Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Thesis (Ph.D. (Chemistry)) - Rhodes University, 2008.

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:49:55Z : No. of bitstreams: 1 zanphorlin_lm_me_sjrp.pdf: 945446 bytes, checksum: 9959e6f0472c3ac5cdce9a3374c74387 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fungos termofílicos têm despertado grande interesse acadêmico e industrial por produzirem uma variedade de enzimas termoestáveis com potenciais aplicações em processos biotecnológicos como biocatálise nas indústrias de couro, farmacêutica, têxtil e alimentícia, e na preparação de produtos de limpeza e cosméticos. Particularmente, as proteases, além de participarem de inúmeros processos fisiológicos vitais como vias metabólicas, hemostasia e sinalização celular, também representam hoje cerca de 60% do mercado mundial de enzimas. Neste trabalho, descrevemos a produção, purificação e caracterização bioquímica de uma serino-protease produzida por um fungo termofílico do gênero Myceliophthora. As taxas de atividade proteolítica foram avaliadas através de fermentação em meio sólido (FES) e submerso (FSM) e observou-se um rendimento na atividade proteolítica 4,5 vezes maior para o meio sólido. A enzima bruta obtida por ambos os procedimentos (FES e FSM) exibiu a mesma temperatura ótima de 50 ºC, porém em relação ao pH ótimo houve um deslocamento de 7 (FSM) para 9 (FES) sugerindo que o perfil enzimático do fungo difere de acordo com suas condições de fermentação. Baseado nesses resultados prosseguiu-se os estudos com o extrato bruto obtido por FES. A imobilização da enzima bruta em esferas de alginato de cálcio resultou no aumento da temperatura ótima e na estabilidade térmica quando comparado com a enzima livre. O extrato bruto obtido por FES foi, então, fracionado por métodos cromatográficos como exclusão molecular e troca-iônica que resultaram na protease pura com peso molecular de 28,2 kDa determinado por espectrometria de massa. A protease pura demonstrou pH ótimo de 9,0 e temperatura ótima de 45 °C que corroboram... / Thermophilic fungi have attracted great academic and industrial interest because they produce a variety of thermostable enzymes with potential applications in biotechnological processes such as biocatalysis in the industries of leather, pharmaceutical, textile and food, and the preparation of detergents and cosmetics. In particular, proteases not only participate in many vital physiological processes such as metabolic pathways, cell signaling and homeostasis, but also currently represent about 60% of the world market of enzymes. In this work, we describe the production, purification and biochemical characterization of a serine protease produced by a thermophilic fungus of the genus Myceliophthora. The levels of proteolytic activity were evaluated either by solid fermentation (SSF) and submerged (SmF). The crude enzyme obtained by both procedures (SSF and SmF) exhibited similar optimum temperature of around 50 ºC, but in relation to the optimum pH was shifted of 7 (SmF) to 9 (SSF), suggesting that the enzymatic profile of the fungus differs from according to its fermentation conditions. Based on these results, the studies were followed with crude extract obtained by SSF. The immobilized enzyme on beads of calcium alginate resulted in increased optimum temperature and thermal stability when compared to the free enzyme. The crude extract obtained by SSF was then fractionated by chromatographic methods including molecular exclusion and ion-exchange that resulted in the pure protease with molecular weight of 28.2 kDa as determined by mass spectrometry. The pure protease showed optimum pH of 9.0 and optimum temperature of 45 °C that corroborate to the preliminary characterization of the crude extract. Inhibition tests resulted in complete inhibition by PMSF, a canonical... (Complete abstract click electronic access below)

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T13:04:06Z No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) / Made available in DSpace on 2017-09-06T13:04:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) Previous issue date: 2015-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microbial communities play a fundamental role in the health of coral and their changes can lead to the onset of disease. Many diseases affect coral reefs in several parts of the world, however, little is known about the pathogenic agents and the factors that trigger the pathological process. In coastal reefs of Carapibus Beach of Conde (Paraiba state, Brazil), a tissue necrosis affects the zoanthid Palythoa caribaeorum. This study aimed to compare the diversity of culturable bacteria in the samples of healthy and necrotic tissue from the P. caribaeorum colony collected from the Carapibus reefs. The present work aimed also to analyze the production of proteolytic enzymes by isolated bacteria. The density of total heterotrophic bacteria in the necrotic tissue was higher than in healthy tissue of P. caribaeorum. Phylogenetic analysis of isolated bacteria based on the partial 16S rRNA gene sequences revealed that the majority of bacterial strains belonged to the Bacilli class of Firmicutes phylum (65.2%), followed by the Gamma Proteobacteria class (34.7%) of Proteobacteria phylum. The genus Bacillus was dominant among the strains of healthy tissue of P. caribaeroum, while among the bacteria of necrotic tissue the Bacillus and Vibrio were the most abundant. The higher number of strains belonged to the Vibrio genus were found in necrotic tissue (42.1%) in comparison with healthy tissue (9.6%). Among bacteria from healthy tissue were found also Staphylococcus sp. isolate and from necrotic tissue Marinobacter spp. and Alteromonas spp. isolates. Among the isolates from healthy tissue only Bacillus spp. showed proteolytic activity, while proteolytic isolates from necrotic tissue belonged to the genera: Bacillus, Vibrio, Alteromonas and Marinobacter. The data suggest that bacteria of the genus Vibrio and proteolytic bacteria may play a role in the development of tissue necrosis of zoanthid studied. / As comunidades microbianas desempenham um papel fundamental na saúde do coral, e suas alterações podem levar ao aparecimento de doenças. Muitas doenças afetam os corais dos recifes em várias partes do mundo, entretanto pouco se sabe sobre os agentes patogênicos e os fatores que desencadeiam o processo patológico. Nos recifes costeiros da praia de Carapibus, Conde (Paraiba, Brasil), uma doença necrosante afeta o zoantideo Palythoa caribaeorum. Em virtude disso, neste trabalho objetivou-se comparar a diversidade de bactérias cultiváveis nas amostras do tecido sadio e necrosado coletadas da colônia de P. caribaeorum dos recifes de Carapibus. Objetivou-se também analisar a produção de enzimas proteolíticas por bactérias isoladas. A densidade de bactérias heterotróficas totais no tecido necrosado foi maior que no tecido sadio. A análise filogenética dos isolados bacterianos realizada na base das sequencias parciais do gene RNAr 16S revelou que o maior número de isolados de bactérias pertenceram a classe Bacilli do filo Firmicutes (65,2%), seguida pela classe Gama-Proteobacteria (34,7%) do filo Proteobacteria. O gênero Bacillus foi predominante entre os isolados do tecido sadio de P. caribaeroum, enquanto entre as bactérias do tecido necrosado os gêneros Bacillus e Vibrio foram dominantes. Um número maior de isolados pertencentes ao gênero Vibrio foi encontrado no tecido necrosado (42,1%) em relação ao tecido sadio (9,6%) de P. caribaeorum. Foram encontradas também as bactérias Staphylococcus sp. no tecido sadio, Marinobacter spp. e Alteromonas spp. no tecido necrosado. Dentre os isolados do tecido sadio de P. caribaeorum, apenas o gênero Bacillus apresentou a atividade proteolítica, enquanto os isolados proteolíticos do tecido necrosado pertenceram aos gêneros: Bacillus, Vibrio, Alteromonas e Marinobacter. Os dados obtidos sugerem que as bactérias do gênero Vibrio e as bactérias proteolíticas podem desempenhar um papel no desenvolvimento da doença necrosante do zoantídeo estudado.

A produção de bacteriocina por Bactérias Ácido Lácticas tem atraído grande atenção por causa do seu status GRAS (Generally Recognized as Safe), e seu uso potencial como aditivo seguro para a conservação de alimentos. Em função de suas características antimicrobianas, as bacteriocinas têm sido testadas como bioconservadores em diversos produtos, mostrando atividade contra microrganismos patogênicos. Porém, o elevado custo de produção ainda tem sido um fator relevante para ampla utilização deste tipo de conservante. Portanto, o objetivo deste trabalho foi avaliar a viabilidade da produção de enterocinas, utilizando soro de leite parcialmente desmineralizado e água de maceração de milho (milhocina) como substrato. Foram avaliados 5 isolados de enterococos (Efm20, Efm22, Efm24, Efm25 e Efs27) produtores de enterocina em meio MRS frente a bactéria Listeria innocua CLIP 12612 e Listeria monocytogenes 2032. O meio de cutura MRS foi substituido na concentração de 25% por soro de leite ou milhocina. O ensaio da ação da enterocina foi realizado pelo método de difusão em poços. O caráter proteico da bacteriocina foi confirmado após incubação com enzimas proteolíticas α-quimiotripsina, protease e proteinase-K. A produção de peróxido de hidrogênio e ácido lático foram descartadas pela utilização de catalase e neutralização com NaOH. O sobrenadante livre de células (CFS) obtido em ambos substratos foram tratados a 80 °C e 100 °C, apresentando termoestabilidade. Os CFS obtidos em MRS com milhocina pelo isolado Efm22 (24 horas) atingiu 6400 UA/mL frente às duas bactérias indicadoras. Já os isolados Efm24 (24 horas) e Efm20 (18 horas) chegaram a 6400 UA/mL frente a Listeria innocua CLIP 12612. Em soro de leite, os maiores valores de UA/mL ocorreram pelos isolados Efm20, Efm25 e Efs27 (24 horas). Em conclusão, constatou-se que os substratos testados apresentam potencial para serem aplicados na fabricação de bioconservantes de produtos alimentícios. / The production of bacteriocin by Lactic Acid Bacteria has attracted great attention because of its GRAS (Generally Recognized as Safe) status, and its potential use as a safe additive for food preservation. Due to their antimicrobial characteristics, bacteriocins have been tested as bioconservatives in several products, showing activity against pathogenic microorganisms. However, the high cost of production has still been a relevant factor for the wide use of this type of preservative. Therefore, the objective of this work was to evaluate the viability of enterocin production using partially demineralized whey and corn steep liquor as substrate. Five Enterococcus isolates (Efm20, Efm22, Efm24, Efm25 and Efs27) were tested on MRS medium against Listeria innocua CLIP 12612 bacterium and Listeria monocytogenes 2032. The MRS culture medium was replaced at 25% concentration by whey or corn steep liquor.The assay of the action of enterocin was performed by the well diffusion method. The proteinic character of bacteriocina was confirmed after incubation with proteolytic enzymes α-chymotrypsin, protease and proteinase-K. The production of hydrogen peroxide and lactic acid were discarded by the use of catalase and neutralization with NaOH. The cell free supernatant (CFS) obtained on both substrates were treated at 80 ° C and 100 ° C, exhibiting thermostability. The CFS obtained in MRS with corn steep liquor by the Efm22 isolate (24 hours) reached 6400 AU / mL against the two indicator bacteria. On the other hand, the isolates Efm24 (24 hours) and Efm20 (18 hours) reached 6400 AU / mL compared to Listeria innocua CLIP 12612. In whey, the highest values of UA / mL occurred with the isolates Efm20, Efm25 and Efs27 (24 hours). In conclusion, it was verified that the substrates tested have potential to be applied in the manufacture of food product bioconservants.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. <p>By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. <p>In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses.<p>Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. <p><p>Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases.<p>Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. <p>Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. <p>The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. <p>To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. <p><p>Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

This study has involved the design, synthesis and evaluation of novel HIV-1 enzyme inhibitors accessed by synthetic elaboration of Baylis-Hillman adducts. Several series of complex coumarin-AZT and cinnamate ester-AZT conjugates have been prepared, in high yields, by exploiting the click reaction between appropriate Baylis-Hillman derived precursors and azidothymidine (AZT), all of which have been fully characterised using spectroscopic techniques. These conjugates, designed as potential dual-action HIV-1 inhibitors, were tested against the appropriate HIV-1 enzymes, i.e. HIV-1 reverse transcriptase and protease or HIV-1 reverse transcriptase and integrase. A number of the ligands have exhibited % inhibition levels and IC50 values comparable to drugs in clinical use, permitting their identification as lead compounds for the development of novel dual-action inhibitors. In silico docking of selected ligands into the active sites of the respective enzymes has provided useful insight into binding conformations and potential hydrogen-bonding interactions with active-site amino acid residues. A series of furocoumarin carboxamide derivatives have been synthesised in four steps starting from resorcinol and these compounds have also been tested for HIV-1 integrase inhibition activity. The structures of unexpected products isolated from Aza-Baylis-Hillman reactions of N-tosylaldimines have been elucidated by spectroscopic analysis, and confirmed by single crystal X-ray analysis. A mechanism for what appears to be an unprecedented transformation has been proposed. Microwave-assisted SeO₂ oxidation of Baylis-Hillman-derived 3-methylcoumarins has provided convenient and efficient access to coumarin-3-carbaldehydes, and a pilot study has revealed the potential of these coumarin-3-carbaldehydes as scaffolds for the construction of tricyclic compounds. The HCl-catalysed reaction of tert-butyl acrylate derived Baylis-Hillman adducts has been shown to afford 3-(chloromethyl)coumarins and α-(chloromethyl)cinnamic acids, the Zstereochemistry of the latter being established by X-ray crystallography. ¹H NMR-based experimental kinetic and DFT-level theoretical studies have been undertaken to establish the reaction sequence and other mechanistic details. Base-catalysed cyclisation on the other hand, has been shown to afford 2H-chromene rather than coumarin derivatives.

A series of chromone-3-carbaldehydes have been prepared using Vilsmeier-Haack methodology while a corresponding series of chromone-2-carbaldeydes have been synthesized via the Kostanecki-Robinson reaction. Baylis-Hillman reactions have been conducted on both series of chromone carbaldehydes using three different catalysts, viz., 1,4-diazabicyclo(2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec- 7-ene (DBU) and 3-hydroxyquinuclidine (3HQ), and acrylonitrile, methyl acrylate and methyl vinyl ketone as the activated alkenes. These reactions have typically (but not always!) afforded both normal Baylis-Hillman and dimeric products. Attention has also been given to the use of 1-methyl-2-pyrrolidine (1-NMP), an ionic liquid, to replace normal organic solvents, and it has been found that, in the presence of DABCO, chromone-3-carbaldehydes afford the dimeric products alone. Reactions of chromone-3-carbaldehydes with methyl vinyl ketone have yielded unexpected, novel adducts, which appear to arise from preferential attack at C(2) in the chromone nucleus. Research on chromone-2-carbaldeydes under Baylis-Hillman conditions has also resulted in the formation of some interesting products instead of the expected Baylis-Hillman adducts. The Baylis-Hillman products have been explored as substrates for aza-Michael reactions using various amino derivatives including protected amino acids in the presence of the tetrabutylammonium bromide (TBAB) and the ionic liquid, 3-butyl-1- methylimidazoleboranetetrafluoride (BmimBF₄), as catalysts. The aza-Michael products have been targeted as truncated ritonavir analogues for investigation as potential HIV -1 protease inhibitors, and representative compounds have been subjected to enzyme inhibition assays to explore the extent and type of inhibition. Lineweaver-Burk and Dixon plots have indicated competitive inhibition in one case as well as non-competitive inhibition in another, and the inhibition constants (Ki) have been compared with that of the ritonavir. Computer modelling studies have also been conducted on selected chromonecontaining derivatives, using the ACCELRYS Cerius² platform. Interactive docking of the chromone-containing ligands into the HIV -1 protease receptor site, using the Ligandfit module, has indicated the importance of hydrogen-bonding interactions mediated by bridging water molecules situated in the receptor cavity. NMR spectroscopy has been used to elucidate complex and competing mechanistic pathways involved in the Baylis-Hillman reactions of selected 2-nitrobenzaldehydes with MVK in the presence of DABCO - reactions which afford the normal BaylisHillman product, the MVK dimer and syn- and anti-Baylis-Hillman type diadducts. The kinetic data confirm the concomitant operation of two pathways and reveal that, in the initial stage of the reaction, the product distribution is kinetically controlled, whereas in the latter stage, thermodynamic control results in the consumption of the normal Baylis-Hillman product and predominance of the anti-diadduct.

Made available in DSpace on 2017-06-01T18:20:40Z (GMT). No. of bitstreams: 1 elisangela_teixeira_silva.pdf: 805083 bytes, checksum: 4f700b752106d3810c1baa4954dc2462 (MD5) Previous issue date: 2013-05-26 / Enzymes are specific biocatalysts that work in wide field of applications as food industry as detergent formulation. The proteases represents an important commercial bioproduct used in industry, managing billion of dollars year by year, producing tons of detergents for different applications. Enzymatic reactions are processed under mild temperature and pressure with great commercial interest, being these catalysts biodegradable. The proteases demand in the brazilian market promoves the researches, as the entrepreneurship in this area althought more investments from government agencies must be necessary. The potenciality in renewable raw material and the increase of development of enzyme technologies are the bases that can promove the enzymes exportation. Hydrogen and disulfide bonds, van der Waals and ionic powers, as well as hydrophobic interactions need to be keeped among these amino acids to manage the spacial conformation of the enzymes, avoiding the inactivation or the protein desnaturation. The formulation process need of physical and chemical managements to promove the stability of the protein chains to try to protect the catalytic site and the spacial structure, adding elements like preservatives, salts, polymers, surfactantes, solvents, detergents and others elements to manage the structure of the enzyme in this process of formulation is necessary. In this work was analyzed the chemical composition of three bioprocts sold in the brazilian market used for domestic laundry, the presence of the main active agents among them like: enzymes, tensioactives and others, and the interaction these additives during the formulation process that could promove the respective differences and characteristics that make these products competitive. / Enzimas são biocatalisadores específicos que são utilizadas em vários campos de atuação, desde a indústria de alimentos, até na formulação de detergentes. As proteases são biocatalisadores de grande interesse comercial na indústria, movimentando bilhões de dólares com produção de toneladas de detergentes para diferentes aplicações. A demana de proteases no mercado brasileiro promove pesquisas como também o empreendorismo nesse segmento embora mais investimentos por parte das agências do governo devem ser necessárias. A potencialidade da matéria prima renovável e o aumento do desenvolviemnto de tecnologias para enzimas, como o conhecimento sobre a conformação protéica e estabilidade com atividades catalítica são as bases que podem promover a exportação de enzimas. Ligações de hidrogênio, forças iônicas e de van der Walls, como também as interações hodrofóbicas precisam ser matindas entre os amimoácidos para gerenciar a conformação espacial das enzimas, evitando desnaturação protéica. No processo de formulação, é necessário investigar a interrrelação de parâmetros físicos e aditivos químicos cujas variáveis são importantes para manter a estabilidade da conformação espacial, adicionando elementos como conservantes, sais, polímeros, surfactantes, solventes, detergentes e outros elementos para manter a estrutura da enzima. Nesse trabalho foram analizadas as composições de três bioprodutos dispostos no mercado brasileiro para lavagem de roupa. A presença de agentes ativos entre eles: enzimas e tensioativos e, a interação entre esses aditivos durante o armazenamento e as condições operacionais promovem as respectivas diferenças e características que impulsionam a competitividade desses produtos.

Made available in DSpace on 2017-06-01T18:20:42Z (GMT). No. of bitstreams: 1 fernanda_cristina_padilha_rocha_silva.pdf: 2431932 bytes, checksum: f7140668c1558241152571bad405e7c7 (MD5) Previous issue date: 2014-02-12 / Oil refineries, as well as other large-scale industrial processes, are potential sources of environmental pollution. Accidents involving spills of oil and oil products in Brazil, in the period 1975-2012, add infective million liters of soil, rivers and sea. In this sense, the process of dissolved air flotation (DAF) is still widely used in industry, both for water supply and for wastewater. The physico-chemical processes such as centrifugation, ultrafiltration and dissolved air flotation (DAF), can be effective when used to separate emulsified oils. The effluent from the oily water type cause many environmental problems, particularly in thermal power plants (TPP s). Thus the aim of the study was to propose the separation water/oil by FAD in pilot scale and to compare the efficiency of the pilot prototype of FAD with and without addition of biosurfactant separation of oily waste waters. According the results, the biosurfactant produced by Candida sphaerica was selected, this being cultivated in using low cost industrial waste. Use of this bioproduct increased the efficiency of the flotation 80.0% to 98.0 %, to provide better determination of the operating conditions. Thus, it is suggested that the use of biosurfactants as auxiliary flotation is a promising alternative for the mitigation of pollution caused by the accumulation of synthetic surfactants in the environment. / As refinarias de petróleo, assim como outros processos industriais em grande escala, são fontes potenciais de poluição ambiental. Os acidentes ocorridos com derramamento de petróleo e seus derivados no Brasil, no período de 1975 a 2012, somam milhões de litros de poluentes que promoveram a contaminação de solos, rios e mar. Os processos fisíco-químicos tais como, a centrifugação, ultrafiltração e flotação por ar dissolvido (FAD), podem ser eficazes quando usados para separar óleos emulsionados. Nesse sentido, o processo de FAD continua sendo amplamente utilizado nas indústrias, tanto para águas de abastecimento como para águas residuárias. A FAD pode ser considerada como uma tecnologia limpa, uma vez que utiliza pequenas quantidades de coagulantes e ar para promover a separação. A utilização de coletores/coagulantes é essencial para melhorar a eficiência do processo, tendo em vista suas características específicas que facilitam a adesão das partículas e, consequentemente, a separação dos poluentes. Por outro lado, esses coletores químicos são tóxicos, fator que representa um agravante no sentido da geração de outros poluentes ambientais. Assim, os surfactantes microbianos ou biossurfactantes, biomoléculas anfipáticas produzidas por bactérias e leveduras, em detrimento dos coagulantes sintéticos, apresentam-se como uma tecnologia sustentável e promissora no aumento de eficiência da flotação. Essas biomoéculas, além de serem muito eficientes, são biodegradáveis e atóxicas, motivando as pesquisas no sentido de produzir e utilizar cada vez mais esses agentes tensoativos. Dessa forma, o presente trabalho foi desenvolvido na busca de uma estratégia para comparar as eficiências de separação água/derivado de petróleo por FAD, em escala piloto, com e sem a adição de um biossurfactante. De acordo com os resultados obtidos, o biossurfactante produzido por Candida sphaerica cultivada em residuos industriais foi considerado adequado como coletor do processo de separação. A utilização da biomolécula elevou a eficiência do processo de FAD de 80,0% para 98,0%, proporcionando a determinação das melhores condições operacionais. Dessa forma, concluiu-se que o uso de biossurfactantes como auxiliares na flotação constitui uma alternativa promissora na mitigação da poluição provocada pelo derramamento de petróleo e derivados em ambientes marinhos.