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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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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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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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Insulin analogues with A-chain extended by the D-domain of IGF-1 and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Insulin and insulin-like growth factors (IGF-1 and -2) together with their receptors take part in a complex system, which affects both basal metabolism of carbohydrates, lipids and proteins as well as cell growth, proliferation, differentiation and apoptosis. Defects in action of insulin or IGFs can lead to serious diseases such as diabetes or cancer. Both of these disorders represent nowadays one of the biggest health threats to the world's population. Insulin and IGFs induce different biological effects through their cognate receptors; two isoforms of the insulin receptor (IR-A and IR-B) and the receptor for IGF-1 (IGF-1R). These receptors bind insulin and IGFs with different affinities and induce different but partially overlapping signalling events leading towards metabolic (especially insulin) or mitogenic responses (IGFs and insulin). To understand the mechanism of action of insulin and IGFs it is important to specify which structural domains of these hormones are responsible for binding to the receptors and exerting specific effects. One region that is missing in insulin is the D-domain of IGF-1 and -2. For this reason, we decided to prepare insulin analogues with the A-chain extended by either the whole D-domain of IGF-1 or IGF-2, or by fragments of the IGF-1 D-domain in order to define the...
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The role of estrogen receptors in prognosis and therapy outcome of breast cancer
Kloudová, Alžběta ; Souček, Pavel (advisor) ; Dračínská, Helena (referee)
Estrogen receptors (ER) are members of nuclear receptor family, which mediate distinct physiological functions after binding a steroid ligand. Apart from that they play a role in many diseases including breast cancer. ER is among proteins routinely evaluated in clinical practice and on the basis of ER expression, patients are treated by endocrine therapy. There are different opinions of the role of ER in cancer cells, but in the future, detection of ER and treatment by ER- and ER-selective ligands could contribute to improvement of cancer therapy. Isoforms, mutations and posttranslational modifications of ER present other important factors, which can influence estrogen signalization and endocrine therapy efficiency and deciphering of their importance for cancer cells could bring better understanding of ER signalization and improvement of the therapy.
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The role of Hippo Signalling pathway in tumor cell metabolism
Lettlová, Sandra ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...
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Mechanism of carcinogenicity and nephrotoxicity of aristolochic acids
Bárta, František ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Aristolochic acids (AA) are human carcinogens which have also very strong nephrotoxic properties. A mixture of AA is present in Aristolochiacae plant species. These plants were and still are used in traditional medicine in some countries, particularly in Asia. Aristolochic acids participate in development of two types of nephropathies. The first disease is designated as Aristolochic Acid Nephropathy (AAN), the second one is Balkan Endemic Nephropathy (BEN). Both nephropathies are associated with urothelial malignancies, which are caused by AA. One of the common features of ANN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumour development. One cause for these different responses may be individual differences in the activities and expression levels of the enzymes catalyzing the biotransformation of AAI, the major toxic component of AA contained in Aristolochia species. Detailed knowledge of enzymes which participate in metabolism of AAI may contribute to elucidation of inter-individual susceptibility to AAN, BEN and later urothelial malignancies. Aristolochic acid I is either oxidative detoxicated or reductive activated by biotransformation enzymes. Reductive bioactiovation of AAI leads to formation of covalent AA-DNA adducts in organism which result in producing of...
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The influence of stress on NADP-dependent enzymes in higher plants.
Kovaľová, Terézia ; Hýsková, Veronika (advisor) ; Dračínská, Helena (referee)
Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...
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Aromatic nitrocompounds in the environment and their effect on organisms
Rohlenová, Terezie ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Aromatic nitro-compounds are contaminants present in environment. They are produced from gas-phase reaction of their parent compound with oxides of nitrogen. Aromatic nitro-compounds are found in an extract of exhausts from diesel and gasoline engines, in a cigarette smoke and in the soot from burning wood. The most of aromatic nitro-compounds are mutagens in bacterial and mammalian systems and the most of them are carcinogens that are connected with induction of cancer. Primarily lung followed by liver and mammary glands are target organs of their carcinogenic activity. This bachelor thesis describes a metabolisms and carcinogenic activity of several aromatic nitro-compounds: 2-nitroanisole, 4- nitroanisole, 3-nitrobenzathrone, 2-nitrobenzanthrone, 2-nitrotoluene, dinitrotoluenes, 2,4,6- trinitrotoluene. 2-Nitroanisole is used primarily as precursor in the synthesis of o-anisidine, an intermediate in the manufacture of many azo dyes and pigments. Both chemicals show strong carcinogenic activity to rats and mice. 4-Nitroanisole is produced by metabolic methylation of 4-nitrophenole that is used for synthesis of dyes and fungicides. 4-Nitrophenole is accumulated in environment. Therefore, it is considered to be an environmental pollutant. 3- Nitrobenzathrone is a strong carcinogen of rodents and...
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The effect of cancerogenic azo dye Sudan I on expression of biotransformation enzymes
Hejduková, Žaneta ; Dračínská, Helena (referee) ; Svášková, Dagmar (advisor)
Sudan I is a widely used azo dye which has the ability to cause carcinomas in organs and tissues of experimental animals. During reactions catalyzed by microsomal monooxygenase enzyme systems or cytoplasmic biotransformation enzymes, Sudan I is oxidized to reactive metabolites that covalently bind to nucleic acids and cause their damage. Sudan I can also be metabolized by reduction, e. g. by a DT-diaphorase enzyme (NQO1). Reduction of Sudan I is considered to be a detoxification reaction. In this work, the in vivo action of Sudan I is examined in terms of its ability to induce an expression of the biotransformation enzyme DT-diaphorase in tissues of rats treated with the azo dye. The aim of this work was to quantify the degree of NQO1 induction at mRNA level. After the isolation of total RNA from organs of rats treated with Sudan I, the RNA was converted to cDNA by reverse transcription using random hexamers as primers. Using specific probes, the abundance of mRNA for the enzyme NQO1 in the organs of treated rats was quantified by "real-time" PCR, relatively to the control gene with a constant expression (β-actin). Through comparing thus determined amounts of mRNA in individual organs of treated and untreated rats, it has been found that Sudan I had caused a significant increase in the expression...
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