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Application for the Data Processing in the Area of Genome Engineering
Brychta, Jan ; Burgetová, Ivana (referee) ; Očenášek, Pavel (advisor)
This masters thesis has a few objectives. One of them is to acquaint with the problems of genome engineering, especially with fragmentation of DNA, the macromolecule DNA, the methods for purification and separation of the nucleic acids, the enzymes used for modification of these acids, amplification and get to know with cluster and gradient analysis as well. The next aim is to peruse the existed application and compare it to the layout of the proposed application, that is the third aim. The last one from the objectives is the implementation and the report how was the application tested by the real data. The results will be discussed as well as the possibilities of the further extension.
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Changes of gluten proteins during beer processing
Porubiaková, Otília ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of thesis was monitoring of changes in the content of gluten proteins in the biotechnological process of beer production. During the production process of wheat and barley beer, the samples were collected and analysed using the electrophoresis and immunoassay method. The results of the analyses were compared with commercial Czech beers. The theoretical part contains description and composition of gluten proteins, malt and beer technology, the changes that occur in this process, and methods of gluten proteins analysis. The experimental part contains procedures for laboratory production of barley and wheat beer and analyses of gluten proteins. To identify the individual gluteal protein fraction acid and SDS electrophoresis methods were used. For quantification, enzyme immunoassay was used and evaluated spectrophotometrically. The identification of the gluten‘s fractions by electrophoretic methods has been shown to be less specific for samples with lower content of gluten proteins and for barley specimens. A decrease in the concentration of gliadins and glutenins in the beer production process was demonstrated. A significant change was found during wort production with 98% decrease of gluten content compared to the feedstock and during the fermentation, when the gluten concentration dropped below 10 mg/kg. This value is acceptable from the legislation for products labelled „gluten-free“.
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Dynamic spot analysis in the 2D electrophoresis gels images
Polášková, Lenka ; Provazník, Ivo (referee) ; Nedvěd, Jiří (advisor)
Práce shrnuje faktory a parametry, které ovlivňují výsledky 2D elektroforézy, se zaměřením na zpracování obrazu jako jeden ze způsobů snížení nesprávné interpretace jejích výstupů. Proces zpracování obrazu využívá jako zdroj dat především obrazů z opakovaných provedení téhož pokusu, neboli víceplik. Pomocí analýzy obrazů víceplik je možno pozorovat nebo korigovat změny jednoho pokusu a také porovnávat je s výstupy jiných pokusů. Cílem práce je poskytnout podporu specialistovi, který má na starosti popsat vlastnosti struktur nacházejících se v elektroforetických obrazech.
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Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
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Pulse proteolysis in evaluation of conformational stability of cytochromes b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...
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Optimization of DNA amplification isolated from buccal swabs for sequencing purposes
ROŠTÍKOVÁ, Kristýna
The topic of my bachelor thesis was the issue of taking primary DNA samples from the buccal mucosa, determination of the ideal length and optimal conditions and subsequent analysis of these samples. In the theoretical part, I focused on the preanalytical phase of the laboratory examination. It was about, the methods of sampling and the type of material that is most often used used for DNA isolation in the molecular biological laboratory. Subsequently, I focused on the methods themselves, which are used for DNA analysis. These included DNA isolation, DNA amplification methods, nucleic acid electrophoresis and DNA sequencing. In the practical part, I processed 42 samples. First, I isolated DNA from the samples and measured its concentration and purity. Then I performed electrophoresis in these samples and based on its results I decided which samples I would process further by PCR. Subsequently, I determined by electrophoresis whether DNA was amplified in the samples. Finally, I selected samples that I sent for sequencing. The aim of my bachelor thesis was to determine the optimal length and storage conditions of samples from the buccal mucosa for further processing. Then I evaluated how these parameters affect the quality, purity and quantity of isolated DNA as well as the course of the polymerase chain reaction. According to the obtained results, the best option is to store the samples in a refrigerator temperature and perform their analysis in the shortest possible time, or to store samples in a refrigerator temperature and process them within weeks to three months.
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Health risk and profit of genetically modified golden rice
KUČEROVÁ, Kateřina
People in developing countries very often suffer from severe vitamin A deficiency due to an insufficient and unbalanced diet. This vitamin is necessary, among other things, for the production of the visual pigment rhodopsin. According to the World Health Organization, up to half a million children go blind each year because of this deficit. Its deficiency also weakens the immune system and thus significantly increases the risk of death from various infectious diseases. The best solution to this deficiency would be if the vitamin was contained directly in the only food that these people get - in husked rice. There is already a special, genetically modified rice, into which has been introduced by a complicated and extensive modification of its DNA the entire metabolic pathway, which ensures the production of betacarotene in this crop. In the theoretical part I deal with the golden rice project and the origin of this genetically modified crop by the method of transgenesis, using bacteria of the genus Agrobacterium tumefaciens. Plasmids of this bacterium are able to incorporate parts of their genetic information into the target organism. Thanks to restriction enzymes, we can insert genes selected by us into plasmids, which we then introduce into a specific plant. But are these products safe? Opinions on these crops are very diverse, but one thing interests everyone: how to safely identify a genetically engineered plant? In the laboratory, these experiments can best be mastered on model material. Therefore, in the experimental part of my bachelor thesis I deal with the transgenesis of the model plant Nicotiana tabacum using a selected strain of Agrobacterium tumefaciens. These bacterial strains were provided to me from the private collections of the Institute of Molecular Plant Biology in České Budějovice. The aim of my work in the laboratory was to master the practical methodology of preparation of genetically modified plant and subsequent verification of the presence of introduced genes in the examined samples of the model organism. Specifically, genes for antibiotic resistance. DNA isolation, PCR amplification and electrophoretic assays were used for this purpose. The signal gene used in agroinfection was verified too. Also, it was monitored how many copies of the transgene were integrated into the research material during agroinfection.
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Human infectious diseases and GMO
KOBLIHOVÁ, Tereza
This bachelor thesis is focused on the detection of the transgenes in DNA of the genetically modified plants, especially on the detection of the transgene nptII. Moreover, it will discuss the determinations of the fission ratio (the amount of copies) of the injected transgenes into the next generation. The theoretical part of this bachelor thesis includes information about bacteria because some of their kinds are the reasons of human infection diseases, however, couple of them are used in genetic engineering for plant transformation at the same time. The diseases which are caused by bacteria are cured by antibiotics. Antibiotics are antimicrobic substances which are used to stop the growth of or exterminate these microorganisms. The theoretical part is especially focused on findings related to antibiotics and gene manipulated organisms (GMO). They are related to the concerns about using the selective gene nptII in the construct of these organisms, because this gene codes enzyme neomycin phosphotransferase II, which deactivates the effects of the Antibiotic called kanamycin. Organisms, where this gene is included are getting resistant to these antibiotics. The aim of the methodical part was the cultivation of own genetic modified plants carrying artificially injected genes for the resistance to kanamycin (nptII) and the signal gene GUS. Furthermore, we were looking for the subsequent determination of presence of the gene ntpII in the samples from these plants. The first necessary step to the determination of the gene nptII in the transgenic plants was multiplying of the DNA section containing this gene. That was accomplished by Polymerase Chain Reaction (PCR). After PCR was adapted, the detection of nptII gene in samples was made by gel electrophoresis. A histochemical test was used for the detection of the presence of the gene nptII in the plant samples and the confirmation of the transformation. As a model organism, the Nicotiana tabacum was chosen, which was transformed by Agrobacterium tumefaciens.
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