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Isolation of bacterial flagellin as an immunogen for the preparation of antibodies for cystic fibrosis patients
Supová, Veronika ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR gene mutation. The consequence of this mutation is an improper function of the chloride channel consisting of CFTR protein. Due to this abnormality, the transport of chloride ions is either reduced or inhibited completely, which leads to a mucus secretion. Mucus is mostly created in the lungs and it is the ideal environment for pathogenic bacteria like Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa is an aerobic, gram-negative conditioned pathogen occurring in patients with weakened immunity such as patients with CF, who are often hospitalized. PA has one polar flagellum which contains filament composed of a protein called flagellin. The flagellum is one of the most important virulence factors of PA bacteria. This thesis focused on the isolation of flagellin from Pseudomonas aeruginosa flagella. The isolated flagellin will serve as an antigen for the preparation of prophylactic antibodies for CF patients. The isolation of flagellin was carried out by four methods using combinations of precipitation and centrifugation. The published methods were optimized to achieve the isolation of flagellin. Final samples and intermediates were analyzed by SDS-electrophoresis on polyacrylamide gel and the presence of flagellin was verified...
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Use of chicken antibodies against PAIIL lectin for prevention of Pseudomonas aeruginosa infections in patients with cystic fibrosis
Kubíčková, Božena ; Hodek, Petr (advisor) ; Anzenbacherová, Eva (referee) ; Eckschlager, Tomáš (referee)
Cystic fibrosis (CF) is one of the relatively common inherited diseases caused by a mutation in the gene encoding for CFTR protein, which forms a chloride channel that significantly affects ion homeostasis and the associated fluid management of the cell. This disease mainly affects the respiratory and digestive systems, being the most life-threatening in the respiratory tract. Patients with CF suffer from frequent and recurrent respiratory infections that lead to the development of chronic inflammation and gradual destruction of lung tissue. These lung infections, which are caused mostly by the opportunistic pathogen Pseudomonas aeruginosa, are the most common cause of morbidity and mortality in these patients. At present, antibiotics are used in the treatment of Pseudomonas aeruginosa infections, but new methods of antibacterial therapy need to be found to overcome the development of resistance. In addition to active immunization of CF patients against Pseudomonas aeruginosa, their passive immunization with specific chicken antibodies directed against this pathogen offers promising possibilities. This dissertation thesis is aimed to verify the prophylactic potential of hen IgY antibodies against the virulence factor Pseudomonas aeruginosa - lectin PAIIL, and to further develop an experimental...
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Study on glycocluster prophylaxis by using a bacterium adherence model
Augustínová, Michaela ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by the mutations of the CFTR gene encoding CFTR protein which serves like a field ion channel in the body. For the patients suffering from CF is a typical increased adherence of bacteria to lung epithelium caused by the accumulation of thickened mucus on the surface of air passages and varied glycosylation in lungs. These conditions increase sensitivity of patients who suffer from CF to bacterial infections of lungs caused by Pseudomonas aeruginosa (PA). A considerable role in the pathogenicity of PA plays its virulent factors such as for example PA-IL and PA-IIL lectins, which enable an adherence of PA on the host cells joining on its surface receptors containing D-galactose (PA-IL) or L-fucose (PA-IIL). In this thesis the main aim was to verify an ability of given anti-PA-IIL chicken antibodies and multivalent PA-IIL inhibitors (specifically of the so-called fucose-based glycoclaters) to prevent PA adherence on lung epithelial cells and to stop from a formation of the PA infections at the patients. A prophylactic impact both antiadhesive agens (anti-PA-IIL antibodies, fucose-based glycoclasters) was studied ex vivo on the model system of immortalized lung epithelial cell lines CuFi-1 isolated from the ill patients with CF and NuLi-1...
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Study the potential of chicken anti-peptide antibodies as means of viral infection prophylaxis
Wojčíková, Lenka ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
In 2020, the WHO declared a global pandemic due to the disease COVID-19, which is caused by the SARS-CoV-2 virus. This virus uses the binding of a surface 'spike' protein to the affected cell's ACE 2 receptor to enter the cell. Vaccination therefore aims at inducing the production of antibodies that inhibit the virus binding. Alternatively, antibodies can be obtained from other organism and used as a means of passive immunisation against COVID-19 disease. The aim of the work was therefore to prepare chicken antibodies, IgY, that would be able to prevent the virus from binding. For the preparation of peptide immunogens, appropriate amino acid sequences have been selected from the RBD domain of SARS-CoV-2 S1 protein, which interacts with the ACE 2 receptor. The synthesized peptides were coupled with the KLH protein carrier, and the resulting conjugate was used to immunize hens. Subsequently, the IgY produced was isolated from the egg yolks and their ability to recognise selected peptides in different types of antigens was demonstrated using the ELISA method. After that, the protective properties of antibodies against the virus were tested ex vivo in cell lines and their efficiency was compared to that of Remdesivir. The antibodies were able to inhibit virus binding to cells in the assays, but the...
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Metabolism of cabozantinib by enzymes of first phase of biotransformation
Jurečka, Tomáš ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cabozantinib is an anticancer drug that inhibit tyrosine kinases which allow signal pathways important for growth and development of tumors. It is used for treatment of medullary thyroid cancer, hepatocellular carcinoma and kidney cancer. The major enzymes of the first phase of biotransformation that metabolize cabozantinib are cytochromes P450. In this thesis it was studied metabolism of cabozantinib and cytochromes P450 that participated on this metabolism. Hepatic microsomes of rat, mouse and rabbit were used for studying metabolism of cabozantinib in this thesis. It was also focused on the impact of particular isoforms of cytochromes P450 on metabolism of cabozantinib in rat microsomes. Time dependence of cabozantinib conversion in hepatic rat microsomes was also studied. Enzyme kinetics of metabolism of cabozantinib in hepatic rat microsomes, as well as impact of cytochromes P450 inhibitors on the metabolism were included. Metabolites were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. Formation of metabolites of cabozantinib increased over time to 30 minutes of incubation and with some others to 40 minutes of incubation. Up to five different metabolites were detected in experiments (M1, desmethyl cabozantinib, M3, monohydroxy cabozantinib and cabozantinib...
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Study of interaction of dihydromyricetin and alcohol in vivo
Vargová, Silvia ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
- Abstrakt Dihydromyricetin (DHM), also known as ampelopsin, is a natural antioxidant found in the plant Ampelopsis grossedentata, traditionally used in Chinese medicine. Dihydromyricetin exhibits health-benefiting activities. Besides its antioxidative, anti-inflammatory, anticancer, and antimicrobial effects, it is said to suppress intoxication by alcohol and mitigate the symptoms of hangover and abstention. The mechanism of the activity of DHM on the metabolism of ethanol has not been fully explored. Thus, the focal point of the presented thesis was to explore the effect of DHM on the expression of CYP2E1, one of the major enzymes participating in the metabolism of ethanol in organisms. Male rats were administered with ethanol (EtOH) or ethanol in combination with DHM, and the EtOH concentration of the blood was determined. The amount of EtOH and acetaldehyde in the blood of individuals indicates that DHM does not affect the rate of EtOH metabolism. Subsequently, the effect of DHM on repeated premedication of EtOH was monitored. The group of rats treated with DHM showed a slower metabolism of EtOH than the group solely premedicated with EtOH. Ultimately, the effect of DHM on CYP2E1 expression was studied. Microsomal fractions were prepared from the rat livers. By use of Western blotting with a...
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Metabolism of inhibitors of tyrosine kinases, the drugs of new generation
Čillíková, Olívia ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cancer is the second major cause of death after heart-attack in the world. In recent years, research has focused on tyrosine kinase inhibitors (TKIs) as part of targeted chemotherapeutic treatment. Vandetanib is a TKI affecting epidermal growth factor receptor (EGFR), rearrangement during transfection (RET) and vascular endothelial growth factor receptor 2 (VEGFR2). It is primary used for treatment of medullary thyroid cancer. Vandetanib is biotransformed by cytochromes P450 and flavin monooxygenases in human organism. Cytochromes P450 (CYPs) oxidaze vandetanib to only one metabolite, N-desmethyl vandetanib, which exhibits similar efficiency as parental molecule. NADPH is the major cofactor of reaction cycle of CYPs. This bachelor thesis studies the effect of various types of cofactors and pH on oxidation of vandetanib by selected human recombinant cytochromes P450, namely CYP2C8 coexpressed with cyt b5, CYP2D6, CYP3A4 and CYP3A4 coexpressed with cyt b5. Here, we investigate the effect of cofactors NADPH, NADH and their mixture in a 1:1 ratio on the amount of N- desmethyl vandetanib formed during the biotransformation of vandetanib. The effect of pH on the oxidation of vandetanib by CYP 3A4 and CYP 3A4 + b5 was also analysed. We analysed the amount of the metabolite formed at the pH range 7 to 8.5...
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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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Study of enzymes secreted by oomycete Pythium oligandrum
Hrdinová, Karolína ; Ryšlavá, Helena (advisor) ; Kubíčková, Božena (referee)
Pythium oligandrum is one of the non-pathogenic microorganisms of Pythium species which found its use as biological control agent. The main modes of action of this oomycete lie in inducing the plant immunity and in mycoparasitism of pathogenic fungi and fungus-like organisms. The oomycete attacks the pathogens by secretion of hydrolytic enzymes into the environment. In the first part of this bachelor's thesis, activity of hydrolytic enzymes endo-1,3-β-glucanase, cellulase, chitinase and proteases was observed in a commercial product based on Pythium oligandrum, called Polyversum-Biogarden. It was conclu- ded that the direct hydrolysis of phytopathogens is probably not the main mechanism of this product because the activity of glycosidases increased only after six hour-long incubation of the Polyversum-Biogarden in water and the proteolytic activity was not detected. In the second part of the bachelor's thesis, properties of proteases secreted by orga- nism Pythium oligandrum were studied. The highest proteolytic activity was observed at pH 6,5. Ovomucoid acted as an inhibitor of secreted proteases. The stability of pro- teases was lowered by SDS, detergents present in liquid soaps and by a solution of a solid soap or NaOH. Only the highest concentrations of urea lowered the activity of proteases....
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