|
|
Proteomic characterization of human NK cell membrane microdomains
Kádek, Alan ; Man, Petr (advisor) ; Martínková, Markéta (referee)
Proteomic characterization of human NK cell membrane microdomains. (in Czech) Bc. Alan Kádek (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Natural killer (NK) cells are one of the important components of innate immune system. Their main function is to fight against tumors, virally infected or otherwise malformed cells. Plasma membranes of NK cells contain regions with specific lipid composition compared to the surrounding membrane (called membrane microdomains or rafts). Because of their lipid composition, microdomains preferentially accommodate some immunologically relevant proteins and play a role during cellular polarization and signalization. Characteristic feature of membrane microdomains is their partial resistance to solubilization by mild non-ionogenic detergents. In this thesis, microdomains were isolated in a detergent-resistant membrane fraction (DRM) from human NK-92MI cell line and from NK cells immunomagnetically enriched from peripheral blood of non-leukemic donors. For the isolation, Triton X-100 or Brij-98 detergent solubilization and ultracentrifugation in a sucrose density gradient was used. Protein composition of isolated DRMs was analyzed by mass spectrometry employing an LC-MALDI-TOF/TOF method. Protein lists generated in these...
|
|
|
The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Vranová, Iveta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
|
|
|
RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
|
|
| |
|
| |
|
|
The comparison of the efficiency of cytochromes P450 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
v anglickém jazyce Cancer disease is a group of clinically diverse diseases characterized by uncontrolled proliferation of cancer cells incapable of differentiation. Tumor therapy consists of a combination of surgery, radiotherapy and chemotherapy utilizing cytostatic agents. The plant alkaloid ellipticine ranks among a group of cytostatic agents. Its mode of action is based on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. Ellipticine is oxidized through cytochromes P450 catalysis into detoxication metabolites 9-hydroxyellipticine and 7-hydroxyellipticine, and into activation metabolites 12-hydroxyellipticine, 13-hydroxyellipticine and ellipticine N2 -oxide. The aim of practical part of this work was to compare the efficiency of human cytochromes P450 1A1/2 and 3A4 expressed in prokaryotic and eukaryotic systems in the process of ellipticine oxidation. Ellipticine metabolites were analysed using high performance liquid chromatography. It was found that cytochrome P450 1A1 expressed in the prokaryotic system catalyses predominantly the formation of 9-hydroxyellipticine and 7-hydroxy- ellipticine. The difference is minimal in the production of ellipticine metabolites catalysed by cytochromes P450 1A2 expressed in both cellular systems. The formation of...
|
|
|
Bilrubin - physicochemical properties and biochemical importance
Kupcová, Kristýna ; Hudeček, Jiří (advisor) ; Martínková, Markéta (referee)
This thesis briefly summarizes the literary data concerning bilirubin. In addititon to this, its experimental part is devoted to comparison of stability of bilirubin and its derivative, ranarubin, towards photolysis in aerobic conditions. The photolytic experiments were carried out at wavelength 366 nm for 4 hours. The resulting photolysates were characterised by absorption spectroscopy (400 - 800 nm) and mass spectrometry. The data obtained show a high degree of similarity for both compounds, which are both relatively stable against photolytic splitting and photooxidation to biliverdin.
|
|
|
Isolation and characterization of catechol 1,2-dioxygenase of Candida tropicalis
Jechová, Jana ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole source of carbon and energy without formation of any secondary waste product. Catechol-1,2- dioxygenase was isolated from cytosolic fraction of this yeast by the procedure consisting of chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G- 100. The catechol-1,2-dioxygenase was purified to homogeneity. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). The activity profiles at different temperatures showed temperature optimum of 30řC. Kinetic characterizations were studying in different values of pH. The values of Km and Vmax of 0,52 mM and 17,2 nM/min for consumption of catechol, respectively, and 0,34 mM and 12,6 nM/min for formation of cis,cis-muconic acid, respectively, were found at optimum pH of the reaction, pH 7,6.
|
|
| |
|
|
Creation of the plan of care for locality Velky Kamen in Novohradske foothills
MARTÍNKOVÁ, Markéta
The Novohradske Mountains and their foothills belong in a group of well-preserved areas of the Czech Republic. The value of this area lies in its unimpaired landscape and in presence of cryogenic mesoforms of the landscape. The nature park Sobenovska Highlands ensures a widespread protection for the Novohradske Mountains. We can find some smaller protected areas too. They mostly exist in order to protect forest districts. The aim of this bachelor thesis is to project a plan of care for a nature sanctuary called Velky Kamen, including other parts of the landscape like cryogenic mesoforms as the object of protection. The work is divided into two basic sections - physical-geographic characteristicof Novohradske Mountains' foothills and the plan of care for the nature sanctuary Velky Kamen. Maps and photographs are included as supplements acquired during a field research in the key locality.
|
guest ::
RSS feed.