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Chromosomal damage and shortening of telomeres in cancer patients and healthy subjects
Kroupa, Michal ; Vodička, Pavel (advisor) ; Kmoníčková, Eva (referee) ; Rössner, Pavel (referee)
Impaired chromosome segregation during mitosis, inaccurate DNA damage response and excessive telomere shortening may all modulate the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL). There is evidence that increased frequency of structural CAs in PBL may be considered as a marker of enhanced cancer risk. In the present Thesis, an effect of variants in genes involved in mitotic checkpoint and DNA damage response on the inter-individual differences in CAs frequency in PBL was investigated. Considering the importance of disrupted telomere structure and its function in cancer biology, a link between telomere length and clinicopathological and molecular features of cancer patients was analysed. Furthermore, the relevance of telomere length and CAs frequency as markers of patients' survival was examined. The major outcomes of the Thesis, fully reported in detail in seven attached Manuscripts, are: I) Increased frequency of structural CAs and/or disrupted telomere length in PBL may be considered as risk factors for the different types of solid cancer; II) Telomere shortening in PBL of healthy subjects increased the frequency of structural CAs; III) Binary interactions of gene variants in mitotic checkpoint and DNA repair pathways may modulate the frequency of structural...
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The source of endogenous DNA damage in neurodegenerative disease
Havelková, Jarmila ; Hanzlíková, Hana (advisor) ; Vodička, Pavel (referee)
DNA single-strand breaks (SSBs) are amongst the most frequent DNA lesions arising in cells and can threaten genetic integrity and cell survival, as indicated by the elevated genetic deletion, embryonic lethality and neurological disease observed when single-strand break repair (SSBR) is attenuated. One of the proteins important for rapid repair of SSBs is XRCC1, which is a molecular scaffold protein that interacts with multiple DNA repair enzymes (e.g. PARP1, PNKP, POLβ, APTX, LIG3) and thus, promotes their stability and/or function. Defects in SSBR have been associated with hereditary neurodegeneration in humans, cerebellar ataxias and seizures. Here, I focus on genetic disease spinocerebellar ataxia autosomal recessive-26 (SCAR26), which has been shown to be linked to mutations in XRCC1. I investigate the amount of XRCC1 protein in XRCC1-defective cells and reveal that cells from patients with mutations in XRCC1 exhibit greatly reduced XRCC1 levels. I show that reduced levels of XRCC1 protein in cells correlate with the increasing number of endogenous SSBs, measured by quantification of ADP-ribose in the chromatin. In addition, I confirm that the most endogenous SSBs arise in S phase of the cells cycle during replication. Moreover, I prove that the main sources of the endogenous SSBs in...
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Molecular biomarkers related to DNA damage and repair: their role in carcinogenesis, patients' treatment and monitoring
Vodenková, Soňa ; Vodička, Pavel (advisor) ; Anděra, Ladislav (referee) ; Černá, Marie (referee)
Genome instability represents one of the leading forces driving the onset and development of cancer. It arises as a consequence of the combined effect of DNA damage and errors made by the DNA repair system. In many cancers, DNA damage tolerance and DNA repair pathways are disrupted or deregulated, thereby promoting cancer progression. DNA repair also appears to play a substantial role in cancer therapy response. This Dissertation Thesis was performed in response to several unclear and unresolved issues of the role of DNA damage and DNA repair in cancer pathogenesis. The aim of the Thesis was to search for potential novel biomarkers and confirmation of the validity of already existing biomarkers related to DNA damage and DNA repair, which may be associated with cancer susceptibility and patient's clinical outcome. We also explored the biological basis of different biomarkers and their associations. The major outcomes of this Thesis are: 1) The elevated chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) may serve as a biomarker of cancer susceptibility and partially affects patients' clinical outcome. While telomere shortening contributes to the formation of CAs in PBLs only in healthy individuals, less efficient DNA double- strand break repair in PBLs is associated with telomere...
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Genetic variability in sporadic colorectal cancer: Searching for novel risk, prognostic and predictive biomarkers.
Jirásková, Kateřina ; Vodička, Pavel (advisor) ; Machoň, Ondřej (referee) ; Eckschlager, Tomáš (referee)
Colorectal cancer (CRC) is a major public health problem worldwide. Despite improvements in the diagnostic process and advancement in the treatment methods, the prognosis remains poor. To improve survival rates, it is important to identify people with the predisposition for CRC and to detect the potentially curable early stage of the disease. Furthermore, identifying those who would have an adverse clinical outcome associated with a particular chemotherapy would help to avoid redundant chemotherapy burden in patients and contribute to enhanced therapeutic efficacy, while minimizing treatment-related toxicity. The aim of the Thesis was to search for novel promising diagnostic, prognostic and predictive DNA-based biomarkers of sporadic form of CRC. As each patient is genetically unique, these biomarkers would aid clinicians in better diagnosis and/or in the selection of an optimal type of therapy for an individual CRC patient based on their molecular profile. In order to explore this issue, we investigated several candidate genes in healthy individuals as well as in newly diagnosed cancer patients. The major outcomes of this PhD study, which were fully reported in seven publications included in the present Thesis, are 1) The observation of several candidate single nucleotide polymorphisms in microRNA...
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The Question of Succession of James, Duke of York, during the Reign of Charles II
Chmelíková, Pavla ; Valkoun, Jaroslav (advisor) ; Vodička, Pavel (referee)
This diploma thesis deals with 70s and 80s in 17the century in England. During this period of the reign of Charles II Stuart comes to the forefront the question of succession, which culminated in the so-called exclusion crisis (1679-1681). The thesis will try to outline in seven chapters, including the introduction and conclusion, the development of the crisis of succession associated with the person of James, Duke of York. The first part of the work will focus on the period before the exclusion crisis and will highlight important moments such as the approval of the Test Act or the Popish Plot. Another part of the thesis will deal with the period of crisis itself until the dissolution of the last Exclusion Parliament in Oxford in 1681 and the defeat the Whig party. The last chapter will close the whole question of succession in 1685, the death of Charles II Stuart and the advent of James II to the English throne.
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Clinical classification of sequence variants in non-coding regulatory regions in breast cancer susceptibility genes.
Bubáková, Eliška ; Ševčík, Jan (advisor) ; Vodička, Pavel (referee)
Inactivation of tumor supressor gene BRCA1 causes a life-long risk of breast carcinoma development. Genetic screenings of indicated individuals from high-risk families help to identify large number of sequence variants in known predisposing genes. Majority of discovered variants doesn't have clinical significance yet which causes a big problem for diagnostics. Some of these variants are found within regulatory non-coding regions of gene. A part of the clinical classification of variants is their functional characterization. The goal of this thesis was to create a model system for functional characterization of variants in non-coding regions and to verify its function. Model system was based on targeted gene manipulation by co-transfecting CRISPR-Cas9 construct and donor construct that contained a portion of BRCA1 gene sequence with analyzed modifications, into U2 OS cells. The cells have stably integrated DR-GFP system which allows the activity of homologous recombination (HR) to be determined. Monoallelic modifications were induced into U2 OS cells. These modifications were in a Kozak sequence region of BRCA1 gene. Expression level of BRCA1 mRNA was determined by qRT-PCR, which showed the same levels of mRNA in all cells with analyzed alterations. Next, expression level of BRCA1 protein was...
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Chromosomal damage and shortening of telomeres in cancer patients and healthy subjects
Kroupa, Michal ; Vodička, Pavel (advisor) ; Kmoníčková, Eva (referee) ; Rössner, Pavel (referee)
Impaired chromosome segregation during mitosis, inaccurate DNA damage response and excessive telomere shortening may all modulate the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL). There is evidence that increased frequency of structural CAs in PBL may be considered as a marker of enhanced cancer risk. In the present Thesis, an effect of variants in genes involved in mitotic checkpoint and DNA damage response on the inter-individual differences in CAs frequency in PBL was investigated. Considering the importance of disrupted telomere structure and its function in cancer biology, a link between telomere length and clinicopathological and molecular features of cancer patients was analysed. Furthermore, the relevance of telomere length and CAs frequency as markers of patients' survival was examined. The major outcomes of the Thesis, fully reported in detail in seven attached Manuscripts, are: I) Increased frequency of structural CAs and/or disrupted telomere length in PBL may be considered as risk factors for the different types of solid cancer; II) Telomere shortening in PBL of healthy subjects increased the frequency of structural CAs; III) Binary interactions of gene variants in mitotic checkpoint and DNA repair pathways may modulate the frequency of structural...
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Molecular mechanisms involved in genotoxicity of industrially important monomers (styrene, 1,3-butadiene)
Kuricová, Miroslava ; Vodička, Pavel (advisor) ; Černá, Marie (referee) ; Machala, Miroslav (referee)
1 ABSTRACT The evaluation of individual health risk in workers occupationally exposed to industrial xenobiotics requires the use of a large number of parameters reflecting external exposure, internal exposure, biological effects and individual susceptibility. Environmental, occupational and life style-related exposure to mutagenic agents may contribute to cancer risk in humans. To prevent the potentially hazardous effects of such agents it is important to understand their mechanisms of action. Styrene is one of the most important monomer for producing polymers and copolymers in plastics, latex paints and together with 1,3-butadiene (BD) in the manufacture of synthetic rubbers. In this thesis, a large set of parameters, including markers of external and internal exposure and biomarkers of biological effects and susceptibility have been studied in relation to the occupational exposure to both styrene and BD. First part of the present study was focused on evaluating the role of various biomarkers to assess genotoxic effects of above mentioned xenobiotics. Biomarkers reflecting styrene- and BD-induced genotoxicity and mutagenicity: O6 -styrene guanine DNA adducts, haemoglobin adducts, single-strand breaks (SSBs), SSB Endo III sites, chromosomal aberrations (CA), hypoxanthine-guanine phosphoribosyltransferase...
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