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Analysis of association of MHC and KIR variants with the course of lentivirus induced disease by next-generation sequencing.
Zelníková, Jana ; Jílek, Petr (advisor) ; Červený, Lukáš (referee)
Jana Zelníková Analysis of association of MHC and KIR variants with the course of lentivirus induced disease by next-generation sequencing Thesis Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové Farmacie Background: The aim of this work was to use the SIV infection in macaques (the only available model of human HIV infection) to show potential differences in MHC I haplotypes in macaques experimentally infected with SIVmac239 (or SHIV clade C) and their correlation with low or high viral loads, using pyrosequencing. Methods: The RNA samples from leukocytes from experimental macaques were reversely transcribed into cDNA and subjected to PCR of the 190 bp fragment of MHC I using fusion primers containing MIDs and FastStart High Fidelity Taq DNA polymerase. Subsequently the PCR generated samples were subjected to pyrosequencing using GS Junior System (Roche) according to the manufacturer`s protocol. The generated data were analyzed by a series of data filtration steps using scripts in Python and subsequently by DNAStar Lasergene 11. Final alleles were identified by ncbi-blast+ 2.25. Results: A total of 98 known aleles and 11 new aleles of MHC class I were found. The analysis identified those aleles, which correlate with fast or slow disease course. The aleles associated with slow...
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Neurogenetický potenciál kmenových buněk u myší
Slabá, Barbora ; Štaud, František (advisor) ; Červený, Lukáš (referee)
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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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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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 ; Štaud, František (advisor) ; Červený, Lukáš (referee)
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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Beneficial Effects of 11β-HSD1 Inhibition on Cognitive Performance in a Mouse Model of Alzheimer's Disease
Červinková, Tereza ; Červený, Lukáš (advisor) ; Musílek, Kamil (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Candidate: Tereza Červinková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title: Beneficial Effects of 11β-HSD1 Inhibition on Cognitive Performance in a Mouse Model of Alzheimer's Disease The increased life expectancy goes hand in hand with ageing-related cognitive impairments. Alzheimer's disease (AD) is the most common type of dementia being an irreversible and progressive brain disorder with loss of cognitive functions. Recent studies suggest that excess of glucocorticoid (GC) action exerts deleterious effects on the hippocampus and causes impaired spatialmemory. In addition, it has been demonstrated that aged mice with cognitive deficits show increased gene expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the hippocampus and parietal cortex. The Senescence-Accelerated Mouse Prone 8 (SAMP8) strain is a spontaneous animal model of accelerated ageing. Many studies indicate that SAMP8 harbour the behavioural and histopathological signatures of AD. In the present study, we evaluated the neuroprotective effects of 11β-HSD1 inhibition by a potent pyrrolidine-based compound RL-118 and/or effects of diet on cognitive performance in different groups of SAMP8 by conducting behavioural and...
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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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Flow-cytometric analysis of inhibitory effect of novel targeted drugs on the activity of ABC drug efflux transporters
Burianová, Gabriela ; Hofman, Jakub (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Pharmacology & Toxicology Student: Gabriela Burianova Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Flow-cytometric analysis of inhibitory effect of novel targeted drugs on the activity of ABC drug efflux transporters Cancer is the second leading cause of death. Cancer treatment often combines conventional chemotherapy, radiation therapy and surgery. More recent approach to treatment is the use of targeted cancer therapy with a greater specificity towards cancer cells. Development of resistance is a major obstacle in the success of chemotherapy. Multidrug resistance (MDR) can be acquired through various mechanisms e.g. overexpression of efflux transporters. ATP binding cassette (ABC) transporters represents a large family of transmembrane proteins that use ATP to pump molecules across the membrane. The three main ABC proteins related to MDR are: P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Use of ABC transporter inhibitors increases the amount of chemotherapeutical substrates accumulated within the cells. In this study we evaluated interactions of six synthetic small molecule inhibitors (alisertib, ensartinib, entrectinib, talazoparib,...
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Beneficial Effects of 11β-HSD1 Inhibition on Cognitive Performance in a Mouse Model of Alzheimer's Disease
Červinková, Tereza ; Červený, Lukáš (advisor) ; Musílek, Kamil (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Candidate: Tereza Červinková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title: Beneficial Effects of 11β-HSD1 Inhibition on Cognitive Performance in a Mouse Model of Alzheimer's Disease The increased life expectancy goes hand in hand with ageing-related cognitive impairments. Alzheimer's disease (AD) is the most common type of dementia being an irreversible and progressive brain disorder with loss of cognitive functions. Recent studies suggest that excess of glucocorticoid (GC) action exerts deleterious effects on the hippocampus and causes impaired spatialmemory. In addition, it has been demonstrated that aged mice with cognitive deficits show increased gene expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the hippocampus and parietal cortex. The Senescence-Accelerated Mouse Prone 8 (SAMP8) strain is a spontaneous animal model of accelerated ageing. Many studies indicate that SAMP8 harbour the behavioural and histopathological signatures of AD. In the present study, we evaluated the neuroprotective effects of 11β-HSD1 inhibition by a potent pyrrolidine-based compound RL-118 and/or effects of diet on cognitive performance in different groups of SAMP8 by conducting behavioural and...
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Study of interactions of antiviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters
Lalinská, Anežka ; Červený, Lukáš (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...
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