Thesis (Ph.D.) - University of St Andrews, January 2008.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jun 1.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 135 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Eric B. Kmiec, Dept. of Biological Sciences. Includes bibliographical references.

Apesar de intensivos estudos e substanciais progressos no entendimento dos fatores de risco e suscetibilidade ao câncer de mama (CM), esta neoplasia permanece como importante causa de morte entre mulheres. Idade, história familiar, menarca precoce, menopausa tardia, ocorrência da primeira gravidez após os 30 anos e da nuliparidade constituem fatores de risco. Além disso, polimorfismos nos genes envolvidos no reparo de danos no DNA, como os genes XRCC1 e XRCC3, podem contribuir para o aumento da suscetibilidade ao CM. Os objetivos do presente trabalho foram avaliar pelo Teste do MN e Ensaio Cometa os danos basais e a resposta celular aos danos induzidos, in vitro, no DNA pelo quimioterápico Etoposido em pacientes com CM, virgens de qualquer tipo tratamento e em mulheres saudáveis utilizadas como controles e além disso, estabelecer as freqüências dos polimorfismos nos genes XRCC1 e XRCC3 na amostra de pacientes com CM e em mulheres saudáveis e associação destes dois polimorfismos com a suscetibilidade ao CM. No Teste do MN, foi observada uma sensibilidade maior do grupo de pacientes aos danos induzidos pelo Etoposido. O Ensaio Cometa mostrou que pacientes e mulheres saudáveis respondem de modo semelhante ao tratamento com o Etoposido. Também foi observado que pacientes acima de 45 anos apresentaram um grau maior de sensibilidade aos danos induzidos pelo Etoposido na concentração de 25 M quando comparadas com pacientes abaixo de 45 anos avaliadas no Ensaio Cometa. Quanto ao hábito tabagista, este se mostrou um fator de contribuição ao aumento de sensibilidade a indução de danos pelo Etoposido no Ensaio Cometa no grupo de mulheres saudáveis, para os tratamentos com esta droga nas concentrações de 10 e 25 M. Na análise molecular, o alelo variante 241Met do gene XRCC3 mostrou-se mais freqüente no grupo de pacientes tanto na amostra estudada na análise citogenética quanto na amostra estudada na análise molecular, sugerindo uma diminuição da capacidade de reparo destas pacientes, o que poderia conferir um risco aumentado ao CM. Quanto ao hábito tabagista, somente as pacientes não fumantes, portadoras do alelo 241Met do gene XRCC3, possuem um risco aumentado para o CM. Não foi encontrada associação do polimorfismo Arg399Gln do gene XRCC1 com o risco ao CM mesmo quando associado à fatores de risco como hábito tabagista e a presença de familiares com câncer. / In spite of intensive studies and substantial improvements in the understanding of the risk factors and breast cancer (BC) susceptibility, this neoplasia remains as an important cause of death among women worldwide. Age, family history of cancer, early menarche, late menopause, the first pregnancy after the age of 30 years and nulliparity are BC risk factors. Furthermore genetic polymorphisms in repair genes like XRCC1 and XRCC3 could contribute to increase BC risk. The aims of the present study were to evaluate, by Micronucleus Test and Comet Assay, the basal damage and the cellular response to DNA damage induced by Etoposide, in vitro, in BC patients without chemotherapy treatment and in healthy women. Also establish the frequencies of polymorphisms of XRCC1 and XRCC3 genes in this sample and the association of these two polymorphisms with the susceptibility to BC. In the Micronucleus Test it was observed increased sensibility to DNA damage induced by Etoposide in patients group. Patients and healthy women exhibited the same repair capacity to DNA damage induced by Etoposide when evaluated by Comet Assay. Patients > 45 years old showed more sensibility to DNA damage induced by Etoposide (25 M) when were compared with patients 45 years old in Comet Assay. Tobacco habits contributed to increased sensibility to damage induced by Etoposide in Comet Assay in healthy women group when treated with Etoposide in 10 and 25 M. In the molecular analysis, the XRCC3 241Met allele was more frequent in patients group in both analysis (cytogenetic and molecular) suggesting a low repair capacity of DNA damage and consequently increase risk to BC. Non-smokers patients, carriers of XRCC3 241Met allele showed an increased risk to BC. The polymorphism Arg399Gln in XRCC1 gene was not associated with BC risk even if associated with risk factors like tobacco habit and family history of cancer.

The integrity of the genome is critical to organisms and it is affected by many factors. Radiation, for example, poses a serious threat to genome stability of human beings. While physical monitors for radiation hazard are present, the biological consequences of long term exposure to radiation are not well understood. With the opportunity as part of the International Caenorhabditis elegans Experiment-1 flight project, several approaches using C. elegans were taken to measure mutational changes that occurred during the spaceflight. Among these methods, the eT1 balancer system was demonstrated to be well-suited as an integrating biological dosimeter for spaceflight. The dog-1 gene in C. elegans is required to prevent mutations at poly-G/poly-C tracts, and previous work has described that in the absence of DOG-1, small deletions initiate within these tracts, most likely as a consequence of improperly repaired replication blocks. The eT1 balancer system was adapted to investigate the broad mutational spectrum of dog-1 mutants. Using this system, I was able to determine a forward mutation rate of approximately 1 x 10-3, 10 fold higher than spontaneous. Both small deletions as reported previously and unreported large chromosome rearrangements were observed, and most of mutations analyzed are associated with G/C tracts. Thus, I propose that following dog-1-induced replication blocks, repair leads to a wide range of mutational events and chromosomal instabilities, similar to those seen in human cancers. The existence of the G/C tracts in C. elegans creates a fortuitous but perplexing problem. They are hotspots for genome instability and need enzymatic protection. In the genome of C. elegans, approximately 400 G/C tracts exist and are distributed along every chromosome in a non-random pattern. G/C tracts are also over-represented in another Caenorhabditis species, C. briggsae. However, the positions and distribution differ from those in C. elegans. Furthermore, in C. elegans, analysis of SAGE data showed that the position of the G/C tracts correlated with the level of gene expression. Although being a threat to genome stability, the genomic distribution of G/C tracts in C. elegans and their effect on regional transcription levels suggest a role for G/C tracts in chromatin structure. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

L’ADN est constamment exposé à des insultes génotoxiques endogènes et exogènes. Plusieurs mécanismes de réparations de l’ADN sont mis en œuvre pour préserver la stabilité du génome et de l’épigénome. La recombinaison homologue (RH) joue un rôle central dans la réparation des cassures double brin de l’ADN (DSBs) et le redémarrage des fourches de réplication en réponse à un stress réplicatif. Ces deux processus sont tous deux couplés à l’assemblage de la chromatine. Le facteur d’assemblage de la chromatine 1 (CAF-1) est un chaperon d’histone conservé au cours de l’évolution qui fonctionne dans le processus d’assemblage des nucléosomes couplé à la réparation de l’ADN et à la réplication, en déposant sur l’ADN les tétramères d’histones (H3-H4)2 nouvellement synthétisés. Chez la levure Schizosaccharomyces pombe, le complexe CAF-1 est constitué de trois sous-unités, Pcf1, Pcf2 et Pcf3. Il a été montré que CAF-1 agit dans l’étape de synthèse de l’ADN durant le processus de réplication dépendante de la recombinaison (RDR) et protège le désassemblage des D-loop par l’hélicase Rqh1, membre de la famille des hélicases RecQ. Dans cette étude, nous avons adressé le rôle de CAF-1 pendant la réparation de l’ADN par recombinaison homologue chez la levure Schizosaccharomyces pombe. Par l’utilisation d’approches in vivo et in vitro, nous avons validé des interactions protéines-protéines au sein d’un complexe contenant Rqh1, CAF-1, PCNA, et l’Histone H3. Nous avons montré que Rqh1 interagit avec Pcf1 et avec Pcf2 indépendamment l’un de l’autre, et que l’interaction Rqh1-Pcf1 est stimulée par des dommages à l’ADN. Nous avons mis en place une méthode d’analyse de liaison à la chromatine pour suivre l’association de CAF-1 à la chromatine en réponse aux dommages à l’ADN. Nous avons observé qu’un stress réplicatif, mais pas l’induction de cassures double brin de l’ADN, favorise l’association de CAF-1 à la chromatine. Nous avons identifié plusieurs facteurs de la RH nécessaire pour l’association de CAF-1 à la chromatine en réponse à un stress réplicatif. De plus, nous avons mis en évidence des interactions physiques entre Pcf1 et des facteurs de la recombinaison homologue, parmi lesquels RPA et Rad51. Nos données suggèrent que CAF-1 pourrait s’associer aux sites de synthèse d’ADN dépendent de la recombinaison via son interaction avec des facteurs de la RH. L’ensemble des données de cette étude contribuent à renforcer le role de CAF-1 couplé à réparation de l’ADN, et révèlent une interconnexion entre les facteurs de la RH et l’assemblage de la chromatine. / DNA is constantly exposed to both endogenous and exogenous genotoxic insults. Multiple DNA repair mechanisms are exploited to guard the genome and epigenome stability. Homologous recombination (HR) plays a major role in repairing DNA double strand breaks (DSBs) and restarting stalled replication forks under replicative stress. These two processes are both coupled to chromatin assembly. Chromatin assembly factor 1 (CAF-1) is a highly conserved histone chaperone known to function in a network of nucleosome assembly coupled to DNA repair and replication, by depositing newly synthesized histone (H3-H4)2 tetramers onto the DNA. The fission yeast CAF-1 complex consists of three subunits Pcf1, Pcf2 and Pcf3. CAF-1 has been previously reported to act at the DNA synthesis step during the process of recombination-dependent replication (RDR) and protects the D-loop from disassembly by the RecQ helicase family member, Rqh1. In this study, we addressed the role of CAF-1 during homologous-recombination-mediated DNA repair in fission yeast.Using in vivo and in vitro approaches, we validated interactions within a complex containing Rqh1, CAF-1, PCNA, and Histone H3. We showed that Rqh1 interacts with both Pcf1 and Pcf2 independently of each other, and the Pcf1-Rqh1 interaction is stimulated by DNA damage. We developed an in vivo chromatin binding assay to monitor the association of CAF-1 to the chromatin upon DNA damage. We observed that replication stress but not double strand break favors CAF-1 association to the chromatin. We identified that several HR factors are required for CAF-1 association to the chromatin upon replication stress. In support of this, we have identified physical interactions between Pcf1 and HR factors, including RPA and Rad51. Our data suggest that CAF-1 would associate with the site of recombination-dependent DNA synthesis through physical interactions with HR factors. Put together, this work contributes to strengthening the role of CAF-1 coupled to DNA repair, and reveals the crosstalk between HR factors and chromatin assembly.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21874号 / 人博第903号 / 新制||人||215(附属図書館) / 2018||人博||903(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 小林 純也, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23086号 / 医博第4713号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 藤田 恭之, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les protéines et les voies impliquées dans la réponse aux dommages de l'ADN (DDR), permettant de maintenir la stabilité génétique et de préserver la fidélité de la réplication, agissent non seulement comme l'initiation de la cancérogenèse mais peuvent également jouer un rôle majeur dans la progression tumorale et dans la résistance aux thérapies. La voie FANC joue un rôle central dans la stabilité génétique lors d'un stress réplicatif. La perte de fonction de cette voie est la cause d'un syndrome de fragilité chromosomique et de prédisposition au cancer appelé Anémie de Fanconi (FA).Nous avons démontré que les protéines FANC était surexprimées et suractivées dans les mélanome métastatiques exprimant le facteur de transcription MITF, un oncogène exprimé dans 80% des cas de mélanome. Nous avons identifié MITF comme un régulateur majeur de l'expression des transcrits codant les protéines de la voie FANC dans les cellules de mélanomes et montré que les cellules déplétées pour MITF présentaient les caractéristiques des cellules FA, i.e., une hypersensibilité aux agents pontants l'ADN. De plus, la voie FANC module également la migration des cellules de mélanome. Nos observations montrent le rôle central de cette voie de réparation dans la résistance des cellules de mélanomes aux dommages de l'ADN. Cette voie serait donc une nouvelle cible thérapeutique dans le traitement du mélanome.Nous avons également observé que la perte de fonction de la voie FANC augmente l'expression de MiTF dans des cellules FA et des cellules déplétées pour les protéines FANC. Nous avons montré que la voie FANC régule négativement l'expression de MITF au niveau transcriptionnel. Des résultats préliminaires montre que FANCD2 pourrait être associés au promoteur de MiTF au niveau d'un site de fixation de NF-kB, où il pourrait empêcher son action. La déplétion de MiTF conduit à une sensibilité aux agents pontants l'ADN dans des cellules contrôles mais pas dans des cellules FA, suggérant que MITF jouerait un rôle dans la DDR en régulant l'expression des protéines FANC. Enfin, nous avons montré que l'expression de MiTF est induite en réponse aux stimuli pro-inflammatoire. Enfin, l'expression altérée de MITF pourrait expliquer des défauts de pigmentation et la microphthalmie rapportés chez les patients FA. L'ensemble de ces données, à la fois validée et préliminaire, supporte l'existence d'une relation épistatique entre MiTF et la voie FANC. Cet voie aurait un rôle important à la fois dans la résistance au mélanome métastatique et dans certaines caractéristiques pathologiques de l'anémie de Fanconi. / Proteins and pathways involved in DNA damage response (DDR), maintaining genetic stability and safeguarding DNA replication, act not only as caretakers against cancer initiation but also play a major role in sustaining cancer progression and resistance to pharmacological-based therapies. The FANC pathway is central in maintaining genetic stability under conditions of replication stress and its loss-of-function is causative of the cancer predisposition and chromosome fragility syndrome Fanconi Anemia (FA).We demonstrate here that FANC proteins are over-expressed and over-activated in metastatic melanoma cells expressing the oncogenic microphthalmia-associated transcription factor (MiTF), which high expression is maintained in 80% of melanoma cases. We identified MiTF as a critical regulator of the expression of the mRNAs coding key proteins of the FANC pathway in melanoma cells and demonstrated that MiTF-silenced cells display the primary characteristics of FA cells, i.e., the cellular and chromosomal hypersensitivity to DNA interstrand crosslink- inducing agents. Moreover, FANC pathway also modulates melanoma cell migration. Our observations point to a central role of the FANC pathway in cellular and chromosomal resistance to DNA damage in melanoma cells. Thus, the FANC pathway appears as a promising new therapeutic target for melanoma treatment.Inversely, we observed that FANC pathway loss-of-function is associated to increased expression of MiTF in both FA patient-derived and siRNA-downregulated cells. We demonstrated that the FANC pathway negatively regulates MiTF expression at the mRNA level and have obtained preliminary data suggesting that FANCD2 associates to the MiTF promoter, impeding the action of the NF-kB transcription factor. MiTF depletion increases MMC sensitivity in FANC pathway proficient cells, but does not modify the sensitivity of FA cells, supporting the hypothesis that MiTF acts on the DDR by regulating the expression of FANC proteins. Finally, we demonstrated that MiTF expression is induced in response to inflammatory stimuli, like TNF-a. Thus, altered MiTF expression in FA could be involved in the pigmentation defects and microphthalmia reported in patients.In conclusion, we will present a corpus of both validated and yet preliminary data that strongly supports the existence of an epistatic relationship between MiTF and the FANC pathway. This circuitry appears to have an important role in melanoma resistance to chemotherapies and in some FA pathological traits.