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Characterization of organic nanoparticles with encapsulated antimicrobial peptides
Vejrostová, Petra ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on characterization of particles containing encapsulated antimicrobial enzyme lysozyme. The theoretical part deals with characterization of antimicrobial peptides and their description. Further part of review was focused on lysozyme, the selected representative used in this thesis, its structure, mechanism of action and possible usage. In the experimental part the Hartree-Lowry method was used for lysozyme detection, determination of encapsulation efficiency and for detecting the amount of lysozyme released after incubation in model physiological environment and in model foods. In process of encapsulation the highest amount of lysozyme was packed into 1% chitosan particles, manually prepared alginate particles and into liposomes. During study of stability of particles in model foods as the least stable manually prepared chitosan particles were found. The released lysozyme exhibited changes probably caused by its degradation. The highest influence on particles proved 3% acetic acid. During studying the stability of the particles in artificial digestive fluids as the most unstable manually prepared chitosan particles were found, while alginate particles were the most stable. The thesis also deals with changes in antimicrobial activity of encapsulated lysozyme in prepared particles and after its application into the model environments. A gram-positive bacteria Bacillus subtilis was used in order to test the antimicrobial activity. Antimicrobial tests showed that after encapsulation antimicrobial activity of lysozyme was substantially decreased in most samples. Size and stability of prepared particles was tested using dynamic light scattering.
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Production and characterization of antimicrobial substances from lactic acid bacteria
Franeková, Eliška ; Smetana, Jan (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on the production of antimicrobial compounds using lactic acid bacteria, their characterization and testing of their antimicrobial activity. The theoretical part of this work deals with the characteristics of lactic acid bacteria and the bacteriocins they produce, the possibilities of bacteriocin production and the factors that influence it. In the experimental part of this work, lactic acid bacteria Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Bifidobacterium bifidum were cultivated, their growth curves and total protein content in supernatants after cultivation were measured. Cell-free culture supernatants were prepared by lyophilization and their antimicrobial activity against gram-positive and gram-negative bacteria was determined. The antimicrobial activity of commercial antimicrobial peptide nisin and a commercial preservative obtained using lactic acid bacteria was also measured. The sample obtained from Bifidobacterium bifidum was selected as the most effective of the isolates. Its antimicrobial activity was further tested on polymeric sausage packaging materials. Antimicrobial substances produced by lactic acid bacteria can be used in the food industry as preservatives or as a part of antimicrobial packaging, and in the pharmaceutical industry in materials for antimicrobial wound dressings.
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Identification of antimicrobila peptides in spider venom
Benýšek, Jakub ; Tichá, Marie (advisor) ; Liberda, Jiří (referee)
Still increasing resistance to antibiotics leads to the need to find new active compounds with antimicrobial properties. This work is focused on the occurrence, chemical and physical description, mechanism of action and biological activity of such substances, found in spider venom. The second part is focused on isolation and identification of compounds with these properties from the venom of wild bees and a one spider. A novel peptide was isolated and identified from venom of bee Trachusa byssina. This novel peptide possess antimicrobial properties and low hemolytic activity. Molecular weight was estimated to 1749,9 ? 0,1contains 16 amino acids and is amidated on its C-terminus. Its primary structure GILSVLKNLLKKHMAS-NH2 was determined by using Edman degradation and ESI-QTOF mass spektrometry.
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New antimcrobial peptides isolated from the bee venom and the study of their action mechanism
Čujová, Sabína ; Čeřovský, Václav (advisor) ; Fusek, Martin (referee) ; Hlaváček, Jan (referee)
EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...
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The effect of antimicrobial peptides on Saccharomyces cerevisiae and other yeast species
Makarova, Anna-Marie ; Hodek, Petr (advisor) ; Malcová, Ivana (referee)
The increased use of antibiotics, antifungal agents and disinfectants in the last decades has resulted in development of microbial resistance to these drugs. Candida species are the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients. Natural antimicrobial peptides are promising candidates for the development of new agents to treat yeast and bacterial infections, as their presumed mechanism of action differs significantly from the mechanism of action of current drugs. This work is focused on several peptides isolated from the venom of wild bees and their synthetic analogues and the identification of the most effective ones against non-pathogenic Saccharomyces cerevisiae and several pathogenic Candida species. Antifungal activity of eight cationic antimicrobial peptides was tested and compared under various conditions. The overall susceptibility of pathogenic yeast species to currently used antifungal drugs and the antimicrobial peptides was screened with the aim to identify potential synergistic and species-specific effects. The effect of antimicrobial peptides on membrane potential was measured by a fluorescent probe (diS-C3(3)), and the relative hyperpolarization of plasma membrane was shown for each peptide. The effect of antimicrobial peptides on...
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Variability of the domestic chicken breeds in selected immunological traits of hen and egg
Bílková, Barbora ; Vinkler, Michal (advisor) ; Plachý, Jiří (referee) ; Trefil, Pavel (referee)
The avian immune system is a complex system of defence mechanisms that protect bird hosts against threats from ubiquitous pathogens. According to the co-evolutionary models, variability in immune traits of hosts is the key component providing ability to adapt and enhance their defence mechanisms in presence of constant selective pathogen pressure. Domestic chicken (Gallus gallus f. domestica) is used as a model organism in avian biology and also is one of the most important food-producing animals, not only for their meat but also for the egg production. Unfortunately, in research usually only inbred chicken lines are used and modern poultry husbandry is tight with unilateral breeding towards highly productive breeds. Those approaches decrease intra-population polymorphism in chickens. However, especially in case of farm animals, searching and extending the pool of immune variability and enhancing pathogen resistance is crucial for sustaining healthy and biologically secure populations and their products. Morphologically highly distinct traditional chicken breeds, which have evolved for hundreds years under different selective pressures, may represent this desirable immunological variability. In my thesis I described variability in chosen immunological traits, haematological parameters and proteomic...
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Capacity of probiotics to affect innate lymphoid cells type 3
Věcek, Jan ; Hrdý, Jiří (advisor) ; Schwarzer, Martin (referee)
Innate lymphoid cells type 3 (ILC3) are a group of relatively newly discovered lymphocytes that lack an antigen-specific receptor. Nevertheless, their important role of immune regulators on mucous membranes is evident. In addition to the development of lymphoid tissue in embryogenesis, and during ontogenesis, postnatally, ILC3 are mainly involved in maintaining intestinal homeostasis and controlling intestinal microbiota. ILC3 produces various cytokines that stimulate surrounding intestinal cells to produce antimicrobial peptides and maintain epithelial wall integrity. The major cytokine produced by ILC3 is IL-22. Th17 lymphocytes and ILC3 are similar in many respects but differ significantly in some functions. ILC3 can regulate adaptive immunity cells towards an antimicrobial response without inducing inflammation. They are also directly connected to cells of the nervous system. Some probiotic bacterial strains produce metabolites that directly affect ILC3. This mechanism could be used in new therapeutic approaches to ameliorate the severity of diseases where changes in microbiota composition and function are inducing proinflammatory responses of the host. Key words: innate lymphoid cells; IL-22; antimicrobial peptides; probiotics; microbiota
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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New antimcrobial peptides isolated from the bee venom and the study of their action mechanism
Čujová, Sabína
EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...
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