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Study of the role of glutathione in the anthracycline-induced cardiomyocyte injury
Vávrová, Anna ; Šimůnek, Tomáš (advisor) ; Kvasničková, Eva (referee)
The clinical usefulness of anthracycline antineoplastic drugs has been limited by the risk of serious cardiotoxicity. Its precise mechanisms have not been fully elucidated, although the oxidative stress is widely believed to play a principal role. The anthracycline toxicity to heart may result from its high accumulation in myocardial tissue, richness on mitochondria producing reactive oxygen species and relatively poor antioxidant systems including superoxide dismutase, catalase and glutathione peroxidase. This study is focused on the role glutathione in daunorubicin (DAU)-induced cardiotoxicity both in vitro and in vivo. Total, reduced and oxidized glutathione (GSH/GSSG) have been determined by a spectrophotometric method using dithiobis(2-nitrobenzoic acid) in DAU (0.1 - 10 µM) - exposed H9c2 cardiomyoblast cell line and heart tissue from daunorubicin-treated rabbits (3mg/kg weekly, 10 weeks). In H9c2 cells, DAU induced significant toxicity (IC50=0.48 µM), levels of total glutathione were slightly but insignificantly decreased, the levels of oxidized glutathione were unafected. Neither buthionine sulfoximine (BSO, an inhibitor of glutathione synthesis) nor 2-oxo-4-thiazolidine-carboxylic acid (OTC, a glutathione synthetic precursor) had any significant effects of DAU cytotoxicity. This contrasted...
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Gene expression profiling after experimental perinatal asphyxia and the effect of complement-derived anaphylatoxin C3a
Šourková, Hana ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
Background: The complement system is involved in neuroprotection and brain repair after brain damage. To understand the molecular mechanisms of these processes, we performed gene expression profiling using quantitative real-time polymerase chain reaction (qPCR), which is the most accurate modern strategy for gene expression analysis. Project: Our project was directly aimed at expression profiling of selected genes potentially involved in loss and rescue of neural tissue during three weeks after hypoxic-ischemic brain injury, an experimental model of perinatal asphyxia. Recent experiments have shown that over-expression of C3a under the control of the GFAP promoter (C3a/GFAP) reduced hippocampal injury after left common carotid artery ligation in neonatal mice by 50%, compared to wild type mice. Here, we assessed how the local expression of C3a/GFAP transgene affects gene expression profiles. Gene expression was measured on samples from hippocampus ipsilateral and contralateral to the injury and ipsilateral part of cortex, taken at the time of injury, 6 and 24 hours; 3, 7 and 21 days after the injury. Results: Our data showed that the regulation of gene expression after hypoxic-ischemic injury differs in timing and intensity and may also be region dependent. The analysed genes belong to families...
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Study of the antipoliferative properties of the series of novel aroylhydrazone iron chelators
Kolbabová, Lucie ; Šimůnek, Tomáš (advisor) ; Jirkovská, Anna (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Kolbabová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of the antipoliferative properties of the series of novel aroylhydrazone iron chelators Aroylhydrazones are tridental lipophilic iron chelators with good cell-membrane permeability. In the last decades some compounds of this chemical group have been demonstrated to have a significant antiproliferative activity. Salicylaldehyde isonicotinoyl hydrazone (SIH), an aroylhydrazone with an oral availability, showed a significant antiproliferative activity in previous experiments in our laboratory as well as the ability to protect cells from the oxidative stress- induced damage. The main disadvantage of SIH is low stability caused by rapid hydrolysis of the hydrazone bond in aqueous environment. To deal with this issue, the Department of Organic and Inorganic Chemistry of the Faculty of Pharmacy (Charles University) designed and synthesized analogues of SIH, derived from aromatic ketones, which showed significantly improved stability in rabbit plasma in comparison with the parent SIH. The aim of this study is to expand the previous experiments and it is focused on further structure modifications of the...
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In vitro study of novel derivatives of iron chelator salicylaldehyde isonicotinoyl hydrazone.
Maťašová, Nikola ; Šimůnek, Tomáš (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Nikola Maťašová Supervisor: prof. PharmDr. Tomáš Šimůnek, Ph.D. Consultants: PharmDr. Hana Jansová, Ph.D. Mgr. Jan Kubeš Title of diploma thesis: In vitro study of novel derivatives of iron chelator salicylaldehyde isonicotinoyl hydrazone Heart disease is one of the most common causes of death and disability worldwide. Oxidative stress, which can also be generated or propagated by iron ions, plays an important role in the development of cardiovascular diseases. Excessive amounts of this metal lead to the apoptosis, necrosis, as well as recently described regulated cell death - ferroptosis. This type of cell death is associated with cardiomyocytes, but studies have also been described linking ferroptosis to other pathological conditions, such as cancer, nervous system disease, ischemia-reperfusion injury, kidney damage and blood disease. Therefore, research on iron chelators capable of forming a non-toxic complex with iron and thus preventing cell death is promising not only from the point of view of cardiovascular diseases. This work is focused on the in vitro study of new potential iron chelators derived from the structure of salicylaldehyde isonicotinoyl hydrazone (SIH). SIH selectively and...
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Cloning and expression of human carbonyl reductase CR-1
Laštovková, Linda ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
The purification of pOTB7 plasmid containing coding sequence of carbonyl reductase1 (CBR1) in cells of Escherichia coli (E. coli) was done by alkali hydrolysis. The sequence of CBR1 was multiplied by polymerase chain reaction (PCR) and synthesized primers with restriction sites were used. The left primer contained sequence for restrictive endonuclease NdeI and the right primer for restrictive endonuclease XhoI. Validation of the first step was confirmed by size measurement of the synthesized fragment with following restrictive analysis realized by restrictive endonuclease BamHI. Prepared sequence CBR1 was cloned into Topo vector, which was transformed into the competent E. coli cells. Topo vector was purified by alkali hydrolysis after innidation of E. coli cells. The size of cyclic Topo vector without CBR1 and Topo vector with CBR1 coding sequence was verified on agar gel by restrictive endonuclease BamHI. This was the validation of the second step. Subcloning of coding sequence CBR1 from Topo vector to express vector pET15b was the last step.
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Cloning, expression and purification of Francisella tularensis disulfide oxidoreductase
Kohlová, Michaela ; Šimůnek, Tomáš (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Kohlová Supervizor: doc. PharmDr. Tomáš Šimůnek Supervizor - konsultant: Mgr. Monika Schmidt Title of diploma thesis: Cloning, expression and purification of Francisella tularensis disulfide oxidoreductase Conserved hypothetical lipoprotein called FTT1103 (for Francisella tularensis subsp. tularensis) is one of the virulence factors of gramnegative intracellular bacteria Francisella tularensis. This protein is homologue to protein E. coli Dsba. Periplasmatic protein DsbA is member of disulfide oxidoreductase family and it is responsible for disulfide bond formation in newly secreted proteins. The disulfide bonds are essential for right conformation and activity in the proteins. Biotransformatic analysis of protein FTT1103 showed, that this protein contains N-terminal FKBP domain. The domain has probably dimerization activity and it is expected to have chaperone activity too. The aim of the presented diploma thesis was to verify, if the domain FKBP_N is really responsible for correct function and activity of DsbA protein. Using molecular biology methods we prepared mutant gene with deleted domain FKBP_N (dsbAΔFKBP_N). We cloned this gene into plasmidplazmid E. coli pET28b,...
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Genetic polymorphism in the HFE gene in the Czech population
Červinková, Barbora ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Barbora Červinková Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Title of diploma work: Genetic polymorphism of the HFE gene in Czech population Outline: The main objective of the thesis was to map the incidence of the three mutations (C282Y, H63D, S65C) in the HFE gene in the Czech population. Consequently, the obtained results were compared with the reported occurrence in the world. Methods: The work involved the isolation of DNA from buccal swabs obtained from 167 donors (65 men, 102 women; average age for men was 31, for women 28). Isolated DNA strings were amplified by PCR methods using 5'-CAG ATC CTC ATC TCA CTG-3' and 5'-CTG GAT AAC CTT GGC TGT ACC CCC-3' primers for C282Y mutation, 5'-GCC ACA TCT GGC TTG AAA TT-3' and 5'-ACA TGG TTA AGG CCT GTT GC GCC ACA- 3' primers for H63D and S65C mutations. DNA samples were treated with restriction enzymes Rsa I, Bcl I and Hinf I, for C282Y, H63D and S65C mutations, respectively. Finally, the restriction fragments were separated by gel electrophoresis (2 % agarose gel). Results: C282Y mutation was present in 0 (0 %) and 19 (11,38 %) samples as homozygote or heterozygote, respectively. Moreover, C282Y mutation was present in 3 (1,80 %)...
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Study of potential pharmacological protection of cardiac cells against oxidative stress and antracycline anticancer drugs
Jansová, Hana ; Šimůnek, Tomáš (advisor) ; Hrdina, Radomír (referee) ; Jun, Daniel (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ DEPARTMENT OF BIOCHEMICAL SCIENCES Candidate: Mgr. Hana Jansová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis: STUDY OF POTENTIAL PHARMACOLOGICAL PROTECTION OF CARDIAC CELLS AGAINST OXIDATIVE STRESS AND ANTRACYCLINE ANTICANCER DRUGS Development of cardiovascular disorders is associated with various risk factors and oxidative stress plays an important role in many of them. Iron-catalysed production of highly toxic and reactive hydroxyl radicals may contribute to oxidative stress. Chelation of free iron seems to be a promising strategy to prevent the propagation of oxidative stress. However, the use of classic iron chelators in pathological conditions without iron overload is associated with the risk of toxicity due to the iron depletion. Hence, this study deals with cardioprotective properties of iron chelators as well as prochelators derived from them. We focused on prochelators with almost no affinity for iron ions until they are activated under disease-specific oxidative stress conditions. For a long time, it has been assumed that oxidative stress is also the main denominator in an anthracycline-induced cardiotoxicity. However, the previous studies suggested alternative mechanism(s). Therefore in the...
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