No description available.

No description available.

No description available.

A rearing methodology for dogwood borer was developed, using standardized procedures at each developmental stage. These methods enabled the establishment of a laboratory colony and efficient production of synchronized cohorts of each of its lifestages throughout the year for specific experimental needs. The behavioral repertoire shown by mated female dogwood borer in an apple orchard was characterized and quantified and the diel periodicity with which those behaviors occurred was determined. Mated females were easily recognized, based on their characteristic casting flight directed toward areas below the graft union of apple trees, and were observed more frequently during the late afternoon and evening. Casting flight, probing with the ovipositor, and oviposition were the most frequent behaviors observed, but the duration of those behaviors was relatively short compared with the much lengthier periods of resting behavior that typically occurred within the canopy. Data from a previous, three-year study in two newly planted apple orchards were subjected to geostatistical analyses to examine the temporal and spatial patterns of infestation by larval dogwood borer and to gain further information about the spatial scales at which oviposition occurs. There were moderate to high degrees of aggregation of dogwood borer infestations on neighboring apple trees, with ranges of spatial dependence from 7.50–19.87 m. No directionality was observed in the spatial autocorrelation of infestation and it appears that females utilized oviposition sites equally along and across orchard rows. The aggregated nature of infestations requires that random, independent samples must be taken from a number of sample pairs at distances greater than the range of spatial dependence to ensure that sample data are not autocorrelated. Alternatively, an efficient sampling program for mapping dogwood borer infestation can be achieved by limiting sample points to distances within the range of spatial dependence. These sample points can be used in interpolating algorithms, such as kriging, to predict infestation at unsampled locations in space for use in site-specific pest management programs. The external morphology of male and female dogwood borer antennae and their sensilla were examined using light and scanning electron microscopy to characterize, measure and compare the types, number, and distribution of sensilla. Although the general shape and size of male and female antennae were similar, those from females possessed a greater number of generally smaller antennal flagellomeres. The flagellum of both male and female antennae contained seven sensillum types including auricillica, basiconica, chaetica, coeloconica, squamiformia, styloconica, and three subtypes of sensilla trichoidea. With the exception of sensilla basiconica, which were present in roughly equal numbers on male and female antennae, all other sensillum types were significantly more abundant on female antennae. The antennae of female dogwood borer appear well equipped to perceive olfactory stimuli, based on the types and number of sensilla present. Coupled gas chromatography and electroantennogram detection (GC-EAD) analyses of headspace collections from damaged and undamaged tissues from apple and dogwood trees were conducted to examine and compare the antennal responsiveness of female dogwood borer to host plant volatiles. A total of 16 and 9 compounds from apple and dogwood tissues, respectively, consistently elicited an antennal response in females. There were no differences in the response of antennae from virgin and mated females, and the amplitude of the female response to host odors was greater than that of males. Six compounds were identified from the headspace collections from apple trees, four of which (octanal, nonanal, decanal, and methyl salicylate) were identified from all apple tissues sampled. A novel compound,α-bergamotene, was identified from injured apple bark, from apple burr knots infested with dogwood borer larvae and from larval dogwood borer frass, and appears to be produced by apple trees in response to injury. Another novel compound, methyl-2,4-decadienoate, was identified from infested burr knot tissue and appears to be produced in response to an insect-plant interaction. Two compounds, hexanoic and nonanoic acid, were identified from headspace collections from dogwood trees. Numerous approaches were used to examine the behavioral response of mated female dogwood borer to host plant headspace collections and to individual compounds from those collections that elicited a strong and repeatable antennal response. Under both natural and semi-natural conditions in the field and in laboratory bioassays, neither attraction/orientation or consistent oviposition were documented and it is apparent that correlating the electrophysiological and behavioral responses of mated female dogwood borer to olfactory stimuli from their host plants will require further research on bioassay development. / Ph. D.

Le procédé innovant d’hydrodiffusion par micro-onde a été testé et validé pour la fabrication de jus de fruit sur des raisins, abricots et prunes. Ce procédé présente des avantages au niveau technique : rapidité d’extraction, pas de préparation de l’échantillon, aucun auxiliaire de fabrication. Il permet l’obtention de jus à partir de fruits difficiles à presser, avec une pasteurisation « in line » et des produits se différenciant nettement des jus commerciaux par leur couleur et texture.Le jus est expulsé du fruit sous l’effet de la vapeur produite in situ. A partir du fruit congelé, la déstructuration liée à la croissance des cristaux de glace facilite l’extraction. Les rendements le plus élevés sont obtenus à partir de fruits congelés, et à basse puissance. Une partie de l’eau des fruits est convertie en vapeur, mais peut être récupérée en utilisant un réfrigérant, ce qui permet d’atteindre des rendements proches des rendements de jus obtenus avec un prétraitement enzymatique et pressurage. Globalement, le jus a une composition proche de celle du jus présent dans le fruit ou obtenus par pressurage. La composition du jus expulsé des fruits change au cours de l’extraction. La concentration des composés présents dans la chair et hydrosolubles (sucres, acides, acides phénoliques) baisse nettement en fin d’extraction, tandis que les anthocyanes, présentes dans l’épiderme des raisins ou des prunes, montrent un pic de concentration lors de la phase d’expulsion rapide des jus. La teneur en procyanidines est nettement plus élevée que pour des jus obtenus par pressurage / The hydrodiffusion process was tested and validated for production of juices from grapes, apricots and plums. Its major advantages are speed, no sample preparation, no processing aid. It allows production of juices from hard-to-press fruits with an in line pasteurisation. The juices present original characteristics notable bright colours, texture, and a fresh fruit aroma.The juice is expulsed from the fruit by the pressure due to in situ vapour generation. The destructuration occuring in frozen fruits due to growth of ice crystals facilitate the juice expulsion. The highest yields are obtained from frozen fruits and at low power densities. Part of the water from the fruits is vaporised, and can be collected by use of a cooler, allowing to reach yields comparable to those obtained by pressing after an enzymatic pre-treatment. Juice composition is close to that of juice extracted by pressing, though with slightly lower concentrations of most soluble solids. The composition of juice changes along the extraction. The highly soluble compounds present in the fruit flesh mostly show a clear exhaustion at the end ofextraction. Anthocyans, present exclusively in the peel of the test fruits, present a maximum during the fast juice extraction phase. Procyanidin concentrations is much higher in juices obtained by hydrodiffusion than in conventional juices

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As lisozimas digestivas de insetos são do tipo c e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pKas do nucleófilo e do doador de prótons. Estruturas cristalográficas das lisozimas digestivas de Musca domestica foram obtidas a 1,9 Å e análise comparativa com a estrutura terciária da HEWL revelou resíduos de aminoácidos no ambiente do nucleófilo (N46) e do doador de prótons (S106 e T107) que podem estar envolvidos na modulação das constantes de ionização dos resíduos essenciais à catálise. Esses resíduos foram substituídos via mutagênese sítio-dirigida por D, V e A respectivamente e três mutantes simples (N46D, S106V e T107A) e um triplo (N46DS106V- T107A) foram produzidos e purificados. Caracterização revelou que as contribuições individuais da N46, S106 e T107 foram pequenas e próximas do limite de detecção da técnica utilizada. Por outro lado, o conjunto dos 3 aminoácidos foi responsável pelo pH ótimo ácido frente ao substrato sintético, elevando os valores de pKas do nucleófilo e doador de prótons para valores muito semelhantes ao da HEWL. Diferentemente, essa tripla mutação não foi suficiente para elevar o pH ótimo da lisozima 2 sobre cápsulas de Micrococcus lysodeikticus para valores próximos àqueles de HEWL, sugerindo que as bases moleculares do pH ótimo frente ao substrato natural e sintético são diferentes. Uma comparação estrutural entre lisozima 1 e HEWL sugere que os resíduos de aminoácidos carregados na superfície dessas lisozimas sejam importantes para determinação do pH ótimo. A investigação dessa hipótese foi feita substituindo 5 aminoácidos neutros e 1 ácido, via mutagênese sítio-dirigida, por resíduos básicos. A caracterização do mutante sêxtuplo revelou um aumento significativo nos valores de pH ótimo da lisozima 1, indicando que a redução da basicidade da superfície das lisozimas digestivas é determinante para seus pHs ótimos ácidos. / Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.

En raison de leur rôle central dans la physiologie et le développement des plantes, les phythormones ont depuis longtemps été considérées comme des médiateurs chimiques déterminants dans la capacité des insectes à contrôler leur environnement végétal. Les mécanismes permettant aux insectes de manipuler la balance phytohormonale permettant ainsi la régulation des apports nutritifs et la modulation des défenses végétales demeurent néanmoins pour la plupart inconnus, en particulier pour les insectes galligènes et mineurs de feuilles. L’objectif de ma thèse visait à caractériser les capacités de modulation phytohormonale par les insectes manipulateurs de plantes avec un accent particulier sur le lépidoptère mineur de feuille Phyllonorycter blancardella. Nous avons ainsi développés une caractérisation spatio-temporelle de la réponse des plantes aux attaques des larves mineuses au niveau moléculaire et biochimique. Une comparaison entre différents systèmes biologiques nous a permis d’évaluer les similitudes entre les stratégies adoptées par les les insectes galligènes et les insectes mineurs de feuilles, d’identifier l’origine possible des phytohormones impliquées dans la manipulation de la plante et le rôle des bactéries endosymbiotiques d’insectes dans ces interactions. / Because phytohormones lie at the very core of molecular mechanisms controlling the plant physiology and development, they have long been hypothesized to be involved in insect-induced plant manipulations. Insects are using phytohormones to manipulate their host plants for their own benefit, regulating nutrient provisioning and plant defenses. However, a mechanistic understanding of how phytohormones operate in plant reconfigurations by plant-manipulating insects, especially by gall-inducing and leaf-minging insects, is lacking. The objective of my Ph.D. was to provide an extensive characterization of how plant-manipulating insects modulate the plant global hormonal balance with a specific focus on the leaf-mining moth Phyllonorycter blancardella. We thus developed a time course characterization of plant transcriptomic and biochemical responses following attack by leaf-mining larvae. A comparative analysis between different biological systems allowed us to estimate similarities in strategies developed leaf-mining and gall-inducing insects, to identify the possible origin of phytohormones involved in the plant manipulation and to estimate the role of insect endosymbiotic bacteria in these interactions.

Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:57:05Z : No. of bitstreams: 1 okino_ch_me_jabo.pdf: 1181305 bytes, checksum: cf91a5205aface873e536d06ccd0eb78 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A bronquite infecciosa das galinhas (BIG) é uma doença infecciosa que está amplamente disseminada entre as criações avícolas brasileiras e é uma das enfermidades virais que mais têm causado perdas econômicas na atualidade. Portanto, a rápida detecção e identificação do agente causal são imprescindíveis para que medidas eficazes de controle sejam prontamente tomadas. Para tanto, é necessário que os métodos de diagnóstico empregados sejam sensíveis, específicos, rápidos e também de baixo custo. Nesse contexto, a técnica de RTPCR em tempo real abordada no presente estudo permitiu a amplificação de duas regiões de hipervariabilidade do gene S1 de 17 estirpes diferentes do vírus da BIG (VBI), que foram testadas, mas não foi capaz de amplificar nenhum dos RNAvírus heterólogos analisados (vírus da doença de Newcastle, pneumovírus aviário e vírus da doença de Gumboro). Com essa mesma técnica foi possível fazer a diferenciação em grupos geneticamente distintos, de estirpes do VBI através de análises das curvas de dissociação de fragmentos amplificados a partir das regiões de hipervariabilidade gênica I e II do gene S1. A RT-PCR em tempo real desenvolvida apresentou maior sensibilidade na detecção do VBI em amostras teciduais, quando comparada à técnica padrão de Isolamento Viral em ovos embrionados de galinha... / The avian infectious bronchitis virus (IBV) is an infectious disease widely spread in Brazilian commercial poultries where causes significant economical losses. Rapid and accurate diagnosis of the IBV strain involved in a field outbreak is necessary to establish an effective control of this disease. The real-time RT-PCR performed in this study to amplify two hypervariable regions of S1 gene, was able to detect 17 IBV strains, e.g., nine reference strains (including Massachussets, Connecticut, JMK, SE 17 and Iowa serotypes) and eight Brazilian field isolates, whilst non-related avian viral pathogens such as Newcastle disease virus, Avian Pneumovirus and Gumboro disease virus were not detected. The differentiation between IBV strains was accomplished using the melting curve analysis of the amplified fragments corresponding to the hypervariable regions I and II of S1 gene. The real-time RT-PCR developed here showed a higher rate of IBV detection in tissue samples of experimentally infected chickens, when compared to the goldstandard technique of Viral Isolation in embryonated chicken eggs, and the same rate of detection was found for the conventional RT-PCR... (Complete abstract, click electronic access below)

Made available in DSpace on 2016-12-08T16:44:38Z (GMT). No. of bitstreams: 1 PGPV11MA076.pdf: 421773 bytes, checksum: 6fbf2a3579e5da5aa39372d361a40b76 (MD5) Previous issue date: 2011-07-22 / The aim of this study was to evaluate effects of spraying apple trees with prohexadione-calcium (ProCa) (na inhibitor of gibberellins synthesis) and gibberellin (GA₃) on vegetative growth, yield potential, fruit quality, and occurrence of bitter pit in the fruit. The experimente was conducted in na orchard located in São Joaquim, Santa catarina State (Southern Brazil), in 2009/2010. catarina and Fuji apple trees were sprayed with water (control), ProCa and gibberellin (both products at the dose of 319 mg L¯¹), at the petal fall stage (October 15, 2009), when shoots were 5-10 cm long, with treatments repeated after 20 days. Leaves were assessed (in termsmof chlorophyll contente, área, dry mater and specific área) in January/2010, and length of current season shoots and weight of shoots removed by winter pruning were assessed in May/2010. The number of flower buds ando f fruit per flower bud were assessed in October/2009 and November/2010, respectively. Fruit were harvest at comercial maturity and then assessed for percentage of red skin área, skin color, density, weight, skin and pulp textures, flesh firmness, titratable acidity (TA), solids soluble contente (SSC) and starch index. Fruit were cold stored (0±0.5°C/90-95% RH) for four months, followed seven days for simulate marketing (20±4°C/60-70% RH) for four months, same evaluation carried out at harvest, as well as for incidence (%) and index of bitter pit. Trees treated with ProCa had higher leaf chlorophyll contente and lower specific leaf área in Fuji , lower leaf área in Catarina , lower shoot growth in both cultivars, and lower weight of shoots removed by winter pruning in Catarina , in the year following the treatment of the trees, both cultivars sprayed with ProCa had lower fruit set. the ProCa incresead the red color in Catarina apples. At harvest, the force for pulp penetratoon in the red side of the fruit was incresead in apple trees of both cultivars sprayed with ProCa. After cold stored, Fuji apples of trees sprayed with GA₃ had lower flesh firmness, while Catarina apples of trees sprayed with ProCa had higher firmness. Fruit Ca contente was not diferente between ProCa and GA₃ and between ProCa e the control in both cultivars. Fuji apples from trees treated with ProCa had lower N/Ca ratio in the skin tissue. Teh treatment with ProCa incresead the flesh firmness in the reddish fruit side and reduced the index of bitter pit during cold storage in both cultuivars. The use of ProCa in apples orchards might represent a new technology to reduce trees vegetative growth and the occurrence of bitter pit in the fruit. The GA₃ sprayed after full bloom increases trees vegetative growth and might cause changes in fruit quality atributes, leading to advanced maturiry, as well as increases the development of bitter pit / Este trabalho foi desenvolvido com o objetivo avaliar os efeitos da pulverização de macieira com um inibidor da síntese de giberelinas, o prohexadiona-cálcio (ProCa), e com giberelina (GA₃), no crescimento vegetativo e potencial produtivo das plantas, e na qualidade e ocorrência de bitter pit nos frutos. O experimento foi conduzido em um pomar localizado no município de São Joaquim, SC, na safra 2009/2010. Macieiras Catarina e Fuji foram pulverizadas com água (tratamento controle), ProCa e GA₃ (ambos os produtos na dose de 319 mg L¯¹), na queda das pétalas (15/10/2009), quando as brotações do ano estavam com 5-10 cm de comprimento, sendo repetidas após 20 dias. Foram feitas avaliações foliares (teor de clorofila, área, matéria seca e área específica), em janeiro/2010, e de comprimento dos ramos do ano e de peso dos ramos podados, em maio/2010. A contagem dos cachos florais e do números de frutos por cacho floral ocorreram em outubro/2009 e novembro/2010, respectivamente. A colheita foi realizada na maturação comercial, ocasião em que foram feitas análises nos frutos do índice de cor vermelha, cor da epiderme, densidade, peso, atributos de textura (força para a penetração da casca e força para a penetração da polpa), firmeza de polpa, acidez titulável, sólidos solúveis totais (SS) e índice de iodo-amido. Os frutos foram armazenados em câmara fria convencional (0±0,5°C/90-95% UR), durante quatro meses, seguido de sete dias de comercialização simulada (20±4°C/60-70 UR), e após submetido as mesmas avaliações feitas na colheita, bem como de incidência (%) e índice de bitter pit . O tratamento com ProCa aumentou o teor de clorofila e reduziu a área foliar específica em macieira Fuji , reduziu a área foliar em Catarina , reduziu o comprimento dos ramos do ano nas duas cultivares e o peso dos ramos podados em Catarina . No ano subsequente ao da aplicação dos tratamentos, macieiras pulverizadas com o ProCa, de ambas as cultivares, apresentaram menor frutificação. O tratamento com ProCa proporcionou maior coloração vermelha em maçãs Catarina . No momento da colheita, maçãs Fuji e Catarina provenientes de plantas pulverizadas com ProCa apresentaram maior força para a penetração da polpa no lado mais vermelho dos frutos. Após o armazenamento, maçãs Fuji de plantas pulverizadas com GA₃ apresentaram menor firmeza de polpa e maçãs Catarina de plantas pulverizadas com ProCa apresentaram maior firmeza de polpa. O teor de Ca nos frutos não diferiu entre os tratamentos ProCa e GA₃ e entre ProCa e controle, em ambas as cultivares. Maçãs Fuji apresentaram menor relação N/Ca no tecido da casca quando pulverizadas com ProCa. O tratamento com ProCa proporcionou aumento na firmeza no lado mais vermelho do fruto e reduziu o índice de bitter pit durante o armazenamento refrigerado em ambas as cultivares. A utilização do ProCa na cultura da macieira pode ser uma nova alternativa para o controle do crescimento vegetativo de da manifestação do bitter pit nos frutos. O GA₃, pulverizado em pós-floração, em macieiras aumenta o crescimento vegetativo e pode ocasionar algumas modificações nos atributos de qualidade nos frutos, indicando um avanço na maturação, e na manifestação do bitter pit

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As lisozimas digestivas de insetos são do tipo c e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pKas do nucleófilo e do doador de prótons. Estruturas cristalográficas das lisozimas digestivas de Musca domestica foram obtidas a 1,9 Å e análise comparativa com a estrutura terciária da HEWL revelou resíduos de aminoácidos no ambiente do nucleófilo (N46) e do doador de prótons (S106 e T107) que podem estar envolvidos na modulação das constantes de ionização dos resíduos essenciais à catálise. Esses resíduos foram substituídos via mutagênese sítio-dirigida por D, V e A respectivamente e três mutantes simples (N46D, S106V e T107A) e um triplo (N46DS106V- T107A) foram produzidos e purificados. Caracterização revelou que as contribuições individuais da N46, S106 e T107 foram pequenas e próximas do limite de detecção da técnica utilizada. Por outro lado, o conjunto dos 3 aminoácidos foi responsável pelo pH ótimo ácido frente ao substrato sintético, elevando os valores de pKas do nucleófilo e doador de prótons para valores muito semelhantes ao da HEWL. Diferentemente, essa tripla mutação não foi suficiente para elevar o pH ótimo da lisozima 2 sobre cápsulas de Micrococcus lysodeikticus para valores próximos àqueles de HEWL, sugerindo que as bases moleculares do pH ótimo frente ao substrato natural e sintético são diferentes. Uma comparação estrutural entre lisozima 1 e HEWL sugere que os resíduos de aminoácidos carregados na superfície dessas lisozimas sejam importantes para determinação do pH ótimo. A investigação dessa hipótese foi feita substituindo 5 aminoácidos neutros e 1 ácido, via mutagênese sítio-dirigida, por resíduos básicos. A caracterização do mutante sêxtuplo revelou um aumento significativo nos valores de pH ótimo da lisozima 1, indicando que a redução da basicidade da superfície das lisozimas digestivas é determinante para seus pHs ótimos ácidos. / Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.