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Asymmetric biotransformation of certain tetralones to tetralols
Andrejch, Jan ; Poljaková, Jitka (advisor) ; Martínek, Václav (referee)
Preparation of pure enantiomers is very important part of chemical synthesis of industrially and pharmaceutically useful compounds. In recent years traditional chemical synthesis is replaced by biotransformations. Biotransformation means the use of isolated enzymes, enzymatic systems or whole cells in catalysis of chemical reactions. In comparison with chemical synthesis it has certain advantages. In this work the activity of yeast strains Saccharomyces uvarum K8, Saccharomyces cerevisiae K12, Torulopsis azyma K43, Torulopsis sphaerica K46 and Candida tropicalis K49, K51 a K52 in catalysis of reduction 6- bromo-2-tetralone, 7-hydroxy-2-tetralone and 7-methoxy-2-tetralone was studied. Products of these reductions are used in preparation of pharmaceutical drugs. Suitable conditions for biotransformations were found and it was proved that the yeast strains Saccharomyces uvarum K8 and Candida tropicalis K52 are able to produce 2- tetralones with ee greater than 90% with conversion 100% after four-hour biotransformation. This thesis is written in Czech.
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T cells labelling by bimodal contrast agent for in vivo studies of stroke
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Kříž, Jan (referee)
Stroke is a serious brain injury, which causes sudden death or terminates in permanent neurological disability. Nowadays, tissue plasminogen activator (tPA) is used as the only effective treatment of stroke. One of the potential targets for novel therapy are T cells. Even though the explicit role of T cells in the pathogenesis of brain injury, amounts and timing of all T cell subtypes infiltrating into brain during the stroke still needs further investigation. The research in this field is complicated by the lack of efficient methods for in vivo cell tracking. Therefore the aim of this thesis was to develop a method of T cells labelling by MRI contrast agent in order to investigate T cells distribution in ischemic mice model using in vivo MR imaging. T cells were isolated from C57/BL6 mice in two step isolation protocol using gradient centrifugation and magnetic separation with the efficiency of 97 %. The isolated cells were labelled with 100 μg Fe/mL of Molday ION Rhodamine B contrast agent. The labelling efficiency after 17 hours of cells incubation was higher than 99 %. The labelled cells were cultured with CD3 and CD28 antibodies resulting into the 74 % viability of labelled T cells compared to 83 % viability of non labelled T cells. The labelled T cells were visualized by fluorescent...
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Differences in histone acetylation in normoxia and hypoxia
Čepek, Pavel ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Histones and their N and C terminal tails undergo different covalent modifications that regulate gene transcription. Among these histone modifications are methylation, ubiquitinilation, SUMOylation, ADP- ribosylation, phosphorylation, proline izomerization, deimination and acetylation. Histone acetylation is regulated by histonacetyltransferases (HATs) and histondeacetylases (HDACs). The balance between acetylation/deacetylation influences chromatin condensation and thus regulates gene transcription. Acetylation balance is disrupted in many human cancers and this fact can contribute to the development of malignant diseases. Histondeacetylase inhibitors (HDACi) can restore this acetylation imbalance. One of these HDACi is valproic acid (VPA) which has been used in treatment of epilepsy for decades. VPA shows antitumour effect in many studies. Decreased expression of n-myc oncoprotein, inhibition of tumour growth and angiogenesis are one of these anticancer effects observed in neuroblastoma cell lines after treatment with VPA. Despite the fact that exact mechanism of antitumour effect of VPA remains unclear, one of the most important mechanisms is hyperacetylation of histone H3 and H4. It is shown in this work that VPA increases acetylation of histones H3 and H4 in human neuroblastoma cell lines...
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Phylogenetic analysis of Rieske dioxygenases large subunits genes in soil contaminated with jet fuel
Ptáček, Jakub ; Bořek Dohalská, Lucie (advisor) ; Poljaková, Jitka (referee)
The former military air-base Hradcany is among the most contaminated with organic pollutants localities in Czech Republic. Main cleanup strategy in the area is the bioremediation taking advantage on the natural potential of the autochthonous soil microorganisms to evolve catabolic pathways for in situ degradation of the pollutant. The diversity and abundance of the pathways, as well as the specificity and activity of the encoded enzymes are priority biotic factors determining the bioremediation efficiency. Main task of this work was to analyze the bacterial diversity in jet fuel contaminated soils based on key catabolic genes encoding the Rieske non-haem iron dioxygenases of the toluene/ biphenyl oxygenase branch. High molecular soil DNA was extracted and the sequences encoding catabolic genes were selectively enriched by hybridization to biotinylated oligonucleotides on magnetic microbeads with covalently bound streptavidin. Fragments of the genes for the -subunits of Rieske non-haem iron oxygenases were amplified and analyzed by restriction analysis, cloning and sequencing. Their evolutionary histories were inferred using the Neighbour-Joining and the maximum likelihood methods. The catabolic genes diversity in the actively bioremediated and highly polluted soil HRB was compared with the diversity in the...
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Antioxidant properties of medicinal plants
Tupec, Michal ; Hýsková, Veronika (advisor) ; Poljaková, Jitka (referee)
During the evolution plants have developed rather efficient mechanisms for the re- moval of harmful compounds, which include e. g. the reactive oxygen species (ROS). These mechanisms are mainly based on the antioxidant effects of ascorbic acid, gluta- thione in intimate cooperation with superoxide dismutase, catalase and various perox- idases - the reduced coenzymes, NADH and NADPH, play a major role in the ROS elimination, too. However, in recent times, the attention has turned from these key compounds of primary metabolism to secondary metabolites. Interestingly, the phenolic com- pounds together with some terpenes and alkaloids exhibit antioxidant effects, too, and apparently they play an irreplaceable role in the life of a plant - besides their antiox- idant action they are also important in the fight against pathogens and herbivores, in attracting the pollinators or in the moderation of UV light effects. Natural products are also studied in their relation to man - a broad variety of effects is known including the antioxidant, anti-inflammatory and anticancer effects for the most part. In this work, extracts comprising mainly of low-molecular-weight compounds were obtained from 32 plants with either hot water, 80 %w aqueous methanol or just by plain juicing. The extracts were then examined by three...
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Analysis of the pathways responsible for the resistance of leukemic cells towards L-asparaginase
Šimčíková, Markéta ; Konvalinka, Jan (advisor) ; Poljaková, Jitka (referee)
Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood. Despite the very successful ALL therapy, relapses occur to 15-20 % of children. One of the possible relaps causes is the resistance to therapeutics. ALL is treated with combined chemotherapy in which cytostatic agent L-asparaginase plays the essen- tial role. L-asparaginase depletes extracellular asparagine and glutamine. Antagonist of the L-asparaginase is asparagine synthetase enzyme, which synthesizes the cellular asparagine. The specific antileukaemic effect of L-asparaginase is believed to be thanks to lower activity of the asparagine synthetase in leukaemic cells comparing to the healthy cells. The asparagine and glutamine deficiency harms the cellular proteosyn- thesis and induces apoptosis. Mechanism of the L-asparaginase cytotoxic effect and mechanism of corresponding resistance is still not fully explained. This bachelor thesis is a part of a project studying mechanisms of leukaemic cells resistance to L-aparaginase. In the model leukaemic REH cell line a deletion del(5)(q34) was discovered, which cannot be found in the resistant clone of these cells. This thesis focuses on proving different sensitivity of leukaemic cells, with or without the deletion, to L-asparaginase. The limiting dilution was used to obtain...
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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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Effects of natural compounds on viability of tumor cell lines
Boleslavská, Barbora ; Poljaková, Jitka (advisor) ; Bořek Dohalská, Lucie (referee)
Cancer is considered to be one of the most serious issues of medicine nowadays. Moreover, its incidence is still rising. Despite the huge progress in modern treatment methods, cancer therapy is still limited by many difficulties. This work focuses on the natural substances such as epigallocatechin gallate, caffeine, Cannabis sativa ethanol extract, Origanum acutidens water extract, Mentha piperita water extract and its effects on the human neuroblastoma cell line UKF-NB-4. The first part of the bachelor thesis deals with determining the viability of human neuroblastoma cell line UKF-NB-4 exposed to tested substances as well as it studies the effects of those substances on the cell cycle and caspase activity. Finally, it was tested, whether those substances are able to induce apoptosis in neuroblastoma cell lines. Tests were undertaken on the MUSETM cell analyzer and on the flow cytometer. The second part of the bachelor thesis focuses on the expression of protein p53 and retinoblastoma protein in neuroblastoma cell lines exposed to tested substances. Detection was carried out by Western blot analysis. Epigallocatechin gallate exhibited the most significant effect on human neuroblastoma cell line. It lowers the expression of retinoblastoma protein as well as it arrests cells between G0/G1 and S...
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