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Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
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Characteristics and gene expression levels of plant transformants modified by one related constructs shearing a gene for proteinase inhibitor.
BEZUNK, František
Tobacco plants were transformed by bacteria Agrobacterium tumefaciens bearing a plasmid construct with a fusion gene SPI2:GFP and selectable marker NPTII coding for resistance of transformants to antibiotic kanamycin. SPI2:GFP gene was created by a fusion of SPI2- (for serine protease inhibitor) and GFP gene (coding for a green fluorescent protein - GFP) sequences. Transformed tobacco plants were selected on a medium with kanamycin. Positively selected plants were selfed and segregation ratios for NPTII determined for each of genotypes. The presence and function of transgenic DNA was verified by methods of PCR, RT-PCR, Western blot and using the stereomicroscopic fluorescence study of transformants of T0 and T1 generations. This Transgenic genotypes of tobacco Nicotiana tabacum, cv. Petit Havana, SR1 WT showing the expression of the fused gene SPI2:GFP on a level of mRNA and GFP protein were obtained. Thesis was perfomed with a support of the project No. M106030 of Ministry of Education, Youth and Sports of the Czech Republic.
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Study of effects of antiretroviral drugs on transmembrane transport of tenofofovir disoproxil fumarate across MDCKII-ABCB1 cell monolayer
Repeľová, Beáta ; Červený, Lukáš (advisor) ; Čečková, Martina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...
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Detekce a kvantifikace inhibitorů proteáz v klíštěti \kur{Ixodes ricinus} pomocí monoklonálních protilátek
VANÍČKOVÁ, Martina
Inhibitors of proteases in tick saliva play an important role during tick feeding. Tick saliva contains a wide range of bioactive components which are able to modulate host imunity. Therefore, ticks are able to feed for a long time and transfer tick-borne diseases pathogens. The risk of transfer can be significantly reduced by deactivation of theese protease inhibitors. In this study I made monoclonal antibodies for detection and quantification of two serine protease inhibitors in tick saliva and other tick-body parts.
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Study of effects of antiretroviral drugs on transmembrane transport of tenofofovir disoproxil fumarate across MDCKII-ABCB1 cell monolayer
Repeľová, Beáta ; Červený, Lukáš (advisor) ; Čečková, Martina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...
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Study of effects of antiretroviral drugs on transmembrane transport of tenofofovir disoproxil fumarate across MDCKII-ABCB1 cell monolayer
Repeľová, Beáta ; Červený, Lukáš (advisor) ; Čečková, Martina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...
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Optimization of methods for evaluation of antimicrobial activity of selected active matters against chosen spectrum of bacterial pathogens
BRZÁKOVÁ, Michaela
The bachelor thesis deals with optimalization of selected methods (e.g. evaluation of bacterial suspension concentration, Resazurin antibacterial assay) needed for evaluation of active substances antibacterial activity against pathogens Pectobacterium carotovorum, Pseudomonas syringae and Clavibacter michiganensis subsp. michiganensis. Literature review contains information about potato tubers proteins, especially proteins with antibacterial activities, as well as a brief overview of mechanisms of action of plant defense system against pathogenic microorganisms and the effect of antimicrobial substances on bacterial pathogens. The experimental part deals with optimalization of methods needed for evaluation of antibacterial activities of selected substances. Optimized were methods of McFarland Turbidity Standards, Most-probable number method and Resazurin antibacterial assay. It was found that increase of optical density, measured by spectrometer at 620 nm, is in direct correlation with increase of bacterial suspension concentration (CFU/ml). It was identified proteins of patatin complex and protease inhibitors by using SDS-PAGE method to evaluated potato tuber proteins of variety Bella and Westamyl and also changes of molar mass of the patatin proteins in relation of their modification were evaluated. Antimicrobial activity of these proteins was verified by resazurin antibacterial assay and ability of modified proteins of cultivars Bella and Westamyl in higher concentration (10 mg/ml) to inhibit growth of bacteria Pseudomonas syringae.
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