Candida parapsilosis is an opportunistic fungal pathogen of humans causing a variety of infections. Immunocompromised individuals represent the most threatened group of patients. The increasing frequency of infections and occurrence of drug resistant strains are the main reasons for research focused on novel antimycotic compounds. Inhibition of secreted aspartic proteases (Sap) of pathogenic Candida spp. appears to be a potential target of therapeutic intervention. The genome of C. parapsilosis contains at least three genes coding for secreted aspartic proteases, denominated SAPP1-3. Protease Sapp1p has been well biochemically and structurally characterized, whereas Sapp2p and Sapp3p have been given less attention. The first part of the thesis is focused on structural analysis of Sapp1p complexes with selected peptidomimetic inhibitors binding to the active site of the enzyme. In addition, complex of the isoenzyme Sapp2p with the well-known secreted aspartate inhibitor Pepstatin A has been analyzed. The second part is related to the fact that C. parapsilosis belongs to the Candida spp. with the unique ability to translate standard leucine CUG codon mostly as serine. Even though it is a non-conservative substitution of hydrophobic amino acids for a hydrophilic one, this unique ability is maintained for more...

Com o intuito de gerar subsídios para melhoria do processo de produção de Metarhizium anisopliae, o presente estudo teve como principais objetivos, determinar os efeitos de inibidores de proteinases de soja no crescimento vegetativo, esporulação e virulência do fungo e comparar a produção, viabilidade e virulência dos conídios produzidos em diferentes tipos de arroz e aditivos. A adição de 5 g.L-1 de inibidores de proteinases semi-purificados de soja ou 0,5 g.L-1 de inibidor purificado do tipo Kunitz no meio de cultura ME resultou em grande aumento na esporulação (de duas a 75 vezes) sem afetar a viabilidade dos conídios de quatro isolados (ESALQ-1037, IBCB348, E9, F20) de M. anisopliae. A presença destes inibidores de proteinases também alterou a morfologia dos conídios produzidos em ME. Os mecanismos responsáveis por estas alterações fisiológicas não foram determinados, mas provavelmente estejam associados à ação anti-nutricional, diminuindo a absorção protéica e estimulando a esporulação. Os conídios produzidos no meio com adição de 0,5 g.L-1 de inibidor de proteinase do tipo Kunitz ou com 2,5 g.L-1 de albumina de soro bovino e no meio BDA apresentaram virulência superior aos conídios produzido no meio ME sem inibidores. Quando o inibidor de proteinase do tipo Kunitz foi adicionado à suspensão de conídios do fungo antes da pulverização de lagartas de Diatraea saccharalis, a eficiência de controle foi 35,1%, inferior ao apresentado pelos demais tratamentos sem a presença deste inibidor. Nos estudos para determinação dos melhores tipos de arroz para produção de M. anisopliae, tentou-se correlacionar à produção de conídios com características destes substratos como valor nutricional, teor de resíduos de agrotóxicos e densidade de microorganismos. O arroz parboilizado foi responsável pela maior produção de conídios (4,38 x 109 conídios.g-1). Este tipo de arroz apresentou teor de proteína bruta menor do que a maioria dos arrozes e o maior teor de umidade (41,3% após a autoclavagem). Além disto, os grãos após autoclavagem ficaram menos gelatinosos e mais soltos, o que facilita o processo de produção do fungo. Enquanto que os arrozes dos tipos brancos polidos, canjicão e integral ficaram pegajosos e formaram grumos, o que provavelmente deve acarretar em menor superfície de desenvolvimento para o fungo. O segundo melhor arroz, o canjicão, com produção de 3,42 x 109 conídios.g-1, teve a maior quantidade de fungos contaminantes nos grãos crus. Quantidades intermediárias de conídios foram produzidas pelos arrozes branco polido irrigado, de terras altas e orgânico. O integral foi o que resultou na menor quantidade de conídios (1,53 x 109 conídios.g-1), sendo o mais rico em minerais, proteína bruta e extrato etéreo. Nenhum dos aditivos (farelo de soja, grãos de soja partidos, extrato de soja, peptona de soja, inibidores de proteinases de soja semipurificados e purificado do tipo Kunitz, polpa cítrica e levedura) resultou em aumento na produção de conídios em comparação com o arroz parboilizado sem aditivos. Os conídios produzidos em todos os arrozes e aditivos apresentam viabilidade superior a 99%. As vantagens da utilização do arroz parboilizado levando-se em consideração custo, facilidade de manuseio e produtividade são discutidas. / In order to optimize the production process of Metarhizium anisopliae, the present study aimed to determine the effects of soybean proteinase inhibitors on growth, sporulation and virulence of the fungus and to compare yield, viability and virulence of M. anisopliae conidia produced in different types of rice and additives. The addition of 5 g.L-1 of semi-purified soybean proteinase inhibitor or 0.5 g.L-1 of Kunitz-type inhibitor purified on the culture medium ME resulted in large increases in sporulation (two to 75 times) without affecting the viability of conidia of four M. anisopliae isolates (ESALQ-1037, IBCB348, E9, F20). The presence of proteinase inhibitors altered morphology of conidia produced in ME. Mechanisms responsible for these physiological changes in the fungus have not been determined, but it is probably associated to an anti-nutritional action, reducing the absorption of protein and stimulating sporulation. Spores produced in the medium with the addition of 0.5 g.L-1 Kunitz-type inhibitor purified or 2.5 g.L-1 of bovine serum albumin (BSA) and PDA medium showed higher virulence of conidia produced in ME without inhibitors. When the Kunitz-type inhibitor was added to conidial suspension of the fungus before spraying larvae of Diatraea saccharalis, control efficiency was 35.1% lower than that presented in other inhibitor-free treatments. In the studies aiming to determine the best types of rice for production of M. anisopliae, we tried to correlate production of conidia with characteristics of these substrates such as nutritional content, pesticide residues and density of microorganisms. The parboiled rice was responsible for greater production of conidia (4.38 x 109 conidia.g-1). This type of rice showed crude protein content lower than most rice and the highest moisture content (41.3% after autoclaving). Besides that, grains became less gelatinous and loose after autoclaving, and these feature favored fungus production. While types of polished white, brown rice and course (broken) rice grain were sticky and formed clumps, providing a smaller area for fungus development. The second best rice, course rice grain, with production of 3.42 x 109 conidia.g-1, had the highest amount of fungal contaminants in raw grains. Intermediate amounts of conidia were produced by white irrigated polished rice, upland rice and organic rice. The brown rice was the kind that resulted in fewer conidia (1.53 x 109 conidia.g-1), being the richest in minerals, protein and lipids. None of the additives (soybean meal, soybean parties, soy extract, soy peptone, semi-purified soybean proteinase inhibitor, Kunitz-type inhibitor purified, citrus pulp and yeast) resulted in increased production of conidia compared to parboiled rice without additives. Conidia produced in all types of rice and additives presented viability greater than 99%. The advantages of the use of parboiled rice taking into consideration the cost, easy handling and productivity are discussed.

Zusätzlich zu seiner Funktion innerhalb des Gerinnungssystems vermittelt Thrombin inflammatorische Reaktionen. Mastzellen (MZ) sind durch die Freisetzung von proinflammatorischen Mediatoren wie Maus-Mastzell Proteasen (MCPTs mouse mast cell proteases auch als mMCPs bekannt), Zytokinen und Chemokinen. charakterisiert. Da Thrombinrezeptoren, auch als Proteinase-aktivierbare Rezeptoren (PAR) bekannt, von MZ exprimiert werden, wurde untersucht ob eine MZ-Aktivierung über die Thrombin/PAR Interaktion bei einer dermalen Entzündung eine Rolle spielt. Die intrakutane Injektion von Thrombin in die Ohren von C57BL/6 Kit+/+ Mäusen löste eine sofortige kutane Entzündung, einhergehend mit einer starken Ohrschwellung, aus. Im Vergleich dazu war diese Schwellung in MZ-defizienten C57BL/6 KitW-sh/W-sh Mäusen deutlich stärker, was darauf hindeutet, dass MZ anti-inflammatorisch wirken. Die lokale Rekonstitution von C57BL/6 KitW-sh/W-sh Mäusen mit knochenmarksgenerierten MZ normalisierte diesen Effekt. Die quantitative histomorphometrische Untersuchung der MZ bestätigte zusätzlich eine starke Degranulation der MZ nach Thrombininjektion nach. PCR-Analysen der MZ wiesen die Expression aller bekannten Thrombin-Rezeptoren. Die Stimulation mit verschiedenen Konzentrationen von Thrombin oder PAR-agonistischen Peptiden führte zu einer dosis-abhängigen Degranulation der MZ, was nahe legt, dass die Degranulation der MZ für die Limitierung der thrombin-induzierten Entzündung nötig ist. Gestützt wird die Hypothese durch die Tatsache, dass Zellkulturüberstand von degranulierten MZ zu einer Inaktivierung der Thrombinaktivität führt. Des Weiteren führte die Injektion von Thrombin in die Ohren von MCTP4-defizienten Mäusen zu einer deutlich erhöhten Ohrschwellung im Vergleich zur korrespondierenden Wildtyp-Maus. Zusammengenommen zeigen die Ergebnisse, dass die sofortige thrombin-induzierte Entzündungsreaktion durch kutane MZ kontrolliert wird. Dieser Mechanismus wird teilweise durch MCPT4 vermittelt. / In addition to its function in the coagulation system, thrombin mediates inflammatory reactions. Mast cells (MCs) are characterized by releasing inflammatory mediators like mouse mast cell proteases (MCPTs, also designated mMCPs), cytokines, and chemokines, upon activation. Since thrombin-receptors, also known as Proteinase-activated receptors (PAR), are expressed by MCs, it was questioned whether MC activation via the thrombin/PAR axis plays a role in skin inflammation. Intracutaneous injection of thrombin in ears of C57BL/6 Kit+/+ mice induced immediate inflammatory skin reactions associated with a distinct ear swelling. This swelling was more pronounced in MC-deficient C57BL/6 KitW-sh/W-sh mice, indicating that MCs are anti-inflammatory, local reconstitution of C57BL/6 KitW-sh/W-sh mice with C57BL/6 Kit+/+ bone marrow-derived MCs normalized this effect. Additionally thrombin injection resulted in a strong degranulation of MCs assessed by quantitative histomorphometry. PCR analysis of MCs displayed expression of all known thrombin receptors. Stimulation with thrombin or PAR agonistic peptides resulted in a dose-dependent degranulation of MC, suggesting MC degranulation could be necessary for the limitation of thrombin-induced inflammatory responses. Supporting this hypothesis, supernatant from degranulated MCs inactivated thrombin activity. Furthermore, injection of thrombin in ears of C57BL/6 MCPT4-deficient mice, resulted in markedly increased ear swelling compared to the corresponding wild type mice. Together our results suggest that thrombin-induced immediate inflammatory skin reactions are controlled by cutaneous MCs, a mechanism partly mediated via MCPT4.

A resistência aos antibióticos adquirida por micro-organismos patogênicos é um problema de saúde mundial e, por isso, o desenvolvimento de novos agentes antimicrobianos vem sendo amplamente estimulado. Sabendo que muitos peptídeos bioativos correspondem a fragmentos peptídicos de proteínas e/ou seus análogos, este trabalho teve o objetivo de desenvolver novos peptídeos antimicrobianos (AMPs) a partir das sequências aminoacídicas e das estruturas 3D de proteínas possivelmente envolvidas na atividade antimicrobiana de venenos de serpentes pouco estudados. As etapas iniciais seguidas foram: a) escolher uma fosfolipase A2 (PLA2) de veneno de serpente peruana do gênero Bothrops da família Viperidae com sequência de aminoácidos conhecida e modelar por homologia a sua estrutura 3D; b) verificar atividade antimicrobiana em venenos de serpentes peruanas dos gêneros Bothrops e Bothriopsis da família Viperidae, selecionar um veneno ativo, fracioná-lo para isolar proteínas provavelmente envolvidas nessa atividade, tripsinizar as proteínas isoladas, sequenciar os fragmentos trípticos para identificá-las, localizar esses fragmentos em sequências aminoacídicas de proteínas com estruturas 3D disponíveis correlatas às proteínas isoladas/identificadas em classe, função e fonte natural. Em seguida, foram escolhidos fragmentos peptídicos da PLA2 (item a) e das proteínas isoladas do veneno ativo (item b) e/ou desenhados análogos que apresentassem características exibidas por AMPs conhecidos. Os peptídeos desenhados foram sintetizados, purificados, caracterizados e testados em suas atividades antimicrobianas. Os modelos estruturais 3D da PLA2 de Bothrops pictus e quatro peptídeos (PLA2-1 a -4) amidados derivados dela foram obtidos, sendo o PLA2-1 ativo frente a Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida krusei e Candida parapsilosis (MICs de 6,25-200 µmol.mL-1). Dos três venenos de serpentes peruanas testados, Bothrops taeniatta, Bothrops barnetti e Bothriopsis oligolepis, os dois últimos inibiram o crescimento de S. aureus (MICs 0,78-50 µmol.mL-1), mas apenas B. oligolepis demonstrou espectro de ação amplo. O seu fracionamento sequencial, acompanhado de ensaios de inibição do crescimento de S. aureus, gerou frações ativas relativamente homogêneas que, tripsinizadas e os fragmentos trípticos sequenciados, continham metalo-peptidases do tipo III, serino-peptidase ou lectinas do tipo C. A verificação de atividade enzimática e de coagulação sanguínea nessas frações confirmaram as naturezas das proteínas isoladas. Dos três peptídeos amidados (Bo-Ser1, Bo-Met1 e Bo-Lec1) desenhados a partir de suas estruturas, um deles foi ativo frente às leveduras C. albicans, C. krusei e C. parapsilosis (Bo-Met1; MIC de 6,25 - 200 µmol.mL-1). Pela primeira vez, foi demonstrado que: a) os venenos das serpentes peruanas B. barnetti e B. oligolepis apresentam ação antimicrobiana, sendo o último de espectro amplo; b) que as proteínas acima citadas, que incluem uma serino-peptidase, estão envolvidas com essa propriedade do veneno de B. oligolepis; c) que as sequências aminoacídicas e modelo 3D de uma PLA2 ácida e de proteínas presentes nos venenos das serpentes peruanas B. pictus e Bothriopsis oligolepis podem funcionar como fontes naturais para o desenvolvimento de novos AMPs de ação potente em micro-organismos de interesse clínico e científico. / Resistance to antibiotics obtained by pathogenic microorganisms is a global health problem, so the search for new antimicrobial agents has been encouraged. Knowing that many protein fragments and analogues are bioactive peptides, the aim of this work was to develop new antimicrobial peptides (AMPs) based on the amino acid sequences and 3D structures of proteins apparently involved in the antimicrobial activity of snake venoms very little or not studied so far. The first steps taken were: a) selection of a phospholipase A2 (PLA2) present in the venom from a Peruvian Bothrops sp. belonging to the family Viperidae, whose amino acid sequence was known, to model by homology its 3D structure; b) detection of antimicrobial activity in venoms from other Peruvian Viperidae Bothrops and Bothriopsis snakes, selection of an active venom, fractionation of it for isolation of proteins possibly involved in the antimicrobial activity, trypsinization of the isolated proteins, sequencing of the tryptic fragments for protein identification, location of such fragments in the amino acid sequences and 3D structures of proteins directly related in class, function and natural source to the isolated proteins. Then, peptide fragments from the chosen PLA2 (item a) and from the isolated proteins (item b) that presented structural features found in the known AMPs were selected and/or their analogues were designed. Finally, synthesis, purification and characterization of the peptides with AMP potential, (viii) verification on whether or not they display antimicrobial activity. The 3D-structure models of Bothrops pictus PLA2 and four amidated peptides (PLA2-1 to -4) derived from it were obtained, being PLA2-1 active against Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as the yeasts Candida albicans, Candida krusei and Candida parapsilosis (MICs de 6.25-200 µmol.mL-1). Among the three Peruvian snake venoms tested Bothrops taeniatta, Bothrops barnetti and Bothriopsis oligolepis, the last two inhibited the growth of S. aureus (MICs 0.78-50 µmol.mL-1) and B. oligolepis presented a wide spectrum of bacterial action. Sequential fractionation followed by S. aureus growth inhibition assays of the main fractions led to active relatively homogeneous ones. Their trypsinization and sequencing of the tryptic fragments indicated that they contained metalloproteinases type III, serine-proteinase or lectins type CTL. Enzymatic activity and blood coagulation assays confirmed the nature of the isolated proteins. From the three amidated peptides (Bo-Ser1, Bo-Met1 e Bo-Lec1) derived from them, Bo-Met1 showed to be active against C. albicans, C. krusei e C. parapsilosis (MIC 6,25 - 200 µmol.mL-1). In summary, for the first time, it was demonstrated that: a) the venoms of the Peruvian snakes B. barnetti and B. oligolepis display antimicrobial activity, being the last of wide spectrum of action, b) the proteins isolated from B. oligolepis snake venom, including a serine-peptidase, are involved in the antimicrobial activity of the B. oligolepis snake venom, c) the amino acid sequences and 3D structures of acidic PLA2 and of other proteins found in the venoms of the Peruvian B. pictus e Bothriopsis oligolepis snakes can be used as safe and natural sources for the development of new AMPs potent against microorganisms of clinical and scientific interest.

Com o intuito de gerar subsídios para melhoria do processo de produção de Metarhizium anisopliae, o presente estudo teve como principais objetivos, determinar os efeitos de inibidores de proteinases de soja no crescimento vegetativo, esporulação e virulência do fungo e comparar a produção, viabilidade e virulência dos conídios produzidos em diferentes tipos de arroz e aditivos. A adição de 5 g.L-1 de inibidores de proteinases semi-purificados de soja ou 0,5 g.L-1 de inibidor purificado do tipo Kunitz no meio de cultura ME resultou em grande aumento na esporulação (de duas a 75 vezes) sem afetar a viabilidade dos conídios de quatro isolados (ESALQ-1037, IBCB348, E9, F20) de M. anisopliae. A presença destes inibidores de proteinases também alterou a morfologia dos conídios produzidos em ME. Os mecanismos responsáveis por estas alterações fisiológicas não foram determinados, mas provavelmente estejam associados à ação anti-nutricional, diminuindo a absorção protéica e estimulando a esporulação. Os conídios produzidos no meio com adição de 0,5 g.L-1 de inibidor de proteinase do tipo Kunitz ou com 2,5 g.L-1 de albumina de soro bovino e no meio BDA apresentaram virulência superior aos conídios produzido no meio ME sem inibidores. Quando o inibidor de proteinase do tipo Kunitz foi adicionado à suspensão de conídios do fungo antes da pulverização de lagartas de Diatraea saccharalis, a eficiência de controle foi 35,1%, inferior ao apresentado pelos demais tratamentos sem a presença deste inibidor. Nos estudos para determinação dos melhores tipos de arroz para produção de M. anisopliae, tentou-se correlacionar à produção de conídios com características destes substratos como valor nutricional, teor de resíduos de agrotóxicos e densidade de microorganismos. O arroz parboilizado foi responsável pela maior produção de conídios (4,38 x 109 conídios.g-1). Este tipo de arroz apresentou teor de proteína bruta menor do que a maioria dos arrozes e o maior teor de umidade (41,3% após a autoclavagem). Além disto, os grãos após autoclavagem ficaram menos gelatinosos e mais soltos, o que facilita o processo de produção do fungo. Enquanto que os arrozes dos tipos brancos polidos, canjicão e integral ficaram pegajosos e formaram grumos, o que provavelmente deve acarretar em menor superfície de desenvolvimento para o fungo. O segundo melhor arroz, o canjicão, com produção de 3,42 x 109 conídios.g-1, teve a maior quantidade de fungos contaminantes nos grãos crus. Quantidades intermediárias de conídios foram produzidas pelos arrozes branco polido irrigado, de terras altas e orgânico. O integral foi o que resultou na menor quantidade de conídios (1,53 x 109 conídios.g-1), sendo o mais rico em minerais, proteína bruta e extrato etéreo. Nenhum dos aditivos (farelo de soja, grãos de soja partidos, extrato de soja, peptona de soja, inibidores de proteinases de soja semipurificados e purificado do tipo Kunitz, polpa cítrica e levedura) resultou em aumento na produção de conídios em comparação com o arroz parboilizado sem aditivos. Os conídios produzidos em todos os arrozes e aditivos apresentam viabilidade superior a 99%. As vantagens da utilização do arroz parboilizado levando-se em consideração custo, facilidade de manuseio e produtividade são discutidas. / In order to optimize the production process of Metarhizium anisopliae, the present study aimed to determine the effects of soybean proteinase inhibitors on growth, sporulation and virulence of the fungus and to compare yield, viability and virulence of M. anisopliae conidia produced in different types of rice and additives. The addition of 5 g.L-1 of semi-purified soybean proteinase inhibitor or 0.5 g.L-1 of Kunitz-type inhibitor purified on the culture medium ME resulted in large increases in sporulation (two to 75 times) without affecting the viability of conidia of four M. anisopliae isolates (ESALQ-1037, IBCB348, E9, F20). The presence of proteinase inhibitors altered morphology of conidia produced in ME. Mechanisms responsible for these physiological changes in the fungus have not been determined, but it is probably associated to an anti-nutritional action, reducing the absorption of protein and stimulating sporulation. Spores produced in the medium with the addition of 0.5 g.L-1 Kunitz-type inhibitor purified or 2.5 g.L-1 of bovine serum albumin (BSA) and PDA medium showed higher virulence of conidia produced in ME without inhibitors. When the Kunitz-type inhibitor was added to conidial suspension of the fungus before spraying larvae of Diatraea saccharalis, control efficiency was 35.1% lower than that presented in other inhibitor-free treatments. In the studies aiming to determine the best types of rice for production of M. anisopliae, we tried to correlate production of conidia with characteristics of these substrates such as nutritional content, pesticide residues and density of microorganisms. The parboiled rice was responsible for greater production of conidia (4.38 x 109 conidia.g-1). This type of rice showed crude protein content lower than most rice and the highest moisture content (41.3% after autoclaving). Besides that, grains became less gelatinous and loose after autoclaving, and these feature favored fungus production. While types of polished white, brown rice and course (broken) rice grain were sticky and formed clumps, providing a smaller area for fungus development. The second best rice, course rice grain, with production of 3.42 x 109 conidia.g-1, had the highest amount of fungal contaminants in raw grains. Intermediate amounts of conidia were produced by white irrigated polished rice, upland rice and organic rice. The brown rice was the kind that resulted in fewer conidia (1.53 x 109 conidia.g-1), being the richest in minerals, protein and lipids. None of the additives (soybean meal, soybean parties, soy extract, soy peptone, semi-purified soybean proteinase inhibitor, Kunitz-type inhibitor purified, citrus pulp and yeast) resulted in increased production of conidia compared to parboiled rice without additives. Conidia produced in all types of rice and additives presented viability greater than 99%. The advantages of the use of parboiled rice taking into consideration the cost, easy handling and productivity are discussed.

Genes de plantas que codificam inibidores de enzimas digestivas de insetos têm sido introduzidos em plantas cultivadas visando o controle de pragas. Os inibidores de proteinases estão presentes nos tecidos vegetais, principalmente nas sementes, e atuam em resposta a ataques por herbívoros e patógenos. Inibidores de serino-proteinases (IPs) dos tipos Bowman-Birk e Kunitz isolados de sementes de soja foram inseridos em variedades de cana-de-açúcar para aumentar a resistência à broca Diatraea saccharalis (Fabr.), principal praga desta cultura. Para utilização de plantas geneticamente modificadas contendo inibidores de proteinases é necessário um conhecimento profundo de sua sustentabilidade e segurança ambiental, determinando a estabilidade da característica inserida e os seus efeitos nos organismos não-alvo. O objetivo desta pesquisa foi avaliar os efeitos diretos e indiretos de inibidores de proteinases de soja em organismos não-alvos: um parasitóide larval, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); um patógeno, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); um polinizador, Apis mellifera L. (Hymenoptera: Apidae) e um decompositor, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae); associados à cultura da cana-de-açúcar. O consumo de Kunitz e BBI não afetou a sobrevivência de S. praeincisus. Por outro lado, a ingestão dos inibidores semi-purificados e purificados do tipo Kunitz diminuiu a duração das fases imaturas de S. praeincisus. A ingestão de folhas de cana GM expressando inibidores de proteinases (Kunitz e BBI) não afetou o tempo de desenvolvimento e a sobrevivência dos imaturos deste oribatídeo quando comparada à ingestão de suas isolinhas. Os inibidores de proteinases semi-purificados e purificados não afetaram a duração dos períodos larval e pupal, o peso e número de pupas e percentual de emergência do parasitóide C. flavipes em D. saccharalis. Por outro lado, a proporção de fêmeas em relação a machos de C. flavipes foi maior no tratamento onde as lagartas foram alimentadas com dieta contendo 0,5% de inibidores semi-purificados comparado-se à testemunha. A proporção fêmea:macho foi significativamente maior também quando os parasitóides foram alimentados com o inibidor do tipo Kunitz em relação ao controle e aos parasitóides alimentados com BBI. A adição de 0,5% (p/v) de inibidores de proteinases semi-purificados e 0,05% (p/v) de inibidores purificados do tipo Kunitz nos meios de cultura MC e BDA resultaram em maiores crescimento vegetativo e produção de conídios de M. anisopliae. Os inibidores purificados do tipo BBI não alteraram a esporulação do fungo. Os resultados dos estudos com A. mellifera não foram conclusivos e novas investigações precisam ser conduzidas para esclarecer os potenciais efeitos de inibidores de proteinases em abelhas. De uma forma geral, observou-se que os inibidores de proteinases (Kunitz e BBI) não afetaram negativamente os organismos não-alvo testados. Por outro lado, a ingestão de inibidor do tipo Kunitz alterou positivamente alguns parâmetros biológicos de C. flavipes, M. anisopliae e S. praeincisus. / Genes of plants expressing insect proteinase inhibitors have been introduced into plants for pest control. Proteases inhibitors are present in plant tissues, mainly in seeds, and act in response to predators and pathogens. The Bowman-Birk type and Kunitz type of serine proteases inhibitors (PI) from soybean seeds are been used to increase resistance of sugarcane to Diatraea saccharalis (Fabr.), the most important pest of this crop. The sustainability and environmental safety of PI crops is still unknown. For these reasons, it is necessary to understand the stability and the non-target effects of this new trait. The objective of this study was to evaluate the direct and indirect effects of soybean PI on the following non-target organisms associated to sugarcane: the larval parasitoid, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); the entomopathogen, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); the pollinator Apis mellifera L. (Hymenoptera: Apidae) and the soil mite involved in the process of recycling organic matter, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae). Kunitz and BBI did not affect S. praeincisus survival. On the other hand, Kunitz semi-purified and purified inhibitor ingestion reduced duration of the immature stages of S. praeincisus. Ingestion of GM senescent leaves did not have an effect on mite immatures development time and survival compared to ingestion of its isolines leaves. The semi-purified and purified proteinases inhibitors did not alter either the duration of larval and pupal stages of C. flavipes on D. saccharalis, or weight and number of pupae and parasitoid emergence. In other hand, the parasitism and proportion of female was higher on the treatment where caterpillars were fed with diet containing 0.5% of semi-purified inhibitors, comparing to control. The ratio female:male was significantly higher also when parasitoids were fed to the Kunitz type inhibitor compared to the control and BBI. The addition of 0.5 % (w/v) of semi- purified proteinase inhibitors and 0.05% (w/v) of Kunitz type purified inhibitors on two culture media (CM and PDA), resulted in increase of vegetative growth and production of conidia. BBI type purified inhibitors did not change the fungus sporulation. The results from the studies with A. mellifera were not conclusive and investigations are needed to clarify the potential impact of proteinase inhibitors on A. mellifera. Overall, proteinase inhibitors (Kunitz and BBI) did not negatively affect the non-target organisms tested. Conversely, ingestion of the Kunitz type of proteinase inhibitors altered positively some biological parameters of C. flavipes, M. anisopliae and S. praeincisus.

Yeasts are traditionally used as part of the surface microflora in surface-ripened cheeses, where they contribute positively to the flavour of the cheese. The primary objective of this study was to investigate the potential of three dairy yeasts to provide attributes as adjuncts in anaerobically ripened cheeses. Geotrichum candidum (B9001), Yarrowia lipolytica (B9014) and Candida kefyr (B9006), obtained from the Fonterra Co-operative Group Ltd, Palmerston North, New Zealand, were studied. They showed diverse metabolic activities in laboratory media, which were influenced by the growth conditions. The metabolic activities of special interest were the lipase and proteinase activities and the production of volatile compounds, as these are important for cheese ripening and flavour development. Lipase activity (p-nitrophenyl butyrate assay) and proteinase activity (fluorescein isothiocyanate β-casein assay) were determined in three fractions prepared from yeast cultures and designated as extracellular fraction, washed-cell fraction and intracellular fraction. Lipase activity of G. candidum was detected only in the extracellular fraction and increased five fold when induced by safflower oil in a shake culture (0.16 µM/min/mL supernatant at 24 h). Lipase expression was delayed in static cultures. Y. lipolytica showed lipase activity in extracellular, washed-cell and intracellular fractions under all conditions. Static cultures in both glucose and safflower oil media showed higher lipase activity than shake cultures. The lipase activity of Y. lipolytica was higher in the late stationary phase than in the log phase under all conditions tested. The highest lipase activity was detected in a 192 h static culture grown in safflower oil medium (0.13 µM/min/mg dry cell weight, 0.3 µM/min/mg dry cell weight and 4.29 µM/min/mL supernatant in the intracellular, washed-cell and extracellular fractions respectively). C. kefyr did not show any lipase activity (< 0.03 µM/min/mL culture) under any of the growth conditions tested. Proteinase activity was detected in the intracellular fraction of 72 h shake cultures of G. candidum grown in both glucose medium and safflower oil medium (154 and 122 RFU/min/mg dry cell weight respectively) but was not detected in static cultures. Proteinase activity was absent in the Y. lipolytica cultures under all conditions tested (< 10 RFU/min/mL culture). C kefyr showed low proteinase activity (12-74 RFU/min/mL supernatant) in the extracellular fraction only in shake cultures grown in glucose medium. Volatile compounds of the headspace were sampled and analysed using solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). The concentrations of volatile compounds were highest in shake cultures grown in glucose medium for all three yeasts. All yeasts produced several alcohols. Several esters were also detected in the G. candidum and C. kefyr cultures whereas aldehydes were detected only in the G. candidum cultures. G. candidum and Y. lipolytica were selected for cheese production trials because of their active cheese ripening enzymes. These yeasts, grown under different conditions, were added to Cheddar cheese (10 L vat). The yeast adjuncts influenced the cheese ripening by lipolysis [in terms of the production of free fatty acids (FFAs) analysed by gas chromatography-flame ionisation detector (GC-FID)] and the production of volatile compounds (SPME-GC-MS), whereas proteolysis (analysed by size-exclusion high performance liquid chromatography) by yeast enzymes was not obvious. The influence of Y. lipolytica as an anaerobic adjunct to cheese ripening was dependent on the growth conditions used during its propagation in laboratory media. The concentration of total FFAs was very high (37.1 mg/g cheese at 6 months) when a 192 h Y. lipolytica culture grown in safflower oil medium was added to a cheese make, whereas the cultures grown in glucose medium did not have any detectable effect. Addition of G. candidum culture to the cheese curd was more effective than its addition to the cheese milk. Both G. candidum and Y. lipolytica lipase(s) selectively hydrolysed the long-chain unsaturated fatty acids from the milk triglyceride in the cheese environment. Also, Y. lipolytica lipase exhibited some selectivity towards hydrolysis of butyric acid from the milk fat in the cheese. 2-Heptanone, 3-methyl-2-butanone and 2-nonanone were detected (1-10 x 106 relative peak area) only in the cheeses with yeast adjuncts but not in the control cheese. Enhancement of the production of both conjugated linoleic acid (CLA) and ethyl esters in a washed-curd, dry-salted cheese (375 L vat), made with G. candidum, Y. lipolytica, Propionibacterium freudenreichii ssp. shermanii, Lactobacillus fermentum and Lb. rhamnosus, was only partially successful. Higher concentrations of ethyl esters (> five fold; analysed by SPME-GC-MS) were produced in the cheeses made with yeast adjuncts. However, the concentration of total CLA (free plus esterified; analysed by GC-FID) did not increase although a higher concentration of free linoleic acid (> 10 fold), the substrate for CLA synthesis, was produced in the cheeses made with yeast adjuncts. A study of the formation of aromatic volatile compounds by C. kefyr in a medium containing L-phenylalanine (L-phe) showed that the yeast's ability to produce phenyl ethanol, phenyl ethyl acetate and benzaldehydc (analysed by SPME-GC-MS) was enhanced with an increase in the initial L-phe concentration (in the experimental range; analysed by enzymatic assay using phenylalanine ammonia lyase), but the yield was very low (20-27%). The initial concentration of glucose (in the experimental range; analysed by enzymatic assay using Peridochrom glucose reagent) did not affect the production of these aromatic volatile compounds. This study successfully showed that the yeasts G. candidum and Y. lipolytica, when used as anaerobic adjuncts, can influence the ripening and flavour development in Cheddar and washed-curd, dry-salted cheeses. The study also showed the capability of C. kefyr to produce aromatic volatile compounds from amino acid fermentation but the yields need to be increased by further manipulation of the medium components and the culture conditions before this capability can be used commercially.

MONTEIRO JUNIOR, José Edvar. Caracterização bioquímica, funcional e estrutural de duas cistatinas recombinantes de feijão-caupi. 2012. 179 f.Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-20T14:56:24Z No. of bitstreams: 1 2012_tese_jemonteirojunior.pdf: 22956458 bytes, checksum: 4912cb5f52b152df0d6dcc912bb7ccef (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:37:33Z (GMT) No. of bitstreams: 1 2012_tese_jemonteirojunior.pdf: 22956458 bytes, checksum: 4912cb5f52b152df0d6dcc912bb7ccef (MD5) / Made available in DSpace on 2016-08-02T20:37:33Z (GMT). No. of bitstreams: 1 2012_tese_jemonteirojunior.pdf: 22956458 bytes, checksum: 4912cb5f52b152df0d6dcc912bb7ccef (MD5) Previous issue date: 2012 / mRNA sequences coding for two cystatins were isolated from leaves of hydroponically growing cowpea plants. The sequences were ligated in the expression vector pET302NT-HIS, which was introduced into Escherichia coli, ArcticExpress (DE3) cells. The expression was induced by addition of 0.5 mM IPTG and the recombinant proteins VuCys1 (Vigna unguiculata one-domain cystatin) and VuCys2 (Vigna unguiculata two-domain cystatin), both fused to an N-terminal 6x-His tag, were purified by homogeneity using an affinity matrix containing Ni2+ immobilized to Sepharose. The apparent molecular masses for VuCys1 (yield of 10 mgP.L-1 culture) and VuCys2 (yield of 22 mgP.mL-1 culture) were 14 and 26 kDa, respectively. Both inhibitors were strongly active against the proteinases papain and chymopapain, while bromelain and particularly cathepsin B were less susceptible to in vitro inhibition. No inhibition was detected against the serine proteinase trypsin. Thermal stability tests revealed that both cystatins are thermostable proteins able to achieve a minimum residual proteolytic inhibition activity against papain of 95% (VuCys1) and 85% (VuCys2) when incubated at 100 C for 60 minutes. Similar stability results were also obtained when the proteins were tested to the ability of to inhibit papain activity after incubation in pH values ranging from 2.0 to 11.0. Circular dichroism spectroscopy measurements demonstrated that the secondary structure arrangement of both cystatins undergoes only fewer alterations when both proteins were incubated in temperatures varying from 10 to 90 C and pH values varying from 2.0 to 11.0, as well. These data are in agreement with the thermal and pH stability results previously obtained on papain inhibition assays. Biological assays conducted with different phytopathogenic fungi didn’t show any negative impact on spore germination as well as on mycelial growth of the tested fungi. Different human pathogens, including the pathogenic yeast Candida albicans were shown to be insensible to VuCys1 and VuCys2. Some scientific reports have proposed the use of cystatins as potential molecules in the control and inhibition of the activity of cysteine proteinases related to carcinogenic process. However, cytotoxicity assays performed on three tumor cell lineages revealed no toxic effects of VuCys1 and VuCys2. Inhibitor activities were also tested against digestive enzymes isolated from third instar larvae of the bruchid insects Callosobruchus maculatus and Zabrotes subfasciatus, two major cowpea plagues. Both cystatins were able to cause high inhibition of C. maculatus enzymes; however they were poorly active against Z. subfasciatus counterpart. Feeding tests were conducted in which both cystatins were added to artificial seeds at final concentrations of 0.025, 0.05 and 0.1% and supplied to C. maculatus. Despite the in vitro inhibition of C. maculatus larvae enzymes, the bioassay data suggest that larvae and adult insects appear to develop adaptive mechanisms which can make them insensible to the ingestion of the inhibitors. Crystallographic studies were carried out in order to solve the tridimensional structure of cystatins. 576 different crystallization conditions were tested in which three were favorable to the formation and growth of diffractable crystals of VuCys1 protein. These crystals belong to the orthorhombic space group P212121 and the unit cell dimension was a = 41.48 Å; b = 64.68 Å and c = 87.91 Å, α = β = γ = 90. V. unguiculata one-domain cystatin presents a typical 3D domain swapped dimmer molecular structure, which was solved at 1.95 Å resolution. No crystals were obtained for VuCys2. The physiological importance of this structure to the plant, the structural stability of both inhibitors and the results raised from biological assays are all discussed in this work. / Sequências de mRNA que codificam para duas cistatinas foram isoladas de folhas de plantas de feijão-caupi cultivadas em sistema hidropônico. As sequências foram ligadas no vetor de expressão pET302NT-His, o qual foi introduzido em células de Escherichia coli, ArcticExpress (DE3). A indução da expressão foi realizada por meio de adição de IPTG (0,5 mM) e as proteínas recombinantes VuCys1 – (cistatina de um domínio de Vigna unguiculata) e VuCys2 – (cistatina de dois domínios de V. unguiculata) ambas fusionadas a uma cauda de histidina N-terminal, foram purificadas por homogeneidade em matriz de afinidade constituída de Ni2+ imobilizado à Sepharose. VuCys1 apresentou uma massa molecular aparente de 14 kDa e um rendimento de 10 mgP.L-1 de meio de cultura, já a proteína VuCys2 mostrou uma massa molecular aparente de 26 kDa e um rendimento de 22 mgP.L-1 de meio de cultura. As duas proteínas foram fortemente ativas contra as proteinases papaína e quimopapaína, moderadamente ativas contra bromelaína e apenas fracamente ativas contra catepsina B, enquanto que nenhuma atividade inibitória foi detectada contra a proteinase serínica tripsina. Ensaios de estabilidade térmica mostraram que as duas cistatinas são proteínas termoestáveis, uma vez que, mesmo após incubação a 100 C por 60 minutos apresentam atividade inibitória residual contra papaína superior a 95% (VuCys1) e superior a 85% (VuCys2). Resultados similares de estabilidade também foram obtidos quando as proteínas foram testadas quanto à capacidade de inibir a atividade de papaína, após incubação em valores de pH variando de 2,0 a 11,0. Análises espectroscópicas de dicroísmo circular revelaram que o padrão de estruturas secundárias de ambos inibidores sofre pouca alteração após incubação em temperaturas variando de 10 a 90 C e em valores de pH variando de 2,0 a 11,0. Estes dados estão de acordo com os resultados de elevada estabilidade térmica e a extremos de pH previamente obtidos nos ensaios de inibição in vitro de papaína. Ensaios biológicos realizados com diferentes espécies de fungos fitopatogênicos não mostraram nenhum efeito negativo das proteínas sobre a germinação de esporos ou crescimento micelial dos fungos testados. Os inibidores também não se mostraram ativos contra diferentes patógenos humanos, incluindo a levedura patogênica Candida albicans. Alguns relatos científicos propõem o uso de cistatinas como agentes em potencial no controle e inibição da atividade de proteinases cisteínicas relacionados a processos carcinogênicos. Contudo, testes de citotoxicidade dos inibidores para três diferentes linhagens de células tumorais não mostraram potencial citotóxico. Os inibidores também foram testados quanto à capacidade de inibir a atividade de enzimas digestivas isoladas do intestino de larvas de terceiro instar dos insetos bruquídeos Callosobruchus maculatus e Zabrotes subfasciatus, duas importantes pragas do feijão-caupi. Ambas as cistatinas apresentaram elevado potencial inibitório contra as enzimas de C. maculatus sendo, porém, fracamente ativas contra as de Z. subfasciatus. Bioensaios foram realizados nos quais as cistatinas foram inseridas em sementes artificiais nas concentrações finais de 0,025; 0,05 e 0,1% e administradas a C. maculatus. A despeito da inibição in vitro das enzimas digestivas das larvas de C. maculatus, os resultados do bioensaio sugerem que, tanto larvas como insetos adultos, parecem desenvolver mecanismos adaptativos à administração dos inibidores que os tornam insensíveis à sua ingestão. Estudos cristalográficos foram realizados na tentativa de solucionar a estrutura tridimensional das cistatinas. 576 condições de cristalização foram testadas das quais três levaram à formação e crescimento de cristais difratáveis de VuCys1. Os cristais pertencem ao grupo espacial ortorrômbico, P212121, e a célula unitária apresentou dimensões de a = 41,48; b = 64,68 e c = 87,91 Å, α = β = γ = 90. VuCys1 apresenta uma estrutura molecular de dímero de domínios trocados a qual foi resolvida a uma resolução de 1,95 Å. Cristais de VuCys2 não foram obtidos nas condições testadas. O significado fisiológico desta estrutura para a planta, a estabilidade estrutural de ambos inibidores e os resultados referentes aos diferentes bioensaios são discutidos no presente trabalho.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-23T14:07:21Z No. of bitstreams: 1 Andresa Cristina Batista de Oliveira.pdf: 456966 bytes, checksum: 512be8b21cb03d0d47d281c2432f28cf (MD5) / Made available in DSpace on 2016-11-23T14:07:21Z (GMT). No. of bitstreams: 1 Andresa Cristina Batista de Oliveira.pdf: 456966 bytes, checksum: 512be8b21cb03d0d47d281c2432f28cf (MD5) Previous issue date: 2011-02-01 / The insects’ midgut pH has a great influence on the activity of Cry toxins. Some toxins are activated under alkaline or acidic conditions. The proteolytic processing of Cry toxins is a crucial step in their activation and information about distribution and interaction of these toxins on target and non-target insects are important to explain how proteolytic activity of digestive enzymes contributes to the tolerance of natural enemies to Cry toxins. Furthermore, studies have shown that insects can adapt to Bacillus thuringiensis (Berliner) toxins under field and laboratory conditions. Because Alabama argillacea (Hübner) (Lep.: Noctuidae), is a monophagous species with short life cycle, it poses a risk of developing resistance to the toxins of B. thuringiensis . The knowledge about the different mechanisms of resistance to these toxins is important to extend the usefulness of commercial products based on Bt. The proteinase activity of the digestive tract of Podisus nigrispinus (Dallas) (Hem.: Pentatomidae), proteolytic capacity of these on Cry1Ac and cellular and humoral immune response of larvae of A. argillacea towards commercial formulations based on B. thuringiensis, were tested. The results indicated that the major enzymes of the digestive tract of P. nigrispinus are serine-proteases and cysteine proteases that work in a neutral to mildly acidic pH and apparently digestive enzymes were not able to process the Cry1Ac toxin within 12 hours of incubation. 4th instar larvae of A. argillacea do not have potential to develop tolerance to Bt formulated based on immunological responses. Dipel® led to quantitative variation in all cell types while XenTari® changed prohemocytes, plasmatocytes, granulocytes and oenocytoids. In insects treated with Dipel® was no increase in the level of nitric oxide. Differences in responses to treatments can be attributed to different composition of the insecticides, Dipel ® had a more effective and faster to control A. argillacea. / O pH do intestino médio dos insetos tem grande influência na atividade das toxinas Cry que podem ser ativadas em condições alcalinas ou ácidas. O processamento proteolítico das toxinas Cry é uma etapa crucial na sua ativação e informações sobre a distribuição e interação dessas toxinas sobre insetos alvo e não-alvo, são de valor para explicar como a ação proteolítica de enzimas digestivas contribui para a tolerância de inimigos naturais às toxinas Cry. Estudos têm mostrado que insetos podem adaptar-se às toxinas de Bacillus thuringiensis (Berliner) sob condições de campo e laboratório. Alabama argillacea (Hübner) (Lep.: Noctuidae), por ser uma espécie monófaga com ciclo de vida curto, apresenta risco de desenvolver resistência às toxinas de B. thuringiensis. O conhecimento dos diferentes mecanismos de resistência a essas toxinas é importante para prolongar a utilidade de produtos comerciais à base de Bt. A atividade de proteinases do tubo digestivo de Podisus nigrispinus (Dallas) (Hem.: Pentatomidae), capacidade proteolítica dessas sobre Cry1Ac e a resposta imunológica celular e humoral de lagartas de A. argillacea frente às formulações comerciais à base de B. thuringiensis, foram testadas. Os resultados indicaram que as principais enzimas do tubo digestivo de P. nigrispinus são serinoproteases e proteases cisteínicas que atuam em pH levemente ácido até neutro e que as enzimas do tubo digestivo não são capazes de degradar Cry1Ac. Lagartas do 4º ínstar de A. argillacea não apresentam potencial para desenvolverem tolerância de natureza imunológica a formulados Bt. Dipel® ocasionou variação quantitativa em todos os tipos celulares enquanto XenTari® alterou prohemócitos, plasmatócitos, granulócitos e oenocitóides. Nos insetos tratados com Dipel® houve aumento no nível de óxido nítrico. As diferenças nas respostas aos tratamentos podem ser atribuídas à diferente composição dos inseticidas testados, onde Dipel® tem uma ação mais efetiva e mais rápida no controle de A. argillacea.

A resistência aos antibióticos adquirida por micro-organismos patogênicos é um problema de saúde mundial e, por isso, o desenvolvimento de novos agentes antimicrobianos vem sendo amplamente estimulado. Sabendo que muitos peptídeos bioativos correspondem a fragmentos peptídicos de proteínas e/ou seus análogos, este trabalho teve o objetivo de desenvolver novos peptídeos antimicrobianos (AMPs) a partir das sequências aminoacídicas e das estruturas 3D de proteínas possivelmente envolvidas na atividade antimicrobiana de venenos de serpentes pouco estudados. As etapas iniciais seguidas foram: a) escolher uma fosfolipase A2 (PLA2) de veneno de serpente peruana do gênero Bothrops da família Viperidae com sequência de aminoácidos conhecida e modelar por homologia a sua estrutura 3D; b) verificar atividade antimicrobiana em venenos de serpentes peruanas dos gêneros Bothrops e Bothriopsis da família Viperidae, selecionar um veneno ativo, fracioná-lo para isolar proteínas provavelmente envolvidas nessa atividade, tripsinizar as proteínas isoladas, sequenciar os fragmentos trípticos para identificá-las, localizar esses fragmentos em sequências aminoacídicas de proteínas com estruturas 3D disponíveis correlatas às proteínas isoladas/identificadas em classe, função e fonte natural. Em seguida, foram escolhidos fragmentos peptídicos da PLA2 (item a) e das proteínas isoladas do veneno ativo (item b) e/ou desenhados análogos que apresentassem características exibidas por AMPs conhecidos. Os peptídeos desenhados foram sintetizados, purificados, caracterizados e testados em suas atividades antimicrobianas. Os modelos estruturais 3D da PLA2 de Bothrops pictus e quatro peptídeos (PLA2-1 a -4) amidados derivados dela foram obtidos, sendo o PLA2-1 ativo frente a Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida krusei e Candida parapsilosis (MICs de 6,25-200 &#181;mol.mL-1). Dos três venenos de serpentes peruanas testados, Bothrops taeniatta, Bothrops barnetti e Bothriopsis oligolepis, os dois últimos inibiram o crescimento de S. aureus (MICs 0,78-50 &#181;mol.mL-1), mas apenas B. oligolepis demonstrou espectro de ação amplo. O seu fracionamento sequencial, acompanhado de ensaios de inibição do crescimento de S. aureus, gerou frações ativas relativamente homogêneas que, tripsinizadas e os fragmentos trípticos sequenciados, continham metalo-peptidases do tipo III, serino-peptidase ou lectinas do tipo C. A verificação de atividade enzimática e de coagulação sanguínea nessas frações confirmaram as naturezas das proteínas isoladas. Dos três peptídeos amidados (Bo-Ser1, Bo-Met1 e Bo-Lec1) desenhados a partir de suas estruturas, um deles foi ativo frente às leveduras C. albicans, C. krusei e C. parapsilosis (Bo-Met1; MIC de 6,25 - 200 &#181;mol.mL-1). Pela primeira vez, foi demonstrado que: a) os venenos das serpentes peruanas B. barnetti e B. oligolepis apresentam ação antimicrobiana, sendo o último de espectro amplo; b) que as proteínas acima citadas, que incluem uma serino-peptidase, estão envolvidas com essa propriedade do veneno de B. oligolepis; c) que as sequências aminoacídicas e modelo 3D de uma PLA2 ácida e de proteínas presentes nos venenos das serpentes peruanas B. pictus e Bothriopsis oligolepis podem funcionar como fontes naturais para o desenvolvimento de novos AMPs de ação potente em micro-organismos de interesse clínico e científico. / Resistance to antibiotics obtained by pathogenic microorganisms is a global health problem, so the search for new antimicrobial agents has been encouraged. Knowing that many protein fragments and analogues are bioactive peptides, the aim of this work was to develop new antimicrobial peptides (AMPs) based on the amino acid sequences and 3D structures of proteins apparently involved in the antimicrobial activity of snake venoms very little or not studied so far. The first steps taken were: a) selection of a phospholipase A2 (PLA2) present in the venom from a Peruvian Bothrops sp. belonging to the family Viperidae, whose amino acid sequence was known, to model by homology its 3D structure; b) detection of antimicrobial activity in venoms from other Peruvian Viperidae Bothrops and Bothriopsis snakes, selection of an active venom, fractionation of it for isolation of proteins possibly involved in the antimicrobial activity, trypsinization of the isolated proteins, sequencing of the tryptic fragments for protein identification, location of such fragments in the amino acid sequences and 3D structures of proteins directly related in class, function and natural source to the isolated proteins. Then, peptide fragments from the chosen PLA2 (item a) and from the isolated proteins (item b) that presented structural features found in the known AMPs were selected and/or their analogues were designed. Finally, synthesis, purification and characterization of the peptides with AMP potential, (viii) verification on whether or not they display antimicrobial activity. The 3D-structure models of Bothrops pictus PLA2 and four amidated peptides (PLA2-1 to -4) derived from it were obtained, being PLA2-1 active against Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as the yeasts Candida albicans, Candida krusei and Candida parapsilosis (MICs de 6.25-200 &#181;mol.mL-1). Among the three Peruvian snake venoms tested Bothrops taeniatta, Bothrops barnetti and Bothriopsis oligolepis, the last two inhibited the growth of S. aureus (MICs 0.78-50 &#181;mol.mL-1) and B. oligolepis presented a wide spectrum of bacterial action. Sequential fractionation followed by S. aureus growth inhibition assays of the main fractions led to active relatively homogeneous ones. Their trypsinization and sequencing of the tryptic fragments indicated that they contained metalloproteinases type III, serine-proteinase or lectins type CTL. Enzymatic activity and blood coagulation assays confirmed the nature of the isolated proteins. From the three amidated peptides (Bo-Ser1, Bo-Met1 e Bo-Lec1) derived from them, Bo-Met1 showed to be active against C. albicans, C. krusei e C. parapsilosis (MIC 6,25 - 200 &#181;mol.mL-1). In summary, for the first time, it was demonstrated that: a) the venoms of the Peruvian snakes B. barnetti and B. oligolepis display antimicrobial activity, being the last of wide spectrum of action, b) the proteins isolated from B. oligolepis snake venom, including a serine-peptidase, are involved in the antimicrobial activity of the B. oligolepis snake venom, c) the amino acid sequences and 3D structures of acidic PLA2 and of other proteins found in the venoms of the Peruvian B. pictus e Bothriopsis oligolepis snakes can be used as safe and natural sources for the development of new AMPs potent against microorganisms of clinical and scientific interest.