A cana-de-açúcar é um alimento bem aceito por toda a população em geral, podendo ser consumido na forma de toletes (in natura) ou na forma de caldo, podendo ser apreciado com gelo somente, ou adicionado de sucos cítricos, mas nem sempre tratada com algum sanitizante ou outro produto para manutenção de sua qualidade. A cana-de-açúcar também pode ser consumida na forma de minimamente processada, sanitizada e embalada, facilitando sua utilização em redes de alimentação. Embora o processamento mínimo de vegetais aumente a suscetibilidade ao ataque de microrganismos e escurecimento enzimático, essas alterações podem ser reduzidas com o uso de tratamentos adequados, como sanitizantes e agentes antioxidantes. Em vista disso, procurou-se nesse trabalho avaliar a qualidade da cana-de-açúcar minimamente processada, tratada com antioxidantes e radiação gama. Os tratamentos aplicados para a conservação dos toletes de cana-de-açúcar foram: ácido cítrico nas concentrações de 1,5% e 3,0%; cloridato de L-cisteína a 1,0% e 2,0%; radiação gama com as doses de 2,0 e 4,0 kGy. Antes de receberem os tratamentos, os toletes de cana-de-açúcar foram processados e sanitizados com solução de hipoclorito de sódio. Após a drenagem, os toletes foram acondicionados em embalagens de polietileno de alta densidade e refrigerados a 4°C. Os produtos foram analisados após 1, 7 e 14 dias de armazenamento. Todos os tratamentos apresentaram-se em condições sanitárias adequadas para o consumo, independente das concentrações e dosagens. Os toletes de canas-de-açúcar tratados com ácido cítrico e cloridrato de L-cisteína apresentaram os melhores resultados quanto à inibição das enzimas de escurecimento, polifenoloxidase e peroxidase, sendo a cana-de-açúcar tratada com L-cisteína a que apresentou o caldo com melhores resultados quanto à inibição de seu escurecimento e níveis de açúcares. Todos os tratamentos inibiram o desenvolvimento de microrganismos / Sugar cane is a well-accepted food for the general population and can be consumed in the form of straw (in natura) or in the form of juice, which can be enjoyed with ice only, or added with citrus juice, but always treated with some sanitizing agent or other product to maintain its quality. Sugarcane can also be consumed in the form of minimally processed, sanitized and packaged sugarcane, improving its use in food networks. Unless, minimal processing of vegetables increases susceptibility to microorganism attack and enzymatic browning, but such changes can be reduced with the use of appropriate treatments such as sanitizers and antioxidants. The aim of this work was to evaluate the quality of minimally processed sugar cane treated with antioxidants or gamma radiation. The treatments applied for the preservation of sugarcane straw were: citric acid at concentrations of 1.5% and 3.0%; L-cysteine at concentrations of 1.0% and 2.0%, and gamma irradiation at doses of 2.0 and 4.0 kGy. Before receiving the treatments, the sugar cane straws were processed and sanitized with sodium hypochlorite solution. After draining, the straws were packed in high-density polyethylene packages and refrigerated at 4°C. The products were analyzed after 1, 7 and 14 days of storage. All treatments were in sanitary conditions suitable for the consumption, independent of the concentrations and dosages. The sugarcane treated with citric acid and L-cysteine presented the best results regarding the inhibition of the darkening enzymes, polyphenoloxidase and peroxidase. Sugarcane treated with L-cysteine presented the juice with better sugar levels and inhibition of browning. All treatments showed effective inhibiton of the microorganisms development

Nos últimos anos tem se intensificado o uso de produtos com a finalidade de aumentar a produtividade na cultura da soja. Dentre estes estão os bioestimulantes que podem possuir em sua constituição extratos de algas, aminoácidos e hormônios. No entanto, pouco se sabe sobre o efeito isolado de cada um destes constituintes. Frente a isto, esta pesquisa teve por objetivos avaliar o efeito da aplicação de aminoácidos isolados em plantas de soja. Para isto, o trabalho foi dividido em três experimentos. No primeiro, foi realizada a aplicação via sementes de glutamato, cisteína, fenilalanina e glicina em doses variáveis. Essa etapa foi conduzida em sistema de canteiros e foi avaliada a emergência, índice de velocidade de emergência, acúmulo de massa de matéria seca, metabolismo antioxidante (enzimas superóxido dismutase - SOD, catalase - CAT, peroxidase - POD, teor de peróxido de hidrogênio - H2O2, prolina e peroxidação lipídica - PL) e produtividade. A partir da seleção das melhores doses obtidas na primeira etapa, foi realizado o segundo experimento, conduzido em casa de vegetação. As aplicações desse experimento foram realizadas no tratamento de semente, via foliar ou em ambas as épocas, além disso, foi realizada a aplicação de todos os aminoácidos em associação. Nesse experimento foram avaliados metabolismo antioxidante, enzimas de resistência (polifenoloxidase - PFO e fenilalanina amônia-liase - PAL), metabolismo do nitrogênio (enzimas nitrato redutase e urease, teor de NO3-, NH4+, N-Aa, ureídeos e N-Total), variáveis de crescimento de raíz, acúmulo de massa de matéria seca e produtividade. Já o experimento III foi realizado em campo, utilizando os mesmos tratamentos do experimento II. Foram avaliados o metabolismo antioxidante, enzimas de resistência, metabolismo do nitrogênio, massa de matéria seca e produtividade. Todos os experimentos foram conduzidos em delineamento em blocos casualizados com quatro repetições para cada tratamento. Todos os aminoácidos proporcionaram efeito positivo em diversas variáveis fisiológicas analisadas. O uso de glutamato, fenilalanina, cisteína e glicina de forma isolada repercutiram em melhores efeitos quando a aplicação é realizada somente no tratamento de sementes. A partir da aplicação desses aminoácidos ocorreu incremento da assimilação de nitrogênio e no acúmulo de massa de matéria seca, o que levou a maior produtividade dessas plantas. O maior efeito na produtividade foi observado por meio da aplicação de fenilalanina em todos os experimentos, quando comparados com os demais aminoácidos. Com relação ao metabolismo antioxidante o uso de cisteína no tratamento de sementes proporcionou aumento da atividade das enzimas SOD e PAL e redução da PL. O uso de fenilalanina no tratamento de sementes induz ao incremento da CAT e SOD e o glutamato induz o aumento de PAL e SOD. A utilização de todos os aminoácidos em associação somente foi eficiente na aplicação foliar, o que proporcionou maior desenvolvimento de raíz, maior assimilação de nitrogênio, acúmulo de massa de matéria seca e produtividade. Portanto, foi possível perceber que o glutamato, cisteína, fenilalanina e glicina apresentam importante papel de sinalização em plantas, pois pequenas doses já são suficientes para induzir ao incremento de parâmetros fisiológicos e, consequentemente aumentar a produtividade. / In recent years, the use of products to increase productivity in soybean has been intensified. Bio-stimulants can have in their constitution algae extracts, amino acids and hormones. However, little is known about the isolated effect of each of these constituents. Facing this problem, this research aimed to evaluate the effect of the application of single amino acids to soybeans. For this, the work was divided into three experiments. In the first, the application of amino acids was performed via glutamate, cysteine, phenylalanine and glycine to seeds. This stage was carried out on planting beds and the following variables were evaluated: emergency, emergency speed index, dry matter accumulation, antioxidant metabolism (superoxide dismutase - SOD, catalase - CAT, peroxidase - POD, hydrogen peroxide - H2O2 - content, proline and lipid peroxidation - PL) and productivity. From the selection of the best rates obtained in the first stage, the second experiment was carried out in a greenhouse. The applications of this experiment were performed as seed treatments, foliar application and both procedures; furthermore, the application of all amino acids in combination was also performed. In this experiment, the following variables were evaluated: antioxidant metabolism, resistance enzymes (polyphenol oxidase - PFO and phenylalanine ammonia lyase - PAL), nitrogen metabolism (nitrate reductase and urease, NO3- content, NH4+, N-Aa, ureide and N-Total), root growth, dry matter accumulation and productivity. The third experiment was carried out in the field using the same treatments of the second experiment. The following variables were evaluated: antioxidant metabolism, resistance enzymes, nitrogen metabolism, dry matter and productivity. All experiments were carried out in a randomized block design with four replications for each treatment. All amino acids provided positive effect on several physiological variables. The use of glutamate, phenylalanine, cysteine, and glycine alone lead to the best effect when the application was done only as seed treatment. From the application of these amino acids, the nitrogen assimilation was increased and the dry matter accumulation, which led to higher productivity of the plants. The greatest effect on productivity was observed by application of phenylalanine in all experiments, when compared with other amino acids. Regarding the antioxidant metabolism, cysteine use in seed treatment increased SOD and PAL activity and PL reduction. The phenylalanine use in seed treatment increased CAT and SOD activities and glutamate induced an increase of PAL and SOD activities. The use of all amino acids in association was only effective in foliar application, which provided further development of root, greater assimilation of nitrogen, dry matter accumulation and productivity. So, it was possible to conclude that glutamate, cysteine, phenylalanine and glycine have an important signaling role in plants, because small rates are enough to induce the increase of physiological parameters and consequently increase productivity.

Chu Ling Hon Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 201-223). / Abstracts in English and Chinese. / Declaration --- p.i / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxiii / List of tables --- p.xxviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical presentation --- p.3 / Chapter 1.1.3 --- Diagnostic tests --- p.5 / Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7 / Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11 / Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13 / Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15 / Chapter 1.3.1 --- SARS-CoV S protein --- p.15 / Chapter 1.3.2 --- S protein-driven infection --- p.17 / Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22 / Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22 / Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25 / Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28 / Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28 / Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30 / Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31 / Chapter 1.6 --- SARS Drug Development --- p.32 / Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32 / Chapter 1.6.2 --- Current anti-SARS drugs --- p.36 / Chapter 1.7 --- Project Objectives --- p.39 / Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39 / Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40 / Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42 / Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42 / Chapter 2.1.2 --- Recombinant protein approach --- p.43 / Chapter 2.1.2.1 --- Plasmids construction --- p.43 / Chapter 2.1.2.2 --- Protein expression and purification --- p.52 / Chapter 2.1.2.3 --- Amino acid analysis --- p.57 / Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58 / Chapter 2.1.2.5 --- Laser light scattering --- p.61 / Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62 / Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62 / Chapter 2.1.3 --- Synthetic peptide approach --- p.64 / Chapter 2.1.3.1 --- Peptide synthesis --- p.64 / Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65 / Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66 / Chapter 2.1.3.4 --- Laser light scattering --- p.66 / Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67 / Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70 / Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70 / Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74 / Chapter 2.2.3 --- Molecular modeling --- p.75 / Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79 / Chapter 2.3.1 --- Protein expression and purification --- p.79 / Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80 / Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83 / Chapter 3 --- Results --- p.84 / Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84 / Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84 / Chapter 3.1.2 --- Recombinant protein approach --- p.87 / Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87 / Chapter 3.1.2.2 --- Protein expression and purification --- p.92 / Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101 / Chapter 3.1.2.4 --- Laser light scattering --- p.103 / Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105 / Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107 / Chapter 3.1.3 --- Synthetic peptide approach --- p.112 / Chapter 3.1.3.1 --- Peptide synthesis --- p.112 / Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116 / Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117 / Chapter 3.1.3.4 --- Laser light scattering --- p.122 / Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124 / Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129 / Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129 / Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131 / Chapter 3.2.3 --- Molecular modeling --- p.135 / Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141 / Chapter 3.3.1 --- Protein expression and purification --- p.141 / Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142 / Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142 / Chapter 4 --- Discussion --- p.149 / Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149 / Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149 / Chapter 4.1.2 --- Recombinant protein approach --- p.151 / Chapter 4.1.3 --- Synthetic peptide approach --- p.153 / Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157 / Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167 / Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167 / Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168 / Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172 / Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183 / Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184 / Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188 / Appendix --- p.191 / Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191 / Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194 / Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195 / Chapter IV. --- Vector maps --- p.196 / Chapter 1. --- Vector map and MCS of pET-28a --- p.196 / Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197 / Chapter V. --- Electrophoresis markers --- p.198 / Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198 / Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198 / Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199 / Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199 / Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200 / References --- p.201

Chong Lin-Tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 76-78). / Abstracts in English and Chinese. / Chapter Chapter 1 --- introduction / Chapter 1.1 --- Severe acute respiratory syndrome Coronavirus (SARS CoV) --- p.13 / Figure 1.1 Genome organization and putative functional ORFs of SARS CoV --- p.14 / Chapter 1.2 --- SARS main protease / Chapter 1.2.1 --- Three dimensional structure --- p.15 / Figure 1.2 Ribbon illustration of the SARS-coronavirus main protease --- p.17 / Figure 1.3 Surface representations of P1 and P2 substrate-binding pocket of main protease --- p.18 / Chapter 1.2.2 --- Substrate specificities --- p.19 / Table 1.1. Eleven predicted cleavage sites of SARS CoV main protease --- p.21 / Chapter 1.3 --- Protein-based FRET assay system --- p.22 / Figure 1.4. The principle of fluorescent resonance energy transfer (FRET) --- p.24 / Chapter 1.4 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5a and23 BL21 (DE3)pLysS --- p.26 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.27 / Chapter 2.1.3 --- Spectrophotometric quantitation DNA --- p.28 / Chapter 2.1.4 --- Agarose gel electrophoresis / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.29 / Chapter 2.1.7 --- Ligation of DNA fragments into vector / Table 2.1. Standard recipe of ligation reaction --- p.30 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.31 / Table 2.2. Standard recipe of separating gel for SDS-PAGE --- p.32 / Table 2.3. Standard recipe of stacking gel for SDS-PAGE --- p.33 / Chapter 2.2 --- Sub-cloning and site-directed mutagenesis / Chapter 2.2.1 --- Sub-cloning of SARS Co V main protease --- p.34 / Chapter 2.2.2 --- Sub-cloning of Substrate / Chapter 2.2.3 --- Site-directed mutagenesis of substrate variant --- p.35 / Table 2.4 Primer sequence for generating substrate variants --- p.36 / Table 2.5. Standard recipe of Polymerase Chain Reaction (PCR) --- p.40 / Table 2.6. Polymerase Chain Reaction (PCR) profile --- p.41 / Chapter 2.3 --- Sample preparation / Chapter 2.3.1 --- Expression of recombinant proteins --- p.42 / SARS CoV main protease / Substrate and substrate variants / Chapter 2.3.2 --- Purification of recombinant proteins / SARS CoV main protease / Substrate and substrate variants / Chapter 2.4 --- Protein-based FRET kinetic analysis --- p.45 / Chapter 2.5 --- A model for substrate-enzyme binding by docking simulation --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Preparation of SARS CoV main protease and substrate / Chapter 3.1.1 --- Expression and purification of SARS main protease --- p.48 / Figure 3.1. Purification profile of SARS CoV main protease --- p.49 / Chapter 3.1.2 --- Expression and purification of substrate and substrate variants --- p.50 / Figure 3.2. Purification profile of substrate and substrate variants --- p.51 / Chapter 3.2 --- A novel protein-based FRET assay system was established / Chapter 3.2.1 --- "With the cleavage of active main protease, absorbance at 528nm dropped while signal at 485nm were slightly increased" --- p.52 / Figure 3.3. Absorbance at 528nm dropped and 485nm increased with the substrate hydrolysis --- p.53 / Chapter 3.2.2 --- FRET efficiency ratio (528/485) decreased over time --- p.54 / Figure 3.4. FRET efficiency ratio (528/485) decreased over time --- p.55 / Chapter 3.2.3 --- Comparable kcat/Km value of SARS CoV main protease was obtained --- p.56 / Figure 3.5. Catalytic parameter (kcat/ Km) was determined from the slope of straight Line --- p.57 / Chapter 3.3 --- Main protease activity towards substrate variants at different substrate-binding sites (S2'-S2) --- p.58 / Table 3.1. Kinetic parameterrs of 76 substrate variants in descending order --- p.59 / Chapter 3.3.1 --- S2'substrate-binding site --- p.60 / Chapter 3.3.2 --- S1' substrate-b inding site / Chapter 3.3.3 --- S1 substrate-binding site / Chapter 3.3.4 --- S2 substrate-binding site / Figure 3.6. Kinetic analysis of some typical substrate variants against main protease --- p.62 / Figure 3.7. SDS-PAGE analysis of some typical substrate variants against main protease --- p.63 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Quantitative and high-throughput analysis by protein-based FRET assay system --- p.64 / Chapter 4.2 --- Substrate specificities of SARS CoV main protease at S2'-S2 subsites / Chapter 4.2.1 --- β-strand conformation was preferred at S2，subsite / Chapter 4.2.2 --- Residues with small aliphatic side chain were preferred at S1 ´ة subsite --- p.65 / Chapter 4.2.3 --- "Glutamine at S1 subsite was absolutely conserved, but alternatives were disclosed" --- p.66 / Figure 4.1. Glutamine was not absolutely conserved in S1 subsite --- p.67 / Chapter 4.2.4 --- Hydrophilic residues were tolerated at S2 subsite --- p.68 / Figure 4.2. Hydrophilic residues were tolerated at S2 subsite --- p.70 / Table 4.1. Summary of types of residues preferred at individual subsites --- p.71 / Chapter 4.3 --- Predicted conformation of substrate towards SARS CoV main protease at S2' and S1' subsites --- p.72 / Figure 4.3. Small residues were preferred at S1´ة subsite and Val at S2' subsite was more favoured than the native one --- p.73 / Chapter Chapter 5 --- Summary --- p.74 / Chapter Chapter 6 --- Future work --- p.75 / References --- p.76

Orientador: Alexandrina Sartori / Resumo: A esclerose múltipla (EM) é uma doença inflamatória crônica e desmielinizante do sistema nervoso central (SNC). A imunopatogênese da EM envolve linfócitos Th1, Th17 e Tc autorreativos e ativação de macrófagos e microglia, os quais liberam vários mediadores pró-inflamatórios e radicais livres. Não há cura para a EM e as terapias disponíveis desencadeiam efeitos colaterais. Neste contexto, testamos o potencial terapêutico da cisteína selenizada (CS), um produto em desenvolvimento pela empresa Biorigin, no controle da encefalomielite autoimune experimental (EAE). O potencial profilático/terapêutico foi testado por administração de CS durante 30 dias (14 dias antes seguido de 16 dias pós-indução da EAE) e o potencial terapêutico por administração da CS a partir de 1 ou 7 dias após a indução da doença. Os 3 procedimentos reduziram escore clínico e incidência da EAE. A estratégia profilática/terapêutica reduziu discretamente a expressão de componentes do inflamassoma NLRP3 e o percentual de macrófagos e microglia ativada no SNC. As duas estratégias terapêuticas determinaram redução significativa no escore clínico, no percentual de macrófagos e microglia ativada e na expressão de MHCII por estas células. A terapia com CS também reduziu a ativação do inflamassoma NLRP3 e a expressão de mRNA para iNOS e CX3CR1.A redução na percentagem de células dendríticas ativadas (DCs) (CD11b+MHCII+) e o aumento no percentual de DCs tolerogênicas (CD11b-CD103+) sugere o estabelecimento deum perfil ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Its immunopathogenesis is complex and involves the interplay of distinct autoreactive T lymphocyte subsets and innate immune cells. The persistent inflammation observed in MS patients is related to oxidative stress and inflammasome activation which have been recently implicated in both, demyelination and axonal damage processes. Since the development of new therapeutic procedures remains necessary, we evaluated the efficacy of an antioxidant product to control experimental autoimmune encephalomyelitis (EAE) development. The selenized cysteine (SC) is an organic selenium, developed by a Brazilian company (Biorigin). For prophylactictherapeutic strategy, oral treatment with SC (45µg of selenium/dose) started 14 days before EAE induction and was daily extended until the acute phase of the disease. The precocious and delayed therapies have begun 1 or 7 days after MOG administration. The three therapeutic approaches reduced EAE incidence and clinical manifestations. Prophylactic-therapeutic strategy attenuated NLRP3 inflammasome activation and reduced the number of macrophages and activated microglia in the CNS. Precocious and delayed therapies were efficient in controlling EAE symptoms, reducing the percentage of macrophages and activated microglia and the MHCII expression. This protection was concomitant by less NLRP3 inflammasome activation and lower expression of mRNA for ... (Complete abstract click electronic access below) / Mestre

Humans are approximately 99% similar with inter-individual differences caused in part by single-nucleotide polymorphisms (SNPs), which poses a challenge for the effective treatment of disease. Bioinformatics resources can help to store and analyse gene and protein information to address this challenge, however these resources have limitations, so the collation and biocuration of gene and protein information is required. Using the large conductance calcium- and voltage-activated potassium channel, also known as the Big Potassium (BK) channel as an example, due to its ubiquitous expression and widespread varied role in human physiology, this study aimed to prioritise SNPs with the potential to affect the function of the channel. Using a BK channel resource created with bioinformatics tools and published literature, mSlo SNPs H55Q and G57A, located in the S0-S1 linker, were prioritised and selected for lab-based verification. These SNPs flank three cysteine residues proven to modulate channel cellular distribution via palmitoylation, a reversible process shown to increase protein association with the cell membrane. The SNPs alter the predicted palmitoylation status of C56, one of the cysteine residues located in the S0-S1 linker. The cellular distribution of BK channels incorporating the SNPs was assessed using confocal microscopy and revealed that the direction and magnitude of SNP mimetic cell membrane expression was closely related to the C56 predicted palmitoylation score; a 'C56 palmitoylation pattern' was observed. It was shown that exposure to metabolic substrates glucose, palmitate and oleate modulated SNP-mimetic cellular distribution and could invert the 'C56 palmitoylation pattern', indicating that there is interplay between the metabolic status of the cell and the amino-acid composition of the channel via palmitoylation. The creation of a novel BK channel resource in this thesis highlighted the limitations, and inter-dependency of bioinformatics and lab based experimentation, whilst SNP verification experiments solidified the link between S0-S1 cysteine residues and BK cellular distribution. BK channel function is linked with a number of physiological processes; thus, the potential clinical consequences of the SNPs prioritised in this thesis require further research.

Orientador: Lauro Tatsuo Kubota / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-09-11T21:18:50Z (GMT). No. of bitstreams: 1 Silva_CeciliadeCarvalhoCastroe_M.pdf: 2199742 bytes, checksum: e0c39714ed0a9045beffe47b579b8438 (MD5) Previous issue date: 2011 / Resumo: Este trabalho descreve o desenvolvimento de um sensor amperométrico para detecção eletrocatalítica de cisteína, através da construção de uma plataforma funcional para a complexação de íons cobre. O material base desta plataforma foi um nanocompósito obtido por meio da modificação de nanotubos de carbono de paredes múltiplas (MWCNTs) com poli(4-vinilpiridina) PVP, através uma reação de polimerização in situ. Foi realizada uma otimização multivariada, empregando um planejamento composto central de face centrada, para a modificação da superfície do eletrodo de carbono vítreo (ECV), sendo a condição ótima obtida quando se utiliza concentração da dispersão de nanocompósito de MWCNTs-PVP de 6,00 mg L, concentração da solução CuCl2 de 50 mmol L e tempo de complexação dos íons cobre de aproximadamente 83 minutos. A plataforma foi caracterizada por microscopia eletrônica de varredura (MEV), espectroscopia de energia dispersiva de raios-X (EDX), espectroscopia de impedância de eletroquímica (EIE), cronoamperometria e voltametria cíclica. Os valores da constante heterogênea de transferência de elétrons (ks) e da constante cinética da reação entre os íons Cu- cisteína (kobs) foram 5,78 s e 6,96 x 10 L mol s respectivamente. A curva analítica apresentou uma faixa linear de 5 a 60 mmol L para a detecção de cisteína. Já os limites de detecção e quantificação foram 1,50 e 5,00 mmol L, respectivamente. Além disso, o ECV/MWCNTs-PVP/Cu apresentou um tempo de resposta extremamente baixo, 0,10 s e quando aplicado para determinação de cisteína em amostras de suplemento alimentar apresentou resultados estatisticamente iguais em um nível de confiança de 95% com os resultados obtidos pelo método oficial / Abstract: This work describes the fabrication of an amperometric sensor for electrocatalytical detection of cysteine. The developed sensor is based on a functional platform for complexing copper ions on multi-walled carbon nanotubes (MWCNTs) modified with poly-4-vinylpyridine through an in situ reaction of polymerization. A multivariate analysis using a central composite design to investigate the surface modification of glassy carbon electrode (GCE) was employed to optimize the experimental variables. The established optimal conditions for the concentration of MWCNTs-PVP dispersed nanocomposite were, 6,00 mg L, 50 mmol L of concentration CuCl2 and around 83 min. for complexation of copper ions. The platform was characterized performing scanning electron microscopy (SEM), energy dispersive x-ray (EDX), electrochemical impedance spectroscopy (EIS), chronoamperometry and cyclic voltammetry analyses. The obtained values for the kinetic constants for heterogeneous electron transfer rate (ks) and for chemical reaction (kobs) between Cu and cysteine were 5.78 s and 6.96 L mol s, respectively. The analytical curve showed a linear range for detecting cysteine in concentrations from 5 to 60 mmol L. The detection and quantification limits obtained were 1.50 and 5.00 mmol L, respectively. Moreover, the response time of the ECV/MWCNTs-PVP/Cu sensor was 0.1 s. The application of the sensor to detect cysteine in nutritional supplement showed results statistically equal (0.95 confidence level) to those obtained with official methods / Mestrado / Quimica Analitica / Mestre em Química

Au cours de ce travail de thèse, les deux approches permettant la caractérisation de nouveaux agents antileishmaniens ont été suivies : d’une part identification de molécules de synthèse originales et actives, par des méthodes de criblage, et d’autre part recherche de nouvelles cibles thérapeutiques leishmaniennes, faisant appel à des outils de biologie moléculaire, et de protéomique.Dans une première partie consacrée à l’optimisation du criblage antileishmanien de molécules de synthèse, nous avons notamment mis au point et validé une nouvelle méthode applicable aux formes promastigotes. Cette méthode reposant sur le principe de la bioluminescence, s’est avérée précise, rapide, répétable, sensible, automatisable et applicable à des isolats cliniques. Nous nous sommes également intéressés à la recherche d’une nouvelle méthode de criblage sur les formes amastigotes intracellulaires remplissant ces mêmes critères.Dans une seconde partie consacrée au lien entre la mort cellulaire programmée et les cystéines peptidases (métacaspase et Raptor) chez Leishmania, nous avons mis en évidence un lien possible entre métacaspase et autophagie chez L. infantum. De plus, nous avons identifié, in vivo, plusieurs substrats protéiques potentiels de ces peptidases : HSP70, ARN-Hélicase ATP-Dépendante et Lmjf09.1010 pour la métacaspase ; NDPKb et la protéine associée à l’ADN du kinétoplaste pour Raptor. Ces différents résultats ont permis de proposer un modèle conciliant les rôles possibles des cystéine-Peptidases métacaspase et Raptor au cours de l’autophagie et de l’apoptose chez Leishmania, rôles potentiellement dus au clivage de substrats protéiques spécifiques. / During this thesis work, two approaches affording the characterization of new antileishmanial agents were followed: on the one hand, the identification of new original antileishmanial synthetic drugs, through screening assays and on the other hand, research of new parasitic therapeutic targets by using molecular biology and proteomic tools. In the first part, dedicated to the optimization of the antileishmanial screening of synthetic compounds, we set up and validated a new bioluminescence-Based screening method for studying anti-Promastigote compounds. This method appears accurate, rapid, repeatable, sensitive and is also transposable to automats and usable on clinical isolates. In parallel, we investigated new screening protocols aiming at improving the screening in intracellular amastigotes which could meet the same criteria. In the second part focusing on the link between programmed cell death and cystein peptidases (metacaspase and Raptor) in Leishmania, our study first highlighted a possible link between metacaspase and autophagy in L. infantum. Moreover, we identified in vivo several potential subtrates for both peptidases: HSP70, ATP-Dependent RNA-Helicase and Lmjf09.1010 for the metacaspase; NDPKb and kinetoplast-DNA-Associated-Protein for Raptor. These results allowed us to propose a model reconciling the possible roles of cystein peptidases metacaspase and Raptor during autophagy and apoptosis in Leishmania, roles potentially due to the cleavage of specific proteic subtrates.

nÃo hà / Studies have shown that latex of plants is a rich source of enzymes with proteolytic activities. Isolation and characterization of cysteine proteases of latex have recently been reported. In this work we report the purification and characterization of three new cysteine proteases of laticifer fluid of Calotropis procera, as well as its activity in plasma coagulation assays. The three proteases, termed CpCP-1, 2-CpCP and CpCP-3 are isoforms of cysteine proteases and were purified using two sequential steps of ion exchange chromatography on CM-Sepharose and Resource S columns, coupled to FPLC system. Their molecular masses were determined by ESI-Q-TOF mass spectrometry: CPCP-1 had mass = 26.213, CPCP-2 = 26.133 and CPCP-3 = 25.086 Da. The amino acid sequences of the N-terminal region was identical for all three enzymes, being composed of 30 amino acid residues. Analysis revealed high sequence identity with others cysteine proteases. The proteolytic activity of these enzymes was tested against different substrates (azocasein, BANA and BApNA) and at different pH and temperature. The three enzymes are capable of degrading azocasein and BANA, substrates nonspecific and specific for cysteine proteases, respectively. CPCP-1 showed proteolytic activity twice that CPCP-3, and this, a little bigger than CPCP-2. Enzymes maintained 60-80% of their activities even when tested at 60 ÂC temperature, and the optimum pH for these activities was 6.0. Circular Dichroism Analysis showed that the secondary structure of the proteases was composed of 15.1 to 19.9% of alpha-helices and 20.6 to 21.3% of beta-sheets. The spectra deconvolution of proteases showed that their structures were altered in the presence of the reducing agent DTT, suggesting the presence of disulfide bridges stabilizing the three dimensional structures. In biological tests proteases were able to strongly inhibit the germination of spores of the fungus Colletotrichum gloeosporioides and also exhibited plasma coagulation activity by thrombin-like mechanism. / Estudos tÃm demonstrado que lÃtex de plantas à uma rica fonte de enzimas com atividades proteolÃticas. O isolamento e a caracterizaÃÃo de proteases cisteÃnicas de lÃtex tÃm sido recentemente relatados. Neste trabalho nÃs reportamos a purificaÃÃo e caracterizaÃÃo de trÃs novas proteases cisteÃnicas do fluido laticÃfero de Calotropis procera, bem como sua atividade em ensaios de coagulaÃÃo plasmÃtica. As trÃs proteases, denominadas CpCP-1, CpCP-2 e CpCP-3 sÃo isoformas de proteases cisteÃnicas e foram purificadas utilizando dois passos sequenciais de cromatografias de troca iÃnica em colunas de CM-Sepharose e Resource S, acoplada a sistema FPLC. Suas massas moleculares foram determinadas por espectrometria de massas em aparelho do tipo ESI-Q-TOF, onde: CpCP-1 apresentou massa=26,213, CpCP-2=26,133 e CpCP-3=25,086. A sequÃncia de aminoÃcidos da regiÃo N-terminal foi idÃntica para as trÃs enzimas, sendo constituÃda de 30 resÃduos de aminoÃcidos. AnÃlises de sequÃncias revelaram alto nÃvel de identidade (88%) com proteases cisteÃnicas A atividade proteolÃtica dessas enzimas foi testada frente a diferentes substratos (AzocaseÃna, BANA e BApNA) e em diferentes valores de pH e temperatura. As trÃs enzimas foram capazes de degradar AzocaseÃna e BANA, substratos inespecÃfico e especÃfico para proteases cisteÃnicas, respectivamente. CpCP-1 apresentou atividade proteolÃtica duas vezes maior que CpCP-3, e esta, um pouco maior que CpCP-2. As enzimas mantiveram 60-80% de suas atividades mesmo quando ensaiadas a 60ÂC de temperatura, e o pH Ãtimo para essas atividades foi 6,0. AnÃlises de DicroÃsmo Circular revelaram que a estrutura secundÃria das proteases era composta de 15,1-19,9% de alfa-hÃlices e 20,6-21,3% de folhas-beta. Os espectros de desconvoluÃÃo das proteases mostrou que suas estruturas foram alteradas na presenÃa do agente redutor DTT, sugerindo a presenÃa de pontes dissulfeto na estabilizaÃÃo das estruturas tridimensionais. Em testes biolÃgicos as proteases foram capazes de inibir fortemente a germinaÃÃo de esporos do fungo Colletotrichum gloesporioides e tambÃm exibiram atividade de coagulaÃÃo plasmÃtica por um mecanismo do tipo trombina.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O Ãxido nÃtrico (NO) à uma espÃcie endÃgena responsÃvel pela dilataÃÃo dos vasos sanguÃneos, sendo tambÃm ativo no cÃrebro e em outros processos fisiolÃgicos. Doadores de NO sÃo substÃncias farmacologicamente ativas que liberam espontaneamente ou sÃo metabolizadas. Nitroprussiato de sÃdio, Na2[Fe(CN)5NO].2H2O, faz parte de uma classe de compostos que liberam NO espontaneamente e à o Ãnico complexo metÃlico usado clinicamente. Problemas associados com o uso de nitroprussiato incluem suscetibilidade a fotÃlise e aÃÃo oxidativa do sistema imune, no qual conduz à liberaÃÃo de cianeto. Neste trabalho foi realizado o estudo e acompanhamento cinÃtico da reaÃÃo dos complexos cis-[Ru(bpy)2LNO](PF6)n (L = imidazol e sulfito) com cisteÃna, glutationa, metionina e histidina, para a obtenÃÃo de dados cinÃticos e espectroscÃpicos que possam contribuir para a elucidaÃÃo de seu mecanismo de aÃÃo. Os resultados cinÃticos para a reaÃÃo dos nitrosilo complexos com a cisteÃna e glutationa sugerem que hà formaÃÃo de dois intermediÃrios: o primeiro com banda de absorÃÃo em 450 nm à referente ao ataque do enxofre dos tiÃis e o Ãxido nÃtrico. O segundo intermediÃrio com banda de absorÃÃo caracterÃsticas em 380 nm se deve ao ataque da segunda molÃcula dos redutores ao aduto formado. As constantes de velocidade da reaÃÃo com cisteÃna apresentaram dependÃncia com relaÃÃo ao pH. Isto ocorre, provavelmente, devido à desprotonaÃÃo no enxofre da cisteÃna, facilitando a interaÃÃo deste tiÃl com o Ãxido nÃtrico coordenado ao rutÃnio (II).As reaÃÃes com metionina e histidina mostram que nÃo hà o aparecimento dos intermediÃrios, devido à ausÃncia do grupo SH nos aminoÃcidos. O acompanhamento realizado com HPLC nos mostra a existÃncia do mesmo mecanismo entre os complexos cis-[Ru(bpy)2SO3NO](PF6) e cis- [Ru(bpy)2ImN(NO)](PF6)3 com cisteÃna e glutationa. No caso da interaÃÃo com metionina e histidina, ocorre à diminuiÃÃo do pico referente aos nitrosilos complexos e o aparecimento do pico atribuÃdo ao aqua complexo. Os resultados obtidos com o eletrodo seletivo de NO, de ressonÃncia paramagnÃtica de elÃtrons e RMN, mostraram que o Ãxido nÃtrico à reduzido e liberado nos complexo sem que haja a formaÃÃo do nitrosotiÃl. Baseado em estudos cinÃticos e no espectro de EPR, a reaÃÃo dos nitrosilo complexos com cisteÃna e glutationa apresenta o seguinte esquema de reduÃÃo e liberaÃÃo do Ãxido nÃtrico: / The oxide nitric (NO) is a responsible endogenous species by dilation of the blood vessels, being also active in the brain and in other physiologic processes. Donors of NO are pathophysiologically active healthy substances that liberate spontaneously or they are metabolized. Sodium nitroprusside, Na2[Fe(CN)5NO].2H2O, is part of a class of compounds that liberate NO spontaneously and it is the only metallic compound used clinically. Associated problems with the use of nitroprusside include susceptibility the photolysis and oxidative action of the immune system, in which it leads to the liberation of cyanide. In this work it was accomplished the study and kinetic monitoring of the reaction of the compounds cis-[Ru(bpy)2LNO](PF6)n (L = imidazole and sulphite) with cysteine, glutathione, methionine and histidine, for the obtaining of kinetic and spectroscopic data that can contribute to the elucidation of your action mechanism. The kinetic results for the reaction of the nitrosyl complex with the cysteine and glutathione suggest that there is two intermediates formation: the first with absorption band in 450 nm is regarding the attack of the sulfur of the thiols and the nitric oxide. The second intermediate with characteristics band of absorption in 380 nm is due to the attack of the second molecule of the reducers to the formed adduct. The rate constants of the reaction with cysteine presented dependence regarding the pH. This occurs, probably, due to the deprotonated in the sulfur of the cisteÃna, facilitating the interaction of this thiol with the coordinated nitric oxide to the ruthenium (II). The reactions with methionine and histidine show that there are not the intermediates, due to the absence of the group SH in the amino acids. The monitoring accomplished with HPLC reveal the existence of the same mechanism among the compounds cis-[Ru(bpy)2SO3NO](PF6) and cis- [Ru(bpy)2ImN(NO)](PF6)3 with cysteine and glutathione. In the case of the interaction with methionine and histidine, occurs the decrease of the peak regarding the nitrosyl complex and the appearance of the peak attributed to the aqua complex. The obtained results with the NO sensor, of electron paramagnetic resonance and RMN, they showed that the nitric oxide is reduced and release in the complex without there is the formation of the nitrosothiol. Based on kinetic studies and in the spectrum of EPR, the reaction of the nitrosyl complex with cysteine and glutathione presents the following reduction scheme and liberation of the nitric oxide: