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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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Použití spektroskopických technik pro studium interakcí platinových komplexů a nanočástic s nukleovými kyselinami =: Interactions study of nanoparticles and platinum complexes with nucleic acids assessed by spectroscopic techniques /
Nejdl, Lukáš
The ability of the metal ions to form a covalent bond with the nucleic acids (DNA or RNA) is critical for their structural properties and functions. In the 60s of the last century the potential of platinum complexes in anticancer therapy was revealed. The success of these complexes in anticancer treatment is given by their ability to bind to the DNA bases to form different types of coordination covalent bonds. The formation of these bonds results in an interference of the DNA secondary structure and thereby blocking of important cellular processes such as replication or transcription. Presented thesis examines the ability of metal and semimetal ions (Zn(III), As(III) and As(V)), platinum nanoparticles (PtNPs) and cadmium-based quantum dots (QDs-CdS) to influence the DNA secondary structure. In this work the interactions of metal ions with DNA were firstly investigated due to the implementation and verification of instrumental methods. Results of these studies served as the basis for subsequent experiments dealing with the effects of nanoparticles on eukaryotic cells with regard to DNA damage. In this work we demonstrated that PtNPs show higher affinity for DNA polymerases than to DNA. For this reason, PtNPs can arrest the cell cycle and trigger apoptosis. The affinity rate of nanoparticle binding to DNA is determined by its size, as was shown by the experiments with variously sized CdS-QDs.
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Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins
Klčová, Silvia ; Jirkovská, Anna (advisor) ; Suchá, Simona (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Silvia Klčová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins. Every single cell of the human body is continuously exposed to a wide range of stress factors, with the consequence of damage to the DNA molecule. The resulting changes represent variety of alterations - from simple alkylation modifications of bases to the most unfavorable double-strand breaks (DSBs). However, the effect of cellular stress and subsequent genotoxic DNA damage is a double-edged sword. On the one hand, typical alterations in the genome can be triggered by mutagenic agents (such as components of tobacco smoke or ionizing radiation). Consequences of their action can accumulate and trigger loss of control over various steps of the cell cycle, which results in tumor cell transformation. On the other hand, however, inducing detrimental impact affecting the genome of tumor cells is one of the fundamental approaches in cytostatic treatment of cancer. Therefore, we focused our research on several antineoplastic drugs widely used in clinical practice (etoposide, daunorubicin, dexrazoxane) or undergoing clinical...
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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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