National Repository of Grey Literature 53 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Interaction of bacterial lectins with human lung epithelium
Vyhnalová, Kateřina ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene encoding the same named chloride channel, which is present on the apical membrane of epithelial cells. As a consequence of these mutations, the transepithelial transport of ions and the regulation of other ion channels are impaired. Cystic fibrosis affects primarily respiratory system, where, as a result of the dysfunction of the CFTR protein, is an accumulation of excessively viscous mucus and altered glycosylation of saccharide structures on the surface of the lung epithelium. This pathological condition predisposes CF patients to bacterial lung infections, the leading cause of death in CF patients. The most common pathogen colonizing the respiratory system of CF patients is Pseudomonas aeruginosa (PA). For adhesion to host tissue, PA uses several virulence factors, including lectin PA-IIL, which exhibits an unusually high affinity for L-fucose. In this work the influence of selected monosaccharides and multivalent fucose-based inhibitors (glycoclusters) on PA-IIL binding to human lung epithelium was investigated. To assess the effect of these agens ex vivo, paraffin-embedded formalin-fixed human lung tissues (FFPE tissues) from a healthy individual and...
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...
Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
The study of the role of biotransformational enzymes in chemical carciogenesis
Kondrová, Eliška ; Souček, Pavel (advisor) ; Skálová, Lenka (referee) ; Vobořilová, Jana (referee) ; Bořek Dohalská, Lucie (referee)
In vitro biotransformation studies are an integral part of both toxicological research and drug development. They allow for a significant reduction of tests on human volunteers and provide detailed information about the metabolism of a given compound. Due to limited availability of human liver tissue, it is necessary to make use of alternative model systems and model animal species in the study of the interactions between biotransformation enzymes and xenobiotics. Since the activities of the most important human biotransformation enzymes are absent in the most commonly used laboratory species, rat, information gained in experiments with minipigs is very valuable. Recombinant human biotransformation enzymes expressed in bacteria can be used in biotransformation studies in the form of isolated bacterial membranes. These model systems provide information about the metabolism of a given xenobiotic by a defined enzyme and thein advantages compared to purified enzymes include low cost and quicker and easier preparation with lower loss of enzymatic activity during isolation. The aims of this study were: 1) To prepare a model system with recombinant human biotransformation enzymes expressed in bacterial membranes and to compare the properties of this system with minipig liver microsomes. To assess the usefulness of...
CFTR-mRNA: the alternative for the gene therapy of cystic fibrosis
Pecková, Kateřina ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by mutation of the CFTR gene coding homonymous protein, whose main function is transport of chloride ions. In this thesis, gene therapy was used for correction of this defect. Two types of stable mRNA was synthetised - both contained at least 200 adenines on the 3' end, 25 % of pseudouridine, 25 % of 5-methylcytidine and classical cap (enzyme mRNA) or cap analogue 3'-O-Me-m7G(5')ppp(5')G (ARCA cap) on the 5' end. Cell lines isolated from healthy volunteer (NuLi-1) and those from patient suffering from cystic fibrosis with F508del mutation (CuFi-1) were used. The mRNA transfection efficiency was determined using different methods. Increased expression of the CFTR protein was confirmed by visualization of this protein by optimized immunofluorescence method in both cell lines while using both ARCA mRNA and enzyme mRNA. CFTR protein function was studied using fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinoline (MQAE), which is quenched by halogen ions. CFTR channel ion transport was verified using CFTR(inh)-172. This inhibitor specifically inhibits this channel through binding on the R domain of the CFTR protein. CFTR protein function was restored after 24h transfection of the CuFi-1 cell line by ARCA mRNA. The bacterial adhesion of Pseudomonas...
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...
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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