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Phylogenetic analysis of Rieske dioxygenases large subunits genes in soil contaminated with jet fuel
Ptáček, Jakub ; Bořek Dohalská, Lucie (advisor) ; Poljaková, Jitka (referee)
The former military air-base Hradcany is among the most contaminated with organic pollutants localities in Czech Republic. Main cleanup strategy in the area is the bioremediation taking advantage on the natural potential of the autochthonous soil microorganisms to evolve catabolic pathways for in situ degradation of the pollutant. The diversity and abundance of the pathways, as well as the specificity and activity of the encoded enzymes are priority biotic factors determining the bioremediation efficiency. Main task of this work was to analyze the bacterial diversity in jet fuel contaminated soils based on key catabolic genes encoding the Rieske non-haem iron dioxygenases of the toluene/ biphenyl oxygenase branch. High molecular soil DNA was extracted and the sequences encoding catabolic genes were selectively enriched by hybridization to biotinylated oligonucleotides on magnetic microbeads with covalently bound streptavidin. Fragments of the genes for the -subunits of Rieske non-haem iron oxygenases were amplified and analyzed by restriction analysis, cloning and sequencing. Their evolutionary histories were inferred using the Neighbour-Joining and the maximum likelihood methods. The catabolic genes diversity in the actively bioremediated and highly polluted soil HRB was compared with the diversity in the...
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Stimulation of pancreatic β-cell proliferation by synthetic mRNA
Volejníková, Anna ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a severe metabolic disease associated either with loss or functional impairment of insulin-producing β-cells. A major goal in diabetes research is to develop effective ways of pancreatic β-cells restoration. One of the possible approaches is the stimulation of β-cell proliferation. Transcription factor c-Myc is one of the promising targets for stimulation of β-cell proliferation. However, the adenovirus-mediated long-term overexpression of this oncoprotein is associated with risk of cellular dedifferentiation, apoptosis and development of cancer. Therefore, the applicability of an alternative approach for the c-Myc ectopic expression, based on the in-vitro transcribed synthetic mRNA, was verified in this thesis. Use of in vitro transcribed c-Myc-mRNA allows achieving short-term overexpression of transcription factor c-Myc in host cells. High level of c-Myc expression by the β-cells, was detected following the transfection with c-Myc-mRNA. However, within the 48 hours the expression level of c-Myc protein declined to the basic level. The physiological degradation of c-Myc-mRNA by the host cells is a key factor that reduces the risk of cancer development. Transfection of rat islet cells with c-Myc-mRNA resulted in a significantly increased β-cell proliferation. Under the...
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The development of a model system for studying chloride ion transport in the epithelial cells of patients with cystic fibrosis
Pecková, Kateřina ; Bořek Dohalská, Lucie (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis is caused by a genetic defect in the CFTR protein, whose main function is chloride transport across epithelial cells. The measurement of CFTR ability to transport chloride is considered a good, and perhaps, the only practical method to assess its activity. In this thesis, the transport of chloride ions across the CFTR channel was studied using airway epithelial cell lines of healthy patients (NuLi-1) and patients with cystic fibrosis (CuFi-1). A fluorescent method using a fluorescent chloride-sensitive probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE) was chosen and optimized. This compound is providing fluorescence in the blue part of the spectrum and has the greatest sensitivity to chloride ions. In the development of an optimal method two approaches of chloride transport measurement were used. In the first experiment the secretion of the chloride ions to the buffer containing MQAE was measured. In the second one the dye had to be loaded into cells before performing experiment. Then, the MQAE fluorescence quenched by intracellular chloride was monitored by a change in the fluorescence intensity of the probe. The second method was considered as a usefull and more reproducible to study chloride transport across cell membranes. Moreover, the influence of the CFTR modulator...
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Possible approaches to the treatment of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by a mutation in the CFTR gene. This leads to an absence or a malfunction of CFTR chloride channel, which is also a crutial regulator of other ion channels. This thesis was aimed at gene therapy of cystic fibrosis using CFTR- mRNA gene transfer. To determine the expression of CFTR protein, methods of indirect immunofluorescence and Western blot immunodetection were utilized. Also relative gene expression levels of CFTR gene were assessed by quantitative PCR. Experiments were carried out on a healthy lung epithelial cell line (NuLi-1), a lung epithelial cell line with F508del mutation (CuFi-1) and an embryonic kidney epithelial cell line (HEK293S). CFTR protein was visualized by previously mentioned methods using six primary antiobodies (432, 450, 570, 596, 769, CF3). Primary antibodies 570 a CF3 were found as optimal for the detection of CFTR protein by the method of indirect immunofluorescence, whereas for the detection of this protein by the method of Western blot only the CF3 antibody was suitable. It was also determined that CFTR gene is expressed in overall small levels. Its relative gene expression in the CuFi-1 cell line was approximately six times higher than in the NuLi-1 cell line. The efficiency of transfection of CuFi-1 cell line by CFTR-mRNA...
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Effects of natural compounds on viability of tumor cell lines
Boleslavská, Barbora ; Poljaková, Jitka (advisor) ; Bořek Dohalská, Lucie (referee)
Cancer is considered to be one of the most serious issues of medicine nowadays. Moreover, its incidence is still rising. Despite the huge progress in modern treatment methods, cancer therapy is still limited by many difficulties. This work focuses on the natural substances such as epigallocatechin gallate, caffeine, Cannabis sativa ethanol extract, Origanum acutidens water extract, Mentha piperita water extract and its effects on the human neuroblastoma cell line UKF-NB-4. The first part of the bachelor thesis deals with determining the viability of human neuroblastoma cell line UKF-NB-4 exposed to tested substances as well as it studies the effects of those substances on the cell cycle and caspase activity. Finally, it was tested, whether those substances are able to induce apoptosis in neuroblastoma cell lines. Tests were undertaken on the MUSETM cell analyzer and on the flow cytometer. The second part of the bachelor thesis focuses on the expression of protein p53 and retinoblastoma protein in neuroblastoma cell lines exposed to tested substances. Detection was carried out by Western blot analysis. Epigallocatechin gallate exhibited the most significant effect on human neuroblastoma cell line. It lowers the expression of retinoblastoma protein as well as it arrests cells between G0/G1 and S...
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Effect of gene optimization on recombinant expression of human cytochrome P450 3A4
Svobodová, Barbora ; Martínek, Václav (advisor) ; Bořek Dohalská, Lucie (referee)
Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis
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The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
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Therapeutic strategies in cystic fibrosis
Křesťan, Jan ; Bořek Dohalská, Lucie (advisor) ; Hudeček, Jiří (referee)
Cystic fibrosis is an autozomal recessive disease caused by mutations in the CFTR gene. The aim of this work is conclusion of therecent knowledgefrom theusage of CFTR modulators in the treatment of cystic fibrosis. The first part of this bachelor thesis is dedicated to the disease itself, its history and symptoms. In the form of description of the CFTR gene and its mutations which lead to the cystic fibrosis disease, the causes of the disease are described. Function of CFTR protein is also mentioned. The second part of this thesis is focused on conclusion of the current knowledge of the CFTR modulators treatment. In the last chapter of the work, possible future changes and ways of treating patients with CFTR modulators therapy are considered.
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