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Made available in DSpace on 2014-06-11T19:33:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:04:09Z : No. of bitstreams: 1 marcondes_jpc_dr_botfm.pdf: 345309 bytes, checksum: 8feb5bc849ad4ec9e213cb66ca068172 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os linfomas representam um grupo heterogêneo de tumores que acometem o tecido linfóide nodal e extranodal. O tratamento, baseado na utilização da poliquimioterapia associada ou não à radioterapia, tem proporcionado altas taxas de cura. Entretanto, é sabido que tais terapias podem induzir mutações genéticas que, mais tarde, podem ser responsáveis pelo desenvolvimento de neoplasias secundárias. Assim sendo, o presente estudo objetivou avaliar os efeitos tardios das terapias antineoplásicas para linfomas. Para isso, foram investigados os danos no DNA e a capacidade de reparo da molécula pelo teste do cometa, e a relação entre polimorfismos e expressão de dois genes de reparo do DNA - XRCC1 (codons 280 e 399) e hOGG1 (codon 326) – com os níveis de lesões genotóxicas. A casuística do estudo incluiu 3 grupos de indivíduos: 14 pacientes recém-diagnosticados com linfoma e antes de qualquer tratamento antineoplásístico (grupo pré-terapia); 29 pacientes com história de linfoma e que haviam finalizado o tratamento há no mínimo 2 anos (histopatologicamente negativos para neoplasia; grupo pós-terapia); 29 indivíduos saudáveis pareados por sexo, idade e hábito tabagista (grupo controle). Os resultados mostraram que os pacientes com diagnótico ou história de linfoma (pré e pós-terapia, respectivamente), apresentavam níveis aumentados de danos no DNA quando comparados aos indivíduos saudáveis. Esses dados evidenciam a relação entre a presença da doença e lesões no DNA, e que mesmo com diagnóstico negativo, os indivíduos com história de linfoma apresentam níveis aumentados de genotoxicidade até, em média, sete anos após o término da terapia. A menor capacidade de reparo do DNA observada nos pacientes do grupo pós-terapia, e o menor nível de expressão dos genes XRCC1 e hOGG1 nos pacientes pré e pós-terapia, poderiam ser explicações para tais achados... / Lymphomas are a heterogenous group of malignancies that arise in nodal sites with or without extranodal involvement. The treatment, based on polychemotherapy associated or not with radiotherapy, has provided high cure rates. However, it is known that such therapies can induce genetic mutations that could be related to development of second malignancies. Therefore, the present study aimed to evaluate the late effects of antienoplastic therapies for lymphomas. DNA damage and repair capability as depicted by the comet assay, and the relationship between DNA repair genes polymorphisms (XRCC1 codons 280 and 399, hOGG1 codon 326) or gene expressions and the levels of DNA lesions were investigated. Three groups were included in this study: pre-therapy, with 14 patients newly diagnosed with lymphoma and before any antienoplastic; post-therapy, with 29 patients with history of lymphoma and who had finished treatment at least three years before blood collection (histopathologically negative for neoplasia); control, with 29 healthy subjects matched for age, sex and smoking habit. The results showed that patients from pre- and post-therapy groups presented higher amount of DNA damage than the healthy subjects. These data first indicated that individuals with lymphoma have high frequency of primary DNA lesion in lymphocytes, then, that even with negative histopathological diagnostic, patients with history of lymphoma presented increased DNA damage until the average of 7 years after the end of therapy. The reduced DNA repair capability and the low XRCC1 and hOGG1 expression observed in the post-therapy group could explain such findings. Furthermore, higher DNA repair capability was observed in those subjects with XRCC399arg/arg, XRCC1280arg/his and hOGG1326ser/ser genotypes. In conclusion, our data demonstrated that lymphomas were associated with high level of damage and low DNA repai... (Complete abstract click electronic access below)

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Male factor infertility is a contributing factor in up to 50% of infertile couples. Increasing numbers of couples undergoing assisted reproductive technology (ART) treatment and reports of a possible decline in male fertility suggest that lifestyle, occupational and environmental exposures might impair semen quality. Sperm DNA contains both DNA strand breaks and base damage that has been associated with poor semen quality but few studies have examined the role of double strand breaks (DSBs), a toxic lesion, or DNA damage such as N7-methyldeoxyguanosine (N7-MedG) arising from alkylating agents that can be toxic and mutagenic. The aim of this research was to examine the relationships between exposures, DNA damage and male fertility. Men were recruited from couples attending for ART treatment and they provided information on lifestyle, occupational and environmental exposures as well as a sperm sample. Semen concentration and motility was determined by standard techniques in the neat sample and the prepared sample that underwent density gradient centrifugation for ART treatment. DSBs were measured in individual sperm cells by the neutral Comet assay and N7-MedG levels in sperm DNA by an immunoslot blot assay. Information on ART outcomes (% fertilisation, % cleavage and clinical pregnancy) was collected and associations between DNA damage, exposures, semen quality and ART outcomes were determined. Expression of individual DNA repair proteins was also examined in individual oocytes. Men in manual work had significantly lower semen volumes and higher % immotile sperm. Exposure to dry cleaning fluids and having a fever were associated with a decrease in sperm number and while non-ionizing radiation was associated with an increase in % immotile sperm, X-ray exposure was correlated to a decrease in % progressively motile sperm. Semen parameters were significantly and negatively correlated with DSBs in neat and prepared sperm, and N7-MedG levels in neat sperm. Density gradient centrifugation improved sperm sample quality and decreased DSBs and N7-MedG levels significantly. Successful fertilization of oocytes was negatively associated with DSB levels in neat and prepared sperm and with N7-MedG levels in neat sperm. Lower DSB levels in men were associated with an increased chance of an achieving clinical pregnancy especially in ICSI couples. N7-MedG levels were significantly correlated with driving a car and exposure to detergent or printing inks and dyestuffs. DSBs were correlated negatively with exercise and positively with eating nuts and almonds or exposure to non-ionizing radiation. DNA repair gene expression in individual oocytes showed significant intra and inter-individual variability. Sperm DNA damage can reduce male fertility, but the causes of such damage remain to be identified. The variable ability of individual oocytes to repair this damage may well affect the chance for a successful pregnancy.

archives@tulane.edu / BRCA1 faithfully repairs damaged DNA by promoting homology-directed repair (HDR). Loss of Brca1 and other HDR genes are incompatible with embryonic viability and cause severe genomic instability. Cells lacking BRCA1 are sensitive to cellular stresses such as DNA damage caused by ionizing radiation (IR). Homozygous loss of Brca1 is embryonic lethal in mice, and the few tissue-specific knockouts generated develop abnormally. Therefore, we created an inducible Cre mouse model to study Brca1 loss in all adult mouse tissues allowing for examination of viability, longevity, and stress response in the absence of HDR and the importance of HDR in different tissues of an adult mouse. After validating the inducible Cre system using a reporter allele in mice, we generated mice with alleles of the inducible Cre system and floxed Brca1 alleles. Cre was induced in adult mice at ten weeks of age, resulting in extensive, widespread deletion of Brca1. Contrary to the embryonic lethality observed in all previously tested germline Brca1 knockout mouse models, adult mice with Brca1 deletion displayed no overt phenotypes. Brca1Δ/Δ mice showed extensive, widespread deletion of Brca1 and survived up to 1 year after Brca1 recombination. Targeted, high-depth sequencing of recombined tissues indicated mutations accumulated in both the mammary gland and the intestine. However, only the mammary gland had an HDR deficiency signature. Next, we examined Brca1Δ/Δ mice survival after exposure to ionizing radiation and mitomycin C (MMC). Surprisingly, Brca1Δ/Δ mice responses are DNA damage specific. Brca1Δ/Δ mice deficient for HDR showed no increased sensitivity to IR but died four to eight days following MMC exposure. Our results show that BRCA1 is not required for long-term viability or DNA double-strand break repair, but BRCA1 is essential for DNA crosslink repair to maintain viability in an adult mouse. / 1 / JoyOlayiwola

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclobutane pyrimidine dimers (CPD) are the predominant DNA lesions formed upon exposure of this biopolymer to sunlight. Given the potentially dire biological consequences of DNA lesions, there is a need to fully characterize their behavior, with an eye towards understanding their complete reactivity and as a possible means to detect and quantify their presence in the genome. The work described in this dissertation describes studies of the alkaline reactivity of CPD lesions generated within dinucleotide & polynucleotide strands. It was found that CPD-TpT is generally inert under alkaline conditions at room temperature, which is in agreement with earlier studies on alkaline hydrolysis of CPD-thymine and CPD-thymidine. However, a re-evaluation of the same reaction in the presence of 18O labelled water demonstrated that, similar to other UV-induced DNA lesions containing a saturated pyrimidine ring, CPD undergoes a water addition at the C4=O group of the nucleobase leading to the formation of a hemiaminal intermediate. This intermediate, however, does not lead to hydrolysis products and completely reverts to starting material under those same conditions. Moreover, the two C4=O groups present on 3′ and 5′-thymines in a CPD molecule show different chemical reactivities, with the 3′ C4=O group having greater affinity towards water addition as compared to the one on 5′ end, a fact reflected in different rates of exchange with the incoming nucleophile leading to the hemiaminal intermediate. The 18O labelling reaction was also investigated in CPD lesions generated within oligonucleotides to probe the cause of asymmetry between the 3′ vs 5′ C4=O groups; ultimately, it was determined that the asymmetric reactivity observed to occur between the two C4=O groups was an intrinsic property of the CPD molecule and did not arise as a result of asymmetry in a dinucleotide setting. In addition to the above studies, during the course of the investigation of the nucleophilic reactivity of CPD, a chemical reaction was observed leading to what appeared to be the rapid and total chemical reversal of CPD lesions to the original TpT (thymine-thymine dinucleotide)! This “repair” reaction occurred when CPD reacted with hydrazine, and appears facilitated by an inert atmosphere under which it rapidly proceeds to completion at room temperature.

Early events occurring during induction of the SOS response in Escherichia coli are poorly understood. In order to understand the early steps in SOS induction more fully, we have isolated several mutations which constitutively express the SOS regulon. Using a Mud(Apᴿ,lac) fusion to the SOS regulated gene sulA, we isolated Lac⁺ colonies as mutants in which RecA protein is constitutively activated for repressor cleavage. The mutations map to four loci: dam, lig, uvrD and recA. The extent of constitutive SOS induction in these mutants varied greatly, indicating different levels or types of signal in the cell. The mutations isolated demonstrate two early steps in SOS induction. The first step in SOS induction is signal generation and includes mutations found in dam, lig and uvrD genes. The mutant gene products presumably alter DNA metabolism to produce an inducing signal. These non-lethal mutations lead to sub-induction and probably generate very specific signals, such as abnormally unwound DNA in the case of DNA helicase II mutants or unsealed DNA nicks that result from deficient ligation in lig mutants. Greater induction may require quantitatively more signal or different types of signal generated by severe defects leading to cell death. These mutations also show that signal is a variable quantity, allowing the cell to fine tune the levels of SOS repair activity according to the amount or type of signal (damage) perceived. In some cases (such as dam mutations), blocking the SOS response by lexA(Ind⁻) alleles leads to cell death. In this type of constitutively activated strain, the increased level of repair from SOS induction is required to allow the cell to tolerate potentially lethal DNA structures generated by the mutant gene product. The second step in induction is the interaction of signal with RecA protein and is shown by isolating 8 recA mutants. Mutant recA alleles caused the strongest SOS induction in any mutants obtained, similar to the level found in strains lacking repressor (lexA(Def) mutants). This full induction in the absence of lethal DNA damage underscores the pivotal role of RecA protein in regulating the SOS response.

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published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy