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Interaction of selected flavonoids with N-acetyltransferases
Veselá, Šárka ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Flavonoids are natural compounds synthesized in plants as secondary metabolites. Due to broad range of their biological effects, for example we can mention antioxidant, hepatoprotective or antibacterial effects, flavonoids are increasingly used as a component in dietary supplements. Besides positive effects, some negative effects were observed, especially mutagenic and pro-oxidative properities. The biotransformation is an important process which is affected by flavonoids. Interactions between flavonoids and biotransformation enzymes in both phases of biotransformation are essential for the metabolism of drugs and carcinogenesis processes. In this bachelor thesis the influence of selected flavonoid compounds, myricetin and dihydromyricetin, on the activity of N-acetyltransferase 1 and 2, enzymes of second phase of biotransformation, was studied. Mainly the mechanism of this influence was tested. The inhibition of human recombinant and of N-acetyltransferase 1 and 2 by myricetin and dihydromyricetin was proved. The more in-depth study of interactions of N-acetyltransferase 1 and 2 with selected flavonoids shows that raising concentration of second substrate (p-aminobenzoic acid, sulfamethazine) does not lead to reduction of inhibition effect. Thus, it is highly probable that both flavonoids already...
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The influence of endocrine disruptors on biotransformation enzymes
Klusoňová, Zuzana ; Bořek Dohalská, Lucie (advisor) ; Linhartová, Lucie (referee)
Endocrine disruptors (ED) are compounds that interfere with the endocrine system by mimicking or antagonising the effects of endogenous hormones. The synthetic estrogen 17-ethinylestradiol (EE2), a major component of oral contraceptives and a human strong carcinogen benzoapyrene (BaP) are assigned as an exogenous ED whereas the natural estrogenic hormone 17-estradiol (E2) figures as an endogenic endocrine disruptor. In this thesis the rats were treated with these ED individually and in combination, and their effect was studied on the gene and protein expression of cytochrome P450 1A1, 2C11 and 2C6 in rat liver, lung and kidney. In addition, the effect of the mentioned ED and their combinations on CYP1A and CYP2C specific enzyme activity and on metabolism of 17-ethinylestradiol in rat liver microsomal samples was studied. Changes in CYP gene and protein expression were analyzed by real-time PCR and Western blotting, CYP specific activity and metabolism of EE2 were investigated by HPLC method. It has been confirmed that BaP acts as a strong inducer of CYP1A1 in rat liver, lung and kidney, whereas EE2 or E2 alone revealed no significant effect on the expression of this CYP. However, when EE2 or E2 were administered to rats together with BaP, they considerably decreased the BaP-mediated CYP1A1...
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Possible approaches to the treatment of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by a mutation in the CFTR gene. This leads to an absence or a malfunction of CFTR chloride channel, which is also a crutial regulator of other ion channels. This thesis was aimed at gene therapy of cystic fibrosis using CFTR- mRNA gene transfer. To determine the expression of CFTR protein, methods of indirect immunofluorescence and Western blot immunodetection were utilized. Also relative gene expression levels of CFTR gene were assessed by quantitative PCR. Experiments were carried out on a healthy lung epithelial cell line (NuLi-1), a lung epithelial cell line with F508del mutation (CuFi-1) and an embryonic kidney epithelial cell line (HEK293S). CFTR protein was visualized by previously mentioned methods using six primary antiobodies (432, 450, 570, 596, 769, CF3). Primary antibodies 570 a CF3 were found as optimal for the detection of CFTR protein by the method of indirect immunofluorescence, whereas for the detection of this protein by the method of Western blot only the CF3 antibody was suitable. It was also determined that CFTR gene is expressed in overall small levels. Its relative gene expression in the CuFi-1 cell line was approximately six times higher than in the NuLi-1 cell line. The efficiency of transfection of CuFi-1 cell line by CFTR-mRNA...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana ; Bořek Dohalská, Lucie (advisor) ; Cajthaml, Tomáš (referee)
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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Gene therapy of cystic fibrosis
Baráčková, Petra ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the membrane protein called cystic fibrosis transmembrane conductance regulator (CFTR). It is the most frequent genetic disease in Caucasian populations. It currently affects approximately 75,000 individuals worldwide. CF as a monogenic disease represents an appropriate target for the gene therapy. Since the sequencing of the CFTR gene in 1989, a large number of preclinical and clinical studies have been performed examining the various principles of this therapy. The aim of this review study is to summarize the findings and introduce the latest strategies of the gene therapy of cystic fibrosis, which represent a promising basis for definitive cure of this disease. The evolving technologies of genomic engineering, deepening knowledge in pathogenesis of this disease and the results of preclinical and clinical trials motivate researches to further testing of the gene therapy strategies, which have the potential to eliminate the cause of the disease and cure it by intervening in human genome. Keywords: cystic fibrosis, CFTR gene, CFTR protein, gene therapy
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Development of a model system to study adhesion of Burkholderia cenocepacia to epithelial lung cells of patients with cystic fibrosis
Volejníková, Anna ; Bořek Dohalská, Lucie (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is a common autosomal recessive disorder that primarily affects epithelial cells in respiratory system. As a consequence of lung damage the patients are chronically colonized by a number of pathogenic microorganisms such as Pseudomonas aeruginosa or Burkholderia cepacia komplex. Due to the seriousness of these bacterial infection and its primary resistance to commonly used antibiotics the suitable therapeutic alternatives are being sought. The passive immunization with yolk antibodies seems to be a suitable alternative. This thesis studied the bacterial adhesion of Burkholderia cepacia, the opportunistic human pathogen, to lung epithelial cells of healthy individuals and patients with CF, respectively. The aim of this study was to develop a model system suitable for this research. Two types of immortalised cell lines have been used for this purpose. CuFi-1 is the cell line derived from patient with CF caused by ∆F508 mutation , NuLi-1 is a healthy epithelial cell line. The strains of B. cenocepacia (ST28 a ST32) were fluorescently labeled with fluorescent cell linker PKH26. Furthermore, the way of fluorescent labeling of cell lines CuFi-1 and NuLi-1 using PKH67 dye was optimized. Using this model system, the adhesion of bacteria to cell line CuFi-1 was up to three times higher...
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Identification and characterization of reductases of fatty acids participating at pheromone biosynthesis
Wenzelová, Petra ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
In the mating season, bumblebee males leave pheromone track on different objects on their flight routes to attract females of the same species. Synthesis of pheromones, which takes place in the labial gland of bumblebee males, involves enzymes called fatty acid reductases (hereinafter reductases). They catalyse the reduction of fatty acids bound in the form of acyl-coenzyme A to the alcohols. In this work, I functionally characterized two reductases highly expressed in labial gland of the two most common species of bumblebees Bombus lucorum and Bombus terrestris in order to verify their role in the biosynthesis of species specific male pheromones. I expressed these reductases in yeast Sacccharomyces cerevisiae. I confirmed the heterologous expression of reductases and prepared lipid extracts from yeast cells, in which we identified by gas chromatography with mass spectrometric detection (GC/MS) alcohols derived from fatty acids with long chain. These were octadecanol, icosanol and docosanol.These results lead us to conclusion that reductases are involved in the biosynthesis of alcohols with long hydrocarbon chains, which are components of marking pheromones of mentioned bumblebee species. These partial results will be used in a future study that wants to reveal the molecular mechanisms of male...
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