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Protein macromolecules immobilization onto polymer carriers
Šitnerová, Michaela ; Holas, Ondřej (advisor) ; Ondrejček, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Michaela Šitnerová Title of Thesis: Protein macromolecules immobilization onto polymer carriers Enzymes are unique biocatalysts because of their properties. They are highly specific, selective and functional even under mild reaction conditions. The method of immobilization is used to increase their operational stability, activity and possible reuse. This process allows the wide use of enzymes in industry, for example in the food industry, analytical chemistry, chemical synthesis and in the pharmaceutical industry. The aim of my thesis was immobilized enzyme acetylcholinesterase (AChE) on the surface pellets of microcrystalline cellulose (MCC). Used method was simple sorption, immobilization using glutaraldehyde, and TEMPO oxidation using MCC. Well known Ellman's method served to measure the activity of AChE. The absorbance of the solution with the immobilized AChE was measured spectrophotometrically at 412 nm.
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Chemo-enzymatic synthesis of antiviral prodrugs
Tupec, Michal ; Stiborová, Marie (advisor) ; Indra, Radek (referee)
Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...
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Preparation of Microbial Metabolites from Waste Materials
Zichová, Miroslava ; Márová, Ivana (referee) ; Stloukal, Radek (referee) ; Rosenberg, Michal (advisor)
In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.
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The influence of therapy by Vojta reflex locomotion method on functional abilities of immobile seniors
Černá, Gabriela ; Smékal, David (advisor) ; Zounková, Irena (referee)
This project assesses effects of four -week active rehabilitation combined with Vojta reflex stimulation on mobility of bed-bound seniors. Sixteen female patients were divided into two groups of eight members, i.e. tested group and control group. Both groups were put through training focused on self-support, maintaining or enhancement of range of movement, strengthening of weak muscle groups, stability training, practicing sitting up and lying down from sitting position, and walking in the high walking frame in the case of stronger patients. Besides that the tested group underwent Vojta reflex stimulation (global model of reflex turning), coordination and stability exercise and exercise with rehabilitation tools (elastic band, over ball, small ball, water bottle). In the beginning and in the end of the project the EMS (Elderly Mobility Scale), BI (Barthel Index), and MMSE (Mini-mental State Examination) scales were tested as well as ability to turn from back to side and stability in the sitting position. Statistical comparability of both groups was proved true in the beginning of the therapy by Mann-Whitney test. There was a noticeable tendency towards higher probability of enhancing measured scores in the tested group compared to the control group after the therapy. In case of the EMS this probability was...
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The use of (nano)oxides for metal and metalloid stabilization in contaminated soils
Michálková, Zuzana ; Komárek, Michael (advisor) ; Luke, Luke (referee)
The main objective of the thesis was to evaluate the potential of selected Mn and Fe (nano)oxides for the stabilization of metals and metalloids in contaminated soils. The research was focused basically on three materials - commercial nanomaghemite (Fe III), nanomagnetite (Fe II,III) and a synthetic amorphous Mn oxide (AMO). The main aim of the work was to provide a complex view on the chosen stabilizing amendments regarding not just their direct influence on contaminants mobility and stabilization mechanisms, but also their stability and alterations in soil conditions together with influence on soil microorganisms and higher plants. Firstly, adsorption properties of the tested materials towards Cd, Cu, Pb and As were investigated. In this context, the most effective material showed to be the AMO reaching one to two orders of magnitude higher adsorption capacities than Fe III and Fe II,III under given experimental conditions. Interestingly, the rate of As(V) adsorption onto AMO was increasing with increasing pH as a result of high pHzpc of the AMO (8.1) and significant dissolution of this phase at lower pH values. As a next step, the influence of (nano)oxides on metal(loid)s mobility and other physico-chemical soil characteristics after application to contaminated soil was examined. Again, the AMO proved to be the most efficient in reducing mobile pools of Cd, Cu, Pb, Zn and As. On the other hand, Fe III and Fe II,III addition had generally less significant effects on contaminants mobility. AMO application further resulted in an increase of soil pH connected with AMO dissolution and unwanted decomposition of soil organic matter. When (nano)oxides alterations in soil conditions were observed, MnCO3 coatings were identified on AMO surface while no significant changes were recorded for Fe III and Fe II,III. As the MnCO3 formation was connected with increased AMO stability, AMO particles synthetically covered with MnCO3 coating (denoted as SM AMO) were prepared. Although the SM-AMO had a lower mass loss in soil than pure AMO, the stabilizing efficiency was almost the same for both materials. The differences in surface composition of both materials were decreasing with time as MnCO3 naturally precipitated on the AMO surface in soils while the SM AMO coating was gradually dissolving. When investigating the effect on soil microbiota, AMO efficiently promoted soil microbial activity while no significant changes were observed in the case of Fe III and Fe II,III. The AMO was also able to reduce the uptake of Cd, Pb and Zn by sunflower (Helianthus annuus L.), eliminate Zn phytotoxicity symptoms and increase biomass yield. On the other hand, toxic levels of Mn released from the AMO in an acidic soil were found in sunflower tissues. AMO application is thus recommended for contaminated neutral or slightly alkaline pH with a higher cation exchange capacity in order to avoid unwanted release of Mn. Finally, various types of AMO-biochar composite sorbents were recently prepared and field experiment focused on stabilization of Cd, Pb, Zn and As using studied materials is currently under preparation. The combined results from the thesis highlight the importance of a complex experimental approach dealing with all parts of the contaminated soil environment in order to obtain complete information about the efficiency and usefulness of any newly developed stabilizing amendment.
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Investigation of nano zero-valent iron and zinc interactions with plants in the hydroponics
Švengrová, Denisa ; Vítková, Martina (advisor) ; Petr, Petr (referee)
The diploma thesis deals with the issue of nanoscale Zero-Valent Iron (nZVI) application for the chemically assisted phytostabilization of zinc (Zn) in order to decrease mobility of this element in the environment. The interactions of nZVI and Zn have been investigated during the experiments using tomato plants (Lycopersicon esculentum L.), which were grown in a hydroponic solution. Furthermore, the effects of the interactions on the uptake of water, macronutrients and iron (Fe) have been assessed. The use of nZVI proved to be an effective solution for the stabilization of lower concentrations of Zn (100 micromol) in the root system or for purposes of the assisted phytoremediation. However, the impact of nZVI was not significant high concentrations of Zn (500 micromol). During the application of Zn at increased concentrations a decrease in water uptake has been observed, which can limit the macronutrient uptake. It is necessary to pay attention to further research and study the effects of the nanoparticles application and their interactions in the environment.
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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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Inhibition of Hazardous Compounds in Alumino-Silicate Systems
Koplík, Jan ; Škvára, František (referee) ; Rovnaníková, Pavla (referee) ; Havlica, Jaromír (advisor)
The ability of alumino-silicate systems to immobilize hazardous compounds has been investigated since 1990s. The aim of this work is to develope alumino-silicate system (matrix) based on industrial waste products with ability to immobilize hazardous compounds (heavy metals). This ability of the matrix was confirmed by leaching tests based on the law 294/2005 Sb. Concentration of heavy metals in leachates was determined by ICP-MS method. Alumino-silicate system prepared in this work consists of high-temperature fly ash and blast furnace slag activated by mixture of alkaline activators (hydroxide and water glass). Matrixes were characterized by suitable analytic methods (XRD, SEM, FT-IR, DTA-TGA-EGA). The same analytic methods were used to describe the mechanism of immobilization of selected elements (Ba, Cu, Pb) in prepared systems.
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Preparation of geopolymeric binders for toxic metals inhibition
Jevická, Lucie ; Kalina, Lukáš (referee) ; Šoukal, František (advisor)
This thesis deals with preparation of geopolymeric binders for inhibition of toxic elements, especially arsenic. It studies influence of content progress of different phases in prepared binders on leachability of As during the setting. The aim of this study is to determine the type of inhibition or the cause of leachability of As in prepared binders respectively. Fly ash from high-temperature coal combustion, slag, cement kiln dust and sodium hydroxide solution were used for experimental works. FT-IR, TG-DTA, XRD analyses after different hydration times were used. The leachates were analyzed by ICP-MS. Calorimetric measurements of tested mixtures fresh pastes were implemented to learn about setting process. Hardened binder specimens were tested for bending and compressive strength.
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Comparision of leachability of heavy metals from various types of alkali-activated alumino-silicate systems
Smolková, Miroslava ; Kalina, Lukáš (referee) ; Koplík, Jan (advisor)
The work deals with the determination and comparison of leaching of heavy metals from various types of alkali-activated alumino-silicate systems (high-temperature fly ash, blast furnace slag). Such alkali activated materials are capable in their structure effectively encapsulate heavy metals, toxic chemicals or other contaminants, which can be beneficial for the environment. The aim of this work is to obtain a mixture with acceptable mechanical properties that could be useful for the inhibition of these substances. Leachability was tested according to norm ČSN EN - 12457-4, followed by analysis by ICP-OES. The structure of the matrix was investigated using SEM equipped with EDS.
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