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Molecular mechanisms of regulation of forkhead transcription factor FOXO4
Bouřa, Evžen ; Obšil, Tomáš (advisor) ; Konvalinka, Jan (referee) ; Schneider, Bohdan (referee)
I 7. Abstract The main goal of this PhD thesis was to investigate the structure of FOXO4-DBD/DNA complex and the molecular mechanism of FOXO4 DNA binding properties. Especially, the role of protein kinase B phosphorylation and the regulatory role of 14-3-3 proteins. This work has been published in four original papers (for full citation see page no. 3). Paper l: The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation- dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of Foxo localization toward the cýoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have...
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Structural studies and tissue distribution of human GCPII and characterization of its rat and porcine orthologs
Rovenská, Miroslava ; Konvalinka, Jan (advisor) ; Jonák, Jiří (referee) ; Martásek, Pavel (referee)
Since GCPII is a potential pharmacological target, it is being extensively snrdied in many labs all around the world and these studies comprise many topics. This is minored also by this PhD thesis. The papers included here concem two major issues: 1. analysis ofGCPII structure and interactions with ligands; 2. study of CCPII distribution in tissues of human and two other species considered as potential animal models and kinetic characterization of the corresponding GCPII orthologs. Structural studies of GCPII active site and substrate binding are driven by the attempt to broaden the information that could help the rational design of novel small GCPII ligands, functioning either as inhibitors in neuronal damage or as imaging agents in cancer diagnosis. Two papers included in this thesis describe ligand binding in the GCPII active site in detail, with particular ernphasis on the Sl'pocket in one case and on the Sl pocket in the other' Based on our findings, we can describe a set of interactions goveming GCPII affinity to a substrate, accommodations that CCPII active site is capable of during ligand binding, the limits imposed on the ligand and tolerance of the enzyme to varying ligand nature. All these pieces of infomation are useful for the design of novel compounds with high affinity to GCPII' sufÍicient...
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Interaction of the adenylate cyclase toxin with complement receptor 3 - Relation of structure and function
Morová, Jana ; Šebo, Peter (advisor) ; Konvalinka, Jan (referee) ; Bezouška, Karel (referee)
4 CoNcl-usroxs PnonucrroN oF tNrEcRtN CDI lb/CDlg F Four fragments of the subunit cDilb were produced and purified and then they were used for immunization of mice and for preparation of specific antibodíes. detected that 52 cells are not able to transport effectively this subunit to cell surface or to secrete its extracellular domain into medium and not even it has a signal peptide specific for 52 cells. Further it has been shorvn, that the level of production of CDl8 subunit was not affected by usage of constitutive or inducible plasmid. ANALYSIS oF THE |NTERÁCTIoN oF RTx ToxINs w|TH p2 INTEGRINS - THE RoLE oF THT, GLYCOSYLATION OF RECEPTORS IN BINDINC ON RTX TOXIN of cell surface glycoproteins, suggesting that cyaA binding to the cell surface- expressed cDllb/cDl8 integrin fully depends on its glycolsylation. It has been a|so demonstrated. that the deglycosylation did not affected ťormation of CD I I b/CD l8 heterodimer or its expression to cell surface. inhibited in the presence ofonly saccharide units that occur in the oligosaccharide chain of integrin molecule. This demonstrates, that cyaA directry recognizes the N-linked oligosaccharide chain ofits p2 integrin recepror. CD l I b/CD I 8-expressing cells. cytotoxic activity of others RTX toxins. l5...
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The effect of deficiency and excess of copper ions on the alga Ostreococcus tauri
Malych, Ronald ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Copper is an essential element for many organisms. Copper ions play an important role in respiration, iron ion transport and cell development. The mechanism of uptake and utilization of copper ions is best described in Saccharomyces cerevisiae. This is a very demanding process, consisting of many regulators that maintain the concentration of copper ions below the toxic level. The toxicity of these ions is manifested by the formation of reactive oxygen species by reaction similar to the Fenton reaction. Algae, being photosynthetic organisms that contain both plastids and mitochondria, are a good model for studying the uptake and utilization of copper ions. As algae model organism has been chosen Ostreococcus tauri, which is the smallest living eukaryote. In this work, we are looking at the question of how cells of O. tauri deal with the deficiency and toxicity of copper ions. An immunochemical analysis revealed that there was no change in the expression of the enzyme cytochrome c oxidase, which is part of the respiratory chain and contains CuA domain that binds atoms of copper. Spectrophotometric assay showed that the lack of copper ions does not affect the formation of photosynthetic pigments, chlorophyll a and b. Proteomic analysis revealed proteins whose expression was affected by the toxicity of...
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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
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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Preparation and enzymatic incorporation of deoxyribonucleic triphosphates derived from pyrimido[4,5-b]indol to DNA using selected polymerases.
Bosáková, Andrea ; Konvalinka, Jan (advisor) ; Hodek, Petr (referee)
This bachelor thesis deals with the synthesis of pyrimido[4,5-b]indole 2'- deoxyribonucleosides and their following trifosforylation. Overall there three new analogues 2'-deoxyadenosine triphosphate with benzen ring were prepared. Furthermore, the ability of DNA polymerase to incorporate in total four modified 2'-deoxyribonucleosides into the oligonucleotide strand by primer extension (PEX) method was observed. All the modified 2'-deoxyribonucleotides were incorporated into the oligonucleotide strand, however the success of the subsequent elongation was different accoring to the DNA polymerase that was used and according to the substitution in position 6 in the structure of substrate. Key words nucleosides, modified nucleotides, DNA polymerase, PEX reaction
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Human glutamate carboxypeptidases II and III
Navrátil, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Pavlíček, Jiří (referee)
The herein presented Ph.D. dissertation describes kinetic and structural characterization of human glutamate carboxypeptidases II and III (GCPII and GCPIII) using a complete panel of their natural substrates. These enzymes hydrolyze C-terminal glutamate from their substrates. They share 67 % sequence identity and also similar enzymatic activities. This thesis quantitatively compares human GCPII and GCPIII in terms of their ability to hydrolyze the substrates N-acetyl-L-aspartyl-L-glutamate (NAAG), folyl-poly-γ-L-glutamic acids (FolGlun) and β-citryl-L-glutamate (BCG). We demonstrated that GCPIII hydrolyzes its substrates in a metal- dependent manner, that BCG is a specific substrate of GCPIII, and that NAAG and FolGlun are specific substrates of GCPII. We also provide indirect biochemical evidence that GCPIII might feature a heterometallic active-site cluster. Additionally, we characterized the relevance of a surface exosite of GCPII, the arene-binding site (ABS), for the hydrolysis of FolGlun substrates using mutagenesis and enzyme kinetics and showed that polymorphic His475Tyr variant of GCPII hydrolyzes FolGlun substrates with the same kinetic parameters as the wild-type enzyme. Furthermore, this thesis focuses on structural aspects of the substrate specificities of GCPII and GCPIII: we present...
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Identification and characterization of reductases of fatty acids participating at pheromone biosynthesis
Wenzelová, Petra ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
In the mating season, bumblebee males leave pheromone track on different objects on their flight routes to attract females of the same species. Synthesis of pheromones, which takes place in the labial gland of bumblebee males, involves enzymes called fatty acid reductases (hereinafter reductases). They catalyse the reduction of fatty acids bound in the form of acyl-coenzyme A to the alcohols. In this work, I functionally characterized two reductases highly expressed in labial gland of the two most common species of bumblebees Bombus lucorum and Bombus terrestris in order to verify their role in the biosynthesis of species specific male pheromones. I expressed these reductases in yeast Sacccharomyces cerevisiae. I confirmed the heterologous expression of reductases and prepared lipid extracts from yeast cells, in which we identified by gas chromatography with mass spectrometric detection (GC/MS) alcohols derived from fatty acids with long chain. These were octadecanol, icosanol and docosanol.These results lead us to conclusion that reductases are involved in the biosynthesis of alcohols with long hydrocarbon chains, which are components of marking pheromones of mentioned bumblebee species. These partial results will be used in a future study that wants to reveal the molecular mechanisms of male...
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