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The estimation of oxidative DNA damage using the single-cell gel electrophoresis (commet assay).
Škubalová, Věra ; Hochmann, Jiří (advisor) ; Slyšková, Jana (referee)
The oxidative cell damage represents one of the most common type of damage, which concerns all cell components. It produces the oxidative modifications of lipid structure, proteins and DNA. We concentrated on the last one in this work. The oxidative changes result in modification of replication cell cycle, which are represented for example by carcinogenesis, mutagenesis or by cell aging. Cells are provided by repairing mechanism. Owing to this mechanism the cells are able to repair this damage and stay viable. The oxidative damage is induced by various factors, for example free oxygen radicals or UV radiation, which we used for producing oxidative damage of DNA in this work. It is possible to measure the oxidative damage by using different methods, the most common way is using the comet assay. It represents an important method used for detection of single and doublestrand breaks, for detection of alcali-labile sites, searching for DNA- DNA/DNAprotein cross-linking, oxidative and alkylate damage of DNA. Only a little number of cells are needed for the measurement, which is the most important advantage of this method. We also used the modification of comet assay. It means the application of the reparative enzymes, which are able to recognize the specific oxidative damage of DNA. We used three...
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IL 57 - Sporadic colorectal cancer: From genetic make-up to complex phenotypic measurement, from risk determination to prognostic markers
Vodička, Pavel ; Slyšková, Jana ; Pardini, B. ; Naccarati, A. ; Souček, P. ; Vodičková, Ludmila ; Vymetálková, Veronika ; Svoboda, Miroslav ; Foersti, A. ; Hemminki, K.
Colorectal carcinogenesis (CRC), is a complex process, resulting in both genomic and chromosomal instabilities. The valid theories of carcinogenesis accent either the role of somatic mutation or the surrounding microenvironment, however neither of them explains all features of cancer. Uncontrolled proliferation and genomic instability point to the DNA damage response and repair as to the key players. In the present study, we will overview several biomarkers in mapping heterogeneous complex CRC disease and providing prognostic information.\nVariants in genes involved in important pathways, such as DNA repair, cell cycle control, folate metabolism and methylation, insulin resistance and obesity, ABC transporters, selenoprotein genes, genes involved in inflammatory/immune response have shown various degree of association with CRC risk. We present also the data on mutations in high risk genes involved in colorectal carcinogenesis. Gene expression levels were determined in relevant pathways and complemented with other important parameters (epigenetic regulators of transcription by methylation). Additionally, the role of post-transcriptional regulation via miRNA or lncRNA was investigated in relation to the risk of CRC and the efficacy of chemotherapy. We have discovered several genetic and epigenetic markers affecting independently the prognosis of CRC. Functional DNA repair tests (complex phenotype) have been implemented as markers of individual susceptibility to sporadic CRC and its prognosis.\nAn application of the whole set of various biomarkers is inevitable to define the phenotypic landscape of the disease and to delineate the individual response to the therapy.\n
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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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The estimation of oxidative DNA damage using the single-cell gel electrophoresis (commet assay).
Škubalová, Věra ; Hochmann, Jiří (advisor) ; Slyšková, Jana (referee)
The oxidative cell damage represents one of the most common type of damage, which concerns all cell components. It produces the oxidative modifications of lipid structure, proteins and DNA. We concentrated on the last one in this work. The oxidative changes result in modification of replication cell cycle, which are represented for example by carcinogenesis, mutagenesis or by cell aging. Cells are provided by repairing mechanism. Owing to this mechanism the cells are able to repair this damage and stay viable. The oxidative damage is induced by various factors, for example free oxygen radicals or UV radiation, which we used for producing oxidative damage of DNA in this work. It is possible to measure the oxidative damage by using different methods, the most common way is using the comet assay. It represents an important method used for detection of single and doublestrand breaks, for detection of alcali-labile sites, searching for DNA- DNA/DNAprotein cross-linking, oxidative and alkylate damage of DNA. Only a little number of cells are needed for the measurement, which is the most important advantage of this method. We also used the modification of comet assay. It means the application of the reparative enzymes, which are able to recognize the specific oxidative damage of DNA. We used three...
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