The use of capillary electrophoresis (CE) has been restricted to applications having high sample concentrations because of its low sensitivity caused by small injection volumes and, when ultraviolet (UV) detection is used, the short optical path length. Sensitivity in CE can be improved by using more sensitive detection systems, or by preconcentration techniques which are based on chromatographic and/or electrophoretic principles. One of the promising strategies to improve sensitivity is solid phase extraction (SPE). Solid Phase Extraction utilizes high sample volumes and a variety of complex matrixes to facilitate trace detection. To increase the specificity of the SPE a selective solid phase must be chosen. Immunosorbents, which are a combination of an antibody and a solid support, have proven to be an excellent option because of high selectivity of the antibody. This thesis is an exploratory study of the application of immunosorbent-SPE combined with CE for trace concentration of benzodiazepines. This research describes the immobilization and performance evaluation of an immunosorbent prepared by immobilizing a benzodiazepine-specific antibody on aminopropyl silica. The binding capacity of the immunosorbent, measured as µg of benzodiazepine/ gram of immunosorbent, was 39 ± 10. The long term stability of the prepared immunosorbent has been improved by capping the remaining aminopropyl groups by reaction with acetic anhydride. The capped immunosorbent retained its binding capacity after several uses.

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.

Orientador: Luiz Carlos Vulcano / Coorientador: Marco Antonio Rodrigues Fernandes / Banca: Alexandre Lima de Andrade / Banca: Sheila Canevese Rahal / Resumo: No Brasil, o uso da radioterapia na medicina veterinária ainda é incipiente, com apenas estudos em nível de pesquisa científica e poucos grupos de especialistas dedicados a esse tipo de trabalho. Centros de radioterapia veterinária já existem em outros países como Estados Unidos, França, Canadá, Inglaterra, e até mesmo na Argentina. Os resultados apresentados nestas clínicas são bastante incentivadores e promissores. Muitos dos estudos desenvolvidos na radioterapia veterinária são derivados e facilitadores da implementação na medicina humana. A Faculdade de Medicina Veterinária e Zootecnia (FMVZ) da UNESP de Botucatu foi contemplada com um novo equipamento do tipo acelerador linear, o qual será instalado no setor de radiologia veterinária. Este trabalho apresenta os protocolos clínicos específicos para a radioterapia veterinária usados em alguns dos centros atualmente em funcionamento. Foram desenvolvidos acessórios para imobilização de pequenos animais, visando o posicionamento durante o planejamento tomográfico e a reprodutibilidade dos procedimentos de radioterapia. Para a produção foi utilizado acrílico liso, madeira do tipo Medium Density Fiberboard (MDF), silicone de condensação (Speedex putty) e um catalizador universal (Speedex). Os acessórios produzidos foram a rampa de MDF, as cunhas de posicionamento e o suporte para o molde dentário, os quais, após testados em um animal durante a simulação, demostrou ser eficaz na utilização em pequenos animais e nos procedimentos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Radiation therapy is a medical modality that uses sources of ionizing radiation for the treatment of diseases, and is already a medical procedure well established in human medicine, however, in Brazil, the use of this technique in veterinary medicine is still in its incipient, with only studies in scientific research level and few groups of specialists dedicated to this type of work. Veterinary radiotherapy centers already exist in other countries like USA, France, Canada, England, and even in Argentina. The results presented in these clinics are very incentive and promising. Many of the studies developed in veterinary radiation and enhancers are derived implementation in human medicine. The Scholl of Veterinary Medicine and Animal Science (FMVZ) of UNESP in Botucatu is funding a new linear accelerator will be installed in the veterinary radiology sector. However, it is necessary to establish specific clinical protocols, in addition to produce a routine guide procedures and techniques that are appropriate to the reality of the veterinary clinic. The success of radiation therapy depends on several factors, among which, the daily reliability of the planned treatment field, which requires immobilization of the target volume irradiation. In this respect, due to the peculiarities of handling routine veterinary patients, most procedures should be monitored anesthesia of the animal. To minimize this fact, methods and immobilization accessories were developed in this work for the application in different types of animals undergoing radiotherapy. / Mestre

8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...

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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This research explores various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), observing the increase of surface area and what effect that has on enzyme binding potential. This study also manipulates incubation and reaction conditions and observes what affect that has on activity and stability of β-galactosidase comparing various solid support materials and free enzyme. Nylon-6 fiber membranes were created using the process of electrospinning and were compared with other materials as solid support materials for enzyme binding. The other materials included polyvinylidene fluoride 5 kD nanofiltration dairy membranes, nylon-6 pellets, silica glass beads, and free—dissolved—enzyme. Scanning electron microscopy images exposed the nylon-6 fiber membrane’s large amount of surface area which coordinated with greater enzyme activity as compared to the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate ortho-Nitrophenyl-β-galactoside. NFM had greater maximum enzyme binding potential than the other solid supports. Across pH conditions ranging from 3.5 to 6.0., enzyme activity was maintained on the membrane immobilized samples whereas free enzyme did not maintain activity. Altering storage temperature (4, 22, and 50 °C) affected enzyme stability, the ability of the enzyme to maintain activity over time, of free and polyvinylidene fluoride membrane samples. However, nylon-6 fiber membrane samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above maximum enzyme binding capacity had no significant effect on enzyme stability for membrane immobilized samples. Although, both had lower mean stability than free enzyme by approximately 74% percent. With further development, β-galactosidase immobilized on nylon-6 fiber membranes, or other membranes, could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis—creating products reduced in lactose and increased in sweetness with no “added sugars” requirement for a nutrition label and no enzyme listed as final product ingredient.

In North Dakota, adoption of conservation tillage practices has resulted in an accumulation of crop residue remaining on the soil surface. North Dakota producers receive a nitrogen credit for long-term no-till but due to previous crop residue this credit may not be realistic for providing partial nutrient needs to subsequent crops in a cool environment with a short growing season. Our objectives were to evaluate the N mineralization potential of common crop residues to determine whether crop residue accumulation in no-till systems can provide sufficient nitrogen quantities needed for subsequent crops. Three lab incubation studies were conducted to provide N mineralization insights for individual crop residues, crop residues over several simulated growing seasons, and crop residue in diversified cropping systems. Differences in soil texture, surface application versus incorporation of residue, freeze and thaw cycles and combinations of residues were all factors examined. Results indicated that crop residue decomposition and N release from the residue treatments generally immobilized N but were not significantly different from the bare soil for nearly all studies. The only exception observed was for the forage radish cover crop which showed the potential to improve soil N mineralization in select three-year rotations. Findings of these studies show that most wide C:N ratio crop residues will immobilize soil N in a no-till system under ideal conditions (i.e. moisture, temperature, and residue particle size). These findings suggestion that a fertilizer N credits may require reevaluation and take into consideration soil moisture with validated data to support the fertilizer N credit.

The objective of this work was the development of an efficient adsorbent for irreversible immobilization of heavy metals in contaminated soils. The adsorbent was prepared by pillaring of montmorillonite with silica followed by grafting of a chelate ligand on its surface. Obtained adsorbent was mesoporous with high content of adsorption sites. Its structure was studied by BET adsorption of N2, dynamic light scattering, and scanning electron microscopy. The adsorption capacity of the organoclay was measured by its mixing with contaminated kaolin and soil samples and by analysis of heavy metal contents in leachate. Deionized water and 50% acetic acid were used for leaching of metals from the samples. As it was demonstrated by the experiments, the adsorbent was efficient in immobilization of heavy metals not only in neutral aqueous media but also in the presence of weak acid. As a result, the adsorbent can be used for reduction of heavy metal leaching from contaminated sites.

The objective of this work was the development of an efficient adsorbent for irreversible immobilization of heavy metals in contaminated soils. The adsorbent was prepared by pillaring of montmorillonite with silica followed by grafting of a chelate ligand on its surface. Obtained adsorbent was mesoporous with high content of adsorption sites. Its structure was studied by BET adsorption of N2, dynamic light scattering, and scanning electron microscopy. The adsorption capacity of the organoclay was measured by its mixing with contaminated kaolin and soil samples and by analysis of heavy metal contents in leachate. Deionized water and 50% acetic acid were used for leaching of metals from the samples. As it was demonstrated by the experiments, the adsorbent was efficient in immobilization of heavy metals not only in neutral aqueous media but also in the presence of weak acid. As a result, the adsorbent can be used for reduction of heavy metal leaching from contaminated sites.