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Introduction of the MST method for the study of interactions between biomolecules
Krátký, Josef ; Szotáková, Barbora (advisor) ; Boušová, Iva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Josef Krátký Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Introduction of the MST method for the study of interactions between biomolecules MicroScale thermophoresis (MST) is currently a recognized method for studying interactions between biomolecules; the method is using directed movement of biomolecules in a temperature gradient, which allows to analyze practically all interactions where changes in analyte properties are possible. This thesis was focused on introduction of the MST method for research of protein interactions with low molecular weight substances focusing on verifying the possibility of using MST in monitoring the interaction of glutathione S-transferase (GST) with selected terpenes ((-)-fenchone, (-)-isopulegole, (+)-isomenthone, (-)-carvone, (+)-carvone, piperitone, and thujone). Another aim was to determinate the properties of terpenes by classical spectrofluorimetric methods and MST. Using spectrofluorimetric methods it was found that terpenes affect the fluorescence of human serum albumin, and they do not affect the fluorescence spectrum of the dye NHS, which is used as a dye for samples for MST. Next part of experimental work was to determine...
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Effect of selected monocyclic monoterpenes on the activity and expression of antioxidant enzymes in human liver
Fišerová, Romana ; Boušová, Iva (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Romana Fišerová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of diploma thesis: Effect of selected monocyclic monoterpenes on the activity and expression of antioxidant enzymes in human liver Monocyclic monoterpenes belong to plant secondary metabolites, which are characterized by their aroma and possess a wide range of use. They attract pollinators and are involved in plant communication as well as in the cosmetic and food industries. They are ingredients of spices, beverages, and food. In some of them, anti-cancer activity has been proven, however, we should keep in mind that some of them exert toxic properties in various organs of the organism, mainly in the liver. The aim of this thesis was to find out what effect have (+)- carvone, (-)-carvone, (+)-isomenthone, (-)-isopulegol, and piperitone on the activity and mRNA expression of antioxidant enzymes in human liver. The liver tissues obtained from patients of both sexes and 50-80 years old were used as biological material. At first, the effect of the selected monoterpenes (100 µM) on the activity of the enzymes glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferase (GST) in subcellular...
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Effect of polyphenon on selected biotransformation enzymes in mice
Skorkovská, Jana ; Skálová, Lenka (advisor) ; Boušová, Iva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Jana Skorkovská Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of Diploma Thesis: Effect of polyphenon on selected biotransformation enzymes in mice The green tee has been used in the world for centuries. Main substances in green tee include polyphenol compounds, for example catechines. Lately, possible positive effects of green tea and its substances on organisms have been studied intensively. Reduction of obesity and obesity related pathological conditions belongs among these effects. The aim of this diploma thesis was to study the effects of green tee abstracts (polyphenon) on activity and expression of selected biotransformational enzymes in normal and in obese mice. Measuring was performed in subcellular fractions of liver and small intestine homogenates of male mice. Out of 8 experimental groups two were the control groups (a group of normal and a group of obese mice who had not polyphenon in diet). Three groups of normal mice and three groups of obese mice were fed (over 3 or 28 days) with diet containing polyphenon in two different concentrations (0,1 and 0,01 %). Results showed that three-day administration of polyphenon (0,1 % concentration in diet) significantly increased...
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Biological activity of antioxidant compounds in monocytes THP-1
Hájek, Jan ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University in Prague University Roma Tre Faculty of Pharmacy in Hradec Králové Department of Biology Department of Biochemical Sciences Candidate: Jan Hájek Supervisor: PharmDr. Iva Boušová, Ph.D. Prof. Sandra Incerpi, Prof. Jens Z. Pedersen Title of diploma thesis: Biological activity of antioxidant compounds in monocytes THP-1 Elaboration of this work is based on the experiments carried out in the Laboratory of Physiology at University Roma Tre in Rome, under the supervision of Prof. Sandra Incerpi. The team of this laboratory studies the antioxidants of different structures and evaluates their ability to prevent the production of reactive oxygen species (ROS). These reactive species are essential for many physiological processes, but they can also have deleterious effects and participate in the development of various diseases. In this work, I dealt with a study of antioxidant properties of different polyphenolic compounds (mosloflavone, negletein, 5,6-dihydroxyflavone, baicalein, gallic acid, and compound 1625) in the cell culture of human monocytes THP-1 and also in the in vitro test. Three different methods were used: intracellular ROS determination (dichlorofluorescein assay), cytotoxicity assay, and electron paramagnetic resonance spectroscopy (EPR). Mosloflavone showed scavenging...
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Modification of selected biotransformation enzymes during ageing (in rat)
Vyskočilová, Erika ; Boušová, Iva (advisor) ; Svobodová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Erika Vyskočilová Supervisor: PharmDr. Iva Boušová, Ph.D. Title of diploma thesis: MODIFICATION OF SELECTED BIOTRANSFORMATION ENZYMES DURING AGEING (IN RAT) Ageing is an inevitable biological process characterized by progressive functional decline of all organ systems with simultaneous increase in oxidative damage as a result of reactive oxygen species (ROS) accumulation. Increased activity of antioxidant enzymes, such as peroxidase and catalase, is related to increased production of ROS during ageing. Activity of biotransformation enzymes such as cytochrome P450 (CYP) and glutathion-S-transferase (GST) changes during ageing too. In general, activity of enzymes is lower in very young and very old individuals than in the period of adulthood. The aim of the present work was to study age-related changes in activities and expression of selected xenobiotic-metabolizing enzymes (GST, CYP 1A, CYP 2B, CYP 3A4) and antioxidant enzymes (catalase, peroxidase, superoxiddismutase) in rat liver. Male Wistar rats were sacrificed by decapitation under ether anesthesia at the age of 6 weeks and 21 months (6 individuals in each group). Subsequently, liver specimens were processed to obtain the subcellular...
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Analysis of arginine and its methylated derivatives in clinical samples using LC-MS/MS
Prokop, Pavel ; Boušová, Iva (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Prokop, DiS. Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of diploma thesis: Analysis of arginine and its methylated derivatives in clinical samples using LC-MS/MS Asymmetric dimethylarginine (ADMA) and its enantiomer, symmetric dimethylarginine (SDMA), are naturally occurring amino acids that are generated intracellularly from L-arginine. ADMA is an endogenous competitive inhibitor of nitric oxide synthase. Increased levels of ADMA and SDMA are associated with endothelial dysfunction, hypertension, cardiovascular and renal diseases. Both compounds are accumulating in chronic kidney disease (CKD) patients. The aim of this diploma thesis was to develop and validate a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of L-arginine, ADMA and SDMA in human plasma. Subsequently, LC-MS/MS method was compared with enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography with fluorescence detection (HPLC-FL) method. Chromatographic separation was performed on Dionex UltiMate 3000 UHPLC liquid chromatograph, followed by tandem mass spectrometric detection on amaZon SL mass spectrometer. L-Arginine-13 C6, ADMA-d6 and...
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Selection and validation of reference genes for relative mRNA quantification in human liver slices
Studenovská, Petra ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Petra Studenovská Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Selection and validation of reference genes for relative mRNA quantification in human liver slices The use of precision-cut liver slices is often employed in vitro system in the biochemical, pharmacological and toxicological studies. This is mainly due to the preservation of the cellular tissue architecture and therefore its functions. The aim of this diploma thesis was to select and validate reference genes for the relative quantification of mRNA in human liver slices. Liver tissues obtained from 3 patients of different age, diagnosis and pharmacotherapy were used as samples. Liver slices, prepared from samples of liver tissue, were exposed to dimethyl sulfoxide (control) or known inducers of cytochrome P450, rifampicin (10 µM) and β-naphthoflavone (10 µM). Subsequently, the stability of the expression of reference genes was verified using RefFinder program, which integrates the programs of the geNorm, BestKeeper, NormFinder and comparative ΔCT method. The optimal number of reference genes that should be used to evaluate the relative gene expression of target genes was determined using the geNorm program....
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Interactions of antiretroviral drugs with membrane transporters
Martinec, Ondřej ; Červený, Lukáš (advisor) ; Boušová, Iva (referee) ; Chládek, Jaroslav (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Ondřej Martinec Supervisor: Assoc. Prof. PharmDr. Lukáš Červený, Ph.D. Title of doctoral thesis: Interactions of antiretroviral drugs with membrane transporters Oral delivery is the most common, convenient, and economical form of drug administration. Absorption of orally administered drugs occurs mainly in the intestine. Intestinal absorption can be reduced by the activity of efflux drug ABC transporters, mainly p-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), located in the apical membrane of the intestinal epithelium. HIV-infected patients are dependent on lifelong pharmacotherapy, which includes a combination of three or more antiretroviral drugs. Hepatitis C (HCV) is a common co-infection of HIV. In addition, the HIV-positive population is aging, which is associated with burden of other comorbidities. This results in an indication of polypharmacy and thus an increased risk of drug-drug interactions. Many antiretroviral drugs used are substrates, inhibitors and /or inducers of ABCB1, so they might quantitatively affect the intestinal absorption of co-administered drugs (ABCB1 substrates), thereby affecting the efficacy/safety of treatment. As part of this...
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Pistacia lenstiscus essential oil: in vitro testing of cytotoxicity and anti-inflammatory activity
Elstnerová, Marie ; Szotáková, Barbora (advisor) ; Boušová, Iva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Marie Elstnerová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Pistacia lentiscus essential oil: in vitro testing of cytotoxicity and anti-inflammatory activity This project, which has been initiated in cooperation with University of Sassari (Italy), is aimed to find out more details about biological activity of essential oil from Pistacia lentiscus. We want to contribute to the decision, if this essential oil can be used as a part of oral health products. The essential oil was prepared from leaves of wild occurring plants growing in North Sardinia. The cytotoxicity and anti-inflammatory activity of not only the pure essential oil but also of the lecithin nanoemulsion with P. lentiscus oil and Tween 80 nanoemulsion with P. lentiscus oil and moreover, the two most abundant terpenes in the essential oil, (−)-α-pinene and (−)-terpinen-4-ol, were tested. The cytotoxicity was measured by the colorimetric assay using tetrazolium salt WST-1 on four oral cell lines (gingival fibroblasts, periodontal ligament fibroblasts, dysplastic oral keratinocytes and primary gingival keratinocytes). The viability of human oral cells was the most diminished by lecithin nanoemulsion, pure...
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In vitro effects of 3-hydroxytyrosol on renal hypoxia and inflammation
Kamasová, Terézia ; Boušová, Iva (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Terézia Kamasová Supervisor: Asst. Prof. PharmDr. Iva Boušová, Ph.D. Prof. Alice Santos-Silva Maria João Valente, Ph.D. Title of diploma thesis: In vitro effects of 3-hydroxytyrosol on renal hypoxia and inflammation Chronic kidney disease (CKD) results from a group of heterogeneous disorders affecting the kidneys. The renal hypoxia and hypoxia-derived oxidative stress, renal fibrosis, and inflammation are highly prevailing conditions appearing in the diseased kidney, contributing to the progression of CKD. Phytochemicals are an essential part of contemporary therapeutic strategies for the treatment of various diseases. 3-Hydroxytyrosol (HT), a phenolic compound extracted from olives and olive-derived products (e.g. olive oil), is believed to carry a potent antioxidant, anti-inflammatory, antithrombotic, bactericidal and bacteriostatic activity. The aim of this work was to determine the preventive effect of HT in hypoxic renal cells and evaluate the effect of HT on hypoxia-related inflammation, fibrosis, and oxidative stress, in order to summarize the value of this phenolic compound as a promising novel remedy in the treatment of CKD. A cell line of human renal proximal tubular cells (HK-2) was...
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