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Optimalizace molekulárně-biologických metod pro detekci kontaminant v koření
Došková, Markéta
Contaminants are described in food generally, with narrower aim to spices in literary part of thesis. Bigger attention is dedicated to mycotoxins: secundary products of molds, basic survey, ways of assessment and legislation connected with their monito-ring. The main part of literary searches is focused on methods for microbial quality as-sessment of foods, then on description of classic and modern methods. Whereas samples of dried capsicum and pepper powders served as a samples for practical part, the end of theoretical part is devoted to individual steps for optimalization of new moleculary-biological method. The outputs of each steps are then parts of results and discussion, as well as other prospects and possible ways to future.
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Preparation of a library of methionine sulfoxide reductase for applications in synthesis of chiral sulfoxides
Havelka, Václav ; Míšek, Jiří (advisor) ; Hlouchová, Klára (referee)
Chiral sulfoxides are important compounds in the pharmaceutical and chemical industries, however, their enantioselective synthesis providing only one desired enantiomer is not fully mastered. Some natural enzymes can be used for the biocatalytic preparation of chiral sulfoxides. One of such enzymes is methionine sulphoxide reductase. Methionine sulphoxide reductase is an enzyme limiting the effects of reactive oxygen radicals in the organism resulting from oxygen metabolism. Its function is the reduction of methionine sulfoxide in proteins to methionine. There are two types of methionine sulphoxide reductase, methionine sulphoxide reductase A reducing only (S)-methionine sulphoxide and methionine sulphoxide reductase B reducing (R)-methionine sulphoxide. Methionesulfoxide reductase B is suitable for the preparation of (S)-sulfoxides, however its catalytic activity is not sufficient for practical use. Using the recombinant DNA and mutagenic PCR techniques, a methionine sulphoxide reductase B mutant library was prepared, and the extent and nature of mutation introduced was determined. This library will serve as a starting point for the controlled evolution of the enzyme to obtain clones with increased activity and reduced substrate specificity.
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The analysis of structural details of the NMDA receptor
Radilová, Kateřina ; Balík, Aleš (advisor) ; Jakubík, Jan (referee)
NMDA receptor is necessary for excitatory transmission in the central nervous system. Altered funtion of the NMDA receptors is associated with many neurodegenerative and neuropsychiatric diseases. All available crystal structures of the NMDAR meant great shift towards our understanding of details of the receptor and its function. Unfortunately, these up- to-date available structures present only certain functional states of receptors and also a few structural data are still missing. For complete comprehension of the process of activation and deactivation of NMDA receptors, we need to supplement the current information with more data. The aim of this thesis was to employ a combination of different approaches (computational modelling, cloning, biochemistry, protein expression and purification and mass spectrometry) to obtain new structural data, by which we would be able to fill in the gaps in current receptor models, especially at various functional states of the receptor. Key words: NMDA receptor, glutamate receptor, computational modelling, structure, cloning, protein expression
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Photobiont diversity in lichen thallus Psora decipiens
Jadrná, Iva ; Škaloud, Pavel (advisor) ; Peksa, Ondřej (referee)
Psora decipiens is a characteristic species of the terricolous lichen community Toninio-Psoretum decipientis distributed mostly on calcareous or basic substrates. The community consists in various modifications of lichens Placidium squamulosum, Toninia sedifolia, T. opuntioides, Fulgensia fulgens, F. bracteata and others. Photobionts of the lichen Psora decipiens were determined. Psora decipiens shared with Placidium sp. the single photobiont species, a common terrestrial alga Myrmecia israeliensis. Cloning of ITS rDNA revealed high intrathalline variability in M. israeliensis within a single lichen thallus. Several genotypes were often found in a thallus, uncovering either a high mutation rate of the algae or constant relichenization processes. Saxicolous Psora species (P. testacea, P. himalayana, P. valesiaca and P. rubiformis) had M. biatorellae as a photobiont, indicating a possible photobiont influence on substrate specifity of Psora lichens. Finally, the proper methodology used for identification of lichen photobionts is discussed. For a correct photobiont identification, morphological investigations of intrathaline diversity combined with coherent molecular techniques are needed. Such procedure was not applied in the former studies of Psora decipiens, resulting in a poor characterization of...
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Functional analysis of the population-specific checkpoint kinase gene CHEK2 sequence variants
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Živný, Jan (referee)
CHEK2 gene codes for serin/threonine kinase Chk2 (Checkpoint kinase 2). In response to genomic DNA damage, Chk2 phosphorylates its substrates (proteins Cdc25C, BRCA1 or p53), whose activation leads either to cell cycle arrest, DNA damage repair or induction of apoptosis. Germline mutations in CHEK2 gene increase risk of cancer development. Analysis of high risk breast cancer patients in Czech Republic reveals rare CHEK2 mutations (mainly missense) with yet unknown clinical significance. This work focuses on functional impact of these variants and analysis of kinase activity of variant isoforms of Chk2 kinase. For this purpose, recombinant constructs were expressed in bacterial cells of E. coli. Enzymatic activity of Chk2 kinase isoforms in crude cell lysates was measured by the phosphorylation of Chk2 arteficial substrate spectrophotometrically. Results of in vitro kinase assay were correlated to the results of in silico prediction software. The results show that from 15 analyzed mutations (together with one in frame deletion), kinase activity was abrogated in all variants affecting the kinase domain of Chk2, in concordance with in silico predictions. The same result has been found for a FHA domain variant p.R145Q. No significant changes in kinase activity were observed in case of two FHA domain variants...
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Cloning, epression and purification of mycobacterial dihydrofolate reductase
Šedivá, Kateřina ; Novotná, Eva (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Šedivá Supervisor: Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Cloning, expression and purification of mycobacterial dihydrofolate reductase Dihydrofolate reductase is an enzyme essential for the metabolism of folic acid - it catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an important cofactor involved in one-cabron transfer reactions. Dihydrofolate reductase plays a key role in the synthesis of DNA, RNA and proteins. Dihydrofolate reductase was also found in M. tuberculosis. This bacterium is the most common causative agent of tuberculosis in humans. Thus dihydrofolate reductase could be a potential target for the design of new antituberculotics. The recombinant protein dihydrofolate reductase was prepared in several steps. The coding sequence of the protein was first amplified by polymerase chain reaction. A recombinant plasmid, obtained by the ligation of an amplified segment of DNA with plasmid pET-28b(+), was transformed into competent cells E. coli strain BH101 by the heat shock method. Cells E. coli strain BL21(DE3) were used for the protein expression. The expression was induced by the addition of isopropyl-β-D-...
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Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family
Mikulová, Barbora ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification. Keywords: NKp65, KACL, NK cell, lectin, receptor, plasmid (in Czech)
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Preparation of the constructs for analysis of expression of nuclear receptor nhr-97 by using transgenic techniques in the model system Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Hudeček, Jiří (referee)
The aim of this work was to prepare two constructs of the promoter of a gene coding for nuclear hormone receptor nhr-97 in C. elegans. Nuclear receptors belong to a large group of genes sharing homologous sequences in some vertebrate nuclear receptors. The first part of the work describes the structure of nuclear hormone receptors, their function and significance in the nematodes C. elegans. The model organism C. elegans, its anatomy, life cycle and genome were also described. The work also discusses the structure and use of green fluorescent protein (GFP), which serves to localize the expression of the nhr-97 gene in C. elegans. In the practical part of the work, the preparation of two constructs of the promoter is described. Isolation of genomic DNA of C. elegans, PCR amplification of the promoters and their subsequent cloning into vector pPD95.67 containing a gene coding for green fluorescent protein were performed. To verify the successful cloning of the promoter constructs, sequencing DNA was performed. Cloned promoters of nhr-97 will be used for microinjetions to C. elegans gonads and the expression of this gene regulated from particular promoters will be subsequently monitored using expression of green fluorescent protein in progeny.
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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