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Influence of makroelements from food on DNA and epigenetic profile
Veselý, Zdeněk ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
The macroelements contained in food have an important function for the human body. They are involved in several of biochemical reactions in the body and their abundance can prevent serious diseases. The theoretical part of the bachelor thesis describes the function of minerals in the human body, the function of DNA and epigenetic mechanisms such as DNA methylation or histone modification. The influence of nutrition and function of selected macroelements – sodium, magnesium, calcium, potassium on epigenetic modifications and on the stability of G-quadruplexes was described. The aim of the experimental part of this work was to study the effect of these substances on DNA structures in vitro and to prepare them experimentally for in vivo studies.
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Influence of storage on the microbial composition of French Saint-nectaire cheese
Šislerová, Lucie ; Mikulíková, Renata (referee) ; Brázda, Václav (advisor)
The aim of my work is the comparison of microbial composition between farmtype and dairytype of Saint-nectaire cheese and the influence of storage time and temperature on the development of microbial composition, content of fatty acids and aromatic substances. Selected microorganisms were identified by RT-PCR. In addition, Penicillium roqueforti and fuscoglaucum have been identified in the Saint-nectaire farm type compared to the dairy type. In both types of cheese, the highest amount of selected microorganisms was detected in fresh cheese. When stored at 20 °C, an increase over fresh cheese occurred in the following microorganisms: Streptococcus thermophilus, Lactobacillus bulgaricus, Cladosporium herbarum and Penicillium commune and camemberti, and the presence of contaminants and pathogens was noted. After one week of storage at 20 °C, they were Micrococcus luteus, Salmonella enterica and Staphylococcus aureus, and after another two weeks of storage, Listeria monocytogenes was identified. The fatty acid and volatile compounds were compared for five samples: fresh cheese, cheese stored in the refrigerator for one week and three weeks and cheese stored at 20 °C for one week and three weeks. The content of bound and free fatty acids was measured, both by GC-FID. The content of bound fatty acids was comparable in all measured samples. The highest content of free fatty acids was in the cheese after three weeks of storage at 20 °C. The most common fatty acid is palmitic acid. Volatiles were determined by HS-SPME-GC-MS. The most volatiles were identified in the cheese after three weeks at 20 °C and in the cheese after one week in the refrigerator. The most represented groups were alcohols, ketones and acids.
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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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Use of molecular techniques to characterize yeasts of the genus Metschnikowia
Schneiderwindová, Nicole ; Brázda, Václav (referee) ; Němcová, Andrea (advisor)
This diploma thesis deals with the possibilities of implementation and use of molecular methods for the characterization of yeasts of the genus Metschnikowia and the application of methods in biotechnology or the food industry. The theoretical part focuses on a brief description of yeast, specially selected species that were used during the practical part of the work, the possibilities of their use, and especially on a detailed description of all molecular techniques used. The practical part focuses on the optimization of the molecular methods, namely the method of pulsed gel electrophoresis and the method of denaturing gradient gel electrophoresis. Initially, yeast was cultured under optimal conditions that are specific to this genus. Furthermore, their DNA was isolated using isolation techniques, which were subsequently processed using PFGE and PCR–DGGE methods. The DNA isolation procedure needed to be optimized the most. Several optimizations of the concentration of lysis enzymes, especially the lyticase enzyme, were performed. It was also necessary to determine the correct ratio of low-melting agarose and isolated DNA, which was essential for the correct consistency of the isolated DNA blocks and their further application in PFGE analysis. Finally, the PFGE method was optimized, which brought the correct distribution of chromosomes, and it was possible to describe the individual chromosomes according to their size according to the standard used CHEF of the yeast Hansenula wingei. To properly optimize the DGGE analysis process itself, it was first necessary to isolate the yeast DNA using a kit, then it was used as a template for the PCR reaction. The annealing temperature was also optimized for the individual groups of primers. The amplicons obtained by this reaction were separated by the DGGE method. This technique mainly required the optimization of basic parameters such as the range of the denaturation gradient or the total separation time. According to the measurement results, it can be determined that the process of yeast DNA isolation and their subsequent analysis using molecular methods of pulsed gel electrophoresis and denaturing gradient gel electrophoresis was successful. We were able to describe the genome and determine the number of chromosomes in all used yeast species of the genus Metschnikowia at least partially.
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DNA Isolation and Analysis Focused on Microorganisms Important in Food Production
Čutová, Michaela ; Obruča, Stanislav (referee) ; Fojtová,, Miloslava (referee) ; Brázda, Václav (advisor)
Identification of bacterial DNA consists from several steps: cell lysis, isolation and purification of DNA, precipitation by ethanol, identification of bacterial strain by PCR or other molecular biology methods. Each step must be optimised. Nucleic acids can be isolated from cells using magnetic particles. The molecules of DNA are bound to the surface of magnetic carriers by electrostatic interaction, and then they are eluted into buffer. The aim of the work will be to optimize individual steps of identification of bacterial DNA: cell lysis, DNA isolation, characterization of solid magnetic carriers functionalized by amino groups for nucleic acids isolation. The presence of DNA will be verified using agarose gel electrophoresis and the amount of eluted DNA will be determined spectrophotometrically. The quality of isolated DNA will be proved by their amplification using polymerase chain reaction (PCR). Furthermore, the thesis focuses on the study of secondary structures of nucleic acids – cruciforms structures and quadruplexes. These structures are involved in the regulation of cellular processes and their appearance is associated with cancer development and neurodegenerative diseases. In silico genome analysis was performed on important food industry microorganisms. The microorganisms genomic sequences were obtained from the NCBI (National Center for Biotechnology) database. The Palindrome Analyzer and G4 Hunter software were used for the analysis.
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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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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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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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Detection of probiotic bacteria in diary food products using PCR technique
Klaška, Dominik ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
In the bachelor thesis, DNA was isolated from commercially available white yogurt. The isolated DNA, which was gained by two different methods, was performer analyse by a spectrophotometer. Both methods provided sufficiently concentrated and high-quality DNA for further analysis by PCR. Precisely defined sections of isolated DNA were amplified using specific primers. The presence of the bacterium domain was detected, and in the case of genus specific amplification, the presence of bacteria of the genus Lactobacillus was detected too, by gel electrophoresis.
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The effects of chemicals on cell lines viability
Zemanová, Anita ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
The subject of this diploma thesis is the influence of selected chemicals on cell lines viability. The theoretical part contains options of cancer treatment by using chemotherapeutics including their mechanism of action and side effects. Additionally, there are described alternative DNA structures with focus on G-quadruplexes and ligands that interact with G-quadruplexes. These compounds are promising drugs in cancer treatment due to their high specificity to G-quadruplexes, which are found in telomeres of chromosomes. G-quadruplex interacting ligands by stabilization of G-quadruplexes can inhibit the enzyme telomerase, which is necessary for telomere lengthening of rapidly dividing cancer cells. Additionally, the possibilities of viability assays are summarized in the theoretical part. The aim of the experimental part was comparing cytotoxic activity between commercially available chemotherapeutics and selected G-quadruplex interacting ligands. Another task was the study of apoptosis and necrosis after the treatment of selected chemicals on cell lines and after the localization of ligands interacting with G-quadruplexes in the cells of the breast cancer cell line. In the experimental part, G-quadruplex interacting ligands have been shown to exhibit similar cytotoxic activity to commercially available chemotherapeutic agents.
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