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Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines
Vagiannis, Dimitrios ; Hofman, Jakub (advisor) ; Čečková, Martina (referee)
4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...
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Analysis of genomic regions bound and regulated by Ataxin-3
Svoreň, Martin ; Čečková, Martina (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martin Svoreň Supervisor: PharmDr. Martina Čečková, Ph.D. Specialized supervisor: PD Dr. Bernd Evert Title of diploma thesis: Analysis of genomic regions bound and regulated by Ataxin-3 Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a dominantly inherited neurodegenerative disease. In SCA3, the disease protein ataxin-3 (ATXN3) contains an abnormally long polyglutamine (polyQ) tract encoded by CAG repeat expansion. ATXN3 binds DNA and interacts with transcriptional regulators pointing toward a direct role of ATXN3 in transcription. It is conceivable that mutant ATXN3 triggers multiple, interconnected pathogenic cascades leading to neurotoxicity, however, the principal molecular pathomechanism remains elusive. Here, PCR analyses of 16 ATXN3-bound genomic regions recently identified by next generation sequencing of immunoprecipitated ATXN3-bound chromatin fragments confirmed enriched binding of ATXN3 to 5 genomic regions next to genes encoding CCAAT/enhancer binding protein delta (CEBPD), period circadian clock-2 (PER2), phosphatase and tensin homolog (PTEN), serine protease inhibitor family F2 (SERPINF2) and thrombospondin-1 (THBS1). To investigate putative...
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Study of the role of nucleoside transporters in intestinal absorption with the use of in vitro transport method based on the Caco-2 cell line.
Ďurišová, Markéta ; Červený, Lukáš (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Mgr. Markéta Ďurišová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of rigorosum thesis: Study of the role of nucleoside transporters in intestinal absorption with the use of in vitro transport method based on the Caco-2 cell line. Perorally used drugs are primarly absorbed in the small intestine. Enterocytes, forming the intestinal barrier, express a large number of transporters, representing both ATP-binding (ABC) and "solute carrier" (SLC) transporters, which can significantly affect the pharmacokinetics of the drugs transmitted by them. For this reason, FDA and EMA are currently focusing on studying these interactions and their effects on drug permeability. The aim of this thesis was to investigate whether the equilibrative nucleoside transporters (ENTs), belonging to the SLC transporters, are involved in the absorption of nucleosides (adenosine and thymidine) and subsequently abacavir, the nucleoside antiretroviral. The in vitro transport method was performed on polarized, Caco-2 cell monolayer. By the statistical analysis of acquired data was found, that although the expression of ENTs was confirmed on the apical side of the Caco-2 cell membrane, the transport of nucleosides [3H]ADE and...
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Reporter gene studies for nanoparticle mediated DNA and siRNA delivery.
Kovářová, Barbora ; Jirkovská, Anna (advisor) ; Hofman, Jakub (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...
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Study of effects of prolonged administration of emtricitabine on expression of ABC efflux transporters in maternal and fetal organs
Havlová, Ivana ; Červený, Lukáš (advisor) ; Hofman, Jakub (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Ivana Havlová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of prolonged administration of emtricitabine on expression of ABC efflux transporters in maternal and fetal organs The aim of this thesis was to evaluate the effect of chronic administration of antiretroviral substance emtricitabine (FTC) on expression of drug efflux ABC-binding cassette (ABC) transporters in the placenta, maternal and fetal organs (brain, liver, kidney, small intestine) of pregnant rats. We focused on two most important representatives of drug efflux ABC transporters, P-glycoprotein (MDR1) and Breast Cancer Resistance Protein (BCRP). Knowledge of FTC effects on gene expression of these transporters is crucial especially when using FTC in combination therapy, known as Highly Active Antiretroviral Therapy (HAART). Changes in MDR1/BCRP expression may lead to significantly altered pharmacokinetics of concomitantly administered drugs. First, bioavailability was analyzed. AUC of FTC (3,5 mg/kg) following i.m. administration was comparable to that one after i.v. administration allowing us to use i.m. application as a route of FTC administration to rats. Subsequently,...
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Effect of cadmium chloride on P-glycoprotein in the blood-brain barrier
Zahradníková, Tereza ; Štaud, František (advisor) ; Hofman, Jakub (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Zahradníková Supervisor: Prof. PharmDr. František Štaud, Ph.D. Consultant: Alexander Zaremba, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany Title of diploma thesis: Effect of cadmium chloride on P-glycoprotein in the blood-brain barrier The blood-brain barrier (BBB) separates the central nervous system (CNS) and the peripheral blood circulation. It regulates the material transport between these compartments due to its specialised structure and cellular constitution. The endothelial cells forming the BBB are characterized by the expression of different multidrug resistance proteins which belong to the ATP-binding cassette (ABC) transporter family. These transmembranous ABC proteins actively transport molecules out of the BBB endothelia into the bloodstream and protect the brain against harmful xenobiotics, toxins and metabolites. On the other hand, ABC export proteins constitute obstacles to the delivery of many therapeutic drugs across the BBB into the CNS, thus the efficacy of CNS pharmacotherapy is limited. One of the most important efflux transporters is P-glycoprotein (P-gp). Cadmium is a heavy metal that is dangerous to human health....
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Preparation of mammalian vectors encoding selected microsomal SDR enzymes
Slezák, Jan ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradci Kralove Department of Biochemical Sciences Candidate: Jan Slezák Superviser: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Preparation of mammalian vectors encoding selected microsomal SDR enzymes Microsomal short-chain dehydrogenases/reductases DHRS1, DHRS7 and HSD11b1 (SDR19C1, SDR34C1, SDR26C1) are membrane-bound NAD(P)H-dependent enzymes metabolizing a broad spectrum of carbonyl-bearing substrates. These enzymes from the superfamily of short-chain dehydrogenases/reductases mediate metabolism of endogenous compounds, such as glucocorticoids, retinoids, sfingolipids as well as various xenobiotics. Apart from their biological roles, they participate in the etiology of severe diseases (e.g cancer, Alzheimer disease, obesity etc.). Knowledge of inhibitory and substrate affinity may lead to better understanding of the functions of these enzymes in organism and to the development of new therapeutic approaches. The goal of the present work was to prepare mammalian vectors encoding DHRS1, DHRS7 and HSD11b1. Primer design and cDNA amplification were among the first steps. Primers beared the sequence recognized by restriction endonucleases which was concomitantly present in the multicloning site of the pCI plasmid. Such primer design allowed...
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Preparation of mammalian vectors encoding selected aldo-keto reductases
Herbolt, Daniel ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
1 Abstract Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Daniel Herbolt Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Preparation of mammalian vectors encoding selected aldo-keto reductases Aldo-keto reductases 1B1, 1B10 and 1C1 (AKR1B1, AKR1B10, AKR1C1) are NADPH-dependent enzymes metabolizing a broad spectrum of carbonyl-bearing substrates. These enzymes from the superfamily of aldo-keto reductases mediate the reduction of endogenous compounds such as hormones and sugars as well as various xenobiotics and drugs. Beside their physiological roles, they significantly participate in the genesis of severe diseases (e.g. cancer, diabetes etc.). Inhibition of AKR1B1, AKR1B10 and AKR1C1 is accepted as promising therapeutic approach for the treatment of these diseases. The goal of the present work was to prepare mammalian vectors encoding AKR1B1, AKR1B10 and AKR1C1. Primer design and cDNA amplification were the first steps. Primers contained the sequence recognized by restriction endonucleases present in the multicloning site of the pCI plasmid which allowed for the generation of cohesive ends and the consequent joining of insert with plasmid by ligation. Prepared vectors were transformed into Escherichia coli HB101 bacteria....
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Interactions of Cyclin-dependent Kinase Inhibitors with ABC Drug Transporters in vitro and in situ
Hofman, Jakub ; Štaud, František (advisor) ; Wsól, Vladimír (referee) ; Kryštof, Vladimír (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Jakub Hofman Supervisor: Prof. PharmDr. František Štaud, Ph.D. Title of Doctoral Thesis: Interactions of cyclin-dependent kinase inhibitors with ABC drug transporters in vitro and in situ In the present work, we focused on the study of pharmacokinetic interactions of cyclin-dependent kinase inhibitors (CDKi) with drug efflux transporters breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Using accumulation and transport methods with MDCKII-ABCG2 cells, we showed purvalanol A, olomoucine II, bohemine and roscovitine to inhibit BCRP. Employing accumulation method with MDCKII-ABCB1 cells, we further observed that purvalanol A potently inhibits P-gp and partial inhibition was recorded in the case of other studied CDKi (olomoucine II, roscovitine, flavopiridol, SNS-032). Transport method with the same cellular models was used for the study of substrate affinity of olomoucine II and purvalanol A when olomoucine II was determined to be a dual P-gp and BCRP substrate. Substrate affinity of purvalanol A toward any of the transporters tested was not observed. These findings were confirmed by the ATPase vesicular assay and using this experimental setup, we further...
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