Protection of the genome from carcinogenic consequences of DNA double-strand breaks (DSBs) is accomplished through the pathways of non-homologous end-joining (NHEJ) or homologous recombinational repair. Five human proteins with homology to Rad51 known as the Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, whose loss of function in cell lines leads to high chromosomal instability. Previous studies have shown Rad51C participate in two paralog protein complexes, one containing Rad51B, Rad51C, Rad51D and XRCC2 (BCDX2) and the other containing only Rad51C and XRCC3 (CX3). However, the only structural data available is the crystal structure of RecA, the bacterial homolog, the determination of the N-terminus of human Rad51 by NMR, and the crystal structure of Pyroccocus furious Rad51. Currently the Alvinlla pompejana Rad51C has been cloned, expressed and is currently being crystallized in the Tainer laboratory (UC Berkeley) since the human Rad51C protein has proven too difficult to be utilized. To test functional association of Hs Rad51B and Hs XRCC3 to Ap Rad51C. The human proteins were heterologously expressed in Pichia pastoris and the other expressed in E. coli. The proteins were extracted and interaction was tested through co-immunoprecipitation. Initial results depict weak binding or an unstable interaction between Hs Rad51B and Ap Rad51C. Hs XRCC3 and Ap Rad51C interaction remains unclear and requires further testing. Additionally, we have utilized a cellular model of HNSCC to identify whether the down-regulation of Rad51 after application of VD 3 is concomitant with the down-regulation of NBS1. NBS1 is a DNA repair protein involved in both pathways of DNA double-strand break repair, non-homologous end-joining and homologous recombinational repair. It has recently been demonstrated that NBS1 binds to Rad51 aiding in its localization to sites of DNA damage. VD 3 is a potential chemopreventive agent in the treatment of head and neck cancer. For the in vitro model Rad51 and NBS1 protein were both extracted from SCC25 and MCF-7 cancer cell lines were treated with 100 nM of VD 3 . For the in vivo model hamsters cheek pouch tissue sections with VD 3 treated and DMBA over the course of 14 weeks were used. Rad51 and NBS1 staining is restricted to the nuclei of the basal cell layer of the epithelium in VD 3 treated animals as compared to untreated controls where staining is evident throughout the dysplastic epithelium and is not restricted to nuclei. Unlike the western blot data of Rad51 that shows similar downregulation as the immunocytochemistry, the western blot analysis of NBS1 is unclear. However, the immunocytochemistry suggests that NBS1 is also downregulated by VD 3 in vivo, and therefore, it may be implied that both the HRR and NHEJ pathways are involved in the cellular effects of VD 3 in HNSCC.

O glioblastoma (GBM) é um tumor extremamente agressivo e resistente aos tratamentos convencionais. Os agentes utilizados na quimio- e radioterapia são indutores de danos no DNA, por induzirem quebras de fita simples (SSBs) e quebras de fita dupla (DSBs), as quais são letais para as células, mas quando eficientemente reparadas pelas células tumorais tornam estas resistentes. As principais vias de reparo de DSBs são: a via por recombinação homológa (Homologous Recombination HR) e por recombinação não- homóloga (Non-homologous end joining NHEJ). As proteínas de reparo participantes dessas vias têm sido estudadas como potenciais alvos moleculares na terapia contra o câncer. A estratégia empregada no presente trabalho foi a de inibir a via de reparo HR em células já comprometidas para a via NHEJ. Para isso foi utilizado o inibidor de RAD51 (uma das principais proteínas da via HR), 3-chloro-1-(3,4-dichlorophenyl)-4- morpholinylo-1H-pyrrole-2,5-dione, conhecido como RI-1(Calbiochem), testado em células de glioma M059J e M059K (deficientes e proficientes para DNA-PK, respectivamente), irradiadas com raios-X. Diferentes ensaios foram realizados para o teste com o inibidor RI-1 em células irradiadas ou não e comparados ao controle sem tratamento. Os resultados dos ensaios de sobrevivência clonogênica mostraram que a concentração de 40 M do inibidor RI-1 exerceu um maior efeito inibitório sobre a capacidade de as células se dividirem e formarem colônias. O RI-1 induziu alterações significativas na cinética do ciclo celular predominantemente na linhagem selvagem M059K, nos tempos de 24 e 72 h. Apesar de a linhagem M059J não mostrar alterações significativas na cinética do ciclo celular, esta demonstrou sensibilidade à irradiação, conforme demonstrado pela cinética de reparo das DSBs, sendo mais lenta em relação à M059K, demonstrando o comprometimento do reparo NHEJ na linhagem mutante para DNA-PK. A expressão das proteínas LIG3, XRCC1 e PARP-1 foram analisadas no tempo de 15 minutos e 24 h após a irradiação. Na presença do inibidor RI-1, a expressão da LIG3 foi aumentada em células M059K (15 min e 24 h) comparadas ao grupo controle. Já a linhagem M059J apresentou uma elevada expressão das proteínas XRCC1 e PARP-1 apenas em 15 min em relação ao controle; esses dados indicaram que um possível reparo de DSBs envolvendo essas proteínas pode ter sido ativado logo nos primeiros minutos após a indução de danos nessas células. O conjunto dos resultados deste trabalho sugere um papel atuante do inibidor RI-1 em células comprometidas para o reparo NHEJ, isto é, a linhagem M059J, levando à sugestão de que vias alternativas de reparo podem possivelmente estar envolvidas na resistência das células tumorais aos tratamentos convencionais. / Glioblastoma (GBM) is an extremely aggressive and resistant tumor to conventional treatments. The agents used in chemotherapy and radiotherapy are inducers of DNA damage, since they induce single strand breaks (SSBs) and doublestrand breaks (DSBs), which are lethal to cells, but when efficiently repaired by tumor cells make them resistant to antitumoral agents. The main repair pathways for DSBs are the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Proteins participating in these processes have been studied as potential molecular targets in cancer therapy. Thus, the strategy employed in this work involved the inhibition of HR repair pathway in cells already committed to the NHEJ pathway, aiming to sensitize irradiated GBM cells. An inhibitor of RAD51 (one of the major HR proteins) was used: 3-chloro-1-(3,4-dichlorophenyl) -4 morpholinylo-1Hpyrrole- 2,5-dione, known as RI-1 (Calbiochem); this compound was tested in GBM cells, M059K and M059J (proficient and deficient for the DNA-PK, respectively) irradiated with X-rays. Various assays were performed to test the inhibitory property of RI-1 in irradiated cells and the combination of the inhibitor with X-irradiation, compared with the untreated control. The results of clonogenic survival showed that 40 M of RI-1 inhibitor exerted a higher inhibitory effect on the ability of cells to divide and form colonies. The RI-1 induced changes in cell cycle kinetics predominantly in the wild-type M059K, at 24 and 72 h. Although M059J did not show significant changes in cell cycle kinetics, these cells showed sensitivity to X-irradiation, as shown by the kinetics of DSB repair (gamma-H2AX foci), which was slower compared to M059K, demonstrating the commitment of the NHEJ repair in M059J (mutant for DNA-PK). The expression of LIG3, PARP-1 and XRCC1 proteins were analyzed at 15 min. and 24 h after irradiation. In the presence of the inhibitor RI-1, LIG 3 expression was increased in M059K cells (15 min. and 24 h) compared to the control group. M059J cells showed a high expression of XRCC1 and PARP-1 only at 15 min., compared to the control. These data indicated that a possible repair of DSBs involving these proteins may have been activated in the first minutes after DNA damage induction. The overall results of this study suggest that RI-1 inhibitor was efficient to influence cellular responses in cells committed to the NHEJ repair, i.e. M059J cell line, leading to the hypothesis that alternative repair pathways may be possibly involved in the resistance of tumor cells.

O glioblastoma (GBM) é um tumor extremamente agressivo e resistente aos tratamentos convencionais. Os agentes utilizados na quimio- e radioterapia são indutores de danos no DNA, por induzirem quebras de fita simples (SSBs) e quebras de fita dupla (DSBs), as quais são letais para as células, mas quando eficientemente reparadas pelas células tumorais tornam estas resistentes. As principais vias de reparo de DSBs são: a via por recombinação homológa (Homologous Recombination HR) e por recombinação não- homóloga (Non-homologous end joining NHEJ). As proteínas de reparo participantes dessas vias têm sido estudadas como potenciais alvos moleculares na terapia contra o câncer. A estratégia empregada no presente trabalho foi a de inibir a via de reparo HR em células já comprometidas para a via NHEJ. Para isso foi utilizado o inibidor de RAD51 (uma das principais proteínas da via HR), 3-chloro-1-(3,4-dichlorophenyl)-4- morpholinylo-1H-pyrrole-2,5-dione, conhecido como RI-1(Calbiochem), testado em células de glioma M059J e M059K (deficientes e proficientes para DNA-PK, respectivamente), irradiadas com raios-X. Diferentes ensaios foram realizados para o teste com o inibidor RI-1 em células irradiadas ou não e comparados ao controle sem tratamento. Os resultados dos ensaios de sobrevivência clonogênica mostraram que a concentração de 40 M do inibidor RI-1 exerceu um maior efeito inibitório sobre a capacidade de as células se dividirem e formarem colônias. O RI-1 induziu alterações significativas na cinética do ciclo celular predominantemente na linhagem selvagem M059K, nos tempos de 24 e 72 h. Apesar de a linhagem M059J não mostrar alterações significativas na cinética do ciclo celular, esta demonstrou sensibilidade à irradiação, conforme demonstrado pela cinética de reparo das DSBs, sendo mais lenta em relação à M059K, demonstrando o comprometimento do reparo NHEJ na linhagem mutante para DNA-PK. A expressão das proteínas LIG3, XRCC1 e PARP-1 foram analisadas no tempo de 15 minutos e 24 h após a irradiação. Na presença do inibidor RI-1, a expressão da LIG3 foi aumentada em células M059K (15 min e 24 h) comparadas ao grupo controle. Já a linhagem M059J apresentou uma elevada expressão das proteínas XRCC1 e PARP-1 apenas em 15 min em relação ao controle; esses dados indicaram que um possível reparo de DSBs envolvendo essas proteínas pode ter sido ativado logo nos primeiros minutos após a indução de danos nessas células. O conjunto dos resultados deste trabalho sugere um papel atuante do inibidor RI-1 em células comprometidas para o reparo NHEJ, isto é, a linhagem M059J, levando à sugestão de que vias alternativas de reparo podem possivelmente estar envolvidas na resistência das células tumorais aos tratamentos convencionais. / Glioblastoma (GBM) is an extremely aggressive and resistant tumor to conventional treatments. The agents used in chemotherapy and radiotherapy are inducers of DNA damage, since they induce single strand breaks (SSBs) and doublestrand breaks (DSBs), which are lethal to cells, but when efficiently repaired by tumor cells make them resistant to antitumoral agents. The main repair pathways for DSBs are the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Proteins participating in these processes have been studied as potential molecular targets in cancer therapy. Thus, the strategy employed in this work involved the inhibition of HR repair pathway in cells already committed to the NHEJ pathway, aiming to sensitize irradiated GBM cells. An inhibitor of RAD51 (one of the major HR proteins) was used: 3-chloro-1-(3,4-dichlorophenyl) -4 morpholinylo-1Hpyrrole- 2,5-dione, known as RI-1 (Calbiochem); this compound was tested in GBM cells, M059K and M059J (proficient and deficient for the DNA-PK, respectively) irradiated with X-rays. Various assays were performed to test the inhibitory property of RI-1 in irradiated cells and the combination of the inhibitor with X-irradiation, compared with the untreated control. The results of clonogenic survival showed that 40 M of RI-1 inhibitor exerted a higher inhibitory effect on the ability of cells to divide and form colonies. The RI-1 induced changes in cell cycle kinetics predominantly in the wild-type M059K, at 24 and 72 h. Although M059J did not show significant changes in cell cycle kinetics, these cells showed sensitivity to X-irradiation, as shown by the kinetics of DSB repair (gamma-H2AX foci), which was slower compared to M059K, demonstrating the commitment of the NHEJ repair in M059J (mutant for DNA-PK). The expression of LIG3, PARP-1 and XRCC1 proteins were analyzed at 15 min. and 24 h after irradiation. In the presence of the inhibitor RI-1, LIG 3 expression was increased in M059K cells (15 min. and 24 h) compared to the control group. M059J cells showed a high expression of XRCC1 and PARP-1 only at 15 min., compared to the control. These data indicated that a possible repair of DSBs involving these proteins may have been activated in the first minutes after DNA damage induction. The overall results of this study suggest that RI-1 inhibitor was efficient to influence cellular responses in cells committed to the NHEJ repair, i.e. M059J cell line, leading to the hypothesis that alternative repair pathways may be possibly involved in the resistance of tumor cells.

The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-10-15 11:06:30.613

Acquisition of invasiveness through extracellular matrix is a crucial characteristic of transition to malignancy in the breast. It was previously shown that Polo-like kinase 1 (PLK-1), a mitotic kinase and genome stability regulator, is involved in acquisition of invasiveness in a breast cell model (HMT-3522 cell line) of pre-invasive to invasive transition. This and other data led to the suggestion that a new class of genes called GISEM for Genome Instability and Extracellular Matrix Invasiveness may exist. Previous lab data show that XRCC3 is found downregulated in progression from preinvasive to invasive phenotype. This led to the hypothesis that XRCC3 may be a negative regulator of invasion. To support this hypothesis, overexpression of XRCC3 in the invasive T4-2 cells downregulated invasion, but also growth. In order to verify the role of XRCC3 in invasiveness, and determine whether it is independent from any effects on growth, we tested the effect of downregulating XRCC3 on the invasiveness of T4-2 cells. Short-term downregulation of XRCC3 using siRNAs produced a significant increase in invasiveness, suggesting a role for XRCC3 as a negative regulator of invasion. During the invasion assay time course, XRCC3 downregulation had no effect on growth or apoptosis supporting the idea that this is a direct effect on invasion and not an artifact of the assay. XRCC3 is one amongst the five members of the RAD51 paralog family, consisting of accessory proteins or RAD51 cofactors (namely RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) which interact with each other to form complexes (BCDX2, BC, DX2 and CX3) that collaboratively assist RAD51 in homologous recombinational repair (HRR) of DNA double-strand breaks. To see if these interactions are important in terms of invasion, as they have been demonstrated for DNA repair, we studied the effect of XRCC3 downregulation on the levels of RAD51 paralogs. We found lowered levels of RAD51C, but not RAD51B or RAD51D, when XRCC3 was downregulated. Since XRCC3 forms the CX3 complex with RAD51C, we downregulated RAD51C using siRNAs in T4-2 cells and found this to significantly increase invasiveness. Consistent with previous findings by other groups, downregulating RAD51C also lead to decreased levels of XRCC3 in invasive T4-2 cells. These results suggest that the XRCC3-RAD51C interaction is important for invasion as well as the previously studied DNA repair function. In delineating the mechanism by which XRCC3 acts as a negative regulator of invasion, we further questioned if XRCC3 alters secreted factors that are important for the invasiveness of T4-2 cells and tested the effects of conditioned medium (CM) from XRCC3 altered T4-2 cells on parental T4-2 cells’ ability to invade. Results show a significant increase in invading T4-2 cells when suspended in CM from XRCC3 siRNA transfected T4-2 cells, suggesting a direct effect of XRCC3 siRNAs on the ability of T4-2 CM to induce invasiveness in T4-2 cells. Furthermore, we investigated the effects of XRCC3 inhibition on cell surface integrins and focal adhesion kinase (FAK). Indirect immunofluorescence results show increased formation of focal adhesions containing two phosphorylated FAK residues- autophosphorylated FAK-Y397 and FAK-Y861 (previously implicated in increased migration and invasion of tumor cells) in XRCC3 siRNA transfected T4-2 cells. Overall, these results support a new role of XRCC3 in invasion, in addition to its previously reported role in DNA repair. These findings imply that loss of XRCC3 function in cancer progression would upregulate invasion as well as downregulate DNA repair and genome stability. Therefore, stabilization of XRCC3 function could provide a promising therapeutic against breast cancer progression. The dual role of XRCC3 in invasion and DNA repair also renders it an attractive candidate risk biomarker of breast pre-cancer to invasive cancer progression.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-25T20:42:57Z No. of bitstreams: 1 EdilmarDeMouraSantos_TESE.pdf: 2404882 bytes, checksum: 5eff55db010b690ecc915ee6c24050f0 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-26T19:45:12Z (GMT) No. of bitstreams: 1 EdilmarDeMouraSantos_TESE.pdf: 2404882 bytes, checksum: 5eff55db010b690ecc915ee6c24050f0 (MD5) / Made available in DSpace on 2016-08-26T19:45:12Z (GMT). No. of bitstreams: 1 EdilmarDeMouraSantos_TESE.pdf: 2404882 bytes, checksum: 5eff55db010b690ecc915ee6c24050f0 (MD5) Previous issue date: 2016-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Falhas nos genes respons?veis por reparos no DNA podem influenciar no surgimento de c?ncer ou afetar a resposta aos tratamentos. Estudos t?m demonstrado que a varia??o na capacidade de reparo do DNA pode ser resultado de polimorfismos funcionais nestes genes, e alguns destes experimentos sugerem que a presen?a de polimorfismos de nucleot?deos simples (SNPs), em genes de reparo, est? relacionada ao desenvolvimento e resposta ao tratamento de v?rios c?nceres, incluindo o Carcinoma Epidermoide Oral (CEO) e o Carcinoma Epidermoide de Orofaringe (CEOR). Nesta pesquisa avaliou-se a frequ?ncia de tr?s SNPs em dois genes de reparo do DNA RAD51 172G>T (c.-61 G>T, rs1801321), RAD51 135G>C (c.-98 G>C, rs1801320) e XRCC3 T241M (c. 722 C>T, rs861539) em indiv?duos saud?veis (n=130) e indiv?duos com CEO e CEOR (n=126) e investigou-se poss?veis rela??es de tais achados com os desfechos cl?nicos: resposta tumoral ao tratamento com radioterapia e quimioterapia, recidiva, e sobrevida global. Constatou-se frequ?ncia al?lica e genot?pica em equil?brio. A presen?a dos SNPs analisados n?o revelou ser um fator de risco para o desenvolvimento de CEO ou CEOR; contudo, quando associado ao h?bito de fumar ou beber, aumentou o risco de desenvolver o c?ncer de tr?s a cento e cinquenta vezes (p<000,1). A resposta tumoral ao tratamento de radioterapia e quimioterapia foi semelhante nos pacientes com ou sem SNPs. Nenhum polimorfismo demonstrou signific?ncia estat?stica em rela??o ? sobrevida livre de recidiva ou sobrevida global. Os gen?tipos AA e AC do SNP rs861539 no gene XRCC3, os gen?tipos CC e CG do SNP rs1801320 e GG e GT do SNP 1801321 no gene RAD51, aumentam o risco do desenvolvimento de carcinoma epidermoide oral e de orofaringe, quando associados ao h?bito de beber ou fumar. Os polimorfismos estudados nos genes XRCC3 e RAD51 n?o est?o associados ? resposta ? radioterapia, sobrevida livre de recidiva ou sobrevida global. / Faults in the genes responsible for repairs to the DNA can influence the onset of cancer or affect the response to treatment. This research evaluated the frequency of three single nucleotide polymorphisms (SNPs) in two repair genes DNA RAD51 172g> T (rs1801321), RAD51 135G> C (rs1801320) and XRCC3 T241M (rs861539) in individuals without cancer (n = 130) and patients with oral squamous cell carcinoma (OSC) and carcinoma oropharyngeal squamous (ORSC) (n = 126) and investigated possible relationships of these findings with clinical and pathological data and clinical outcomes: tumor response to radiotherapy and chemotherapy, disease-free survival, and overall survival. It was found that the allele and genotype frequencies were in equilibrium Hard-Weinberg equilibrium. The presence of at least one polymorphic allele in XRCC3 (rs861539) gene is associated with histological grade (WHO) higher (p = 0.007). We observed a higher recurrence rate trend (p = 0.08) and more advanced stage (p = 0.08) in the group that had at least one polymorphic allele of RAD51 gene (rs1801321). The presence of the analyzed SNPs not proved to be a risk factor for the development of CEO or CEOR; however, when combined with smoking or drinking, increased the risk of developing cancer from three to one hundred and fifty times. The tumor response to radiotherapy and chemotherapy was similar in patients with and without SNPs. No polymorphism showed statistical significance in relation to recurrence-free survival or overall survival. We conclude that the presence of at least one polymorphic allele of the SNPs rs861539 in XRCC3 gene, rs1801320 and rs1801321 in the RAD51 gene increase the risk of development of OSC and ORSC, when associated with the habit of drinking or smoking. Polymorphisms studied in XRCC3 and RAD51 genes are not associated with response to radiation therapy, relapse-free survival or overall survival.

Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.

Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests the involvement of DNA repair mechanism defects in the pathogenesis of this disorder. The first part of this work was focused on monitoring of gene expression of DNA repair genes in MDS patients and on their alterations during disease progression. In the second part, next generation sequencing was used to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with MDS development was evaluated. Methods: Expression profiling of 84 DNA repair genes was performed on bone marrow CD34+ cells of patients with MDS. Screening cohort consisted of 28 patients and expression of selected genes was further validated on larger cohort of 122 patients with all subtypes of MDS. Paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 recombinase protein was done on samples acquired by trephine-biopsy. Targeted enrichment resequencing of exonic parts of 84 DNA repair genes was performed on the screening cohort of MDS patients. Real-time PCR was used for genotyping of selected SNPs in the population study. Results: RAD51 and XRCC2 genes showed...

No description available.

During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...