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Preparation of plasmids for expression of interleukin 2-fusion monoclonal antibody
Slavata, Lukáš ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Aim of the thesis was optimization of production of monoclonal antibody against interleu-kin 2 (IL 2) as fusion construct with IL 2 by preparation of new expression vectors based on pTW5 plasmid and by replacing the native signal sequence with new one, originally from secreted alkaline phosphatase. This fusion antibody has interesting biological activity with therapeutic potential - prolonging the half-life of IL 2 in blood circulation. (The thesis is written in Czech.) Powered by TCPDF (www.tcpdf.org)
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Determination of cortisol in saliva
Žánová, Magdalena ; Martínková, Markéta (advisor) ; Milichovský, Jan (referee)
This thesis deals with salivary cortisol levels measured by the ADVIA: Centaur CP Immunoassay System. Experimental saliva sampling was performed on 141 pro- bands from age 7 to 76. Volunteers were divided to groups according to their sex: males, females using hormonal contraceptives (HC) and females not using HC. Re- ference intervals of morning and evening salivary cortisol were defined: females not using HC 13,2 - 55,5 nmol/l and females using HC 15,5 - 44,2 nmol/l for mor- ning salivary cortisol. Females not using HC 4,0 - 16,6 nmol/l and females using HC 7,9 - 22,6 nmol/l for afternoon salivary cortisol. Males 15,8 - 47,7 nmol/l for morning salivary cortisol and 5,2 - 25,4 nmol/l for afternoon salivary cortisol. Dif- ferences in stated intervals were imperceptible in all monitored groups. However, it is necessary to maintain different reference intervals for both morning and evening sampling. Reference interval for morning sampling was 14,3 - 46,2 nmol/l and refe- rence interval for afternoon sampling was set at 4,0 - 22,2 nmol/l. Daily profiles of salivary cortisol were determined in 6 females and 4 males in four different parts of a day. The course of salivary cortisol levels corresponded in females not using HC, females using HC and males with circadian rhythm, which is subject to cortisol. Sa-...
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Molecular mechanisms of apoptosis induction in cancer cells.
Fenclová, Tereza ; Novák, Petr (advisor) ; Milichovský, Jan (referee)
Cancer diseases are now the third leading cause of death (20% of all deaths). It is therefore important to find new ways of getting tumor cells effectively and specifically disposed of and a promising path is targeted therapy. One of the most frequently deregulated protooncogenes in cancer is C-MYC, which makes it suitable as an effective target for treatment. However, the development of such targeted active ingredients is very expensive, so in this thesis we investigate natural substances that have been used for the treatment of cancer in ancient China. We examined the substances shikonin, cnicin and artemisinin. The results show that shikonin induces apoptosis of tumor cells by reducing the expression of C-MYC and activating tumor suppressor kinase MST1. Cnicin reduces the expression of C-MYC as well, but activates MST1 only weakly. Artemisinin, on the other hand, increases expression of C-MYC and doesn't activate MST1, thus operates on inducing apoptosis of tumor cells by a completely different mechanism. (In Czech)
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Heterologous expression and isolation of human cytochromes P450 1A1/2
Milichovský, Jan ; Martínek, Václav (advisor) ; Hodek, Petr (referee)
Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...
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Assessment of laboratory tests efficiency in case of exocrine pancreas function determination
Šubčíková, Lenka ; Martínková, Markéta (advisor) ; Milichovský, Jan (referee)
Chronic pancreatitis is complex disease with complicated diagnosis. Nowadays there are the diagnosis and classification of chronic pancreatitis primarily based on imaging methods. In this study the results of two indirect tests of pancreatic exocrine function with different arrangement were compared. The pancreatic fecal elastase 1 was determinate by immunoassay with monoclonal antibody, as a simple screening test. The dynamics and kinetics of pancreatic exocrine secretion was observed by a breath test with 13 C-labeled substrate. The group of four volunteers was monitored for six months and analyzed each month. The mutual variability of these tests and their correlation was studied. The patients' (suspected suffering by chronic pancreatitis) anonymous data for these tests created by the Institute of laboratory biochemistry and laboratory diagnostics between 1999-2012 were statistical processed. We found, that the indirect tests of pancreatic exocrine function and determination of pancreatic enzymes does not correlate. Both of these tests have the specific diagnostic value mainly for diagnosis of pancreatic exocrine insufficiency. 1
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