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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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Optimization of plasmid DNA isolation by magnetic particles
Chlopková, Barbora ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
The theoretical part summarizes information on the isolation and purification of plasmid DNA and nucleic acids as. Plasmid DNA is often used in gene engineering as a vector for the transfer of a particular gene. Its insulation and transportation in sufficient quality is crucial for other processes associated with it. Isolation and survival of pDNA using magnetic carriers of different concentrations of PEG 8000 in combination with 1M NaCl was investigated in experimental parts. Furthermore, the isolation of pDNA using commercial kits was examined.
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Preparation and expression of p53 protein isoforms using the GATEWAY expression system
Wikarská, Monika ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor)
The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.
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Infuence of natural and synthetic G4-ligands to p53 transactivatio
Perná, Kristýna ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Secondary DNA structures, such as G-quadruplexes, occur in the promoters of human genes. Dysfunctions of these quadruplex structures have been observed in several cases of cancer, and therefore these structures are the subject of research for the design of new anticancer drugs. The p53 protein is an important regulatory protein in the process of cell cycle control and DNA repair. Mutations in the gene encoding this protein occur in more than 50 % of cancer cases. In this thesis, the influence of natural ligands binding to G-quadruplexes on the binding and activity of the p53 protein was investigated. The theoretical part of the thesis describes the structure and binding properties of p53 protein, the structure and role of G-quadruplexes and describes selected G4-ligands occurring in food - curcumin, quercetin, berberine and ellagic acid. The aim of the experimental part of this thesis was to determine the effect of these substances on the transactivation activity of the p53 protein in vivo based on their interaction with G-quadruplexes using yeast isogenic systems. The interaction between selected G-quadruplex structures and ligands was first verified in vitro using the ThT assay and circular dichroism.
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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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Analysis of C. necator genome changes after evolutionary adaptation
Vojsovič, Matúš ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
The p53 protein is a transcription factor that belongs to the group of the tumour suppressor proteins. p53 functions include cell cycle regulation, interaction with DNA, and responses to damaged DNA that may lead to apoptosis or even senescence may occur. The theoretical part summarizes the latest information about the chemical structure and functions of the protein p53 and allosteric modifications that the p53 protein may possess in cells. It also describes selected methods of isolation and characterizes proteins. The aim of the experimental part was to isolate and characterize -isoforms of the p53 protein, which were isolated from the microorganism E.coli. Protein production was preceded by preparation and transformation of plasmids into production cells. From the production cells, 4 -isoforms of p53 were isolated by affinity chromatography, due to the polyhistidine anchor that the proteins contain. The isolated proteins were subsequently characterized by SDS-PAGE and western blotting. Moreover, the transactivation potential in the yeast system was monitored by luciferase assay under conditions involving the use of various culture media, as well as expressed or coexpressed under the inducible GAL1 promoter and the constitutive GPD promoter.
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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Tumor cell lines viability testing after exposure to chemicals and chemotherapeutics
Horáčková, Lucie ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
Individual types of viability tests based on colorimetric changes of the solution are desribed in the theoretical part. Furthermore, HSP proteins are characterized, which are not connected only by heat shock, but also during other cell stresses such as exposure to UV, cold, extreme pH or heavy metals. They are important for the cell, because they help to reformulate proteins that have been damaged by cellular stress and also bind to new unpacked proteins and ensure their correct folding. Proteins that are affected by molecular chaperones are collectively called client proteins. Some HSPs also contribute to membrane transport or degradation. These proteins are co-operative with the cochaperones, which are important for heat shock proteins because they help them to pack protein, in particular by catalyzing the hydrolysis of ATP to ADP. Herein is also described cisplatin and its derivatives, including mechanism of action and adverse effects. This work was focused on detection cytotoxicity of cisplatin and its derivatives. Cells were exposed to stress condition induced by cytostatics and huge changes in heat shock proteins and cochaperon levels were observed. There was also observed colocalization of heat shock proteins and their client protein p53 by confocal microscopy in these stressing conditions.
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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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