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Using PCR to study the DNA damage
Jansová, Adéla ; Jirkovská, Anna (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Bc. Adéla Jansová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: Using PCR to study the DNA damage Key words: functions of DNA, DNA damage, PCR, SINE elements The transmission of genetic information to future generations is possible thanks to DNA replication using enzymes, mainly DNA polymerase. The most important function of DNA is the biosynthesis of proteins that perform specific functions throughout the cell. The coding DNA sequences are the source for protein synthesis. These are produced by transcription of a DNA sequence using RNA polymerase and then translated into amino acids by translation. Non-coding sequences have mainly regulatory functions, they are functional DNA molecules (rRNA, tRNA, snRNA) and regulatory regions (promoters, enhancers and silencers) as well as transposons (SINE, LINE) and pseudogenes. DNA damage is caused by UV radiation, ionizing radiation, chemicals (cisplatin-based drugs, alkylating agents, etc.), reactive oxygen species, and DNA damage by base deamination. When damage occurs, repair processes are activated to remove mismatches, adducts and breaks. If the damage is not repaired by repair processes, the damage leads to mutation formation, senescence...
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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Novel Approaches in Electrochemical Determination of Xenobiotic Compounds and in Study of Their Interaction with DNA
Hájková, Andrea ; Vyskočil, Vlastimil (advisor) ; Trnková, Libuše (referee) ; Labuda, Ján (referee)
Presented Ph.D. Thesis is focused on the development of analytical methods applicable for determination of selected xenobiotic compounds and for monitoring DNA damage they can induce. The main attention has been paid to the development and testing of non-toxic electrode materials for preparation of miniaturized electrochemical devices and novel electrochemical DNA biosensors. 2-Aminofluoren-9-one (2-AFN) was selected as a model environmental pollutant, which belongs to the group of hazardous genotoxic substances. Its carcinogenic and mutagenic effects may represent a risk to living and working environment. 2-AFN has one oxo group, where the cathodic reduction occurs, and one amino group, where the anodic oxidation occurs. The voltammetric behavior of 2-AFN in the negative potential region was investigated at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) representing a non-toxic and more mechanically robust alternative to mercury electrodes. This working electrode was subsequently used for the development of a newly designed miniaturized electrode system (MES), which has many benefits as the possibility of simple field measurements, easy portability, and the measurement in sample volume 100 µL. Moreover, a glassy carbon electrode (GCE) was used for further investigation of...
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Mercury Electrodes as Tools for Voltammetric Determination of Biologically Active Organic Compounds and for Detection of Their Interaction with DNA
Horáková, Eva ; Vyskočil, Vlastimil (advisor) ; Ludvík, Jiří (referee) ; Vytřas, Karel (referee)
The main aim of this work was to use traditional mercury electrodes for the development of voltammetric methods of determination of organic xenobiotics and for the electrochemical study of the interaction between double-stranded deoxyribonucleic acid (DNA) and these compounds. In relation to my previous research work (conducted in the framework of my diploma thesis), firstly, 4-nitrobiphenyl (4-NBP), the suspected carcinogen, was studied. Interaction of DNA with 4-NBP was studied using differential pulse voltammetry (DPV), cyclic voltammetry (CV), and chronocoulometry at a hanging mercury drop electrode (HMDE), and using CV and alternating current voltammetry at a DNA modified HMDE. Using CV, the reduction mechanism was investigated. The interaction of DNA with 4-aminobiphenyl (4-ABP), a metabolite of 4-NBP, and 4-NBP reduction intermediates was studied. It was found that the interaction of DNA with 4-NBP or 4-ABP results in a formation of a DNA aggregate with these analytes. The second studied analyte was methyl violet 2B (MV). For determination of MV in a buffered solution were used: direct current tast polarography and differential pulse polarography at a dropping mercury electrode, and direct current voltammetry, DPV, and differential pulse adsorptive stripping voltammetry (DPAdSV) at HMDE. The...
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DNA-protein covalent complexes detection as the means for the assessment of the DNA damage induced by topoisomerase poisons.
Karešová, Aneta ; Jirkovská, Anna (advisor) ; Fikrová, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Aneta Karešová Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: DNA-protein covalent complexes detection as the means for the assessment of the DNA damage induced by topoisomerase poisons. Topoisomerase II is essential cellular enzyme, which modifies the secondary structure of DNA. By introducing a temporary double strand break to DNA it relieves a structural tension raised during transcription and translation. Absolutely indispensable is the role of topoisomerase II in the separation of sister chromatids synthesized in the S-phase of the cell cycle. The mechanism of DNA cleavage involves a covalent bond formed between active site tyrosine and 5' phosphate on both of the DNA strands and through the formed break the other strand or the other DNA molecule can pass. After that, the DNA strands are rejoined and topoisomerase II is detached. The indispensability of topoisomerase II mainly for proliferating cells makes it a great target for the antineoplastic drugs and the molecules belonging to the class of topoisomerase II inhibitors (etoposide, anthracyclines) are amongst the most useful anticancer drugs in the clinical practice. These clinically used "topoisomerase...
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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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Defects in DNA repair and RNA metabolism associated with human neurological disorders
Cihlářová, Zuzana ; Hanzlíková, Hana (advisor) ; Čermák, Lukáš (referee) ; Roithová, Adriana (referee)
The human genome is constantly under the attack by various damaging agents, leading to the breakage of one or both strands of DNA that might interfere with RNA processing. Importantly, our cells have evolved diverse mechanisms to rapidly repair various DNA lesions, highlighting the importance of genetic integrity. Defects in DNA repair and/or RNA metabolism can lead to a variety of human hereditary diseases, with pathologies including growth and developmental defects, immunodeficiency, predisposition to cancer, and neurodegeneration. Mutations in the BRAT1 (BRCA1-associated ATM activator-1) protein have been associated with neurological disorders characterized by heterogenous phenotypes with varying levels of clinical severity ranging from microcephaly, hypertonia, epilepsy, seizures, and early death in the first two years of life to mild cerebellar atrophy and ataxia. Previously, BRAT1 protein has been implicated in the cellular response to DNA double-strand breaks and ATM signalling. However, the exact mechanism/s by which mutations in BRAT1 gene trigger neurological disorders are largely unknown. Recently, we have identified a homozygous missense c.185T>A (p.Val62Glu) variant in BRAT1 that markedly reduced the level of BRAT1 protein in patient-derived cell lines. Surprisingly, our data show that...
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Mercury Electrodes as Tools for Voltammetric Determination of Biologically Active Organic Compounds and for Detection of Their Interaction with DNA
Horáková, Eva
The main aim of this work was to use traditional mercury electrodes for the development of voltammetric methods of determination of organic xenobiotics and for the electrochemical study of the interaction between double-stranded deoxyribonucleic acid (DNA) and these compounds. In relation to my previous research work (conducted in the framework of my diploma thesis), firstly, 4-nitrobiphenyl (4-NBP), the suspected carcinogen, was studied. Interaction of DNA with 4-NBP was studied using differential pulse voltammetry (DPV), cyclic voltammetry (CV), and chronocoulometry at a hanging mercury drop electrode (HMDE), and using CV and alternating current voltammetry at a DNA modified HMDE. Using CV, the reduction mechanism was investigated. The interaction of DNA with 4-aminobiphenyl (4-ABP), a metabolite of 4-NBP, and 4-NBP reduction intermediates was studied. It was found that the interaction of DNA with 4-NBP or 4-ABP results in a formation of a DNA aggregate with these analytes. The second studied analyte was methyl violet 2B (MV). For determination of MV in a buffered solution were used: direct current tast polarography and differential pulse polarography at a dropping mercury electrode, and direct current voltammetry, DPV, and differential pulse adsorptive stripping voltammetry (DPAdSV) at HMDE. The...
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