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Side-effects of siRNA - induced gene silencing
Feřtek, Marina ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Vedlejší účinky siRNA byly testovány na buněčných liniích po "umlčení" genu kódujícího cyklooxygenázu. Teoretickým základem tohoto experimentu byl poznatek, že siRNA mohou vyvolat imunitní odpověď. Z tohoto důvodu byla testována exprese IFN-indukovaných genů v intaktní Hep2 buněčné linii a ve třech liniích odvozených od Hep2 buněk, kde byla různými metodami vnesena siRNA. Buněčné linie byly odvozeny z Hep2 pomocí různých metod transfekce siRNA (tj. nuzkleofekce, effectene technologie a klonování). RNA byla izolována z těchto buněčných linií a cDNA byla získaná reverzní transkripcí. Genová exprese byla měřena pomocí QRT PCR a kvantifikována jako počet kopií/mikrogram RNA. Stupeň exprese byl porovnáván s intaktní linií Hep2. Jako houskeeping gen byl použit NUP. Výsledky byly vyhodnoceny pomocí softwarů REST verze 2 a rotoru Gene verze 6. Mělo být testováno devět IFN-indukovaných genů, RNL, PKR, IRF1, IRF3, IFITM, IFIT1, OAS1, OAS2 a OAS3. Z nich OAS1 a OAS2 nebylo možné izolovat a tedy měřit. Exprese PKR byla up-regulovaná; exprese všech ostatních testovaných genů byly down-regulovány. Další rozsáhlé studie jsou nezbytné pro vysvětlení těchto výsledků.
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Effect of combined statin therapy MDOCTM and the atherogenic / / process in an experimental model of atherosclerosis
Metelková, Marie ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
The effects of combination treatment of MDOCTM and statin on atherogenic process in experimental model of atherosclerosis. Mgr. Marie Metelková MDOC™ is micro dispersed derivatives of oxidized cellulose (polyanhydroglucuronic acid - PAGA). This semi-natural, biocompatible, bioabsorbable, non-acidic, sterile powder has been in use as a topical haemostatic agent since 1998. In this rigorous work we wanted to evaluate whether MDOC™ affects cholesterol levels, inflammatory markers and cell adhesion molecule expression in both blood and vessel wall in cholesterol fed apoE-deficient mice. Male apoE-/- mice were divided into 4 groups. Control group (n=8) of mice consumed an atherogenic diet for 4 weeks. The same diet was used in other animals except MDOC™, atorvastatin and MDOC™ together with atorvastatin were added to the diet. Analysis of blood cholesterol, IL-6 in blood and ICAM-1 endothelial expression in aortic sinus were performed by means of biochemical, ELISA and immunohistochemical analysis. LDL cholesterol levels were significantly decreased in all drug treated groups when compared with control animals. Moreover MDOC™ treatment significantly increased HDL cholesterol in comparision to control group. ELISA analysis revealed significant decrease of IL-6 levels MDOC™ group and atorvastatin group....
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The study of expression of various markers of aterogenesis in apoE/LDLr-deficient.
Rambousková, Kristýna ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
The aim of this diploma thesis was to set immunohistochemical detection of nuclear transcription factor kappaB (NF-κB). We studied its expression in aortic sinus in 8 and 16 weeks old apoE/LDL receptor deficient mice. Female apoE/LDL receptor mice were divided into two groups. The first group (n=8) was represented by eight weeks old mice. In the 16 weeks group (n=8) mice were fed with cholesterol enriched diet (0.15% of cholesterol) for the period of eight weeks. Indirect fluorescence immunohistochemistry with detection of antibody reaction with CY3 red fluorochrome was used for the detection of active form of NF-κB. In 8 weeks old mice the NF-κB expression was detected in endothelial cells and in some smooth muscle cells in vessel media. In 16 weeks old mice the NF-κB expression was detected inside atherosclerotic plaques and in vessel media under these plaques predominantly by macrophages and smooth muscle cells. In conclusion, the results of immunohistochemical analysis showed induction of inflammatory reaction by various cells during atherogenesis in blood vessels of apoE/LDL receptor deficient mice which will be used in prospective studies.
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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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Monitoring expression of selected cytokines in murine models of atherosclerosis.
Tlapalová, Barbora ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
Atherosclerosis, or sclerosis of arteries, is a degenerative disease of arteries. Sometimes it is called "the disease of 20th century". ApoE/LDL - receptor double knockout mice represent a new animal model for study of atherogenesis, which is characterized by severe hyperlipidaemia and atherosclerosis. Statins (or competitive inhibitors 3-hydroxyl-3-methyl-glutaryl-coenzym Areductase) currently belong to the most efficient and the most useful hypolipidemic drugs for all over the world. They decrease mainly levels of total cholesterol and LDL cholesterol. The aim of this rigorous thesis was to describe the expression of endoglin and SMAD 2 in atherosclerotic plaques in apoE/LDL-receptor deficient mice. Moreover we wanted to determine the effect of atorvastatin treatment on the expression of both endoglin and SMAD 2. ApoE/LDLR-deficient mice on were subdivided into 2 groups. The control group of animals was fed with the western type diet. The same atherogenic diet was used in ATV group, where atorvastatin was added to the atherogenic diet at the dosage of 100 mg/kg per day. The results of this thesis confirmed the expression of endoglin and SMAD 2 in atherosclerotic lesions in ApoE/LDLR-deficient mice. The expression of endoglin was located on the aortic vascular endothelium and in other smaller...
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Transport nesteroidních antiflogistik přes hematoencefalickou bariéru in vitro
Nováková, Iveta ; Štaud, František (advisor) ; Červený, Lukáš (referee)
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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Modulace exprese alfa-synukleinu pomocí působení 5-S-cysteinyldopaminu na buňky lidského neuroblastonu SH-SY5Y: možná úloha katecholthioetherů při neurodegeneraci
Hrabáková, Rita ; Štaud, František (advisor) ; Mladěnka, Přemysl (referee)
-Synuclein is a presynaptic protein which has been demonstrated to be involved in PD etiopathogenesis.It can regulate DA homeostasis by inhibition of TH activity, by regulation of the DAT activity and finally by potential role in vesicular storage. - Synuclein is a natively unfolded protein, which can undergo overexpression and aggregation due to toxic insults or oxidative stress. The aggregation of α-synuclein leads to a loss of function, which in PD neurons may determine a dysregulation of the DA pathways with subsequent excess of cytosolic DA, that can enhance the neurotoxic effect of α-synuclein aggregates. In recent years, a catecholthioether metabolite of DA, 5-S-cysteinyl-dopamine, has been identified in certain dopaminergic regions of the brain, notably the Substantia nigra. Cys-DA seems to have a possible significance as an index of oxidative stress in aging and in neurodegenerative processes and it was recently hypothesized that this substance or its metabolites may be the endogenous neurotoxins responsible for neurodegeneration in PD. Hence, the aim of this experimental work was to determine whether Cys-DA is able to cause overexpression of -synuclein both at transcriptional and translational levels in a cellular model of PD, the human neuroblastoma dopaminergic cell line SH-SY5Y....
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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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Monitoring of Selected Markers of Damage to the Colon after Induction of Chrohnic Colotis
Římalová, Pavla ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
Monitoring of selected markers of damage to the colon after induction of chrohnic colotis Pavla Římalová 2010 Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Crohn disease and Colitis belong to the Infammatory Bowel Diseases. AHR is Arylhydrocarbon receptor, which was suggested to affect the activity of immune system. Bilirubin is endogenous substance with significant antioxidative and anti-inflammatory effects. In this pilot thesis, we wanted to study the expression of AHR and large intestine wall after the induction of chronic colitis in normobilirubenimic (Wistar) and hyperbilirubinemic (Gunn) rats. Sixteen rats for Wistar and sixteen rats for Gunn were used in the experiment. Eight rats from each strain were administered by dextran sulphate (DSS) in drinking water for 7 days. All animals were on drinking water for another 14 days. This cycle was repeated three times. Immunohistochemical staining revealed the expression of AHR muscular layer and muscularis mucosa in all experimental groups. Administration of DSS resulted in increased expression of AHR in epithelial cells in Wistar rats. AHR expression was higher in non-treated Gunn rats when compared with non-treated Wistar. DSS treatment resulted in decreased expression of AHR in...
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