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Processing peptidases of M16B family and their evolutionary relationships
Hanušová, Iva ; Stiborová, Marie (advisor) ; Šulc, Miroslav (referee)
The theoretical part of this bachelor thesis deals with the group of processing peptidase of the M16B family. The main focus is on the structure and the evolution of mitochondrial and hydrogenosomal processing peptidase and also the hypothetical peptidase from the bacterium Rickettsia prowazekii. In the practical part of this work, the constructs coding the α-subunit of hydrogenosomal processing peptidase (α-HPP) with the substituted tryptophan residue in the position 236 for phenylalanine and tyrosine were prepared using the site-directed mutagenesis. Subsequently, the new reporter tryptophan residue was introduced in α-HPP in positions 256, 260, 267 or 271 in the so-called glycine-rich loop.
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Enzymatic modification of Glc-NAc-thiazoline inhibitors of hexosaminidase
Šmeringaiová, Ingrida ; Weignerová, Lenka (advisor) ; Šulc, Miroslav (referee)
This work deals with the problem of searching for effective derivatives of 1,2-dideoxy-2'- methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NGT), potential inhibitors of human β-N- acetylhexosaminidases. The work is targeted to production of inhibitors derived from NGT by the modification of its structure by free and immobilised lipases. Potential inhibitors of β-N- acetylhexosaminidases may be potent tools for studying of enzymes in cell processes and also open possibility to the discovery of new possible drugs for the treatment of some neurodegenerative diseases, such as Alzheimer disease. The thesis is focused on brief characterization of β-N-acetylhexosaminidases, e.g., glycosidases from enzymatic groups GH 20 and GH 84. β-N-Acetylhexosaminidases from microorganisms Bacteroides thetaiotaomicron, Aspergillus oryzae, Streptomyces plicatus, Talaromyces flavus and humans1 were used in the experiments. The enzymes produced were purified and then tested for their activity. We also tested inhibitory activity of potential inhibitor 6-O-acetyl-1,2-dideoxy-2'-methyl-α- D-glucopyranoso-[2,1-d]-Δ2'-thiazoline. Starting compound, NGT, was synthesized by the modified process (originally created by prof. Knapp2 ) using 2-acetamido-1,3,4,6-tetra-O- acetyl-2-deoxy-α-D-glucopyranose in the reaction with...
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Recombinant production of bovine NK cell receptors
Böerová, Nikola ; Kavan, Daniel (advisor) ; Šulc, Miroslav (referee)
NK cells, which are part of the innate immune system, are increasingly gaining attention, especially due to their cytotoxic ability to kill tumor cells of certain lines and certain viral, bacterial or parasitic infestation of the body. They lay a role in organ transplantation, the fight against HIV and other autoimmune diseases. NK cells have been studied since the 70th of the 20th century, but the structures and physiological ligands of their receptors remain only partially understood, as does the exact role of these cells in the organism. They communicate with others through their receptors, that recognize the lack of expression of MHC class I glycoproteins on the surface of target cells, thereby preventing cell recognition by cytotoxic lymphocytes. This diploma thesis deals with the research of receptors from cattle (Bos taurus), which is not a traditional laboratory animal and my task was to contribute to research of the structure of this group of proteins. I dealt with the recombinant production of some of the most important representatives of NK receptors CD69, NKRP1 and NKG2D in bacterial cells. The findings published in this thesis are a continuation of my bachelor thesis and together can be beneficial for further research into structural proteins and thus may help as in veterinary medicine...
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Interaction of apoprotein with heme side chains: A comparison of cytochromes P450 and NO-synthases
Fastová, Dagmar ; Hudeček, Jiří (advisor) ; Šulc, Miroslav (referee)
Cytochromes P450 and NO-synthases are two classes of heme-thiolate proteins, participating in catalysis of important biochemical reactions. In this work we analysed and compared both enzyme groups from the viewpoint of torsional angles of vinyl side chains of heme in positions 2 and 4. We used the Yasara programme to analyse the available crystalographic data. Whereas the torsional angle distributions for both vinyl groups in NO-synthases are pointing to a rather "conservative" conformations of both vinyls (between -30ř and 90ř for position 2, -150ř to 120ř for position 4, resp.) with little differences for individual forms of these enzymes, the mammalian cytochromes P450 display considerable differences between different families. In accordance with previous analyses is the distribution of torsional angles of 2-vinyls in most cases narrower (-120ř to 150ř) compared to 4-vinyls, which display more conformational flexibility (with torsional angles between -30ř and 180ř). In the second part of the Thesis we analysed the interaction of cytochrome P450 with its reaction partner, cytochrome P450 reductase. On the basis of crystalographic data we tried to prove, if the spatial positions of basic aminoacids, corresponding in the primary structure of various mammalian forms the those in the isoform 2B4...
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Isolation and purification of the environmental DNA from the horizon of the capping: stratification study
Zahradník, Jiří ; Šulc, Miroslav (advisor) ; Weignerová, Lenka (referee)
According to our contemporary knowledge soil is the most abundant source of microbial biomass. Unfortunately, only one percent of the microorganism species is available by classical cultivation techniques. Soil metagenomic DNA is a collection of the whole DNA including also uncultivated microorganism in the soil sample and provides information to study molecular aspects of microorganism and their DNA sequences inaccessible by other techniques. This work is focused on characterization and isolation of soil metagenomic DNA from deep horizon of capping. Evaluation in term of the isolation and techniques of recombination of DNA and stratification study are included in this work. Obtained collection of samples was preliminary characterized with the view of quality - content of clay, humic compounds and crude number of microorganism. The purified soil metagenomic DNA was quantitatively and qualitatively characterized for each sample. The quantification method and DNA quality determined the next applications and procedures of DNA techniques. Also the soil quality was discussed from this point of view. According to the results of DNA analysis, the three selected DNA samples were processed to DNA library with 16S rRNA DNA loci and after DNA sequencing analysis the phylogenetic study was performed. This...
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Search for an Axion in OSQAR Experiment at CERN
Husek, Tomáš ; Finger, Miroslav (advisor) ; Šulc, Miroslav (referee)
In the present work, OSQAR experiment at CERN is being introduced. The goal of this rather small experiment is a search for a hypothetical particle called axion, which could play an important role in modern physical theories. It is necessary to say, that the experimental setup as well as particular devices are still being developed or calibrated and measurement modes optimized. That is why desired functionality and reliability of the presently used equipment with a variety of different operating procedures are discussed. Preliminary results, simulations and estimation of sensitivity limitations of the current setup are also presented.
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Novel antibody array validation and application to determine new prognostic markers in childhood acute leukemia
Černá, Daniela ; Šulc, Miroslav (advisor) ; Poljaková, Jitka (referee)
v anglickém jazyce Leukemia is the most common malignant childhood disease, which occurs in Czech Republic every year in about 100 cases. That's why has been devoted over the last 5 decades much attention and funding to this research and the children with leukemia have a high chance for achieving complete remission. Nevertheless, for about 10% of patients leukemia still remains lethal and these cases are subjects of current studies. Leukemia is a complex disease in its pathology involving lots of aberrations, which pathologically manifest at the DNA, mRNA and protein level. Nevertheless some symptoms of this disease are only at the protein grade (and not DNA or mRNA). Since a broad part of the protein profile in leukemic cell changes, it is necessary to develop newer, more sensitive and especially more efficient methods, which are able to follow up changes in large sets of proteins and detect prognostic and diagnostic markers. Within diploma study a new method - multiplex antibody array - has been tested. Our laboratory tests this method in collaboration with a Norwegian laboratory (Rikshospitalet University Hospital, University of Oslo) under leading by Dr. Lund-Johansen, who is the author of this method. Antibody array presents a set of 1152 latex bead populations labeled with fluorescent dyes in...
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Lysosomal inherited disorders - pathobiochemistry of the Gaucher disease
Illner, Jan ; Hudeček, Jiří (advisor) ; Šulc, Miroslav (referee)
Gaucher disease is one of the lysosomal storage disorders belonging to inherited defects of catabolism of sphingolipids. These defects are caused by mutation in genes of sphingolipid hydrolases or their protein activators. Subsequent storage of non-degraded sphingolipids leads to severe clinical phenotypes in patients. Gaucher disease is caused by deficiency of lysosomal β-glucocerebrosidase (GBA1) activity. Non-degraded glycosphingolipids are glucosylceramide (GlcCer) and glucosylpsychosine (lyso-GlcCer). Accumulation of these glycosphingolipids is related to Gaucher cells which are derived from macrophages and are abundant in spleen, liver or lung. The objective of the diploma thesis is pathobiochemistry of above mentioned glycosphingolipids. One of the topics of this work was optimization of mass spectrometry method for determination of activity of lysosomal β-glucocerebrosidase, using a natural substrate. The method was successfully optimized and can be effectively used for diagnostic purpose, instead of methods utilizing artificial substrates. In the next step, we performed analysis of pH profiles of lysosomal β-glucocerebrosidase activity focusing to search for non-lysosomal enzyme, which is able to degrade glucosylceramide. Evaluation of pH profiles did not confirm the existence of such an...
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