Submitted by Automação e Estatística (sst@bczm.ufrn.br) on 2018-06-05T21:48:08Z No. of bitstreams: 1 DeborahGondimLambertMoreira_DISSERT.pdf: 3428451 bytes, checksum: d076c04fe64f94b94e5ebeea08f6c085 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-06-11T18:55:00Z (GMT) No. of bitstreams: 1 DeborahGondimLambertMoreira_DISSERT.pdf: 3428451 bytes, checksum: d076c04fe64f94b94e5ebeea08f6c085 (MD5) / Made available in DSpace on 2018-06-11T18:55:00Z (GMT). No. of bitstreams: 1 DeborahGondimLambertMoreira_DISSERT.pdf: 3428451 bytes, checksum: d076c04fe64f94b94e5ebeea08f6c085 (MD5) Previous issue date: 2018-02-23 / O sistema de reparo do DNA envolve genes e proteínas essenciais à manutenção da integridade do genoma e consequente controle de diversos processos celulares. Alterações nesses genes e proteínas estão envolvidas no desenvolvimento de tumores, em sua evolução e na resistência a tratamentos radio e quimioterápicos. O objetivo deste trabalho foi analisar a imunoexpressão das proteínas de reparo do DNA, XPF e XRCC1 em carcinoma epidermoide de lábio inferior (CELI) e de língua oral (CELO) e investigar possíveis associações com os parâmetros clínicos e histopatológicos. Foram selecionados 80 casos de CELI e CELO analisados morfologicamete por meio dos sistemas de gradação propostos por BrandweinGensler et al. (2005) e Almangush et al. (2014). A expressão imuno-histoquímica foi realizada de forma semi-quantitativa, utilizando para a análise estatística os testes de Quiquadrado, ou quando aplicável, o teste exato de Fisher, para investigar a associação entre as expressões das proteínas com as características clínico-patológicas. A alta expressão nuclear de XPF foi observada em 72,5% dos casos de CELO e em 70% dos casos de CELI. Quanto à imunoexpressão de XRCC1 nos casos de CELO, alta expressão foi observada em 60% dos casos e nos CELI 70% apresentaram alta expressão nuclear, não sendo observada expressão citoplasmática. Foram observadas associações estatisticamente significativas (p < 0,05) entre a imunoexpressão de proteínas com alguns parâmetros clínicos e histopatológicos, no entanto, não se verificaram associações com o prognóstico dos pacientes. A alta expressão das proteínas XPF e XRCC1 nos casos de CELO e CELI sugere sua importância no desenvolvimento e progressão destes tumores. Os resultados desta pesquisa indicam que a imunoexpressão dessas proteínas não está associada a parâmetros clínicos e histopatológicos determinantes para o comportamento biológico tumoral. / The DNA repair system involves genes and proteins essential for the maintenance of genome integrity and consequent control of various cellular processes. Alterations in these genes and proteins are involved in the development of tumors, in their evolution and resistance to radio and chemotherapy treatments. The aim of this study was to analyze the immunoexpression of DNA repair proteins, XPF and XRCC1 in squamous cell carcinoma of the lower lip (SCCLL) and oral tongue (SCCOT) and investigate possible associations with the clinical and histopathological groups. For this purpose, 80 cases of SCCLL and SCCOT were selected and evaluated by grading systems proposed by Brandwein-Gensler et al. (2005) and Almangush et al. (2014). The immunohistochemical expression was performed semi-quantitatively, was used for statistical analysis the Chi-square tests, or when applicable, Fisher's exact test, to evaluate an association between the expressions of the proteins as clinical-pathological characteristics. A high nuclear expression of XPF was observed in 72.5% of cases of SCCOT and in 70% of cases of SCCLL. Regarding the XRCC1 immunoexpression in cases of SCCOT, high expression was observed in 60% of the cases and in the SCCLL 70% presented high nuclear expression, and no cytoplasmic expression was observed. Statistically significant associations were observed between immunoexpression of proteins with some clinical and histopathological parameters (p<0.05), however, there were no associations with the prognosis of the patients. The high expression of XPF and XRCC1 proteins in the cases of SCCOT and SCCLL suggest its importance in development and progression of these tumors. The results of this research indicate that immunoexpression of the studied proteins is not associated with a clinical and histopathological characteristics determinant for the biological tumor behavior.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-21T17:20:00Z No. of bitstreams: 1 MelkaCoelhoSa_TESE.pdf: 3257998 bytes, checksum: 6feeeb452aad7be3dee1941b26d85af8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-22T11:49:09Z (GMT) No. of bitstreams: 1 MelkaCoelhoSa_TESE.pdf: 3257998 bytes, checksum: 6feeeb452aad7be3dee1941b26d85af8 (MD5) / Made available in DSpace on 2016-07-22T11:49:09Z (GMT). No. of bitstreams: 1 MelkaCoelhoSa_TESE.pdf: 3257998 bytes, checksum: 6feeeb452aad7be3dee1941b26d85af8 (MD5) Previous issue date: 2016-02-19 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Os genes de reparo do DNA s?o essenciais para manuten??o da integridade do genoma, evitando graves doen?as como o c?ncer. O papel de v?rias prote?nas codificadas por esses genes vem sendo associado tanto ao risco do desenvolvimento, como na evolu??o de variados c?nceres humanos, dentre eles, o carcinoma epidermoide oral. O objetivo deste trabalho foi analisar a imunoexpress?o das prote?nas de reparo do DNA, XRCC1, THIIF e XPF em carcinoma epidermoide de l?ngua oral e investigar associa??o com par?metros cl?nicos, histopatol?gicos e de desfecho. Tamanho do tumor, comprometimento linfonodal, est?gio do tumor, profundidade de invas?o >4mm e o sistema de grada??o de Almangush, mostraram-se como fatores progn?sticos. Evidenciou-se de uma maneira geral, alta express?o imuno-histoqu?mica das prote?nas de reparo nas c?lulas parenquimatosas; no entanto, apenas verificou-se associa??o significativa da elevada express?o de XRCC1 com melhor estadiamento cl?nico. Os resultados deste experimento sugerem que as prote?nas XRCC1, TFIIH e XPF participam do processo de tumorig?nese, entretanto a imunoexpress?o das mesmas n?o pode ser utilizada como indicador progn?stico para o carcinoma epidermoide de l?ngua oral. / DNA repair systems, genes and proteins are essential for genome integrity maintenance, avoiding serious diseases such as cancer. Deregulation in the expression of those proteins has been associated with both the risk of development and evolution of various human cancers, including oral squamous cell carcinoma. The purpose of this study was to analyze the immunoreactivity of the DNA repair proteins XRCC1, THIIF and XPF in oral tongue squamous cell carcinoma (OTSCC) and to investigate its association with clinical and histopathological parameters, outcome and 5-year survival rate. Seventy-four cases of OTSCC were analyzed semi-quantitatively through immunohistochemistry. We observed that DNA repair proteins were highly expressed in parenchymal cells; however, we only observed a significant association between XRCC1 high expression and better clinical staging (p=0,02). Cox regression showed that tumor size (p<0,01), lymph node involvement (p=0,04), tumor stage (p=0,02) and depth of invasion> 4mm (p=0,05) were prognostic factors. The results of this experiment suggest that XRCC1, TFIIH and XPF participate in the tumorigenic process, however, their immunoexpression may not be used as an independent prognostic indicator for OTSCC.

Les télomères sont des structures nucléoprotéiques spécialisées dont l'un des rôles est d'empêcher le raccourcissement progressif de l'extrémité des chromosomes suite à la réplication et à l'instabilité génomique due à la recombinaison de l'extrémité de chromosomes. Malgré le rôle des télomères dans la protection de l'extrémité des chromosomes contre les mécanismes de réparation de l'ADN et de recombinaison, de nombreuses protéines de ces voies jouent des rôles essentiels dans l'homéostasie télomérique et la stabilité des chromosomes. Parmi elles, la protéine RAD50 appartenant au complexe MRE11/RAD50/XRS25(NBS1) et l'endonucléase structure spécifique XPF/ERCC1 sont localisées aux télomères ; ces deux complexes connus pour leur rôle dans les voies de réparation de l'ADN ainsi que dans les études sur la recombinaison. Nous avons identifié deux rôles différents pour la protéine RAD50 dans la maintenance télomérique et dans la protection des extrémités des chromosomes, en contexte de présence et absence de la télomérase. L'absence d'AtRAD50 augmente significativement le nombre de fusions chromosomiques impliquant des télomères raccourcis. Nous proposons que ce rôle protecteur des télomères raccourcis de RAD50 est le résultat de sa fonction de contraindre la recombinaison entre chromatides soeurs et ainsi d'éviter les évènements de fusions par les extrémités. Nous avons recherché le ou des mécanismes impliqué(s) dans ces évènements de fusions chromosomiques chez les mutants atrad50 en réalisant des croisements entre les plantes déficientes pour ATRAD50 et des plantes déficientes pour des gènes codant des protéines des voies de réparation par recombinaison non-homologue et homologue. Au contraire de la situation en cellules de mammifères, nous n'avons pas observé d'instabilité chromosomique chez les plantes mutantes correspondantes pour XPF (AtRAD1) or ERCC1 (AtERCC1). Cependant, en absence de la télomérase, la mutation de l'un de ces deux gènes entraîne une augmentation précose et significative de l'instabilité chromosomique sans accélération générale de la perte des répétitions télomériques, mais associée à la présence de fragments ADN extrachromatiques visibles en cytologie. Une analyse intensive par FISH a permis d'identifier ces ADN comme des bras entiers spécifiques de deux chromosomes. Nos données indiquent un rôle protecteur de RAD1/ERCC1 comme l'invasion de l'ADN simple brin télométrique dans des séquences télomériques interstitielles. Le fait que les mutations de rad1 (ou ercc1) augmentent dramatiquement l'instabilité chromosomique des mutants télomérase a des implications très importantes pour les modèles des rôles de la recombinaison aux télomères.

BACKGROUND:Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations.PURPOSE:To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer.RESULTS:Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million.CONCLUSIONS:The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-29T22:55:50Z No. of bitstreams: 1 StefaniaJeronimoFerreira_TESE.pdf: 1963411 bytes, checksum: 83b197f2ea9b0bdbd5ac4b4732412733 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-03-02T19:57:46Z (GMT) No. of bitstreams: 1 StefaniaJeronimoFerreira_TESE.pdf: 1963411 bytes, checksum: 83b197f2ea9b0bdbd5ac4b4732412733 (MD5) / Made available in DSpace on 2016-03-02T19:57:46Z (GMT). No. of bitstreams: 1 StefaniaJeronimoFerreira_TESE.pdf: 1963411 bytes, checksum: 83b197f2ea9b0bdbd5ac4b4732412733 (MD5) Previous issue date: 2015-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / As vias de reparo por excis?o de base (BER) e por excis?o de nucleot?deo (NER) desempenham um papel crucial na manuten??o da integridade gen?mica. Polimorfismos em genes das vias BER e NER, que modulam a capacidade de reparo do DNA, podem estar relacionados ao risco de desenvolvimento e progn?stico do c?ncer oral. O presente trabalho teve como objetivo investigar a frequ?ncia de polimorfismos de nucleot?deos simples, em dois genes da via de reparo do DNA por excis?o de base (XRCC1 ? rs25487 e APEX1 ? rs1130409) e dois genes da via de reparo por excis?o de nucleot?deo (XPD ? rs13181 e XPF ? rs1799797), em pacientes com carcinoma de c?lulas escamosas oral (CCEO), buscando associa??es com o risco de desenvolver esta neoplasia maligna e o seu progn?stico. Um total de 92 amostras de DNA de pacientes com CCEO e 130 controles foram genotipadas utilizando o m?todo da rea??o em cadeia da polimerase em tempo real. O software estat?stico GraphPad Prism version 6.0.1. foi utilizado para a aplica??o dos testes apropriados. Odds ratio (OR) e hazard ratio (HR), e seus intervalos de confian?a (IC) de 95%, foram calculados pela regress?o log?stica. A avalia??o do progn?stico foi realizada por meio da curva de Kaplan-Meier e an?lise multivariada de Cox. A presen?a das variantes polim?rficas nos genes XRCC1, APEX1, XPD, e XPF n?o foram associadas ao risco de desenvolver CCEO. A intera??o da presen?a da variante polim?rfica com o h?bito de fumar n?o foi significativa para nenhum dos polimorfismos analisados. J? a presen?a do polimorfismo em XPD, somada ao h?bito de beber, aumentou o risco de desenvolver CCEO (OR 1,86, 95% IC: 0,86 ? 4,01, p=0,03). Apenas o SNP do APEX1 (rs1130409) esteve associado a uma diminui??o da sobrevida espec?fica (HR 3,94, 95% IC: 1,31 ? 11,88, p=0,01). O presente estudo sugere uma intera??o entre o consumo de ?lcool e a presen?a do polimorfismo estudado no gene XPD. Al?m disso, indica um pior progn?stico para pacientes que possuem o polimorfismo estudado em APEX1. / Base excision repair (BER) and nucleotide excision repair (NER) pathways play critical role in maintaining genome integrity. Polymorphisms in BER and NER genes which modulate the DNA repair capacity may affect the susceptibility and prognosis of oral cancer. This study was conducted with genomic DNA from 92 patients with oral squamous cell carcinomas (OSCC) and 130 controls. The cases were followed up to explore the associations between BER and NER genes polymorphisms and the risk and prognosis of OSCC. Four single-nucleotide polymorphisms (SNPs) in XRCC1 (rs25487), APEX1 (rs1130409), XPD (rs13181) and XPF (rs1799797) genes were tested by polymerase chain reaction ? quantitative real time method. The GraphPad Prism version 6.0.1 statistical software was applied for statistical analysis of association. Odds ratio (OR), hazard ratio (HR), and their 95 % confidence intervals (CIs) were calculated by logistic regression. Kaplan-Meier curve and Cox proportional hazard model were used for prognostic analysis. The presence of polymorphic variants in XRCC1, APEX1, XPD and XPF genes were not associated with an increased risk of OSCC. Gene-environment interactions with smoking were not significant for any polymorphism. The presence of polymorphic variants of the XPD gene in association with alcohol consumption conferred an increased risk of 1.86 (95% CI: 0.86 ? 4.01, p=0.03) for OSCC. Only APEX1 was associated with decreased specific survival (HR 3.94, 95% CI: 1.31 ? 11.88, p=0.01). These results suggest an interaction between polymorphic variants of the XPF gene and alcohol consumption. Additionally APEX1 may represent a prognostic marker for OSCC.

La réparation des cassures double brin (CDB) de l'ADN par recombinaison est essentielle au maintien de l'intégrité du génome de tous les être vivants. Ce processus doit cependant être finement régulé puisque la recombinaison peut générer des mutations ou des réarrangements chromosomiques, parfois extrêmement délétères pour la cellule. Les CDB peuvent être réparées par deux mécanismes : la recombinaison non homologue (ou jonction des extrémités d'ADN) ou la recombinaison homologue (impliquant une homologie de séquence entre les molécules recombinantes). Dans les cellules somatiques, les deux voies principales de recombinaison homologue (RH) sont la voie Synthesis Dependent Strand Annealing (SDSA) dépendante de la recombinase RAD51 et la voie Single Strand Annealing (SSA) indépendante de RAD51. Nos résultats ont d'abord mis en évidence un rôle inattendu de XRCC2, RAD51B et RAD51D - trois paralogues de RAD51 - dans la voie SSA. Nous avons confirmé que la fonction de la protéine XRCC2 dans la voie SSA ne dépend pas de RAD51, ce qui démontre que certains paralogues de RAD51 ont acquis des fonctions indépendantes de la recombinase. La différence de sévérité des phénotypes des mutants individuels ainsi que les analyses d'épistasie menées sur le double et le triple mutant suggèrent des fonctions individuelles de ces protéines au cours du SSA. Nous proposons qu'elles facilitent l'étape d'hybridation des deux séquences complémentaires situées de part et d'autre de la cassure, bien que ceci reste à confirmer par des études in vitro. L'étude des fonctions de l'hétérodimère XPF-ERCC1 - un complexe impliqué dans le clivage des extrémités d'ADN non homologues au cours des voies de RH - a révélé un rôle inhibiteur de ce complexe sur la voie SDSA. Cette action est dépendante de son activité endonucléasique et serait liée au clivage des longues extrémités 3' sortantes réalisant l'invasion d'un duplex d'ADN homologue, l'étape initiale de la voie SDSA. Notre étude a de plus confirmé que le rôle du complexe dépend de la longueur des extrémités non homologues chez Arabidopsis, comme chez les mammifères et la levure. Bien que le complexe XPF-ERCC1 soit essentiel au clivage des longues extrémités d'ADN non homologue, il n'est pas requis à l'élimination des courtes extrémités au cours de la RH. / The repair of DNA double-strand breaks (DSB) by recombination is essential for the maintenance of genome integrity of all living organisms. However, recombination must be finely regulated as it can generate mutations or chromosomal rearrangements, sometimes extremely deleterious to the cell. DSB can be repaired by two classes of recombination mechanism: non-homologous recombination (or DNA End Joining) or homologous recombination (implicating DNA sequence homology between the recombining molecules). In somatic cells, the two main pathways of homologous recombination (HR) are RAD51-dependent Synthesis Dependent Strand Annealing (SDSA) and RAD51-independent Single Strand Annealing (SSA). Our results have demonstrated an unexpected role of XRCC2, RAD51B and RAD51D - three RAD51 paralogues – in the SSA pathway. We confirmed that the function of XRCC2 in SSA does not depend upon RAD51, thus demonstrating that some RAD51 paralogues have acquired RAD51 recombinase-independent functions. The different severities of individual mutant phenotypes and epistasis analyses carried out on the double and triple mutants suggest individual functions of these proteins in SSA recombination. We propose that they facilitate hybridization of the two complementary sequences located on both sides of the break, although this remains to be confirmed by in vitro experiments. Study of the roles of XPF-ERCC1 - a complex involved in the cleavage of non-homologous DNA ends during HR - revealed an inhibitory role of this complex on the SDSA pathway. This is dependent on its endonuclease activity and is probably due to the cleavage of long 3' ends performing the homologous DNA duplex invasion, the initial step of the SDSA pathway. Our analyses also confirmed that the role of the complex depends on the length of the nonhomologous ends, as seen in mammals and yeasts. Although XPF-ERCC1 is essential for the cleavage of long nonhomologous DNA ends, it is not required for the elimination of short ends during HR.