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Magnetic biocomposite materials for removal of significant xenobiotics from water systems
BALDÍKOVÁ, Eva
The theoretical part of this doctoral thesis provides a comprehensive overview on the topic of preparation and subsequent utilization of magnetic derivatives of biological materials for xenobiotic separation from water. Main attention is paid to magnetic modification of waste materials and by-products originating from agricultural and food industry, which represent widely available and low-cost materials, and also to magnetic modification of microbial cells. In addition to the description of magnetic particle preparation and individual developed techniques of magnetic modification, a brief characterization of selected pollutants and a detailed table overview on utilization of magnetically responsive biomaterials for biosorption of organic dyes, heavy metals, pharmaceutical and personal care products together with ubiquitous industrial endocrine disruptors and also of crude oil derivatives is presented. Experimental part of this thesis is focused on the preparation and optimization of new types of magnetic materials. Emphasis is placed on the employment of simple, fast and simultaneously low-cost magnetic modification techniques (e.g., postmagnetization using microwave-synthesized magnetic iron oxides or one-step modification by magnetic fluids). Selected plant materials (barley and rye straw) were chemically modified to significantly (up to five-times) increase the maximum adsorption capacities for tested dyes. All prepared biomaterials exhibited a great magnetic response and simultaneously relatively high adsorption capacity for selected xenobiotics under experimental conditions used. Factors substantially affecting adsorption process, such as pH, initial concentration, incubation time or temperature were also studied. Adsorption equilibrium data were assessed using Langmuir, Freundlich or Sips isotherm models. Experimental data from time dependence study were analyzed by chosen kinetic models, namely the pseudo-first-order and pseudo-second-order ones and by intraparticle diffusion model. Thermodynamic parameters (Gibbs free energy, enthalpy and entropy) describing the nature of adsorption were also included in study.
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Peptide inhibitors immobilized on magnetic particles and Sepharose used for separation of stomach aspartate proteinases
Rajčanová, Michaela ; Kučerová, Zdenka (advisor) ; Fusek, Martin (referee) ; Pacáková, Věra (referee)
IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...
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Magteic particles and their applications in biotechnology
Knápková, Monika ; Konečná, Jana (referee) ; Trachtová, Štěpánka (advisor)
The thesis is focused on the magnetic particles which are used in several biotechnological applications. The theoretical part deals with the specific properties of these nanoparticles and materials of which the nanoparticles can be made. There are also mentioned some of the biotechnological applications of magnetic particles. During the experimental part, selected types of magnetic particles were used to isolate nucleic acid. The quality of the isolated DNA with respect to purity was evaluated using the polymerase chain reaction and its modifications. High resolution analysis (HRM analysis) was also used to verify the quality of the isolated DNA and to resolution Lactobacillus casei and Lactobacillus rhamnosus. DNA isolation using magnetic carriers was successful. Commercially available MPG microcarriers and magnetic microparticles Fkol 77ox were the most suitable. In terms of purity magnetic nanoparticles F79/L3-PLL were the most suitable for the DNA isolation. The resolution of bacterial strains of Lactobacillus casei and Lactobacillus rhamnosus was not successful.
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Characterization of enzyme reactor with immobilized alkaline phosphatase
Plecitá, Denisa ; Kučerová, Zdenka (advisor) ; Miarková, Eva (referee)
Phosphorylation is one of the most common of all post-translational modifications of proteins and has been found in nearly all cellular processes. Abnormal phosphorylation is associated with many serious human diseases. One of the approaches used for the identification of protein phosphorylation sites is based on the application phosphatases and the comparison of MS analysis of samples before and after the sample treatment with the enzyme. The use of phosphatase immobilized to magnetic carriers is advantageous in comparison with the application of soluble enzyme: e.g. easy manipulation of samples, an increase of enzyme stability and a possibility of repeated use of immobilized enzyme. Investigation of properties of enzyme reactor - bovine alkaline phosphatase from intestinal mucosa immobilized to magnetic particles is a subject of this Bachelor Thesis. The enzyme was coupled to cellulose magnetic particles after activation with divinyl sulfone via the protein free amino groups. p-Nitrophenylphosphate was used as a substrate for the phosphatase activity determination. The effect of different conditions on the activity of soluble and immobilized forms of alkaline phosphatase was compared: the effect of pH and Mg2+ ions, storage stability and thermostability and possibility of repeated use of the...
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Immobilization of protease V8 on magnetic particles for application to proteolytic cleavage of pepsin A
Čepa, Adam ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
This thesis is part of a long-standing research in the field of diagnosis of the stomach diseases, which is based on the gastric enzyme pepsin A mapping. It was found that a phosphorylation in the primary structure of this enzyme may serve as a marker of incipient stage of carcinogenesis. This thesis is focused on the immobilization of protease V8 isolated from microorganism Stafylococcus aureus to magnetic agarose beads. Protease V8 is a promising candidate for producing peptide maps of pepsin A. The influence of pH, temperature and reaction time on the enzyme to activity has been studied and the optimal conditions for hydrolytic catalysis of formation of peptide fragments of pepsin A.
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Application of magnetic particles for isolation and purification of DNA
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With a development of molecular biology methods it is an increasing interest in new procedures of DNA isolation of high quality. DNA isolation is performed on crude cell lysates by many techniques e.g. phenol extraction, salting out or adsorption on solid phase. Classical DNA isolation, such as phenol extraction is quite complicated and time consuming. New alternative methods of DNA isolation was development using reverse immobilizing DNA to a solid phase. Widespread is the use of the magnetic particles as carriers, which allow the isolation of DNA in high quality directly from crude cells lysates of complex samples. The current method of DNA adsorption onto the surface of magnetic particles does not provided sufficiently pure DNA for analysis of some comlex samples (e.g. food). Some inhibitors of the polymerase chain reaction (PCR) are apparently adsorbed onto the tube wall and the next step of DNA elution leads to their release into the solution and cpnsequent negative effect on quality of DNA (e.g. decreasing of PCR amplification). The principle of the developed procedure is design a device, which utilizes transfer of magnetic particles by paramagnetic newddle from one to another Eppendorf tube, in which further processing of the sample extends. Transfer of magnetic particles with DNA using needle prevents transmission of contaminating impurities. The proposed device allows to realize above-mentioned procedure. The functionality of the device being tested in the isolation of plasmid pUC19 DNA from crude lysates of E. Coli JM 109 (pUC19).
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The application of magnetic particle for DNA isolation from selekted probiotic products for children
Vozárová, Petra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In the food industry, it is important to correctly identify the species of bacteria and thier properties so that they can be used as a probiotic in dietary supplements. This is performed using DNA diagnostics. In the experimental part, the DNA from four probiotic dietary supplements for children was isolated. Magnetic particles P(HEMA-co-GMA) were tested for isolation. Isolated DNA was amplified by PCR and the presence of DNA of genus Lactobacillus, Bifidobacterium and Bacillus was demonstrated in the products according to the data declared by the manufacturer. The presence of species L.acidophilus, B.animalis in accordance with the data on the product has been demonstrated by PCR with species specific primers. Using PCR, the presence of L.casei, which was declared by the manufacturer, has not been proven in one product at given experimental conditions.
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The application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
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The use of magnetic particles for DNA isolation from selected spices
Gaňová, Martina ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of DNA from plant tissue of the required quality is very complicated, especially because of the presence of substances that can interfere during amplification of DNA. These substances are mainly polyphenols, polysaccharides, proteins and various dyes. The chemical diversity of such materials can have a significant effect on the yield and quality of DNA using one isolation procedure. The main aim of the work was to evaluate the use of microisolation protocol for related matrices to the quality of the isolated DNA as well as the evaluation of the effect of inhibitors isolated with the nucleic acid to the amplification in the PCR. DNA was isolated from dried paprika (Capsicum annuum). In the first step, the samples were homogenized using a lysis reagent with cetyltrimethylammonium bromide. Subsequently, the DNA was purified by reversible adsorption on magnetic particles. It was tested six different modified particles. The concentration and purity of the obtained DNA was determined by spectrophotometry measuring the absorbance of the DNA solution in TE buffer. The quality of the DNA was confirmed by amplification in PCR. For the PCR were used primers specific for plant ribosomal DNA (rDNA). The presence of PCR products was detected by agarose gel electrophoresis. It was found out that used microassay is suitable for isolating of the DNA of the corresponding purity that is suitable for the genetic analysis by PCR. The differences were found between the magnetic particles that were tested for DNA isolation.
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Biofyzikální studium malých RNA
Šmerková, Kristýna
Thanks to the prove of connection between the aberrant occurrence of small RNA and various diseases and their potential in diagnostics and treatment led to discovery of new methods and materials facilitating their detection and targeted transport during gene therapy. This work summarizes present knowledge about chosen groups of small RNA, their significance in medical science and the possibilities of their detection. This work primarily concentrates on combination of magnetic separation with electrochemical detection. Magnetic particles (MPs) with different surface modifications were used for isolation. Non-specific isolation was carried out using silanol-coated MPs; streptavidin-coated MPs modified with specific biotinylated probe were used for specific separation. Square wave voltammetry (SWV) was used as a very sensitive electrochemical detection method. Optimized method based on specific magnetic separation with SWV was able to reach nanomolar detection limit (4 nM) with microRNA. The method was applied on human embryonic cells for specific isolation and detection of miR-124. The CdTe quantum dots (QDs) were studied as a nanomaterial tool for nucleic acid detection. The QDs were modified with streptavidin for their bioconjugation with biotinylated molecules were used. Interaction of QDs with nucleic acids was studied using capillary electrophoresis.
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