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Study of mechanism of signal transduction in case of two model heme-containing sensor proteins
Mihalčin, Peter ; Martínková, Markéta (advisor) ; Kavan, Daniel (referee)
Heme-based gas sensing proteins belong to a group of proteins that are present in signalling pathways of bacteria. A precise regulation of physiological functions, such as intercellular communication or biofilm production, is essential for the survival of these bacteria and their adaptation to the changing surrounding conditions. Heme-based gas sensors are able to detect the concentration of gas molecules in the local environment via their sensory domain (which contains a heme molecule as the intrinsic detection site) and transmit the signal to the functional domain helping to regulate the adaptation of many processes. These, often pathogenic, processes contribute to extended resistance of bacteria against antibiotics. Heme-based sensors are thus potentially a new therapeutic object of interest in antimicrobial treatment. In order to provide this type of treatment, it is crucial to understand the exact mechanism of intramolecular signal transduction facilitated by heme-based sensors. One of the approaches to unravel these mechanisms is further study of model sensory proteins. This thesis focuses on the analysis of a signal transduction performed by two model globin-coupled heme-based oxygen sensors.
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The use of mass spectrometer for aminoacid analysis
Matoušková, Zuzana ; Kavan, Daniel (advisor) ; Kukačka, Zdeněk (referee)
Amino acid analysis is used across many disciplines. However, it carries certain problems such as missing asparagine, glutamine and tryptophan, due to the primary purpose for which it was developer. Nevertheless, there are many applications in which these missing amino acids need to be quantified as well. A faster but less sensitive alternative would be the mass spectrometry detection, which also makes it possible to analyze other metabolites and non-amino compounds. It is necessary to know all the possibilities and limits of the newly developed method to be applicable to the full benefit. This bachelor thesis involves both a method in which individual amino acids are separated using hydrophilic interaction chromatography and subsequently detected by mass spectrometry as well as its basic characterization and comparison with a commonly used fluorescence method commercially available from Waters.
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Multivalent carbohydrate ligands of galectins
Hovorková, Michaela ; Křen, Vladimír (advisor) ; Kavan, Daniel (referee)
Galectins are proteins, wich belong to a group of lectins that are able to bind to saccharide units and they specifically recognize glycans exposed to the surface of the cells. Galectins participate in vivo, for example, in carcinogenesis, angiogenesis or fibrosis. Their occurrence increases significantly in connection with a number of pathogenic processes, therefore they can be used as markers for some types of cancer or cardiopathology and also for the targeted binding of therapeutics and/ or imaging agents in diagnosis and therapy. Galectin-3 has a specific structure known as chimeric and it is capable of forming multivalent oligomers. The natural ligands of galectins are glycans containing terminal β-galactosides, especially N-acetyllactosamine, but the binding of monovalent glycans is very weak. Glycoconjugates with high affinity to galectin receptors are optimally multivalent, biocompatible and stable in vivo. These criteria accomplish carbohydrate ligands conjugated to soluble and structurally flexible N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In this work two types of functionalized disaccharides based on N-acetyllactosamine (Galβ4GlcNAc) and its structural analogue of N,N'-diacetyllactosamine (GalNAcβ4GlcNAc) was prepared by enzymatic synthesis. For the synthesis were used...
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Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for "decoy" surface receptor - RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less...
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Structure and interaction of human 14-3-3 regulatory protein: photoaffinity labelling in vitro and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
This work is focused on the interactome study of 14-3-3ζ protein, a regulatory protein found in all eucaryotic cells. An important 14-3-3 protein feature is the ability to bind a number of structurally and functionally distinct protein ligands. This link is usually implemented through phosphorylated serine and threonine motifs. The first aim of this work is the preparation of sufficient amount of recombinant 14-3-3ζ protein with incorporated photoactivatable analogue of methionine (foto-Met, L-2-amino-5,5- azihexan acid). The four different conditions of recombinant expression in auxotrophic E. coli B834 (DE3) strain were tested to obtain a protein with a maximal rate of photoactivatable methionine analogue incorporation into the sequence 14-3-3 protein. The second aim is to study the methionine 121, 160 and 218 participation in the 14-3-3ζ protein binding groove and finding of potential covalent bond with the phosphorylated peptide 251-266 of Raf-1 kinase (phosphorylation on Ser259). The photo-initiated cross-linking method was used (photolysis), to form a reactive biradical of methionine analogue capable to attack any amino acid residues in close vicinity (till 5Å). Finally, the products of photo-initiated cross-linking were analyzed by cross-linking reactions using MALDI-TOF MS, LC-MS and...
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Heterologous expression and purification of human NADPH: cytochrome P450 oxidoreductase
Kostelanská, Marie ; Černá, Věra (advisor) ; Kavan, Daniel (referee)
NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...
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Recombinant expresion and purification of nitrilase from Neurospora crassa
Zawadová, Dorota ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Nitrilases are enzymes able to convert toxic nitriles to corresponding carboxylic acids or amides. Thus they might be used in the detoxification of dyes, herbicides and pharmaceutical intermediates and byproducts. They can be used also for enzymatic syntheses of carboxylic acids not available by standard procedures. The aim of this diploma thesis is a recombinant expression of nitrilases from Neurospora crassa and the optimization of their purification. Cells of E. coli (BL 21 Gold) were utilized as an expression system. The purification was performed by ion-exchange chromatography, chelation chromatography and gel filtration - all under reducing conditions. Purified enzymes were studied by sedimentation analysis in an analytical ultracentrifuge. They were also used for searching of optimal conditions for their crystallization. Keywords: nitrilase, Neurospora crassa, recombinant expression
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Structural analysis of filamentous hemagglutinin (FhaB) from Bordetella pertusis
Jurnečka, David ; Kavan, Daniel (advisor) ; Man, Petr (referee)
: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA
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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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