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Stady of apple proliferation phytoplasma diversity in the Czech Republic.
STREJČKOVÁ, Monika
Phytoplasmas are prokaryote organisms of the Mollicutes class. So far they have been described on more than 800 plant species. Apple proliferation phytoplasma, which classifies in the ribosomal subgroup 16Sr X-A (Apple proliferation, AP, ?Candidatus Phytoplasma mali?), belongs amongst the agriculturally most important phytoplasmas. AP is considered a quarantine organism in Europe and in North America. Common symptoms caused by AP are as follows: notably enlarged stipules, which are morphologically like real leaves ? they have petiole and lamina; the edge of the stipules is irregularly dentate small fruits with fewer seeds and reduced flavour quality premature reddening of leaves, witches' broom appearance, abnormal growth of shoots in the autumn, repeated blossoming DNA isolated from trees suspected to have AP present was tested in the presented thesis. The trees originated from the gene pool, mass production planting as well as from planting of small producers. To detect phytoplasma, the polymerase chain reaction (PCR) was used with the subsequent analysis of restriction fragment length polymorphism (RFLP). To detect and differentiate AP phytoplasma subtypes, direct PCR with primers rpAP15f/rpAP15r amplifying the DNA of ribosomal proteins rpS3 and rpl 22 with subsequent restriction breakdown by AluI enzyme.
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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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Aspects of Gene Regulation of CYP3A4 in Hepatic Tissue.
Krausová, Lucie ; Štaud, František (advisor) ; Mičuda, Stanislav (referee) ; Skálová, Lenka (referee)
1 Summary CYP3A4 is an important enzyme involved in elimination of majority of metabolized xenobiotics. It plays a major role in the detoxification system of the human body, therefore it is responsible for many drug-drug interactions (DDIs). DDI present a complication of current pharmacotherapy, in the extreme they can lead in failure of therapy or in life-threatening toxic effects. DDIs are caused by changes in enzymatic activity of CYP3A4, which is highly variable among individuals. An important mechanism of modulating CAP3A4 activity is the regulation of inducible transcription by nuclear receptors, especially PXR, CAR and GR. The structure of CYP3A4 promoter and mechanisms of transcriptional regulation has been studding intensively for many years, but the research of relationship of nuclear receptors and transcriptional cofactors in CYP3A4 transactivation is still incomplete. Present work contributes to elucidation of some questions concerning the effects of azole antimycotics on CYP3A4 transcription via PXR, potency of valproic acid to activate PXR and CAR or determinants of CYP3A4 expression via GR in placental cells. The experiments were performed with up-to-date molecular biology methods and using in vitromodels of the primary human hepatocytes and hepatoma cell lines. To the aims of the doctoral...
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Study of the Function of Reproductive Barriers for Use in Vivo and in Vitro Models
Pospěchová, Kateřina ; Semecký, Vladimír (advisor) ; Jirásek, Jan Evangelista (referee) ; Stingl, Josef (referee)
Summary: The main limiting factor of distribution of the drugs in the body is the presence of physiological barriers. Placental and blood-testis-barrier are two barriers that were studied in this thesis. The placenta is an important endocrine organ bringing maternal and fetal blood into proximity, allowing exchange of nutrients and waste products. In addition, the placenta acts as the barrier between the mother and fetus, and plays an important role in fetal protection. The key rate-limiting layer in the placenta is the single layer of syncytiotrophoblast. The placental trophoblast contains multiple drug transporters and metabolizing enzymes that control maternofetal exchange of nutrients and hormones, or which form placental "metabolic" barrier. Moreover, several members of the nuclear receptor superfamily are expressed in placental trophoblasts. They regulate transcription of the genes involved in nutrient transport, mineral metabolism, proliferation and differentiation. Moreover it was suggested that they control expression of placental drug transporters and xenobiotic metabolizing enzymes. Germ cells in the testis are protected mainly by the presence of blood testis barrier, formed by the tight or adherens junctions between Sertoli cells. Moreover, intercellular junctions between Sertoli and germ cells...
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Study of biodegradation of poly(hydroxy alkanoates).
Wurstová, Agáta ; Přikryl, Radek (referee) ; Obruča, Stanislav (advisor)
The master‘s thesis is focused on the study of biodegradation of polyhydroxyalkanoates, namely polymer polyhydroxybutyrate. The first part of the thesis is focused on the study of biodegradation of polyhydroxybutyrate in the form of crystalline granules of PHB and PHB films using selected species of microorganisms from bacteria, yeasts and fungi. As a representative of bacteria was chosen microorganism Delftia acidorovans, as yeast was selected Aureobasidium pullulans and Aspergillus fumigatus as fungi. PHB depolymerase activity was measured employing turbidemtiric method with suspension of PHB granules as substrate. The results showed that D. acidorovans can partially degrade PHB. On the contrary A. pullulans cannot effectively degrade PHB. The most significant degradation ability revealed A. fumigatus, which was able to degrade PHB completely. Extracellular enzymes excreted by these microorganisms when cultivated on PHB materials as sole carbon sources were analyzed by SDS-PAGE. The second part of the thesis deals with the biodegradation of PHB in the form of PHB film, PHB hardened foil and PHB Nanoul fabric using standard composting test. Semi-solid cultivation showed positive results. In the interval from 14 days to two months were all forms of the PHB completely biodegraded. With semi-solid cultivation was also studied biodegradation rate of the polyurethane elastomeric films which were modified by partial replacement of polyester polyol by PHB. The test samples were prepared using PHB from Sigma and the PHB samples prepared at the Faculty of chemistry VUT. Samples with different concentrations of the dispersed PHB (1 %, 5 % and 10 %) in the polyurethane were also object of the study. At the end of the cultivation (after 2 months) were measured mechanical properties in tension of the material, then efficiency of biodegradation by gravimetric analysis and modification of the material surface by microscopic analysis.
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Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (referee) ; Hermanová, Soňa (advisor)
Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.
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The tools for study of cholesterol in biological membranes
Koukalová, Alena ; Nováková, Olga (referee) ; Černý, Jan (advisor)
Cholesterol is one of the major membrane component in most eukaryotic cells, where it performs multiple functions. Cholesterol regulates the fluidity of cell membranes, influences the activity of several membrane proteins and is the precursor of many steroid hormones and bile acids. Cholesterol is not a fluorophor or chromophor on its own, therefore it is necessary to visualize it to study its localization and distribution in cells. Reporter molecules that can be used for this purpose can be classified in two groups: specific cholesterol binding probes and cholesterol analogues. Among specific binding cholesterol probes are cholesterol oxidase enzyme that catalyses the reaction of cholesterol and oxygen into the products cholest-4-en-3-one and hydrogen peroxide and is used to study membrane organization and cholesterol flip-flop kinetics in plasma membrane, the polyene filipin forms fluorescent complex with cholesterol and is usually used to gain information about cholesterol distribution and localization in cells. Thiol-activated cytolysins and a derivate of perfringolysin O - BCθ-toxin - are suitable for recognizing cholesterol-rich domains and localization cholesterol in membrane. Cyclodextrins (especially methyl-β-cyclodextrin) selectively extract cholesterol from membranes and they can be used as...
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Study of β-N-acetylhexosaminidase in tobacco plants
Valenta, Robert ; Tichá, Marie (referee) ; Ryšlavá, Helena (advisor)
β-N-acetylhexosaminidases are ubiquitous in all living organisms, but there is little information about these enzymes in plants, especially in leaves. Suggested functions of plant β-N-acetylhexosaminidases are participation in defence responses against fungal and other pathogens and also in degradation of storage glycoproteins. β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was purified to final specific activity 190 nmol min-1 mg-1 with p-NP-GlcNAc as substrate. Precipitation by ammonium sulphate, gel filtration chromatography on Sephacryl S-300 column and affinity chromatography on Con A- Sepharose column were used. Michaelis constant and maximal reaction rate of β-N- acetylhexosaminidases determined for p-NP-GlcNAc was 0.33 mM and 414 nmol min-1 mg-1 , respectively. The cleavage of diacetylchitobiose catalyzed by β-N-acetylhexosaminidase was studied using capillary electrophoresis. Determined activity of β-N-acetylhexosaminidase for diacetylchitobiose was more than 10-times lower compared to β-N-acetylhexosaminidase activity for p-NP-GlcNAc. These results indicate that chitobiose and probably other chitooligomers are not natural substrates of β-N-acetylhexosaminidase and thus this enzyme is most likely not involved in protection against fungal pathogens. Natural substrate...
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Study of plant stress responces in presence of pharmaceuticals in cultivation medium
Bystroňová, Jana ; Soudek, Petr (advisor) ; Podlipná, Radka (referee)
The aim of this study was to verify the possibility of ibuprofen degradation by selected plant cultures and determination of activities of antioxidant enzymes (peroxidase, catalase, ascorbate peroxidase and glutathione-S-transferase) as markers of oxidative stress caused by ibuprofen. Nicotiana tabaccum (cv. La Burley 21, cv. SR 1 and their GMOs) and Nicotiana glauca were used as experimental plants. The rate of removal of ibuprofen tested by tobacco was decreasing in the following order: N. tabaccum SR1 > N. tabaccum Zm-P60-1-T4 > N. tabaccum TRI 2T2 > N. glauca > N. tabaccum TRI 2T1 > N. tabaccum cv. La Burley > N. tabaccum Zm-P60-1-T5. As the most suitable tobacco for the removal of ibuprofen seemed untransformed N. tabaccum SR1. The long-term experiment showed that plant stress is being manifested even after longtime. N. tabaccum cv. La Burley 21 seemed to be the most tolerant to ibuprofen in compare with the total enzyme activities in cultures with the presence of ibuprofen and controls. N.glauca was the least tolerant cultivar. Keywords: phytoremediation, ibuprofen, Nicotiana tabaccum, Nicotiana glauca, HPLC, peroxidase, catalase, ascorbate peroxidase, glutathion-S-transferase
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