Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

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Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

WT1 has two main isoforms: WT1(-KTS) and WT1(+KTS). Both are known to bind to a DNA consensus sequence with different affinities, and are thus postulated to play overlapping but distinct functional roles in the cell. WT1 is also known to bind to certain RNA moieties as well as to various protein partners (e.g. Ciao-1). This study focuses on the development of large scale protein purification protocols for WT1 zinc finger (ZF) proteins as well as Ciao-1. By using a combination of his-tag affinity and size exclusion chromatography we were able to purify milligram quantities of these proteins. It was also the intention to obtain crystals of the WT1 ZF protein in complex with any one of its known binding partners, in particular the protein Ciao-1 (a WD40 protein) and the 14 mer consensus sequence of DNA (known as WTE). In conjunction with structural studies it was determined that a previously made SELEX RNA library was not selective for the (+KTS) isoform of WT1 ZF, and therefore no RNA candidate could be identified for future structural studies.

L'ubiquitination est une modification post-traductionnelle qui joue un rôle central dans divers processus biologiques. Elle peut être contrecarrée par les déubiquitinases (DUBs). "BRCA1-Associated Protein 1" (BAP1) est une déubiquitinase, qui fait partie de complexes multiprotéiques, possèdant une fonction de suppression tumorale ainsi qu'un potentiel anti-métastatique. De plus, BAP1 est phosphorylée suite aux dommages à l’ADN par les kinases ATM/ATR. En nous basant sur ces données, nous avons purifié les protéines associées à BAP1 dans des conditions de stress génotoxique. Bien que la composition du complexe et l’activité DUB semblent inchangées, nous avons pu identifier des changements critiques dans les niveaux et les sites de phosphorylation, confirmant la régulation de BAP1 suite aux dommages à l’ADN. En déplétant BAP1 par ARNi et en utilisant des mutants dominants négatifs, nous avons obtenu des résultats suggèrant que suite au stress génotoxique, cette DUB est requise pour prolonger le point de contrôle en G2/M et ce, en retardant la reprise du cycle cellulaire. D'un autre côté, l'expression de BAP1 dans des cellules cancéreuses qui en sont déficientes restore une ploïdie normale et diminue la fréquence d'aberrations nucléaires, suggérant que cette protéine joue un rôle dans la stabilité génomique. Nos résultats suggèrent fortement que BAP1 joue un rôle dans la réponse des cellules au stress génotoxique et la stabilité génomique. Nos travaux permettront ainsi d’identifier et de caractériser les voies de signalisation cellulaire régulant l’activité et la fonction de BAP1 durant les périodes d’exposition à des agents qui endommagent l’ADN. Les connaissances acquises seront donc d’une valeur tangible pour nôtre compréhension de la mutagenèse induite par des agents carcinogènes, un déterminant clé de la formation des tumeurs. / Ubiquitination is a reversible, covalent post-translational modification that regulates protein function and as such plays crucial roles in a wide range of physiological processes. Importantly, gain- or loss-of-function mutations in components of the ubiquitin system have been causally linked to tumorigenesis. The reverse reaction of ubiquitination is catalyzed by deubiquitinases (DUBs), a family of enzymes that removes ubiquitin from proteins. BRCA1-Associated Protein 1 (BAP1) is a deubiquitinase known to be a tumor suppressor and anti-metastatic protein since deletions and rearrangements are observed in a wide range of tumors. However, little is known about how BAP1 works into the cells. Here, we show that BAP1 is hyperphosphorylated after DNA damage by gamma radiations and ultraviolet light, probably by ATM and/or ATR. Moreover, we found that BAP1 depletion cause a defect in the maintenance of the G2/M checkpoint after gamma radiation, suggesting that BAP1 is required to maintain the arrest after DNA damage. This delay is important to allow DNA repair and to prevent genomic instability. Consistently, we found that BAP1 expression in BAP1 deficient cells restore normal diploidy and prevent nuclear aberrations, suggesting that BAP1 links DNA damage induced checkpoint regulation to genomic stability: two important processes for carcinogenesis. These findings provide new insights into the role of deubiquitination in cell signaling and neoplastic transformation.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 2 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online in PDF format.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 87 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 128 pages. Includes vita. Includes bibliographical references.