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Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes
Mizerovská, Jana ; Stiborová, Marie (advisor) ; Mareš, Jaroslav (referee) ; Hýsková, Veronika (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY Study on metabolism of 3-aminobenzanthrone and induction of biotransformation enzymes Summary of PhD Thesis RNDr. Jana Mizerovská Supervisor: Prof. RNDr. Marie Stiborová, DrSc. Prague 2010 RNDr. Jana Mizerovská Introduction 1 INTRODUCTION 3-Nitrobenzanthron, precursor of 3-ABA The nitroaromatic 3-nitrobenzanthrone (3-nitro-7H-benz de anthracen-7-one, 3-NBA) occurs in diesel exhaust and in airborne particulate matter(8, 9, 14) . 3-NBA is most likely formed during the atmospheric reaction of benzanthrone with nitrogen oxides, especially in the presence of ozone, or during imperfect burning of diesel. 3-NBA exhibits extremely high mutagenic activity(9, 14) and is also a genotoxic carcinogen causing lung tumors in rats(14) . 3-NBA is also evaluated to be a potential carcinogen for humans(14, 1, 9, 19) . The genotoxicity of 3-NBA was documented by the detection of specific 3-NBA-derived DNA adducts in vitro, in human cell lines and also in vivo in rats and mice(12, 13, 15, 2) . The predominant DNA adducts formed by 3-NBA after its metabolic activation by reduction of the nitro group are 2-(2'-deoxyguanosin-N2 -yl)-3- aminobenzanthrone and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone(9, 14) and these are most probably responsible for the...
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Study of tyrosin kinase inhibitor vandetanibe bound in apoferritin and liposomes
Jáklová, Kateřina ; Indra, Radek (advisor) ; Hýsková, Veronika (referee)
5 Abstract In this thesis the anticancer drug vandetanib was studied. Vandetanib is a tyrosine kinase inhibitor affecting signalling of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) or RET protooncogene (REarranged during Transfection). It is primarily used for the treatment of advanced tumors of the thyroid gland. Unfortunately, the usage of vandetanib in the cancer treatment is significantly limited by its toxicity and cardiotoxicity (one of the adverse effects is connected with long QT interval). One way, how to minimize these side effects, is binding a drug into a suitable transporter. Apoferritin and liposomes were used as a transport nanoparticles in this study. The aim of this thesis was to study the stability of the complex of nanoparticle apoferritin with vandetanib molecules (ApoVan) and to study the effect of pH on the release of inhibitor from the ApoVan form. Experiments have shown that ApoVan complex is relatively stable after its storage at 4 řC and - 20 řC for up to 8 weeks. Unfortunately after monitoring the effect of pH on the release of vandetanib from ApoVan, it was found that vandetanib is gradually released from its ApoVan form into the neutral environment at pH 7,4 as well as into the acidic environment at pH 6,5 and the way ApoVan is...
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Biochemical characterization of tomato plants infected with fungal pathogens
Oušková, Marie ; Hýsková, Veronika (advisor) ; Kubíčková, Božena (referee)
Fungal pathogens including Verticillium longisporum which causes verticillium wilt are among the serious diseases of crops that easily spread worldwide. One of the ways to prevent fungal infection is to use fungal biocontrol agents applied as a seed coating. This control agent, the non-pathogenic oomycete Pythium oligandrum, enters the soil together with the seed and acts symbiotically in the plant's root system. On the one hand, it stimulates the plant's defence mechanism by secreting elicitors, and on the other hand, by providing tryptamine, it stimulates growth and increases the plant's fitness with auxin. In this work we studied the effect of seed treatment of Solanum lycopersicum L. cv. Micro-Tom with three different isolates of the genus Pythium (including the commercially used isolate M1 and two yet unused isolates X42 and X48) on the activities of antioxidant and NADP(H)-dependent enzymes in the leaves of plants infected with fungal pathogen V. longisporum. Two weeks after pathogen inoculation, no significant difference was found in the studied enzymes except for increased glucose-6phosphate dehydrogenase and NADP-malate dehydrogenase (oxaloacetate decarboxylation) activity in plants treated with X42 isolate and increased shikimate dehydrogenase activity and antioxidant capacity in plants...
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Nanoparticle forms of anticancer drugs and the mechanisms influencing their efficiency
Urbanová, Tereza ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Currently, cancer is one of the major diseases of civilization. The disadvantage of conventional chemotherapy, which began in the 1940s, is its non-specific effect, so the cytostatics are toxic to healthy cells. However, if the cytostatic is inserted into a nanotransporter, it increases its specific efficacy and reduces the negative side effects. One of the possible nanotransporters is protein called apoferritin (a protein component of ferritin, an iron-carrying protein) that contains light and heavy subunits differing in their function in iron uptake. In this bachelor thesis, the ability of apoferritin to encapsulate two cytostatics (ellipticine and doxorubicin), depending on its origin and the proportion of light and heavy apoferritin subunits, was studied.
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The inhibitory effect of estrogenic endocrine disruptors on cytochrome P450 activity
Otáhalová, Barbora ; Dračínská, Helena (advisor) ; Hýsková, Veronika (referee)
Endocrine disruptors are exogenous and endogenous compounds that interfere with the production, signaling and metabolism of natural hormones, thereby disturbing the balance of the endocrine system. Exogenous endocrine disruptors include 17α-ethinylestradiol and endogenous endocrine disruptors include 17β-estradiol. This thesis examinates effects of these endocrine disruptors on the specific activities of rat cytochromes P450 1A1 and 3A1. The enzyme specific activity of CYP1A1 is determined by the marker reaction O-deethylation 7-ethoxyresorufin and the activity of CYP3A1 is determined by the marker reaction 6β-hydroxylation of testosterone. It has been confirmed that both estrogens 17β-estradiol and 17α-ethinylestradiol inhibit activity of CYP1A1 and CYP3A1. The stronger inhibitor of CYP1A1 was 17β-estradiol and of CYP3A1 was 17α-ethinylestradiol. The concentration of estrogens causing 50% inhibition (IC50) of enzymes has been determined, for 17β-estradiol it was 4,6 μM and for 17α-ethinylestradiol 7,9 μM. CYP1A1 is a important enzyme for the biotransformation of carcinogens, it can be said that estrogens can modulate the genotoxicity of CYP1A1 activated carcinogens. The results show that CYP1A1 inhibition increased after pre-incubation of estrogens with NADPH (cofactor of cytochromes P450), that...
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Detection of Hsp70 protein in plants exposed to various stress factors.
Lengálová, Alžběta ; Hýsková, Veronika (advisor) ; Liberda, Jiří (referee)
! Plants are continuously exposed to various stress conditions. Being sessile, they are not able to escape from adverse conditions. Therefore, they have developed specific defence mechanisms. Most studies focus on plant responses to a single type of stress. However, plants in nature must cope with a variety of stresses at the same time. In this work, the effects of heat shock on the interaction of tabaco (Nicotiana tabacum L.) with the Potato virus Y (PVY) were investigated. Obviously, heat stress is associated with the synthesis of Hsp70 protein, which has many important functions alleviating adverse effects of stress conditions (e.g. Hsp70 participates in refolding or degradation of damaged proteins and protein syntetis de novo). The effect of Hsp70 during viral infection is still not fully understood, some studies revealed Hsp70 as a part of viral multiplication and transport processes in plant. In the first experiment performed in this work, higher levels of PVYNTN virus was found in tobacco plants that have been exposed to heat shock after inoculation than in plants only infected. The amount of the virus corresponded with the amount of Hsp70 protein detected immunochemically using a specific antibody. It seems that the plant response to combination of heat stress with viral infection is a...
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Enzyme kinetics and regulation of shikimate dehydrogenase from parsley roots.
Šmeringaiová, Ingrida ; Hýsková, Veronika (advisor) ; Kalendová, Alžběta (referee)
In plants the shikimate pathway followed by the phenylpropanoid pathway leads to the formation of not only aromatic amino acids but also a plenty of secondary metabolites. The crucial enzyme shikimate dehydrogenase is there a part of bi-functional protein 3-dehydroquinate dehy- dratase/shikimate dehydrogenase (DHD/SDH; EC 4.2.1.10 and EC 1.1.1.25). Whereas the regulation of the shikimic pathway is complex, little is known about the feedback regulation of plant SDH. The aim of this project was to find plant source with high SDH activity and to find out, if and in which way this SDH is regulated by compounds of phenylpropanoid metabolism, specifically by simple polyphenols. SDH from parsley root (Petroselinum crispum) has been prepared by 3-step purification to final specific activity 470 mol.min-1 .mg-1 . The enzyme exhibited one band after both isoelectric focusing and red native electrophoresis using detection of the activity. Relative molecular mass of native enzyme was determined by gel chromatography (Mr 60 000) and red native electrophoresis (Mr 63 000). Isoelectric point pI 4.5 was established by isoelectric focusing. Optimal pH for the reaction catalyzed by SDH was determined in rage pH 9.5 - 10.0. The kinetic parameters (Km, Vmax) of SDH for substrate shikimate, and coenzyme NADP+ were...
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The effect of Hsp70 protein on the infection caused by Potato virus Y
Doričová, Vlasta ; Hýsková, Veronika (advisor) ; Hoffmeisterová, Hana (referee)
Whithin their natural environment, plants are subjected to a combination of stress conditions. Since potential interactions between signal pathways, plants respond to multiple stresses differently from how they do to individual stresses, activating a specific programme. Heat shock proteins (HSP70) overexpressed after heat shock influence the viral infection. On one side HSP70 can participate on refolding of aggregated or partially denaturated proteins, on the other side HSP70 can interact with viral proteins and facilitate propagation of viral replication complexes. In this work the effect of heat shock (42řC, 2. hours) applied before or after the inoculation of plants Nicotiana tabacum L. cv. Petit Havana SR1 with Potato virus Y on viral infection was detected. This effect was studied in two biological experiments. The amount of coat protein of PVYNTN and protein HSP70 were detected simultaneously with the activity assays of Hatch-Slack cycle enzymes, glycosidases and peroxidase. Both experimental approaches (heat shock applied before or after the inoculation by PVYNTN ) enhanced amount of the virus and in the 2nd experiment it accelerated infection development. Immediately after application of heat shock the amount of HSP70 was increased. The enhancement of HSP70 by viral infection occurred...
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