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Tufarelli, Cristina. "Activation and silencing of α globin expression." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365741.
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Staines, Daniel Michael. "Characterisation of the 3'-boundary of the β-globin chromosomal domain in adult chicken erythrocytes." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625070.
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Gardiner, T. J. "Functional characterisation of an evolutionary conserved domain of non-coding Y RNA in human chromosomal DNA replication." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599307.
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In order to investigate the function of the Y RNAs, I employed a human cell-free DNA replication assay. Firstly, I used this assay to verify the original observations that showed Y RNAs to be required for human chromosomal DNA replication in vitro. I then carried out density substitution experiments to show that Y RNA mediated DNA replication in human nuclei was semi-conservative. I also used a mouse cell-free system to show that Y RNA function in DNA replication can act independently of the chemical synchronisation used to prepare human nuclei and to show that Y RNA function is conserved between mouse and man. The Y RNAs are highly conserved both in terms of sequence and structure in the vertebrates. The Y RNAs are also found in some non-vertebrate organisms such as C. elegans, B. floridae and D. radiodurans although they are not found in yeast or flies. I subsequently studied the evolutionary conservation of the Y RNAs further by using the cell free system to test the functionality of several different species’ Y RNAs. I concluded that Y RNA function is conserved throughout vertebrate evolution. RNA to promote DNA replication appeared to correlate with conserved sequence domains in the double stranded stem of the RNA. From this information I generated a library of mutant hY1 RNAs containing alterations to their sequence and structure to investigate further which domains were essential for DNA replication. Mutant Y RNA with deletions of the loop and secondary stems promoted DNA replication as efficiently as wild-type human Y1 RNA. Deletions in the upper-stem of the RNA, however, did not allow DNA replication to proceed. Further alterations to the sequence of the upper stem in most cases abrogated function in DNA replication. The upper part of this double-stranded stem of hY1 RNA was not only necessary, but also sufficient for Y RNA function in DNA replication. DNA oligonucleotides were not able to substitute for human Y RNA in vitro.
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Szabo, Quentin. "Étude du repliement tridimensionnel de la chromatine en domaines topologiques." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT064.
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Mon projet de thèse a consisté à étudier les mécanismes du repliement tridimensionnel du génome dans les cellules eucaryotes. L’organisation des chromosomes est étroitement liée à la régulation de nombreuses fonctions biologiques, telles que le contrôle de l’expression génique, la réplication de l’ADN ou encore la stabilité génomique. La méthode de « chromosome conformation capture » Hi-C, qui permet la cartographie des interactions entre régions d’ADN, a révélé que chez de nombreuses espèces, le génome est organisé en domaines enrichis en interactions chromatiniennes, les « Topologically Associating Domains » (TADs). Les TADs sont apparus être des acteurs majeurs de la régulation du génome par leur capacité à définir spatialement des domaines fonctionnels. Cependant, les méthodes de chromosome conformation capture générèrent des profils d’interactions généralement moyennés à partir d’ensemble de cellules. Déterminer la nature du repliement des TADs en cellules individuelles est donc crucial pour comprendre la relation structure-fonction de ces domaines génomiques. Au cours de ma thèse, j’ai utilisé des techniques de marquage fluorescent d'ADN combinés à de la microscopie en super-résolution afin d’étudier l’organisation des chromosomes en cellules uniques. Chez la drosophile, les TADs coïncident avec le partitionnement de la chromatine en domaines épigénétiques distincts. Nous avons pu caractériser chez cette espèce que les chromosomes sont organisés en une série d’unités discrètes qui correspondent aux TADs, reflétant l’exclusion mutuelle de régions transcriptionnellement actives et inactives. Ces résultats indiquent que les TADs de drosophile forment des domaines physiques qui caractérisent un niveau d’organisation structurale des chromosomes en cellules uniques. Chez les mammifères, la majorité des TADs est formée grâce à l’action du complexe cohésine et à la présence de la protéine CCCTC-binding factor (CTCF) à leurs frontières. L'application de l'imagerie à super-résolution dans des cellules souches embryonnaires et des cellules progénitrices neuronales de souris nous a permis de caractériser l’hétérogénéité du repliement des TAD d’une cellule à l’autre. Nous avons notamment pu observer leur organisation en sous-domaines globulaires qui semblent représenter une propriété générale du repliement de la chromatine à l’échelle de la centaine de nanomètres. De plus, nos résultats indiquent que les interactions chromatiniennes sont fortement favorisées à l’intérieur des TADs dans la majorité des cellules. La déplétion de CTCF abolie l’organisation spatiale de la fibre de chromatine associée aux TAD, soulignant le rôle de cette protéine dans la génération de barrières physiques entre TAD adjacents. Ces données démontrent que le repliement dynamique des TAD est compatible avec l'établissement d'environnements chromosomiques dans lesquels les contacts sont privilégiés, et réconcilient ainsi la nature probabiliste du repliement de la chromatine avec le rôle proposé des TAD dans la définition spatiale d’unités génomiques fonctionnellesMy thesis project consisted in studying the mechanisms of the three-dimensional genome folding in eukaryotic cells. The organization of chromosomes is closely related to the regulation of many biological processes, such as gene expression control, DNA replication or genomic stability. The Hi-C "chromosome conformation capture" method, which allows the mapping of interactions between DNA regions, has revealed that the genome of many species is organized into domains enriched in chromatin interactions, the "Topologically Associating Domains" (TADs). TADs have emerged as major players of genome regulation by their ability to spatially define functional domains. However, chromosome conformation capture methods generate averaged interaction profiles that generally come from an ensemble of cells. Determining the nature and the folding of TADs in individual cells is therefore crucial to better understand the structure-function relationship of these domains. During my thesis, I used a combination of fluorescent DNA labeling and super-resolution microscopy to characterize the organization of chromosomes in single cells. In Drosophila, TADs coincide with the partitioning of the chromatin into distinct epigenetic domains. In this species, we could characterize the folding of the chromosomes into a series of discrete units that correspond to TADs, reflecting the mutual exclusion of transcriptionally active and inactive regions. These results indicate that Drosophila TADs form physical domains that characterize a higher-order layer of chromosome folding in individual cells. In mammals, the majority of TADs emerge through the action of the cohesin complex and the CCCTC-binding factor (CTCF) bound at their borders. The application of super-resolution imaging in mouse embryonic stem cells and neuronal progenitor cells revealed the high degree of cell-to-cell heterogeneity of TAD folding, ranging from condensed and globular objects to dispersed and stretched conformations. We were able to observe their organization into discrete subdomains which seem to represent a general property of the folding of the chromatin fiber at the nanoscale. Furthermore, our data indicate that the physical intermingling of the chromatin is highly favored within TADs in a large majority of cells. Depletion of CTCF abolishes the TAD-dependent spatial organization of the chromatin fiber, highlighting the role of this protein in generating physical barriers between adjacent TADs. Altogether, our results demonstrate that the dynamic folding of TAD is compatible with the establishment of chromosomal environments in which contacts are privileged, and thus reconcile the probabilistic nature of chromatin folding with the proposed role of TADs in the spatial definition of functional genomic units
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Hocher, Antoine. "Exploring the plasticity of chromosomal domains upon overexpression of silencing factors in Saccharomyces cerevisiae." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066335.
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La présence de domaines chromosomiques heterochromatiniens associé à des effets de position est une propriété communes à de nombreux génomes eukaryotes. L'intensité et l'étendue de la variégation liée aux effets de position sont généralement sensibles à la dose des protéines effectrices de l'hétérochromatine. Les propriétés d'auto-propagation des complexes d'hétérochromatine a un cout, qui est la nécessité d'établir des mécanismes stoppant la propagation de la répression transcriptionelle. Cette thèse explore la dose-dépendance de l'effet de position télomérique en étudiant le complexe SIR de la levure du boulanger. La caractérisation du groupement des télomères en foyers, de la localisation de Sir3 et de la transcription dans des souches sur-exprimant Sir3 a permis d'établir l'étendue maximale des domaines silencieux présent aux subtelomeres. L'étude de jeux de données publiés a révélé que ces domaines terminent généralement au niveau de zones correspondant où les propriétés de la chromatine montrent une transition importante. Ces transitions chromatiniennes sont requises pour survivre en présence d'un excès de protéines Sir3 puisque nous avons démontré que les mutants dot1 ne survivent pas un tel excès. En outre nous avons conduit un crible génétique qui a révélé de nombreux gènes requis pour la survie en présence d'une surdose de Sir3. Ce travail caractérise la réponse du génome à une surdose d'hétérochromatine et a permis de révéler des domaines subtélomeriques associés à des propriétés chromatiniennes particulières. En conséquence nous démontrons comment l'effet de position télomerique est efficacement restreint au subtelomere chez la levureA shared property of several eukaryotic genomes is the presence of heterochromatic chromosomal domains experiencing transcriptional variegation. The intensity and the extent of position effect variegation are sensitive to the dosage of silencing effectors in many systems. The self-propagating properties of heterochromatin machineries come with a cost, which is the requirement for mechanisms preventing ectopic spreading of silencing. This thesis explores the dose-dependency of telomere position effect, using the budding yeast SIR system as a model for chromatin based heterochromatic silencing. To assess the dose-dependency of telomere position effect in budding yeast, we systematically characterized the impact of Sir3 overexpression by quantifying the clustering of telomeres, the genome wide binding of Sir3 and its impact on coding and non coding transcription. Analysis of published data sets enabled to uncover candidates potentially responsible for the limitation of subtelomeric silent domains. Our study reveals that extension of silent domains can reach saturation, associated with the anti-silencing properties of histone marks deposited by the conserved enzyme Dot1. In addition we discovered genes required for viability upon SIR3 overexpression by conducting a genetic screen. Our work describes the dynamics of the dose dependency of heterochromatin propagation in budding yeast. It uncovers previously uncharacterized discrete chromosomal domains associated with specific chromatin features and demonstrates how telomere position effect is efficiently restricted to subtelomeres by the preexisting chromatin landscape
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Ea, Vuthy. "Dynamique et organisation supérieure de la chromatine : exploration des domaines d’association topologique." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON1T024.
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La chromatine sert de support à de multiples processus biologiques, cependant son organisation spatiale diffère fortement selon l'échelle considérée. L'expression des gènes est ainsi coordonnée par des éléments régulateurs dispersés dans le génome mais capables d'interagir entre eux. Chez les métazoaires, des expériences de capture de conformation de chromosome (3C) combinées au séquençage haut-débit (Hi-C) ont permis la découverte de domaines d'association topologique (TAD), à l'échelle de la mégabase. Puisque la résolution du Hi-C reste limitée, nous avons utilisé la 3C-qPCR pour explorer, dans des cellules souches embryonnaires murines, la dynamique chromatinienne à l'intérieur de ces domaines ainsi qu'à leurs bordures. Nous identifions ainsi une modulation des fréquences de contacts, sur quelques centaines de kilobases. Cette modulation est plus ou moins importante en fonction du contenu en gènes des domaines, mais elle semble néanmoins universelle. Des modèles dérivés de la physique des polymères permettent de décrire cette modulation sous la forme d'une hélice statistique, que la chromatine adopterait en moyenne et en l'absence d'interactions spécifiques, à l'intérieur des TAD. Cette hélice reflète certaines contraintes que la chromatine subit à l'échelle supranucléosomale. Elle est très affectée par les bordures, qui bloquent la modulation, mais elle l'est beaucoup moins par le contenu en histone de liaison H1. Par ailleurs, grâce à des résultats de Hi-C à haute résolution, nous montrons que la modulation observée chez les souris n'est pas retrouvée chez la drosophile, où les caractéristiques des TAD semblent avant tout liées au paysage épigénétique local. Pour ces deux organismes, la dynamique chromatinienne à l'intérieur des domaines est donc sous le contrôle de phénomènes différentsThe chromatin hosts various biological processes. However, its organization differs considerably depending on the scale. For example, gene expression is coordinated by regulatory elements that are dispersed in the genome but that are able to interact within the tridimensional space of the nucleus. In the Metazoa, chromosome conformation capture (3C) assays combined with high-throughput sequencing (Hi-C) uncovered the existence of topologically associating domains (TADs), at the mégabase scale. Due to the limited resolution of Hi-C, we used the 3C-qPCR method to explore, in murine embryonic stem cells, the chromatin dynamics inside TADs as well as at their borders. We found that contact frequencies undergo a periodic modulation over large genomic distances (few hundred kilobases). This modulation is weaker in gene-deserts than in gene-containing domains but it seems nevertheless to be universal. Using models derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt statistically, when no strong locus-specific interaction takes place, within the TADs. This statistical helix reflects some constraints that the chromatin undergoes at the supranucleosomal scale. It is affected by TADs borders, which disrupt the modulation, but linker histone H1 depletion only leads to subtle changes in the helix characteristics. Furthermore, using high-resolution Hi-C data, we found that chromatin dynamics is unconstrained in Drosophila where it seems mainly linked to the local epigenetics landscape. Therefore, distinct genome organization principles govern chromatin dynamics within mouse and Drosophila topologically associating domains
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Mitra, Robi David. "Polony sequencing : DNA sequencing technology and a computational analysis reveals chromosomal domains of gene expression." Thesis, Massachusetts Institute of Technology, 2000. http://hdl.handle.net/1721.1/8797.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2000.Includes bibliographical references.
The first part of this thesis describes the development of polony sequencing, a sequencing technology in which DNA is cloned, amplified and sequenced in a polymer matrix. A complex library of one to ten million linear DNA molecules is amplified by performing polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the DNA molecules so that each amplification product remains localized near its parent molecule. At the end of the reaction, a number of polymerase colonies, or "polonies", have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. By including an acrydite modification at the 5' end of one of the PCR primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. Also described in this thesis is my progress in development of a protocol to sequence the polonies by repeated cycles of extension with fluorescent deoxynucleotide. Because polony sequencing is inherently parallel, and sub-picoliter volumes are used for each reaction, the technology should be substantially faster and cheaper than existing methods. Applications for polony sequencing such as gene expression analysis, SNP discovery, and SNP screening will also be discussed. The second part of this thesis describes a computational analysis that tests the hypothesis that chromosomal position affects gene expression. It is shown that, throughout the genome, genes lying close together on the same chromosome often show significant coexpression. This coexpression is independent of the orientation of genes to each other, but is dependent on the distance between genes. In several cases where adjacent genes show highly correlated expression, the promoter of only one of the genes contains an upstream activating sequence (UAS) known to be associated with the expression pattern. These results suggest that in certain regions of the genome a single transcription factor binding site may regulate several genes. It is also shown that evolution may take advantage of this phenomenon by keeping genes with similar functions in adjacent positions along the chromosomes. The techniques that are presented provide a computational method to delineate the locations of chromosomal domains and identify the boundary elements that flank them.
Robi David Mitra.
Ph.D.
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Lin, Shau-Ping. "Regulation of genomic imprinting at the Dlk1-Gtl2 imprinted domain on mouse chromosome 12." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616241.
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Becking, Thomas. "Impact des bactéries féminisantes du genre Wolbachia sur l'évolution des chromosomes sexuels d'isopodes terrestres." Thesis, Poitiers, 2017. http://www.theses.fr/2017POIT2299/document.
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Les Oniscidea présentent une diversité remarquable de systèmes chromosomiques de déterminisme du sexe (hétérogamétie mâle XX/XY ou hétérogamétie femelle ZW/ZZ), dont l'origine reste encore largement inconnue à ce jour. Il a été proposé que ces différents systèmes puissent être le produit de la coévolution entre les isopodes terrestres et Wolbachia, une bactérie endosymbiotique féminisante transmise verticalement par voie ovocytaire. Dans le but de caractériser l'impact de l'endosymbiose à Wolbachia sur l'évolution des mécanismes de déterminisme du sexe, nous avons utilisé une combinaison d'approches génomique, transcriptomique et d'expression de gènes. Tout d'abord, le génome de l'espèce Armadillidium nasatum (caractérisée par un système XX/XY) a été généré et ensuite annoté structurellement et fonctionnellement. A partir de ce génome, des approches de génomique comparatives ont permis la caractérisation de séquences liées au chromosomes Y, afin de mieux comprendre les processus impliqués dans la dégénérescence des gonosomes. Afin d'identifier des effecteurs liés au déterminisme ou à la différenciation du sexe, une approche par gènes candidats a permis de caractériser des gènes à domaines DM, connus pour être impliqués dans le déterminisme du sexe de nombreuses espèces, et d'en mesurer l'expression au cours du temps. Enfin, une phylogénie des Oniscidea a été réalisée en parallèle d'expériences de réversion de sexe afin d'estimer le nombre et la direction des transitions de systèmes d'hétérogamétie au cours de l'évolution des isopodes terrestres. Ces travaux contribuent à illustrer l'impact de l'endosymbiose sur l'évolution des mécanismes de déterminisme du sexe de l'hôteOniscidea show a remarkable diversity of chromosomal sex determination systems (male heterogamety XX/XY or female heterogamety ZW / ZZ). However, the origin of such diversity is still largely unknown to date. It has been proposed that these different systems may be the product of the coevolution between terrestrial isopods and Wolbachia, a feminizing endosymbiotic bacteria transmitted vertically through oocytes. In order to characterize the impact of Wolbachia endosymbiosis on the evolution of sex determination mechanisms, we used a combination of genomic, transcriptomic and gene expression approaches. First, the genome of the species Armadillidium nasatum (characterized by an XX/XY system) was generated and then structurally and functionally annotated. From this genome, a comparative genomic approach allowed us to characterize sequences Y-linked, in order to better understand the processes involved in the sex chromosome degeneration. In order to identify effectors potentially related to sex determination or differentiation, a candidate gene approach has been used to characterize DM-domain genes, known to be involved in the sex determination pathways of many species, and then to measure their expression over development. Finally, a Oniscidea phylogeny was generated in parallel with sex-reversal experiments in order to characterize the number and the direction of the transitions of heterogenetic systems during the terrestrial isopods evolution. This work emphasize the impact of endosymbiosis on the evolution of host sex determination mechanisms
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Hamperl, Stephan [Verfasser], and Joachim [Akademischer Betreuer] Griesenbeck. "Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae / Stephan Hamperl. Betreuer: Joachim Griesenbeck." Regensburg : Universitätsbibliothek Regensburg, 2012. http://d-nb.info/1027850340/34.
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Fung, King-leung. "Molecular study of the deleted in liver cancer 2 (DLC2)h[electronic resource] : solution structure of the SAM domain and interaction with MCM7 /." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36218716.
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Bentley, Louise. "Molecular analysis of the imprinted domain at chromosome 7q32 : a candidate region for Silver-Russell syndrome." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408768.
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Sabbaghian, Nelly. "Structure-function analysis of three widely dispersed point mutations in the hormone-binding domain of the androgen receptor." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68254.
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Three point mutations have been found in the hormone-binding domain (HBD) of the human androgen receptor (hAR): one in the N-terminal end (Ile663Asn in a family with partial androgen insensitivity syndrome (PAIS)); one in the middle, (Leu820Val in a family with PAIS); and one in the C-terminal end (Pro903Ser, in a family with complete AIS). The positions 663 and 903 were the most terminal mutation sites in the HBD found to date. The three mutant hARs have been previously characterized biochemically in genital skin fibroblasts. In the family with the Leu820Val substitution, the mother and the grandmother were found to be carriers for the same mutation. To prove their pathogenicity, each of the three mutations has been reproduced in an hAR expression vector that was transfected into COS-1 cells. In COS-1 cells, the complexes from Pro903Ser and Leu820Val had: increased thermolability; increased dissociation rates; decreased affinity; and abnormal transactivation. There was a hierarchy in the severity of the mutations expressed in kinetic and transactivation assays that correlated with the severity of the clinical phenotype. The pathogenicity of the Pro903Ser and the Leu820Val mutations was thereby confirmed. In COS-1 cells, the AR with Ile663Asn had normal thermolability, normal dissociation rates, and normal transactivation, but a decreased affinity. Although this sequence alteration has only been found in a PAIS patient, its pathogenicity is not considered to be proven. More sensitive assays are needed for this purpose.
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Fung, King-leung, and 馮景良. "Molecular study of the deleted in liver cancer 2 (DLC2)h[electronic resource]: solution structure of the SAM domain and interaction withMCM7." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36218716.
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Kanhoush, Rasha. "Etude moléculaire et biochimique des interactions de la protéine hnRNP G avec l’ARN." Paris 6, 2010. http://www.theses.fr/2010PA066054.
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La protéine hnRNP G est impliquée dans l’épissage alternatif de plusieurs précurseurs d’ARN messager. Pour mieux comprendre la spécificité de cette protéine, j’ai étudié ses interactions avec les ARNs in vitro et in vivo. Un motif d’ARN structuré dans une tige-boucle qui contient la séquence GGAAA a été identifié in vitro comme une cible potentielle d’hnRNP G. Cet ARN m’a permis d’identifier un nouveau domaine d’interaction avec l’ARN dans la structure de l’hnRNP G, situé dans sa région C-terminale et désigné « C-ter RBD ». Le modèle des chromosomes en écouvillon chez l’amphibien m’a permis de montrer qu’un nouveau domaine situé au centre de la structure de la protéine et distinct de ses domaines d’interaction avec l’ARN est responsable de son recrutement au niveau des transcrits naissants des chromosomes. Ce domaine a été désigné Nascent transcripts Targeting Domain (NTD). Cette étude montre que l’hnRNP G s’associe avec la majorité des ARNs transcrits par l’ARN polymérase II tout en montrant un recrutement plus important au niveau de quelques boucles des chromosomes. En particulier, l’association de l’hnRNP G avec les transcrits du bivalent sexuel ZW chez Pleurodeles waltl, montre un recrutement hétéromorphe au niveau de certains sites de ce bivalent. Au cours de ma thèse, j’ai participé à la mise en évidence d’une activité différentielle de liaison à l’ARN liée au génotype sexuel, pour les isoformes de l’hnRNP G dans l’ovocyte de P. Waltl. Les résultats suggèrent la présence d’une famille multigénique de l’hnRNP G répartie sur un autosome et sur les chromosomes sexuels. Cette étude contribue à la caractérisation de l’hnRNP G et à la compréhension de ses fonctions.
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Xiang, Wanqing [Verfasser], and Jan [Akademischer Betreuer] Ellenberg. "Structure and Dynamics of Replication Domains in Single Chromosome Territories of Interphase Nuclei / Wanqing Xiang ; Betreuer: Jan Ellenberg." Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/1178010597/34.
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Awal, Sushil. "Targeting ubiquitin receptor protein UBASH3B for future cancer therapies." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ094.
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Des anomalies dans la ségrégation des chromosomes pendant la mitose entraînent une aneuploïdie ou une polyploïdie. La protéine UBASH3B, une protéine de liaison à l’ubiquitine, joue un rôle crucial dans la transmission de l’Aurora B aux microtubules avant l’apparition de l’anaphase pour une ségrégation chromosomique appropriée. Nous avons effectué un dépistage à haut débit pour cribler un nouvel inhibiteur de l’UBASH3B à petite molécule, le FD-E09, à l’aide de la protéine recombinante UBASH3. Le traitement par l’inhibiteur de l’UBASH3B a entraîné la propagation d’Aurora B sur les chromosomes pendant la prométaphase, affectant ainsi le moment et la fidélité de la mitose. Il affecte également le complexe UBASH3B-Aurora B-Mklp2. Mes données décrit également que le domaine 2 Histidine phosphoesterase dans UBASH3B entre le domaine UBA et SH3 joue un rôle dans la localisation correcte d’Aurora B, la progression mitotique, ainsi que l’interaction avec Aurora B et Mklp2. Nous avons également criblé les lignées cellulaires cancéreuses qui réagissent à l’inhibition de l’UBASH3B. Par conséquent, nos résultats découvrent l’inhibiteur de la petite molécule UBASH3B qui pourrait cibler l’UBASH3B pour traiter des cellules cancéreuses spécifiques et également découvrir le domaine de la phosphoésterase 2H nouvellement identifié dans UBASH3B qui régule la localisation d’Aurora B en mitoseDefects in proper segregation of chromosomes during mitosis result in aneuploidy or polyploidy. UBASH3B protein, a ubiquitin-binding protein, plays a crucial role in driving Aurora B to microtubules before anaphase onset for proper chromosome segregation. We here performed a high-throughput screening to screen out a novel small-molecule UBASH3B inhibitor, FD-E09 using recombinant UBASH3 protein. Treatment with UBASH3B inhibitor led to the spreading of Aurora B to chromosomal arms during prometaphase, thus affecting the timing and fidelity of mitosis. It also affects the UBASH3B-Aurora B-MKlp2 complex. My data also unfold the 2 Histidine phosphoesterase domain in UBASH3B between UBA and SH3 domain that plays a role in proper localization of Aurora B, mitotic progression, as well as interaction with Aurora B and MKlp2. We also screened out the cancer cell lines that are responsive to the UBASH3B inhibition. Hence, our findings uncover the small-molecule UBASH3B inhibitor that could target UBASH3B to treat specific cancer cells and also uncover newly identified 2H phosphoesterase domain in UBASH3B that regulates Aurora B localization in mitosis
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Grouche, Lakhdar. "Traitement d'images par morphologie mathematique : applications aux domaines medical et industriel." Clermont-Ferrand 2, 1987. http://www.theses.fr/1987CLF2D207.
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Ghandil, Pegah. "Genetic study of the type 1 diabetes : search for susceptibility genes on chromosom 16." Paris 6, 2005. http://www.theses.fr/2005PA066593.
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De, Benedittis Caterina <1983>. "Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6753/.
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In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients
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Carpentier, Anne-Sophie. "Le transcriptome : un domaine d'application pour lesstatistiques, de nouveaux horizons pour la biologie." Phd thesis, Université d'Evry-Val d'Essonne, 2006. http://tel.archives-ouvertes.fr/tel-00067855.
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Les mesures des niveaux d'expression de tous les gènes d'un génome requièrent une analysestatistique afin d'obtenir des conclusions fiables. Les biologistes ont du mal à faire un choix dans la
foule de méthodes existantes. Afin de déterminer quelle méthode est la plus adéquate pour la
problématique abordée, des comparaisons de méthodes d'analyse disponibles sont nécessaires.
Actuellement les critères de comparaison se révèlent soit lacunaires ou soit non pertinents du point de
vue biologique.
Nous avons introduit un nouveau critère biologique de comparaison des méthodes d'analyse du
transcriptome fondé sur une structure des génomes bactériens : les opérons. Les gènes d'un opéron
sont généralement transcrits sur un même ARNm. Si un gène d'un opéron bactérien est identifié, les
autres gènes de l'opéron devraient l'être également. Nous avons ainsi comparé des méthodes
d'analyse appliquées au transcriptome : l'ACP et l'ACI, respectivement analyses en composantes
principales et indépendantes, l'ANOVA, analyse de variance, la régression des moindres carrés
partiels PLS et différents t-tests. Chaque méthode aborde le nuage de données d'un point de vue
différent ce qui donne des résultats complémentaires. Globalement, l'ACI a fourni les meilleurs
résultats tant en sensibilité qu'en terme de précision.
Un autre aspect, en plein développement, de l'analyse du transcriptome est la méta-analyse de
données d'origines diverses malgré les biais inhérents à cette technologie. Généralement ces métaanalyses
visent à préciser les résultats concernant des gènes différentiellement exprimés ou coexprimés.
Elles ouvrent également la possibilité d'étudier de nouveaux champs en biologie. Nous
avons utilisé des données de transcriptome indépendantes afin d'étudier l'organisation de l'expression
des gènes et, ainsi, celle du chromosome bactérien. L'étude du transcriptome de trois bactéries, B.
subtilis, E. coli et S. meliloti a révélé des corrélations d'expression à longue distance valables quel que
soit le gène étudié. Les structures en opéron se manifestent clairement au travers de cette étude, qui
a également permis de préciser que la co-expression de gènes proches s'étend au-delà des opérons
dans une région qui se répand jusqu'à une centaine de gènes.
Pour conclure, l'analyse du transcriptome n'a pas réellement nécessité la mise au point de méthodes
d'analyse statistiques spécifiques. Cependant, elle permet d'aborder de nouveaux horizons dans la
biologie, et notamment l'organisation chromosomique du génome bactérien.
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Pöpsel, Juliane. "Characterization of a novel lysine acetylation site in the N-terminal domain of the centromeric histone variant Cse4 in Saccharomyces cerevisiae." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17343.
Full textAbstract:
Die zentromerischen Regionen eukaryotischer Chromosomen sind notwendig für die Segregation der Schwesternchromatiden während der Zellteilung. In den Nukleosomen in diesen Regionen ist das kanonische Histon H3 durch die zentromerische Histon H3 Variante CENP-A ersetzt. Diese wird in Saccharomyces cerevisiae Cse4 genannt. Indem CENP-A die geordnete Assemblierung einzelner Untereinheiten des Kinetochors vermittelt, spezifiziert es die zentromerische Chromosomenregion und ist damit essentiell für die korrekte Segregation der Chromosomen während der Zellteilung. Kürzlich wurde gezeigt, dass Cse4 posttranslational modifiziert wird. Von Bedeutung für diese Arbeit sind die in der essentiellen Domäne des N-terminus liegenden Methylierung von Arginin 37 und die Acetylierung von Lysin 49, die durch phänotypische Suppression eine genetische Interaktion und eine antagonistische Funktion bei der epigenetischen Regulation in der korrekten Assemblierung der Kinetochoruntereinheiten zeigen. In dieser Arbeit wurde gezeigt, dass die Acetylierung an Cse4K49 von der Histonacetyltransferase Gcn5 abhängt, und dass dieses Enzym Komponenten des SAGA-Komplexes, aber nicht des ADA-Komplexes benötigt. Außerdem konnte in dieser Arbeit gezeigt werden, dass die Acetylierung von K49 in der frühen S-Phase ansteigt und zum Ende der S-Phase abnimmt. Es konnte weiterhin eine biochemische Interaktion der N-terminalen Domäne von Cse4 mit der COMA-Untereinheit Ctf19, der zentralen Region des Kinetochors, nachgewiesen werden, möglicherweise mit einer Präferenz zu einer nicht acetylierten Form von Cse4K49. In dieser Arbeit konnte gezeigt werden, dass der Transkriptions-Co-Aktivator Komplex SAGA eine Funktion an der zentromerischen Region in S. cerevisiae aufweist. Des Weiteren ist die Acetylierung an Cse4K49 in die Rekrutierung der Kinetochoruntereinheit Ctf19 involviert, und gibt damit einen Hinweis auf einen epigenetischen Regulationsmechanismus während der Chromosomensegregation.The centromeric regions of eukaryotic chromosomes are essential for proper chromosome segregation. The nucleosomal composition in these regions differs from that of canonical nucleosomes in that histone H3 is replaced by the H3 variant CENP-A, which is termed Cse4 in Saccharomyces cerevisiae. CENP-A is the most important epigenetic mark on centromeres and essential for accurate kinetochore establishment at centromeric sites, which in turn are necessary to connect the centromeric sites to the microtubules. Whereas the histone fold domain of Cse4 shares a sequence homology with canonical histone H3, the N-terminal domain is rather long as compared to canonical histone tails and the centromeric histone variants of higher eukaryotes. One part of this flexible, positively charged histone tail has been shown to represent an essential N-terminal domain (END). Lysine 49 was defined as an acetylation site in this region, but the modification remains to be characterized in detail. In this study, we found that the Cse4K49 acetylation is dependent on the histone acetyltransferase Gcn5, and that the enzyme in this context acts within the SAGA/SLIK complex, whereas an involvement of the smaller ADA complex was not observed. Furthermore, we addressed the cell-cycle dependence of the K49 acetylation and found an increase in early S-phase and a decrease in late S-phase, whereas the R37 methylation persisted throughout S-phase. Ctf19 was found to bind acetylated and unacetylated Cse4K49 with a preference for unacetylated Cse4. The findings presented here show that the transcriptional co-activator complex SAGA functions at centromeric regions in S. cerevisiae, and that the Cse4K49 acetylation has an influence on the recruitment of the kinetochore subunit Ctf19. This suggests an epigenetic regulatory mechanism involving a specific acetylation on the N-terminus of Cse4 in chromosome segregation.
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Rupon, Jeremy William. "The Role of DNA Methylation and Methyl Binding Domain Protein 2 in the Regulation of Human Embryonic and Fetal Beta Type Globin Genes." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1833.
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Griese, Julia Johanna. "Structures and DNA-Binding Activities of the Hinge Domains from the Structural Maintenance of Chromosomes Proteins of Pyrococcus furiosus and the Mouse Condensin Complex." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-122003.
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Mitra, Sayantan. "Arabidopsis Cohesin proteins: WAPL, CTF7 and PHD finger proteins: MMDL1, MMDL2 are essential for proper meiosis, gamete development and plant growth." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami1517605898967702.
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Abramo, Kristin N. "Building the Interphase Nucleus: A study on the kinetics of 3D chromosome formation, temporal relation to active transcription, and the role of nuclear RNAs." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1099.
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Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood.
The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.
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Jean, Laetitia. "La superfamille de l'inter-alpha-inhibiteur : approche des fonctions par l'analyse de l'expression des gènes et l'identification de domaines protéiques." Rouen, 1999. http://www.theses.fr/2000ROUES026.
Full textAbstract:
La famille de l'inter-α-inhibiteur (IαI) recouvre un ensemble de protéines plasmatiques pour la plupart inhibitrices de protéases. Ces molécules sont structurées à partir de combinaisons variées entre la bikunine et une ou plusieurs des chaînes lourdes homologues H. Les 4 chaînes H sont les produits de gènes ITIH distincts mais très conservés entre espèces. Toutes ces chaînes sont synthétisées sous forme de précurseurs, AMBP et HxP, donnant naissance à la bikunine et aux chaînes H matures après modifications post-traductionnelles. Ce travail de thèse réalisé dans un modèle rongeur a visé à progresser dans la connaissance des fonctions biologiques de la famille par l'étude de ses gènes et de ses protéines. Nous avons localisé le gène ITIH 4 sur des régions homologues du chromosome 3 humain et du chromosome 14 de souris. Par RT-PCR quantitative et hybridation in situ en situation physiologique (ontogénèse) ou pathologique (phase aiguë de l'inflammation) nous avons précisé et comparé les régulations d'expression de ces gènes selon les tissus et entre espèces. En particulier, nous avons identifié les aires cérébrales exprimant le gène ITIH3 et défini le profil d'expression développemental du transcrit H3 dans le cerveau. D'autres sites d'expression extrahépatiques (estomac, intestin) ont été mis en évidence par RT-PCR. Les protéines de la famille IαI joueraient un rôle dans la stabilisation de matrices extracellulaires via la capacité des chaînes H à lier l'acide hyaluronique (HA). Par des techniques d'expression de protéines recombinantes et de mutagénèse dirigée, nous avons cartographié sur la chaîne H3P de souris le site de liaison à HA. Identifié dans la région la plus N-terminale, il inclut un motif B(X)7B critique pour l'interaction. Enfin, nous avons identifié in silico une protéine nommée PH5P à l'interface des familles IαI et PARP. PH5P qui aurait une fonction potentielle de réparation de l'ADN permet l'élargissement de la famille IαI en une superfamille.
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Leveziel, Nicolas. "Génétique de la dégénérescence maculaire liée à l'âge variants majeurs de prédisposition à la forme exsudative." Paris 6, 2008. http://www.theses.fr/2008PA066183.
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Rege, Mayuri. "RNA Exosome & Chromatin: The Yin & Yang of Transcription: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/812.
Full textAbstract:
Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood.
We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome.
Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis.
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Sessa, Gaetana. "Role of the Interaction of BRCA2 and DDX5 in the DNA Damage Response BRCA2 promotes DNA-RNA hybrid resolution by DDX5 at DNA double strand breaks to facilitate homologous recombination Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS116.
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Un nombre croissant d’études soutiennent le fait que les protéines majeures du métabolisme des ARN, telles que les hélicases ARN, sont impliquées dans la réponse aux dommages à l’ADN. Cette activité est généralement accomplie par leur interaction avec des facteurs de réparation de l’ADN. BRCA2, une protéine suppressive de tumeurs, joue un rôle crucial dans la réparation des cassures double-brin (CDB) de l'ADN par recombinaison homologue (RH) et donc, est un facteur essentiel pour l’intégrité du génome. Les cellules déficientes pour BRCA2 accumulent des hybrides ADN-ARN ou R-loops, une source de dommage à l'ADN, suggérant ainsi l’importance de cette protéine dans la prévention ou la suppression de ces structures. Toutefois, le rôle spécifique de BRCA2 dans la résolution des hybrides ADN-ARN reste inconnu.Afin de connaître des potentiels partenaires de BRCA2, une analyse par spectrométrie de masse réalisée dans notre laboratoire a révélé un enrichissement en protéines impliquées dans le métabolisme de l'ARN, comme les hélicases ARN. Ces résultats nous ont menés à examiner la coopération entre BRCA2 et les hélicases ARN dans la séparation des structures ADN-ARN. Nous avons d’abord confirmé l'interaction entre l'hélicase ARN DDX5 et BRCA2, qui est améliorée dans les cellules exposées à γ-irradiation. Ensuite, nous avons réduit l’interaction aux premiers 250 aa de BRCA2 (BRCA2T1) et avons constaté que celle-ci est directe en utilisant des protéines purifiées. En collaboration avec le laboratoire du docteur A. Aguilera (Cabimer, SP), nous avons montré que la déplétion de DDX5 conduit à une accumulation des hybrides ADN-ARN dans l’entièreté du génome, particulièrement aux sites de dommages à l’ADN. De plus, nos résultats indiquent que DDX5 localise aussi aux hybrides ARN-ADN qui se forment à proximité de CDB.De manière intéressante, nous avons constaté que BRCA2 est important pour la rétention de DDX5 aux sites de dommage à l’ADN induit par l’irradiation laser. Notamment, des tests de déroulement de brins in vitro en utilisant les protéines purifiées DDX5 et BRCA2 ont révélé que BRCA2 stimule l’activité de déroulement des R-loops de DDX5.Un variant de signification inconnue (VSI) trouvé dans de patients atteints de cancer du sein situé dans la région BRCA2T1 (T207A) réduit l’interaction de BRCA2 avec DDX5 et conduit à l’accumulation des hybrides ADN-ARN. Les cellules exprimant stablement BRCA2-T207A montrent également une diminution de l’association de DDX5 avec les hybrides ARN-ADN, en particulier lors d’une exposition de cellules à l’irradiation. L’analyse de l’efficacité de la réparation des CDB par RH dans les cellules déficientes en DDX5 ou exprimant BRCA2-T207A, montre une cinétique retardée de l’apparition des foyers de réparation RAD51 lors de l’irradiation, ce qui suggère un rôle actif de l’interaction BRCA2-DDX5 pour assurer la réparation par RH efficacement. En accord avec cette hypothèse, la ribonucléase RNAseH1, qui dégrade spécifiquement la fraction d’ARN dans les structures d’ADN-ARN, restaure partiellement le phénotype de cinétique des foyers RAD51 dans les cellules BRCA2 T207A. De plus, les cellules portant le variant BRCA2-T207A ont également montré un nombre réduit de foyers RPA par rapport aux cellules qui expriment BRCA2 sauvage, témoins d’un défaut dans l’étape qui précède le chargement de RAD51 aux CDB.Ensemble, nos résultats suggèrent que les hybrides ADN-ARN représentent un obstacle à la réparation des CDB par RH et révèlent BRCA2 et DDX5 en tant que facteurs actifs dans leur suppressionIncreasing evidence support the idea that proteins involved in RNA metabolism such as RNA binding proteins (RBPs) and RNA helicases are directly implicated in the DNA damage response (DDR). This activity is generally achieved through their interaction with DNA repair factors.BRCA2 is a tumor suppressor protein that plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) as well as protecting stalled replication forks from unscheduled degradation; therefore, it is essential to maintain genome integrity. Interestingly, BRCA2 deficient cells accumulate DNA-RNA hybrids or R-loops, a known source of DNA damage and genome instability, providing evidence for its role in either R-loop prevention or processing. However, the specific role of BRCA2 on these structures remains poorly understood.A mass spectrometry screen to identify partners of BRCA2 performed in our laboratory revealed an enrichment of proteins involved in RNA metabolism such as RNA helicases. These findings led us to investigate whether BRCA2 could cooperate with these candidate interacting RNA helicases in processing DNA-RNA structures. First, we confirmed the interaction of BRCA2 and the DEAD-box RNA helicase DDX5, which we found is enhanced in cells exposed to -irradiation. Then, we narrowed down the interaction to the first 250 aa of BRCA2 (BRCA2T1) and found that it is direct using purified proteins. In collaboration with A. Aguilera lab (Cabimer, SP), we could show that depletion of DDX5 leads to a genome-wide accumulation of DNA-RNA hybrids that is particularly enriched at DNA damage sites. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs. Interestingly, we found that BRCA2 is important for the retention of DDX5 at laser irradiation-induced DNA damage. Notably, in vitro R-loop unwinding assays using purified DDX5 and BRCA2 proteins revealed that BRCA2 stimulates the R-loop helicase activity of DDX5.A breast cancer variant of unknown clinical significance (VUS) located in BRCA2T1 (T207A) reduced the interaction between BRCA2 and DDX5 and led to the accumulation of DNA-RNA hybrids. Cells stably expressing BRCA2-T207A also showed a decreased association of DDX5 with DNA-RNA hybrids, especially upon irradiation. Notably, monitoring RAD51 foci to evaluate HR-mediated DSBs repair efficiency in either DDX5-depleted cells or in BRCA2-T207A cells resulted in a delayed kinetics of appearance of RAD51 foci upon irradiation suggesting an active role of BRCA2-DDX5 interaction in ensuring timely HR repair. In agreement with this, overexpression of the RNAseH1 ribonuclease, that specifically degrades the RNA moiety in DNA-RNA structures, partially restored RAD51 kinetics phenotype of BRCA2-T207A cells. Moreover, cells bearing BRCA2-T207A variant also showed a reduced number of RPA foci compared to BRCA2 WT expressing cells, a step that precedes RAD51 loading at DSBs.Taken together, our results are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by HR and reveal BRCA2 and DDX5 as active players in their removal
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Domínguez, Solà David. "Mecanismes de regulació en l'activitat biològica del factor de transcripció Snail." Doctoral thesis, Universitat Pompeu Fabra, 2003. http://hdl.handle.net/10803/7065.
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Els factors de transcripció de la família Snail són fonamentals en la "transició epiteli-mesènquima", procés morfogènic essencial en el desenvolupament embrionari i en els fenòmens metastàsics tumorals.En els mamífers l'activitat d'Snail és modulada per dos mecanismes. (i) En el promotor humà es troben regions definides de resposta a factors repressors, predominants en les cèl·lules epitelials, i elements diferenciats de resposta a inductors de la "transició epiteli-mesènquima". (ii) L'activitat d'Snail és condicionada també per la seva localització subcel·lular, modulada per mecanismes no transcripcionals: la fosforilació d'Snail determina si és o no exclós del nucli. Al citosol no pot actuar com a repressor transcripcional però pot interaccionar amb la xarxa microtubular, que estabilitza i en condiciona el dinamisme. Això coincideix amb l'activació de la GTPasa RhoA i la reorientació dels filaments de vimentina, fets associats a l'adquisició de capacitat migratòria. L'efecte com a repressor transcripcional i la modulació del dinamisme microtubular són possiblement esdeveniments coordinats necessaris per al rol biològic d'Snail en mamífers.
Snail family of transcription factors is fundamental to the "epithelial-mesenchymal transition", morphogenic process essential to embryonic development and metastatic phenomena in tumors.
Snail's activity is modulated in two ways in mammals. (i) The human promoter harbors definite regions that respond to repressor factors, which prevail in epithelial cells; and differentiated elements that respond to known inducers of the "epithelial-mesenchymal transition". (ii) Snail's activity is also conditioned by its subcellular localization, mechanism not dependent on its transcriptional control: Snail phosphorylation determines whether Snail is excluded or not from the nucleus. When in the cytosol, Snail is unable to act as a transcriptional repressor, but however binds to the microtubular meshwork, which becomes stabilized and whose dynamism is conditioned as a result. This fact coincides with the activation of the RhoA GTPase and reorientation of vimentin filaments, both phenomena being related to the acquisition of cell motility. The transcriptional repressor and the microtubule dynamics effects are probably two coordinated events necessary to Snail's biological role in mammals.
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Lou, Zheng active 2012. "Transposable prophage Mu exists as an independent chromosomal domain in E. coli." 2012. http://hdl.handle.net/2152/22180.
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The 4.6 Mb circular E. coli chromosome is compacted by segregation into 400-500 supercoiled domains, created by both active and passive mechanisms like transcription and DNA-binding proteins. We find that transposable prophage Mu, transcriptionally silent by definition, is organized into an independent domain as determined by the close proximity of Mu termini L and R separated by a 37 kb Mu genome. Cre-loxP recombination is used in this study in vivo and in vitro. Critical to formation/maintenance of the Mu 'domain' configuration are a strong gyrase site SGS at the center of Mu, the Mu L end, the MuB protein, and the E. coli nucleoid-associated proteins IHF, Fis and HU. The Mu domain was observed at two structurally different chromosomal locations, and was specific to the Mu prophage, i.e. was not observed for the [mathematical symbol] prophage. A model is proposed that by employing its cis-elements to create a domain barrier for segregation and compaction of its genome, the large selfish DNA element Mu profits from the transposition-ready arrangement of its ends, while simultaneously providing a fitness advantage to the host.text
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Valente, Luís Paulo Ferreira. "MYB domain proteins and the maintenance of chromosome integrity in S. Pombe." Doctoral thesis, 2010. http://hdl.handle.net/10316/13891.
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Tese de doutoramento em Bioquímica (Biologia Molecular), apresentada à Fac. de Ciências e Tecnologia da Universidade de CoimbraProteins containing Myb domains are present in a wide variety of organisms, performing different tasks. A subset of them has been related to the telomere, as they are able to bind to the telomere repeats. In this work, while characterizing several of these Myb domain proteins it was found that they can be involved in regulating both the transcription and post transcriptional modification of genes implicated in essential processes, most notably the assembly of key chromatin structures including centromeres. More precisely, in this thesis it will be shown that the essential fission yeast Myb protein Laz1 binds to promoters throughout the genome and regulates the transcription of several groups of genes, like the histones. Moreover, the same protein is also in control of the post-transcriptional clipping of the histone H3 that occurs upon G1 arrest. In addition to these roles, it will be shown that Laz1 is involved in loading the histone variant CENP-A histone into the centromeres. In this work the function of another Myb protein, Tbf1, was also studied. This protein is essential for viability and localizes to the nucleus. Truncation of Tbf1 shows that its C-terminal part, which includes the Myb domain, is fundamental for survival of the cell. It is also shown that this protein may have a telomere function, as its overexpression leads to telomere elongation. Finally, it is here described how a well characterized telomere protein containing Myb domains, Rap1, is crucial for the maintenance of the telomere chromatin structure. In summary, results from this thesis show how several proteins that share a similar domain can perform different essential tasks during the cell cycle, allowing the preservation of S. pombe chromosome integrity.
Proteínas que contêm domínios Myb estão presentes numa grande variedade de organismos, efectuando diferentes funções. Um subgrupo destas proteínas tem sido relacionado com os telómeros, pois os seus membros são capazes de se ligar às repetições teloméricas. Neste trabalho, ao caracterizar-se diversas proteínas contendo domínios Myb, descobriu-se que estas podem estar envolvidas em regular não só a transcrição, como também modificações pós-transcripcionais de genes associados a processos essenciais. Entre os processos abrangidos encontra-se a montagem de estruturas chave na cromatina, como os centrómeros. Mais precisamente, nesta tese vai ser demonstrado que a proteína Myb chamada Laz1 se liga a promotores presentes no genoma e regula a transcrição de diferentes grupos de genes, como as histonas. Além disso, a mesma proteína também controla o corte pós-transcripcional da histona H3 que ocorre após paragem na fase G1. Para além de descrever essas funções, este trabalho demonstra que Laz1 está envolvida no carregamento da variante de histonas CENP-A para o centrómero. A função de Tbf1, outra proteína Myb, também é aqui estudada. Esta proteína é essencial e encontra-se no núcleo da célula. A supressão de partes de Tbf1 demonstra que a sua parte C-terminal, que inclui o domínio Myb, é fundamental para a sobrevivência da célula. É também demonstrado que esta proteína poderá ter uma função telomérica, pois a sua sobre-expressão leva a um elongamento dos telómeros. Por último, é aqui descrito como a já caracterizada proteína Myb Rap1 é fundamental para a manutenção da estrutura da cromatina dos telómeros. Em resumo, os resultados apresentados nesta tese demonstram como diversas proteínas que partilham um domínio semelhante podem efectuar diferentes tarefas essenciais durante o ciclo celular, permitindo a preservação da integridade cromossómica da levedura de fissão.
Trabalho realizado no Telomere Biology Laboratory, London Research Institute, Cancer Research UK com o apoio financeiro da Fundação para a Ciência e Tecnologia (SFRH/BD/15232/2004), co-financiada pelo POCI2010 e pelo FSR
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Wani, Saima Masood. "Elucidation of the Role of Nse1, a RING Domain Containing Component of Smc5/6 complex, in Maintenance of Chromosome Stability in Saccharomyces cerevisiae." Thesis, 2017. http://etd.iisc.ernet.in/2005/3570.
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Structural Maintenance of Chromosomes (SMC) proteins are a highly conserved class of proteins required for the maintenance of genome stability and regulate nearly all aspects of chromosome biology. Eukaryotes, such as the budding yeast Saccharomyces cerevisiae, have six Smc proteins that form three SMC complexes in association with non-SMC proteins, i.e., the cohesin complex, the condensin complex and the Smc5/6 complex. The yeast Smc5/6 complex consists of Smc5, Smc6 and six non-Smc elements (Nse1-6) that are all essential for the survival of cells.
Nse1 is the first non-smcelement that was identified associated with the Smc5/6 complex. Nse1 has a C-terminal RING-domain, which is a characteristic feature of some E3 ubiquitin ligases. A RING domain consists of eight conserved Zn-coordinating residues arranged in a cross-brace conformation. To understand the importance of this domain, we created site directed mutations in conserved residues identified by sequence alignment of the budding yeast Nse1 RING domain with that of other species. We found a new RING domain mutant nse1-103that was temperature sensitive at 37°C and showed an increased sensitivity towards genotoxic agents such as hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. Thense1-103 mutant cells are slow growing and show delayed chromosomal replication at the restrictive temperature. Genetic interactions with replication factors such as RRM3, TOF1 etc. revealed thatnse1-103shows a synthetic sick growth defect in combination with rrm3∆ that is partially suppressed by deletion of TOF1. We found an enhancement in chromosome loss in nse1-103 compared to wild type cells. This was accompanied by a slight reduction in cohesion between the sister chromatids in nse1-103,suggesting a plausible mechanism for the chromosome destabilization observed in the mutant.
Since Nse1 forms part of a trimeric sub-complex with Nse3 and Nse4 in the Smc5/6 complex, we performed a yeast two hybrid assay to test the interaction of nse1-103 with Nse3 or Nse4, and found a defect in interaction of nse1-103 with Nse3 and Nse4. In addition, a defect in association of nse1-103 with Smc5 or Smc6 could be observed by performing co-immunoprecipitation from yeast cell lysates, suggesting that the integrity of the RING-domain is critical for the interaction of Nse1 with other subunits of the Smc5/6 complex. However, there was no defect in the interaction between Nse3 and Smc5 in nse1-103, indicating that the interaction of these components within the complex isindependent of Nse1.
We also identified a novel sequence motif near the RING domain of Nse1, deletion of which leads to an increased sensitivity towards genotoxic stressors and higher temperature. Biochemical characterization of this mutant also suggests a defect ininteraction with Nse3 or Nse4, and also with Smc5. The nse1 mutants also showed defects in post translational modification of Smc5 and other proteins.
Since the Smc5/6 complex also has a SUMO E3 ligase, Mms21/Nse2, we also investigated genetic interactions between the RING domain mutant,nse1-103 and the SUMO ligase RING domain defective mutant,mms21∆sl, and found an exacerbation of the drug sensitive phenotypes in thense1-103 mms21∆sl double mutant relative to either of the single mutants nse1-103 or mms21∆sl, indicating that the two proteins contribute independently to the function of Smc5/6 complex in resisting genotoxic stress.
In conclusion, the present study emphasizes the role of the RING domain of budding yeast Nse1 in resisting genotoxic stress and maintaining chromosome stability and reveals that the integrity of the RING-domain is critical for interactions of Nse1 with Nse3 and other Smc5/6 complex components. In addition, we report identification of another novel sequence motif in Nse1 that is also crucial for its interaction with other subunits of the Smc5/6 complex and for maintenance of post-translational modifications of some cellular proteins.
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Vadakkan, Kunjumon Ittira. "Clustering of centromeric domains in cerebellar Purkinje and granule neurons is chromosome-specific and cell-type specific." 2004. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=80982&T=F.
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Griese, Julia Johanna [Verfasser]. "Structures and DNA-binding activities of the hinge domains from the structural maintenance of chromosomes proteins of Pyrococcus furiosus and the mouse condensin complex / Julia Johanna Griese." 2010. http://d-nb.info/1008342513/34.
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Abd, Rabbo Diala. "Étude du transcriptome des cellules non tumorales de l’épithélium de surface de l’ovaire des femmes porteuses d’une mutation des gènes BRCA1 et BRCA2." Thèse, 2009. http://hdl.handle.net/1866/4384.
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Nous avons étudié le transcriptome de neuf échantillons d'ARN extraits de cultures primaires de cellules non tumorales de l’épithélium de surface de l’ovaire (NOSE) provenant de quatre donneuses non porteuses de mutation, deux mutées sur BRCA1 et trois sur BRCA2, ainsi que de quatre échantillons d’ARN extraits de cultures primaires de cellules tumorales de l’ovaire (TOV) provenant de trois donneuses porteuses de mutation sur BRCA1 et une sur BRCA2. Nous avons identifié, pour la première fois, les signatures moléculaires associées à la présence d’une mutation de BRCA1 et BRCA2 dans les cellules NOSEs ainsi que la signature associée à la transformation tumorale des cellules NOSEs en TOVs chez les porteuses de mutation de BRCA1. Nous avons également localisé les domaines chromosomiques comportant des gènes corégulés en association avec la présence d’une mutation de BRCA1 dans les cellules NOSEs. Les allèles sauvage et muté de BRCA2 étaient exprimés dans les cellules TOVs provenant des porteuses de la mutation 8765delAG sur BRCA2. Nous avons observé que le niveau d’expression des transcrits de BRCA2 était plus élevé dans les cellules provenant des tumeurs ovariennes les plus agressives chez les femmes porteuses de la mutation 8765delAG sur BRCA2, les transcrits correspondants à l’allèle muté contribuant avec un pourcentage élevé du niveau d’expression total du gène. Le phénotype tumoral observé chez les Canadiennes Françaises porteuses de cette mutation pourrait résulter d’un effet de dosage de l’allèle muté.We analyzed the transcriptome of nine primary cultures of non-tumor ovarian surface epithelium cells (NOSE) from four non-carriers, two BRCA1 and three BRCA2 carriers, and four primary cultures of tumor ovarian cells (TOV) from three BRCA1 and one BRCA2 carriers. We identified the first molecular signatures associated with the presence of BRCA1 and BRCA2 mutations in NOSEs and the first molecular signature associated with the transformation from NOSEs to TOVs in French Canadian women carriers of BRCA1 mutation. Moreover, we localized some co-regulated chromosomal domains associated with the presence of a BRCA1 mutation in NOSE cells. Wild-type and mutated BRCA2 allelic transcripts were expressed in tumor cells from 8765delAG BRCA2 mutation carriers, with the highest level of BRCA2 transcript expression and the highest contribution of the mutated allele in cells originating from the most aggressive ovarian tumors. The observed phenotype in BRCA2-mutated cells as well as the aggressiveness of the tumor could result from a dosage effect of the BRCA2 mutated allele.