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Dans nos sociétés modernes, la presbyacousie, perte de l'audition liée au vieillissement, prend une place de plus en plus importante. Outre le vieillissement de la population, la prévalence de la presbyacousie est accentuée par l'exposition à des bruits toujours plus forts (concerts, baladeurs, environnement de travail, ...) et la prise de médicaments ototoxiques (cisplatine, aminoglycosides, ...). À ce jour, le lien entre l'endommagement de l'ADN, le stress oxydant et l'inflammation avec l'apparition précoce de certaines maladies liées au vieillissement (Alzheimer, démence, Parkinson, …) a été démontré. Cependant, il n'existe aucune donnée concernant le rôle des dommages de l'ADN dans la dégénérescence des cellules cochléaires et trop peu d'études témoignent de l'existence d'un stress oxydant dans la presbyacousie.Le premier objet de ce travail a donc été d'élucider le rôle des dommages de l'ADN dans la dégénérescence des cellules cochléaires. Pour ce faire, nous avons utilisé des approches de biologie moléculaire et cellulaire pour identifier des voies de signalisation associées aux lésions de l'ADN dans des explants cochléaires issus de souris âgées de 3 jours traités au cisplatine (CDDP). Cet antinéoplasique tire sa cytotoxicité de sa capacité à causer directement des dommages dans l'ADN et est connu pour ses effets nocifs sur l'audition en induisant la dégénérescence des cellules cochléaires. Enfin, nous avons étudié l'implication de p53, un des effecteurs clés de signalisation des dommages de l'ADN, in vivo en traitant avec le CDDP des souris dont le gène codant pour ce facteur de transcription a été invalidé. Nos résultats montrent que le CDDP induit des cassures double brin dans l'ADN des cellules ciliées qui sont à l'origine de l'activation de la voie ATM/DNA¬PK-Chk2-p53, de la formation de foyers βH2AX et 53BP1 et, in fine, de la mort de ces cellules par apoptose. Les cellules ciliées internes, plus résistantes au CDDP que les cellules ciliées externes, présentent une signalisation moins intense et un nombre inférieur de cassures double brin, un phénomène qui pourrait expliquer leur plus faible sensibilité. Nous avons également montré que l'absence de p53 in vivo prévient les pertes d'audition et la dégénérescence des cellules ciliées externes après injection intrapéritonéale de CDDP. Le second objectif a porté sur l'étude des effets délétères du vieillissement sur l'audition et les mécanismes moléculaires associés à cette pathologie. Pour ce faire, nous avons choisi les souris SAMP8 (senescence accelerated mice prone 8), un modèle bien établi de sénescence précoce et des maladies liées au vieillissement. Nous avons combiné des approches fonctionnelles, morphologiques, moléculaires et cellulaires pour phénotyper ces souris et identifier l'origine de l'atteinte de leur audition au cours du vieillissement. L'étude des souris SAMP8 nous a permis de montrer qu'elles sont un excellent modèle de presbyacousie mixte (atteinte de la strie vasculaire, de l'organe de Corti et du ganglion spiral), résumant la pathologie humaine. La dégénérescence des structures cochléaires que nous avons observée chez ces souris provient d'une profonde dysfonction mitochondriale, de l'augmentation du stress oxydant et des processus inflammatoires, d'un stress autophagique et de l'endommagement de l'ADN. Les mécanismes moléculaires aboutissant à la perte des cellules cochléaires constituent autant de cibles thérapeutiques à explorer dans l'avenir afin de tenter de prévenir les troubles de l'audition imputables à l'exposition au bruit ou aux médicaments ototoxiques et au vieillissement. / Our modern society is confronted with a dramatic increase in the number of patients suffering from presbycusis or age related hearing loss. Besides aging, presbycusis prevalence increases with exposition to loud noise (concerts, Walkman, work environment …) and ototoxic drugs (cisplatin, aminoglycosides …). It was reported that the early onset of some aging related diseases (Alzheimer, dementia, Parkinson …) are linked mechanistically to DNA damage, oxidative stress and inflammation. However, the role of DNA damages in cochlear cells degeneration is totally unknown and only few studies have investigated the implication of oxidative stress in presbycusis.The first goal of this study consisted in clarifying the role of DNA damage in cochlear cell degeneration. For this purpose, we used molecular and cellular biology approaches to identify the activation of DNA damage response pathways in cisplatin (CDDP) treated 3 days postnatal mouse cochlear explants in culture. Indeed, the cytotoxicity of CDDP arises from its capacity to directly damage DNA. It is also well known that one of the major dose limiting side effects of CDDP is its ototoxicity. Finally, we investigated the role of p53, a key effector of the DNA damage response pathway, in vivo by treating p53 knockout mice with CDDP. Our results show that CDDP induces double strand breaks leading to the activation of ATM-/DNA PK¬ Chk2 p53 pathway, βH2AX and 53BP1 foci formation and, in fine, apoptotic cell death. Inner hair cells, which are more resistant to CDDP treatment than outer hair cells, show a less intense signaling and fewer double strand breaks. This phenomenon could explain their weaker sensitivity to CDDP treatment. In vivo, p53 deletion prevents hearing loss and outer hair cells degeneration induced bay intraperitoneal injection of CDDP.The second goal consisted in studying the deleterious effects of aging on hearing and the molecular mechanisms involved in this pathology. Here, we studied the mechanism of presbycusis using the senescence-accelerated mouse prone 8 (SAMP8) which is a useful model to probe the effects of aging on biological processes. Based on complementary approaches combining functional, morphological, biochemistry, cellular and molecular biology, we found that the SAMP8 strain displays premature hearing loss and cochlear degeneration recapitulating the processes observed in human presbycusis (i.e. strial, sensory and neural degeneration). The molecular mechanisms associated with premature presbycusis in SAMP8 mice involve oxidative stress, mitochondrial dysfunction, chronic inflammation, autophagic stress and DNA damages. Molecular mechanisms leading to cochlear cells loss represent therapeutic targets of interest to explore in the future in order to prevent hearing impairments due to loud sound or ototoxic drugs exposure and due to aging.

Made available in DSpace on 2014-10-09T12:54:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:14Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:54:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:14Z (GMT). No. of bitstreams: 0 / Câncer de mama é considerado uma das malignidades mais comuns que acometem as mulheres, representando cerca de uma em cada três de todas as neoplasias femininas. Aproximadamente, 90% dos casos de câncer de mama são esporádicos, atribuíveis aos eventos somáticos e cerca de 10% estão associados com a história familial e destes somente 4-5% são decorrentes de fatores hereditários. Em clínica, a radiação ionizante é a principal ferramenta utilizada no controle do crescimento tumoral, além da intervenção cirúrgica e quimioterapia. Há, no entanto, poucas infomnações no que diz respeito a resposta celular frente à ação da radiação ionizante em células-alvo, isto é, em linhagens celulares originárias de câncer de mama. O presente estudo foi proposto para analisar a radiossensibilidade de células humanas tumorals (T-47D e MCF-7) e não tumorals (MCF-10), originárias de mama, submetidas a várias doses (0,5 a 30 Gy) de radiação y de 60Co (0,72 - 1,50 Gy/min). Para tanto, foram utilizados como parâmetros de radiossensibilidade, dano radioinduzido ao DNA, capacidade de reparo e morte celular, por meio das técnicas do micronúcleo, eletroforese de microgel (teste do cometa) e viabilidade celular. Os dados obtidos mostraram que as linhagens tumorais (T-47D e MCF-7) foram mais radiossensíveis que a linhagem não tumoral (MCF-10) para todos os testes utilizados. A linhagem T-47D foi a que apresentou uma maior quantidade de dano radioinduzido, um ciclo celular mais acelerado e uma maior taxa de morte celular. As três linhagens celulares apresentaram uma capacidade de reparo relativamente eficiente, tendo em vista que uma hora após a irradiação, todas elas exibiram uma redução considerável de dano radioinduzido quando comparadas logo após as exposições. Os testes empregados mostraram ser seguros, sensíveis e reprodutíveis e permitiram quantificar e avaliar danos induzidos ao DNA, capacidade de reparo e morte celular, nas três linhagens originárias de mama humana. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Ionising radiation potentially produces a broad spectrum of damage in DNA including single and double strand breaks (ssb and dsb) and base damages. It has been hypothesised that sites of complex damage within cellular DNA have particular biological significance due to an associated decreased efficiency in repair. The aim of this study is to gain further understanding of the formation of complex DNA damage. Irradiations of plasmid DNA illustrate that an increase in ionising density of the radiation results in a decrease in ssb yields/Gy but an increase in dsb per ssb, indicative of an increase in the number of complex damage sites per simple isolated damage site. As the mechanism for damage formation shifts from purely indirect at low scavenging capacities to a significant proportion of direct at higher scavenging capacities the proportion of complex damage increases. Comparisons with the yields of ssb and dsb simulated by Monte-Carlo calculations for AIK USX and a-particles also indicate this correspondence. The ionisation density of low energy, secondary electrons produced by photons was assessed experimentally from the dependence of the yield of OH radicals escaping intra-track recombination on photon energy. As energy decreases the OH radical yield initially decreases reflecting an increased ionisation density. However, with further decrease in photon energy the yield of OH radicals increases in line with theoretical calculations. Base damage yields were determined for low and high ionising density radiation over a range of scavenging capacities. As scavenging capacity increases the base damage:ssb ratios increases implying a contribution from electrons to base damage. It is proposed that base damage contributes to DNA damage complexity. Complex damage analysis reveals that at cell mimetic scavenging capacities, 23% and 72% of ssb have an additional spatially close damage site following y-ray and a-particle irradiation respectively.

TRF1 est une protéine télomérique essentielle pour la stabilité et la régulation de la longueur des télomères. Son expression est altérée dans de nombreux cancers humains, et son inhibition, dans un contexte p53 déficient, favorise le développement de tumeurs chez la souris. Nous montrons ici que l'inhibition de TRF1 dans les fibroblastes primaires humains conduit à une accumulation télomérique de γ-H2AX et à une activation de la voie de réponse aux dommages de l'ADN dépendante des kinases ATR/Chk1, menant rapidement les cellules vers la sénescence. En revanche, lorsque les voies p53 et pRb sont défaillantes, les cellules échappent à la sénescence. L'érosion accrue des télomères engendre alors une fragilité télomérique et une instabilité chromosomique, caractérisées par la présence de fusions entres chromatides soeurs et de signaux multi-télomériques (MTS). Un niveau élevé de MTS, associés à la présence de télomères courts, est également retrouvé après la surexpression de TRF1. Cette fragilité télomérique conduit à une extension de la capacité proliférative des cellules, due à une stabilisation de la longueur des télomères par réactivation de la télomérase. Nous proposons que la fragilité des télomères, induit par l'altération de la charge télomérique de TRF1, conduit à une instabilité chromosomique qui facilite la réactivation de la télomérase et à des anomalies chromosomiques comparables à celles retrouvées dans les tumeurs. La dérégulation de l'expression de TRF1 joueraient un rôle dans la progression tumorale des cellules p53 et pRb déficientes. / TRF1 is a telomere-binding protein which is essential for both telomere stability and telomere length regulation. TRF1 depletion in the context of p53 deficiency promotes tumor development in the mouse, and TRF1 expression is altered in some human cancers. We report here that inhibition of TRF1 in human primary fibroblast results in rapid induction of senescence, which is concomitant with telomeric accumulation of γ-H2AX and phosphorylation of the ATR downstream checkpoint kinase Chk1. Abrogation of p53 and pRb pathways bypasses senescence but leads to accelerated telomere shortening and early onset of chromosomal instability, including sister chromatid fusions and the occurrence of multi-telomeric signals (MTS) related to telomere fragility. MTS are also elevated in TRF1-overexpressing cells and are coincident with the presence of short telomeres. Elevated telomere fragility was associated with greater immortalization potential and resultant cells maintained their telomeres via telomerase reactivation. We propose that changes in TRF1 occupancy at telomeres lead to telomere-fragility driven chromosome instability, which facilitates the reactivation of telomerase and engenders cancer-relevant chromosomal aberrations. These events would occur at early stages of the tumor progression process in the context of an impaired p53 and pRb response.

Les centrosomes sont les centres organisateurs des microtubules et jouent un rôle crucial dans l’organisation du fuseau bipolaire pendant la mitose. Plus récemment, le rôle des centrosomes dans la régulation de l’entrée en mitose a été mis en évidence. Les centrosomes semblent également contribuer à l’activation du point de contrôle en G2/M en réponse aux lésions de l’ADN en servant de point de rencontre pour les régulateurs du cycle cellulaire et les gènes de réponse aux dommages à l’ADN. L’amplification du nombre de centrosomes est une caractéristique des cellules tumorales mais de façon intéressante, elle constitue aussi une réponse des cellules aux dommages à l’ADN. Les mécanismes qui régulent l’homéostasie et la dynamique des centrosomes sont encore mal compris. Pour mieux comprendre le rôle des centrosomes dans la régulation du point de contrôle en G2/M en réponse aux dommages à l’ADN, le recrutement et/ou l’activation au niveau des centrosomes des kinases impliquées dans les voies de signalisation de ce point de contrôle ont été étudiés par immunofluorescence indirecte sur cellules HeLaS3 ou par Western blot sur des fractions enrichies en centrosomes. Nos résultats montrent que les kinases ATM, ATR, CHK1 et CHK2 sont actives dans les centrosomes de cellules en phase G2. En réponse à l’activation du point de contrôle en G2/M, les formes actives de ces kinases diminuent au niveau des centrosomes. Pour identifier de nouveaux acteurs centrosomaux potentiellement impliqués dans la régulation de ce point de contrôle, une analyse comparative des protéomes de centrosomes purifiés a également été réalisée par spectrométrie de masse. Pour étudier plus particulièrement la fonction de CHK2 au niveau des centrosomes, nous avons développer des outils moléculaires qui serviront à déterminer le rôle de la sous population de CHK2 localisée aux centrosomes 1) dans la régulation de l’entrée en mitose au cours d’un cycle normal 2) dans l’activation et la stabilité du point de contrôle en G2/M en réponse aux lésions l’ADN et 3) dans l’homéostasie et la dynamiques des centrosomes en réponse aux dommages à l’ADN. Cette étude permettra de mieux comprendre la fonction des centrosomes dans la réponse cellulaire au stress génotoxiques anti-cancereux et de révéler de nouvelles fonctions potentielles pour la kinase CHK2. / Centrosomes function primarily as microtubule-organizing centres that play a crucial rôle in the equal segregation of chromosomes by organizing the bipolar spindle during mitosis. Recent studies have revealed the involvement of centrosomes in regulating G2/M transition during normal cell cycle progression. Moreover, increasing evidence suggests that centrosomes also play roles in the DNA damage response and cell cycle checkpoint signalling by serving as “meeting points” where DNA-damage-responsive genes and cell cycle regulators communicate. Numerical centrosome aberrations, or centrosome amplification, is a common feature of most human cancers that promotes aneuploidy and is involved in tumorigenesis as well as tumor progression. Interestingly, centrosome amplification and fragmentation have also been shown to constitute a cellular response to impaired DNA integrity that triggers cell death by mitotic failure. Although their roles are critical in tumorigenesis and the DNA damage response, the mechanisms that regulate centrosome homeostasis and dynamics remain poorly understood. To gain a better understanding of the role of the centrosomes in checkpoint regulation at G2/M transition in response to DNA damage; the recruitment and/or centrosomal activation of the kinases implicated in this checkpoint pathways were studied by indirect immunofluorescence on HeLaS3 cells or western blot on purified centrosomal fractions. Our results showed that the kinases CHK1, CHK2, ATM and ATR are activated at the centrosomes in cell synchronised in G2. However, after activation of the G2/M checkpoint, these activated kinases moved from centrosomes. Finally, to identify new centrosomal actors potentially involved in the regulation of this checkpoint, a comparative analysis of the proteome of purified centrosomes was also realized by mass spectrometry. To study more specifically the function of CHK2 at the centrosomes, we developped molecular tools wich will serve to determine the role of the sub-population of CHK2 localized at the centrosomes in 1) in regulating entry into mitosis during unperturbed cell cycle progression, 2) in checkpoint regulation at G2/M transition in response to DNA damage induced by ionizing radiations and genotoxic drugs and 3) in centrosomal homeostasis and dynamics by regulating centrosomal amplification, fragmentation and clustering during normal cell cycle progression and in response to genotoxic drugs. This study will further elucidate the importance of centrosomes in regulating the cell response to genotoxic stress induced by anti-cancer treatments and reveal potential new functions for the kinase CHK2 in unperturbed cell cycle progression and in response to DNA damage.

Câncer de mama é considerado uma das malignidades mais comuns que acometem as mulheres, representando cerca de uma em cada três de todas as neoplasias femininas. Aproximadamente, 90% dos casos de câncer de mama são esporádicos, atribuíveis aos eventos somáticos e cerca de 10% estão associados com a história familial e destes somente 4-5% são decorrentes de fatores hereditários. Em clínica, a radiação ionizante é a principal ferramenta utilizada no controle do crescimento tumoral, além da intervenção cirúrgica e quimioterapia. Há, no entanto, poucas infomnações no que diz respeito a resposta celular frente à ação da radiação ionizante em células-alvo, isto é, em linhagens celulares originárias de câncer de mama. O presente estudo foi proposto para analisar a radiossensibilidade de células humanas tumorals (T-47D e MCF-7) e não tumorals (MCF-10), originárias de mama, submetidas a várias doses (0,5 a 30 Gy) de radiação y de 60Co (0,72 - 1,50 Gy/min). Para tanto, foram utilizados como parâmetros de radiossensibilidade, dano radioinduzido ao DNA, capacidade de reparo e morte celular, por meio das técnicas do micronúcleo, eletroforese de microgel (teste do cometa) e viabilidade celular. Os dados obtidos mostraram que as linhagens tumorais (T-47D e MCF-7) foram mais radiossensíveis que a linhagem não tumoral (MCF-10) para todos os testes utilizados. A linhagem T-47D foi a que apresentou uma maior quantidade de dano radioinduzido, um ciclo celular mais acelerado e uma maior taxa de morte celular. As três linhagens celulares apresentaram uma capacidade de reparo relativamente eficiente, tendo em vista que uma hora após a irradiação, todas elas exibiram uma redução considerável de dano radioinduzido quando comparadas logo após as exposições. Os testes empregados mostraram ser seguros, sensíveis e reprodutíveis e permitiram quantificar e avaliar danos induzidos ao DNA, capacidade de reparo e morte celular, nas três linhagens originárias de mama humana. / Breast cancer is one of the most common malignancies that account women, representing about one in three of all female neoplasm. Approximately, 90% of cases are considered sporadic, attributed to somatic events and about 10% have a family history and this only 4 - 5 % is decurrent of hereditary factors. In the clinic, ionizing radiation is a major tool utilized in the control of tumour growth, besides surgery and chemotherapy. There is, however, little information concerning cellular response to the action of ionizing radiation in the target cells, i.e., cell lines originating from breast cancer. The present study proposed to analyze the radiosensitivity of the human tumorigenic (T-47D and MCF-7) and nontumorigenic (MCF-10) cell lines, originating from breast and submitted to various doses (0.5 to 30 Gy) of 60Co rays (0.72 - 1.50 Gy/min). For this purpose, DNA radioinduced damage, repair capacity and cell death were utilized as parameters of radiosensitivity by micronucleus, single cell gel electrophoresis (Comet assay) and cell viability techniques. The data obtained showed that tumorigenic cell lines were more radiosensitive than nontumorigenic breast cells in all assays here utilized. The T-47D cell line was presenting the highest amount of radioinduced damage, a more accelerated proliferation rate and a higher rate of cell death. The three cell lines presented a relatively efficient repair capacity, since one hour after the irradiation all of them showed a considerable reduction of radioinduced damage. The techniques employed showed to be secure, sensitive and reproducible, allowing to quantify and evaluate DNA damage, repair capacity and cell death in the three human breast cell lines.

Câncer de mama é considerado uma das malignidades mais comuns que acometem as mulheres, representando cerca de uma em cada três de todas as neoplasias femininas. Aproximadamente, 90% dos casos de câncer de mama são esporádicos, atribuíveis aos eventos somáticos e cerca de 10% estão associados com a história familial e destes somente 4-5% são decorrentes de fatores hereditários. Em clínica, a radiação ionizante é a principal ferramenta utilizada no controle do crescimento tumoral, além da intervenção cirúrgica e quimioterapia. Há, no entanto, poucas infomnações no que diz respeito a resposta celular frente à ação da radiação ionizante em células-alvo, isto é, em linhagens celulares originárias de câncer de mama. O presente estudo foi proposto para analisar a radiossensibilidade de células humanas tumorals (T-47D e MCF-7) e não tumorals (MCF-10), originárias de mama, submetidas a várias doses (0,5 a 30 Gy) de radiação y de 60Co (0,72 - 1,50 Gy/min). Para tanto, foram utilizados como parâmetros de radiossensibilidade, dano radioinduzido ao DNA, capacidade de reparo e morte celular, por meio das técnicas do micronúcleo, eletroforese de microgel (teste do cometa) e viabilidade celular. Os dados obtidos mostraram que as linhagens tumorais (T-47D e MCF-7) foram mais radiossensíveis que a linhagem não tumoral (MCF-10) para todos os testes utilizados. A linhagem T-47D foi a que apresentou uma maior quantidade de dano radioinduzido, um ciclo celular mais acelerado e uma maior taxa de morte celular. As três linhagens celulares apresentaram uma capacidade de reparo relativamente eficiente, tendo em vista que uma hora após a irradiação, todas elas exibiram uma redução considerável de dano radioinduzido quando comparadas logo após as exposições. Os testes empregados mostraram ser seguros, sensíveis e reprodutíveis e permitiram quantificar e avaliar danos induzidos ao DNA, capacidade de reparo e morte celular, nas três linhagens originárias de mama humana. / Breast cancer is one of the most common malignancies that account women, representing about one in three of all female neoplasm. Approximately, 90% of cases are considered sporadic, attributed to somatic events and about 10% have a family history and this only 4 - 5 % is decurrent of hereditary factors. In the clinic, ionizing radiation is a major tool utilized in the control of tumour growth, besides surgery and chemotherapy. There is, however, little information concerning cellular response to the action of ionizing radiation in the target cells, i.e., cell lines originating from breast cancer. The present study proposed to analyze the radiosensitivity of the human tumorigenic (T-47D and MCF-7) and nontumorigenic (MCF-10) cell lines, originating from breast and submitted to various doses (0.5 to 30 Gy) of 60Co rays (0.72 - 1.50 Gy/min). For this purpose, DNA radioinduced damage, repair capacity and cell death were utilized as parameters of radiosensitivity by micronucleus, single cell gel electrophoresis (Comet assay) and cell viability techniques. The data obtained showed that tumorigenic cell lines were more radiosensitive than nontumorigenic breast cells in all assays here utilized. The T-47D cell line was presenting the highest amount of radioinduced damage, a more accelerated proliferation rate and a higher rate of cell death. The three cell lines presented a relatively efficient repair capacity, since one hour after the irradiation all of them showed a considerable reduction of radioinduced damage. The techniques employed showed to be secure, sensitive and reproducible, allowing to quantify and evaluate DNA damage, repair capacity and cell death in the three human breast cell lines.