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Influence of nanoparticles and polymers on the amyloid fibril formation
Holubová, Monika ; Štěpánek, Petr (advisor) ; Štěpánek, Miroslav (referee) ; Nardin, Corrine (referee)
The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...
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Influence of nanoparticles and polymers on the amyloid fibril formation
Holubová, Monika ; Štěpánek, Petr (advisor) ; Štěpánek, Miroslav (referee) ; Nardin, Corrine (referee)
The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...
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Detekce patogenů pomocí molekulárně imprintovaných polymerů
Hutařová, Jitka
The theoretical part of this diploma thesis deals with molecularly imprinted polymers. Properties of the monomers and templates used for the polymerization are described. Part is devoted to the preparation of molecularly imprinted polymers and interactions leading to the formation of imprinted polymers. Imprinting technologies and strategies, template removal methods, detection methods, and the advantages and disadvantages of the molecular imprinting are included. Also the applications in various fields, focusing mainly on the area of imprinting proteins and pathogens are discussed in details. The experimental part deals with optimization of the preparation of a molecularly imprinted polymer based on dopamine subsequently used especially for the isolation and detection of Staphylococcus aureus and Enterococcus faecalis. The polymer was coated over a microscope slide, into the 96-well microplate and on a surface of magnetic particles. Fluorescence spectrometry and fluorescence microscopy were used for analyte detection. The developed technique was capable of isolating the target (Staphylococcus aureus and Enterococcus faecalis) at relatively low concentrations (1.103 CFU.ml-1).
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Interactions of prosthetic material with proteins
Osifová, Zuzana ; Hudeček, Jiří (advisor) ; Martínek, Václav (referee)
This bachelor thesis deals with the problem of ocular prostheses. This problem is in thesis widely researched. Emphasis is placed on protein adsorption onto synthetic surface. Experimental part of the thesis focuses on lysozyme adsorption onto polymethylmethacrylate surface. Protein adsorption was lower than the specified limit of detection. According to experimental data was this amount lower than 5,37 μg/cm2 . In the thesis was conducted survey on secondary education students' awareness of ocular prosthesis. The results show slightly greater awareness of science-oriented students.
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Use of antimicrobial products of animal origin in cosmetics
Puškárová, Radka ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
The Bachelor thesis deals with characterization a whey in free and encapsulated form and its application to cosmetic industry. The theoretical part is focused on characterization of whey, especially on its antimicrobial properties, and use of this effect in selected cosmetics preparatives. Further, methods of encapsulation and particles characterization were described. In the experimental part, antimicrobial properties of whey were tested by two different methods. Whey was encapsulated into three different types of particles: liposomes, alginate and chitosan. Long-term colloid stability of particles during one and three weeks of storage in model conditions was tested. Further, particles were added into model skin creams. Long-term sedimentation stability of these emulsions was analyzed by analytical centrifuge.
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Proteomic analysis of lysozyme and lysozyme-like proteins of synanthropic mites
Chum, Tomáš ; Erban, Tomáš (advisor) ; Mikeš, Libor (referee)
This diploma thesis was focused on the study of lysozyme and lysozyme-like proteins, either of similar function (antibacterial) or molecular weight (14 - 17 kDa), of synanthropic acaroid mites. In general, animals utilize lysozymes for defensive (antimicrobial) or digestive purposes but also as a digestive enzyme. Some chitinases or other enzymes that act similarly to lysozyme can be utilized for similar purposes. Chitinases belong to house dust mite allergens. One of major mite are historically named lysozyme-like proteins which name relates to their size similar to lysozyme. Bacteriolytic activity has also 14.5 kDa (UniprotKB Q8MWR6) protein. The species selected for the study were domestic mites Dermatophagoides farinae, D. pteronyssinus and Lepidoglyphus destructor. Presence of lysozyme was detected by direct detection with polyclonal antibody using immunohistochemistry and dot blots. Immunohistochemistry proved presence of lysozyme epitopes in the feces of D. farinae, D pteronyssinus a L. destructor. Dot blot analysis demonstrated the presence of imunoreactivity of antibody in spent growth medium extracts (SGME) of all three species. This implies that lysozyme is synthesized in the midgut. The presence of lysozyme and lysozyme-like proteins was proved using 2D electrophoresis and MALDI TOF/TOF...
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Function of antimicrobial proteins in albumen of precocial birds
Krkavcová, Eva ; Kreisinger, Jakub (advisor) ; Kratochvíl, Lukáš (referee)
Antimicrobial proteins contained in the albumen represent maternal effects, including the non- genetic component allocated into the egg during its oogenesis. Especially for species, whose broods are exposed to environmental influences until completation, these proteins play a crucial role in the viability of embryos due to their potential to influence the risk of microbial infection, which is considered one of the main causes of reduced hatchability. Also, it is assumed that these proteins, beacause of their specific traits, may influence phenotype of chicks, especially its size and immunity in the early postembryonal stage. In my thesis I focused on three antimicrobial proteins of avian egg white - avidin, lysozyme and ovotransferrin, which vary in their antimicrobial activity. For a better understanding of causal relationships between the concentrations of these proteins in the albumen and their effect on hatching success or offspring phenotype, a series of manipulation experiments and correlative measurements were performed. These experiments were held on the eggs of two precocial species - Japanese Quail (Coturnix japonica) and Mallard (Anas platyrhynchos). Our results indicate a crucial role of antimicrobial proteins in reducing the risk of bacterial infection and their natural concentration...
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Use of some encapsulation techniques to controlled release of active substances in food and cosmetics products.
Skoumalová, Petra ; Rittich, Bohuslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation, characterization and application of organic micro- and nanoparticles as transport systems for active components and some their complex natural sources. Active component were packed into liposomes and polysaccharide particles. As active components were used caffeine, some drugs – clotrimazole and ibuprofen, further antioxidants and vitamins. Antimicrobial herbs and spices extract, antimicrobial peptides lysozyme, nisin and other antimicrobial ingredients were encapsulated too. Encapsulation of selected hydrolytic enzymes was tested, too. Particles were also used for encapsulation of probiotic strains Bifidobacterium breve and Lactobacillus acidophilus and prebiotic components. These prebiotics were co-encapsulated into capsules with probiotic cells. Natural extracts were encapsulated e.g. extracts of guarana, ginseng, goji, green barley, propolis, black, green and white tea, coffee, fruit and vegetable extracts. The efficiency of encapsulation was determined by HPLC/PDA and by spectrophotometry. Long-term stability of particles and amount of released component in model/real foods, in model cosmetic conditions and in a model physiological environment were monitored too. Size of prepared liposomes and polysaccharide particles was determined by dynamic light scattering and by light microscopy and electron microscopy, respectively. Stability of the particles was measured using a zeta potential. Also, analytical centrifugation was used to measurement of sedimentation velocity and stability of the prepared particles. The antimicrobial activity were tested using two Gram-positive (Bacillus subtilis, Micrococcus luteus), two Gram-negative (Escherichia coli, Serratia marcescens) bacteria and one fungal strains (Candida glabrata). For determining the antimicrobial properties of active component and prepared particles two the most widely used methods were used - agar diffusion method and broth dilution method. The viability of probiotic strains were performed using flow cytometry and fluorescence microscopy. Encapsulation of active component was successful in all types of particles. Liposome showed a very good long-term stability mainly in water conditions with neutral pH and polysaccharide particles were stable in acidic conditions. Prepared particles showed a very good stability in model stomach environment, while in model intestines environments particles were disintegrated and active component were released. Prepared particles with encapsulated caffeine as well as other tested antioxidants and vitamins could be used to modern types of energy drinks, food supplements and also for some cosmetics applications. Encapsulated antimicrobial components could be used for food application as well as for cosmetics and pharmaceutical application like antimicrobial wound formulation. Encapsulated enzymes can be used for controlled release of proteases in wound healing, as delivery systems in digestive tract and as a part of pharmaceutical preparative and food supplements for enzyme therapy. The study revealed that encapsulation of probiotics and also co-encapsulation of probiotics with prebiotics exhibited longer stability of particles and survival bacterial cells. So, prepared particles are suitable for use to food product with beneficial effects on the human body.
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Characterization of specific proteins form selected animal products.
Janhuba, Filip ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.
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Preparation of PCR-ready DNA
Čuta, Robert ; Vojtíšková, Marie (referee) ; Rittich, Bohuslav (advisor)
In these days are probiotic lactic acid bacteria (LAB) very often used in food processing industry, such as milk products, cheese and fermented salami production. As well as the food conservation agent. Except of the industry usage of the LAB there are microbiological aspects. Identification of the bacterial species methods based on the isolation and amplification of DNA are very often used last few years. Diploma thesis deal with the bacterial cell of Lactobacillus species lysis and it´s optimalization. At first it was tested the optimal concentration of lysozyme (3mg/ml, 5mg/ml, 10mg/ml) and the exposure times (3, 5 a 10 hours). Another testing was aimed to the use and suitability of washing powder to the bacterial cell of Lactobacillus species lysis. I tested the Amway washing powder optimal concentration (1%, 2%, 3% a 4%). Four of another comercial washing powders were tested too. All these tests were performed at the pure Lactobacillus bacterial culture. To ensure the results I tested the washing powders at the real food matrix (Acidified milk, yogurt mango, yogurt white). All the methods were evaluated at the amplification method PCR with specific primers for the Lactobacillus genus. The DNA isolation was performed with the paramagnetic microsperes P(HEMA-co-GMA) and the amount of the DNA was quantified spectrophotometrically. The PCR products detection was performed with the agarose gel electrophoresis.
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