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Investigation of sugar modified pyrimidine nucleosides as potential inhibitors of Thymidine (TP) and Uridine (UP) phosphorylasis
Růžičková, Markéta ; Wsól, Vladimír (advisor) ; Boušová, Iva (referee)
1 Abstract of the thesis RŮŽIČKOVÁ M.; INVESTIGATION OF SUGAR MODIFIED PYRIMIDINE NUCLEOSIDES AS POTENTIAL INHIBITORS OF THYMIDINE (TP) AND URIDINE (UP) PHOSPHORYLASE Key words: uridine phosphorylase, thymidine phosphorylase, nucleoside, inhibitor, anticancer chemotherapy, substrate specifity The work deals with the investigation of novel potential inhibitors of recombinant thymidine and uridine phosphorylases from E. coli and Salmonella typhimurium. The main goals of this study are the determination of kinetic parameters for natural substrates (thymidine and uridine) for recombinant TP and UP, assessment of the role of phosphate anion in the enzymatic transformations and search for new inhibitors of TP and UP. Study of UP and TP substrate specifity and activity under the various reaction conditions is desired to obtain better view to their functions and to design novel structures of potencial inhibitors. The determination of the Michaelis constant KM and reaction rate Vmax was performed at the spectrophotometer Varian Cary-300 Bio. The representation of obtained kinetic data we used the Lineweawer-Burk plot. The next point of my work was the determination of the enzymatic activity and their substrate specifity for some pyrimidine analogues by HPLC analysis of reaction mixtures. Tested compounds...
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora
This paper deals with the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and its use in tissue engineering of vascular wall. During the work interaction of endothelial cells with the modified fibrin-based biomaterial into which vascular endothelial growth factor (VEGF-A121) has been incorporated was monitored. This modification supported the adhesion and growth of endothelial cells. Vascular endothelial growth factor VEGF-A121 is signal glycoprotein that activates transmembrane receptors on endothelial cells. VEGF-A121 is a key regulator in vasculogenesis, angiogenesis, proliferation, migration and survival of endothelial cells. In this work, this protein was heterologously expressed at a thioredoxin fusion partner in an expression system of E. coli Origami B (DE3). Recombinant VEGF-A121 was additionally coexpressed with bacterial chaperones GroEL/GroES for potential increase of its solubility and biological activity. In the next part of this work thin fibrin network was prepared by catalytic action of thrombin on the polystyrene-bound monolayer of fibrinogen. This network has been further enriched by vascular endothelial growth factor (VEGF-A121), which was covalently incorporated in it by enzyme activity of transglutaminase (factor XIIIa). The last...
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Effect of gene optimization on recombinant expression of human cytochrome P450 3A4
Svobodová, Barbora ; Martínek, Václav (advisor) ; Bořek Dohalská, Lucie (referee)
Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis
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Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry
Kavan, Daniel
CD69 is considered as the marker receptor of activated lymphocytes and is expressed at sites of active immune response. Physiologically it appears in the form of covalently bound homodimer, however after examining its three- dimensional structure we suggested Q93 on one subunit and D88 with E87 on the other one to participate on the inter-subunit interactions. Even more profound intertwining was observed in case of R134 of one subunit with A136 and Y135 on the other one. Therefore Q93, R134 or both were mutated into alanines and showed the monomeric form just in case of double-mutant. This fact influenced significantly also the binding of ligands. While the Kd values for binding of GlcNAc was approximately 10-5 M in case of monomeric form, in case of dimeric form it was 10 times lower and even 100 times lower in case of the longest covalently bound dimers. Although the gel filtration retention time decrease was observed, which could indicate a change in molecular fold, the value of experimentally determined sedimentation coefficient was identical. Moreover neither the comparison of HSQC NMR spectra before and after ligand saturation revealed any significant shifts. Hydrogen deuterium exchange is a chemical process in which a covalently bonded hydrogen atom is replaced by deuterium or vice versa. As...
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Screening for the HCV IRES interacting proteins
Roučová, Kristina ; Pospíšek, Martin (advisor) ; Kuthan, Martin (referee)
Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...
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Development of transient transfection protocol for HEK293 EBNA1 cells
Šmíd, Jiří ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.
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Characterization of excretory-secretory proteins of liver fluke Fascioloides magna.
Beránková, Kateřina ; Kašný, Martin (advisor) ; Horn, Martin (referee)
Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...
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Recombinant probiotics
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Theoretical part of this thesis focuses on present state and research of recombinant probiotics and their use in food industry and health care. It also concentrates on their beneficial effects on the health of individuals. The experimental part focuses on the identification of specific bacterial strain in a probiotics food supplements. The DNA was isolated from these products by use of magnetic microparticles and obtained DNA was subsequently analysed through polymerase chain reaction.
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Preparation of X. tropicalis recombinant growth factors and their characterization in testicular tissue culture.
Borecká, Marianna ; Krylov, Vladimír (advisor) ; Drobná Krejčí, Eliška (referee)
In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells
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