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Measurement of signalling pathway activation in mouse macrophage line IC-21 and primery dendritic cells after infection with tick-borne encephalitis virus.
Kožantová, Jana ; Lieskovská, Jaroslava (advisor) ; Drbal, Karel (referee)
Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.
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Comparison of methods of ECLIA and ELISA in determining the beta-CrossLaps
KOŽANTOVÁ, Jana
Osteoporosis is a very old disease affecting a large majority of population. Its occurrence rate has been growing permanently. It is a systemic disease of bones for which the decrease in bone mass, i.e. of inorganic as well as organic bone part is characteristic. Typical symptoms are osteoporotic fractures. To perform an in time diagnostics of the disease, it is necessary to execute the necessary examinations. One of them is also the laboratory determination of markers of bone resorption and new creation for finding out the state of bone metabolism. The target of my bachelor thesis was to find out the level of the bone resorption marker (beta-CrossLaps) by means of ELISA and ECLIA methods on the same serum from patients with the diagnosed osteoporosis and to process statistically the results. In the next part I dealt with the structure and functions of bones. Then I focused on markers of bone formation and resorption. Finally I mentioned the bone metabolism and its control mechanisms. I concentrated mainly on the metabolism of calcium, D vitamin, parathormone and calcitonin. I carried out the practical part of my bachelor's thesis in the accredited private laboratory STAFILA, spol. s r. o. in České Budějovice. The procedure of my work followed the standard operating procedure of the laboratory. In the research, in total 66 serums of patients with osteoporosis were analysed by me. At first, I determined the concentration by beta-CrossLaps method based on the principle of sandwich ELISA on the appliance NexGen Four from TestLine company. Then I analysed the same samples on the automatic analyser IDS-iSYS from IDS company, this time by ECLIA method. Both appliances on which the determination was executed, principles of both methods, applied analytic sets and the procedures of analysis are described in the methodical part. In the next part of my bachelor's thesis, the results of my measuring are stated. At first I entered the concentrations beta-CrossLaps in ng/ml measured by both methods into the table and for the respective samples I stated the sex and years of birth of individual patients. Moreover I prepared a graph illustrating the share of men and women of various age categories in my research. The second graph shows the differences in values of beta-CrossLaps concentrations between both methods. Resulting concentrations were subject to regression analysis. I applied linear regression and Grubbs test for finding out the outlying values excluded from the further statistical processing. I performed Passing-Bablok regression based on the remaining values. As a result of regression equation and 95% CI of the slope B and intercept A, the proportional error of measuring was rejected but on the contrary, the systematic error was confirmed. Correlation coefficient R = 0,97 shows a very good linear dependency between both methods. It turned out by practice in the laboratory that the evaluation of concentrations beta-CrossLaps from the same patients on various appliances may show partially different values. I worked at two different analysers, each of them was based on another principle, with a different sensitivity and calibration. All of this could have the influence on the resulting concentration of individual samples. The advantage of automatic analyser IDS-iSYS is a quicker determination. Moreover it is possible to perform on this apparatus all the possible examination concerning the bone metabolism from D vitamin, parathormone, osteocalcin up to bone markers. It is advantage for the patients with osteoporosis as well as with other metabolic skeleton diseases. It follows from this that all the necessary examinations may be carried out on one apparatus.
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