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Molecular characteristics of mismatch repair pathway in ovarian cancer
Burócziová, Monika ; Vodička, Pavel (advisor) ; Schierová, Michaela (referee)
In humans, multi enzymatic processes are involved in maintaining DNA stability and cellular homeostasis. Cells undergo several episodes to survive and protect itself in daily basis. Accumulation of DNA errors and breaks are repaired by dynamic machinery, such as mismatch repair (MMR), replication-related process. In presented diploma thesis, we report the studied MMR pathway and its involvement in malignancy of epithelial ovarian cancer (EOC). Our working hypothesis postulated that core genes of MMR, such as MLH1 and MSH2 are down-regulated in malignant cells. Cells therefore become incapable to repair accumulating DNA damage, undergo apoptosis or most likely uncontrolled proliferation. Above mentioned genes may also be silenced in cancer patients at transcription, translation or epigenetic levels. Our aims were to clarify and to investigate the importance of MMR based on mRNA transcription, protein stability and promoter hypermethylation on a set of major MMR genes, particularly MLH1, MSH2, PMS1, MLH3, MSH6, MSH3, and PMS2. In our study, we analysed samples from 63 epithelial ovarian cancer patients and 12 non-malignant reference tissues using RT-qPCR, MS-HRM, and Western Blotting methods. Consequently, our results show down-regulation of all MMR genes except for MSH2 (up-regulated) in tumor...
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English royal court and its changes in the context of first half of the 17th century (1603-1640)
Vodička, Pavel ; Soukup, Jaromír (advisor) ; Kovář, Martin (referee) ; Skřivan, Aleš (referee)
The aim of the dissertation is a comparative analysis of the English royal court in the first half of the 17th century. The analytical part of the dissertation focuses on researching processes that influenced the structure and roles in the royal court in regards to the political, religious, economic, social and cultural development of the world. The benchmarks represent the personality of the ruler, institutional structure and personnel composition of the court, its financing and its culture. The comparison is a defined period of time between the beginning of James I (1603) and the end of the personal rule of Charles I (1640). The dissertation is based on critical analysis of the sources and studies of secondary literature. One of the features of the Royal Court during the rule of James I was the rivalry of various factions. In the interest of retaining a balance in power, the monarch revealed selected offices only to members of his Scottish clubs. Targeted strengthening of the influences of selected institutions of court, especially Bedchamber, ended up contributing to a significant weakening of the unitary system of the government, where the Privy Council played a key role up until then. In addition, between 1603 and 1625, there became a strong concentration of power in the hands of the royal...
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The application of functional tests to measure DNA repair capacity in molecular epidemiological studies
Slyšková, Jana ; Vodička, Pavel (advisor) ; Hampl, Aleš (referee) ; Kment, Milan (referee)
DNA repair is a vital process of a living organism. Inherited or acquired defects in DNA repair systems and cellular surveillance mechanisms are expected to be important, if not crucial factors in the development of human cancers. DNA repair is a multigene and multifactorial process which is most comprehensively characterized by the phenotypic evaluation of DNA repair capacity (DRC). DRC represents a complex marker with high informative value, as it comprises all genetic, epigenetic and non-genetic factors, by which it is modulated. Accordingly, DRC reflects the actual capability of the cell, tissue or organism to protect its DNA integrity. The present PhD study was focused on investigating DRC, which specifically involves base and nucleotide excision repair pathways, in human populations with different characteristics. The main aim was to answer substantial questions on the possible use of DRC as biomarkers in epidemiological studies. The study was in fact designed to understand the extent of physiological variability of DRC in a population, its modulation by genetic and non-genetic factors, tentative adaptability to high genotoxic stress and, finally, its involvement in cancer aetiology. In order to explore these issues, DRC, in respect to genetic and environmental variability, was investigated...
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Vztah mezi genetickými polymorfismy DNA reparačních genů a jejich expresí u zdravé populace (s výhledem na stanovení u onkologických pacientů).
Hánová, Monika ; Vodička, Pavel (advisor) ; Bencko, Vladimír (referee) ; Černá, Marie (referee)
DNA damage response is a complex system responsible for protection of a cell against internal and external DNA damaging agents and in maintaining genome integrity. Many of genes participating in DNA damage response pathways are polymorphic. Genetic polymorphisms in coding and regulatory regions may have impact on the function of proteins encoded by the genes. Phenotypic effect of single nucleotide polymorphisms (SNPs) is subject of investigation in connection with the ability of a cell to manage genotoxic stress and subsequently, in relation to cancer susceptibility. The aim of this thesis was to evaluate the association between SNPs in DNA repair genes (hOGG1, XRCC1, XPC) and cell cycle genes (TP53, p21CDKN1A , BCL2 and BAX) and their mRNA expression in peripheral blood lymphocytes from individuals occupationally exposed to styrene and control individuals. The aim was extended to analyses of relationships between mRNA expression levels of the above-mentioned genes and markers of exposure to styrene (concentration of styrene in blood and in air), markers of DNA damage (single strand breaks - SSBs, and endonuclease III specific sites - Endo III sites) and the base excision repair (BER) capacity, by means of γ-irradiation specific DNA repair rates and oxidative repair. Study on the group of healthy...
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Chromosomal damage and DNA repair capacity in blood lymphocytes as transient markers in carcinogenesis.
Kroupa, Michal ; Vodička, Pavel (advisor) ; Štětina, Rudolf (referee)
Recent knowledge suggests that the onset of cancer is modulated by the interplay of internal and external environmental factors along with numerous gene variants. Structural chromsomal aberrations in peripheral blood lymphocytes are considered as biomarkers of effect of genotoxic carcinogens and reflect elevated risk of cancer. Incomplete or deficient repair of double-strand breaks in DNA underlie chromosomal aberrations and the measurement of cytogenetic alterations may reflect interindividual differences in the response towards the mutagen. In this study the expected deficiences in the DNA repair capacity have been determined in incident oncological patients with breast, colorectal and urogenital cancers. The determination of chromosomal aberrations have been supplemented by the measurement of variants in genes involved in double-strand breaks repair (XRCC3, rs861539; RAD54L, rs1048771). Methodologically, we employed conventional cytogenetic analysis, cytogenetic analysis following the induction of chromocomal damage by bleomycin ("Challenge assay"), TaqMan discrimination analysis for the detection of allelic variants and statistical analyses. By using these methods we did not observe statistically signifiant differences either in chromosomal breaks (p=0,354) or in a percentage of cells with...
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Mutations in MLH1 gene and MSI status as molecular characteristics of sporadic colorectal cancer
Čaja, Fabián ; Vodička, Pavel (advisor) ; Kadlecová, Jitka (referee)
Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...
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From the search for new oncogenes to the effort of redefining the cancerogenesis phenomenon
Pajer, Petr ; Dvořák, Michal (advisor) ; Vodička, Pavel (referee) ; Eckschlager, Tomáš (referee)
The described experimental model of clonal tumors induced through the insertional mutagenesis with MAV-2 proved to be a valid and rich source of information describing the process of transformation of normal into tumor cell. We have mapped more than 2000 individual clonal VISs from several hundreds of tumor tissue samples. We have analyzed five tumor types of different histology and tissue of origin along with their derivative tissue cultures. Furthermore, we have discovered the industasis phenomenon and described it during the course of the study. The goal of my study was to uncover common reasons for neoplastic transformation of the cell. The results of my study led me to the paradoxical conclusion that the significance of genetic changes as the primary cause of induction of neoplastic transformation is being overestimated. Although studying the functions of individual genes and search for new tumor markers and therapeutical targets are still beneficial, I believe that the traditional perception of tumor formation as a function/result of mutation accumulation and selection is becoming a serious drawback in further investigations. These conclusions are further discussed in the last section of the presented Ph.D. thesis.
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Methylation profile in malignancy
Stojčeva, Nina ; Vodička, Pavel (advisor) ; Vondrejs, Vladimír (referee)
Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.
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Mutagenic and Antimutagenic Effect of Environmental Substances
Langová, Martina ; Vodička, Pavel (advisor) ; Sedmíková, Markéta (referee) ; Novotná, Božena (referee)
In the present study were used reference mutagens which effects mutagenic activity in prokaryotic and eukaryotic testing systems. Aflatoxin B1 (AFB1) is one of the most thoroughly studied and the well known mycotoxin with carcinogenic activity. 2-Amino-3-methylimidazol[4,5-f] quinoline (IQ) in isolated form has been used as another reference mutagen. These compounds are indirect acting genotoxins, i.e. need metabolic activation to exert genotoxic effect. As a third reference mutagen in the present study there has been chosen N-nitroso-N-methylurea (MNU) which is direct acting carcinogenic N-nitroso compound. The aim of the study was to investigate antimutagenic effects of ellagic acide (EA), resveratrol (RES), diallyl sulfide (DAS) and phenetyl isothiocyanate (PEITC) on the mutagenicity of the mutagens. The Ames test with Salmonella typhimurium TA98 and TA100 strains was used for the evaluation of antimutagenic effect of EA, RES, DAS and PEITC in vitro.
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Fanconi anemia and pancreatic cancer
Hucl, Tomáš ; Špičák, Julius (advisor) ; Nečas, Emanuel (referee) ; Vodička, Pavel (referee)
Inactivation of the Fanconi anemia (FA) pathway occurs in diverse human tumors including pancreatic cancer and renders those tumors hypersensitive to DNA interstrand-cross-linking agents (ICL). How to treat specificly pancreatic and other cancers harboring FA mutations has recently raised great interest, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in an adenokarcinoma cell line and observed a typical phenotype of FA pathway deficiency (abrogation of FANCD2 monoubiquitination; chromosomal instability, G2M arrest and decreased proliferation upon treatement with ICL, spontaneous chromosomal breakage). Homozygous deletion was achieved for FANCC and FANCG but not for FANCD2 and BRCA2/FANCD1 in RKO cells, suggesting a detrimental phenotype. It provided direct evidence for the paradoxical assumption that their inactivation could be predominantly selected against in cancer cells. Using high-throughput screening, we assessed the growth of our isogenic FANCC and FANCG cells upon treatment with 880 active drugs and 40 000 diverse compounds. The compound having the stronges effect, named 80136342, had a distinct mechanism of action from that of ICL agents. When applied in combination with ICL agents, 80136342 had at least...
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