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The study of properties of anticancer drugs ellipticine, etoposide and doxorubicin in the forms of nanocarriers
Lengálová, Alžběta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...
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Comparison of the determination of hormones (Follicle stimulating hormone, Luteinizing hormone, Prolactin, Testosterone, Progesteron) by two analytical systems. Converting accredited method and its verification.
Kucejová, Soňa ; Martínková, Markéta (advisor) ; Mrízová, Iveta (referee)
Analytical system ARCHITECT i2000SR was verified according to requirements of ÚLBLD VFN and 1. LF UK laboratory in Prague. Repeatability, intermediate precision, and measurement uncertainty were determined as performance parameters for verification of analytical assays for testosterone, progesterone, luteinizing hormone, follicule stimulating hormone and prolactin. Results of Lyphochek control samples, which were measured, were consistent with values given by manufacture. Repeatability: coefficients of variation for testosterone Lyphochek 1 6,81%, for Lyphochek 3 6,40%, progesterone 2,4% and 1,8%, luteinizing hormone 5,38% and 1,89%, follicle stimulating hormone 5,12% and 3,24% prolactin 1,45% a 1,83%. Intermediate precision: coefficients of variation for testosterone Lyphochek 1 6,02%, Lyphochek 2 3,60%, Lyphochek 3 3,07%, progesterone 7,9%, 4,9% and 5,8%, luteinizing hormone 4,50%, 5,51% and 5,83%, follicle stimulating hormone 4,00%, 3,72% and 4,87%, prolactin 4,60%, 4,20% and 5,00%. Measurement uncertainty: testosterone 6,02%, progesterone 7,9%, luteinizing hormone 5,83%, follicle stimulating hormone 4,87%, prolactin 5,00%. Analytical System Architect i2000SR was compared with previously used ADVIA Centaur system to find out, whether it is possible to convert the method Centaur Testosterone,...
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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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Production of recombinant cathepsin C from human blood fluke
Illichová, Hana ; Konvalinka, Jan (advisor) ; Martínková, Markéta (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, a serious parasitic disease occurring in tropical and subtropical areas. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes which participates in the degradation of hemoglobin through its dipeptidyl aminopeptidase activity. This enzyme is critical for metabolism of the parasite and represents a potential target for the development of antischistosomal drugs. Cathepsin C has not yet been studied in detail. This bachelor thesis is focused on the development of expression systems for production of recombinant cathepsin C of Schistosoma mansoni (SmCC). The yeast Pichia pastoris system was used for the expression of an inactive SmCC precursor (zymogen) whose proteolytic stability was analysed. Furthermore, the expression system for SmCC in the protozoan Leishmania tarentolae was employed, and four different SmCC constructs were prepared to optimize production.
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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...
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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.
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Laboratory tests for thyroid function assessment from the economic point of view
Stehlíková, Petra ; Martínková, Markéta (advisor) ; Hýsková, Veronika (referee)
In 2011 Recommendation for laboratory diagnostics of functional and autoimmune thyroid diseases was published. Its target is to recommend correct method when choosing thyroid examination parameters to general practitians and thus find a way how to save money on useless examinations which then can be used by hospital in other area. To verify physicians adhere to these recommendations file of VFN patients in Prague for year 2011 was used and statistically evaluated. Watched parameters included thyrotropin (TSH), free triiodothyronine (FT3), total triiodothyronine (T3), free thyroxine (FT4), total thyroxine (T4), thyroid peroxidase antibodies (anti - TPO), thyroglobulin antibodies (anti - Tg), thyroglobulin (Tg) and thyroxine - binding globulin (TBG). Basic test according to published recommendation should include only TSH and if out of reference limits then it should be followed by FT4 test or antidote assessment. Other parameters are unnecessary for initial examination, some of them are not used anymore or should be examined only by clinics specialized in endocrinology. Based on the file records many physicians are still ordering unnecessary examination parameters. In one year 13,8 % of unnecessary parameters out of 44 415 thyroid function examinations were determined. This is 1 095 013 points that could by...
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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...
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