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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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Inhibice histon-deacatyláz a mitochondriální pohyb v neuronech
Prokopová, Iva ; Pávek, Petr (advisor) ; Červený, Lukáš (referee)
1 Abstract Histone Deacetylase Inhibition and Mitochondrial Trafficking in Living Neurons Mitochondrial trafficking is necessary for proper neuronal function and its impairment leads to neurodegeneration. We have found significant differences in mitochondrial movement between cortical and striatal neurons derived from rat brain. In striatum, mitochondria move with lower average speeds and decreased overal mitochondrial dynamics than those in cortex, exhibiting the same mitochondrial fractional occupancy in neuronal processes. How this could contribute to high striatal vulnerability in neurodegeneration is discussed. We used two different methods of trafficking analysis: manual and semi-automatic. These analyses determine average movement speeds of single mitochondria, as well as overal mitochondrial dynamics in particular field. Only neurons with established synaptic connections were analyzed, mitochondria travelling in both directions were found to move with equivalent average speeds, thus not further distinguished. Our preliminary results also reveal that histone deacetylase inhibition with trichostatin A increases mitochondrial trafficking in striatal neurons, independently of mitochondrial fractional occupancy and Ca2+ levels in neuronal processes. We suggest that possible mechanism of this effect is...
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Modification of real-time PCR method and its application for detection of microorganisms of the genus Bacillus
Hubálková, Lenka ; Kroča, Michal (advisor) ; Červený, Lukáš (referee)
The real-time polymerase chain reaction (PCR) is one of the most widely used techniques in modern molecular biology. This method is based on fluorescent monitoring of DNA amplification by using some detection system specific for reaction product. Since its development in the 1990s many different detection formats have been developed. These include dsDNA specific dyes, which are very simple and cheap, but not sufficiently specific, and various types of sequence- specific fluorescent oligonucleotide probes or modified primers, which provide a high-level of specifity, but are relatively expensive and require careful optimization of reaction conditions. An alternative approach to monitoring of real-time PCR reactions is presented in this study. Its function is based on the observation that guanine nucleotide can quench fluorescence of some fluorescent labels. The approach makes use of an oligonucleotide primer containing a labelled cytosine nucleotide at its 5' end. When such a primer is incorporated into the product of amplification, its fluorescence is quenched by the quanine nucleotide complementary to the modified cytidine. This system of "5' labelled primers" is easy and low-cost like the dsDNA specific dyes, but it is more specific, because non-specific products of amplification are not detected....
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Characterisation of multidrug resistant Klebsiella pneumoniae and Enterococcus faecium isolates by spectroscopic and genotypic method
Bavlovič, Jan ; Červený, Lukáš (advisor) ; Mladěnka, Přemysl (referee)
Charles University in Prague Faculty of Pharmacy Hradec Králové Department of Pharmacology and Toxicology Student: Jan Bavlovič Supervisors: PharmDr. Lukáš Červený, Ph.D. Prof. Doutora Luísa Peixe Co-advisors: Doutora Ângela Novais Doutora Ana Freitas Title: Characterisation of multidrug resistant Klebsiella pneumoniae and Enterococcus faecium isolates by spectroscopic and genotypic methods. Ever increasing antimicrobial resistance is currently a worldwide problem, traditionally addressed by DNA-based approaches. This study aimed to evaluate the potential of Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) for the characterization of multidrug resistant carbapenemase-producing K. pneumoniae and E. faecium isolates. We analysed 20 clinical K. pneumoniae isolates obtained from different community laboratories from Portugal between March 2014 and September 2015 and 143 previously characterized vancomycin-resistant E. faecium isolates obtained from humans, animals, and the environment in 26 countries between 1992 and 2015. Isolates were primarily characterized by genotypic methods including antimicrobial susceptibility testing, detection of carbapenemases and extended-spectrum β-lactamases (ESBLs), identification of antibiotic resistance coding transposons (Tn) and...
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