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Panels of steroid receptor reporter cell lines for compound profiling and development of selective ligands for estrogen receptor alpha and beta
Sedlák, David ; Bartůněk, Petr (advisor) ; Anzenbacher, Pavel (referee) ; Kasal, Alexander (referee)
of the Ph.D. thesis Steroid hormone receptors represent a major target in the drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. The first part of this work describes a preparation of two panels of potent, selective and robust luciferase reporter cell lines on the unified cellular background in U2OS osteosarcoma cell line. This system consists of two panels of stable luciferase reporter cell lines for estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR). The first panel of reporter cell lines relies on the expression of the chimeric steroid receptors created by the replacement of the N-terminal part of the steroid receptor molecule by Gal4 DNA binding domain (Gal4 DBD) binding to 9 copies of Gal4 upstream activation sequences (Gal4 UAS) in the promoter of the pGL4 luciferase reporter vector. In the second panel of reporter cell lines the activation of either synthetic promoter containing multiple hormone response elements or viral promoter derived from MMTV LTR is mediated by full- length exogenously expressed steroid receptors. We have extensively validated both panels using 28 well established ligands, carefully...
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The Usage of Separation Methods for Research of Biologically Active Substances in Waters
Vydrová, Lucie ; Friedl, Zdeněk (referee) ; Opatřilová, Radka (referee) ; Chýlková,, Jaromíra (referee) ; Vávrová, Milada (advisor)
Pharmaceuticals are biological active compounds with different functional groups, physico-chemical and biological properties. These chemical compounds are called as “new contaminants” which cumulate in various environmental components. These contaminants input to environment from industrial processes, hospitals and health care institutions or household sources and these pollutants can to negatively interact with environmental components. Pharmaceuticals are separated according to structure and their effects to live organisms. On the basis of drug consumption in Czech Republic the non-steroidal anti-inflammatory drugs are the most using pharmaceuticals, because these drugs can be obtain without prescriptions. The choosing analgesics for study were monitored in waste water from waste waters treatment plant (WWTP Brno – Modřice) and in surface water from two sampling sites of river Kretinka. Pharmaceuticals were monitored in surface waters in Czech Republic, in Scotland (river Thurso) and in Taiwan (river Erren, Agongdian, Yanshuei and canal of Tainan) Solid phase extraction (SPE) is used for extraction of study pharmaceutical from water system. The high performance liquid chromatography (HPLC) with diode array detection or mass spectrometry was used for determination of drugs in surface waters from Czech Republic. The water samples from Scotland and Taiwan were analysed and the high performance liquid chromatography with mass spectrometry (HPLC/MS) was used for determination of pharmaceuticals. The method of HPLC/MS enables the determination of study compounds in the very low range of concentrations (ng ? l-1). All monitored pharmaceuticals were identified and quantified in water samples from river Kretinka in Czech Republic, river Thurso (Scotland) and rivers Erren, Agongdian, Yanshuei and canal of Tainan in Taiwan and the obtained data were compared.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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Vliv vybraných kardiovaskulárních léčiv nalézaných ve vodním prostředí na ryby
STEINBACH, Christoph Antonius
Cardiovascular pharmaceuticals are among the most prescribed drugs. As a result of the high consumption, these pharmaceuticals have been frequently detected in waste and surface waters. Verapamil, diltiazem and atenolol are very important representatives of cardiovascular pharmaceuticals; therefore, the present research focused on their acute and sub-chronic effects, bioconcentration, half-life time and metabolism in fish. Moreover, unified protocol for the quantitative assessment of histopathological alterations on the heart ventricle and coronary blood vessels employing heart index calculation was developed with the aim to better assess histopathological changes in fish heart which is one of the targets of cardiovascular pharmaceuticals and other chemicals. The effects caused by high concentrations of the studied substances, verapamil, diltiazem and atenolol, in fish can be considered similar to the therapeutic effects and side effects that are found in humans. The acute exposure to verapamil at the human therapeutic plasma level reduced the heart rate in common carp embryos and larvae. In addition, the acute and chronic exposure to this substance caused peripheral edema and gastrointestinal haemorrhage in carp. Similarly, the histological changes in heart and the blood vessels of the liver in diltiazem exposed rainbow trout suggested vasodilatation similar to the pharmacological effect of diltiazem in the human body. In rainbow trout sub-chronically exposed to atenolol at a human therapeutic blood plasma concentration, histopathological changes in the cardiovascular system were found. The bioconcentration of verapamil, diltiazem and atenolol in fish can be classified as low. Bioconcentration factor (BCF) of verapamil in whole body homogenates of common carp ranged between 6.6 and 16.6. The BCF of diltiazem was also relatively low (0.5-194) in analysed tissues of trout, following the order kidney liver muscle blood plasma. BCF of atenolol in rainbow trout tissues was the lowest among the tested substances (BCF = 0.002-0.27), following the order of liver > kidney > muscle. In the blood plasma, the concentration of atenolol was below the limit of quantification. Verapamil showed a longer half-life time (10.6 days) in fish compared to the human body, indicating the slow rate of biotransformation and/or elimination of verapamil in fish. Estimated half-life times of diltiazem in liver (1.5 h) and kidney (6.2 h) were in the same order of magnitudes as those determined for the human blood plasma. The half-life time of atenolol in trout was not studied, because of its very low bioconcentration. In diltiazem exposed rainbow trout, 8 groups of metabolites of diltiazem with 17 different isoforms were identified using liquid chromatography/high resolution mass spectrometry method. Diltiazem was found to undergo a biotransformation involving desmethylation, desacethylation and hydroxylation in fish. These results showed that diltiazem was metabolised in fish in a similar way like in the human body by desmethylation and desacethylation. On the other hand, hydroxylation, which was involved to a minor extent, seemed to be species specific. Verapamil had no effect on early life stages of common carp at the environmentally relevant concentration after one month lasting exposure. On the other hand, atenolol and diltiazem in environmentally realistic concentrations caused after 42-day exposure some physiological changes in rainbow trout. Namely, atenolol affected haematological and biochemical parameters of the blood in exposed rainbow trout and diltiazem caused changes in the activity of antioxidant enzymes in trout liver and gills. These data indicated that atenolol and diltiazem, when present in the aquatic environment, could be a source of sub-lethal detrimental effects in fish.
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Derivatives of Rhodanine as Potential Antifungal Drugs
El Zein, Mariana ; Opletalová, Veronika (advisor) ; Kopecký, Kamil (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Mariana El-Zein Consultant: Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. Title of Thesis: Derivatives of Rhodanine as Potential Antifungal Drugs Rhodanine (2-thioxo-1,3-thiazolidin-4-one) forms the basic skeleton of many biologically active substances and potential drugs. Antifungal properties of rhodanine derivatives have been studied since the early 1950s. Theoretical part of the thesis deals with mycoses and drugs that are currently used to treat them. The aim of the experimental work was to find suitable reaction conditions for Mannich reaction of (5Z)-5-(subst.)benzylidenes-2-thioxo-1,3-thiazolidin-4-ones with various amines. The attempts to prepare Mannich bases using diethylamine as the basic component were unsuccessful. (5Z)-5-benzylidene-3-(morpholin-4-yl-methyl)-2-thioxo-1,3-thiazolidin- 4-one was successfully obtained by treating (5Z)-5-benzylidene-2-thioxo-1,3-thiazolidin- 4-one with formaldehyde and morpholine in dimethylformamide. This compound was reported previously and exhibited antibacterial activity and acceptable toxicity. Hence, Mannich bases derived from (5Z)-5-arylalkylidene-2-thioxo-1,3-thiazolidin-4-ones deserve further attention as potential...
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Dipentaerythritol branched oligoesters as adhesive carriers of drugs
Doležalová, Kateřina ; Šnejdrová, Eva (advisor) ; Dittrich, Milan (referee)
The aim of this work was to study disolution and adhesive characteristics of branched lactic and glycolic acid oligoesters. The theoretical part describes biomaterials and bioadhezion. Dipenthaerythritol branched oligoester carriers were plastified 20% triethylcitrate. Adhezive properties were measured by material testing machine Zwick/Roel T1-FR050TH.A1K. The maximum force (Fmax), required to detach the polymer carrier from substrate, was measured for studying this material. Solution of mucin was used as a substate for adhesion. All studied oligoesters had sufficient adhesive properties. There was not any significant influence of incorporated drug. Acyclovir and fluconazole release from plastified carriers was tested. Liberation was tested at 37řC to the phosphate-citrate medium. Concentration of drug in medium was measured by spectrophotometer. There was an increase of drug liberation caused by decreasing of molar weight Mw. Oligoester 3D shown advantageous properties, it liberated acyclovir for 21 days with zeroth order kinetics
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Arts educations as a part of prevention of drug addiction among children in kindergarten
Vohradská, Dana ; Jebavá, Jana (advisor) ; Bednář, Miloš (referee)
Title: Arts education as a part of prevention of drug addiction among children in kindergarten. Objectives: The aim of this thesis is to evaluate the degree of importance of education among preschool children through art activities. This form of education can prevent potential drug addiction. Prepare and implement a short project on the theme - fairy tales and games against drug addiction. The output is a methodical list for teachers in kindergartens. Method: Study of literature, Literature review, Compilation, Analysis, Consultations in primary prevention centre Filia, Build-up of Primary Prevention Project, its implementation, evaluation and preparation of methodological material for teachers in kindergartens. Results: The prevention of drug addiction must begin within pre-school children. Art educations, as art, literary, dramatic, musical and dancing, form the main axis of primary prevention. Evaluation criteria are the attention, level of concentration, reaction of children and their artifacts. Keywords: The drug, Pre-school age and its specifics, Games and Rituals in a kindergarten, Arts Education, Primary Prevention.
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A study of the properties of tablets made of directly compressible sorbitol.
Zemanová, Eva ; Mužíková, Jitka (advisor) ; Šnejdrová, Eva (referee)
A study of the properties of tablets made of directly compressible sorbitol. SUMMARY : The thesis deals with the study of the properties of tablets from two types of directly compressible sorbitol, namely SorbitabTM SD 250 a SorbitabTM SD 500. The focus of the study was the dependence of the tensile strength and disintegration time of the tablets on the compression force, the addition of lubricants magnesium stearate and sodium stearyl fumarate (Pruv ), with concentrations of 0,5% and 1% and a 50% addition of model active ingredients, namely acetylsalicylic acid and ascorbic acid. The compression forces were used 3, 4,5 and 6 kN; tablets containing the drugs were compressed only with a force of 6 kN. The tensile strength of tablets from SorbitabTM SD 500 was higher than of tablets from SorbitabTM SD 250. SorbitabTM SD 500 has demonstrated sensitivity to addition of both lubricants in both concentrations, unlike SorbitabTM SD 250, when the tensile strength of tablets decreased only with the addition of 1% magnesium stearate. The disintegration time of tablets was prolonged by the lubricants, in the case of tablets with SorbitabTM SD 500 most with 1% magnesium stearate at all compression forces. Higher tensile strength was found for tablets containing acetylsalicylic acid and for both drugs with SorbitabTM...
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Acyclovir release from the plasticized branched polyesters
Černohousová, Lenka ; Šnejdrová, Eva (advisor) ; Dittrich, Milan (referee)
This diploma thesis aims to study the liberation profile of the acyclovir from plasticized branched polyesters. Polyesters of D,L-lactic acid and glycolic acid, branched using pentaerythritol, dipentaerythritol or tripentaerythritol in different concentrations, plasticized by 30% trietylcitrate were examined. The theoretical part summarizes the liberation of drugs from different modified drug systems and properties of natural and synthetic degradable polymers. Modified static dissolution test was done at 37řC using phosphate citrate buffer. The acyclovir was released within 24 hours at the tested carriers branched by dipentaerythritol with low molecular weight. Therefore it is suitable for short-term liberation. Carriers branched by pentaerythritol or tripentaerythritol with higher molecular weight proved to be optimal for extended release. Keywords: drug release, acyclovir, branched oligoesters, plasticizers
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Study of chiral aspects of drug metabolism using instrumental analytic methods
Holmanová, Veronika ; Nobilis, Milan (advisor) ; Kovaříková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Candidate: Bc. Veronika Holmanová Supervisor: Doc. PharmDr. Milan Nobilis, CSc. Title of diploma thesis: Study of chiral aspects of drug metabolism using instrumental analytic methods For the evaluation of stereospecificity of rat and human cytosolic carbonyl reductases and activity of microsomal oxidases involved in nabumetone biotransformation, a new chiral high-performance liquid chromatographic method was developed. The prepared LLE-HPLC-PDA method enabled extraction, separation and ultraviolet detection of prochiral nabumetone and its five phase I metabolites including enantiomers of two chiral biotransformation products of carbonyl reduction. Methyl ester of naproxen served as an internal standard. Diethyl ether was used for liquid-liquid extraction (LLE) of biomatrices. Chiralcel OD-R 250 mm×4.6 mm column with a mobile phase methanol-1M NaClO4/HClO4 aqueous solution pH=3 (75:25, v/v) were employed in isocratic sufficient separation of nine analytes. The whole analysis lasted 60 minutes at the flow rate of 0.5 ml/min. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at λ=265 nm). The results of in vitro nabumetone...
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