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Biodegradation of environmental pollutans - Crystallization haloalkan dehalogenase DpcA of Psychrobacter cryohalolentis K5
JUNG, Jakub
Knowledge of protein structures is necessary to clarify their structure-function relationships. Haloalkane dehalogenases are microbial enzymes that catalyze the hydrolysis of carbon-halogen in the halogenated hydrocarbons. Based on their properties these enzymes are able to degrade halogenated compounds. As the environment is burdened by a lot of different pollutants, biodegradation is a necessary step to improve the quality of environment. Haloalkane dehalogenases can be used as biosensors since they can detect contamination by halogenated compounds. This thesis is focused on crystallization of the model protein lysozyme and haloalkane dehalogenase DpcA isolated from the organism Psychrobacter cryohalolentis K5. The aim of this work was to prepare crystals suitable for X-ray diffraction analysis. After verification of protein?s purity, both proteins were crystallized by the use of basic and advanced crystallization methods. Single crystals of suitable size and quality for X-ray diffraction analysis were obtained and diffraction data were used to solve the 3D atomic structure of the DpcA. Since haloalkane dehalogenases have a low activity for halogenated substrates, the main aim of protein engineering is to increase their activity so thus further research is focused on modification of substrate specificity.
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Analysis and characterization of BRCA1 splicing variants.
Hojný, Jan ; Kleibl, Zdeněk (advisor) ; Souček, Pavel (referee)
The Breast cancer gene 1 (BRCA1) codes for nuclear phosphoprotein with a key function in the regulation of DNA damage response. The BRCA1 protein contributes to the formation and regulation of protein supercomplexes that participates on the DNA double-strand break repair. These protein supercomplexes are formed by the protein-protein interactions between highly conservative protein motives in BRCA1 and its binding partners. Except to the wild type form of BRCA1 mRNA containing entire set of 22 exons coding for the 220 kD protein, numerous alternative splicing variants (ASVs) BRCA1 mRNA has been described. These ASVs code for BRCA1 isoforms lacking several critical functional domains. It has been proposed, that formation of BRCA1's ASVs represent a tool for regulation of BRCA1 function. Only poorly has been characterized a complex catalogue of in various human tissues and their expression. This study aims to address these questions. We optimized the identification of BRCA1's ASVs including those covering the entire transcripts of the wt BRCA1 mRNA with length exceeding 5.5 kb. In further analysis, we characterized 13 BRCA1's ASVs in RNA samples isolated from peripheral blood mononuclear cells (PBMNC) obtained from patients with breast cancer (BC) and control subjects. The majority of the identified...
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Preparation and characterization of LmbX protein involved in lincomycin biosynthesis
Jiráčková, Petra ; Janata, Jiří (advisor) ; Janderová, Blanka (referee)
Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...
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The search for novel interaction partners of SH3 domain of an adaptor protein p130Cas
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Forstová, Jitka (referee)
Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...
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Characterization and functional analysis of IST2 gene in yeast Saccharomyces cerevisiae.
Andršová, Markéta ; Sychrová, Hana (advisor) ; Hlaváček, Otakar (referee)
IST2 is known as a gene encoding in the model yeast Saccharomyces cerevisiae a membrane protein, that is studied thanks to a unique way of biogenesis and trafficking that apparently does not use classical secretory pathway. Although the gene was named more than ten years ago according to the phenotype of cells with its deletion (Increased Sodium Tolerance), the role of this protein in cell tolerance to toxic sodium has not been elucidated. Our searches in databases revealed that similar proteins are encoded in the genomes of other species of yeast, but none of them has been studied so far. In this work, four new strains lacking IST2 have been constructed in the genetic backgrounds differing by the presence of genes encoding transport systems for accumulation of potassium (Trk1, Trk2), for export of surplus potassium cations (Tok1, Ena1-5, Nha1) and for export of toxic cations lithium and sodium (Ena1-5, Nha1). Plasmid carrying the gene coding IST2 sequence has also been conctructed. The effect of IST2 deletion in different genetic backgrounds was studied by phenotypic tests on solid and liquid media. It was found that IST2 probably does not play a role in osmotolerance in general (absence of the phenotype of IST2 deletion on high concentrations of KCl), but its presence affects ability of the cells...
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(Strept)avidin-biotin interactions at amalgam electrodes covered by thiol monolayer
Josypčuk, Bohdan ; Mareček, Vladimír ; Yosypchuk, O.
Carboxylic group of 11-mercaptoundecanoic acid (MUA) can be used to creat a peptide bond with species containing amino group, e.g., peptides, and proteins. By the help of EDC-NHS technology, streptavidin or avidin was covalently bonded with MUA-monolayer at a silver solid amalgam electrode. Such prepared electrode was used for detecting biotin and biotinylated albumin in the supporting electrolyte (0.15 M NaCl, 0.05 M TRIS, pH 7.0). Electrochemical impendance spectroscopy was performed for the biosensor response monitored by impedance spectroscopy. Binding of biotin or biotinylated albumin with (strept)avidin a change in the resistance of the sensor in the concentration range of 0.5-20 μg mL-1. Electrochemical regeneration of the amalgam electrode permits simply to renew its surface and to create the new biosensor.
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Clustering of Protein Sequences Based on Primary Structure of Proteins
Jurásek, Petr ; Stryka, Lukáš (referee) ; Burgetová, Ivana (advisor)
This master's thesis consider clustering of protein sequences based on primary structure of proteins. Studies the protein sequences from they primary structure. Describes methods for similarities in the amino acid sequences of proteins, cluster analysis and clustering algorithms. This thesis presents concept of distance function based on similarity of protein sequences and implements clustering algorithms ANGES, k-means, k-medoids in Python programming language.
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Molecular dynamics simulations of biomolecular complexes consisting of proteins and nucleic acids
Melcr, Josef ; Barvík, Ivan (advisor) ; Bok, Jiří (referee)
Literature search on the Elongation factor Tu (EF-Tu), which is involved in the process of translation of genetic information, was performed. Further, computational methods as molecular dynamics (MD) and Monte Carlo (MC) were studied. Then, computer programs for MD and MC simulations of a Lennard-Jones gas were developed. MD simulations were further applied to EF-Tu using the NAMD and ACEMD software packages. Multiprocessor PC clusters and programmable NVIDIA GPUs were used. MD simulations of EF-Tu uncovered binding of monovalent ions in nearby of the EF-Tu active site. The impact of Na$^+$ binding on evolutionarily conserved residues (His85, Val20, Ile61, Asp21, Tyr47, Asp87, etc.) was studied in detail.
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První údaj o resistenci Digitaria sanguinalis vůči atrazinu v České republice
Salava, J. ; Chodová, D. ; Kočová, M. ; Krahulec, František
A survey on the occurrence of weeds surviving treatment with atrazine was carried out at the railways in the Czech Republic in 2000-05. Large crabgrass was widely distributed at the surveyed railway junctions. Seeds of large crabgrass were collected from plants growing in the surroundings of the railway station Prague-Bubny in 2003 and 2004. Resistance or susceptibility to atrazine was verified by the whole-plant response and chlorophyll fluorescence assays. A region of the psbA gene encoding D1 protein of Photosystem 2 was amplified by PCR and sequenced to determine the molecular basis for the atrazine resistance. It was found out that the resistance in the Prague-Bubny large crabgrass biotype was conferred by a glycine for serine substitution at residue 264 of the Dl protein of Photosystem 2. There was an excellent correspondence between the presence of the mutation and phenotypic resistance to atrazine of individual plants.
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Identification of interacting partners of Discs overgrown in vivo
MAREŠOVÁ, Petra
The mutated forms of the Discs overgrown gene causes overproliferation of imaginal discs of Drosophila melanogaster. Somatic mutations in its human counterpart, casein kinase I epsilon, were strongly associated with human breast cancer. Using the advantage of a high conservancy between fly's dco and human casein kinase I epsilon genes we have chosen D. melanogaster as a model organism to provide a list of probable Dco interaction partners via tandem affinity purification and mass spectrometry analysis. However, these proteins need to be independently verified as true Dco interaction partners.
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