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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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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav ; Ledvinová, Jana (advisor) ; Stiborová, Marie (referee) ; Holčapek, Michal (referee)
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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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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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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Hydrogels of Humic Acids - Experimental Model and Application Form
Sedláček, Petr ; Klučáková, Martina (advisor)
The thesis deals with a utilization of hydrogels made of humic acids in both basic and applied research of this valuable natural material. The attention is paid to an interaction between the humic gel and cupric ions as the model heavy metal. The main experimental part focuses on an optimation of simple laboratory diffusion methods which serve as an innovative tool for modeling pollutants’ transport in natural humic environments. Various techniques were used in order to determine a diffusion coefficient of cupric ions in humic gel; the value is closely linked with the studied interaction between solid content of the gel and the diffusing species. Consequently, the diffusivity can be used as a standard parameter for basic reactivity mapping studies concerning humic substances. The final chapter of the thesis deals with a preparation of mixed reversible hydrogels formed by a reaction between humic acids and chitosan. These materials represent a suitable colloidal form for humic acids’ industrial and agricultural applications.
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Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (referee) ; Hermanová, Soňa (advisor)
Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.
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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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