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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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Interactions of antiretrovirals with drug efflux transporters and their role in the transplacental pharmacokinetics
Ptáčková, Zuzana ; Štaud, František (advisor) ; Trejtnar, František (referee) ; Ulčová-Gallová, Zdeňka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Zuzana Ptáčková Supervisor: prof. PharmDr. František Štaud, Ph.D. Consultant: PharmDr. Lukáš Červený, Ph.D. Title of doctoral thesis: Interactions of antiretrovirals with drug efflux transporters and their role in the transplacental pharmacokinetics The combination antiretroviral therapy that should be administered during the whole pregnancy is the backbone of prevention of mother-to-child transmission of HIV infection. One of the prophylactic mechanisms of such treatment is the presence of antiretrovirals in the fetal circulation. However this can be associated with the potentially harmful effects of drugs on the developing fetus. To select optimal therapy while minimizing risks it is inevitable to have detailed knowledge of all the factors affecting transplacental transport of drugs. The aim of this study was to detect whether drug efflux transporters are able to protect fetus against xenobiotics can affect the transplacental pharmacokinetics of the selected antiretroviral drugs. Employing variety of in vitro, in vivo, in situ and ex vivo methods we determined the role of the drug efflux transporters in the distribution of drugs between mother and fetus. We suggested that...
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Transport Mechanisms of Xenobiotics (Caco-2 Cell Monolayer Model) in Relation to the Biopharmaceutics Classification System (BCS)
Smetanová, Libuše ; Květina, Jaroslav (advisor) ; Štaud, František (referee) ; Suchý, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: MUDr. Libuše Smetanová Supervisor: Prof. RNDr. Dr.h.c. Jaroslav Květina, DrSc., FCMA Title of Doctoral Thesis: Transport mechanisms of xenobiotics (Caco-2 cell mono- layer model) in relation to the Biopharmaceutics Classi- fication System (BCS) Current technological levels enable to study transbarrier transports of xenobiotics (including gastrointestinal absorption) on in vitro, in situ and in vivo models. In situ and in vivo approaches enable to assess feedbacks at the organ or whole body level, in in vitro models extra barrier factors are eliminated and the models are aimed at the mechanisms of transport processes. In vitro models for studying of transport mechanisms through the intestinal wall are derived either from cell lines (e.g., Caco-2, MDCK, 2/4/A1 cells) or intestinal tissues (e.g., Ussing chamber). The Caco-2 cell monolayer model is one of the relatively most frequently used in vitro models. Caco-2 cells are a cell line derived from colorectal adenocarcinoma. The main characteristic is their spontaneous differentiation with the creation of a monolayer of fully differentiated and polarized cells showing typical brush border and tight junctions under normal culture...
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Cytotoxicita beauvericinu, citrininu, deoxynivalenolu a T-2 toxinu stanovena metodou in vitro v buňkách Vero
Macáková, Petra ; Štaud, František (advisor) ; Vopršalová, Marie (referee)
Mycotoxins are secondary metabolites of moulds. Contamination of food and feed by mycotoxins is a major problem for human and animal health. Ingestion of mycotoxins may cause a range of toxic responses, from acute toxicity to long term or chronic health disorders. Several mycotoxins, either from the same or from different fungal species, occur simultaneously in plant products. However, its implication for food safety assessment is generally not known, as there is relatively little information on the interaction between concomitantly occurring mycotoxins and the consequence for the toxicity. Mycotoxins with similar mode of action would be expected to have at least additive effects. Conversely, some interactions could have subtractive effects. An understanding mode of action in simple in vitro systems can provide a rational basses for predicting interactions between mycotoxins. The aim of this study was to obtain cytotoxicity data (EC50 values) of Penicillium and Fusarium mycotoxins, nominally BEA, CIT, DON and T-2 toxin. For this purpose, Vero cells viability was evaluated in the presence of these four mycotoxins using the NR assay. All mycotoxins tested diminished cell viability in a concentration and incubation time-dependent manner on Vero cells. Individual mycotoxins increase cytotoxicity as...
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DIFFERENTIAL RESISTANCE OF MELANOMA TO VINCA - ALKALOIDS
Rozkydalová, Lucie ; Štaud, František (advisor) ; Čečková, Martina (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxicology Student: Lucie Rozkydalová Supervisor of Diploma thesis: Prof. PharmDr. František Štaud, PhD. Specialized supervisor: Pr. Pierre Cuq PharmD. PhD., Laure-Anaïs Vincent Title of diploma thesis: Differential resistance of melanoma to vinca-alkaloids Malignant melanoma (MM) represents the most dangerous and very aggressive skin tumor with fast development of drug resistance which is the main obstacle in successful treatment of MM. According to previous studies (microarray data analysis), KIT gene, which plays key role in melanoma pathophysiology, was chosen as one of the potential causes of failure of treatment by vinca alkaloids (VAs) because of its complete underexpression in melanoma CAL1 resistant cells (CAL1R-VAs) in comparison with parental cells (CAL1-wt). Moreover, KIT also interacted with NF-κB and cyclin D1-2 proteins involved in chemoresistance of melanoma - inside molecular network built using IPA software. Although KIT underexpression in resistant CAL1 R-VAs cell lines were confirmed (qRTPCR), KIT repression using specific siRNA transfection did not show any effect on in vitro sensibility of CAL1-wt cells to VAs. It signifies that KIT is not directly involved in melanoma resistance...
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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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Investigating the role of zinc transporter ZIP 6 and STAT3 in mitosis
Burgetová, Lenka ; Čečková, Martina (advisor) ; Štaud, František (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lenka Burgetová Supervisor: PharmDr. Martina Čečková PhD. Specialized supervisor: Dr. Kathryn Taylor PhD. Title of diploma thesis: Investigating the role of zinc transporter ZIP 6 and STAT3 in mitosis It has been shown that STAT3 (signal transducer and activator of transcription 3) plays a role in the development of cancer. ZIP6 is the downstream target of this transcription factor. Previous research has focused mainly on the activation of STAT3 by tyrosine phosphorylation, while the effect of phosphorylation at a second site, serine 727, remained relatively uninvestigated. In this study, it is proposed that serine-phosphorylated STAT3 is activated throughout mitosis in tamoxifen-resistant (TamR) breast cancer cells and that zinc transporter ZIP6 and serine-phosphorylated STAT3 are involved in a zinc-mediated mitotic mechanism. After using nocodazole to induce mitotic arrest, the expression of tyrosine- phosphorylated STAT3 protein was observed to be reduced while the expression of serine- phosphorylated STAT3 was increased. Zinc supplementation after nocodazole treatment appeared to push cells through mitotic-arrest and cause proteolytic cleavage of STAT3 suggesting a novel...
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Pharmacological and physiological characteristics of organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs)
Gregorová, Pavla ; Štaud, František (advisor) ; Čečková, Martina (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavla Gregorová Supervisor: Prof. PharmDr. František Štaud, Ph.D. Title of diploma thesis: Pharmacological and physiological characteristics of organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) Abstract: Membrane transporters are important and life-enabling proteins of the body, which facilitate the absorption, distribution, and elimination of nutrients, metabolic waste products, drugs, and xenobiotics. Multidrug and toxin extrusion proteins (MATEs) and organic cation transporters (OCTs) belong to polyspecific transporters of the solute carrier (SLC) family and form a cooperating system of excretion in the kidney and liver. The transporters are expressed in many tissues throughout the body primarily in the kidneys, liver, heart, brain, small intestine, and placenta and transmit countless molecules from the natural neurotransmitters and hormones to exogenous compounds, such as metformine, cimetidine, 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA) or acyclovir. Furthermore, there are described the most recent studies in knockout mice models and genetic polymorphisms that help identify transporters activity and pharmacokinetics with altered...
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Pharmacological treatment of sclerosis multiplex
Martincová, Petra ; Štaud, František (advisor) ; Červený, Lukáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Mgr. Petra Martincová Supervisor: Prof. PharmDr. František Štaud, Ph. D. Title of diploma thesis: Pharmacology treatment of multiple sclerosis Multiple sclerosis is a neurological inflammatory disease of central nervous system of an autoimmune origin with several subtypes according to different clinical courses. The main philosophy of a treatment is based on an early administration of anti-inflammatory treatment. This can delay irreversible neurodegeneration and disability of patients. In treatment of an acute symptomatic attack which may develop at any stage of disease methylprednisolone is most often administered. Long-term therapy is based on immunomodulatory drugs: interferon-β and glatiramer acetate. This treatment is intended for parenteral administration and reduces both the number of attacks and disease progression. However its effect is insufficient in most patients. For escalation of treatment some cytostatics may be used however they have many side effects. The first oral product registered in the Czech Republic is fingolimod. It is used for treatment of remitting form of multiple sclerosis and has a very good pharmaceutical effect. A new option in treatment is provided...
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Organic Cation Transporter 3 (OCT3/SLC22A3) and Multidrug and Toxin Extrusion 1 (MATE1/SLC47A1) Protein in the Placenta: Expression, Localization and Function
Ahmadimoghaddam, Davoud ; Štaud, František (advisor) ; Trejtnar, František (referee) ; Večeřa, Rostislav (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Davoud Ahmadimoghaddam Supervisor Prof. PharmDr. František Štaud, Ph.D. Title of Doctoral Thesis Organic Cation Transporter 3 (OCT3/SLC22A3) and Multidrug and Toxin Extrusion 1 (MATE1/SLC47A1) Protein in the Placenta: Expression, Localization and Function. The aim of the present study was to investigate the expression, localization, and function of organic cation transporter 3 (OCT3, Slc22a3) and multidrug and toxin extrusion protein 1 (MATE1, Slc47a1) in the rat placenta. Using qRT-PCR, Western blotting and immunohistochemical techniques, we demonstrated abundant expression of OCT3 on the basolateral, i.e., fetus-facing side of the placenta, and MATE1 on the apical, i.e., maternal side of the placenta. To investigate the role of these transporters in the transplacental pharmacokinetics, the in situ method of dually perfused rat term placenta was employed in open- and closed-circuit arrangements; 1-methyl-4-phenylpyridinium (MPP+) was used as a model substrate of both OCT3 and MATE1. We provide evidence that OCT3 and MATE1 cause considerable asymmetry between maternal-to-fetal and fetal-to-maternal transport of MPP+ in favor of fetomaternal direction. Using closed- circuit...
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