κB, un TF che controlla importanti funzioni delle cellule immunitarie durante i processi infiammatori, e che NF-κB regola l’attivazione delle piastrine.
In questo lavoro, abbiamo investigato la possibilità che altri TF oltre a NF-κB possano avere un ruolo regolatorio durante l’attivazione piastrinica. Abbiamo osservato che le piastrine contengono un altro importante TF tipicamente espresso dalle cellule immunitarie: il fattore nucleare delle cellule T attivate (NFAT). In particolare, abbiamo osservato che, a seguito dell’attivazione del recettore PAR4, la via calcineurina/NFATc2 viene attivata e regola varie funzioni piastriniche. Questa via viene attivata sia nelle piastrine murine sia umane e la sua inibizione determina un aumento dell’aggregazione piastrinica, dell’attivazione dell’integrina, del rilascio di granule e dell’adesione su fibrinogeno. Attraverso modelli murini in vivo, abbiamo dimostrato che l’attivazione di NFATc2 nelle piastrine modula l’emostasi e la trombosi. Inoltre, l’attivazione di NFATc2 nelle piastrine regola l’interazione tra piastrine e neutrofili, influenzando la severità dello sviluppo di sepsi batterica. I nostril esperimenti in vitro supportano la capacità di NFATc2 di agire a valle del recettore PAR4 attraverso un meccanismo che coinvolge il recettore dell’ADP e l’outside-in signaling dell’integrina α2bβ3.
Infine, abbiamo osservato che l’inibizione della via calcineurina/NFATc2 è in grado di compensare in parte i difetti di attivazione piastrinica in due rare patologie, la sindrome di Hermansky-Pudlack e la trombastenia di Glanzmann. I nostri dati suggeriscono che la via calcineurina/NFATc2 può essere bersaglio per lo sviluppo di approcci terapeutici innovativi per il trattamento di difetti piastrinici con prognosi infausta.
Il nostro lavoro mostra per la prima volta che l’attivatione della via calcineurina/NFATc2 nelle piastrine ha un ruolo non-trascrizionale e è un regolatore negativo delle risposte piastriniche. Ulteriori studi sono necessari per caratterizzare il meccanismo di azione attraverso il quale NFATc2 agisce nel modulare l’attivazione piastrinica e per capire se NFATc2 possa avere un ruolo non-trascrizionale anche in cellule nucleate.<br>Platelets play a critical role in hemostasis but, more recently, it was demonstrated that they also modulate the inflammatory response by establishing direct and indirect interactions with immune cells. Although platelets are anucleated cells, they contain functional transcription factors (TFs) that modulate their activation via non-transcriptional functions. In particular, it has been shown that platelets contain NF-κB, a TF that controls important functions of immune cells during the inflammatory process, and that NF-κB regulates platelet activation.
We investigated the possibility that other TFs different from NF-κB might play regulatory roles during platelet activation. We found that platelets contain another TF typically expressed by immune cells: nuclear factor of activated T cells (NFAT). In particular, we found that upon PAR4 ligation, the calcineurin/NFATc2 pathway is activated and regulates platelet functions. This pathway is activated both in murine and human platelets, and its inhibition results in enhancement of platelet aggregation, integrin activation, granules release and spreading on fibrinogen. By using murine in vivo models, we demonstrated that NFATc2 activation in platelets modulates hemostasis and thrombosis. Moreover, platelet NFATc2 activation regulates the interaction between platelets and neutrophils and impacts the severity of bacterial sepsis development. Our in vitro experiments support the capacity of NFATc2 to act downstream of PAR4 via a mechanism that involves both the ADP receptor and the outside-in integrin α2bβ3 pathway.
Finally, we found that inhibition of the calcineurin/NFATc2 pathway partially rescues platelet activation defects in two rare human platelet pathologies, the Hermansky-Pudlak syndrome and the Glanzmann thrombasthenia. Our data suggest that the calcineurin/NFATc2 pathway can be targeted to develop innovative therapeutic approaches to treat platelet defects with poor prognosis.
Our work reveals for the first time that the activation of the calcineurin/NFATc2 pathway in platelets has a non-transcriptional role and it is a key negative regulator of platelet responses. Further studies are needed to characterize the mechanism of action through which NFATc2 exerts its non-transcriptional function in modulating platelets activation and to understand if NFATc2 could have a non-transcriptional role also in nucleated cells.
7
Evans, C. M. "Non-coding RNA and transcriptional regulation in CD4 T cell lineages." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1466165/.
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CD4 T cell lineage choice epitomises the ability of the immune system to become tailored to a specific threat and provides a framework for understanding the mechanisms behind cell specification. The differentiation of T effectors from naïve cells gives rise to pro-inflammatory lineages including T helper 1 (Th1) and Th2 and anti-inflammatory regulatory T cells (Treg). An additional lineage of Treg also exits the thymus in parallel to naïve T cells and together these Treg are required for prevention of autoimmunity. These T cell lineages are distinct in terms of their cytokine production and functional effects but also through their differences in gene expression and its regulation, which are orchestrated by the presence of lineage-specifying transcription factors specific for each lineage. In addition, post-translational modification of histones also provide insights into this transcriptional regulation and more recently the pervasive and tissue-specific transcription of multiple classes of RNA species without protein coding capacity, non-coding RNA (ncRNA), has been found to play a role in cell differentiation and function. In this thesis I identify several ncRNAs with differential expression different T cell lineages. This includes ncRNAs upregulated Treg compared to T responders. The characterisation of these, including their expression in the autoimmune context of systemic lupus erythematosus (SLE), is presented and their possible biological functions are examined. The relevance of histone modifications for influencing Treg identity in SLE is also investigated. An additional class of ncRNAs that originate from gene enhancer regions, eRNA, is also investigated in the context of Th1 versus Th2 lineage choice. This enhancer transcription is increased genome-wide in Th1 cells at enhancers with high density T-bet binding in, termed ‘super-enhancers’. The functional relevance of these eRNAs, including at the super-enhancer upstream of the Th1 signature cytokine gene, IFNG, is also investigated in knockdown experiments.
8
Murfitt, Kenneth John. "Post-transcriptional regulation of miRNA activity and expression in C. elegans." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648749.
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Chery, Alicia. "Rôle de la transcription pervasive antisens chez Saccharomyces cerevisiae dans la régulation de l'expression des gènes." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066191/document.
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L'expression des gènes est finement régulée dans la cellule et soumise à de multiples contrôles-qualité. Cette régulation intervient à différents niveaux, de façon à garantir une synthèse efficace des produits fonctionnels de l'expression génique, et pour assurer une adaptation à un changement environnemental. Notamment, les régulations transcriptionnelles sont cruciales pour contrôler la cinétique et le niveau d'expression des gènes. La transcription pervasive est une transcription généralisée non-codante et instable qui fut révélée chez la levure Saccharomyces cerevisiae. Bien que son potentiel régulateur ait été démontré de façon ponctuelle, la question de sa fonctionnalité globale restait ouverte. Lors de ma thèse, j'ai pu montrer l'existence de phénomènes multiples d'interférence transcriptionnelle liés à la transcription pervasive, pour co-réguler un ensemble de gènes entre la phase exponentielle et la quiescence. En effet, la transcription non-codante en antisens des gènes concernés conduit à leur répression, dans des conditions où ils ne doivent pas être exprimés. Le mécanisme de répression fait intervenir des modifications de la chromatine. La levure bourgeonnante, dépourvue de la machinerie d'ARN interférence, présente donc un système fin de régulation de l'expression génique utilisant la transcription pervasive<br>In the cell, gene expression is finely tuned and is submitted to different quality-controls. Gene are regulated at different expression levels in order to guarantee a proper synthesis of functional products, and to ensure an optimal adaptation to environmental changes. In particular, transcriptional regulations are critical for gene expression level and kinetics.Pervasive transcription, defined as a generalized non-coding and unstable transcription, was discovered in the yeast Saccharomyces cerevisiae. Although its regulatory potential was punctually shown, the question of its global functionality still remained. During my PhD, I could show the existence of numerous transcriptional interference mechanisms involved in the co-regulation of a group of genes between exponential phase and quiescence. Indeed, non-coding transcription in antisense to genes promoter leads to its repression in conditions where they have to be switched off. The repression mechanism is allowed by chromatin modifications.Hence, budding yeast that lacks RNA interference machinery has developed a fine regulation system using pervasive transcription
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Jones, Amy Madeline. "Post-transcriptional regulation of rpoS and HemA in salmonella." Morgantown, W. Va. : [West Virginia University Libraries], 2009. http://hdl.handle.net/10450/10826.
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Farhadi, Hooman F. "Evolutionarily conserved non-coding sequences confer transcriptional regulation to the Myelin Basic Protein gene." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100359.
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Myelin formation is an evolutionarily late acquisition that contributes to fast conduction in vertebrate axons. Continuous bidirectional signaling is required for the acquisition and maintenance of the mature nerve fiber phenotype. Myelin Basic Protein (MBP) is a representative structural component of compact myelin that is co-ordinately regulated with other myelin protein genes, primarily at the transcriptional level. In an effort to define the regulatory network controlling MBP expression, I located and functionally characterized conserved regulatory sequences in 5' non-coding sequence. First, I identified four widely-spaced segments of preferential conservation in orthologous human-mouse genomic sequences (termed modules 1-4) that range from 0.1 to 0.4 kb. In order to compare qualitative and quantitative regulatory programs mediated by modular and inter-modular sequences, a novel in vivo transgenesis system was adopted and validated. Targeted insertions of single-copy reporter constructs at a predetermined location within the hprt locus allowed for direct inter-construct regulatory program comparisons. The proximal modules M1 and M2 confer relatively low-level expression in oligodendrocytes, primarily during early postnatal development. The upstream M3 module confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. M3 also confers expression to Schwann cells but only transiently during active myelin elaboration and only when isolated from surrounding MBP sequences. These observations define the regulatory roles played by a complex network of conserved non-coding MBP sequences and lead to a combinatorial model in which specific permutations of regulatory sequences are engaged differentially in various glial cell states. This experimental system also was shown to provide sufficient quantitative resolution to reveal the regulatory functions contributed by particular modular sub-domains and individual enhancer elements. M4 regulatory activity requires simultaneous contributions from elements located in both a core targeting as well as surrounding enhancing sub-domains with Sox10 and Krox-20 M4 binding appearing to mediate targeting and enhancer function, respectively. From this investigation, the complex network of elements and transcription factors that control expression of one important myelin gene has begun to emerge. This knowledge should lead to a deeper understanding of the regulatory mechanisms controlling the overall myelination program and, ultimately, to novel therapeutic strategies effective in ameliorating the consequences of inherited or acquired demyelinating disease.
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REY, FEDERICA. "TRANSCRIPTIONAL CHARACTERIZATION OF OBESITY AND TYPE 2 DIABETES: A FOCUS ON NON-CODING RNAS." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/785913.
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L’obesità è definita dall’Organizzazione Mondiale della Sanità come una condizione di accumulo di grasso corporeo anormale o eccessivo che può presentare un rischio per la salute. Questa malattia può portare all’insorgenza di comorbidità associate quali il diabete di tipo 2, l’ipertensione, malattie cardiovascolari, dislipidemia, infarto, artrite e anche alcuni tipi di cancro, con un conseguente aumento della mortalità. Il contributo relativo di fattori genetici o dell’ambiente nell’insorgenza dell’obesità non è ancora chiaramente definito, e gli RNA regolatori potrebbero avere una funzione fondamentale nell’identificazione di nuovi meccanismi di malattia. Lo scopo di questo lavoro è la caratterizzazione delle differenze trascrizionali presenti nel tessuto adiposo sottocutaneo di pazienti obesi e obesi con diabete di tipo 2. Il lavoro si è inoltre focalizzato sull’analisi del ruolo del genoma non codificante nello sviluppo della malattia, per la loro rilevanza in numerosi processi fisiologici e patologici.
A questo scopo è stato effettuato il sequenziamento dell’RNA presente nel tessuto adiposo sottocutaneo di 5 donne normopeso, 5 donne obese e 5 donne obese con diabete di tipo 2. Tre condizioni sperimentali sono state analizzate: le differenze presenti tra le donne obese e le normopeso, quelle tra le donne obese con il diabete di tipo 2 e le normopeso e quelle tra le donne obese con diabete di tipo 2 e le donne obese senza questa comorbidità. Per ogni condizione è stata effettuata un’analisi bioinformatica globale. Questa analisi ha previsto una caratterizzazione estensiva dei trascritti differenzialmente espressi, indicandone la loro localizzazione, interazione e regolazione trascrizionale. L’analisi di ontologia genica ha permesso di identificare le funzioni molecolari specifiche e i processi biologici in cui essi sono coinvolti, insieme ai loro processi di appartenenza. Sono stati consultati inoltre specifici database contenenti informazioni su numerose malattie, in modo da identificare l’implicazione dei trascritti in malattie immunologiche, oncologiche e metaboliche. Un’attenzione particolare è stata posta sul ruolo degli RNA non codificanti, la cui presenza aumenta significativamente passando da una condizione di obesità “pura” a quella di un’obesità associata alla compresenza di diabete di tipo 2. I trascritti differenzialmente espressi non codificanti sono il 6,43% dei deregolati nella condizione di obesità versus normopeso, il 32,43% nei soggetti diabetici versus normopeso e addirittura più del 50% nell’analisi di soggetti obesi diabetici versus obesi. Questo dimostra come il genoma non codificante potrebbe essere di importanza fondamentale nell’insorgenza di specifiche comorbidità e potrebbe rappresentare un bersaglio per futuri interventi terapeutici e di prevenzione. In questo lavoro sono stati svolti esperimenti funzionali in modo da caratterizzare il ruolo di alcuni RNA non codificanti “a catena lunga” (long non-coding RNAs) nell’obesità, e i risultati hanno dimostrato come questi sono espressi negli adipociti e strettamente regolati da fattori trascrizionali implicati nell’adipogenesi quali PPARy, C/EBPa, C/EBPb e C/EBPd.
I risultati dimostrano chiaramente il ruolo della componente non codificante del genoma nello sviluppo della comorbidità diabetica e un’analisi futura di queste molecole potrebbe essere di cruciale rilevanza nella comprensione di nuovi meccanismi di malattia mai prima caratterizzati. I risultati potrebbero inoltre spiegare perché alcune classi di pazienti obesi hanno un rischio maggiore di sviluppo di comorbidità specifiche, aprendo le porte a possibili strategie terapeutiche preventive e di medicina di precisione.<br>Obesity is defined by the World Health Organization as a condition of abnormal or excessive accumulation of body fat that presents a risk to health. This disease can lead to an increase in associated morbidities for many chronic diseases such as type 2 diabetes, hypertension, coronary artery disease, dyslipidemia, stroke, osteoarthritis, and even certain forms of cancer, with a subsequent increase in mortality rate. The relative contribution of either genetic or the environment in obesity onset and co-morbidities development is not yet completely defined, and RNA biology might play a central role in elucidating new targetable pathways. The aim of this research was the characterization of the transcriptional differences present in the sottocutaneous adipose tissue of obese and obese with type 2 diabetes subjects. Moreover, the work focused on the role of the non-coding part of the genome in disease development, as these molecules are becoming more and more relevant for their function in physiological processes and disease mechanisms. To this aim, RNA-sequencing was performed on sottocutaneous adipose tissue obtained from 5 healthy normal weight females, 5 obese females, and 5 obese females with type 2 diabetes. Three experimental conditions were subsequently analyzed: the differences occurring between obese and healthy subjects, the differences occurring between obese with type 2 diabetes and healthy subjects, and moreover the differences occurring between obese with type 2 diabetes and obese subjects. For each condition, a global bioinformatics characterization of the differentially expressed RNAs and the pathways in which they are involved was performed. These analyses extensively characterized the differentially expressed RNAs, highlighting their localization, interaction, and transcriptional regulation. Gene ontology analysis highlighted the gene-specific molecular functions and biological processes involved, and the most significant pathways in which the differentially expressed RNAs are involved were identified. Moreover, disease-related databases were interrogated and a screening of the gene relations with immunological, oncogenic and metabolic diseases were characterized.
Moreover, a special attention was given to non-coding RNAs, whose prevalence increases when switching from a “pure” obesogenic condition to a comorbidity with diabetes. Specifically, whilst non-coding genes are 6.43% of the differentially expressed RNAs in obese subjects, this percentage increases to up to 32.43% in diabetic subjects, and when considering the molecular underlining responsible for the additional diabetic phenotype (diabetic vs. obese), more than 50% of the differentially expressed RNAs are non-coding RNAs. This highlights how the non-coding epigenome could be of crucial relevance in the development of specific comorbidities, highlighting the possibility of new markers and targets for future therapeutic intervention and prevention. Lastly, functional experiments were performed on long-non coding RNAs deregulated in sottocutaneous adipose tissue from obese subjects, and results highlight how these are highly expressed in differentiated adipocytes, and predominantly regulated by adipogenesis transcription factors such as PPARy, C/EBPa, C/EBPb and C/EBPd. The results clearly highlight the role of the non-coding component in the development of the diabetic co-morbidity, and the investigation of this molecules could be of crucial relevance in understanding a new disease-mechanism never before analyzed, and even highlight why certain patients present a higher risk for diabetes development, paving the way for early intervention and precision medicine strategies.
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Souslova, Tatiana. "Transcriptional Regulatory Mechanisms of Freud-1, a Novel Mental Retardation Gene." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20044.
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The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.
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Kolar-Znika, Lorena. "Study of the organisation and the transcriptional activity of mouse major satellites." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066156/document.
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Dans les cellules de souris, l'hétérochromatine péricentromérique, caractérisée par les répétitions des satellites majeurs et une signature épigénétique spécifique, la triméthylation de l'histone H3 sur la lysine 9 (H3K9me3), est organisée en structures nucléaires particulières appelées chromocentres. Cette région est transcriptionnellement active, produisant des ARN non-codants. Pour caractériser le profil transcriptionnel des satellites majeurs, nous avons utilisé des oligonucléotides LNA séquence spécifiques, pour des expériences de northern blot. Nous avons mis en évidence un profil de transcription complexe, révélé avec les sondes conçues pour cibler les deux brins des répétitions des satellites majeurs. Ce profil est modulé en réponse au choc thermique, condition dans laquelle un court ARN transcrit par l'ARN polymérase III, est surexprimé. Cependant, des problèmes de spécificité inhérents à l'utilisation de ces sondes LNA, ne nous ont pas permis de confirmer que les transcrits détectés ont pour origine les satellites majeurs. La seconde partie de ce travail a consisté en l'étude de l'impact de la modification ciblée de H3K9me3 aux satellites majeurs par une protéine TALE fusionnée à l'histone déméthylase mJMJD2D. Nous avons montré que le signal H3K9me3 est aboli dans les cellules transfectées avec cette protéine TALE. La déméthylation provoque des changements morphologiques des chromocentres, tels que l'augmentation de la taille des foci de satellites majeurs, accompagnés par la diminution de leur nombre, suggérant la fusion de plusieurs chromocentres<br>In mouse cells, pericentromeric heterochromatin, characterized by major satellite repeats and a specific epigenetic signature, the trimethylation of the histone H3 at lysine 9 (H3K9me3) is organised in particular nuclear structures called chromocenters. This region is actively transcribed, producing non-coding RNA. To investigate the transcriptional profile of major satellites, we made used of the sequence specific LNA modified oligonucleotides in northern blot experiments. We have shown that a complex transcriptional pattern is revealed with the probes designed to target both strands of the major satellite repeat. This pattern is modified in response to heat shock, in which we reveal that a short, RNA polymerase III-transcribed RNA is overexpressed. However, specificity problems encountered with the use of these LNA probes inabled us to confirm with certainty the major satellite origin of the detected transcripts. The second part of this work consisted in the studying of the impact of the targeted modification of the H3K9me3 at the major satellites by a TALE protein fused to a histone demethylase, mJMJD2D. We have shown that the H3K9me3 signal is abolished in the cells transfected with this TALE protein. The demethylation triggers morphological changes of the chromocenters such as the increase of the major satellite foci size, that are accompanied by the decrease in the foci number, suggesting the merging of several chromocenters
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Fraser, Sherri D. "Post-transcriptional regulation by non-coding sequences of the c-myc transcript in Xenopus laevis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq20736.pdf.
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Chen, Dongsheng. "Transcriptional regulation of the p9Ka gene in metastatic and non-metastatic rat mammary epithelial cells." Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266488.
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Niemczyk, Malwina. "Epigenetic and transcriptional relationship between a novel long non-coding RNA GNG12-AS1 and imprinted DIRAS3." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708234.
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Siouda, Maha. "Transcriptional regulation and epigenetic repression of the tumor suppressor DOK1 in viral- and non viral-related carcinogenesis." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10163.
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Le suppresseur de tumeur DOK1 (downstream of tyrosine kinases1) est une protéine régulatrice de voies de signalisation impliquées dans des processus cellulaires tel que la prolifération, la migration et l'apoptose. Le rôle suppresseur de tumeur de DOK1 a été démontré dans des modèles animaux. Les souris knock-out pour DOK1 présentent une forte susceptibilité de développer des leucémies, des tumeurs malignes hématologiques, des adénocarcinomes pulmonaires, ainsi que des sarcomes histiocytaires agressifs. En outre, nous avons rapporté précédemment que le gène DOK1 peut être muté et son expression réprimée dans différentes tumeurs malignes humaines, telles que les lignées cellulaires de lymphome de Burkitt (BL) et la leucémie lymphoïde chronique (LLC). Cependant, les mécanismes de dérégulation de DOK1 restent inconnus, notamment dans les processus de carcinogenèse induite ou non par des oncovirus. Dans ce projet de thèse, nous avons d'abord caractérisé le promoteur de DOK1 et le rôle du facteur de transcription E2F1 comme le principal régulateur de l'expression de DOK1. Nous avons démontré pour la première fois la contribution de DOK1 dans la réponse cellulaire au stress par son rôle suppresseur de prolifération cellulaire et promoteur d'apoptose. Nous avons trouvé que l'expression du gène DOK1 est réprimée dans une variété de cancers humains, y compris le cancer de la tête et du cou, les lymphomes de Burkitt et les cancers du poumon. Cette répression est due à l'hyperméthylation aberrante de DOK1. Nous avons donc étudié les événements épigénétiques, qui sont souvent altérés dans les cancers, et leurs implications dans la répression de DOK1 dans les lignes cellulaire cancéreuses de la tête et du cou. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation de DOK1 par le virus d'Epstein Barr dans le cadre de sa propriété oncogénique dans les lymphocytes B humains ainsi que dans les lignes cancéreuses du lymphome de Burkitt. Nos résultats apportent de nouvelles informations sur les mécanismes de régulation de l'expression de DOK1 dans la carcinogenèse induite ou non par des oncovirus, ce qui pourrait le définir comme un biomarqueur potentiel de cancer et comme une cible intéressante pour des thérapies épigénétiques<br>The newly identified tumor suppressor DOK1 (downstream of tyrosine kinases1) inhibits cell proliferation, negatively regulates MAP kinase activity, opposes leukemogenesis, and promotes cell spreading, motility, and apoptosis. DOK1 also plays a role in the regulation of immune cell activation, including B cells. The tumor suppressor role of DOK1 was demonstrated in animal models. DOK1 knockout mice show a high susceptibility to develop leukemia, hematological malignancies as well as lung adenocarcinomas and aggressive histiocytic sarcoma. In addition, we previously reported that the DOK1 gene can be mutated and its expression is down-regulated in human malignancies such as Burkitt’s lymphoma cell lines (BL) and chronic lymphocytic leukemia (CLL). However, very little is known about the mechanisms underlying DOK1 gene regulation and silencing in viral- and non viral-related tumorigenesis. In the present project, we first characterized the DOK1 promoter. We have shown the role of E2F1 transcription factor as the major regulator of DOK1 expression and how DOK1 plays a role in DNA stress response though opposing cell proliferation and promoting apoptosis. We demonstrated that DOK1 gene expression is repressed in a variety of human cancers, including head and neck, Burkitt’s lymphoma and lung cancers, as a result of aberrant hypermethylation. We investigated the link between the epigenetic events and DOK1 silencing in non viral head and neck cancer cell lines, and by Epstein Barr virus in relation to its oncogenic activity in human B cells and neoplasia such as Burkitt’s lymphoma. These data provide novel insights into the regulation of DOK1 in viral and non viral-related carcinogenesis, and could define it as a potential cancer biomarker and an attractive target for epigeneticbased therapy
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Wang, Xiangting. "Stress-induced non-coding RNAs allosterically regulate TLS, a HAT-inhibitory RNA binding protein, to mediate transcriptional repression." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3254429.
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Thesis (Ph. D.)--University of California, San Diego, 2007.<br>Title from first page of PDF file (viewed May 2, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 69-83).
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Tran, Van Giang. "Régulation de l'expression du gène Igf2 : nouveaux promoteurs et implication de longs ARN non-codants." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20022/document.
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L'expression du gène Igf2, qui est soumis à l'empreinte génomique parentale chez les mammifères, est hautement régulée au cours du développement embryonnaire et de la période périnatale grâce à divers mécanismes transcriptionnels et post-transcriptionnels. Ces mécanismes mettent à contribution de longs ARN non codants produits au sein même du locus, dont le plus connu est l'ARN H19. En utilisant une approche de complémentation génétique par un transgène H19 dans myoblastes H19 KO de souris, nous démontrons l'existence de plusieurs nouveaux promoteurs d'Igf2. L'un de ces promoteurs, qui est conservé chez l'homme, peut être activé par un ARN ectopique antisens d'H19 (lncARN 91H) en dépit d'une méthylation complète de la région de contrôle empreinte située en cis sur le même allèle. Nous montrons également que les lncARN 91H présentent une certaine spécificité tissulaire et que leur transcription peut être initiée à partir des séquences conservées CS4, CS5 et CS9 situées en aval du gène H19. Quant à l'ARN H19, qui est l'ARN non codant majeur du locus, il semble pouvoir réguler ses transcrits antisens dans les myoblastes H19 KO complémentés par le transgène H19, mais surtout il participe activement à la régulation post-transcriptionnelle du gène Igf2 chez la souris. Nous observons en effet qu'il favorise la coupure endoribonucléolytique de l'ARN Igf2 par un mécanisme qui reste à découvrir. Enfin, nous mettons en évidence l'existence d'un l'arrêt de l'élongation de la transcription du gène d'Igf2, pour lequel nous proposons un modèle de régulation faisant intervenir un autre long ARN non codant du locus: le lncARN PIHit. Au-delà des mécanismes qui restent à explorer, nos résultats renforcent l'idée que la structure tridimensionnelle de la chromatine participe à la régulation de l'expression des gènes chez les mammifères<br>In mammals, the expression of the Igf2 gene, which is subject to parental genomic imprinting, is tightly regulated during embryonic development and the perinatal period through several transcriptional and post-transcriptional mechanisms. These mechanisms are involving long non-coding RNAs (lncRNAs) produced within the locus; among them the best known is probably the H19 RNA. Using a genetic complementation assay consisting in transfections of an H19 transgene into H19 KO myoblasts, we discovered several novel Igf2 promoters in the mouse. One of these promoters, that is conserved in the human, can be activated by ectopic H19 antisens RNAs (91H lncRNAs) despite a complete methylation of the Imprinting-Control Region located in cis on the same allele. We also show that the 91H lncRNAs possess some tissue-specific features and that their transcription can be initiated from the CS4, CS5 and CS9 conserved sequences located downstream of the H19 gene. On the other hand, the H19 RNA, that is the major lncRNA of the locus, appears to regulate its antisense transcripts in H19 KO myoblasts complemented with the H19 transgene, but its major function seems to be in regulating post-transcriptionally the Igf2 gene expression. Indeed, we have observed that it favours the endoribonucleolytic cleavage of the Igf2 messenger RNAs through a mechanism that remains to be elucidated. Finally, we reveal the existence of a premature transcriptional elongation stop of the Igf2 gene, for which we propose a regulation model involving another lncRNA of the locus: the PIHit lncRNA. Beyond the mechanisms that remain to be explored, our results strengthen the idea that, in mammals, the three-dimensional organization of the chromatin is involved in regulating gene expression
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Musahl, Anne-Susann [Verfasser]. "Transcriptional characterization and functional analysis of long non-coding RNA/protein-coding gene pairs encoded in the human genome / Anne-Susann Musahl." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1075493706/34.
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Dam, Sushovan. "Post-transcriptional regulation of porin expression in Escherichia coli and its impact on antibiotic resistance." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0641/document.
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Chez les bactéries à Gram-négatif, l’imperméabilité de la membrane externe est un facteur majeur contribuant au développement de la résistance. Chez Escherichia coli, les porines OmpF et OmpC sont des protéines de la membrane externe qui forment des canaux pour la diffusion de petites molécules hydrophiles tels que les antibiotiques. L’expression des porines est soumise à une régulation fine, et des petits ARN non-codants (sRNAs, small RNAs) jouent un rôle important au niveau post-transcriptionnel. Dans ce cadre, et en utilisant E. coli comme bactérie modèle, les objectifs de mon travail de thèse étaient : (1) de caractériser la régulation du sRNA MicC et la co-régulation putative de la porine quiescente OmpN; (2) d’examiner l'effet global de MicC sur le transcriptome; (3) d’analyser l'impact de l'expression de MicC sur la sensibilité aux antibiotiques. Les résultats obtenus montrent l’induction de MicC en présence d'antibiotiques de la famille des β-lactamines, ou en l’absence du facteur sigma de réponse au stress de l’enveloppe sigmaE. Ces mêmes conditions activent aussi l'activité d'une fusion ompN-lacZ, indiquant une régulation transcriptionnelle commune de micC et ompN. Etant donnée la conservation de MicC chez les entérobactéries, nous avons effectué une étude par RNASeq pour déterminer l'impact de la surexpression de MicC sur le transcriptome d’E. coli et identifié 60 ARNm régulés par MicC en plus de sa cible initiale ompC. L'identification des spectres cibles globaux des sRNAs est importante pour comprendre leur importance dans la physiologie bactérienne, ici celui de MicC dans la résistance aux antibiotiques<br>A major factor contributing to antimicrobial resistance is the inability of antibiotics to penetrate the bacterial outer membrane to reach their target. In Escherichia coli, the two abundantly expressed porins OmpF and OmpC form channels for diffusion of small hydrophilic molecules including antibiotics. The expression of porins is under complex regulation and the small regulatory RNAs (sRNAs) fine tune the porin expression level at post-transcriptional level. MicF and MicC are the two major sRNAs that negatively regulate expression of OmpF and OmpC, respectively. Interestingly, these two sRNAs are encoded next to porin gene, i.e. micF-ompC and micC-ompN, suggesting a dual regulation. Our goals in this work were: (1) to characterize the regulation of the sRNA MicC and the putative co-regulation of the quiescent porin OmpN in E. coli; (2) to examine the global effect of MicC on the E. coli transcriptome; (3) to analyze the impact of MicC expression on antibiotic susceptibility. Our work shows that the expression of micC was increased in the presence of carbapenems and cephalosporins and in an rpoE depleted mutant. The same conditions enhanced the expression of OmpN, suggesting a dual regulation of micC and ompN. We also performed RNA sequencing to determine the impact of MicC overexpression on E. coli transcriptome. This identified 60 mRNA targets negatively regulated by MicC apart from its original target. Identification of the global target spectra of MicC is of importance to understand its importance on the overall bacterial physiology, and more specifically on AMR
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Brown, Robert Vincent. "The Regulatory Significance and Molecular Targeting of Novel Non-B-DNA Secondary Structures Formed from the PDGFR-Beta Core Promoter Nuclease Hypersensitivity Element." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/337361.
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Vitsios, Dimitrios. "Elucidating the function and biogenesis of small non-coding RNAs using novel computational methods & machine learning." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/273493.
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The discovery of RNA in 1868 by Friedrich Miescher was meant to be the prologue to an exciting new era in Biology full of scientific breakthroughs and accomplishments. Since then, RNAs have been proven to play an indispensable role in biological processes such as coding, decoding, regulation and expression of genes. In particular, the discovery of small non-coding RNAs and especially miRNAs, in C. elegans first and thereafter to almost all animals and plants, started to fill in the puzzle of a complex gene regulatory network present within cells. The aim of this thesis is to shed more light on the features and functionality of small RNAs. In particular, we will focus on the function and biogenesis of miRNAs and piRNAs, across multiple species, by employing advanced computational methods and machine learning. We first introduce a novel method (Chimira) for the identification of miRNAs from sets of animal and plant hairpin precursors along with post-transcriptional terminal modifications that are not encoded by the genome. This method allows the characterisation of the prevalence of miRNA isoforms within different cell types and/or conditions. We have applied Chimira within a larger study that examines the effect of terminal uridylation in RNA degradation in oocytes and cells in either embryonic or adult stage. This study showed that uridylation is the predominant transcriptional regulation mechanism in oocytes while it does not retain the same functionality on mRNAs and miRNAs, both in embryonic and adult cells. We then move on to a large-scale analysis of small RNA-Seq datasets in order to identify potential modification signatures across specific conditions and cell types or tissues in Human and Mouse. We extracted the full modification profiles across 461 samples, unveiling the high prevalence of modification signatures of mainly 1 to 4 nucleotides. Additionally, samples of the same cell type and/or condition tend to cluster together based on their miRNA modification profiles while miRNA gene precursors with close genomic proximity showed a significant degree of co-expression. Finally, we elucidate the determinant factors in strand selection during miRNA biogenesis as well as update the miRBase annotation with corrected miRNA isoform sequences. Next, we introduce a novel computational method (mirnovo) for miRNA prediction from RNA-Seq data with or without a reference genome using machine learning. We demonstrate its efficiency by applying it to multiple datasets, including single cells and RNaseIII deficient samples, supporting previous studies for the existence of non-canonical miRNA biogenesis pathways. Following this, we explore and justify a novel piRNA biogenesis pathway in Mouse which is independent of the MILI enzyme. Finally, we explore the efficiency of CRISPR/Cas9 induced editing of miRNA targets based on the computationally predicted accessibility of the targeted regions in the genome. We have publicly released two web-based novel computational methods and one on-line resource with results regarding miRNA biogenesis and function. All findings presented in this study comprise another step forward within the journey of elucidation of RNA functionality and we believe they will be of benefit to the scientific community.
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Dequivre, Magali. "Implication des ARN non codant dans la virulence du phytopathogène Agrobacterium fabrum C58." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10016/document.
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L'une des caractéristiques majeures des microorganismes, et donc des bactéries, est qu'ils sont en contact direct avec l'environnement et doivent donc percevoir et répondre rapidement à ses variations. Pour cela, plusieurs niveaux de contrôle existent, et récemment le rôle des ARN non codants régulateurs, ou riborégulateurs, a été mis en lumière comme un mécanisme de contrôle peu couteux et rapide pour la cellule. Chez le phytopathogène Agrobacterium fabrum (anciennement appelé Agrobaterium tumefaciens), la virulence est principalement régulée au niveau transcriptionnel par le système à deux composants VirANirG. L'implication des riborégulateurs dans la virulence d'A.fabrum est encore mal connue et ces travaux de thèse ont eu pour objectif de déterminer l'implication de riborégulateurs dans le cycle infectieux de cette bactérie.Pour cela, nous avons identifié l'ensemble des transcrits d 'A. fabrum C58 en combinant l'utilisation de plusieurs méthodes d'analyses globales et nous avons étudié la fonction de différents candidats transcrits à partir du plasmide Ti (plasmide de virulence). Des souches modifiées dans la production des riborégulateurs candidats ont été construites, Jeurs ARNm cibles ont été prédits et validés, et des tests phénotypiques, en particulier des tests de virulence, ont été réalisés. Ainsi, le séquençage des transcrits de petite taille a permis d'identifier plus d'un millier de riborégulateurs potentiels dont plusieurs sont exprimés à partir de régions en relation avec le cycle infectieux. Nous avons validé 4 de ces petits transcrits comme étant des riborégulateurs puisqu'ils sont de petite taille, non traduits en protéine et fortement structurés (RNAI 111, RNA1083, RNA1059 et RNA1051) . Plus particulièrement, nous avons montré que le riborégulateur RNAI 111 était nécessaire pour la virulence d'A.fabrum C58, et que son action semblait se faire au travers du contrôle post-transcriptionnel de gènes impliqués dans les fonctions de virulence et de transfert du plasmide Ti. Un rôle plus modéré du riborégulateur RNA1083 dans le contrôle du cycle infectieux a également été observé, potentiellement au travers de la modulation de la mobilité et du transfert conjugatif du plasmide Ti. D'autre part, nous avons mis en évidence deux autres riborégulateurs, RNA1059 et RNA1051, qui sont impliqués dans le contrôle du maintien du plasmide Ti via une implication dans la réplication du plasmide (RNA 1059) et via une implication dans un nouveau system de toxine-antitoxine présent sur le plasmide Ti (RNA1051). Ainsi à partir d'une analyse globale nous avons mis en évidence le rôle des riboregulateurs dans les systèmes de mise en place de l'infection bactérienne , soit via le contrôle de facteurs de virulence, soit via le contrôle de la persistance du plasmide responsable de la virulence<br>One of the main characteristics of microorganisms, including bacteria., is their direct interaction with their environment. They thus need to perceive and quickly answer to its variations. Several steps of control exist, and recently the role of regulatory non-coding RNA, or riboregulator, was highlighted as a fast and economic mechanism of regulation. In the phytopathogen Agrobacterium fabrum (previously named Agrobacterium tumefaciens), the virulence is mainly controlled transcriptionally by the two components system VirANirG. The implication of riboregulators in the virulence of this bacterium is still unknown . The objectives of this thesis were to identify A .fabrum riboregulators and to determine their involvement in the infectious cycle of the bacteria. To this end, we identified small transcripts of A . fabrum C58 strain by combining several global analyses, and we studied the function of different candidates transcribed from the Ti plasmid (the virulence plasmid). Strains modified in the production of these candidates were constructed, their mRNA targets were predicted and validated, and phenotypic analyses -especially virulence tests were realized.Thereby, small transcript deep-sequencing allowed the identification of a thousand potential riboregulators, some of them being transcribed from regions related to the infectious cycle. We validated 4 of these transcripts as riboregulators according to their small size, their strong secondary structure and their non-translation into protein (RNAIOS I, RNA1059, RNA1083 and RNAl ll l). In particular, we showed that RNA 1111 was necessary for the virulence of A. fabrum C58, and that it seems to act through the posttranscriptional control of genes implicated in virulence functions and in Ti plasmid conjugation. A more moderated role of RNA 1083 was also observed, potentially by the modulation of the bacterial mobility and of the plasmid conjugation. Furthermore, we highlighted two riboregulators, RNA1059 and RNA1051, involved in the control of the Ti plasmid persistence, through their implication in the replication of the plasmid (RNA1059) and in a toxin-antitoxin system present on the Ti plasmid (RNA1051) .Thus, from a global analysis, we brought out the role of riboregulators in the control of several steps of the infectious cycle of A. fabrum C58, through the control of virulence factors, or through the contrai of the persistence of the main actor of the virulence, the Ti plasmid
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Dangin, Mathieu. "Contrôle de la différenciation sexuelle de la levure Schizosaccharomyces pombe par un ARN non-codant et la protéine de liaison à l’ARN Mmi1." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV079/document.
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Au cours des cinq dernières années l’existence d’un contrôle de la transcription par les ARN non-codants longs (lncRNAs) a été décrite dans une large variété d’eucaryotes. Cependant, les mécanismes par lesquels les lncRNAs régulent la transcription restent en grande partie méconnus. Les premiers travaux effectués dans le cadre de cette thèse ont participé à la caractérisation du mécanisme mis en jeu par un lncRNA, nommé nam1, dans le contrôle de l’entrée en différenciation sexuelle chez la levure Schizosaccharomyces pombe. Il a ainsi été montré qu’au cours de sa synthèse le lncRNA nam1 est ciblé par la protéine de liaison à l’ARN Mmi1 et une machinerie de surveillance des ARN qui comprend l’exosome, un complexe de dégradation des ARN conservé au cours de l’évolution. La fixation de Mmi1 au lncRNA nam1 contrôle la terminaison de la transcription de nam1 et empêche ainsi la transcription de se poursuivre et d’interférer alors avec la transcription du gène situé en aval (codant pour une MAP kinase essentielle à l’entrée en différenciation). Les travaux suivant montrent l’implication dans ce mécanisme de la protéine Cti1, un des co-facteurs connus de l’exosome. Fait marquant, ces travaux rapportent aussi l’existence d’un mode de production inédit pour un lncRNA. En effet, ils révèlent que la transcription non-interrompue d’un gène codant conduirait à la production d’un ARN bi-cistronique. La maturation co-transcriptionnelle de cet ARN bi-cistronique produirait, d’un côté, un ARN messager et, de l’autre, le lncRNA nam1. Enfin, ils ont permit la caractérisation initiale d’un nouveau composant de la machinerie de surveillance des ARN recrutée sur nam1 par Mmi1. Ainsi, dans leur ensemble, ces travaux contribuent à une meilleure connaissance des mécanismes pouvant être mis en jeu par un lncRNA et agissant en cis pour réguler l’expression génique et, à travers elle, des processus cellulaires majeurs, tel que la différenciation cellulaire. De plus, ils décrivent un nouveau mécanisme de biogénèse d’un lncRNA<br>Over the last five years, the control of transcription mediated by long non-coding RNAs (lncRNAs) has been reported to take place in a wide variety of eukaryotes. However, the mechanisms by which lncRNAs regulate transcription remain relatively poorly described. The first work conducted in the context of this PhD thesis has contributed to the characterization of the mechanism used by a lncRNA, named nam1, to control entry into sexual differentiation of the fission yeast Schizosaccharomyces pombe. It was shown that, while the lncRNA nam1 is being produced, it is targeted by the RNA binding protein Mmi1 and a RNA surveillance machinery that includes the exosome, a conserved complex throughout evolution. The binding of Mmi1 to nam1 lncRNA controls the termination of transcription of nam1, which prevents this non-coding transcription from interfering with the transcription of the downstream gene, coding for a MAP kinase essential to entry into differentiation. The following work shows the importance of the protein Cti1, one of the known co-factor of the exosome, in the nam1-dependent control of sexual differentiation. Remarkably, it also strongly suggests the existence of a new way of producing a lncRNA. Indeed, it reveals that read-through transcription of a protein-coding gene leads to the production of a bi-cistronic RNA, which is co-transcriptionally matured to produce on one side a messenger RNA and on the other side the lncRNA nam1. Finally, this work initiated the characterization of a new component of the RNA surveillance machinery targeting nam1. Collectively, this work brings several insights into the mechanisms used by cis-acting lncRNAs to regulate gene expression and, thereby, major cellular processes such as cell differentiation. Moreover, it also provides insights into the biogenesis of lncRNAs by reporting a new mode of production of lncRNAs
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Richard, Audrey. "Oncolytic H-1 parvovirus NS1 protein : identifying and characterizing new transcriptional and posttranslational regulatory elements." Phd thesis, Université du Droit et de la Santé - Lille II, 2011. http://tel.archives-ouvertes.fr/tel-00826936.
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H 1 parvovirus (H-1PV) is a little single stranded DNA virus that preferentially replicates in a lytic manner in transformed cells due to their expression profile that meets the requirements for the activation of H¬ 1 PV life cycle unlike normal cells. This feature is known as oncotropism. H 1PV genome is constituted by two transcriptional units. The first one is driven by the proliferation and transformation dependent P4 promoter and allows the expression of both non structural proteins NS1 and NS2, and the second one controls the expression of both capsid proteins VP1 and VP2 through the activation of P38 promoter. H-1PV life cycle tightly depends on NS1 protein that is involved in crucial events, including viral DNA replication, P38 promoter activation as well as cytotoxicity.NS1 protein is regulated at both transcriptional and post translational levels.My thesis aimed at identifying new determining elements for both of these regulations and characterizing their involvement in both H-1PV life cycle and oncotropism.
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Frapporti, Andrea. "Programmed genome rearrangements in Paramecium tetraurelia : identification of Ezl1, a dual histone H3 lysine 9 and 27 methyltransferase." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC250.
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Chez les eucaryotes, le génome est organisé en chromatine, une structure nucléoprotéique essentielle pour la régulation de l’expression génique ainsi que pour le maintien de la stabilité du génome. Les ciliés sont d’excellents organismes modèles pour étudier les mécanismes généraux qui maintiennent l’intégrité du génomes eucaryote. Chez Paramecium tetraurelia, la différentiation du génome somatique à partir du génome germinal est caractérisée par des événements massifs et reproductibles d’élimination d’ADN. D’une part, des éléments répétés (transposons,régions minisatellites), de plusieurs kilobases de long, sont imprécisément éliminés.D’autre part, 45000 séquences courtes et uniques, appelées IES, sont précisément éliminées au nucléotide près. Une classe de petits ARN, appelé scnRNAs, est impliquée dans la régulation epigénétique de l’élimination d’ADN, mais comment les scnRNA contrôlent l’élimination d’ADN reste mystérieux. Nous avons testé l’hypothèse selon laquelle une organisation particulière de la chromatine, en particulier des modifications post-traductionelles des histones associées à des formes répressives de la chromatine, est impliquée dans le processus d’élimination d’ADN. Nous avons montré que la triméthylation de l’histone H3 sur la lysine 9 et la lysine 27 (H3K9me3 et H3K27me3)apparaît transitoirement dans le noyau somatique en développement au moment où se produisent les événements d’élimination d’ADN. Nous avons identifié la protéine de type Polycomb, Ezl1, et montré qu’elle est une histone methyltransferase qui présente une dualité de substrat et catalyse à la fois la mise en place de K9me3 et K27me3 sur l’histone H3. Nous avons montré que la déposition de H3K9me3 et H3K27me3 dans le noyau en développement requiert les scnRNAs. Des analyses de séquençage haut débit ont montré que Ezl1 est requise pour l’élimination des longues séquences répétées germinales, suggérant que les scnRNA guident la déposition des marques d’histones au niveau de ces séquences. Au contraire des régions répétées du génome, les IES montrent une sensibilité différente aux scnRNAs et à Ezl1, suggérant que plusieurs voies partiellement chevauchantes sont impliquées dans leur élimination. Notre étude montre que des caractéristiques intrinsèques des séquences d’ADN, telles que leur taille, peut contribuer à la définition des séquences germinales à éliminer. De manière intéressante, nous avons aussi montré que Ezl1 est requise pour la répression transcriptionnelle des éléments transposables. Nous suggérons que les voies H3K9me3et H3K27me3 coopèrent et contribuent à préserver le génome somatique de Paramecium des parasites génomiques<br>Eukaryotic genomes are organized into chromatin, a complex nucleoprotein structureessential for the regulation of gene expression and for maintaining genome stability.Ciliates provide excellent model organisms with which to gain better understandinginto the regulation of genome stability in eukaryotes. In the ciliate Parameciumtetraurelia, differentiation of the somatic genome from the germline genome ischaracterized by massive and reproducible programmed DNA elimination events. Longregions of several kilobases in length, containing repeated sequences and transposableelements are imprecisely eliminated, whereas 45,000 short, dispersed, single-copyInternal Eliminated Sequences (IESs) are precisely excised at the nucleotide level. Aspecific class of small RNAs, called scnRNAs, is involved in the epigenetic regulation ofDNA deletion. How scnRNAs may guide DNA elimination in Paramecium remains tobe discovered. Here, we investigated whether chromatin structure, in particular histonepost-translational modifications known to be associated with repressive chromatin,might control DNA elimination. We showed that trimethylated lysine 9 and 27 onhistone H3 (H3K9me3 and H3K27me3) appear in the developing somaticmacronucleus when DNA elimination occurs. We identified the Polycomb-groupprotein, Ezl1, and showed that it is a dual histone methyltransferase that catalyzes bothH3K9me3 and H3K27me3 in vitro and in vivo. Genome-wide analyses show thatscnRNA-mediated H3K9me3 and H3K27me3 deposition is necessary for theelimination of long, repeated germline DNA. Conversely, single copy IESs displaydifferential sensitivity to depletion of scnRNAs and Ezl1, unveiling the existence ofpartially overlapping pathways in programmed DNA elimination. Our study revealsthat cis-acting determinants, such as DNA length, also contribute to the definition ofgermline sequences to delete. We further showed that Ezl1 is required fortranscriptional repression of transposable elements. We suggest that H3K9me3 andH3K27me3 pathways cooperate and contribute to safeguard the Paramecium somaticgenome against intragenomic parasites
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Ding, Shuai. "Characterization of P.falciparum histone methyltransferases : biological role and possible targets for new intervention strategies." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066578/document.
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On a montré que les PTM jouaient un rôle significatif de P. falciparum dans l'année de contrôle de la régulation transcriptionnelle, de l'expression monoaléique et de la différenciation sexuelle. Dix SET contenant des HKMTs contenant du domaine-ont été prédits; Six d'entre eux être essentiels pour le développement de stages de sang asexués. Le projet de thèse est centré sur la caractérisation biologique de PfSET7 et PfSET6. Nous avons observé l'échange de localisation cellulaire dynamique Pendant le cycle de vie: PfSET7 se trouvent dans de multiples foyers cytoplasmiques dans les stades érythrocytaires asexués et le stage de foie, et plus frappante, dans la membrane du parasite enrichie en gamétocytes. PfSET6 EXPOSÉ une localisation nucléaire dans les anneaux et un modèle ponctué dans le cytoplasme des trophozoites matures et schizontes, et est enrichi dans les structures de foyers dans le cytoplasme des gamétocytes. Pris ensemble, notre étude suggère que la méthylation non histone est beaucoup plus significative chez P. falciparum que précédemment attendu. La méthylation à médiation par PfSET7 Peut être une extension du code histone à - d'autres protéines cytosoliques; Partiellement PfSET6 s'associe à des voies de répression transcriptionnelle dans le noyau et des régulateurs post-transcriptionnels dans le cytoplasme. Une étude plus approfondie vise à identifier des cibles de domaine SET contenant des protéines dans le gène inducible knockout mutant parasite lignes. Le fait que PfSET7 et PfSET6 sont exprimés à différents stades du cycle de vie, les fait comme de nouvelles cibles pour le développement de médicaments contre cette maladie grave et de bloquer la transmission<br>In P. falciparum, PTMs have been shown to play an important role in the control of transcriptional regulation, monoallelic expression, and sexual differentiation. Ten SET domain-containing HKMTs have been predicted; six of them appear to be essential for asexual blood stage development. My lab has expressed and purified two enzymatically active recombinant methyltransferase PfSET7 and PfSET6. In vitro enzyme kinetics assays shows they can methylate histones. The dissertation project is centered around the biological characterization of PfSET7 and PfSET6. We observed the dynamic changes of cellular localization during life cycle: PfSET7 are found in multiple cytoplasmic foci in asexual erythrocytic stages and liver stage, and more strikingly, enriched in parasite membrane in gametocytes. PfSET6 exhibited a nuclear localization in rings and a punctuated pattern in the cytoplasm of mature trophozoites and schizonts, and is enriched within foci-like structures in the cytoplasm of gametocytes. Taken together, our study suggests that non-histone methylation is much more important in P. falciparum than previously anticipated. PfSET7-mediated methylation may be an extension of the histone code to other cytosol proteins; PfSET6 partially associates with transcriptional repression pathways in the nucleus and post-transcriptional regulators in the cytoplasm. Further study aims to identify targets of SET domain containing proteins within the inducible gene knock out mutant parasite lines. The fact that PfSET7 and PfSET6 are expressed in different life cycle stages, makes them as novel targets for drug development that could against severe disease and to block pathogen transmission
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Bardou, Florian. "Les AtNSRs, protéines régulatrices de l’épissage alternatif et du silencing post transcriptionnel." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112054/document.
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Chez les eucaryotes, plusieurs protéines liant l'ARN ou RBPs agissent sur l'ARNm à différents niveaux, de l'épissage à la traduction. Récemment, un grand nombre d’ARN non-codant des protéines (npcRNAs) ont été identifiés chez les eucaryotes et ont été montré comme interagissant avec une variété de ribonucléoprotéines (RNP) pour contrôler l'expression des gènes au niveau post-transcriptionnel. Nous avons identifié une Nuclear-Speckle RBP (ou NSR) qui interagit avec le npcRNA, ENOD40, un lncARN qui s'accumule au cours de la formation des racines latérales et des nodules chez les légumineuses. Durant cette thèse nous avons analysé le rôle des NSR d’Arabidopsis thaliana ainsi que leur lien avec les npcARN.Deux gènes AtNSRs homologues existent chez Arabidopsis nommés NSRa et NSRb, ces gènes codent des protéines localisées dans des speckles nucléaires avec certaines protéines apparentées à l’épissage. Fait intéressant, les fusions AtNSR-GFP sont relocalisées dans des granules cytoplasmiques dans certaines cellules des racines différenciées ainsi que lors d’une co-expression éctopique de ENOD40. Le gène AtNSRb est régulé par l'auxine alors AtNSRa est constitutif. Les simples mutants Atnsr ne montrent pas de phénotype, mais la croissance des racines des doubles mutants est partiellement insensible à l'auxine, ce qui suggère une fonction redondante de ces protéines dans les racines. La localisation observée pour ces protéines nous a mené à explorer un rôle des NSRs dans l’épissage, nous avons donc analysé le profil d'épissage de 288 gènes en réponse à l'auxine chez Arabidopsis et comparé ces profils entre le WT et les mutants nsra/nsrb. Tout d’abord nous avons remarqué que l’épissage général ne variait pas, en revanche, l’analyse de 288 gènes alternativement épissés montre que le profil d'épissage de 77 gènes semble être modifié durant la réponse à l'auxine et 51 gènes nécessitent les protéines AtNSR pour ce changement. Afin de vérifier l’interaction des NSRs avec les cibles d’AS et avec les npcARN nous avons co-immunoprécipité les NSRs et nous avons identifié au moins 5 cible d’AS et 2 npcARN. L’expression de l’ARN ENOD40 ainsi que du partenaire npcARN module L’AS chez Arabidopsis. Dans un deuxième chapitre, nous avons exploré le rôle des NSRs dans le PTGS déclenché par un transgène contenant un intron ce qui nous a permis de lier l’épissage alternatif et le silencing. Nous proposons donc que les NSRs pourraient lier l’épissage alternatif et l’action des ARN non codants, notamment lors de la croissance de la racine<br>In eukaryotes, several RNA binding proteins (RBPs) act on mRNA at various levels from splicing to translation. Recently a large number of non-protein coding RNAs (npcRNAs) have been identified in eukaryotes and shown to integrate into a variety of ribonucleoproteins (RNP) to control posttranscriptional gene expression. Our laboratory has identified a plant Nuclear-Speckle RBP (or NSR) that interacts with an npcRNA, ENOD40 that accumulates during lateral root and nodule formation in legumes. NSR is relocalised into a cytoplasmic RNP in the ENOD40-expressing cells. During this PhD, we have analysed the role of NSRs in Arabidopsis thaliana and its link with npcRNAs. Two AtNSR homologs from Arabidopsis thaliana, named AtNSRa and AtNSRb, code for proteins also localised in nuclear speckles together with certain splicing-related proteins. Interestingly, AtNSR-GFP fusions are relocalised into cytoplasmic granules in certain differentiated root cells and by ectopic expression of the ENOD40 RNA. The AtNSRb gene is regulated by auxin whereas AtNSRa is constitutive. Root growth and lateral root formation of double nsra/nsrb mutants is partially insensitive to auxin. The localisation of these proteins prompted us to explore roles in splicing. No defects in general splicing were observed however analysis of 288 alternatively spliced genes in WT and nsra/nsrb roots in response to auxin revealed 77 changes in splicing profiles in response to auxin from which 51 required AtNSRs. In order to validate the interaction of NSRs with alternatively spliced mRNAs and npcRNAs, we have co-immunoprecipitated NSRs and identified at least 5 interacting alternatively spliced mRNAs and 2 npcRNAs. Expression of the ENOD40 RNA or one interacting ncRNA modulate alternatively splicing in Arabidopsis. In a second chapter, we explored the role of NSRs in the modulation of PTGS triggered by intron-containing transgenes allowing us to link alternatively splicing and silencing. We propose that NSRs may link alternative splicing and the action of non-coding RNA, notably during root growth and development
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Cedrone, L. "THE ROLE OF ENHANCED POLYCOMB REPRESSIVE COMPLEX 2 ACTIVITY IN TUMORIGENESIS." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/468289.
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Polycomb Group of proteins are essential factors present in cells’ nuclei. These multiprotein complexes are key repressive chromatin factors that regulate cellular differentiation during development, contributing to the correct establishment of lineage-specific transcriptional programs. Moreover, they represent key factors of proliferation and deregulation of their levels and activity have been linked to the onset and development of several human cancers.
Recently, gain of function heterozygous EZH2 mutations have been discovered in non-Hodgkin lymphomas and melanomas. These mutations cause an aminoacidic substitution within the EZH2 catalytic SET domain (Y641), resulting in increased H3K27me3 deposition. Very little is known about this mutated enzyme, therefore the aim of my thesis is trying to unravel the tumorigenic mechanisms underlying these mutations. To understand a general oncogenic role for this mutated enzyme, we used MEF as an alternative, simpler model system. We observed increased deposition of H3K27me3 without any relevant transcriptional alteration at steady state, confirming our results also in lymphoma cell lines. To investigate a cooperative transcriptional deregulation for mutant EZH2, we then subjected MEFs to three different stimuli (starvation, myc upregulation and reprogramming to pluripotency). Since we found this to be true only during cell-fate transition, we proposed a model in which the levels of the H3K27me3 are increasingly deposited where the mark is already present at steady state. This could be relevant in lymphomas, impeding centroblasts differentiation and resulting in tumorigenesis in the presence of concomitant oncogenic mutations. This observation could shed light on the molecular mechanisms underlying lymphomagenesis in patients.
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Rasschaert, Perrine. "Régulation transcriptionnelle et post-transcriptionnelle des gênes LAT et ICP4 du virus de la maladie de Marek." Thesis, Tours, 2015. http://www.theses.fr/2015TOUR4011/document.
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Le virus de la maladie de Marek (MDV) est un virus oncogène responsable des lymphomes T chez les poulets. L´infection par ce virus est divisée en une phase lytique dépendante de l´expression du gène très précoce ICP4 et une phase latente, caractérisée par l´expression de l’ARN long non codant LAT localisé en antisens. Nous avons montré que l’expression différentielle des miARN du cluster mdv1-miR-M8-M10 était directement corrélée à l’épissage alternatif de l’intron 1 du LAT et plus particulièrement à la biogenèse par le splicéosome du premier mirtron viral. La présence du mirtron mdv1-miR-M6 au milieu du cluster est associée à une cinétique d’expression des miARN. En parallèle, nous avons identifié deux promoteurs alternatifs de type Sp1, quatre signaux poly-A et trois exons associés à la régulation de la transcription du transcrit ICP4. Nous avons prédits cinq isoformes potentielles pour la protéine ICP4 et avons pu observer par immunodétection que la protéine était exprimée principalement dans le cytoplasme des cellules infectées en phase lytique ou de réactivation<br>The Marek disease virus (MDV) is an oncogenic herpesvirus responsible of T-cell lymphoma in chicken. MDV infections are divided into a lytic phase, depending on the expression of immediate early gene like ICP4, and a latent phase characterized by the expression of the long non-coding RNA LAT localized in antisense. In this study, we have shown the differential expression of the cluster of miRNA mdv1-miR-M8-M10 was directly correlated with the alternative splicing of LAT’s intron 1 and more specifically with the first viral mirtron biogenesis by the spliceosome. The location of the mirtron mdv1-miR-M6 inside of the cluster is associated with a two-step biogenesis of the miARN of the cluster. On the other hand, we have identified a dual promoter that responded to Sp1, four poly-A signals and three exons that are responsible of transcriptional regulation of ICP4 transcript. We also have predicted five potential isoproteines for ICP4 and were able to observe by immunodetection that ICP4 was mainly expressed in the cytoplasm of infected cells during the lytic phase or the reactivation one
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Bovolenta, Luiz Augusto. "Análise exploratória em larga escala de microRNAs expressos em tilápia do Nilo utilizando ferramentas de bioinformática." Botucatu, 2016. http://hdl.handle.net/11449/148567.
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Orientador: Ney Lemke<br>Resumo: MicroRNAs (miRNAs) são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão de genes, modelando o transcriptoma e a produção de proteínas. Em geral, os miRNAs são conservados no genoma de eucariotos, sendo considerados elementos vitais em diversos processos biológicos durante o desenvolvimento, tais como crescimento, diferenciação e morte celular. A grande diversidade de miRNAs identificados está restrita a poucas espécies e apenas uma parte do total de alvos de miRNAs preditos foi caracterizada funcionalmente. Nesse contexto, o uso da tecnologia de sequenciamento de alto rendimento (high throughput sequencing) atrelada à análise de nível transcricional por RT-qPCR possibilitam a identificação do microRNoma. A tilápia do Nilo, Oreochromis niloticus, é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e evolutiva. O presente trabalho teve como objetivos: organizar os dados do sequenciamento dos miRNAs da tilapia do Nilo; disponibilizá-los em forma de uma base de dados para a comunidade científica; integrar as informações dos miRNAs identificados com outros bancos de dados de miRNAs; analisar os dados através de análises de bioinformática para determinação de agrupamentos definidos pelo nível de expressão de cada miRNA em seis tipos de tecido (músculo branco, músculo vermelho, testículo, ovário, fígado, olho, cérebro e coração) com distinção entre os gêneros e nas fases do desenvolvimento (2,... (Resumo completo, clicar acesso eletrônico abaixo)<br>Doutor
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Saadeh, Bashir. "Characterization and search for virulence-related factors in “Classical” and “New” Brucella species." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T010/document.
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L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella<br>We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella
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Otto, Wolfgang. "Transcriptional Regulatory Elements." Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-78960.
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A major challenge in life sciences is the understanding of mechanisms that regulate the expression of genes. An important step towards this goal is the ability to identify transcriptional regulatory elements like binding sites for transcription factors. In computational biology, a popular approach for this task is comparative sequence analysis using both distantly as well as closely related species. Although this method has successfully identified conserved regulatory regions, the majority of binding sites can change rapidly even between closely related species. This makes it difficult to detect them using DNA sequences alone. In this thesis, we introduce two new approaches for the detection and evolutionary analysis of transcriptional elements that consider the challenges of binding site turnover.
In the first part, we develop a method for detecting homologous motifs in a given set of sequences in order to obtain evidence for evolutionary events and turnover. Based on a detailed theoretical scaffold, we develop a simple, but effective and efficient heuristic for assembling local pairwise sequence alignments into a local multiple sequence alignment. This kind of multiple alignment only contains conserved motifs represented in columns which satisfy the order implied by the underlying sequences. By favoring motifs that are contained in a great range of sequences, our method is additionally able to detect even small conserved motifs. Furthermore, the calculation of the initial local pairwise alignments is generic. This allows the use of fast heuristic methods in case of large data sets while exact alignment programs can be used for small data sets where detailed information is needed. Application to artificial as well as biological data sets demonstrate the capabilities of our algorithm.
In the second part, we propose a conceptually simple, but mathematically non-trivial, phenomenological model for the binding site turnover at a genomic locus. The model is based on the assumption that binding sites have a constant rate of origination and a constant decay rate per binding site. The elementary derivation of the transient probability distribution is affirmed by simulations of sequence evolution as well as biological data. Based on the derived distribution, we develop a phenomenological model of binding site number dynamics in order to detect changes in selective constraints acting on transcription factor binding sites. Using a maximum likelihood implementation as well as exploratory data analysis, we show the functionality of the model by identifying functionally important changes in the evolutionary turnover rates on biological data.
Each part of this thesis leads to the development of a new program. While Tracker allows the computation of conserved homologous motifs and their representation in a local multiple alignment, Creto determines the evolutionary turnover rates for arbitrary clades of a phylogenetic tree with given binding site numbers at the final taxa. Both software tools are freely available to the scientific community for further research in this important and exciting field.
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Li, Witharana Wing Kar. "Non-Boolean characterization of Homer1a intranuclear transcription foci." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Neuroscience, c2011, 2011. http://hdl.handle.net/10133/3402.
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Activity-induced immediate-early gene (IEG) transcription foci can be labelled with fluorescent probes, permitting high temporal and spatial resolution in mapping neuronal circuits. Previous quantification approaches have assumed a Boolean function of transcription foci, assuming that cells are either active or inactive. Due to multiple amplification steps in the in situ hybridization process, it was thought that information relating to magnitudes of firing rates was lost. However, the current data suggest that transcription foci actually exhibit non-Boolean intensity and size values which vary according to behavioural condition. Systematic characterization of transcription foci intensity and size revealed incremental variations such that: home-cage < one-environment exposure < five-environment exposure < maximal electroconvulsive shock. Visual differences in transcription foci may result from a quantifiable relationship between spiking patterns and transcription rates. The exact stoichiometry between neuronal spiking and transcription is not yet clear, but these results suggest that Boolean applications of IEG imaging may neglect accurate neuronal activation properties.<br>xvi, 125 leaves : ill. ; 29 cm
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Nguyen, Tania. "Complex transcription units in Saccharomyces cerevisiae." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711667.
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Dermawan, Josephine Kam Tai. "From NF-κB to FACT: Mechanisms and Translational Applications of EGFR-mediated NF-κB Regulation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1436292263.
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Ard, Ryan Anthony. "Functional long non-coding RNA transcription in Schizosaccharomyces pombe." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/20396.
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Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.
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Vujovic, Filip. "Bimodal Transcription Regulates Non-Canonical Signaling of Notch 1." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/21166.
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Notch is a critical determinant of tissue organisation in metazoan development. The activation of Notch signaling is based on ligand-receptor interaction, consequentially the activated intracellular Notch domain is chaperoned into the nuclear compartment where it acts as a transcriptional activator for Notch target genes, leading to a biological outcome that may or may not be predictable. Upon activation, downstream signals contribute to resolution of dichotomies such as proliferation/differentiation or sub-lineage differentiation outcome. Idiosyncratic outcomes are common, leading experimentalists to describe a ‘non-canonical’ pathway for Notch signaling. It has been proposed that inappropriate activation of Notch could drive abnormal biological response and pathological disruption. Herein, we sought evidence for uncharacteristic activation of Notch signaling and a possible biological outcome. We provide an alternative interpretation of the Notch cascade and have identified an unrecognized intragenic enhancer responsible for independent transcription of the 3ʹ aspect of the gene that encodes a truncated intracellular domain (NICDtr). This novel form of Notch 1 was demonstrated to regulate differentiation fate choice in neural precursors. Moreover, we propose a novel understanding of non-canonical activation and unforeseen interplay with the nonsense-mediated decay (NMD) degradation complex.
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Cheng, Tian. "Exploiting piano acoustics in automatic transcription." Thesis, Queen Mary, University of London, 2016. http://qmro.qmul.ac.uk/xmlui/handle/123456789/18421.
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In this thesis we exploit piano acoustics to automatically transcribe piano recordings into a symbolic representation: the pitch and timing of each detected note. To do so we use approaches based on non-negative matrix factorisation (NMF). To motivate the main contributions of this thesis, we provide two preparatory studies: a study of using a deterministic annealing EM algorithm in a matrix factorisation-based system, and a study of decay patterns of partials in real-word piano tones. Based on these studies, we propose two generative NMF-based models which explicitly model different piano acoustical features. The first is an attack/decay model, that takes into account the time-varying timbre and decaying energy of piano sounds. The system divides a piano note into percussive attack and harmonic decay stages, and separately models the two parts using two sets of templates and amplitude envelopes. The two parts are coupled by the note activations. We simplify the decay envelope by an exponentially decaying function. The proposed method improves the performance of supervised piano transcription. The second model aims at using the spectral width of partials as an independent indicator of the duration of piano notes. Each partial is represented by a Gaussian function, with the spectral width indicated by the standard deviation. The spectral width is large in the attack part, but gradually decreases to a stable value and remains constant in the decay part. The model provides a new aspect to understand the time-varying timbre of piano notes, but furtherinvestigation is needed to use it effectively to improve piano transcription. We demonstrate the utility of the proposed systems in piano music transcription and analysis. Results show that explicitly modelling piano acoustical features, especially temporal features, can improve the transcription performance.
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Balthasar, Lukas. "Interaction audio-visuelle : théorie pragma-linguistique et transcription." Paris, EHESS, 2001. http://www.theses.fr/2001EHES0087.
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Holmqvist, Erik. "Macromolecular Matchmaking : Mechanisms and Biology of Bacterial Small RNAs." Doctoral thesis, Uppsala universitet, Mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171642.
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Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.
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Herzel, Lydia. "Co-transcriptional splicing in two yeasts." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-179274.
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Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs.
Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered.
In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing.
I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes:
(1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs.
(2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data.
I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing.
The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS.
For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination.
Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.
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Böhm, Stefanie. "Non-protein-coding RNA : Transcription and regulation of ribosomal RNA." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-102718.
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Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.<br><p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript</p>
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Warthi, Ganesh Daulatrao. "Exploration de mécanismes de transcription et de traduction non-canoniques." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0587.
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Le séquencage d'ARN et la spectrométrie de masse génèrent des RNA reads et des spectres peptidiques non-homologues à l'ADN. Les ignorer cause la perte d'informations génétiques. Nous explorons deux transcription noncanoniques, identifiant et expliquant RNAs et peptides mitochondriaux humains (non prédits par le genome nucléaire). 1) Échanges nucléotidiques systématiques: les échanges systématiques de nucleotides dans l'ARN (swinger RNA, 23 échanges, 9 symmétriques (X↔Y) et 14 asymmétriques (X→Y→Z→X). Les résultats sur les swinger ARNs sont reproductibles entre transcriptomes de souches mitochondriales pures et de la cellule entire, pour deux méthodes différentes de séquencage. Nous identifions des ARNs précédemment inconnus dans les non-Hodgkin lymphomes associés à HIV et des cellules cancéreuses. 2) Délétions nucléotidiques systématiques: des nombres spécifiques de nucléotides sont systématiquement délétés après chaque trinucléotide transcript, produisant des ARNs délétés (delRNAs). Au total 227 delRNAs (1-12 nucléotides délétés) sont confirmés dans trois bases de donnees, les transcriptomes de cellules humaines entières et de mitochondries pures, et la base de EST de GenBank. Les résultats indiquent que des peptides mitochondriaux sont produits à partir de traduction normale de delRNAs et de traduction de mRNAs normaux par des anticodons rallongés. Nous détectons aussi des peptides chimériques, codés par des codons normaux avec des parties contigües codées par des codons rallongés. Cette thèse détecte des transcriptions et traductions noncanoniques dans la mitochondrie humaine, et la transcription swinger de chromosomes humains nucléaires<br>RNA sequencing and mass spectrometry generate RNA reads and peptide spectra nonhomologous to the template DNA. Ignoring them results in losing valuable genetic information. We explored two non-canonical transcriptions, identified and explained RNA reads and peptides from human mitochondria (unpredicted by nuclear chromosomes). 1) Systematic nucleotide exchanges: systematic nucleotide exchanges in RNAs (called swinger RNAs) (23 exchanges, i.e. 9 symmetric (X↔Y) and 14 asymmetric (X→Y→Z→X)). Swinger RNA results are reproducable: data from purified human mitochondrial lines confirm whole-cell transcriptome data obtained by different sequencing methods. We identified previously unknown RNAs in HIV-associated non-Hodgkin's lymphoma and cancer genomes. 2) Systematic nucleotide deletions: specific nucleotide numbers are systematically deleted after each transcribed trinucleotide, producing deletion RNAs (delRNAs). A total of 227 delRNAs (1-12 deleted nucleotides) were confirmed in 3 datasets, human whole-cell and purified mitochondrial transcriptomes, and the Genbank human EST database. These map on mitochondrial protein-coding genes & the hypervariable 2 dloop. Results indicate mitochondrial peptides produced by regular delRNA translation and translation of regular mRNAs by expanded anticodons. We also detected chimeric peptides partly encoded by regular codons, and contiguous parts by expanded codons. Results show non-canonical transcriptions and translations in human mitochondria, and swinger transcription of human nuclear chromosomes
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Bothe, Anna Melissa [Verfasser]. "Investigating the Genomic Effects of Glucocorticoid Receptor Activation : An Analysis of Transcriptional Memory and Mechanisms That Direct Divergent Genomic Occupancy of Related Transcription Factors / Anna Melissa Bothe." Berlin : Freie Universität Berlin, 2021. http://nbn-resolving.de/urn:nbn:de:kobv:188-refubium-31999-3.
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Xiao, Lei. "Transcriptional Regulation of the Xenopus MyoD Gene." Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-11960.
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Sunami, Yoshiaki. "Molecular analysis of the non-canonical NF-kappaB pathway." Université Louis Pasteur (Strasbourg) (1971-2008), 2006. http://www.theses.fr/2006STR13177.
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On a montré ici que les voies canonique/non-canonique de NF-B ne sont pas indépendantes et qu’un rôle principal de la voie canonique est l’activation des acteurs de la voie non-canonique. La traduction est requise et la stimulation par LPS régule positivement un éventuel intermédiaire dans la signalisation par LPS/CD40 qui supporte l'activation de la voie non-canonique. De plus, la voie non-canonique est constitutivement activée dans les cellules de HL, impliquant la phosphorylation persistante de p100. L'activation transitoire et constitutive de la voie non-canonique implique l'incorporation de p100 et p52 dans un complexe « megadalton ». TAP a permis d’identifier des partenaires interagissant avec p100. EDD (une molécule identifiée) induit le processing de p100 par co-expression. La formation du complexe de TRAF3 (autre facteur identifié) avec p100 est médiée par NIK et requiert son activité kinase. L'expression de NIK favorise le recrutement de TRAF3/p100 au signalosome p100<br>It is shown here that the canonical/non-canonical NF-B pathways are not independent and a master regulatory role of the canonical pathway is to upregulate activators of the non-canonical pathway. The data further implicate a translation requirement and that LPS stimulation upregulates a potential intermediate in LPS/CD40 signaling which supports activation of the non-canonical pathway. Further, the non-canonical pathway is constitutively activated in HL cells, involving persistent p100 phosphorylation. It was found that transient and constitutive activation of the non-canonical pathway involves incorporation of p100 and p52 into a megadalton complex. TAP was employed to identify novel p100 interacting partners. EDD (an identified molecule) induces processing of p100 upon co-expression. The complex formation of TRAF3 (another p100 interactor) with p100 is mediated by NIK and requires its kinase activity. The expression of NIK promotes recruitment of TRAF3/p100 to the p100 signalosome
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Kotovic, Kimberly Marie. "Co-transcriptional recruitment of the U1 snRNP." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2004. http://nbn-resolving.de/urn:nbn:de:swb:14-1103190658062-33439.
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It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.