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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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Screening of extremozyme production of selected extremophilic PHA producers
Dyagilev, Dmitry ; Obruča, Stanislav (referee) ; Pernicová, Iva (advisor)
This bachelor thesis deals with the screening of the production of extracellular hydrolytic enzymes in thermophilic microorganisms of the genera Aneurinibacillus, Brevibacillus, Chelatococcus, Pseudomonas, Schlegelella, Tepidimonas and Caldimonas. The ability of selected enzymes, namely proteases, lipases, amylases, xylanases, cellulases and pectinases, was tested in the investigated microorganisms. Such testing made it possible to assess in which microorganisms the production of specific enzymes can be observed. Based on the results of the screening, it was found that Schlegelella aquatica LMG 23380, Tepidimonas fonticaldi LMG 26746 and the investigated microorganisms of the genus Chelatococcus did not show the ability to produce any of the tested enzymes extracellularly. In natural isolates of Brevibacillus borstelensis LK 99 and Aneurinibacillus thermoaerophilus LK 102, only the ability to produce lipolytic enzymes was detected. The isolate Brevibacillus borstelensis Bz acts as a universal producer of all selected extremozymes. Enzyme activity was determined for selected producers. The bacterium Brevibacillus borstelensis Bz proved the ability to produce all six selected hydrolytic enzymes and has the highest activity of lipases, xylanases, cellulases and pectinases from the tested microorganisms. The highest proteolytic activity was measured in Thermomonas hydrothermalis DSM 14834 when cultured on skimmed milk powder.
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Incorporation of glucose oxidase into hydrogel structures
Suchá, Klára ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the incorporation of the enzyme glucose oxidase into hydrogel structures, while the activity of the enzyme after the incorporation into the hydrogel structure was monitored. Glucose oxidase was incorporated into the agarose hydrogel at various concentrations (1 and 2 wt. %). Glucose oxidase activity was determined using the resulting hydrogen peroxide, the concentration of which was measured by fluorescence spectroscopy using an Amplex Red fluorescence probe. The enzyme was found to be active even after incorporation into the hydrogel, but the concentrations of hydrogen peroxide formed were lower compared to the free enzyme. Furthermore, it was found that the enzyme reacts with agarose itself, but this reaction did not significantly affect the rheological properties of the hydrogel.
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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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Incorporation of glucose oxidase into hydrogel structures
Suchá, Klára ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the incorporation of the enzyme glucose oxidase into hydrogel structures, while the activity of the enzyme after the incorporation into the hydrogel structure was monitored. Glucose oxidase was incorporated into the agarose hydrogel at various concentrations (1 and 2 wt. %). Glucose oxidase activity was determined using the resulting hydrogen peroxide, the concentration of which was measured by fluorescence spectroscopy using an Amplex Red fluorescence probe. The enzyme was found to be active even after incorporation into the hydrogel, but the concentrations of hydrogen peroxide formed were lower compared to the free enzyme. Furthermore, it was found that the enzyme reacts with agarose itself, but this reaction did not significantly affect the rheological properties of the hydrogel.
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Nanofibrous scaffolds in controlled delivery of autologous growth factors
Buzgo, Matej ; Amler, Evžen (advisor) ; Gášková, Dana (referee)
Platelet preparations are a source of various autologous growth factors and have numerous applications in tissues engineering. The aim of this work was to development electrospun nanofiber scaffolds with platelet preparations. Scaffolds based on the adhesion of platelets on nanofiber meshes were developed. The scaffolds were able to enhance chondrocyte proliferation in vitro. The main disadvantage of this system is the burst release of growth factors immediately after adhesion. To overcome this, we developed coaxially electrospun scaffolds with incorporated alpha granules. Alpha granules are novel platelet preparations with high amounts of growth factors. This system was able to stimulate chondrocyte proliferation and maintain TGF- 1 concentrations for 7 days. Additionally, a novel drug delivery system with coaxially incorporated liposomes was developed. Liposomes incorporated into nanofibers remain intact and can be used for the delivery of various molecules. The ability to maintain HRP activity was compared to systems based on coaxial electrospinning with liposomes, coaxial electrospinning without liposomes and blend electrospinning. When compared to other systems, coaxial electrospinning with liposomes preserves enzyme activity twice as long. These results clearly indicate the potential of...
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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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