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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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Antitumor activity of the polymeric conjugates bearing derivatives of protease inhibitors and conventional cytostatics for the treatment of head and neck tumors
Běhalová, Kateřina ; Kovář, Marek (advisor) ; Vodička, Pavel (referee)
Head and neck cancers account for about 4,6 % of all malignancies worldwide and their incidence is increasing. However, the development of chemotherapeutics in this field is rather stagnating. One promising approach seems to be the repurposing of drugs originally developed and clinically used as HIV protease inhibitors, which have also been described to have anticancer activity. Esterification of the OH group of these drugs with 5-methyl-4-oxohexanoic acid allows their attachment via pH-sensitive hydrazone linkage to a hydrophilic and biocompatible HPMA copolymer carrier. This binding provides an improvement in the pharmacokinetics of the drug, prolongs its circulation time in the bloodstream, lowers its side effects and it also allows passive accumulation of the drug in the tumor tissue due to the EPR effect. Six protease inhibitors in total (ritonavir, lopinavir, saquinavir, indinavir, nelfinavir and atazanavir) were derivatized as described above. Ritonavir and its derivative had been tested in a previous project and were used as reference substances. After initial determination of in vitro cytostatic and cytotoxic activity in FaDu (human head and neck carcinoma), SCC7 (murine squamous cell carcinoma), 4T1 (murine breast carcinoma) and CT26 (murine colon carcinoma) cell lines, lopinavir derivative was...
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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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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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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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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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Posttranslational modification of the adapter protein DAXX in the cellular response to genotoxic stress
Bražina, Jan ; Anděra, Ladislav (advisor) ; Černý, Jan (referee) ; Vodička, Pavel (referee)
Maintaining the chromosome continuity and complete genetic information in human cells is crucial for cell survival and the whole organism. It prevents life-threatening pathologies and preserves genetic continuity. However, cellular DNA is exposed to both endogenous and exogenous stress damaging its content and integrity. This stress activates mechanisms involving detection and repair of these damaged sites (DDR). One of the most serious types of DNA damage double-stranded breaks (DSB) occuring when both strands are severed. DSBs trigger wave of PTMs that regulate protein interactions, nuclear localization and catalytic activity of hundreds of proteins. Such modifications include acetylation, methylation, SUMOylation, ubiquitinylation and especially phosphorylation. The most important kinases involved in DDR kinases are ATM, ATR and DNA-PK. These kinases are activated immediately after the detection of the damaged area. DAXX (Death-associated protein 6) is an adapter and predominantly nuclear protein, which is involved in chromatin remodeling, gene expression modulation, antiviral response and depositing histone H3.3 variants into chromatin or telomeres. Daxx is essential for murine embryogenesis, since the homozygous deletion is lethal in E9.5-10. In 2006 a study mapping the substrates of kinases...
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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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Validity and inter-instrumental reliability of the model GT1M Actigraph accelerometer
Souček, Filip ; Psotta, Rudolf (advisor) ; Vodička, Pavel (referee) ; Buzek, Mario (referee)
Název práce: Validita a interistrumentálnf reliabilita akcelerometru Actigraph, model GT1M. Cíle práce: Ověřit validitu a interinstrumentálnf reliabilitu nového typu akcelerometru Actigraph, model GT1M v krátkodobé pohybové činnosti. Metoda: Práce se zaměřením na zkoumání : Validity Actigraphu GT1M: Pro odhad energetického výdeje - na základě analýzy vztahů sumy zrychlení (tzv. counts) na straně jedné a srdeční frekvence na straně druhé. Interinstrumentálnf spolehlivosti Actigraphu GT1M. Spolehlivost měření mezi 2 přístroji. Testovaným souborem je 12 hráčů kopané ve věku 7 a 1llet. Výsledky: Platnost odhadu energetického výdeje kritériem srdeční frekvence. Pro toto kritérium byl zjištěn korelační koeficient r = -0,751 až 0,883. Interinstrumentální spolehlivost 2 přístojů kritériem sumy zrychlení a kritériem sumy kroků. Korelační koeficient spolehlivosti sum zrychlení 2 přístrojů se pohyboval v rozmezí 0,984 až 0,997. Pro kritérium kroků byl zjištěn korelační koeficient 0,981 až 0,997. Klíčová slova: Akcelerometr - suma zrychlení - kroky - validita - reliabilita - srdeční frekvence - terénní podmínky
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