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Zinc induced activation of breast cancer cell lines and the involvement of Map kinase
Králová, Jarmila ; Čečková, Martina (advisor) ; Červený, Lukáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Jarmila Králová Supervisor: Dr. Kathryn Taylor, Ph.D., PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Zinc induced activation of breast cancer cell lines and the involvement of MAP kinase The aim of this work was to investigate the effect of zinc on various signalling pathways in breast carcinoma cell lines MCF7 and TamR cells. The differences between signalling pathways in MCF7 cell line and TamR cells were evaluated with a special focus on a role of MAP kinase, which activation is believed to be linked with malignant diseases. An effect of zinc on various cellular kinases in 0, 2, 5, 10, 15 and 20 minute of zinc treatment was analyzed in MCF7 cells transfected by wild and mutant type of ZIP 7, TamR cells and TamR cells pre-treated with MAP kinase inhibitor (PD) using the methods of western blotting and fluorescent microscopy. We show here the dependence of activation of pMAP kinase and other important oncogenic kinases (such as Lyn, Src and STAT3) on zinc release into cytoplasm. According to our results, MAP kinase is activated very upstream and it can stimulate many important protein kinases as Src Y418 , STAT3 S727 and Lyn Y396 in tamoxifen-resistant breast cancer...
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Neurogenetický potenciál kmenových buněk u myší
Slabá, Barbora ; Červený, Lukáš (referee) ; Štaud, František (advisor)
Neural stem cells (NSCs) are present in the mammalian adult nervous system and are found in two specific regions: the subventricular zone of the forebrain and the subgranular zone in the dentate gyrus of the hippocampus. NSCs undergo fate specification, maturation and functional integration before they can take part in neuronal circuits. Less is known about the mechanisms controlling these processes or the function of the newly born neurons in the adult brain. A large number of molecular and environmental factors can regulate various aspects of proliferation, maturation and differentiation of adult stem cells in adult nervous system. NSCs can give rise to three main types of neural cells: neurons, astrocytes and oligodendrocytes. It is suggested that astrocytes may play critical role in regulating neurogenesis in both the intact adult brain and after injury. Therefore, determining how astrocytes regulate adult neurogenesis at the molecular level is an essential step toward understanding the regulation of adult neurogenesis. To find out how genetic alteration of astrocytic intermediate filament will affect neural stem cells of postnatal mice, the neurosphere assay was performed in order to compare the proliferative and differentiation characteristics of neural stem/progenitor cells in GFAP-/- Vim-/-...
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Prepsration of recombinant cDNA of drug transporters
Svobodová, Iveta ; Štaud, František (advisor) ; Červený, Lukáš (referee)
CDS for ABCC4 and ABCC5 transporters were amplified. For ABCC5 two systems were outlined, however the only first one was used. The optimal annealing temperature was 64 řC; optimal concentration of magnesium was 2mM. TOPO XL have proved best results in our research in between cloning kits we tried. The CDS for ABCC4 and ABCC5 were cloned into the vector. Inserts were rended with the use of restrictive endonucleasis. ABCC4 was gashed by Spe I and Not I and ABCC5 by EcoR I and Hind III. With the same endonucleasis, the process of rending was repeated for pZeoSV2(-). Afterwards, the inserts were cloned into pZeoSV2(-).
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Interactions of selected antiepileptic drugs with MRP2 transporter
Garajová, Katarína ; Pávek, Petr (advisor) ; Červený, Lukáš (referee)
Interaction of the MRP2 transporter with selected anti-epileptic drugs Epilepsy is a common neurological disorder affecting approximately 1-2% of the population. In most of the patiens with epilepsy, seizures are well-controlled with currently available anti-epileptic drugs (AEDs). It is estimated that 30% of the patients fail to achieve seizure termination despite carefully optimized drug treatment. Most of these patients with refractory epilepsy are resistant to several AEDs with different mechanisms of action, which suggests that resistance in epilepsy is a multifactorial and drug-nonspecific phenomenon. Based on experimental and clinical studies, two major theories have been put forward to explain the development of pharmacoresistance in epilepsy. The target hypothesis holds that changes in properties of drug targets for AEDs may result into their reduced drug sensitivity. The transporter hypothesis contends that the over-expression of efflux multidrug-transporters in the brain leads to impaired access of AEDs to their targets. The largest and the most important family of multidrug-transporters expressed in the brain, is the ABC (ATP-binding cassette) transporter family. One of the transporters in this family, recently under study, is the multidrug resistance-associated protein 2 (MRP2). The MRP2...
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Preparation of recombinant cDNA of drug transporters
Hadrabová, Zdeňka ; Červený, Lukáš (referee) ; Štaud, František (advisor)
We have prepared suitable plasmid containing coding sequence of MRP3, for subsequent preparation of cell line overexpressing this transporter. Composition of reaction mixture and temperature profile of PCR reaction for MRP3 coding sequence amplification was determined experimentally. Adenosine was added to 3' ends of PCR product to allow cloning into PCR® -XL-TOPO® vector. pCR® -XL-TOPO® vector containing cloned sequence and pZeoSV2(-) plasmid were cleaved using restriction endonucleases EcoRV and SpeI. Afterwards it was possible to clone cleaved coding sequence into pZeoSV2(-) plasmid due to complementary ends. Prepared plasmid is suitable for transfection of eukaryotic cell lines. The cloning was successful.
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Complementation of Francisella tularensis dsbA mutant strain and analysis of potential binding partners of DsbA protein
Šenitková, Iva ; Červený, Lukáš (referee) ; Šimůnek, Tomáš (advisor)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Science Candidate: Bc. Iva Šenitková Supervisor: doc. PharmDr. Tomáš Šimůnek, Ph.D. Supervisor - consultant: RNDr. Petra Špidlová, Ph.D. Title of diploma thesis: Complementation of Francisella tularensis dsbA mutant strain and analysis of potential binding partners of DsbA protein Conserved hypothetical lipoprotein FTT1103 is a virulence factor of gram- negative intracellular bacterium F. tularensis. This protein shares homology with proteins of disulfide oxidoreductase DsbA family. These proteins catalyse formation of disulfide bonds, which are essential for forming proteins conformation, for activity and stability of proteins. The aim of this study was to identify binding partners of lipoprotein FTT1103, commonly known as DsbA. The knowledge of these partners could help us in understanding a role of lipoprotein DsbA in virulence F. tularensis. We prepared fused ftt1103 gene with sequence coding FLAG® tag and cloned this fused gene into plasmid vector which can be replicated in F. tularensis. We used electroporation for introducing plasmid vector into the in frame deletion mutant F. tularensis subsp. holarctica strain FSC200/∆1103 thereby we complemented this deletion mutant in trans. Imunoprecipitation by...
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Transport nesteroidních antiflogistik přes hematoencefalickou bariéru in vitro
Nováková, Iveta ; Červený, Lukáš (referee) ; Štaud, František (advisor)
The migration of substances between the blood circulation and the central nervous system (CNS) is regulated by the blood-brain barrier (BBB). Small, lipophilic molecules such as carbon dioxide, oxygen or ethanol can pass the BBB by passive, transcellular diffusion, while the paracellular transport of hydrophilic substances is restricted by intercellular tight junctions. Due to accessory transport systems, the BBB is able to regulate specifically the permeation of substances (e.g. nutrients) (Ballabh et al., 2004). Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used substances world-wide, yet little is known about their ability to cross the BBB. Since NSAIDs may exhibit CNS side effects including dizziness, headaches and drowsiness we sought to study the transport of several NSAIDs (celecoxib, diclofenac, ibuprofen, lornoxicam, meloxicam, piroxicam and tenoxicam). Both single studies and group studies were carried out applying either a single substance or several substances simultaneously across the BBB in vitro model based on the human cell line ECV304. The permeability data were normalized to the internal standards diazepam and carboxyfluorescein to account for cell layer's variabilities. According to our studies, it was confirmed that crossing of ibuprofen and...
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The effect of genetic factors on the pharmacokinetics of drugs
Šlosárková, Petra ; Červený, Lukáš (referee) ; Štaud, František (advisor)
The interindividual differences of activity of biotransformation enzymes play an important role in drug's pharmacokinetics; their are significantly influenced both by external factors (such as age, gender, weight, diet) and genetic disposition. The presence of single nucleotide polymorphisms (SNPs) and mutations at a genes coding drug-metabolising enzymes can cause a major change of the metabolism and drug effects. In this study was evaluated an influence of enzyme CYP2C9 polymorphisms on the pharmacokinetic parameters of Nurofen forte containing racemic mixture of ibuprofen in 20 healthy Czech volunteers (men, Caucasian population, age 21-40 years). The presence of the CYP2C9*1/*1 genotype was found in 17 individuals, the CYP2C9*1/*2 genotype in 2 individuals and the CYP2C9*3/*3 genotype in 1 individual. No statistically significant difference of pharmacokinetic parameters was observed between CYP2C9*1/*1 and CYP2C9*1/*2 genotypes. AUC(0-inf) and t1/2 were higher by 113% and 90%, respectively and clearance was lower by 54% in subject with CYP2C9*3/*3 genotype compared to subjects CYP2C9*1/*1 and CYP2C9*1/*2. The individuals with CYP2C9*3/*3 genotype have increased risk of adverse drug reaction (ulceration, gastroduodenal bleeding) after long time nonsteroidal anti-inflamatory drugs use. The...
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