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Metabolism of Bacterial Cells and the Effect of Stress on Biosynthesis of PHA
Kučera, Dan ; Kráčmar, Stanislav (referee) ; Ondrejovič,, Miroslav (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of polyhydroxyalkanoate biosynthesis as a microbial product with the potential to replace current conventional plastics made from petroleum. The dissertation thesis is elaborated in the form of a discussed set of already published publications, which are then part of the thesis in the form of appendices. The work builds on relatively extensive knowledge in the field of polyhydroxyalkanoate production and brings new facts and possible strategies. Various possibilities of analysis of polyhydroxyalkanoates using modern methods were tested in this work, which brings especially speed, which can be crucial in real-time evaluation of production biotechnological process. Raman spectroscopy has proven to be a very promising technique for rapid quantification of PHA. Furthermore, the work deals with valorisation of waste of food and agricultural origin. Emphasis is placed on methods of detoxification of lignocellulose hydrolysates. In this context, adsorption of inhibitors to lignin was first used as an alternative to other detoxification techniques. Due to detoxification, selected production strains Burkholederia cepacia and B. sacchari were able to utilize softwood hydrolyzate for PHA production. In the next part of the work was also tested the possibility of using chicken feathers as a complex source of nitrogen. Evolutionary engineering was also used as a possible strategy to eliminate the inhibitory effect of levulic acid as a microbial inhibitor that results from the hydrolysis of lignocellulosic materials. Adaptation experiments were used to develop strains exhibiting higher resistance to levulic acid and the ability to accumulate a higher 3HV copolymer from the original wild-type C. necator strain. Another promising approach tested in the work was the use of extremophilic microbial strain, which leads to a reduction in the cost of biotechnological production. Selected Halomonas species have shown high potential as halophilic PHA producers. The final part of the thesis was devoted to the selection of the production strain with regard to the properties of the resulting PHA. The Cupriavidus malaysiensis strain was selected to produce a P(3HB-co-3HV-co-4HB) terpolymer which revealed significant differences in material properties over P3HB.
Use of Molecular Biology Techniques for Identification and Analysis of Probiotic Bacteria
Konečná, Jana ; Doškař, Jiří (referee) ; Kráčmar, Stanislav (referee) ; Obruča, Stanislav (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
Bokrová, Jitka ; Pekař, Miloslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation of nanoparticles and nanofibers with natural active ingredient and testing their biological effects. Modern types of application forms were prepared from biomaterials based on one or more natural polymers. Chitosan particles were prepared from cross-linked polymer using ultrasonication. A mixture of soy lecithin and cholesterol was used for preparation of liposomes. Poly-3-hydroxybutyrate was used for preparation of combined liposomes, too. All liposome particles were prepared by ultrasonication. Nanofibers were obtained from polyhydroxybutyrate using electrospinning. Mixtures of low-molecular antioxidants obtained by extraction from natural sources were used as active ingredients. Different types of teas, barks, herbs, spices, fruits and vegetables were selected as sources of natural antioxidants. Total phenolic and flavonoid content and total antioxidant activity of extracts were determined using spectrophotometrical methods. Obtained natural extracts were subsequently used for encapsulation. Prepared application forms were characterized in terms of their physicochemical properties. Particle size was monitored by dynamic light scattering. Colloidal stability of particles in suspension was determined using zeta potential. Spectrophotometry was used to evaluate the efficiency of encapsulation of active compounds into particles. The morphology of the new type of combined PHB liposomes was monitored by electron microscopy. Chromatography was used for quantification of individual components of particles. Morphology of nanofibers and incorporation of active agent into their structure were monitored using FTIR-ATR spectroscopy and electron microscopy. Afterwards, antimicrobial, cytotoxic and genotoxic effects of preparations were evaluated. It was found that the most suitable types of extracts for liposome preparation are aqueous and lipid extracts of natural antioxidants. Prepared particles showed excellent stability and good encapsulation efficiency. The study confirmed that incorporation of polydroxybutyrate into liposome structure does not reduce neither the colloidal stability of the particle, nor the efficiency of encapsulation process. Antimicrobial and antimycotic effect of preparations against model microorganisms Micrococcus lutues, Serratia marcescens and Candida glabrata was detected. It was found that process of encapsulation increases the inhibitory effect of natural extracts of antioxidants. The safety of preparations was assessed using two human cell cultures: epidermal keratinocytes and HaCaT cell line. Assays of cell viability and plasma membrane integrity were used to determine cytotoxicity of preparations. Low toxicity of liposome particles was confirmed by a series of cytotoxic tests. Obtained data showed that association of phospholipid with PHB polymer does not cause a significant increase in cytotoxicity in human skin cells. Genotoxicity testing on model procaryotic organism confirmed zero genotoxic potential of preparations. The new type of combined particles and polymeric fibers cant thus be used as a carrier for active ingredients, complex natural extracts, antimicrobial agents and many others.
Hudečková, Helena ; Kráčmar, Stanislav (referee) ; Buňka, František (referee) ; Márová, Ivana (advisor)
The doctoral thesis is focused on the microbial production of lactic acid and ethanol using food processing waste as substrate. Coffee processing waste (spent coffee grounds), wine production waste (grape pomace) and orange processing waste (orange peel) were chosen as substrates for experiments. The theoretical part is dedicated to summarizing current knowledge about waste from food production and possibilities of its processing. It also deals with selected metabolites (lactic acid, ethanol) to which these wastes can be used. Part of the experiments was focused on the characterization and optimization of hydrolysis to maximize the amount of fermentable saccharides. Different combinations of chemical, physical and enzymatic hydrolysis of selected substrates have been tested. Subsequently, a suitable strain for lactic acid and ethanol production was searched for. In the case of lactic acid production, 7 bacterial strains were selected (Lactobacillus casei CCM 4798, Bacillus coagulans CCM 2013, Bacillus coagulans CCM 2658, Lactobacillus rhamnosus CCM 1825T, Lactobacillus delbruckii subsp. bulgaricus CCM 7190, Lactobacillus plantarum CCM 7039T, Streptococcus thermophilus CCM 4757). These strains were first cultivated on the synthetic media containing different kind of saccharides. Afterward, the cultivation on the waste biomass hydrolysates were tested. In the case of ethanol production, 2 yeast strains kmeny (S. cerevisiae CNCTC 6646 a S. cerevisiae CNCTC 6651) were cultivated on hydrolysates of individual waste substrates. Subsequently, the experiments focused on the production of lactic acid and ethanol on hydrolysates of waste biomass in bioreactor were done. The last part of this doctoral thesis deals with the microaerobic pretreatment of lignocellulosic biomass to increase the production of organic acids during the acetogenic phase of anaerobic digestion.
Controlled Drug Release from Biodegradable Hydrogels.
Oborná, Jana ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Kučerík, Jiří (referee) ; Vávrová, Milada (advisor)
This dissertation is focused on the controlled release of drugs from a biodegradable amphiphilic hydrogel based on hydrophobic poly(lactic acid), poly(glycolic acid) and hydrophilic poly(ethylene glycol) (PLGA-PEG-PLGA, ABA) and its modification with itaconic anhydride (ITA). The resulting ,-itaconyl(PLGA-PEG-PLGA) copolymer is referred to as ITA/PLGA-PEG-PLGA/ITA or ITA/ABA/ITA. Itaconic acid provides reactive double bonds and a functional carboxyl group at the ends of the PLGA-PEG-PLGA copolymer chain, thereby rendering the modified ITA/ABA/ITA copolymer less hydrophobic and offering the possibility of forming a carrier for hydrophilic drug substances. These functional copolymers are thermosensitive and change in the external environment (e.g. temperature) causes a sol-gel phase transition due to the formation of micellar structure. The bioactive substances can thus be mixed with a copolymer which is in a low viscous phase (sol phase) and subsequently the mixture can be injected into patient's body at the target site where it forms a gel at 37 °C. This hydrogel becomes a drug depot, which gradually releases the active substance. Prediction of the substance’s release profile from the hydrogel is an effective tool to determine the frequency of administration, potentially enhancing efficacy, and assessment of side effects associated with dosing. The analgesic paracetamol and the sulfonamide antibiotic sulfathiazole were used as model drugs, representing hydrophilic and hydrophobic substances, respectively. The active substances had a significant effect on the resulting hydrogel stiffness. Type of solvent, incubation medium and nanohydroxyapatite also influenced on the gel stiffness and subsequent stability of the hydrogel-drug system. Controlled release of drugs took place in simulated conditions of the human body. Verification of Korsmeyer-Peppas (KP) drug-release model is also discussed in this thesis. The KP model was found suitable for simulating the release of sulfathiazole from ABA and ITA/ABA/ITA hydrogels. On the contrary, the performance of KP model was not suitable for describing the release of paracetamol from the ABA hydrogels. Therefore, a new regression model suitable for both buffered simulated media and water has been proposed. The proposed model fitted better the release of both sulfathiazole and paracetamol from composite material prepared from ABA hydrogel and nanohydroxyapatite.
Ecotoxicological Assessment of Point Pollution in the Ecosystem
Doležalová, Šárka ; Beklová, Miroslava (referee) ; Kráčmar, Stanislav (referee) ; Koutný, Marek (referee) ; Vávrová, Milada (advisor)
The main subject of this dissertation is the ecotoxicological evaluation of significant point sources of pollution. These include wildfires and wide fires of factories and also industrial and urban wastewater treatment plants. The local application of pyrolytic product obtained from sewage sludge as the point source of pollution was also included in the thesis. Tests in aquatic and contact arrangement with using both water and soil organisms for ecotoxicity evaluation of selected matrices were chosen. Crustaceans Thamnocephalus platyurus, Daphnia magna Straus and sea bacteria Vibrio fischeri were selected as representative organisms of water animals. Duckweed Lemna minor was chosen as water plant. Further collembola Folsomia candida and earthworm Eisenia fetida were chosen as representatives of soil animals. Finally onion Allium cepa L. and mustard Sinapis alba are representatives of terrestrial plants. Submitted thesis deals about analytes of interest, their properties and ways of distribution to the environments. The output of the thesis is a comprehensive assessment of the effects of selected point sources of pollution on aquatic and terrestrial ecosystems organisms and ecotoxicological risks assessment. The fire extinguishing agents have been proven to be the most stressful substances for all selecteded organisms.
Complex characterisation of medical herbs and study of their utilization in food industry as a source of functional components
Burdějová, Lenka ; Kráčmar, Stanislav (referee) ; Šilhár, Stanislav (referee) ; Polovka, Martin (advisor)
Presented thesis deals with the complex characterization of extracts from medical plants using spectroscopic and chromatographic techniques, with emphasis on the influence of solvent, post-harvest treatment of the sample, location and year of production on selected parameters. Part of the work is devoted to application of selected extracts from medical plants in suitable form into beverages. The thesis is divided into two parts. At the first part of the work combination of electron paramagnetic resonance, ultraviolet visible spectroscopy, liquid chromatography and inductively coupled plasma optical emission spectrometry was applied for characterization of aqueous, ethanol and dimethylsulfoxide extracts from 10 selected medical plants, which were collected from two different locations during two years (2015, 2016) and post-harvestly treated by two ways (freezing, drying). In total, 39 parameters were determinated: namely total phenolic compounds content, total flavonoids content, 14 specific phenolic compounds, colour characteristics in the CIE L*a*b* system (L *, a*, b *, C*, h°, BI), radical-quenching activity using the cation radical of the 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid), antioxidant/pro-oxidant activity using the spin trapping technique in the presence of 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO)/potassium peroxodisulphate and content of 15 minerals (Al, As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, Zn). The results confirmed that the studied parameters of medical plants are influenced by the extraction system, post-harvest treatment of samples, year of production, origin of samples (locality) and species composition. It has been shown that the type of extraction solvent significantly affects the composition of the extracts. On the basis of these analyzes, for food application – the isolation of the main functional components (phenolic compounds, flavonoids) –50% ethanol, post harvest treatment by drying and samples of Mentha piperita, Melissa officinalis, Hypericum perforatum and Salvia officinalis were selected. The second part of the thesis was focused on the application of selected extracts of medical plants to beverages (syrups). The above mentioned plants, 25% ethanol instead of 50% ethanol (due to the limitation of the ethanol content in final raw material), were selected for the application. At first, extraction of medical plants was optimized. Suitable ratio of material and solvent (1:10), extraction time (8 h) and herb absorbency (50–62 %) were determined. The prepared extract after filtration was applied to the syrup concentrate in different ratios to produce one-species syrups and further sensorially tested to select the suitable recipe, the best flavour and basis for the production of two-species syrups. The mint syrup was the most tasty and generally the most acceptable one-species syrup, and sage syrup was selected as the basis for two-species syrups. Further, the most appropriate recipes of the two-species syrups were chosen, the most sensorially acceptable one was selected and further tested if it would be appropriate to colour them. Preferentially, it has been found that it is not necessary to colour two-species syrups. On the basis of the sensory analysis, it has been found that peppermint and sage-peppermint syrups were the most tasty and acceptable syrups, which could be, after further testing, produced industrially to enrich the food market.
Ecotoxicological Evaluation of Polymers and Biologically Active Substances in Aquatic Environments
Kašpar, Otakar ; Beklová, Miroslava (referee) ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
To determine the ecotoxicity of analgetics, first the individual ecotoxicity values of individual analgetics are determined and then a mixture of two analgetics is tested. To determine the toxicity, both standard and alternative toxicity tests are used (daphnia magna, sinapsis alba, scenedesmus subspicatum, vibrio fischeri, thamnotoxkit FTM a daphnotoxkit FTM magna). The analgetics being whish tested are the commonly used medicines ibuprofen, ASA, diclofenac and paracetamol, which are the most frequently used medicines in the Czech Republic and whole Europe. To determine the ecotoxicity of the polymers, I‘m using an indirect method of examination, in which I determine the antagonistic or synergistic effects of the mixture of monomers from which the polymer is prepared and into which it slowly decomposes in nature. For the determination both standard and alternative toxicity tests are used. The polymers the toxicity of which is being determined are the habitually used polymer PET and the formaldehyde resine known as bakelite in Eastern Europe.
The Introduction of Contact Ecotoxicity Tests for the Assessment of Terrestrial Ecosystems
Modlitbová, Pavlína ; Kráčmar, Stanislav (referee) ; Beklová, Miroslava (referee) ; Demnerová, Kateřina (referee) ; Vávrová, Milada (advisor)
This dissertation thesis is focus on using toxicity tests with testing organism - terrestrial isopod Porcellio scaber. This organism is use for toxicity assessment of selected anorganic compunds which could contaminate terrestrial ecosystem. This organism was selected for several reasons - sophisticated test methodology; well known biology of these animals and their ecological relevance. Besides classical endpoints such as mortality, change in mass of test specimens and the effect of the presence and concentration of contaminants onto food consumption; we monitored changes in the morphometric characteristics at the level of tissues and cells, the cytotoxicity and possibly bioaccumulation in various parts of the body of the organism. Selected test substance in this thesis were nanoparticles and inorganic salts. Wherein for chosen gold nanoparticles was carried out a complete study presents several levels of organization of living matter (organism, tissue, cell). For selected salts compound, this study was carried out on the behavior of organisms and classical endpoints.
Physicochemical Aspects of Ecological Heat-transfer Fluids
Skolil, Jan ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (referee) ; Čorňák, Štefan (referee) ; Čáslavský, Josef (advisor)
This thesis develops composition of heat transfer fluids with antifreeze properties next to traditional one component glycols produced from propene and ethene. Based on assessment of physical properties it considers application of progressive antifreeze compounds of glycerine and propan-1,3-diole and their mixtures with classic glycols, which do not have negative influence on the environment and which are produced in last years from renewable sources. Due to the necessity of selection of certain compromise between their heat transfer properties, price, solubility and other factors physical properties of selected mixtures are determined together with synergy of each compound, including formulas with glycols: ethan 1,2 diol and propan 1,2 diol Another evaluated factor of studied formulations in addition to their heat transfer properties was anticorrosive protection of common corrosion inhibitors mixtures. The result of whole work is the proposal of several most perspective formulations based on mixtures of polyhydric alcohols in terms of their features enabling them heat transfer, reasonable costs, and the restrictions of impact of their use, particularly in heat transfer systems, to human health and the environment. The knowledge of physical properties of water solutions of polyhydric alcohols mixtures also helps to better using of recycled materials from planning recycling plant for glycols.

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2 Kračmar, S.
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