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Production of auxin transporter PIN1 in HEK293 cell line
Petermannová, Romana ; Vaněk, Ondřej (advisor) ; Hýsková, Veronika (referee)
PIN1 protein belongs to a PIN family of transporters. This group of transporters is significantly involved in auxin transports outside of the cell. The auxin transport among plant cells gives an important role in plant growth, gravitropism, etc. The principle of auxin transport by this carrier protein is still not explored. This work deals with the production of PIN1 carrier protein by recombinant expression in human embryonic kidney 293 cell line. Production in these cells was checked by the accumulation of radioactive auxin and by the immunofluorescence.
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Protease of bovine follicular fluid
Drahokoupil, Marek ; Liberda, Jiří (advisor) ; Hýsková, Veronika (referee)
This bachelor thesis deals with isolation and partial characterisation of cow protease follicular fluids. At first, the follicular fluid was isolated according to molecular weights by means of gel chromatography on Sephadex G-100. As another method of separation there was selected the ion exchange chromatography on Sephadex DEAE. For the characterization of resulting fractions SDS electrophoresis, assessment of proteolytic activity of the proteolytic substrate azakasein, zymography and differential SDS electrophoresis were used. The collected fractions of the follicular fluid defined by the gel chromatography method had the relative molecular weights raging 114 000 - 131 000 in fraction pattern I, 44 000 - 51 000 in fraction pattern II, 151 000 - 204 000 in fraction pattern III, 57 000 - 99 in fraction pattern IV, 14 000 - 38 000 in fraction pattern V and 180 - 500 in fraction pattern VI. From the results of the proteolytic activity I decided to work further only with fraction patterns III, IV and V collected from the gel chromatography and with the pattern of the delayed fraction PBS II collected from the ion exchange chromatography. These patterns reported the highest specific activity. Inhibitors were used to determine which type of protease occurs in thee chosen fractions. In the fraction...
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Antioxidant properties of medicinal plants
Tupec, Michal ; Hýsková, Veronika (advisor) ; Poljaková, Jitka (referee)
During the evolution plants have developed rather efficient mechanisms for the re- moval of harmful compounds, which include e. g. the reactive oxygen species (ROS). These mechanisms are mainly based on the antioxidant effects of ascorbic acid, gluta- thione in intimate cooperation with superoxide dismutase, catalase and various perox- idases - the reduced coenzymes, NADH and NADPH, play a major role in the ROS elimination, too. However, in recent times, the attention has turned from these key compounds of primary metabolism to secondary metabolites. Interestingly, the phenolic com- pounds together with some terpenes and alkaloids exhibit antioxidant effects, too, and apparently they play an irreplaceable role in the life of a plant - besides their antiox- idant action they are also important in the fight against pathogens and herbivores, in attracting the pollinators or in the moderation of UV light effects. Natural products are also studied in their relation to man - a broad variety of effects is known including the antioxidant, anti-inflammatory and anticancer effects for the most part. In this work, extracts comprising mainly of low-molecular-weight compounds were obtained from 32 plants with either hot water, 80 %w aqueous methanol or just by plain juicing. The extracts were then examined by three...
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Laboratory tests for thyroid function assessment from the economic point of view
Stehlíková, Petra ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
In 2011 Recommendation for laboratory diagnostics of functional and autoimmune thyroid diseases was published. Its target is to recommend correct method when choosing thyroid examination parameters to general practitians and thus find a way how to save money on useless examinations which then can be used by hospital in other area. To verify physicians adhere to these recommendations file of VFN patients in Prague for year 2011 was used and statistically evaluated. Watched parameters included thyrotropin (TSH), free triiodothyronine (FT3), total triiodothyronine (T3), free thyroxine (FT4), total thyroxine (T4), thyroid peroxidase antibodies (anti - TPO), thyroglobulin antibodies (anti - Tg), thyroglobulin (Tg) and thyroxine - binding globulin (TBG). Basic test according to published recommendation should include only TSH and if out of reference limits then it should be followed by FT4 test or antidote assessment. Other parameters are unnecessary for initial examination, some of them are not used anymore or should be examined only by clinics specialized in endocrinology. Based on the file records many physicians are still ordering unnecessary examination parameters. In one year 13,8 % of unnecessary parameters out of 44 415 thyroid function examinations were determined. This is 1 095 013 points that could by...
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The effect of abiotic stress on cucumber plants (Cucumis sativa L.)
Plisková, Veronika ; Hýsková, Veronika (advisor) ; Müller, Karel (referee)
The exposure of plants to high salt concentrations causes accumulation of sodium ions. This leads to the inability of the plants to uptake water, a disturbance of ion homeostasis, a decrease in photosynthesis and oxidative stress. As a result of the salt stress, the availability of NADPH decreases. The adaptation to the concentrations of salt depends on plant's ability to compensate for the decreased availability of NADPH, which can be further used in antioxidative cycles and the synthesis of antioxidative compounds and osmoprotectants. In this work, the reduction of relative water content, a decrease in the Rubisco enzyme activity, an increase of Hsp70 in the leaves and an increase in the accumulation of sodium ions was shown in cucumber plants (Cucumis sativa L. convar. Jogger F1) exposed to salt stress (100 mM NaCl). As a consequence of salt stress, an increase in the activity of NADPH providing enzymes was found. Particularly on the second and third day of salt stress, an increase in the activity (up to 270 %) of: NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves was detected. The activity of less abundant NADP-dehydrogenases (glucose 1-dehydrogenase, gluconate 2-dehydrogenase, galactose...
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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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Aggregation of bull seminal plasma protein
Boháček, Hanuš ; Liberda, Jiří (advisor) ; Hýsková, Veronika (referee)
Mammalian fertilization is a sequence of unique and fascinating events, during which seminal proteins are of crucial role. In case of bull (Bos taurus), proteins of seminal plasma (BSP), especially its major component PDC-109, are known to be in aggregated forms, but little is known about mechanism of forming aggregates and their biological function. In present thesis we discovered some interesting properties of PDC-109 and BSP proteins. We found that concentration of these proteins influences their aggregation state significantly, which can be of great biological importance. Separation of seminal proteins by size exclusion chromatography revealed three main fractions denoted I, II and III, with apparent molecular weights of Mr > 150 000, Mr = 30 000 and Mr = 13 000, respectively. In case of PDC-109, molecular weights of theese fractions were retained even after purification procedure, which implies very stable interactions in forming of aggregates. In addition, there was a difference in distribution of PDC-109 glycoforms among fractions, which can be related to the fact, that theese fractions have different sperm membrane binding patterns as we determined by fluorescence microscopy. However, further experiments are needed for better understanding this issue.
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Study of mechanism of fibrin network formation
Božíková, Paulína ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
Serine protease thrombin plays an important role in the process of fibrin network formation by converting fibrinogen into fibrin monomer which spontaneously polymerizes to form fibrin network. The aim of this work was to characterize interactions between thrombin and surface adsorbed fibrin(ogen) or fibrin network to which thrombin can bind and initiate grow of the fibrin network. Activity of thrombin bound on fibrinogen or fibrin was determined spectrophotometrically in a relation to cleaved chromogenic substrate. Using the method of surface plasmon resonance fibrin network formation initiated by thrombin bound to fibrinogen or fibrin was observed. These networks were also visualized by atomic force microscopy. Determined value of affinity constant KD for interaction of fibrinogen in solution with a fibrin network prepared on surface is in agreement with previous experiments in which KD was determined from interaction of surface covalently bound fibrinogen with fibrin monomers in solution.
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The effect of heat shock stress on enzyme activities in plants.
Doričová, Vlasta ; Hýsková, Veronika (advisor) ; Liberda, Jiří (referee)
Heat stress is one of abiotic stress factors, which fundamentally influences the growth and development of plants. Plants response to heat stress by series of cell and metabolic changes, the specificity of heat stress is synthesis of molecular chaperons, called heat- shock proteins (HSP). The influence of heat shock (in the form of 1 hour application of 40řC from 20řC) on the activity of NADP-dependent enzymes, enzymes of Hatch-Slack cycle, glycosidases and the activity of peroxidase in tobacco plants (Nicotiana tabacum L. cv. Petit Havana SR1) was studied in this work. Since HSP are involved in proper folding of immature, misfolded or partly denaturated proteins, in degradation of denaturated proteins and in induction of thermotolerance of plants, the aim of this work was to find out, if the activity of studied enzymes will be maintained or modulated within the plant defense response. The highest amount of HSP70 detected immunochemically together with higher activities of NADP-malic enzyme, phosphoenolpyruvate carboxylase, β-hexosaminidase and α- mannosidase compared to control plants was found 1 hour after application of heat shock. Peroxidase activity was most increased 1 day after HS compared to controls. Using native electrophoresis the differences in isoform content between control and...
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Investigation of the β-N-Acetylhexosaminidase Stucture from Penicillium oxalicum.
Krunclová, Tereza ; Novák, Petr (advisor) ; Hýsková, Veronika (referee)
in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.
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