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Hydrogels with incorporated enzymes
Geistová, Karolína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of incorporation of enzymes into phase separated hydrogels. The aim of this work is to determine the enzyme activity in phase separated gels. Gels were prepared by the dry-way based on the interaction of negatively charged polyelectrolyte (hyaluronan) with positively charged surfactant (Septonex). Two enzymes, bromelain and collagenase, were incorporated into the hydrogels. To determine enzyme activity, the modified albumin protein with bound sulfanilamide group (azoalbumin) was used as a substrate. The enzyme activity of the enzyme itself, the enzyme activity affected by one of the two components of the system as well as the activity of the enzyme directly in the hydrogel was determined on UV-VIS spectrophotometry. The enzyme was found to be incorporated in the hydrogel. Furthermore, a significant effect of the positively charged surfactant on the enzyme activity was detected in phase-separated hydrogels.
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Characterization and stabilization of pancreatin
Wurstová, Agáta ; Němcová, Andrea (referee) ; Obruča, Stanislav (advisor)
This work focuses on a study of enzyme mixture pancreatin, its characterization and subsequent encapsulation into liposomes. As a reference proteins bovine serum albumin and trypsin were used. Characterization of pancreatin consisted of two parts. The first part focuses on optimization of methods for the concentration determination by absorption spectrophotometry using basic methods for identifying proteins (Biuret method, Hartree-Lowry method and Bradford method). Moreover, UV spectrums of the protein were measured. As a method for identification of protein´s molecular weight, SDS-PAGE was used. To identify components of pancreatin, LPLC was employed in two modifications, ion-exchange chromatography and size exclusion chromatography. The second part is dedicated to the characterization of pancreatin as enzyme in terms of pH and temperature optimum for the enzyme activities of protease (pH 9, 8 and 50 °C), amylase (pH 7 and 40 °C) and lipase (pH 7 and 50 °C). The last part of this work aimed at an encapsulation of pancreatin into liposomes and DLS analysis of distribution of particles and their zeta potential. Liposomes did not spontaneously release encapsulated enzyme. To confirm that proteins were successfully entrapped into liposomes, their structure was disrupted by application of phospholipase D. In conclusion, liposomes can be utilized as delivery systems for native enzymes.
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Factors influencing the quality of red wine
Zechmeisterová, Lucie ; Vránová, Dana (referee) ; Omelková, Jiřina (advisor)
In my thesis, I focused on monitoring of microorganisms in the sample of red grape juice and on the interactions between yeasts, bacteria and filamentous fungi. Three different media were applied for the cultivation of microorganisms; firstly for monitoring of total volume of microorganisms, secondly for yeasts and third time for lactic acid bacteria. The indirect method was used for the determination of the amount of viable cells. This method consists in enumerating of visible macroscopic colonies grown up on agar plates. When the cells grew up, the forms of colonies were analyzed visually and the morphology of microorganisms was detected microscopically. The operating time of enzymes in grape juice in the production of red wine was monitored after application of commercial enzymatic preparation. The enzym action in grape juice was observed on the basis of the process of degradation of high – molecular substrate by enzymes through the use of Ubbelohd´s viscometer. The research findings provided a lot of knowledge about the occurance of microflora in the process of production of red wine. The commercial preparations added to grape juice played a significant role.
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Phytoextraction of mixed drug samples from aqueous solutions
Hájková, Eliška ; Smrček, Stanislav (advisor) ; Soudek, Petr (referee)
A number of contaminants have been detected in the environment, including pharmaceuticals. Their presence in soil, water sources can have a toxic effect on organisms due to their constantly increasing concentration. The method of phytoremediation uses the ability of plants to absorb these contaminants and detoxify them by various mechanisms. The aim of this work was the phytoextraction of ibuprofen using maize (Zea mays) from aqueous solutions. Phytoextraction of ibuprofen after cultivation with added dextromethorphan was also carried out. The quantitative amount of extracted ibuprofen was detected by HPLC with UV detection. Ibuprofen has been very well extracted by plants. In the presence of dextromethorphan, the efficiency of phytoextraction was decreased. The phytoextraction efficiency of ibuprofen was 0.10 mg per gram of leaf fresh weight, while in the presence of dextromethorphan, the phytoextraction efficiency was 0.09 mg per gram of leaf fresh weight. After phytoextraction of ibuprofen, the antioxidant capacity was determined by FRAP method of leaf and root extracts of the plants, of which the highest values were observed in the roots of plant grown with ibuprofen in combination with dextromethorphan. The phenolic compounds were also detected in leaves and roots, where the highest percentage...
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Modulation of cytochrome P450 activity by the anticancer drug lenvatinib
Ivančík, Martin ; Dračínská, Helena (advisor) ; Mrízová, Iveta (referee)
Lenvatinib, commercially marketed as Lenvima®, is an oral drug approved for the treatment of thyroid cancer, hepatocellular carcinoma and renal cell carcinoma that acts as a tyrosine kinase inhibitor. In vitro and in vivo studies have shown that lenvatinib is in the human body metabolised in liver and kidney by the cytochrome P450 enzyme system and aldehyde oxidase. Therefore, the aim of this bachelor thesis was to determine the effect of lenvatinib on the activity of individual isoforms of human cytochrome P450. Among the isoforms studied, those that ensure the metabolism of majority of foreign substances in the human body were selected. Measurements were performed in vitro using recombinant CYPs expressed in SupersomesTM and using marker reactions that are provided by individual cytochrome P450 isoforms. The activity of the enzyme in the reaction mixtures containing lenvatinib was compared with the activity of the enzyme in the reaction mixtures where only the solvent DMSO was added instead of lenvatinib. The concentration of lenvatinib corresponded to the concentration of the given substrate or was 10 times higher. Based on these measurements, the percentage activity of cytochrome P450 isoforms 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2E1 and 3A4 in the presence of lenvatinib was calculated. A decrease in...
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Inhibitory effect of endocrine disruptor 17α-ethinylestradiol on cytochrome P450 subfamily 1A
Kurshakova, Evgeniia ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
17-ethinylestradiol (EE2) is a synthetic derivative of the natural estrogen 17-estradiol. It is used in medicine as a key component of oral contraceptives. Due to its ability to modulate the functions of the endocrine system, EE2 belongs to the group of endocrine disruptors. The ability to bioaccumulate and affect the reproduction and development of wildlife makes EE2 a compound that possesses a potential environmental risk. Cytochromes P450 1A1 and 1A2 catalyze the oxidative reactions in metabolism of exogenous compounds, including EE2. 17-Ethinylestradiol is known to have a strong inhibitory effect on the isoform CYP1A1. Within the framework of the present thesis, the inhibition effect of EE2 on the activity of cytochromes P450 of subfamily 1A was studied in vitro via three marker reactions: 7-methoxyresorfin O-demethylation, 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation, which had to be pre-optimised for the following uses. A highly selective inhibitory effect of 17-ethinylestradiol on the human and rat isoform CYP1A1 was confirmed. An inhibitory effect on the activity of the CYP1A2 isoform was not observed by any of marker reactions. Using phenacetin O-deethylation, the concentration of EE2 causing 50% inhibition of CYP1A1 (IC50) was also determined, being of 3,4 M, at a...
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Protease Sapp3p of the pathogenic yeast Candida parapsilosis
Sochor, Richard ; Heidingsfeld, Olga (advisor) ; Zábranská, Helena (referee)
Pathogenic yeasts of the genus Candida can cause systemic diseases which, in patients suffering from immunosuppression due to a disease such as AIDS, can cause serious pathological conditions, which can lead to the patient's death. One such yeast is the pathogenic yeast Candida parapsilosis. For the colonization and penetration of host tissues this yeast uses various virulence factors. One of these virulence factors are secreted aspartic proteases. The pathogenic yeast C. parapsilosis contains three secreted aspartic proteases Sapp1p, Sapp2p and Sapp3p, which are paralogs. The first two aspartic proteases are responsible for increasing the virulence of C. parapsilosis. They help the yeast survive in the body, by degrading important components of the host's immune system. However, Sapp3p doesn't exhibit these properties, except that it helps the yeast to adhere to abiotic surfaces to some extent. This work is focused on clarification the functions and localization of Sapp3p in the yeast C. parapsilosis. To clarify the function, precursor of Sapp3p (pro-Sapp3p) was recombinantly prepared in E. coli cells. The protein thus prepared was further tested for its autocatalytic activation and assisted activation by trypsin and Kex2p protease, under various conditions. Under the conditions tested, it was not...
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Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
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Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Lukačiková, Karolína ; Vokřál, Ivan (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Karolína Lukačiková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The resistance of parasitic helminths to anthelmintic drugs is a growing worldwide phenomenon and a concerning issue. Xenobiotic metabolizing enzymes play an important role in drug resistance development as they can lower the concentration of the anthelmintics in the parasite's body and therefore protect the parasite from the anthelmintic effect. The role of drug metabolizing enzymes in drug resistance development has been already described in the group of roundworms and flukes. Limited information is available about this topic in tapeworms. In our study we decided to test the possibility of the anthelmintic mebendazole to affect the activity of these enzymes and possibly to influence the drug resistance development in rat tapeworm (Hymenolepis diminuta). Our first goal was the isolation of adult tapeworms from the definitive host (rat, Rattus norvegicus). We used mealworm beetle (Tenebrio molitor) as an intermediate host. After the successful isolation, adult tapeworms were incubated with the mebendazole (1 and 10µM) in...
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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