|
|
Effect of the subchronic and early-life permethrin exposures on rat liver: oxidative stress and endogenous antioxidant responses
Palan, Matyáš ; Zemanová, Lucie (advisor) ; Dršata, Jaroslav (referee)
Charles University in Prague University of Camerino Faculty of Pharmacy in Hradec Králové Faculty of Pharmacy Department of Biochemical Sciences Department of Molecular Biology Candidate: Matyáš Palan Supervisor: Associate Prof. of Biochemistry Rosita Gabbianelli, Ph.D. RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: Effect of the subchronic and early-life permethrin exposures on rat liver: oxidative stress and endogenous antioxidant responses Permethrin is a widely used insecticide with a low acute toxicity to mammals; however, there are concerns about consequences of a long term and an early life exposure to this compound. Two different animal models were chosen in order to study effects of permethrin on liver. In the model of subchronic treatment, adolescent rats treated for 60 days with permethrin (150 mg/kg body weight/day) were used and sacrificed immediately after the treatment. As far as the model of early life is concerned, the treatment with permethrin (34.05 mg/kg body weight/day) lasted for 15 consecutive days in early life and sacrifice took place after the interval of 300 days since the beginning of the treatment. To evaluate the impact of permethrin treatment on liver tissue, markers of oxidative stress (lipid peroxidation, content of carbonyl groups), amount of superoxide...
|
|
|
HPLC method for determination of testesterone and its use for chatacterization of carbonyl reductases
Plucha, Tomáš ; Zemanová, Lucie (advisor) ; Skarka, Adam (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tomáš Plucha Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: HPLC method for determination of testesterone and its use for chatacterization of carbonyl reductases Prostate cancer belong to hormone-dependent tumors, the proliferation of transformed cells is dependent on activation of the androgen receptor by testosterone. Androgen deprivatin therapy is complicated by productin of active androgens directly in prostate cells. It was proved that among other enzymes also carbonyl-reducing enzymes participate in androgen production, especially enzyme aldo-keto reductase 1C3 (AKR1C3). The aim was to develop a HPLC method for the determination of testosterone and use this method for in vitro screening of activity of carbonyl-reducing enzymes in formation of potent androgen testosterone from its inactive precursor androstenedione. Enzyme samples were incubated with androstenedione c = 20-120 µmol/l and after incubation were extracted by ethyl acetate. Testosterone formed during the incubation was determined by HPLC analysis (column BDS Hypersil C18, 5µm, 250×4.6 mm, mobile phase metanol:voda, 70:30, detection: DAD při 240 nm). Samples of human liver subcelular fractions,...
|
|
|
Enzyme activity of recombinant enzymes. Influence of histidine tag.
Navrátil, Václav ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Václav Navrátil Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Enzymatic activity of recombinant enzymes. Evaluation of His- tag influence. AKR1B1, AKR1C1 and AKR1C3 are three representatives from 15 human enzymes of AKR superfamily, so far described. In the body they take part in a number of biochemical pathways and their substrates include both xenobiotics and endogenous compounds. Reduction of glucose to sorbitol catalyzed by AKR1B1 is associated with the development of secondary diabetic complications. AKR1B1 is also involved in cardiotoxic effect of the cytostatic drug daunorubicine. AKR1C1 is physiologically responsible for the conversion of progesterone to less efficient 20α- hydroxyprogesterone, thereby regulates progestational activity. Overexpression of AKR1C1 may be connected with resistance to certain cytostatic agents. AKR1C3 in human catalyzes a reaction in which testosterone and 5α-DHT is formed and it is also associated with development of several cancer types. Significant involvement of these enzymes in a number of pathological processes led to their closer study. The aim of this work was to evaluate influence of His-tag and chosen buffer on the...
|
|
|
Inhibition study of human 17β-HSD3
Poláková, Lenka ; Štambergová, Hana (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Poláková Supervisor: PharmDr. Hana Štambergová, Ph.D. Title of diploma thesis: Inhibition study of human 17β-HSD3 Enzyme 17β-hydroxysteroid dehydrogenase 3 belonging to the short-chain dehydrogenase/reductase superfamily is involved mainly in reduction of 4-androstane-3,17- dione to testosterone. Any changes in amount of testosterone can result in illness, such as the male pseudhermaphroditism or the prostate cancer. For this reason studies of inhibion effects of various substances have been doing as they could be used in the therapy such as illnesses. Identification of the pharmacologically interesting substances was performed in vitro through the model system. Cell line Sf9 was used to prepare a recombinant form of the target enzyme. Reduction activity of this enzyme was verified towards androstene-4,17-dione. Then screening of potential inhibitors, natural substances: flavonoids and alkaloids, was performed. IC50 value was defined at substances with 100% inhibition effect (2-hydroxyflavanone, biochanine A, fumariline).
|
|
|
Determination of mRNA of DHRS3 enzyme in human tissues by real-time SYBR green qPCR
Rusnáková, Pavlína ; Zemanová, Lucie (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Pavlína Rusnáková Supervisor: RNDr. Lucie Zemanová, PhD. Title of diploma thesis: Determination of mRNA of DHRS3 enzyme in human tissues by real-time SYBR green qPCR Dehydrogenase/reductase (SDR family) member 3 (DHRS3, also known as SDR16C1, retSDR1) is NADPH dependent carbonyl reductase, member of the short-chain dehydrogenases/reductases superfamily. DHRS3 was characterized for the first time by Haeseleer et al. as an enzyme that reduces all-trans-retinal to all-trans-retinol in the visual cycle that is localised in the outer segment of the cone photoreceptors in bovine retina. However, expression of DHRS3 was shown in some other tissues. It was proven that DHRS3 plays an important role in embryogenesis and tumour development. It was also established that DHRS3 participates in the reduction of some endogenous compounds (androstendione, estrone, DL-glyceraldehyde) and xenobiotics (acetohexamide, 4-nitroacetophenone, benzil) in vitro. DHRS3 seems to be an important enzyme with participation in significant metabolic pathways. Nevertheless, knowledge of some DHRS3 properties, including expression in human tissues, is quite poor. The aim of this study was to determine expression of DHRS3...
|
|
|
Carbonyl Reductases Role in the Biotransformation of Drug Bupropion
Tomanová, Radana ; Zemanová, Lucie (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of thesis: Carbonyl reductases role in the biotransformation of drug bupropion Bupropion is a second-generation antidepressant that is also used at lower doses for smoking cessation. Structurally it is a monocyclic aminoketone, which is metabolized in human liver to three major pharmacologically active metabolites. Bupropion is hydroxylated to hydroxybupropion, reduction leads to formation of threohydrobupropion and also minor metabolite erythrohydrobupropion. The aim of this study was to determine in vitro the activity of human liver subcellular fractions and human carbonyl-reducing enzymes with bupropion and to discover the participation of enzymes in the liver metabolism of bupropion. Initially the role of subcellular fractions and recombinant carbonyl-reducing enzymes belonging to the superfamily AKR, family CBR and 11β-HSD 1 in in vitro biotransformation of bupropion was examined. Determination of formed metabolites was shown that all liver subcellular fractions and also 11β-HSD 1, AKR1C1, AKR1C2, AKR1C3 and CBR1 enzymes are active in the reduction of bupropion. The major in vitro liver metabolite was...
|
|
|
Cloning and expression of human AKR1A1
Kořínková, Petra ; Wsól, Vladimír (advisor) ; Zemanová, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Kořínková Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning and expression of human AKR1A1 The aldo-keto reductase 1A1 (AKR1A1) is monomeric cytosolic enzyme from aldo- keto reductase superfamily (AKR) with carbonyl reducing activity. The members of AKR superfamily have an important role in reductive reactions in metabolism of some endogenous substrates and in the first phase of biotransformation of xenobiotics. Fifteen members of this large family have been found in the human body. These proteins are expressed in different tissues within all our organism. The highest concentrations of proteins have been detected in the hepatocytes and in the renal cells. The function of AKR1A1 is to catalyse reductive reactions of aromatic and aliphatic aldehydes to the respective alcohols. Among its important substrates belong mevalonate (endogenous substance) and some drugs like anthracykline antibiotics - doxorubicin and daunorubicin. AKR1A1 also participates in biotransformations of sorbitol and in the development of diabetes complications. Preparation of recombinant protein was performed in E. coli with using of pET28b(+) as an expression vector. Isolated cDNA was...
|
|
|
Detection of His-tag and FLAG-tag recombinant proteins
Javorská, Lenka ; Zemanová, Lucie (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Javorská Supervisor: RNDr. Lucie Škarydová, Ph.D Title of diploma thesis: Detection of His-tag and FLAG-tag recombinant proteins Recombinant proteins are proteins produced in genetically modified organism by recombinant DNA technology that enable insert DNA sequence of target protein in expression cell. This technology allows preparation of actually any protein in any quantity without difficulties associated with demanding purification of native protein from tissues. Recombinant proteins are produced often in form of fusion proteins with suitable peptide tag (e.g. His-tag, FLAG- tag) that can help with their detection and enable simple purification. The aim of this diploma thesis was to introduce and optimize universal method for the detection of recombinant proteins labelled with His-tag (6x histidine) and FLAG-tag. The model recombinant protein with His-tag was enzyme carbonylreductase 1 (CBR1) produced by expression system Escherichia coli. For FLAG-tag labelled protein were chosen enzymes DHRS12, HSD11β1, RODH4, DHRS3, DHRS7, DHRS8 produced by insect cells (strain Sf9). SDS polyacrylamide electrophoresis for protein separation and immunochemical detection after Western...
|
|
| |
|
|
Methods of mounting of acoustic emission sensors on the surface of tested structures
Ščasný, Jakub ; Zemanová, Lucie (referee) ; Mazal, Pavel (advisor)
This bachelor thesis is focused on the issue of mounting acoustic emission sensor to cylindrical and sferical surface of tested tube segments which are made of non-ferromagnetic material. This work includes overview of current state of cognition in the basics of acoustic emission testing needed for understanding the issue. The folowing part includes the overview of commonly used practices for installing transducers. In the part of mechanical design drafts of mounting fixtures are discussed along with testing and comparision of their characteristics.
|
guest ::
