Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, January 2007.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

No description available.

Les quadruplexes de guanines (G4) sont des structures polymorphiques adoptées in vitro par les séquences d’ADN et d’ARN riches en guanines. L’utilisation d’anticorps et de ligands spécifiques des structures G4 a permis leur détection au niveau cellulaire. Des études computationnelles ont prédit des séquences possédant une signature G4 au niveau de régions génomiques capitales comme les télomères ou les promoteurs de certains oncogènes. De plus, de nombreuses protéines impliquées dans des processus cellulaires comme la réplication, la transcription ou encore la réparation, peuvent interagir directement avec des G4, en facilitant leur formation ou au contraire leur dénaturation. C’est notamment le cas d’hélicases impliquées dans des pathologies humaines, comme BLM, WRN, FANC J ou PIF1. Ce sont des enzymes capables de dénaturer des G4 et dont l'inactivation induit une instabilité génomique, en particulier au niveau de régions susceptibles de former un G4. Dans ce travail, nous présentons la mise au point d’un test de criblage à moyen débit pour le suivi des interactions G4/hélicases en temps réel. Ce test nous a permis de définir les conditions favorisant ou inhibant l’interaction d’une hélicase vis-à-vis de son substrat G4. Nous avons démontré que ces conditions pouvaient différer d’une hélicase à une autre, notamment les conditions salines optimales nécessaires aux activités hélicases de ScPif1 et de RHAU. Nous avons également prouvé, à travers ce test, que l’utilisation de ligands capables de stabiliser les G4 n’induisait pas forcément d’inhibition de l’activité hélicase de ScPif1. Enfin, nous avons également pu définir la directionnalité de la protéine RPA, ce qui fait de notre test une technique prometteuse pour la caractérisation de nouvelles protéines pouvant dérouler des structures G4. / G-quadruplexes are highly polymorphic non-canonical nucleic acid structures adopted by both DNA and RNA guanine-rich sequences in vitro. They have been detected at the cellular level using structure specific antibodies and small molecule ligands. Computational studies demonstrated that G4-prone sequences are located in key genomic regions such as telomeres and oncogene promoters. Numerous studies showed that G4 sequences can interact with proteins involved in cellular processes, including replication, transcription or reparation. Those interactions include binding, G4 folding promotion or in contrary unwinding. Indeed, WRN, BLM, FANC J or Pif1 are helicases associated with human-diseases. They can unwind G4 forming sequences; mutation of these helicases lead to genomic instability of G4-prone motifs when mutated. Here, we present a medium-throughput technique to monitor G4-helicase interactions in real time. We were able to determine both favourable and deleterious conditions for G4 unwinding by a given helicase. We show that these conditions differ from one helicase to another as exemplified with the optimal salt conditions required for both ScPif1 and RHAU activities. We also reveal that the G4 ligands that stabilize G4 structures do not necessarily induce an inhibition of their unwinding by ScPif1 helicase. Finally, we also prove that our assay is adapted to clear up RPA directionality, making it an attractive technique to screen for new proteins able to unwind G4 structures.

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24946号 / 生博第508号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 松本 智裕, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

Natural killer cells are innate immune effectors that kill virally infected or malignant cells. Natural killer cell deficiency (NKD) occurs when NK cell development or function are impaired, and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies result in NKD, including variants in MCM4, GINS1, MCM10 and GINS4, which are components of the CDC45-MCM-GINS (CMG) helicase. The CMG helicase unwinds DNA during replication and is expressed in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this susceptibility are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. We found that activated NK cells undergo apoptosis and autophagy in response to this stress, unlike activated T cells. We also identified a patient with a damaging variant in CDC45 to further support these findings of the effects of replication stress on NK cells. This individual, due to broader involvement of the immune system, requires a wider definition of natural killer cell disease, termed NK IEI. However, this CDC45-deficient individual’s cells display disrupted cell cycle, increased DNA damage and replication stress, with upregulation of apoptosis genes in NK cells. These findings show that sensitivity to replication stress affects human NK cell survival and function and can contribute to NK cell deficiency and human disease.

No description available.