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Interaction of non-thermal plasma at atmospheric pressure (CAPP) with non-pathogenic bacteria
Chobotská, Barbora ; Brázda, Václav (referee) ; Kozáková, Zdenka (advisor)
The subject of this bachelor thesis is the study of decontamination effects of cold atmospheric pressure plasma (CAP) on selected bacteria Escherichia coli and Staphylococcus epidermidis. A non-thermal microwave plasma torch was used. The plasma torch was connected to the software in the computer, which allowed its movement over the treated area. The used power fluctuated between 12–13 W and argon (gas purity 4.6) was chosen as the working gas with a constant flow rate of 5 l/min Inhibition efficacy was observed depending on the type of the selected treatment. The chosen treatment parameters included the effect of speed, direction, and treatment time for both studied bacteria. The aim was to achieve the highest decontamination of the treated area and to determine which of these parameters appeared to be the most significant. No obvious difference (between the treatment directions) was found in the overall decontamination of the treated area for studied bacteria Escherichia coli. The negative effect of speed was only observed for the fastest speed used in the case of gram-positive Staphylococcus epidermidis. The most significant parameter was found to be the treatment time, where a significant decrease in colony growth was observed with increasing treatment time. Furthermore, a repeated treatment was done, where the inoculum was prepared from the already treated bacteria. By this set of experiments, possible development of microbial resistance against the plasma treatment was tested. The results showed that there was no significant increase in the number of colonies even after the repeated treatment. It was also observed that the gram-positive bacteria Staphylococcus epidermidis showed lower decontamination effect evaluated via the number of colonies than the gram-negative bacteria Escherichia coli for all treatment types studied.
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Isolation and characterization of antibodies specific to the DNA quadruplex structure
Oršel, Jiří ; Stříž, Radim (referee) ; Brázda, Václav (advisor)
The aim of present bachelor thesis has been to isolate guanine quadruplex (G-quadruplex) binding antibodies and characterise them by the means of SDS-PAGE, western blot and immunostaining with a subsequent evaluation of their binding capabilities using an electrophoretic mobility shift assay. G quadruplexes are non-canonical secondary DNA structures formed by guanine rich sequences. They are commonly found in telomeres or oncogene promoters where they fulfil important regulatory functions and contribute to the onset of some tumours. The first part of this thesis is dedicated to the isolation process of BG4, SG4 and SG4 R105A antibodies from the E. coli BL21 DE3 expression system, their determination using the Bradford protein assay followed by the aforementioned western blot and immunostaining, confirming their purity. Their binding capabilities have been assessed using the electrophoretic mobility shift assay in the second part. On the one hand, SG4 has been found to bind G-quadruplexes with high specificity especially in lower DNA:protein molar ratios (1:5), which is consistent with available literature. BG4 on the other hand, has shown comparable affinity to both guanine quadruplexes and double stranded DNA. A surprising finding has been a relatively large affinity of SG4 R105A, a mutant of SG4 engineered to have a low affinity to G-quadruplexes, towards single stranded DNA. Furthermore, it’s measured affinity to G-quadruplexes has been comparable to that of BG4 in lower DNA:protein ratios.
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Comparison of the microbial composition of English Blue Stilton cheese with related cheeses
Druláková, Tereza ; Vodička, Juraj (referee) ; Brázda, Václav (advisor)
This bachelor’s thesis focuses on the comparison of English blue-veined cheese Blue Stilton with commonly available cheeses of the same type, Roquefort and Niva, in terms of microbial composition, aromatic volatile content and also by sensory analysis. These methods distinguish the expensive Blue Stilton cheese from common and cheaper cheeses of the same type in the Czech Republic. The presence of 15 selected species of microorganisms was determined by RT-PCR. Three of them proved to be specific for Blue Stilton, specifically bacteria Brevibacterium linens and the yeasts Geotrichum candidum and Kluyveromyces lactis. Aromatic volatile compounds were determined by HS-SPME-GC-MS. Heptan-2-one, nonan-2-one, non-8-en-2-one and hexanoic and octanoic acids were the most important substances contributing to the aromatic profile of all cheeses. The cheeses can be distinguished according to the content of these substances in their aromatic profile or according to the amount of these substances in the individual samples. The sensory evaluation revealed that in most parameters like appearance, consistency and taste, Niva cheese was the most acceptable for Czech consumers while Blue Stilton was the most popular in the aroma category. On the basis of the obtained data, we are able to distinguish English Blue Stilton from other blue-veined cheeses according to specific microorganisms and the unique aromatic profile.
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Local structures in retrovirus genomes
Sádlová, Adéla ; Kollerová, Silvia (referee) ; Brázda, Václav (advisor)
This bachelor thesis focuses on the study of local structures in the genomes of retroviruses. Retroviruses are a group of viruses with an RNA genome that have the ability to integrate their genome into the host DNA. Local structures in the retroviral genome play an important role in the processes of replication, gene expression regulation, and interactions with host proteins. The aim of this work was to analyze the local structures in selected retroviruses using bioinformatics methods, with G4 Hunter being the key tool for identifying G quadruplexes in the genome. G-quadruplexes are DNA or RNA structures composed of guanine bases arranged into quartets and are connected by stacking guanine tetrads. G quadruplexes are often located in telomeres, gene promoter regions, and can play an important role in regulating gene expression, DNA replication, and other cellular processes. The analysis focused primarily on the presence of potential G-quadruplex-forming sequences (PQS). The results of this work provide important information on the distribution and frequency of PQS in the genomes of selected retroviruses and contribute to a better understanding of their role in viral biology. The results show that G-quadruplexes are significant local structures and are often found in regulatory areas of the genome, indicating their key role in the viral infection process.
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Evaluation of the presence of probiotic bacteria in a food supplement using molecular biological methods
Dvornyi, Nikolai ; Brázda, Václav (referee) ; Smetana, Jan (advisor)
Probiotics are beneficial microorganisms that, when administered properly, can provide health benefits to the host, such as aiding digestion and boosting the immune system. Their effectiveness and benefits depend on the specific strain of microorganism. These strains are often from the genera Lactobacillus, Bifidobacterium, and represent natural hosts of the gastrointestinal tract. This bachelor thesis focuses on the identification of probiotic bacteria in a commercially available dietary supplement Linex® Forte using molecular biological methods, mainly polymerase chain reaction (PCR). The aim was to verify the presence of the claimed probiotic cultures of Lactobacillus acidophilus, Bifidobacterium animalis, and to compare the results with the manufacturer's claims. Therefore, two DNA isolation methods were used: phenolchloroform extraction and the commercial purification kit OMNI International. The isolated DNA was then analyzed by domain, genus and species-specific PCR. The results show that the methods used are suitable for the identification of probiotic bacteria and confirm their presence in the selected product.
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Influence of production environment on the occurrence of contaminating Penicillium in smear ripened cheese
Veselá, Vendula ; Brázda, Václav (referee) ; Vítová, Eva (advisor)
The topic of this thesis is the monitoring of the environment in a cheese production plant specializing in cheese ripened under a smear rind. The aim is to evaluate the occurrence of Penicillium mold as the main contaminant microorganism, depending on the sampling location and production time. Air samples were collected throughout the production process using the sedimentation method at 21 designated locations. The total number of microorganisms, selected groups of microorganisms, and the target microorganism (Penicillium mold) were monitored in the collected samples using cultivation (according to ČSN standards), microscopic, and molecular diagnostic techniques. The aim was to evaluate the occurrence of Penicillium citrinum, chrysogenum and commune species in dependence on the sampling location and production time. As part of the production screening, the broader microflora was first evaluated, followed by monitoring for contaminating molds. Using macroscopic and microscopic methods, 36 morphologically distinct colonies were selected. Microscopic analysis confirmed the presence of bacteria, yeasts and molds of the genera Penicillium, Cladosporium and Aspergillus. In order to identify the microorganisms present, an analysis using the polymerase chain reaction (PCR) method was performed, preceded by DNA isolation using the NucleoSpin Microbial DNA kit and phenol extraction. Based on real-time PCR results, the occurrence of contaminating Penicillium and Cladosporium molds was detected even in the early stages of production. Molds from these genera are found throughout the entire facility except for the single room that serves as a curd cooler before forming. Aspergillus mold, in the form of yellow mold with white mycelium on the edge of the colony, was detected only once in the drying room where cheese ripening occurs, and confirmed by microscopic observation. The genus Cladosporium was found in five morphologically distinct forms in the production environment. In addition, DNA was isolated from 11 morphologically distinct bacteria. Real-time PCR identified this DNA as belonging to coryneforms, with four of them belonging to the genus Brevibacterium. The genus Penicillium was represented by 13 morphologically distinct colonies, real-time PCR confirmed the presence of Penicillium commune and Penicillium chrysogenum. In addition to the mentioned molds, 11 morphologically distinct bacteria were detected, from which DNA was isolated. Real-time PCR identified this DNA as belonging to coryneforms, with 4 of them belonging to the genus Brevibacterium. The results of this work completely map the contamination of the production environment of the plant, and based on these results, methods for environmental sanitation were proposed to ensure the decontamination of the production process.
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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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