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The role of V-ATPase in resistence to cytostatics and the possibility of its inhibition
Belhajová, Marie ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Cancers belong among the most serious problems of modern medicine and their occurrence is constantly increasing. Neuroblastoma is a malignant embryonal tumor in children, emerging from the peripheral nervous system and is the most frequent tumor in infants. Despite the significant development of therapeutic methods during recent years, this disease remains difficult to treat. It is treated surgically and also with chemotherapy using cytostatic drugs. The cytostatic drugs such as doxorubicin, ellipticine, cisplatin and vincristine have become very significant in treating cancer. However, they induced drug resistance in these neuroblastoma cells. This study investigates expression of the vacuolar H+ -ATPase (V-ATPase), in neuroblastoma cells and its role in the development of drug resistance. V-ATPase is a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. A sensitive neuroblastoma cell line (UKF-NB-4) and cells resistant to doxorubicin, ellipticine and cisplatin (UKF-NB-4DOX , UKF-NB-4ELLI , UKF-NB-4CDDP ) were exposed to these agents and the expression of the V-ATPase was studied by Western blot analysis and real-time quantitative reverse tarnscription polymerase chain reaction (RT-PCR). Ellipticine induces an increase in expression of the V-ATPase in neuroblastoma...
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Flavonoids and other biologically active compound in sea buckthorn
Michalíková, Adéla ; Hudeček, Jiří (advisor) ; Moserová, Michaela (referee)
This bachelor thesis summarizes information about sea buckthorn (Hippophae rhamnoides), about chemical composition of different parts and products based on this species, and about their biologically active contents. A special emphasis is given to the oil prepared from sea buckthorn fruits. Sea buckthorn is a rich source of antioxidants such as vitamin C, vitamin E, flavonoids and carotenoids. The oil contains high levels of unsaturated fatty acids, in particular of palmitooleic acid. The beneficial effects on human health make sea buckthorn and products from this plant popular food supplements. The second part of this thesis describes analytical methods used for determination of bioactive compounds in sea buckthorn.The protocols based on high performance liquid chromatography, gas chromatography or capillary electrophoresis are described in more detail. In the experimental part, four samples of sea buckthorn oil (two from Czech and two from Mongolian producers) were compared using absorption spectrophotometry and colorimetric evaluation using the CIELab method. Both samples of Czech origin displayed somewhat shifted absorption maxima and much weaker absorption, their coloration being much weaker. This may suggest some adulteration of these products Keywords: Sea buckthorn, Hippophae rhamnoides,...
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Study of effect of dihydromyricetine on cytochrome P450 1A1 expression in organs of the rat
Feriančiková, Barbara ; Moserová, Michaela (advisor) ; Dračínská, Helena (referee)
Polyphenols are one of the largest groups of substances in the plant kingdom. They are widely represented in fruits, vegetables and many beverages. They are included up to 80% of higher plants. Moreover, there is an increasing interest to know of their metabolism. Polyphenols are considered as substances with generally positive effects. However, it is necessary to look at them as xenobiotics. However, it is necessary to regard these compounds as xenobiotics. Polyphenols are able to interact with key phase I biotransformation enzymes such as cytochrome P450. In the present work we studied the influence of dihydromyricetine on induction of CYP1A1 in the body of the rat, particularly in the organs involved in the metabolism of xenobiotics, such as liver and small intestine. The small intestine was divided into three sections - a proximal, middle and distal. The evaluation was performed by real-time polymerase chain reaction. It was found dihydromyricetine did not significantly affect gene expression of CYP1A1 in liver. On the other hand, the change of gene expression on mRNA level was observed in small intestine; the significant increase was found in the proximal part, and small decrease in the middle part. Key words: dihydromyricetine, cytochrome P450 1A1, mRNA, RT-PCR
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Phytohormones produced by Leptosphaeria maculans as effectors manipulating plant signaling pathways
Krutinová, Hana ; Ryšlavá, Helena (advisor) ; Moserová, Michaela (referee)
Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus. In this thesis the ability of L. maculans to produce certain phytohormones was established. The thesis focused on auxins and brassinosteroids. Most commonly occurring natural auxin, IAA, and its inactive oxidation product, OxIAA, were found in highest concentration. In plants infected with L. maculans the concentration of OxIAA was higher when compared to control water- treated plants. A surprising difference in IAA production between two sister isolates of L. maculans was discovered. In chemically defined cultivation medium Gamborg JN2 isolate did not produce any IAA. JN3 on the other hand produced IAA in concentration around 1000 pmol/g FW. This difference was used for studying L. maculans putative auxin synthesizing genes. The candidate genes were identified as orthologs of Arabidopsis thaliana genes (YUCCA1.1, NIT1.2) and Ustilago maydis (IAD1.2, TAM1-2.1). An increased transcription level of the auxin biosynthesis candidate genes was observed in JN2 treated in vitro with auxin precursors (tryptophan, tryptamine). Increased IAA concentration was observed as well. No such effect was observed in JN3. Surprisingly, an increased candidate gene transcription and IAA concentration was observed also in JN2 treated with...
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Preparation of glycosylated form of human immunoreceptor NKp30
Kalousková, Barbora ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
NK cells (natural killer cells) play a key role in innate immunity. Their function is to recognize and kill infected, stressed or malignantly transformed cells. A range of surface receptors promotes this recognition. Cytotoxic mechanisms, lead to induction of apoptosis in the target cell. Receptor NKp30 is one of cytotoxic reaction triggers. It belongs, with NKp46 and NKp44, to NCR (natural cytotoxicity receptors) family. This work describes preparation of NKp30 receptor with natural and simple glycosylation in expression system of human embryonic kidney cell line 293 (HEK293). It was found that glycosylated receptor NKp30 forms noncovalent oligomers. Equilibrium is formed in solution between oligomers and monomers. Oligomerization depends on glycosylation, deglycosylated protein doesn't form oligomers. A recombinant endoglycosidase ENDO F1 was prepared for purposes of deglycosylation.
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Study of effect of carcinogenic benzo[a]pyrene on cytochromes P450 and cytochrome b5 expression in organs of the rat
Blecha, Tomáš ; Moserová, Michaela (advisor) ; Dračínská, Helena (referee)
Polycyclic aromatic hydrocarbons are a large group of organic compounds which consist solely of carbon and hydrogen atoms, two or more fused aromatic rings. They represent group of persistent organic pollutants (POPs) present in all components and fields of the environment. Benzo[a]pyrene is characteristic compound of polycyclic aromatic hydrocarbons formed by five fused aromatic rings. Benzo[a]pyrene is procarcinogen with genotoxic effects, which is metabolically activated by variety of enzyme systems such as cytochrome P450 and epoxide hydrolase to reactive metabolite of BaP-7,8-diol-9,10-epoxide (BPDE) and 9-hydroxy-4,5-epoxy-BaP. These reactive metabolites can form covalent DNA adducts. In the present work, we studied the influence of carcinogenic benzo[a]pyrene on the expression of cytochrome P450 (CYP) 1A1, 1A2 and cytochrome b5 in livers, kidneys and lungs of laboratory rats. Total RNA was isolated and afterwards converted into cDNA with the participation of random primers. Using polymerase chain reaction in real time (RT-PCR) the relatively gene expression of CYP1A1, CYP1A2 and cytochrome b5 was quantified in the organs of rats treated with BaP and control (untreated) animals to a reference gene (β-actin). It was found that benzo[a]pyrene significantly increases expression of CYP1A1 and...
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Heterologous expression and isolation of human cytochrome P450 1B1
Sojka, Pavel ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochromes P450 are heme enzymes with very broad substrate specificity, they can oxidize tens to hundreds of different substrates including both eobiotics and xenobiotics, but with varying efficiencies and kinetics. They are responsible for the metabolic activation of many carcinogens resulting in formation of reactive intermediates, these reactive species participate in the formation of DNA adducts and also increase of oxidative stress. Eukaryotic cytochromes P450 are membrane bound enzymes found mostly in the endoplasmic reticulum. In vertebrates, they are expressed in a variety of tissues mostly in the liver, but also in kidney, lung, skin and others. The cytochrome P450 1B1 is an inducible enzyme which occurs mainly in the lung and skin. Its expression is induced predominantly by the presence of polycyclic aromatic hydrocarbons, dioxins and heterocyclic amines. The human cytochromes P450 are typically obtained using heterologous expression and isolated as a C-terminally modified hexa-histaq constructs using immobilized metal affinity chromatography. This thesis focuses on effect of C-terminal modifications on activity of human cytochrome P450 1B1. First, the two expression vectors encoding the human form of cytochrome P450 1B1 were prepared, one contained a classical C-terminal hexa-histaq...
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Preparation of the human NK cell receptor KACL
Nový, Jiří ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
NK buňky neboli přirození buněční zabíječi jsou důležitou součástí imunitního systému organismů. Jejich specifickou vlastností je to, že dokážou rozeznat a zneškodnit některé nádorové buňky a buňky infikované virem bez jakéhokoliv předchozího signálu. Jejich funkce je závislá na aktivitě povrchových stimulačních a inhibičních receptorů, které se aktivují interakcí s ligandy na povrchu cílových buněk. Interakce lidského NK receptoru NKp65 a ligandu KACL je velmi specifická, což potvrzuje velmi vysoká afinita interakce, která je oproti jiným příbuzným komplexům až 400× vyšší. O tomto komplexu je zatím známo jen málo informací, ale je jisté, že se účastní imunitních procesů v kůži, neboť KACL je produkován výhradně keranocyty. Náplní této bakalářské práce byla příprava rozpustné formy receptoru KACL za pomoci rekombinantní exprese proteinů v lidských embryonálních ledvinných buňkách s homogenní glykosylací (linie HEK293S GnTI- ). Protein byl následně charakterizován gelovou chromatografií a SDS elektroforézou. Správné zapojení čtyř cysteinů do dvou disulfidických můstků bylo ověřeno hmotnostní spektrometrií. S připraveným proteinem KACL bylo zahájeno strukturní studium metodou krystalizace proteinů.
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Role of benzo[a]pyrene in cancer development
Vaňátková, Petra ; Moserová, Michaela (advisor) ; Kubíčková, Božena (referee)
4 ABSTRACT Cancer is nowadays one of the most serious diseases. Tumor development is a multistage process in which the effect of internal and external factors lead to failure of regulatory and defense mechanisms of the organism and to the accumulation of mutations which are generated by these organisms. Chemical carcinogens and also biological and physical factors can be regarded as the main external factors. Polycyclic aromatic hydrocarbons are large group of chemical carcinogens. One of them, benzo[a]pyren is the most studied polycyclic aromatic hydrocarbon. Carcinogenic, mutagenic and teratogenic effects of benzo[a]pyrene had been shown on laboratory animals. Benzo[a]pyrene is considered as the main carcinogen in tobacco smoke and is connected with lung cancer development among smokers. Benzo[a]pyrene is metabolized in activation or detoxication pathways by enzymes of mixed function monooxygenase systeme of cytochromes P450. The most important enzymes involved in the activation of these compounds are CYP1A1 and CYP1B1 with cooperation of epoxide hydrolase. The reactive species generated in its activation pathway are able to form covalent adducts with DNA. The most important carcinogenic product of benzo[a]pyrene is benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which can caused irreversible ganges in...
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