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Municipal Hall
Pecková, Kateřina ; Šárka, Justová (referee) ; Balíková, Jitka (advisor)
The aim of the diploma work is a design of the Municipal House in the municipality of Habrovany u Vyškova. The building is designed in a gentle slope, the ground plan is irregular, the height of the object is different in the individual parts. The model of the building is a linear wall system.The building consists of three work sets which are the municipal authority, a fire station, and a cultural social hall. The municipal authority is a two-floor structure, the other rooms are one-floor ones. The fire station has a garage for two vehicles.
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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...
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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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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...
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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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Municipal Hall
Pecková, Kateřina ; Šárka, Justová (referee) ; Balíková, Jitka (advisor)
The aim of the diploma work is a design of the Municipal House in the municipality of Habrovany u Vyškova. The building is designed in a gentle slope, the ground plan is irregular, the height of the object is different in the individual parts. The model of the building is a linear wall system.The building consists of three work sets which are the municipal authority, a fire station, and a cultural social hall. The municipal authority is a two-floor structure, the other rooms are one-floor ones. The fire station has a garage for two vehicles.
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