Carboxylesterases are glycosylated general detoxification enzymes belonging to the serine esterase superfamily and play a critical role in the hydrolysis of numerous ester- and amide- containing molecules, including active metabolites, drugs and prodrugs. Three functionally active carboxyleterases have been identified in man (CES1-3), which all show differential tissue expression and critically overlapping, yet specific substrate selectivities. Elucidating the basis of their exact substrate preference would help facilitate the design of clinical prodrugs which are activated by carboxylesterases. Because of their widespread applications, carboxylesterases have attracted much attention in recent years, with CES1 being the most extensively studied human carboxylesterase to date. The work presented here addresses the structure-function relationship of the three human carboxylesterases using a combination of X-ray crystallography, kinetic analysis and biophysical techniques. Recombinant proteins were successfully produced using a mammalian expression system in high yield (5.0 – 84.0 mg/ L cell culture). Analytic ultracentrifugation and size-exclusion chromatography coupled to multi-angle laser light scattering were used to investigate the proteins in solution. These results showed CES1 exists primarily in a trimeric arrangement, whilst CES2 and CES3 are monomeric. Interestingly, atypical mechanisms of substrate inhibition, positive cooperativity and biphasic kinetics were observed for both CES1 and CES2. Three structures of CES1 were solved: wild type, an aglycosylated form and a catalytically inactive form, to 1.48, 1.86 and 2.01 Å respectively. The novel structure of CES2 was solved to 2.04 Å, which revealed that the enzyme forms a strand exchange dimer in contrast to the trimeric CES1. To summarise, this thesis documents a platform that has been generated for the production, characterisation and crystallization of human carboxylesterases. This will aid future structural work for protein ligand binding studies.

Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-29Bitstream added on 2014-06-13T19:12:51Z : No. of bitstreams: 1 thomazine_vb_me_sjrp.pdf: 300929 bytes, checksum: 8a345195b89148d41da69659555f43dd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Vários trabalhos na literatura têm discutido a importância dos polimorfismos enzimáticos em relação ao estado de ser adaptado dos organismos. Nesse trabalho foram estudadas algumas propriedades bioquímicas das aloenzimas EST-5S e EST-5F de Drosophila mulleri, que poderiam estar relacionadas às diferenças em valores adaptativos em indivíduos que apresentam essas variantes enzimáticas. Os resultados sugerem que a aloenzima EST-5S apresenta maior resistência à desnaturação térmica e a agentes caotrópicos, bem como maior atividade em altas concentrações salinas. Os resultados do experimento de produtividade a 29º C mostram que os indivíduos homozigotos para o alelo Est-5S deixam mais descendentes que os indivíduos homozigotos para o alelo Est-5F. Quanto as caractetísticas cinéticas, essas aloenzimas não diferem de forma significante. Os valores de Vmax e Km obtidos para as enzimas não purificadas foram, respectivamente, para a EST-5S: 32,54 µM/min e 1,43 mM, e para a EST-5F: 33,34 µM/min e 1,54 mM. A purificação das aloenzimas foi obtida pela separação em SDS-PAGE seguida da remoção dos géis, eluição em agitador magnético para a difusão das enzimas e posterior concentração desse material em membrana filtrante de 50 kDa (Millipore). O concentrado foi então analisado quanto à atividade esterásica e quanto à pureza (SDS-PAGE). Ambas as aleloenzimas apresentaram uma massa molecular entre 66 e 68 kDa para a subunidade, em SDS-PAGE. Os resultados do presente estudo não são concordantes com a hipótese de neutralidade para a manutenção dos polimorfismos, tanto em populações naturais como de laboratório, indicando a atuação da seleção natural sobre as aleloenzimas estudadas. / Several works in literature have discussed the importance of the enzymatic polymorphisms in respect to the state of being adapted of the organisms. In this work, some biochemical properties of Drosophila mulleri of the allozymes EST-5S and EST-5F, which could be related to the differences in fitness values in individuals that present those enzymatic variants, were studied. The results suggest that the EST-5S allozyme presents larger resistance on the thermic denaturation and the chaotropics agents, as well as higher activity in elevated salt concentrations. The results of the productivity experiment at 29°C show that the homozygous individuals to the Est-5S allele let more offspring than the homozygous individuals to the Est -5F allele. Regarding the kinetic characteristics, those allozymes do not differ in a significant way. The Km and Vmax values obtained for crude extracts allozymes were, respectively, to the EST-5S: 32,54 µM/min and 1,43 mM, and to the EST-5F: 33,34 µM/min and 1,54 mM. The purification of the allozymes was obtained by the separation on SDS-PAGE followed by their removal from gels, elution in magnetic shaker for the diffusion of the enzymes and concentration of this material in filtrant membranes of 50 kDa (Millipore). The concentrated materials were then analyzed regarding to their enzymatic activity and to the purity (SDS PAGE). Both allozymes presented subunit molecular weights around 66 and 68 kDa on SDS PAGE. The results of the present study do not agree to neutrality hypothesis for polymorphisms maintenance, in natural and laboratory populations, indicating a natural selection action of the on the studied allozymes.

Made available in DSpace on 2015-04-09T12:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-19Bitstream added on 2015-04-09T12:47:49Z : No. of bitstreams: 1 000817170.pdf: 893124 bytes, checksum: ec79ee6704498c3749ce75120e54f120 (MD5) / A metagenômica é uma ferramenta poderosa na descoberta de novos genes microbianos com potencial biotecnológico, pois não são necessárias as técnicas tradicionais de cultivo. Para suprir as necessidades de enzimas no mercado esta técnica tem sido muito utilizada para a descoberta de novos genes codificadores de lipases e esterases microbianas muito utilizadas em processos biotecnológicos como produção de detergentes, biodiesel e biorremediação. Em trabalhos anteriores, foram prospectadas sequências codificadoras para enzimas lipolíticas em uma biblioteca metagenômica de DNA com 4224 clones, de um consórcio microbiano obtido de solo contaminado com hidrocarbonetos de petróleo, localizado na cidade de Ribeirão Preto – São Paulo - Brasil, obtendo-se 30 clones com possível atividade lipolítica em Tributirina. No presente trabalho, um dos clones foi selecionado após ensaio em placa de Petri com Tributirina para a construção de uma sub-biblioteca, com 480 subclones. O sequenciamento foi realizado em aparelho ABI Prism 3100 e o “contig” obtido analisado no ORF Finder do National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA). Foi possível identificar um gene de 1032 pb, 344 aminoácidos , denominado ORF2, que codifica uma enzima lipolítica com identidade de 94 % com uma hidrolase de Pseudomonas denitrificans (YP_007656829.1). As sequências que representam as oito famílias de enzimas lipolíticas propostas por Arpying e Jaeger (1999) foram extraídas do banco de dados do NCBI e comparadas com a sequência da ORF2, possibilitando afirmar que esta enzima é um novo membro da família V de enzimas lipolíticas bacteriana. O gene codificador da ORF2 foi clonado em vetor de expressão pET28a e superexpressado em Escherichia coli BL21(D3). A fração solúvel da proteína foi analisada em gel de poliacrilamida (SDS-PAGE), em condição desnaturante. A atividade lipolítica das bandas da proteína ... / The metagenomics is a powerful tool in the discovery of new microbial genes with biotechnological potential, they are not necessary traditional cultivation techniques. With increasing demand for enzymes in the market this technique has been widely used for the discovery of new genes encoding microbial lipases and esterases widely used in biotechnological processes such as the production of detergents, biodiesel and bioremediation. In previous work, coding sequences for lipolytic enzymes were prospected in 4224 clones from a metagenomic DNA library from a microbial consortium obtained from soil contaminated with petroleum hydrocarbons, located in the city of Ribeirão Preto - São Paulo - Brazil, obtaining 30 clones with possible lipase activity on tributyrin. In this study, one clone was selected after testing in a Petri dish with Tributyrin to build a sub-library, with 480 subclones. Sequencing was performed on ABI PRISM 3100 instrument and analyzed “contig”s obtained in the ORF finder from the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA). It was possible to identify a gene of 1032 bp, 344 amino acids, termed ORF2, encoding a lipolytic enzyme with 94% identity with a hydrolase from Pseudomonas denitrificans (YP_007656829.1). The sequences representing eight families of lipolytic enzymes and Arpying proposed by Jaeger (1999) were extracted from the NCBI database and compared with the sequence of ORF2, allowing affirm that this enzyme is a new member of the family V bacterial lipolytic enzymes . The encoder ORF2 gene was cloned into pET28a expression vector and overexpressed in Escherichia coli BL21 (D3). The soluble protein fraction was analyzed by polyacrylamide (SDS-PAGE) gel in denaturing condition. The lipolytic activity of the recombinant protein bands on the SDS-PAGE gel was confirmed by a zymogram indicating its functionality in a specific substrate. Three-dimensional modeling was also ...

Orientador: Eliana Gertrudes de Macedo Lemos / Banca: Janete Apparecida Desidério / Banca: Mariana Carina Frigieri Salaro / Resumo: A metagenômica é uma ferramenta poderosa na descoberta de novos genes microbianos com potencial biotecnológico, pois não são necessárias as técnicas tradicionais de cultivo. Para suprir as necessidades de enzimas no mercado esta técnica tem sido muito utilizada para a descoberta de novos genes codificadores de lipases e esterases microbianas muito utilizadas em processos biotecnológicos como produção de detergentes, biodiesel e biorremediação. Em trabalhos anteriores, foram prospectadas sequências codificadoras para enzimas lipolíticas em uma biblioteca metagenômica de DNA com 4224 clones, de um consórcio microbiano obtido de solo contaminado com hidrocarbonetos de petróleo, localizado na cidade de Ribeirão Preto - São Paulo - Brasil, obtendo-se 30 clones com possível atividade lipolítica em Tributirina. No presente trabalho, um dos clones foi selecionado após ensaio em placa de Petri com Tributirina para a construção de uma sub-biblioteca, com 480 subclones. O sequenciamento foi realizado em aparelho ABI Prism 3100 e o "contig" obtido analisado no ORF Finder do National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA). Foi possível identificar um gene de 1032 pb, 344 aminoácidos , denominado ORF2, que codifica uma enzima lipolítica com identidade de 94 % com uma hidrolase de Pseudomonas denitrificans (YP_007656829.1). As sequências que representam as oito famílias de enzimas lipolíticas propostas por Arpying e Jaeger (1999) foram extraídas do banco de dados do NCBI e comparadas com a sequência da ORF2, possibilitando afirmar que esta enzima é um novo membro da família V de enzimas lipolíticas bacteriana. O gene codificador da ORF2 foi clonado em vetor de expressão pET28a e superexpressado em Escherichia coli BL21(D3). A fração solúvel da proteína foi analisada em gel de poliacrilamida (SDS-PAGE), em condição desnaturante. A atividade lipolítica das bandas da proteína ... / Abstract: The metagenomics is a powerful tool in the discovery of new microbial genes with biotechnological potential, they are not necessary traditional cultivation techniques. With increasing demand for enzymes in the market this technique has been widely used for the discovery of new genes encoding microbial lipases and esterases widely used in biotechnological processes such as the production of detergents, biodiesel and bioremediation. In previous work, coding sequences for lipolytic enzymes were prospected in 4224 clones from a metagenomic DNA library from a microbial consortium obtained from soil contaminated with petroleum hydrocarbons, located in the city of Ribeirão Preto - São Paulo - Brazil, obtaining 30 clones with possible lipase activity on tributyrin. In this study, one clone was selected after testing in a Petri dish with Tributyrin to build a sub-library, with 480 subclones. Sequencing was performed on ABI PRISM 3100 instrument and analyzed "contig"s obtained in the ORF finder from the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA). It was possible to identify a gene of 1032 bp, 344 amino acids, termed ORF2, encoding a lipolytic enzyme with 94% identity with a hydrolase from Pseudomonas denitrificans (YP_007656829.1). The sequences representing eight families of lipolytic enzymes and Arpying proposed by Jaeger (1999) were extracted from the NCBI database and compared with the sequence of ORF2, allowing affirm that this enzyme is a new member of the family V bacterial lipolytic enzymes . The encoder ORF2 gene was cloned into pET28a expression vector and overexpressed in Escherichia coli BL21 (D3). The soluble protein fraction was analyzed by polyacrylamide (SDS-PAGE) gel in denaturing condition. The lipolytic activity of the recombinant protein bands on the SDS-PAGE gel was confirmed by a zymogram indicating its functionality in a specific substrate. Three-dimensional modeling was also ... / Mestre

No description available.

Orientador: Carlos Alberto Mathias Azania / Resumo: As espécies de Mucuna aterrima, Ricinus communis, Merremia aegyptia, Ipomoea hederifolia e Ipomoea quamoclit comumente infestam os canaviais brasileiros e seu manejo depende da integração de métodos e modalidades de aplicação de herbicidas. O objetivo da pesquisa foi de avaliar a seletividade e eficácia de herbicidas em programas de manejo com aplicações antes e após o plantio da cana-de-açúcar. O experimento foi conduzido no Brasil, município Ribeirão Preto, SP, em canavial cultivado em Latossolo Vermelho de textura argilosa, localizado sob clima Cwa de Koppen, entre janeiro/2018 a março/2019. O delineamento experimental foi em blocos casualisados com dez tratamentos em quatro repetições. As parcelas foram constituídas por cinco linhas de cana-de-açúcar com 5 m de comprimento e espaçadas de 1,5 m. As parcelas foram semeadas com as espécies de M. aterrima, R. communis, M. aegyptia, I. hederifolia e I. quamoclit, sempre antes da primeira aplicação de herbicida. Os tratamentos foram constituídos pela testemunha capinada (Tcapinada) e sem capina (Tpds), além de oito programas de manejo químico (P) constituídos por aplicações em plantio pré-incorporado (PPI), pré-emergência (PRE) e pós-emergência tardia das plantas daninhas e cultura após plantio (POS-t), conforme: P1- PRE (diuron + sulfentrazone); P2 -PPI (imazapyr) + PRE (diuron + sulfentrazone); P3 - PPI (amicarbazone) + PRE (diuron + sulfentrazone); P4 - PPI (sulfentrazone) + PRE (diuron + sulfentrazone); P5 - PRE (indaziflam... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of Mucuna aterrima, Ricinus communis, Merremia aegyptia, Ipomoea hederifolia and Ipomoea quamoclit commonly infest the Brazilian sugarcane plantations and their effective control depends on the integration of herbicide application methods and modalities. The objective of the research was to evaluate the selectivity and efficacy of herbicides in management programs with applications before and after sugarcane planting. The experiment was conducted in Ribeirão Preto, SP, Brazil, in a sugarcane planted in a clayey Red Latosol texture, located under Cwa of Koppen climate, from January/2018 to March/2019. The experimental delineation was performed in randomized blocks with ten treatments in four replicates. The plots were constituted by five lines of sugarcane with 5 m of length and spaced out of 1.5 m. The plots were planted with M. aterrima, R. communis, M. aegyptia, I. hederifolia and I. quamoclit, always before the first application of herbicide. The treatments consisted of the weed control (Tcapinada) and no weed (Tpds), as well as eight chemical management programs (P) consisting of pre-incorporated (PPI), pre-emergence (PRE) and late post-emergence applications weeds and post-planting crop (POS-t) as follows: P1- PRE (diuron + sulfentrazone); P2 -PPI (imazapyr) + PRE (diuron + sulfentrazone); P3 - PPI (amicarbazone) + PRE (diuron + sulfentrazone); P4 - PPI (sulfentrazone) + PRE (diuron + sulfentrazone); P5-PRE (indaziflam); P6-PPI (imazapyr) + PRE (indaziflam); ... (Complete abstract click electronic access below) / Mestre

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular metabolism, signal transduction to defence mechanisms in plants. One aspect of esterases that recently is receiving more attention is the role esterases play in the .degradation of plant material. With fossil fuels (coal and oil) estimated to run out in the next 20 to 30 years, renewable sources such as plant biomass are becoming increasingly important. Plant biomass contains hemicellulosic and cellulosic materials that need to be degraded to their different constituents before they can be optimally used for the production of commodities. Although the enzymes needed to hydrolyse the xylan backbone (xylanases and P-xylosidases) are important, enzymes that remove side chains from the polymer are equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will improve the availability of monomeric sugars for utilisation when used in conjunction with other xylanolytic enzymes. Many of these side-chains are esters and they need to be removed through the action of esterases, either directly from the xylan backbone or from shorter xylo-oligomers. An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was screened for the presence of an acetyl esterase encoding gene. Positive clones were identified by the formation of clearing zones on plates containing glucose pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA insert was sequenced and found to contain two open reading frames, one of which encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223- 3) for expression in E. coli. The plasmid was introduced into E coli and the esterase activity determined, using the chromogenic substrate a-naphthyl acetate. Activity levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature stability of the enzyme was determined. The enzyme exhibited optimal activity at pH 6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was cloned into a yeast expression vector between the PGK promoter and terminator sequences for expression of the gene in Saccharomyces cerevisiae. The estA open reading frame was also fused to the MFa 1 secretion signal for secretion of the protein from S. cerevisiae. The expression vector was successfully transformed into S. cerevisiae, but no extracellular activity was detected. Only low intracellular activity of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20 tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van produkte aangewend kan word. Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word, (xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en ~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede. 'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer. Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal. Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C. Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer. Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae. Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26 nanokatal per mililiter was bepaal.

Orientador: Hermione Elly Melara de Campos Bicudo / Banca: Lílian Castiglioni / Banca: Marcelo Ferreira Lourenço / Banca: Eduardo Alves de Almeida / Banca: Lilian Madi Ravazzi / Resumo: Os padrões de esterases de 19 linhagens de 10 espécies do grupo saltans foram analisados usando eletroforese em gel de poliacrilamida e α e β-naftil acetatos como substratos. Cinqüenta e uma bandas esterásicas foram detectadas e classificadas como 31 α-esterases, 18 β-esterases e duas α/β-esterases. Com base nos padrões de inibição usando Malathion e sulfato de eserina, 34 bandas foram classificadas como carboxilesterases, 14 como acetilesterases e três como colinesterases. Dez loci gênicos foram tentativamente estabelecidos com base nos dados de posição da banda no gel, preferência ao substrato e padrões de inibição. Vinte bandas foram espécie-específicas, as restantes foram compartilhadas por espécie de um mesmo ou diferentes subgrupos. Sete bandas α-esterásicas foram observadas exclusivamente em machos. Bandas com diferentes freqüências ou grau de expressão entre os sexos também foram detectadas. Nos géis preparados para análise da expressão dos genes nas partes do corpo (cabeça, tórax e abdome), o grau de expressão das β- esterases foi alto no tórax, enquanto as α-esterases expressaram-se predominantemente no abdome e tórax. Uma visão global dos dados, atualmente disponíveis, quanto às esterases das espécies do grupo saltans é apresentada neste trabalho. As hipóteses filogenéticas geradas à partir dos dados deste estudo foram comparadas com filogenias anteriores baseadas na morfologia, bioquímica e características moleculares. / Abstract: The esterase patterns of 19 strains from 10 species in the saltans group were analyzed using polyacrylamide gel electrophoresis and α- and β-naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 α-esterases, 18 β-esterases and two α/β- esterases. On the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Seven α-esterase bands were observed exclusively in males. Bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the β- esterases was higher in the thorax, while the α-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of species from the saltans group is presented. Phylogenetic hypotheses generated from data in this study are compared with prior phylogenies based on morphological, biochemical and molecular characteristics. / Doutor

Orientador: Hermione Elly Melara de Campos Bicudo / Coorientador: Lílian Madi-Ravazzi / Banca: Alba Regina de Abreu Lima Catelani / Banca: Francisco Chiaravalloti Neto / Banca: Maria Tercília Vilela de Azeredo Oliveira / Banca: Eduardo Alves de Almeida / Resumo: Hoje se sabe que as enzimas esterásicas estão relacionadas com o desenvolvimento de resistência a inseticidas, em muitos organismos. Em Aedes aegypti, a dedução desse envolvimento tem resultado mais de testes que permitem a avaliação da atividade das esterases no extrato total dos mosquitos (mostrando valores maiores nas populações resistentes) do que de estudos mais profundos de padrões e bandas individualizadas e sua relação com a resistência. Com o objetivo básico de contribuir para o conhecimento desse aspecto, no presente trabalho 11 populações geográficas daquele vetor, sendo seis classificadas como resistentes, três como suscetíveis e duas como tendo suscetibilidade diminuída, foram analisadas quanto ao polimorfismo de esterases, por eletroforese em géis de poliacrilamida. O resultado do estudo de cerca de 30 amostras de larvas e adultos de cada população mostrou 24 bandas que foram tentativamente associadas com oito loci genéticos. Considerando também os dados de Lima-Catelani et al. (2004) e de Sousa-Polezzi & Bicudo (2005), temos o total de 25 bandas esterásicas, incluídas em 12 supostos loci, em 15 populações daquele vetor, até a presente data. A população de São José do Rio Preto, analisada em intervalos de cinco anos entre aqueles dois estudos, e sete anos entre Sousa-Polezzi & Bicudo (2005) e o presente trabalho, mostrou modificações no padrão de esterases, que ocorreram ao longo do tempo paralelamente ao surgimento e aumento da resistência aos inseticidas utilizados no controle, nessa população. Essas modificações abrangeram, basicamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is presently known that the esterases are involved in the process of resistance to insecticides, in several organisms. In Aedes aegypti, the conclusion about such involvement resulted rather from tests in which the total amount of esterases is computed in extracts of the mosquitoes (showing greater quantities in the resistant ones) than from deeper studies of esterase patterns or particular esterase bands and their relationship with resistance. With the basic aim to contribute to the knowledge of this relationship, in the present study the esterase polymorphism of 11 geographic populations of that vector, being six classified as resistant, three as susceptible and two as presenting decreased susceptibility, was studied by electrophoresis in polyacrylamide gels. The results of the analysis of about 30 individual samples of larvae and adults of each population showed 24 esterase bands which were tentatively associated to eight loci. Considering also the data from Lima-Catelani et al. (2004) and Sousa-Polezzi & Bicudo (2005), a total of 25 bands and 12 loci, in 15 populations, was obtained. The population from São José do Rio Preto, analyzed at intervals of five years between those two studies and seven years between Sousa-Polezzi & Bicudo (2005) and the present study, showed changes in the esterase pattern, which occurred along time concomitant to the increase of insecticide resistance in that population, including frequency increase or decrease of some bands and absence of bands previously detected. The search for specific esterase patterns related to the resistance development indicated some bands and combinations of bands as deserving a deeper study. They are EST-1 alone, due to high frequency in five of the six resistant populations, or the combination of EST-1 with EST-4, both occurring... (Complete abstract click electronic access below) / Doutor

Two natural enzymatic browning inhibitors, copper-metallothionein (Cu-MT) and polyphenol esterase (PPE), were obtained from A. niger and investigated. Reflectance measurements, expressed as L (lightness variable) and a (red to green degree of color) were used to compare, over extended periods of time, the relative inhibitory effectiveness of Cu-MT and PPE to those observed with the use of selected chemicals including ascorbic acid (AA), citric acid (CA), ethylenediaminetetraacetic acid (EDTA), sodium bisulfite (NaHSO3) and 4-hexylresorcinol (4HR), in the prevention of browning on the cut surfaces of selected food products such as apple and potato slices as well as freshly prepared apple juice. Treatment of each food product required an optimum concentration of the selected inhibitor for the inhibition of browning. (Abstract shortened by UMI.)