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Preparation of DNA with local DNA structures
Lofítková, Ellen ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
In this thesis I focused on quadruplexes and cruciforms formed by DNA. Plasmids pBluescript and others derived from it by inserting oligonucleotid sequences were studied. Sequences forming quadruplexes and cruciforms were found by in silico analysis by QGRS Mapper and Palindrome analyser. Plasmids were transformed into E. coli, isolated and then cleaved with enzymes S1 nuclease and restriction endonuclease ScaI. Cleaving with S1 nuclease predicated presence of local structure. Combined S1/ScaI cleavage did not bring satisfying results due to lost of DNA during purification.
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Impact of temperature and drought on gliadins content in two varieties of wheat
Seidlová, Kateřina ; Brázda, Václav (referee) ; Hrstka, Miroslav (advisor)
This bachelor’s thesis focuses on the effect of high temperature and drought on protein content of gliadin fraction on two varieties of wheat. Chosen varieties were Hyfi and Julie, cultivated at 26, 29, 32, 35 and 38 °C during flowering in watering controlled conditions. The condition for ‘wet’ samples was at least 70 % soil moisture and for ‘dry’ samples less than 30 % soil moisture. After harvesting, the seeds were milled into flour from which the gliadins were extracted with 2-chlorethanol. A-PAGE method was used for gliadin separation, quantification was carried out through computer densitometry. A significant genotype effect was discovered. Whilst temperature ranging from 26-38 °C with simultaneous drought stress had no significant effect on gliadin content of Hyfi variation, gliadin content of Julie variation shown obvious maximum at 32 °C. Therefore, Hyfi variation shown better resistance to heat stress than Julie variation. Both variations had higher gliadin content under drought stress than under good watering conditions.
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Preparation of constructs for protein isolation and its testing
Osadchuk, Olha ; Kostovová, Iveta (referee) ; Brázda, Václav (advisor)
This study is focused on describing of recombinant protein production. Protein p53 was chosen, as one of the most important tumor suppressor proteins, for studying this issue. The p53 protein is responsible for the gene regulation, control of cell cycle and DNA replication. P53 is the most mutated gene in human cancer. Several point mutations of p53 protein was chosen for work with. The theoretical part describes main properties of protein, expression systems, Gateway cloning system and methods of protein purification. In the experimental part are described the procedures of preparing of the expression vectors by Gateway technology, cell transformation and DNA plasmid isolation. Using cloning technology were prepared three expression clones, they were transformed into competent cells and after was done DNA isolation.
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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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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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Biochemical analyses of the DNA interaction partners
Valchová, Michaela ; Coufal,, Jan (referee) ; Brázda, Václav (advisor)
This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.
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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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Biological effects of substances isolated from Isoptera species
Dušková, Simona ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor)
This thesis was focused on monitoring the viability of eukaryotic and prokaryotic cells after exposure of termites-isolated chemicals. Recently, evidence of antibacterial and antifungal properties of these defense substances has grown, and they can find a wide range of uses not only in the pharmaceutical industry. In this work, three defensive substances from termites were studied: nerolidol, nitropentadecene and methylanthranilate. Their antibacterial effects, minimal inhibitory concentrations and minimal bactericidal concentrations against Escherichia coli STBL3 strain were monitored. Further, their cytotoxic effects on eukaryotic non-tumor (HEK293FT) and tumor cells (MCF7) as well as their effect on plasmid DNA were studied. Antibiotic ampicillin and cytostatic cisplatin were used as control substances for antibacterial and cytotoxic effects, respectively. In the case of the action of nerolidol, nitropentadecene and methylanthranilate on the STBL3 strain, antibacterial activity was not demonstrated. Cytotoxic effects were observed nerolidol and nitropentadecene. None of the examined substances modified the plasmid DNA.
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Earth-space path propagation characteristics in the climatic conditions of the Czech Republic from Alphasat Ka/Q band experiment
Fišer, Ondřej ; Brázda, Vladimír ; Pešice, Petr ; Grabner, M.
The objective of the activity is to prepare, realize and conduct experimental research of the influence of atmospheric phenomena on the Earth-space path propagation characteristics at 20 and 40 GHz (Ka/Q) bands in the specific climatic conditions of the Czech Republic (CR). The scientific approach is based on the comprehensive measurements of physical parameters of atmosphere together with concurrent measurements of propagation characteristics on the Earth-space path and on 10 collocated terrestrial paths with star configuration. The proposed activity will utilize the ESA Alphasat Mission Ka/Q experiment that is designed to investigate the propagation characteristics on slant paths in Europe and elsewhere.
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