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Study of oligandrin protein secreted by oomycete Pythium oligandrum
Neykulova, Anastasia ; Bělonožníková, Kateřina (advisor) ; Adámková, Lyubina (referee)
Oomycete Pythium oligandrum acts as a mycoparasite of pathogenic fungi, bacteria and oomycetes in soil due to the production of a number of hydrolytic enzymes for the degradation of polysaccharides and proteins in the cell wall of the host. At the same time, P. oligandrum can interact with plant roots through specific elicitors and stimulate plant defense and growth. Thus, P. oligandrum is a successfully used environmentally friendly biological control agent of plants. Among the secreted elicitors P. oligandrum to the low molecular weight proteins belong oligandrins (~10 kDa), which have a characteristic structure and a conserved sequence among the group of so-called elicitins, and at the same time are not homologous to plant proteins. In the framework of this bachelor thesis, was analyzed the production of oligandrin in various types of growing media after cultivation of P. oligandrum. The total proteolytic activity and the content of phenolic substances as other possible elicitors of plant defense reactions were also observed in culture media. Further, this work focused on the possibility of recombinant oligandrin expression in E. coli and its subsequent purification. Key words: Pythium oligandrum, oligandrin, elicitors, cultivation, recombinant expression [IN CZECH]
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Preparation of a polymeric therapeutic targeting CD20 positive B-lymphomas and NK cells
Hejl, Maxmilián ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
Malignant transformation of B-cells is manifested by a marked increase in the number of surface markers 20 (CD20, cluster of differentiation 20). In studying this trend, chimeric monoclonal antibodies targeting CD20 were introduced to induce apoptosis in B-lymphomas. Since the introduction of the first therapeutic monoclonal antibody rituximab, many others have been developed, with some still used to treat B-lymphomas today. Unfortunately, in many cases, resistance to these drugs is developing, and therefore the development of new types of therapeutics is still relevant. This work aims to develop a polymer-protein macromolecular conjugate capable of inducing apoptosis in CD20 positive leukemia cell lines. For this purpose, we work with biologically active vectors, so-called anti-CD20 nanobodies. This is a variable binding domain derived from the "heavy chain only" antibodies found in, e.g., llamas or camels. Compared to conventional antibodies, nanobodies are approximately ten times smaller, but their binding affinity for the antigen is not altered. For this reason, nanobodies are ideal candidates for attachment to a polymeric carrier, where poly-N-[2-(hydroxypropyl)methacrylamide] (pHPMA) was chosen in this work. The transpeptidase reaction catalyzed by recombinant sortase A, which recognizes and...
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Changes in plant metabolism due to phytoremediation of pharmaceuticals
Polívková, Linda ; Bělonožníková, Kateřina (advisor) ; Chmelík, Josef (referee)
As a result of human activity, the rate and amount at which various pollutants enter the environment are increasing. Among them, pharmaceuticals and their metabolites, whose fate in nature has not yet been fully understood, have a key role to play. Phytoremediation, as an alternative method to the traditionally established methods of remediation of pollutant- contaminated wastewater and soils, has an increasing potential to become an important and ecologically friendly tool for environmental restoration and conservation. In this bachelor thesis, the effect of the pharmaceuticals buspirone, dextromethorphan, ibuprofen, the combination of dextromethorphan and ibuprofen, paroxetine and sulpiride on the antioxidant system of maize (Zea mays L., DKC 3969) cultivated in vitro in hydroponics under sterile conditions was investigated. The activity and isoenzyme composition of peroxidases, which are among the key components of the antioxidant system, were determined in maize plants. The amount of total soluble proteins in plants was reduced in most cases due to the influence of the aforementioned pharmaceuticals. In contrast, the specific activity of total peroxidases, ascorbate peroxidase and guaiacol peroxidase was increased in most cases, most notably by buspirone, sulpiride and the combination of...
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Phytoremediation of pharmaceuticals
Forróvá, Barbora ; Bělonožníková, Kateřina (advisor) ; Kubíčková, Božena (referee)
Anxiolytics, sedatives, hypnotics and antidepressants are some of the most prescribed pharmaceuticals today. As their consumption increases, there is a growing risk of them leaking into surface water and soil, where they can be accumulated, interfere with key biological processes in animal cells or contaminate the food chain. With this is related an increasing need to find ways to ensure the removal of these substances from contaminated sites. There is a wide range of methods for decontamination, however not all of them are affordable and sufficiently environmentally friendly. Phytoremediation is one of the most environmentally and economically friendly ways used for removing pharmaceuticals and other xenobiotics from the environment. In this work, the effects of the drugs diazepam, rohypnol and trazodone on the antioxidant system of maize (Zea mays, L., cultivar DKC 3969) plants grown under sterile conditions hydroponically in vitro were studied. The effects these drugs had on the plants were monitored through the activity of important antioxidant enzymes, peroxidases and glutathione-S-transferases, as well as through non-enzymatic markers of antioxidant capacity and the content of substances indicative of oxidative stress, malondialdehyde and H2O2. A significant increase in phenolic content was...
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The role of N-glycosylation in homooligomerization of natural cytotoxicity receptor NKp30
Tulpa, Matouš ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells play a key role in the defence against cells that have been infected by a virus, a protozoan or have undergone malignant transformation. In addition, they also regulate the activity and quantity of other cells of the immune system. Target cells are recognized using their activating and inhibitory receptors, from which they receive activating and inhibitory signals, on which the cytotoxic response of NK cells depends. There is a dynamic balance between the signals that determines the life and death of the target cell. If activation signals prevail, the target cell will be eliminated. If inhibitory signals prevail, then a cytotoxic response will not be triggered. The NKp30 receptor, which belongs to the immunoglobulin-like receptor superfamily, is an important activating receptor that recognizes a number of ligands, including hemagglutinin of vaccinia and ectromelia virus, human cytomegalovirus pp65 protein, B7-H6, BAG-6, and galectin-3. The extracellular domain of the NKp30 receptor is capable of homooligomerization in solution under certain conditions. The first requirement is the presence of N-glycosylation, the second requirement is the presence of a 15 amino acid long "stalk" domain that connects the ligand binding domain with the transmembrane α-helix. The aim of this thesis was to...
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Optimization of a loop-mediated isothermal amplification method for testing samples of patients with acute upper respiratory tract infection.
Čížová, Kateřina ; Jeřábek, Petr (advisor) ; Bělonožníková, Kateřina (referee)
The bacterium Staphylococcus aureus is currently one of the most frequently detected pathogenic bacteria in laboratory practice. It is particularly problematic when it comes to antimicrobial resistance. An important tool in the fight against this pathogenic microorganism is a suitable method of its detection, which could be the loop-mediated isothermal amplification method. The presented diploma thesis deals with the optimization of this method for the detection of the nuc gene of the Staphylococcus aureus bacterium. A protocol was established for the detection of Staphylococcus aureus bacteria using loop-mediated isothermal amplification method using newly designed "primers" targeting the nuc gene. To improve the detection limit of the method and to combat false positives of the negative control, steps were taken to optimize the method. Using the method, we were able to safely distinguish samples containing template DNA with a concentration of 0.05 pg/μl from a negative sample within 20 minutes. Using an optimized protocol, it was possible to reliably identify the bacterium Staphylococcus aureus in clinical samples taken from the breath of patients. The result of the work was a new, fast, and reliable method of detecting the bacterium Staphylococcus aureus using the loop mediated isothermal...
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Preparation of mutated forms of protein B7-H6
Malá, Viktorie ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells are an essential part of the immune system. They are the so-called natural killers and carry several receptors on their surface. Two types of receptors are crucial for immune surveillance, according to their function, called inhibitory and activating. Inhibitory receptors provide auto-tolerance. The second type is activating receptors, whose activation signals toward initiating a cytotoxic response or involving other immune system components (e.g., macrophages). The activating receptor NKp30 (also known as NCR3 or CD337) is a protein of the Natural Cytotoxicity Receptors (NCR) family. It is one of the primary human NK cell activating receptors with potential use in targeted anticancer therapy. NKp30 has an activating ligand protein B7-H6, which is not found on the surface of healthy cells. It is exclusively found on highly stressed cells, e.g., infected or malignantly transformed cells. If a malignantly transformed cell carries B7-H6 on its surface, in most cases, it is recognized and eliminated almost at its formation. Not all tumour cells are B7-H6 positive, so it is possible to visualize them to the immune system through surface decoration with B7- H6. Moreover, such a ligand can be modified to amplify the immune response. This work is based on Pekar L. et al. (2020) publication, which...
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Use of biochemical methods in the study of plant metabolism with emphasis on the defense system
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Luhová, Lenka (referee) ; Plchová, Helena (referee)
As sessile organisms, plants cannot avoid being exposed to various biotic and abiotic stress factors. To defend themselves plants have developed complex and sophisticated defense mechanisms. In agriculture, pesticides are used for plant protection, but they can have undesirable side effects, and therefore attention is being paid to biological control agents. One such agent is Pythium oligandrum, a soil oomycete that has a positive effect on plants at several levels. On the one hand, it secretes elicitins, which stimulate defensive responses and prepare the plant for possible infection (i.e., priming effect), and on the other hand, it produces tryptamine, a precursor of auxin, thus supporting the plant's growth. Furthermore, P. oligandrum directly acts as pathogen mycoparasite or evokes antibiosis, or competition for nutrients and space with other microorganisms. The key research topic of this thesis was the analysis of the biochemical properties of eleven Pythium strains and their impact on plant metabolism. Our results showed that even closely related P. oligandrum strains significantly differ in the content of compounds secreted into the medium, including proteins, amino acids, tryptamine, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase),...
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Properties of plant extracts
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Plants synthesize a wide variety of compounds called secondary metabolites. These compounds comprise products of the phenylpropanoid pathway (flavonoids, stilbens, anthocyanines and tannins), isoprenoids, and alkaloids. Since plant secondary metabolites seem to affect human health in a positive way, decrease the risk of some diseases such as cancer, diabetes type 2 and disorders of the cardiovascular system, their studies attract a lot of attention. The mechanism of the effect of individual compounds on human health has not been clarified yet, the positive effect is probably caused by their antioxidant capacity. In this work 22 extracts from different kinds of fruit and vegetables were prepared. The total content of phenolic compounds and flavonoids, the antioxidant capacity by methods FRAP (ferric ion reducing antioxidant power) and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6- sulphonic acid)] were determined. High content of phenolic compounds was established in the extract from strawberry, cherry and kiwi, the highest antioxidant capacity was found in the extract from strawberry. Another studied attribute of extracts from fruit and vegetables was their influence on the activity of digestive enzymes pancreatic lipase and α-amylase. The extracts from lemon, red currant, jostaberry, lime and...
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Use of biochemical methods in the study of plant metabolism with emphasis on the defense system
Bělonožníková, Kateřina
As sessile organisms, plants cannot avoid being exposed to various biotic and abiotic stress factors. To defend themselves plants have developed complex and sophisticated defense mechanisms. In agriculture, pesticides are used for plant protection, but they can have undesirable side effects, and therefore attention is being paid to biological control agents. One such agent is Pythium oligandrum, a soil oomycete that has a positive effect on plants at several levels. On the one hand, it secretes elicitins, which stimulate defensive responses and prepare the plant for possible infection (i.e., priming effect), and on the other hand, it produces tryptamine, a precursor of auxin, thus supporting the plant's growth. Furthermore, P. oligandrum directly acts as pathogen mycoparasite or evokes antibiosis, or competition for nutrients and space with other microorganisms. The key research topic of this thesis was the analysis of the biochemical properties of eleven Pythium strains and their impact on plant metabolism. Our results showed that even closely related P. oligandrum strains significantly differ in the content of compounds secreted into the medium, including proteins, amino acids, tryptamine, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase),...
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