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The repair of the oxidative DNA damage and its relationship to the cytotoxic effect of the oxidants.
Havlínová, Alena ; Herink, Josef (advisor) ; Hochmann, Jiří (referee)
H2O2 is a strong oxidant and therefore affects all cell structures. Especially the oxidative damage of DNA is known as being very severe. It is considered to be contributing cause of carcinogenesis, mutagenesis and aging. Our work is focused on the relationship between cytotoxic effect of H2O2 and the repair of oxidative DNA damage caused by H2O2. We also studied the effects of epigallocatechin gallate (EGCG). Several studies have already proven its protective activity on DNA in particular concentrations. In our experiments we used the test of cytotoxicity to demonstrate the colony forming inhibition and the comet assay test which measures the number of single strand breaks (SSB) in 109 daltons of DNA. Generally we used cells of line AA8 and in one experiment also NER deficient cells of line UV-20. We found out that EGCG in concentrations of 12,5 and 25 μg/ml significantly protects the cells against cytotoxic effects of H2O2. When we followed the repair of the oxidative DNA damage, we have found that EGCG did not affect the repair of direct DNA breaks, but the repair of oxidised pyrimidines and purines was accelerated. And this acceleration is probably largely responsible for the protection against cytotoxic effect of H2O2. However, the EGCG function of free radical scavenger and iron ions chelator...
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The oxidative DNA damage and its relationship to the cytotoxic effect.
Blahová, Hana ; Hochmann, Jiří (advisor) ; Polívková, Zdeňka (referee)
This work has been focused on the study of oxidative DNA damage induced with hydrogen peroxide and on the relationship of this damage to cytotoxic effect . The repair of the oxidative DNA damage and the cell survival was followed both in normal Chinese hamster cells and their mutant derivative UV-20. The modified single cell gel electrophoresis (the comet assay,) was used to estimate the DNA damage. The cytotoxiciy was measured using the inhibition of colony forming capacity of cells. Both normal AA8 and mutant UV-20 cells repair the oxidative DNA damage with approximately the same velocity, and they show also the same sensitivity towards the hydrogen peroxide. These results indicate, that the defect in the nucleotide excision DNA repair in UV-20 cells does not play any important role in the response of these cells to the oxidative DNA damage, and therefore this mechanism is not involved in the removal of this damage. This may indicate, that the main mechanism of the repair of oxidative DNA damage is the base excision repair. Keywords: Oxidative DNA damage, hydrogen peroxide, comet assay, excision repair.
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The repair of the oxidative DNA damage in Chinese hamster cells deficient in nucleotide excision DNA repair.
Strejčková, Lada ; Hochmann, Jiří (advisor) ; Polívková, Zdeňka (referee)
Nucleotide excision repair (NER) is one of the mechanisms of how to repair DNA damaged by the external influences. First of all, it plays the role when removing the results caused by UV radiation. The damaged bases in the form of pyrimidine dimers are enzymatically splitted out from DNA as part of oligonucleotides. The effect of the UV light on DNA is however manifested also indirectly, by oxidation of the bases. In our experiment we tried to find out whether the cells deficient in nucleotide excision repair are able to repair the damages induced only by oxidative agent. We stated the extent of DNA damages in the cells in vitro and the ability of their re- paration after treatment with the dilution of hydrogen peroxide in different concentrations. During the experiments we used the cell lines of a Chinese hamster, namely the parental cell line AA8 and their derivative UV-20, which is defective in gene ERCC1. This gene takes part in creation of nucleases participating in NER. For measuring of DNA lesions we used the method of alkaline comet assay, the ba- sis of which is alkaline single-cell gel electrophoresis. The principle of this method consists in the detection of DNA strand breaks originating in the damaged alkali-labile sites. We evaluated the number of DNA single-strand breaks. We detected...
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Inaktivace ICAM-1 genu metodou oligonukleotidů modifikovaných lysinem
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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The repair of DNA damage induced with suphur mustard and its relationship to the cytotoxicity
Jílková, Martina ; Vopršalová, Marie (advisor) ; Herink, Josef (referee)
The aim of this study was to investigate the induction and repair mechanisms of DNA damage caused by sulphur mustard in specific cell lines and their relationship to cytotoxicity of sulphur mustard. Sulphur mustard is a chemical warfare agent of the blistering agent category, which can be misused in local conflicts, terrorist attacks and during liquidation of its storage. Sulphur mustard is an alkylating agent, which interacts with a wide range of cellular macromolecules including DNA, RNA and proteins. Sulphur mustard forms single - strand breaks, monofunctional guanine and adenine adducts, as well as interstrand cross-links involving the two guanines in interaction with DNA. This study is aimed at cross-links. We used the Single Cell Gel Electrophoresis (SCGE, comet assay) for their detection. It is a method of evaluation of single cells ambeded in agarose. It is used for detection of DNA single strand breaks in a single cell. In our modification of the method we determined cross-links, which make the alkaline DNA unwinding impossible. That is why we had to induce a standard number of single strand breaks in DNA by styreneoxide (at a certain concentration and exposure time) before the comet assay. We studied DNA repair mechanisms of specific DNA lesions using specific cell lines with clearly...
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Inheritance of coat color in horses
Krejčová, Lucie ; Sedláková, Vladimíra (advisor) ; Hofmanová, Barbora (referee)
Genetic side of horse´s coloring is an interesting topic, which has been a subject of many researches for a long time. The DNA testing has shown that the original horses were brown or buckskin and due to changes in environmental conditions also black. During the domestication circulating more colors, quickly began, helped by human selection. The color of the coat is a quality feature and is subjected to Mendel's rules, for each color of the horses are therefore responsible genes of large effect. To most of the color is already assigned to a specific gene mutation and it is identified that the gene can find and change the phenotype. An exception is the color white, roan and tobiano, known only, that are associated with the KIT gene, dun color and leopard are currently only assigned to chromosomes ECA8 and ECA1. Mutations on genes are of different character, in the MC1R gene, MATP, PMEL17 and SLC36A1 is a change of a single base pair in a gene EDNRB is a dinucleotide mutation. ASIP gene mutation is caused by deletion of 11 nucleotide pairs and on the other hand is caused by mutations STX17 duplication 4.6 kilobytes Knowledge of gene structure and mutations is necessary for testing the color of horses at the molecular level. DNA testing is still in development, but currently it is already possible to test horses to most of the colors, which is important in the accurate identification of colors, but also in preventing the development of diseases which are associated with some genotypes. The aim of this work is to organize and unify information on various horse´s colours and the manner of their inheritance, due to the increasing interest of the people and farmers on this issue.
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Histone code and its regulation during early embryonic development in pigs
Jelínková, Pavla ; Žalmanová, Tereza (advisor) ; Miriama, Miriama (referee)
Both pronuclei of the zygote undergo epigenetic changes after fertilization, which determines the quality of the zygote and successful early mammalian embryonic development. Shortly after fertilization epigenetic asymmetry among the pronuclei of the zygote is evident, while the paternal pronucleus undergoes active DNA demethylation, the DNA of the maternal pronucleus remains methylated. The male pronucleus in addition undergoes histone acetylation, whereas the histones of the female pronucleus remain methylated. Asymmetry of pronuclei and their epigenetic status predicts successful reprogramming of the genome, and thus the success of embryonic development. For the successful development of the embryo is therefore required correct formation of both of these pronuclei of the zygote and this formation of pronuclei is regulated by post-translational histone modifications called histone code. It was hypothesized that the histone code is regulated by the activity of NADP+ - dependent histone deacetylases, sirtuins.
In the experiment were used fully grown in vitro maturated pig oocytes that were fertilized with pig spermatozoa in vitro. After isolation of zygotes cultured with addition of the activator sirtuin resveratrol was performed immunofluorescence analysis of acetylated and methylated histone H3 at lysine K9 of pronuclei of the zygotes. From the results of control group asymmetry between the pronuclei of the zygote is evident; wherein the male pronucleus exhibits higher acetylation intensity contrast female pronucleus exhibits higher methylation intensity. After adding resveratrol to all experimental groups female pronucleus showed a significant increase of the methylated histone H3 at lysine K9, and contrary to the male pronucleus significant decrease of acetylated histone H3 at lysine K9.
Sirtuins are involved in the regulation of histone code in porcine zygote and it can be assumed that they also play a role during subsequent embryonic development, which is the subject of further study.
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Molecular dynamics simulations of complexes consisting of proteins and nucleic acids
Hammer, Jiří ; Barvík, Ivan (advisor) ; Obšil, Tomáš (referee)
The goal of this diploma thesis was to study interactions of Argonaute (Ago) protein in a complex with nucleic acids. Based on the available crystal structures of full length Argonaute (from A. aeolicus, Aa-Ago) and/or its domains (human PAZ domain, Hs-PAZ), twelve different simulations were computed. Two initial simulations used model of Aa-Ago with either a duplex of DNA/RNA or RNA/RNA. Major difference was in behavior of the PAZ domain (especially its arginine residues), which tolerated the guide DNA in one simulation, but was disturbing the RNA guide strand in the second. Such an interaction could serve as a mechanism of the substrate recognition. In additional simulations (3-9) employing the Hs-PAZ domain, where no disturbance was found in the DNA/RNA hetero-duplex. Different arrangements of the active site geometry as well as empirical parameterizations of Mg2+ ion were probed and analyzed. The DD-catalytic motif plus D683 in Aa-Ago (equivalent to H807 in human Argonaute2) was observed to coordinate the Mg2+ ion in one and two metal ion dependent catalysis models. Highly conserved R570 and E578 created mutual hydrogen bonds and hence stabilized the active site. To make the cleavage irreversible, a role for the first (unpaired) nucleotide from 5'-end of the guide strand was suggested. It lies in a...
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DNA Extraction of Sweet Cherry (Prunus avium L.)
Hřebcová, Kateřina ; Sedlák, Petr (advisor) ; Korecký, Jiří (referee)
Isolation of high quality DNA in satisfactory yield and purity is a fundamental and essential step for all molecular-biological studies and analyses. The process of its extraction can be complicated by many of materials like are polyphenols, polysaccharides, proteins and other metabolites that can be co-isolated with nucleic acids and can act as inhibitors of PCR and cause deterioration of samples for further analyses.
In this thesis, mostly used methods of plant DNA isolation were mapped, and, in experimental part, results, regarded to the yield and purity, of selected plant DNA isolation methods were compared. DNA was obtained from various tissues of Prunus avium L. species, namely from fresh leaves, buds and from frozen embryos of several varieties. Comparison of the two commercial isolation kits (DNeasy Plant Mini Kit by Qiagen and GeneEluteTM Plant Genomic DNA Miniprep Kit, Sigma Aldrich) was the original intention. The first of the kits was replaced by simple and quick DEP-25 DNA Extraction Kit, Top-Bio and the experiment was extended with CTAB DNA isolation protocol, both with and without application of RNase into the protocol.
The results obtained proved quite significant differences between the methods used, both in yield and purity. The original assumption, supported by several studies, that commercial kits not always gain relevant results, regarded to ability to provide pure DNA, was not accurately proven, the assumption that the CTAB protocol can gain satisfactory results according to the DNA yield and purity was proved only with some tissues. The results of the spectrophotometry were supported with polymerase chain reaction (PCR) analyses conducted with the isolated DNA samples and after statistical evaluation were discussed.
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