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Diagnostic potential of piwi-RNA molecules in the process of in vitro fertilization
Šeršeň, Dominika ; Skálová, Lenka (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Dominika Šeršeň, DiS. Supervisor: doc. RNDr. Lenka Skálová, PhD. Consultant: doc. RNDr. Miroslava Rabajdová, PhD. Title of diploma thesis: The diagnostic potentional of piwiRNA molecules in IVF procedure Infertility is currently a prevalent health problem affecting millions of couples of reproductive age worldwide and the use of in vitro fertilization techniques is increasing worldwide every year. For successful in vitro fertilization, it is essential to select a good quality embryo. Despite advances in in vitro fertilization, there is still an insufficient amount of non-invasive and reliable biomarkers for the selection of embryos with the highest developmental and implantation potential. Small non-coding RNAs, specifically piwiRNAs (piRNAs) and microRNAs (miRNAs), secreted into the extracellular environment, which is the culture medium, appear to be suitable biomarkers. This work describes the evaluation of the quality of the embryos, their morphological features on individual days as well as the annotation system itself using the TimeLapse system. The next chapter describes the composition of the media suitable for the cultivation of embryos and their analysis at the omic level, focusing on...
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Metabolism and transport of 6-prenylnaringenin in vitro
Horní, Martin ; Boušová, Iva (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martin Horni Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Consultant: Mgr. Michaela Šadibolová Title of diploma thesis: Metabolism and transport of 6-prenylnaringenin in vitro Prenylflavonoids constitute a group of plant secondary metabolites with various positive effects on human health. Hops (Humulus lupulus L.) represent a rich source of prenylflavonoids including 6-prenylnaringenin (6-PN). Recently, 6-PN has been found to possess various biological effects including analgesic, antiproliferative, and estrogen-modulating activity. Despite its potential to be used as a nutraceutical, the pharmacokinetic properties of 6-PN are still unknown. This thesis had therefore two objectives. The first objective was to determine the hepatic and intestinal metabolism of 6-PN in humans as well as its hepatic metabolism in rats and mice in vitro using precision-cut tissue slices (PCTS). Samples were analyzed using UHPLC-MS/MS. 6-PN was efficiently metabolized predominantly into phase II metabolites with four glucuronides and four sulfates identified in the studied PCTS. For the first time, a novel metabolite of 6-PN formed as a result of concurrent glucuronidation and sulfation has been identified, which...
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Diagnostic potential of piwi-RNA molecules in the process of in vitro fertilization
Šeršeň, Dominika ; Skálová, Lenka (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Dominika Šeršeň, DiS. Supervisor: doc. RNDr. Lenka Skálová, PhD. Consultant: doc. RNDr. Miroslava Rabajdová, PhD. Title of diploma thesis: The diagnostic potentional of piwiRNA molecules in IVF procedure Infertility is currently a prevalent health problem affecting millions of couples of reproductive age worldwide and the use of in vitro fertilization techniques is increasing worldwide every year. For successful in vitro fertilization, it is essential to select a good quality embryo. Despite advances in in vitro fertilization, there is still an insufficient amount of non-invasive and reliable biomarkers for the selection of embryos with the highest developmental and implantation potential. Small non-coding RNAs, specifically piwiRNAs (piRNAs) and microRNAs (miRNAs), secreted into the extracellular environment, which is the culture medium, appear to be suitable biomarkers. This work describes the evaluation of the quality of the embryos, their morphological features on individual days as well as the annotation system itself using the TimeLapse system. The next chapter describes the composition of the media suitable for the cultivation of embryos and their analysis at the omic level, focusing on...
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The optimization of fenbendazole extraction from environmental samples
Brodská, Denisa ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Denisa Brodská Supervisor: doc. Ing. Petra Matoušková, Ph.D. Consultant: Mgr. Martina Navrátilová Title of bachelor thesis: The optimization of fenbendazole extraction from environmental samples Anthelmintics are drugs used for the treatment and prophylaxis of infectious diseases caused by parasitic worms. In the Czech Republic, they are mainly used in veterinary medicine. During the biotransformation of the active substance, metabolites are formed, which are excreted together with the parent substance in faeces and urine. Fenbendazole and other similar substances can be absorbed into the soil in this way and subsequently act on non-target organisms in the environment. Fenbendazole is widely used in livestock farming, and since animal excrements are often further used for fertilization, the risk of the content of medicinal substances should be strictly monitored. For this purpose, it was necessary to develop and optimize the extraction method and also to carry out the corresponding validation of the analytical method, which will subsequently be applicable to real samples in the environment. The assumption of absorption of fenbendazole and its metabolic products, fenbendazole sulfoxide and...
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Metabolomic analysis of bile acids in various biological samples.
Hadravská, Pavlína ; Skálová, Lenka (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Pavlína Hadravská Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: Metabolomic analysis of bile acids in various biological samples This thesis aimed to establish a suitable qualitative and quantitative analysis of selected bile acids in mouse liver, human plasma and especially in human hair. Nine selected bile acids were analyzed (cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, taurocholic acid, taurodeoxycholic acid, glycocholic acid and glycochenodeoxycholic acid). An identification and a quantification of the bile acids have been performed by UHPLC with two types of hybrid mass spectrometers (Quadrupole connected to Time of Flight and Quadrupole connected to Orbitrap). We tried to develop a method that allows a detection of both unconjugated bile acids and their glycine- and taurine-conjugates in biological samples. To develop and optimize this method, mouse liver and human plasma were used. Afterwards, the method was used to detect bile acids in human hair, because the bile acids could serve as...
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The impact of selected sesquiterpenes on antioxidant enzymes in HT-29 cell line
Junková, Martina ; Raisová Stuchlíková, Lucie (advisor) ; Jansová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martina Junková Tutor: RNDr. Lucie Raisová Stuchlíková, Ph.D. Title: Effect of selected sesquiterpenes on antioxidant enzymes in the HT-29 cell line Colorectal cancer is one of the most common cancer type in Czech republic. The oncological treatment causes frequent side effects, what result in hard accepting by patients. There is also emerging resistance to classical chemotherapy (5-fluorouracil). During searching new alternatives, some promising natural substances became a subject of researches. The subtropical tree Myrica rubra and its extracts contain many biologically active substances, which are studied among others for their anti-cancer and antiproliferative effects. β-caryophyllene (BCP), β-caryophyllenoxid (CAO), α- humulene (HUM), trans-nerolidol (NER) and valencen (Val) belong to main sesquiterpenes in this essential oil Myrica rubra. In our study, effect of essential oil Myrica rubra, HUM and NER, in concentrations of 25 and 50 μg/ml, were examined after a 24-hours incubation of the HT-29 cell line on the activity of antioxidant enzymes, their transcription and amount of proteins. Monitored enzymes were glutathione peroxidase (GPx), glutathione reductase (GR),...
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HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone
Novák, Filip ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Filip Novák Supervisor: Ing. Petra Matoušková, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone Scoparone is an active ingredient of Artemisia scoparia, a medicinal plant used in traditional Chinese medicine. It has been studied for various pharmacological effects such as upregulation of conjugation enzymes included in excretion of bilirubin, reduction of proinflammatory cytokines, lowering of plasma lipids levels and inhibition of platelet aggregation. In this thesis, metabolism of scoparone was studied by LC-MS method using Q-ToF device. Scoparone was incubated with liver microsomes obtained from 6 different mammal species to study in vitro oxidation. In total, six metabolites were detected in the incubation samples. Scopoletin and isoscopoletin were identified as major metabolites in every species, however, the rates of scoparone oxidation as well as a ratio of formed isoscopoletin and scopoletin varied. Furthermore, in vivo metabolites in human were studied in urine samples obtained from...
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Effect of monepantel on the expression of selected enzymes in Haemonchus contortus
Jelenková, Klára ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Jelenková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of monepantel on the expression of selected enzymes in Haemonchus Contortus Barber's pole worm (Haemonchus contortus) is parasitic nematode which is the cause of haemonchosis, worldwide distributed disease of small ruminants leading to decrease in productivity and in many cases even to animals death. For prevention and treatment of this disease are used drugs for a long time - anthelmintics. Due to their frequent use and lack of innovation in this group of drugs parasites have gradually built an extensive resistance to these drugs. New hope in therapy of haemonchoses is monepantel, drug belonging to aminoacetonitrile derivatives which have unique mechanism of action. This drug was launched more than 25 years after registration of the last drug from the group of anthelmintics. Despite the initial great success information about occurrence of resistance often appear in recent years. Current knowledge about the mechanisms of resistance development and the factors that influence them is as well as information about drug action at the molecular level still insufficient. To limit the development of...
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HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone
Novák, Filip ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Filip Novák Supervisor: Ing. Petra Matoušková, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone Scoparone is an active ingredient of Artemisia scoparia, a medicinal plant used in traditional Chinese medicine. It has been studied for various pharmacological effects such as upregulation of conjugation enzymes included in excretion of bilirubin, reduction of proinflammatory cytokines, lowering of plasma lipids levels and inhibition of platelet aggregation. In this thesis, metabolism of scoparone was studied by LC-MS method using Q-ToF device. Scoparone was incubated with liver microsomes obtained from 6 different mammal species to study in vitro oxidation. In total, six metabolites were detected in the incubation samples. Scopoletin and isoscopoletin were identified as major metabolites in every species, however, the rates of scoparone oxidation as well as a ratio of formed isoscopoletin and scopoletin varied. Furthermore, in vivo metabolites in human were studied in urine samples obtained from...
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