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Expression and activity of rat cytochromes P450 3A after exposure to benzo[a]pyrene and Sudan I
Ličko, Vojtech ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
The aim of this Bachelor thesis is the study of the effect of two carcinogenic compounds, benzo[a]pyrene and Sudan I, co-administered to rats individually or in combination, on the expression and the activity of important biotransformation enzymes cytochromes P450 of subfamily 3A in liver - a main organ of xenobiotic metabolism, in which the amount of CYP3A is especially high. Using the quantitative PCR method, the decrease of the gene expression of CYP3A1/2 in the livers of rats exposed to benzo[a]pyrene and Sudan I individually or in combination, was observed. Using the Western Blot method with a consecutive immunodetection, we found the decrease of the protein expression of CYP3A in the livers of rats treated with benzo[a]pyrene and Sudan I alone. Specific activity of CYP3A, determined by marker reaction of CYP3A, which is 6β-hydroxylation of testosterone, did endorse the previous results only in some of the premedicated groups of rats. It can be concluded that the exposure of rats to both studied compounds with carcinogenic potential resulted in a decrease in the expression of hepatic CYP3A in vivo. (In Czech) Keywords: cytochromes P450, benzo[a]pyrene, Sudan I, expression, enzyme activity
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Characterization of hapt oglobin glycoforms in human serum
Darebná, Petra ; Pompach, Petr (advisor) ; Ptáčková, Renata (referee)
Characterization of haptoglobin glycoforms in human serum Petra Darebná (Katedra biochemie, Přírodovědecká fakulta, Univerzita Karlova v Praze, Česká republika) Changes in glycosylation of proteins are associated with several types of cancer, including hepatocelular carcinoma and colorectal carcinoma. This project deals with data independent analysis using ion cyclotron resonance with Fourier transform and tandem mass spectrometry with liquid chromatogramy and multiple reaction monitoring to quantify these changes in hepatocelular cancinoma and colorectal carcinoma with liver metastases. In the first part of the project the haptoglobin was enriched from pooled serum samples of pacients with hepatocellular carcinoma, colorectal cancer and colorectal carcinoma with metastases using hemoglobin immobilized on CNBr-activated Sepharose 4B and then purified by high pressure liquid chromatography. Individual haptoglobin glycopeptides were analyzed using ion cyclotron resonance with Fourier transform. In the second part of the project we analyzed changes in glycosylation depending on diferent tumor diseases in partially depleted serum of individual patients using ethanol precipitation. Individual fucosylated glycoforms of N-glycopeptides of serum proteins were compared with their nonfucosylated forms. In...
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Application potential of modification approaches (chemical agents, photo-nanoprobes) and mass spectrometry to study protein structure and protein-protein interaction
Ptáčková, Renata ; Šulc, Miroslav (advisor) ; Levová, Kateřina (referee) ; Osička, Radim (referee)
A comprehensive understanding of physiological role of proteins requires knowledge of their three-dimensional structure, dynamics and protein-protein interactions. Chemical cross-linking in combination with mass spectrometry represents an alternative approach to standard methods for protein structure elucidation (X-ray crystalography, NMR spectroscopy) and enables characterization of interaction interface within protein complexes in their native states. The presented thesis is mainly focused on novel cross-linking methodology based on the in vivo incorporation of methionine analog with photo-reactive functional group (photo-Met) into the sequence of studied protein (so called protein photo-nanoprobe). Interaction between two molecules of 14-3-3zeta protein was used as a model to test and optimize the protein photo-nanoprobe production. The findings confirmed usefulness of this approach for mapping the protein-protein interactions. The photo-initiated cross-linking was used to detect the heterooligomeric membrane structures of cytochromes P450 2B4 and b5 and the molar ratio of cytochromes within individual complexes was assessed. The chemical cross-linking in combination with mass spectrometry was employed to characterize the interaction of their catalytic domains and two mutual orientations of...
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Structural characterization of Nkr-p1f/Clrg interaction
Tůmová, Lucie ; Kavan, Daniel (advisor) ; Ptáčková, Renata (referee)
NK cells are ranked among lymphocytes, which are involved in primary immune responses to virus infected and tumour cells. They produce cytokines at the same time and are involved in the formation of secondary immune responses together with T and B lymphocytes. This diploma thesis is engaged in the study of the structure and interaction of the transmembrane receptor Nkr-p1f with its ligand Clr-g. This receptor Nkr-p1f has an activation function and is able to initiate the cytotoxic function of the NK cells. The three-dimensional structure has been solved as a Clr-g homodimer, but its interaction with the Nkr-p1f receptor remains mysterious. The aim was to prepare an expression vector coding the entire extracellular region of the receptor NKR-P1F and carry out the production of the receptor Nkr-p1f and Clr-g in prokaryotic expression system, subsequently renaturation and purification of the proteins in vitro. This way prepared proteins were analyzed by electrophoresis and mass spectrometry. Their interaction was studied with biophysical method, surface plasmon resonance (SPR). However, the interaction between them was not revealed by SPR technology. (In Czech)
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Preparation of expression construct of NKp44 receptor
Hajná, Vladimíra ; Kavan, Daniel (advisor) ; Ptáčková, Renata (referee)
NK cells are part of the innate immune system. Their immediate response to pathogen plays a role mainly in the elimination of tumor or virus-infected cells. Recently, NK cells, especially their receptors, are increasingly the subject of various studies. One group of NK cell receptors are known as natural cytotoxicity receptors (NCRs). Nowadays three receptors are belonging to this group, one of them is NKp44. NCR receptors play important role in the activation of NK cells. NKp44 is considered as the first specific receptor for activated NK cells. Its ligands are not yet fully known. Recent research has highlighted one of the potential ligands, the protein PCNA, which surprisingly causes inhibition of NK cell function. The aim of this thesis was to prepare plasmid encoding the extracellular part of the human receptor NKp44. DNA insert was obtained by RT-PCR reaction from isolated NK- 92MI cell line RNA. Subsequently it was ligated into the cloning vector pBSSK(+) and then into production pET30-a(+) vector, resulting in the plasmid for expression of the extacellular portion of NKp44.
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Pošta pro tebe-reality TV and social motivation of participations
Ptáčková, Renáta ; Bednařík, Petr (advisor) ; Gerbery, Juraj (referee)
Abstrakt_anglicky _Diplomova_prace_Ptackova_Renata The diploma thesis "Pošta pro Tebe - reality TV and social motivation of participants" focus on the use of social television. It describes in detail the reality TV genre, its brief history of broadcasting, focusing on the motivational elements of the program participants Pošta pro Tebe. It investigates the impact and effect of media and mediated relationship to the contents of a reality. It analysis the audience base, explores reasons for visiting the TV studio spectators, why people looking for this TV show and why they watch it by questionnaires, interviews. It's looking for the credibility of the program and asking whether the audience would become program participants. Further, the work captures the audience ratings and popularity in each year of its broadcast. Finally, describes the creation of the show and behind the scenes. The diploma thesis is divided into 4 chapters: Theoretical introduction thesis (clarifying the concepts that are crucial for the work related), Ratings (analyzing rating charts of the first channel of Czech Television and the demographic composition of the audience), Behind the Scenes program (described shooting of, the role of interested participants, technical background of the show), Audience (defines who is the audience and...
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