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Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
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Study of mechanism of fibrin network formation
Božíková, Paulína ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
Serine protease thrombin plays an important role in the process of fibrin network formation by converting fibrinogen into fibrin monomer which spontaneously polymerizes to form fibrin network. The aim of this work was to characterize interactions between thrombin and surface adsorbed fibrin(ogen) or fibrin network to which thrombin can bind and initiate grow of the fibrin network. Activity of thrombin bound on fibrinogen or fibrin was determined spectrophotometrically in a relation to cleaved chromogenic substrate. Using the method of surface plasmon resonance fibrin network formation initiated by thrombin bound to fibrinogen or fibrin was observed. These networks were also visualized by atomic force microscopy. Determined value of affinity constant KD for interaction of fibrinogen in solution with a fibrin network prepared on surface is in agreement with previous experiments in which KD was determined from interaction of surface covalently bound fibrinogen with fibrin monomers in solution.
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Comparison of adsorption capacity and adsorption efficiency of diosmectite and charcoal on selected compounds that often cause acute intoxication in adult population of the Czech Republic
Mináriková, Michaela ; Martínková, Markéta (advisor) ; Mrízová, Iveta (referee)
In the treatment of acute intoxications, one of the treatment procedures is an antidote submission, e.g. diosmectite and activated charcoal, where an antidote is a substance which acts antagonistically and disturbingly with the toxic effect of a toxic substance. The aim of this work was to compare the adsorption capabilities of activated charcoal and diosmectite in selected model compounds which are the most common originators of acute intoxications in the adult population of the Czech Republic. The actual comparison of adsorption capabilities of these sorbents was preceded by issues search processing of ten groups of substances that cause the most acute intoxications and subsequent testing of the proposed method for future detailed testing of adsorption and adsorption efficiency of different sorbents. Of the ten groups of substances five model compounds were selected: dosulepin, acetylsalicylic acid, ibuprofen, promethazine and phenobarbital, on which adsorption of diosmectite, activated charcoal, and mixture of these sorbents was compared in the experimental part of this work. This comparison of the adsorption capabilities of sorbents was carried out not only under neutral conditions, but also in an alkaline and acidic environment which simulated physiological conditions in different parts of the...
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Comparison of different molecular-biological approaches for detecting the presence of DNA of the pathogenic bacterium Streptococcus pneumoniae
Čížová, Kateřina ; Martínková, Markéta (advisor) ; Ryšlavá, Helena (referee)
Bacterium Streptococcus pneumoniae is currently one of the most serious pathogenic microorganisms posing a threat to children under the age of 5, especially in developing countries. A suitable detection method is a tool in the fight against this pathogenic microorganism. The presented bachelor thesis deals with the comparison of molecular- biological methods of detection of the bacterium Streptococcus pneumoniae, specifically by quantitative polymerase chain reaction and loop mediated isothermal amplification of DNA. A standard protocol for the detection of Streptococcus pneumoniae using the "golden standard" of molecular diagnostics, that is the quantitative polymerase chain reaction, has been established. An alternative to quantitative polymerase chain reaction detection has been the practical laboratory introduction of a loop mediated isothermal DNA amplification method using published "primers". In order to expand the diagnostic window of the loop mediated isothermal amplification method, and to combat the false positive of the negative control, a set of new "primers" for the detection of Streptococcus pneumoniae was designed and tested. The result of the work was a new, rapid and reliable protocol for the detection of Streptococcus pneumoniae using a combination of newly designed and published...
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The utilization of the uracil-DNA glycosylase in the elimination of carry-over contamination by products of the previous DNA amplifications
Zrnová, Adéla ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
DNA amplification is an important tool in diagnosis of infectious diseases. The most commonly used method in laboratories is the polymerase chain reaction, which, does not meet the need for a fast and inexpensive method. It was therefore necessary to develop a method that would be sensitive, fast, specific and accessible, for example, in developing countries with limited access to instrumentation. It appears that LAMP could be this method. Unfortunately, the LAMP method encounters considerable false positivity, which must be prevented in order to be used in practice. The subject of the presented bachelor thesis is the introduction of a unique LAMP method to detect the presence of DNA Streptococcus pneumoniae and the study of the use of uracil- DNA glycosylase to prevent contamination of samples with DNA formed in previous amplification reactions. The LAMP method of DNA detection Streptococcus pneumoniae was successfully introduced, then the method was optimized even in the presence of deoxyuridine triphosphate. After determining the appropriate deoxyuridine triphosphate concentration for LAMP amplification, the resulting Streptococcus pneumoniae DNA amplification products were treated with uracil-DNA glycosylase. This procedure was shown to have the potential to prevent false positivity of other...
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Comparison of different molecular-biological approaches for detecting the presence of DNA of the pathogenic bacterium Haemophilus influenzae
Smrčka, Tomáš ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Haemophilus influenzae is one of the main initiators of meningitis and pneumonia in children. Implementation of fast, cheap and instrumentally accessible method for detection of this pathogen would enable an early and targeted treatment of patients. The development of amplification methods in the last decades enables, apart from commonly used PCR method, application of alternative approaches, such as the LAMP. The focus of this Bachelor thesis was the study (research) of the alternative method LAMP as a tool for detection of Haemophilus influenzae. The LAMP method was successfully implemented for Haemophilus influenzae, however, it has contended with the false positive results of negative control in case of longer incubation times. Therefore, the optimized LAMP method was designed in presence of deoxyuridine triphosphate and uracil-DNA glycosylase. Its aim was to change the structure of LAMP products via the incorporation of uracils to amplified regions of DNA and subsequent removal of uracils with influence of uracil-DNA glycosylase, and therefore prevent their replication during potential contamination of reaction mixtures and consequently reduce the risk of false positive results of negative controls to minimum. The concentration of deoxyuridine triphosphate in reaction mixtures was optimized...
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Preparation and preliminary characterization of the eukaryotic initiation factor 2α and its heme regulated kinase
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
Heme sensor proteins perform a variety of important functions in both prokaryotic and eukaryotic organisms. Heme-regulated inhibitor (HRI) is an example of a eukaryotic heme-sensor protein, which catalyzes the phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α). In this bachelor thesis, the pET-21c(+)/eIF2α plasmid was amplified and its authenticity for the eIF2α expression was verified with the use of two independent methods. Next, HRI and eIF2α were produced using the recombinant expression in E. coli BL-21(DE3) cells transformed with the pET- 21c(+)/eIF2α and pET-21c(+)/HRI plasmid, respectively. Both proteins were then isolated from the cells and purified with the use of affinity chromatography and gel permeation chromatography. eIF2α was obtained in sufficient yield (560 μg out of 1 l of TB medium) and purity (90%). A lower yield (25 μg out of 1 l of TB medium) and purity (20%) was reached in the case of HRI. On the other hand, the authenticity of the HRI product was confirmed using spectrophotometric characterization and its enzyme activity was verified as well. Pilot experiments showed that GTP may replace ATP in the process of eIF2α phosphorylation, while UTP and CTP may not.
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Heme sensor proteins sensing both heme and CO
Andrlová, Dominika ; Martínková, Markéta (advisor) ; Libus, Jiří (referee)
Heme, a protoporhyrin IX iron complex, is an important component of many proteins necessary for oxygen transfer, storage and activation, as well as for electron transfer. Another group of hemoproteins includes heme sensor proteins. They are either capable of detecting heme itself, which can regulate in turn the sensor function (heme-responsive sensors) or heme forms a binding site for small gas molecules (O2, CO and NO) and the heme-based gas sensors are regulated by these diatomic gases. However, in the case of some proteins their classification is not clear showing a properties of both heme sensor proteins families. Their functions are regulated by heme interaction and a further change in their function after binding of a gas molecule to heme was observed. This summary search is focused on specific representatives of heme-responsive sensors (which function is regulated by heme binding), in which the further influence of the CO molecule on their functions have recently been observed. It is discussed whether some heme-responsive sensors are also heme-based CO sensors aiming the most recent findings about the selected specific heme sensors representatives. Key words: heme, heme sensor proteins, heme-based gas sensors, CO sensors, heme-responsive sensors, heme redox sensors
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