|
|
Mechanism of signal transduction in a model representative of prokaryotic heme-containing oxygen sensors
Smrčka, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
A two-component system, consisting of a histidine kinase and a response regulator, is a crucial molecular-biology tool for many bacteria to react to an environmental changes. An important step in activation of the two-component system is an autophosphorylation reaction on the dimeric histidine kinase, which involves the transfer of a phosphate group from ATP in the catalytic domain to a conserved histidine molecule. Depending on whether the transfer of the phosphate group occurs within one subunit of the dimer or from one subunit to another, we distinguish a cis- or trans-autophosphorylation, respectively. Here we study the autophosphorylation reaction of globin coupled histidine kinase from soil bacteria Anaeromyxobacter sp. Fw109-5 (AfGcHK), which uses heme to detect gaseous molecules. Using a phosphorylation analysis of a heterodimer of AfGcHK composed of a subunit with a defective ATP-binding site and a subunit with a phosphorylatable histidine substituted for alanine, the trans-mechanism of autophosphorylation was identified for AfGcHK. Key words: two component signal systems, histidine kinase, heme-containing oxygen sensors, Phos-tag, AfGcHK [IN CZECH]
|
|
|
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...
|
guest ::
