Dissertations / Theses on the topic 'Biological reagents'

Immunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

No presente trabalho são apresentadas análises de amostras de proteínas totais e aminoácidos empregando-se os reagentes p-benzoquinona e tetraamin cobre (II). No caso do reagente p-benzoquinona, as análises foram feitas através de um sistema inédito em fluxo, no qual a mistura reacional passava através de um reator composto de um tubo capilar de aço inoxidável aquecido num banho de glicerina. Os derivados formados passavam através de uma cela de fluxo acoplada a um espectrofotômetro, onde eram feitas as leituras das absorbâncias. Este método se mostrou mais rápido em relação àquele feito em banho-maria. A utilização de um sistema de análise por injeção em fluxo (FIA) com o reagente tetraamin cobre (II), permitiu que as análises fossem feitas de forma rápida, a baixo custo e com a vantagem de não necessitarem de aquecimento, como no caso da p-benzoquinona. Os resultados das análises de amostras de albumina humana obtidos em ambos os métodos acima apresentaram boa concordância quando comparados àqueles obtidos pelo método Kjeldahl para as mesmas amostras.
This work describes two methods for analysis of total proteins and aminoacids using both p-benzoquinone (PBQ) and tetraamin copper (II) reagents. With the p-benzoquinone reagent the method was performed by an original flow system made with stainless steel capillary tubing, conveniently heated into a glycerin bath, where the reactional mixture travels in its way to the detector. The derivate products are carried out to a flow cell adjusted to a spectrophotometer where the absorbances are measured. This method was efficient, with a fair cost, and providing results very faster than in the batch mode of operation. The second method, using the tetraamin copper (II) reagent, was performed by a common flow injection system (FIA). When compared with the PBQ method, the FIA tetramin copper (II) method was also rapid, efficient, with a fair cost and with the great advantage of simplicity, once it is performed at room temperature. The results obtained with human albumine samples using both methods are in agreement with those ones obtained by the Kjeldahl method for the same samples.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Ebselen is a seleno compound whose antioxidant properties have been attributed to its thiol-peroxidase and thioredoxin-like activity: it decomposes peroxides at the expense of reduced thiols. However, the excessive oxidation of thiols can be potentially toxic when it is not associated with peroxides degradation. Thus, this work investigated if LDH can be a possible in vitro target to toxicity of ebselen in comparison with diphenyl diselenide and diphenyl ditelluride, two antioxidant organochalcogens that can easily interact with thiol in exchange reaction. LDH inhibition was tested in homogenates from rat liver and heart, and in purified LDH from rabbit muscle. Ebselen was the most potent inhibitor of LDH. A maximal inhibitory effect was obtained at 2 μM to LDH purified and at 20 μM to LDH from heart and liver homogenates. Moreover, diphenyl diselenide followed by diphenyl ditelluride also presented a significant inhibitory effect on LDH activity. In addition, we observe that DTT was able to revert the inhibition of LDH induced by all compounds tested, confirming the involvement of essential thiol groups on LDH inhibition by organocalchogens. In conclusion, our results show that liver and heart LDH may be a possible target for toxicity of organochalcogens at relative low concentrations. However, the protection afforded by substrates may hide this potential molecular target of organochalcogenides. Our results also indicate that the use of LDH as a marker of cell viability may be biased by a direct inhibitory effect of ebselen or other chalcogenides on LDH, resulting in false protection in in vitro system.
O Ebselen é um composto de selênio o qual tem suas propriedades antioxidantes atribuídas à sua atividade mimética da tiorredoxina e tiolperoxidase: ele decompõe peróxidos à custa de tióis reduzidos. Contudo, a oxidação excessiva de tióis pode ser potencialmente tóxica quando não esta associada com a degradação de peróxidos. Assim, este trabalho investiga se a LDH pode ser um possível alvo à toxicidade do ebselen, em comparação com o disseleneto de difenila e o ditelureto de difenila, dois organocalcogênios antioxidantes que podem facilmente interagir com grupos tiol. A inibição da LDH foi testada em homogeneizados de fígado e coração de ratos, e em LDH purificada de músculo de coelhos. O Ebselen foi o mais potente inibidor da LDH. O seu efeito inibitório máximo foi obtido com 2 μM para a LDH purificada e 20 μM para a LDH de homogeneizados de fígado e coração de ratos. Além disso, o disseleneto de difenila, seguido do ditelureto de difenila, também apresentaram efeito inibitório significativo sobre a atividade da LDH. Em adição, observou-se que o DTT foi capaz de reverter a inibição da LDH induzida pelos compostos testados, confirmando o envolvimento de grupos tiol essenciais da LDH no processo de inibição pelos organocalcogênios. Em conclusão, estes resultados mostram que a LDH de fígado e coração pode ser um possível alvo para a toxicologia de organocalcogênios a doses relativamente baixas. Nossos resultados também indicam que o uso da LDH como um marcador de viabilidade celular pode ser mascarada por um efeito inibitório direto do ebselen, ou outros calcogênios, sobre a LDH, resultando em uma falsa proteção em um sistema in vitro.

This dissertation is about treatment of the nonbiodegradable organic content of landfill leachate by chemical oxidation combined with biological treatment. It is divided into three parts. In the first part, ferrate was compared to Fenton's reagent for the purpose of removing non-biodegradable organic compounds from mature leachate. Oxidation conditions (time, pH, and dose) were optimized to yield maximum organic removal using two leachate samples from 20 and 12-year old solid waste cells. Results from this research demonstrated that ferrate and Fenton's reagent had similar optimum pH ranges (3-5), but different organic removal capacities, ranging from 54 to 79 % of initial leachate organic contents. An advantage of ferrate was that it was relatively effective over a wide pH range (Fenton's reagent lost its reactivity outside optimum pH range). Advantages associated with Fenton's reagent include a higher organic removal capacity, production of more oxidized organic compounds (measured as chemical oxygen demand/dissolved organic carbon), and production of more biodegradable byproducts (measured as 5-day biochemical oxygen demand/chemical oxygen demand). Finally, both treatments were found to oxidize larger molecules (>1000 dalton) and produce smaller molecules, as indicated by an increase in smaller molecule contribution to organic carbon. In part two, effects of Fenton's reagent treatment on biodegradability of three landfill leachates collected from a Florida landfill were evaluated using biochemical oxygen demand (BOD), biochemical methane potential (BMP), and tertamethylammonium hydroxide (TMAH) thermochemolysis gas chromatography/mass spectrometry (GC/MS). The hypothesis was that Fenton's reagent will remove refractory compounds that inhibit biodegradation and will produce smaller, more biodegradable organic molecules which will result in an increase in BOD and BMP values. Both BOD and BMP results demonstrated that Fenton's reagent treatment did not convert mature leachate to biodegradable leachate, as indicated by a low BOD5 expressed as C /dissolved organic carbon (DOC) ratio of almost 0.15 in treated samples and a low net methane production / theoretical methane potential (less than 0.15). Ultimate BOD only slightly increased. However the first-order BOD reaction rate increased by more than five fold, suggesting that Fenton's reagent removed refractory and inhibitory compounds. BMP results demonstrated that the ratio of CO2/CH4 produced during anaerobic biodegradation did not increase in treated leachate (compared to untreated), indicating that small biodegradable organic acids produced by oxidation were removed by coagulation promoted by Fenton's reagent. Finally, the TMAH thermochemolysis results showed that several of the refractory and inhibitory compounds were detected fewer times in treated samples and that carboxylic acids did not appear in treated samples. In the third part of this dissertation the application of flushing/Fenton's reagent oxidation to produce sustainable solid waste cells was evaluated. A treatment similar to pump and treat process utilizing Fenton's reagent on-site treated leachate combined with in-situ aeration was proposed. Treated leachate would be recycled to the landfill cell flushes releasable nonbiodegradable carbon from the cell and oxidizes it externally. This technique was demonstrated to have treatment cost and time benefits over other alternatives for producing completely stable solid waste cells such as anaerobic flushing and biological and/or mechanical pretreatment of solid waste (used in the EU).
Ph.D.
Department of Civil and Environmental Engineering
Engineering and Computer Science
Environmental Engineering

Biological reagents that recognize target molecules with high affinity and specificity are widely used as capture agents, diagnostic reagents, and therapeutics. Through their ability to adopt structures that confer binding affinity for a target, aptamers represent one major class of such reagents. However, their use is limited by the general inability of current selection methods to reliably discover high-quality aptamers. Inefficiencies in their selection are due in part to a lack of fundamental understanding of the mechanisms underpinning each step in the screening process. This thesis reports on a series of studies conducted to define the factors and mechanisms currently limiting aptamer selections. That knowledge is then used to create highly effective strategies and technologies for ameliorating each limitation affecting their selection. The resulting collection of improvements is integrated into a novel selection workflow termed “Hi-Fi SELEX”. Those improvements include i) application of a novel “competent library” that eliminates fixed-region interference effects during selection, ii) development of effective chemistries to optimally retain desirable library members, iii) invention of simple methods to accurately quantify retained library diversity and mean binding affinity after each selection round, and iv) development of emulsion PCR methods to eliminate generation of amplification artifacts and v) achieve stoichiometric recovery of the desired single-stranded aptamer library. The resulting discovery platform greatly improves the reliability and speed in which useful panels of lead aptamers against several clinically-relevant targets are discovered. Following initial selection of candidate aptamers based on binding affinity, further screening is typically required, in part to ensure target-specific binding – a performance need shared by antibodies selected against specific targets. However, moderate to high-throughput methods to efficiently screen panels of candidates for binding specificity are lacking. A new technology enabling label-free specificity screening of antibody or aptamer populations at suitable throughputs was therefore established at the proof-of-concept level. The novel microfluidic SPRi arrays described permit multiplexed detection of lead candidates by quantifying both equilibrium binding constants and binding kinetics for each interaction in an element-addressable fashion. The technology offers the ability to independently interrogate candidate affinity reagents and then recover those samples for downstream analysis.
Applied Science, Faculty of
Graduate

A Proteína Verde Fluorescente recombinante, GFPuv, é um sistema marcador atrativo pois, sua presença pode ser visualizada através da intensidade de fluorescência emitida, sem o uso de substratos ou meios complexos. Sendo uma molécula estável à presença de substâncias orgânicas, temperaturas acima de 70°C e ampla faixa de pH, é um potencial Indicador Biológico (IH) para diversas aplicações. A estabilidade térmica da GFPuv, foi avaliada pela medida da perda de intensidade de fluorescência, expressa em valores D (min), tempo de exposição necessário para redução de 90% da intensidade de fluorescência inicial da GFPuv. GFPuv (3,5-9,0 µg/mL), expressa por E. coli e isolada por extração de Partição em Três Fases (TPP) e purificação por Cromatografia de Interação Hidrofóbica (IDC), foi diluída nas soluções parenterais preparadas em tampão (10 mM cada: Tris-EDTA, pH 8; Fosfato, pH 6 e 7, e Acetato, pH 5) e em água para injeção, WFI; pH = 6,70±0,40), e expostas a temperaturas de 25°C e ao intervalo entre 80°C e 100°C. A 95°C, os valores D para a GFPuv em soluções de 1,5% a 50% de glicose variaram de: (i) 1,63 (±0,23) min em acetato pH 5; (ii) 2,64 ± 0,26 min em WFI; (iii) 2,50 ± 0,18 min em fosfato pH 6; (iv) 3,24 ± 0,28 min em fosfato pH 7 e, (v) 2,89 ± 0,44 min em Tris-EDTA pH 8. Cloreto de sódio associado aos tampões proporcionou influência positiva na estabilidade da GFPuv, sendo que em soluções de Tris-EDTA, a adição de 15-20% de NaCl dobrou a estabilidade térmica da GFPuv (valores D de 65,79 min e 18,12 min a 80 °C e 85°C) em relação à solução sem cloreto de sódio. Nos processos de esterilização por óxido de etileno (45°C-60°C), a GFPuv pode ser utilizada como IB para monitorar a distribuição de gás dentro da câmara, pois, apresentou variação na concentração remanescente de até 80%, após processamento, estabelecendo áreas distintas dentro da câmara. No tratamento em autoclave, a GFPuv em solução apresentou resistência térmica em solução de fosfato pH 7,0 (valor F = 2,53 min (± 0,12)). Quando expressa por esporos de Bacillus subtilis, a intensidade de fluorescência emitida por esporos sobreviventes se manteve. A estabilidade térmica da GFPuv atestou sua potencialidade como indicador biológico fluorescente da garantia da eficácia de tratamento de soluções e materiais expostos ao calor.
The recombinant Green Fluorescent Protein, GFPuv is an attractive system marker due to its ability to emit fluorescence when exposed to ultraviolet light, without use of substrates or complex environment. Being a stable molecule even in the presence of organic substances, temperatures above 70°C and wide range of pH, it is a potential Biological Indicator, BI, for many applications, including thermal processes. GFPuv thermal stability was evaluated by the loss of fluorescence intensity expressed in decimal reduction time (D-value, min), the exposure time required to reduce 90% of the GFPuv initial fluorescence intensity. GFPuv (3.5-9.0 µg/mL), expressed by E. coli and isolated by Three Phases Partitioning, TPP extraction with Hidrophobic Interaction Chromatography, HIC, was diluted in buffered solutions (each 10 mM: Tris-EDTA, pH 8; phosphate, pH 6 and 7, and acetate, pH 5) and in water for injection, WFI; pH = 6.70 (± 0.40), and exposed to temperatures of 25°C and between 80°C and 95°C. At 95°C, the D-value for GFPuv in 1.5%-50% glucose, ranged from: (i) 1.63 ± 0.23 min in acetate pH 5; (ii) 2.64 ± 0.26 min in WFI; (iii) 2.50 ± 0.18 min in phosphate, pH 6; (iv) 3.24 ± 0.28 min in phosphate, pH 7, (v) 2.89 ± 0.44 min in Tris-EDTA, pH 8. Sodium cloride provided a positive influence over GFPuv stability. In Tris-EDTA solutions, the addition of 15% and 20% of NaCl doubled the thermal stability of GFPuv (D = 65.79 min and D = 18.12 min at 80°C, and 85°C, respectively, in relation to the solutions without NaCl. For ethylene oxide sterilization processes (45°C-60°C), GFPuv can be used as biological indicator to monitor gas distribution into the chamber. After processing, the protein concentration varied by 80%, showing distinct areas into the chamber. In autoclave, GFPuv in solution showed thermal resistance in phosphate pH 7.0 solution (F-value = 2.53 (± 0.12) min. When expressed by Bacillus subtilis spores, the fluorescence intensity was kept constant after thermal processing. The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam.

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Molecular Biology shown to be an effective tool in forensic laboratories for the ability to identify an individual from minute amounts of biological samples such as blood, bones, semen, hair, teeth, nails, spittle, urine and other biological fluids recovered from the crime scene. One of the main biological evidence found at a crime scene are traces of bloodstains. This study aimed to analyze the molecular profiles of biological samples exposed to the chemiluminescent reagent luminol based on different storage times (48 hours and 30 days). With the measurement of all samples can be inferred that in the first 48 hours of storage, there was obtained DNA and varying concentrations after application of the chemiluminescent reagent. The samples were amplified by PCR using a multiplex system AmpFlSTR ® Select NGM and capillary electrophoresis. Molecular profiles were obtained complete and incomplete denoting specificity as the quality and quantity of the sample analyzed. The selection of molecular markers mini-STRs greatly contributed to the success of these profiles, allowing the study and analysis of degraded material. Our data suggest that the degradation of the DNA molecule exposed to the chemiluminescent reagent was higher in the samples compared to those containing diluted whole blood impregnation. Therefore, analyzing the bloodstains samples exposed to luminol in shorter storage provide molecular profiles compatible to clash samples.
A Biologia Molecular mostrou-se uma ferramenta efetiva nos laboratórios forenses pela capacidade de identificar um indivíduo a partir de quantidades ínfimas de amostras biológicas como sangue, ossos, sêmen, cabelo, dentes, unhas, saliva, urina, entre outros fluidos biológicos recuperados no local do crime. Uma das principais evidências biológicas encontradas em local de crime são vestígios de substâncias hematóides. Esta pesquisa visou a análise de perfis moleculares de amostras biológicas expostas ao reagente quimioluminescente a base de luminol em diferentes tempos de armazenamento (48 horas e 30 dias). Com a quantificação de todas as amostras pode-se inferir que nas primeiras 48 horas de armazenamento, obtiveram-se concentrações variáveis de DNA e após a aplicação do reagente quimioluminescente. As amostras foram amplificadas por PCR utilizando um sistema multiplex AmpFlSTR® NGM SElect e eletroforese capilar. Foram obtidos perfis moleculares completos e incompletos denotando inespecificidade conforme a qualidade e quantidade da amostra analisada. A seleção dos 17 marcadores moleculares mini-STRs em muito contribuiu para o sucesso desses perfis, permitindo estudo e análise de material degradado. Os dados indicam que a degradação da molécula de DNA exposta ao reagente quimioluminescente foi maior nas amostras diluídas em relação àquelas contendo impregnação com sangue total. A análise estatística das amostras com e sem exposição ao reagente quimioluminescente comparado ao grupo controle e em diferentes concentrações, mostrou não haver diferença estatísticamente significativa entre os valores quantificados obtidos. Ainda assim, analisar as amostras hematóides expostas ao luminol em menor tempo de armazenamento proporcionarão perfis moleculares compatíveis ao confronto de amostras.

Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a class of natural products obtained from various actinomycetes which have both anti-tumor and antibiotic activities and can bind to specific sequences of DNA. PBD-dilactam was initially produced using isatoic anhydride and (L)-proline which was then converted to the PBD-thiolactam using Lawesson's reagent. Reaction of thiolactam with hydrazine in ethanol afforded PBD-11-hydrazinyl. Condensation of 11-hydrazinyl PBD with aldehydes possessing various substitutions was performed to obtain (S,E)-11-[2-(arylmethylene) hydrazono] pyrrolo [2,1-c] [1,4] benzodiazepine derivatives. 1HNMR, 13C-NMR, DEPT, IR, GC-MS and X-ray crystallography were used for the characterization. Inhibition activity of the products were carried out using TEM-1, AmpC and P99 β-lactamases. A minimal inhibition growth of 25% was observed for one of the selected PBDs on cancer cell line. A promising result was observed on preliminary cannabinoid binding activity test on one of the compounds.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
This study aimed to evaluate at bench-scale the removal of microcystin-LR from water for public supply using Fenton's reagent, coagulation, flocculation, sedimentation and filtration by columns of granular activated carbon (GAC). The water used in this study was prepared by adding 20 mL of microcystin-LR extract (obtained from a pure culture of Microcystis aeruginosa after three consecutive freeze / thaw cycles), in 1L of untreated water from the Acauã reservoir and which corresponded to a concentration of microcystin-LR of aproximately 19 µg.L . The experiments were conducted in three stages. The first step was to determine the best conditions for coagulation obtained via diagrams of coagulation based on remaining turbidity and apparent colour after treatment with Fenton's reagent at concentrations between 5 mg.L-1 and 100 mg.L-1 f eSO4.7H2O and 1.83 mg.L-1 to 36.70 mg.L of H2O and pH varying between 3 and 9 at a settling time of 5 min. In the second stage the fastest sedimentation time was determined after coagulation based on the control parameters of remaining turbidity, apparent colour and microcystin-LR concentration. The third stage evaluated the adsorption of microcystin-LR in columns of GAC (particle size between 1.40 mm and 0.42 mm) after the conditions defined in steps I and II. The GAC columns were constructed from PVC tubing with an internal diameter of 21 mm and a functional height of GAC of 15 cm and 20 cm, corresponding to two different contact times. The fixed flow rate for each GAC column was 2 L.h2-1. The optimum conditions for coagulation were 15 mg.L FeSO4.7H2O and 5.5 g.L-1 H2O, at pH 8.4 and coagulation and sedimentation times of 15 min. After coagulation, flocculation and sedimentation turbidity decreased from 5.8 to 3.0 uT, apparent color of 115 uH to 81 uH and a reduction in microcystinLR concentration from 18.52 µg.L2-1 to 9.59 µg.L-1, with percentage removals of 48%, 30% and 48%, respectively. Break-through occurred in the shorter length column of GAC with the shorter contact time (CC1) after 2 hours of operation, resulting in a smaller q e (1.85 μg.g-1) and higher usage rate (3.82 g.L-1) compared o the GAC column with the greater contact time (CC2), with a break-through after 6 hours operation. Thus the longer GAC column (CC2) gave a better performance both in terms of q e (4.15 μg.g-1) and rate of use (1.70 g.L-1), ensuring effluent oncentration below the maximum allowable value of 1 µg.L-1 required by Ordinance 2914/11, Ministry of Health, with a longer operational life and an economy in GAC usage.
Este trabalho teve como objetivo avaliar em escala de bancada a remoção de microcistina-LR de água destinada ao abastecimento público utilizando coagulação com reagente de Fenton, floculação, decantação e filtração seguido de colunas de carvão ativado granular (CAG). A água de estudo foi preparada pela adição de 20 mL de extrato de microcistina-LR (após congelamento/descongelamento por três vezes consecutivas da cultura pura de Microcystis aeruginosa) em 1 L de água bruta do reservatório de Acauã, que correspondeu a concentração de microcistina-LR em torno de 19 μg.L-1 . O experimento foi realizado em três etapas. Na primeira etapa foi definida a melhor condição de coagulação através de diagramas de coagulação para turbidez remanescente e cor aparente remanescente com concentração de reagente de Fenton entre 5 mg.L-1 - 100 mg.L-1 de FeSO4.7H2O e 1,83 mg.L de H2O para pH de coagulação entre 3 e 9 com tempo de sedimentação de 5 min. Na segunda etapa foi definido o melhor tempo de sedimentação conforme as condições de coagulação estabelecidas na etapa I e os parâmetros de controle foram turbidez remanescente, cor aparente remanescente e microcistina-LR remanescente. Na terceira etapa foi avaliada a adsorção da microcistina-LR em colunas de CAG (granulometria entre 1,40 mm e 0,42 mm) utilizando as condições definidas nas etapas I e II. As colunas de CAG foram construídas com tubos de PVC com diâmetro interno de 21 mm e altura útil de CAG de 15 cm e 20 cm, correspondendo a dois tempos de contato distintos. A vazão fixada para cada coluna de CAG foi de 2 L.h2-1-1. A melhor condição de coagulação foi 15 mg.L de FeSO4.7H2O; 5,5 mg.L-1 de H2O , pH de coagulação de 8,4 e tempo de sedimentação de 15 min. Após a coagulação, floculação e sedimentação a turbidez reduziu de 5,8 uT a 3,0 uT, cor aparente de 115 uH a 81 uH e concentração de microcistina-LR de 18,52 µg.L-12 a 9,59 µg.L-1, com percentuais de remoção de 48%, 30% e 48%, respectivamente. O transpasse na coluna de CAG de menor tempo de contato (CC1) ocorreu após 2 horas de funcionamento do sistema, refletindo em menor qe (1,85 μg.g-) e maior taxa de uso (3,82 g.L-1) quando comparada a coluna de CAG de maior tempo de contato (CC2), que ocorreu o transpasse após 6 horas de funcionamento do sistema, a qual apresentou melhor desempenho tanto em relação ao qe (4,15 g.g-1) como em relação a taxa de uso (1,70 g.L), garantindo efluente com concentração inferior ao valor máximo permitido de 1 µg.L exigido pela Portaria 2914/11 do Ministério da Saúde por mais tempo e utilizando uma menor quantidade de CAG. - 36,70 mg.L

Orientador: Jose Roberto Guimaraes, Pedro Sergio Fadini
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil
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Resumo: Nesse trabalho foram realizados ensaios de tratabilidade de soluções e efluentes contendo formol conjugando-se processos físico-químicos com biológicos. Dentre os vários processos, o UV/H2O2 foi o que apresentou a melhor eficácia no tratamento do efluente do Laboratório de Anatomia/IB-UNICAMP. A redução dos teores de CH2O, COD, DQO e DBO, foram 91, 48, 46 e 53 % para o UV/H2O2 e, 94, 38, 38 e 43 % para o Foto-Fenton, respectivamente, ao final de 420 minutos de ensaio. Para uma solução de formol com concentração inicial de 12.000 mg L-1, as reduções de COD foram de 65 e 61 %, para Foto-Fenton e UV/H2O2, respectivamente, ao final de 390 minutos de ensaio. Para soluções com 400 mg L-1 em formol as redu?es em COD foram 65 e 98 % nos processos Foto-Fenton e UV/H2O2, respectivamente, em ensaios com 120 minutos de duração. O sistema Lodo Ativado por Batelada convencional (?c 7 dias) apresentou reduções nos valores de COD, DQO e DBO de 88, 83 e 96 % e 88, 86 e 98 % para sistemas com aeração prolongada (?c 20 dias), alimentados com efluente tratado por POA
Abstract: In this work assays of treatment of solutions had been carried through and effluent I contend formaldehyde conjugating processes physicist-chemistries with biological. Amongst the some processes, the UV/H2O2 was what it presented the best efficiency in the treatment of the effluent one of the "Laboratorio de Anatomia/IB-UNICAMP. The reduction of the values of the CH2O, COD, DQO and DBO, had been 91, 48, 46 and 53 % for the UV/H2O2 and, 94, 38, 38 and 43 % for Photo- Fenton, in the 420 minutes of assay, respectively. Solution of formaldehyde with initial concentration of 12.000 mg L-1, the TOC reductions had been of 65 and 61 %, for Photo-Fenton and UV/H2O2, respectively, for 390 minutes of assay. For solutions with 400 mg L-1 in formaldehyde the reductions in TOC had been 65 and 98 % in processes Photo-Fenton and UV/H2O2, respectively, for 120 minutes of assay. Systems Activated Sludge had presented reductions in the values of TOC, COD and BOD of 88, 83 and 96 % for conventional systems (?c 7 days) and 88, 86 and 98 % for systems with drawn out aeration (?c 20 days), using effluent treated by POA
Doutorado
Saneamento e Ambiente
Doutor em Engenharia Civil

A metodologia genético-molecular destaca-se como uma técnica apurada para os processos de identificação humana e, dentre as fontes de evidência biológica, o uso de elementos dentais é de grande interesse. A manutenção da integridade do material enviado ao laboratório é imprescindível para o sucesso dos resultados obtidos e uma das principais dificuldades encontradas é com relação ao armazenamento da amostra, que geralmente é realizado em baixas temperaturas. O presente trabalho avaliou a eficácia do produto Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) na estabilização do DNA extraído de tecidos dentais humanos armazenados em diferentes condições. Para tal, foram utilizados 165 elementos dentais, os quais foram distribuídos em dois grupos distintos: dente íntegro e tecido pulpar dental isolado. As amostras foram armazenadas com ou sem a utilização do referido produto, variando o período de tempo (1, 7, 30 e 180 dias) e temperatura (ambiente e refrigeração). Além desses grupos, foi formado um grupo controle positivo composto por cinco elementos dentais armazenados a -20ºC durante 180 dias. Após o armazenamento foi realizada extração do DNA, eletroforese em gel de agarose, quantificação do DNA genômico por PCR Tempo Real e análise de fragmentos de 37 amostras. Os fragmentos de 32 amostras que representavam cada condição possível e as cinco amostras do grupo controle positivo foram analisados, a fim de verificar quatro marcadores pré-selecionados. O gel de agarose mostrou evidências da presença de DNA genômico. Os valores da quantificação foram analisados estatisticamente pelos testes Kruscal-Wallis e Mann-Whitney. Os resultados mostraram valores que variaram de 0,01 a 10246,88ng/L de DNA. Houve diminuição da concentração de DNA nas amostras de dente armazenadas em temperatura ambiente por 30 e 180 dias em relação às que ficaram armazenadas por 1 e 7 dias. Além do fator tempo, a temperatura também influenciou na concentração de DNA, sendo maior nos dentes que ficaram por 30 dias e na polpa dental mantida por 180 dias, quando refrigerados. Em relação à utilização do produto Allprotect Tissue Reagent® (Qiagen, Hilden, Germany), o mesmo mostrou diferença significativa na estabilização dos dentes que foram armazenados em temperatura ambiente durante 30 e 180 dias. A análise de fragmentos foi possível nas 37 amostras selecionadas, independente da quantidade de DNA, confirmando a importância das reações de amplificação e da análise de STR utilizando método automático. Conclui-se que a utilização do produto Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) mostrou diferença significativa na estabilização do DNA das amostras de dentes íntegros armazenadas em temperatura ambiente durante 30 e 180 dias, enquanto que nas demais condições testadas, os resultados não evidenciaram justificativas para o uso do produto.
The genetic-molecular methodology stands out as an accurate technique for human identification process and among the sources of biological evidence, the use of teeth is of great interest in Forensic Dentistry. Maintaining integrity of the material sent to laboratory is essential for success of the analysis, and one of the main difficulties is related to sample storage, which is usually carried out at low temperatures. This study evaluated the effectiveness of the Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) in stabilizing DNA extracted from human dental tissues stored under different conditions. In this study were used 165 teeth, distributed in two groups: intact teeth and isolated pulp tissue. The samples were stored with or without the product and varying the storage time (1, 7, 30 and 180 days) and temperature (room temperature and under refrigeration). In addition to these groups, was formed a positive control group, composed by five teeth, which was stored at -20ºC for 180 days. After storage, DNA extraction, electrophoresis on agarose gel and genomic DNA quantification by Real-Time PCR and fragments of 37 samples were performed. The fragments of 32 samples representing every possible condition and five positive control group samples were analyzed to verify four pre-selected markers. The agarose gel showed evidences of genomic DNA presence. Quantification results were statistically analyzed with the tests Kruscal-Wallis and Mann-Whitney. Quantification results showed values ranging from 0.01 to 10,246.88 ng/L of DNA. There was a decrease in DNA concentration in stored tooth samples at room temperature for 30 and 180 days compared to those stored for 1 and 7 days. Besides the time factor, temperature also influenced the DNA concentration, being higher in teeth that remained for 30 days and in tooth pulp maintained for 180 days, under refrigeration. Regarding the use of Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) it showed a significant difference in stabilization of stored teeth at room temperature for 30 and 180 days. The analysis of fragments was possible in 37 selected samples, regardless of the DNA quantity variation, confirming that amplification reactions and STR analysis using automated methods provides good results. It was concluded that the use of Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) showed a significant difference in stabilizing DNA samples of intact human teeth stored at room temperature for 30 and 180 days, while in the other test conditions the results showed no justification for using this product.

Some latent fingerprint development techniques rely on the reaction with amino acids within the fingerprint and then either change in color or fluoresce to help visualize this fingerprint. Amino acids are the building blocks of proteins and are present in all biological fluid. Thus, these developers should be able to also locate biological stains. In a previous study, ninhydrin was shown to be able to locate biological stains. Two more latent fingerprint developers are introduced as possible universal biological stain detectors: 1,8-diazafluoren-9-one (DFO), and 1,2-indanedione (1,2-IND). Five biological stains were used to test these chemicals: 1:500 diluted blood, saliva, semen, sweat, and urine. A new heating method was also introduced for a more portable application. The hair dryer heating method was optimized for the three chemicals with two traditional oven heating methods: the oven setting at 70oC and the oven setting at 100oC. These chemicals were also examined for their effectiveness on aged samples. Samples aged for three different time intervals were used: 4 weeks, 8 weeks, and 16 weeks. The hair dryer heating method was found to be viable for all three chemicals for each of the biological stains except the 1:500 diluted blood. With the application of the hair dryer for less than 3 minutes, most stains were visible for all three chemicals. 1,2-IND gave slightly different color changes for sweat and the other biological stains. This property can possibly be used to guide subsequent specific body fluids testing. All three chemicals lost their effectiveness as the stain became older. One-month-old stains still gave similar results as fresh stains, but after 2 months, the color became fainter and was barely visible after 4 months. The next stage of this study applied these chemicals as a guide for wearer DNA extraction from worn clothing. Sampling for wearer DNA has mostly been an educated guess with little guidance as to where an abundance of DNA is located. Fingerprint developers can react with amino acids, and cells contain abundant amino acids. Thus, these chemicals may react more to areas with abundant cells. Wearer DNA was extracted from collars of donated shirts before and after the chemical applications to determine the effectiveness of these chemicals as DNA detectors. Of the three collars tested, ninhydrin reacted completely with two of the collars, making any distinction between areas with abundant DNA and areas with no DNA difficult. In addition, the quantitation data of the ninhydrin samples showed no advantage in using ninhydrin as a wearer DNA locator. DFO was shown to have some detrimental effects on the DNA or the DNA extraction and quantitation process. The quantitation data for DFO also showed no advantage in using DFO as a wearer DNA locator. 1,2-IND showed promising results and was the most likely candidate as a wearer DNA locator. All areas that reacted with 1,2-IND produced at least one sample having higher than 0.01 nanograms per microliter of DNA and would be considered viable for DNA profiling.

M.Sc.
It is the widely accepted view that the human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and that South Africa harbors mainly HIV type 1 subtype C (HIV-1 C). Extensively characterized biological isolates (especially of HIV-1 subtype C) for use in HIV/AIDS vaccine and drug development are not readily available. This study evaluated three different protocols for the expansion of virus from infected PBMC's of 68 HIV/AIDS patients (designated HJ1 — 22 and INN1 — 97). Factors influencing the success of a protocol for the expansion of HIV-1 were 1) the amount and time of addition of IL-2 and PHA to the culture media; 2) the fact that freshly isolated clean PBMC's (treated with PHA prior to co-cultivation) was necessary while infected PBMC's could be used fresh or frozen; 3) whether the absence or presence of polybrene as a tissue culture additive had any effect. The I-11V-status of patients could be confirmed with rapid tests and/or NASBA assays, while successful expansion of the virus could be confirmed or refuted by determining p24 levels of sera or culture supematant (with values ranging from <7.8pg/ml to about 280pg/ml). Less sensitive assays like the reverse transcriptase (RT) and gpl 20 ELISA's give much lower absorbance values when compared to the p24 ELISA. Using expanded virus to infect PBMC's and T-cell lines (PM1, U87.CD4-CCR5, U87.CD4-CXCR4 and CEM.NKR-CCR5) and then measuring p24 levels showed that the final protocol chosen was capable of producing high titre, biologically active virus. To further test the biological activity of the isolates, the virus was used in assays evaluating the potential inhibitory ability of natural products and neutralizing antibodies. PCR using universal primers (SK22/SK38/SK39) was not consistently successful in amplifying out the correct sized region of gag (for SK22/SK39 a fragment of 600bp and a 115bp fragment for SK38/SK39 was obtained but not for all the samples). Primers (Cgag189(+/-)) were designed during this project to specifically amplify an 189bp region of gag from subtype C, which proved (in some instances) to be more successful. PCR amplification of the proviral DNA fragments was confirmed by sequencing of selected PCR products. Virus titre was determined by calculating TCID50 (login: 1.054). By modifying expansion and detection protocols it is possible to standardize the process to suit a particular isolate and/or circumstance. This production of large volumes of high titre, biologically active isolates has filled a desperate need for reagents to aid HIV researchers in the development of an effective vaccine or other drug therapy.

The homologation chemistry pertains the possibility of obtaining from an organic substrate with n carbon atoms, the corresponding homologue with n + 1 carbon atoms and, among the most useful methods to obtain such derivatives, organometallic chemistry occupies a prominent role. The aim of this PhD thesis is to provide a detailed overview of the new methodologies developed for the homologation of organic substrates using organometallic reagents such as lithium halocarbenoids and potassium halocarbanion, mainly implicated in the synthesis of halodrins, primary alkyl halides, fluoromethylketones and difluoromethyl ketones. Because of the great importance of fluoroketones associated to the Medicinal Chemistry, the synthesis of difluoromethylketones has been applied to the design and development of peptide difluoromethylketones as new reversible/irreversible cysteine protease inhibitors.

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic agents. We have developed a set of second generation diazo reagents with small steric footprints, namely an alpha-trifluoroethyl (HTFB) diazo reagent, for simultaneous arming and SAR studies of bioactive natural products. The Rh(II)-catalyzed O-H insertions of several alcohol-containing natural products, including the potent translation inhibitor lactimidomycin, are investigated and useful reactivity and both chemo- and site- (chemosite) selectivities are observed. The alpha-trifluoroethyl diazo reagents (HTFB) shows clear differences in the IL-2 reporter assay with FK506 derivatives and provides greater retention of biological activity in a hMetAP2 proliferation assay of fumagillol derivatives compared to the first generation pbromophenyl diazo reagent (HBPA). The synthetic utilities of the new alpha-trifluoroethyl diazo reagent (HTFB) provide a great new tool for basic cellular studies facilitating the discovery of new drug candidates for human disease. Furthermore, we are interested in methodologies for beta-lactone synthesis and transformations. In this study, we demonstrated synthetic versatilities of beta-lactones for the synthesis of beta-lactam congeners of orlistat as fatty acid synthase inhibitors via SnCl4- promoted tandem Mukaiyama aldol-lactonization (TMAL) reaction and a one-pot, mild conversion of beta-lactones to beta-lactams. The inhibitory activities of the derived beta-lactam derivatives are determined in a biochemical fluorogenic assay using recombinant FASTE, and the micro-molar range FAS-TE inhibitory activities were observed. Additionally, we pursued synthetic studies toward the total synthesis of spongiolactone, which is a unique beta-lactone-containing marine diterpenoid, isolated from the marine sponge Spongionella gracilis. This natural product bears a unique tricyclic beta-lactone core possessing four contiguous stereogenic centers and an additional stereogenic quaternary carbon on a cyclohexyl appendage. We completed the total synthesis of 6,15-bis-epi-spongiolactone by employing an intramolecular nucleophilecatalyzed aldol-lactonization (NCAL) process as the key step to construct the fused tricyclic beta-lactone core. Importantly, we developed a double diastereoselective and, for the first time, a kinetic resolution via the NCAL process that enables an enantioselective strategy to the tricyclic beta-lactone core of (+)-spongiolactone.

碩士
中國醫藥大學
藥物化學研究所碩士班
102
Various halomethyleniminium salts as novel Vilsmeier reagents were synthesized from the reaction of formamide or N-methylformamide with phosphoryl chloride. Treatment of N-1-substituted-aminopyrazoles including, N-1-(2-pyridinyl)-5-aminopyrazoles and N-1-(2-quinolinyl)-5-aminopyrazoles with these Vilsmeier reagents to obtain the corresponding pyrazolo[3,4-d]pyrimidine, N-(1H-pyrazol-5-yl)formamide, or N-(1H-pyrazol-5-yl)formamidine products. The experimentant results were different with our previous data which the formylated amidylpyazole and formamidine products were obtained under the similar reaction conditions.

碩士
國立高雄大學
土木與環境工程學系碩士班
107
This study mainly tests the transmission of auxiliary biological reagents and the degradation of chlorine-containing organic compounds by Fe/Al composite metal oxide electrode by applying the electrodynamic system to the field test, and then the biological reagent transmission efficiency and the degradation of chlorine-containing organic substances. Efficiency, as an evaluation of the parameters of the electro-dynamic field test, determine the optimal operating conditions (potential slope, oxidant, electrode distance and electrode arrangement), and then use Mann–Kendall test to analyze the change trend of pollutant concentration and conduct electric Force to deal with economic benefit analysis. This study is used in the transmission test of auxiliary biological reagents for electrodynamic systems (A1, A2 and A3). The parameters include potential slope (0.1~0.3 V/cm), electrode distance (2~4 m) and electroosmotic flow and groundwater. The same direction or not. It was found that the electrokinetic force applied a potential slope of 0.3 V/cm and a well spacing of 2 m through the A1 experiment. The results of this group show that the electroosmotic flow can overcome the groundwater flow; the A2 transmission result can be seen to provide 0.3 V/cm. The potential slope of the potential is 4m, and the transmission of the bioreagent is confirmed by the electrodynamic technology. The transmission of the A3 is shown in the A2 test. 1/3, but its electrodynamically effective transmission distance is also 4m. The concentration of biological reagents in this group is distributed in wells T2 and T3, because its potential slope is lower and the transmission rate is slower, resulting in the accumulation of biological reagents in T2. The reason for the well, and T4 is the downstream of the groundwater flow. This well verified that the groundwater flow could not transfer the biological reagent to the T4 well within 27 days, indicating that its transmission was the contribution of electric power, while the T5 well was used to check the power impact of the side. The results show that the side impact radius of the electrodynamic experiment is within 2m. This study was conducted in the remediation test of Fe/Al composite metal oxide electrode (B1~B5). The parameters including potential slope (0.1~0.3 V/cm), oxidant (NaCO3 and 0.06M Na2S2O8), electrode distance (2m and 4m). And the electrode arrangement (single axis and double axis), the results are shown in the B1 remediation experiment (without adding oxidant), applying a potential slope of 0.3 V/cm, TCE falling below the regulatory standard value in 21 days, anode well (R1) The TCE degradation was completed on the 27th day. The degradation rate of the intermediate well (R2) and the cathode well (R3) was 85.5 and 91.1%, and the VC degradation rate was between 26.8 and 34.5%. The B2 and B3 remediation tests were carried out by adding different oxidants (0.06M Na2CO3 and 0.06M Na2S2O8), while B3 and B4 were oxidants of Na2S2O8 but different concentrations (0.06M and 0.3M). The degradation rate of T2 of B2~B3 was Both of them were 100% in 15 days, while B4 completed TCE degradation in 27 days, indicating that the Fe/Al electrode system of this test has a good degradation effect on TCE. Although the concentration of B4 is higher, the degradation rate is slower than that of B2 and B3. This is because the initial concentration of B4 is 1.8 to 4.1 times that of B2 and B3. The average degradation rate of each test in VC, the degradation rate of B2 was 8%, the degradation rate of B3 was 14.9%, and the degradation rate of B4 was 31.7%. The results showed that the B4 test was the best parameter mentioned above. The experimental parameters of B4 were used in the B5 remediation test, but the electrode arrangement was changed to a biaxial arrangement. The results showed that the degradation of TCE in the R1~R6 well was completed in 24 days compared with the 27-day rate of B4, and at 27 The by-products such as DCE and VC were not generated in the day, so the degradation of TCE and VC showed that the degradation effect of the electrode well biaxial alignment (B5) was better than that of the single axis alignment (B4). Monitoring wells (R4, R5 and R6) were installed around the test. The test results show that the side impact radius of the electric power system with 0.3 V/cm is between 25% and 50% of the well spacing. In this study, the data of MK statistical analysis and remediation test showed that the concentration of TCE in B1~B5 decreased significantly, while the degradation trend of VC except B1 group showed a possible increase in concentration, B1, B2, B4 and B5 were all The trend of reduction, MK statistics show that the decrease in TCE and VC concentration is not due to the concentration of pollutants in the background environment, but is contributed by the Fe/Al oxidation electrode system. The calculation of economic cost in this paper is based on the construction cost of the mold field, the electrode material cost in the operation stage, the operating liquid medicine cost and the electric energy cost. The operation cost of the transmission test (A1~A3) is 2.02×104~2.66× 104 NTD/month, in which the operating cost of the A1 test is the highest, due to the large number of anode wells; and the operating cost of the remediation test (B1~B5) is 1.96×104~4.24×104 NTD/month, of which the operating cost of B5 The highest, this is due to the arrangement of the two-axis electrodes. The main expenses of each group were the highest proportion of installation cost and electrode cost. The A1, A3, B1, B4 and B5 tests all had the highest electrode cost (46.47~65.13%), followed by the setting cost (20.02~39.88%). The cost ratios of the A2, B2 and B3 tests to the experimental group in the opposite direction are reversed, the setting cost is the highest (45.97~47.67%), and the second is the electrode cost (40.46~46.89%), which is caused by the difference of experimental time. Lengthening, the monthly cost of the electrode will drop. This study confirmed in the transmission test that the biological reagent transmission efficiency can be improved. In the future, it can be combined with biological treatment to carry out remediation. The result shows that the electroosmotic flow can overcome the groundwater flow direction and can be applied to containment in the future. It has been confirmed in the remediation test that the Fe/Al oxidation electrode electrodynamic system can effectively degrade chlorinated contaminants. By adjusting the electrode arrangement, well spacing, oxidant type and concentration, the degradation efficiency can be improved.

博士
國立臺灣師範大學
化學系
96
In the first part of this dissertation, we have focused on the use of relatively low toxic, inexpensive reagents such as N-bromosuccinimide for carrying out the catalytic synthetic methodology. Various biologically important 1,5-benzodiazepine derivatives were efficiently synthesized in excellent yields. Furthermore, 3-indolyl-nitroalkane derivatives can be synthesized utilizing this newly development catalytic system as well. The second part is focused on ethyl dichlorophosphate mediates functional group transormation from primary amides into nitriles under mild condition. However, phenyl dichlorophosphate also promotes Beckmann rearrangement at room temperature with highly efficiently to afford products in good to high yields. Finally, a novel series of 1,2,4-oxadiazole as potent and selective CB1 cannabinoid receptor antagonists has been developed. Replacing the conventional pyrazole 3-aryl substituent of rimonabant with the 1,2,4-oxadiazole moiety, a novel class of 1,2,4-oxadiazole-pyrazole derivatives, behaving as highly potent CB1 receptor antagonists with good CB2/1 selectivity, was discovered.