Orientador: Humberto de Araujo Rangel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-14T08:16:40Z (GMT). No. of bitstreams: 1 Costa_MarcosGarcia_M.pdf: 1468128 bytes, checksum: 509b553e635606c76700ca3af2c9578d (MD5) Previous issue date: 1977 / Resumo: A atividade proteinásica de uma fração solúvel, rotulada como FS, obtida a partir de lisados da forma epimastigota de T. cruzi foi investigada em diferentes pH, utilizando diferentes tampões e o método de Anson modificado. A atividade proteolítica em pH 7,0 foi particularmente investigada utilizando-se diferentes substratos protéicos. A caracterização da proteinase neutra foi tentada utilizando-se os métodos de detecção de atividade proteolítica em gel de agarose, após eletroforese, imunoeletroforese simples e imunoeletroforese cruzada. Os resultados obtidos mostram que: 1. A FS exibe atividade proteolítica em uma ampla faixa de pH (entre 1,0 e 9,0), ocorrendo picos de atividade na zona ácida (pH 2,6 a 3,6), na zona alcalina (pH 8,5) e a zona neutra. 2. A atividade proteinásica da zona neutra apresentou um ótimo de atividade em pH 7,0, quando se utilizou tampão fosfato e homoglobina bovina ou humana como substratos. Um mesmo Km foi encontrado para as duas hemoglobinas que quando testadas frente a diferentes concentrações de FS, obteve-se uma relação praticamente linear entre dose e efeito quando os valores de 'delta¿DO se situam entre 0 e 0,500. 3. A proteinase neutra é capaz de agir sobre SNC, SAB, SAH e GGH, não alterando contudo o motivo antigênico desses substratos... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Not informed. / Mestrado / Mestre em Ciências Biológicas

The aim of this thesis was to study the regulation of proteinase secretion by epithelial cells (E-cells) derived from the epithelial cell rests of Malassez. Since these epithelial cell rests are present only in small numbers in-vivo, E-cells derived from porcine cell rests were cultured according to Brunette et al. (1976) and conditions chosen so that detectable amounts of the proteinases, neutral proteinase and plasminogen activator, could be obtained. The regulation of the secretion of these enzymes was investigated by varying the cell population density, adding E.Coli lipopolysaccharide to the cultures and altering the shape of the E-cells by both chemical and physical means. Cell population density modulated both neutral proteinase and plasminogen activator secretion. Neutral proteinase secretion was highest at low cell population densities and the activity decreased with increasing cell population density. Plasminogen activator secretion followed a similar pattern. Escherichia coli lipopolysaccharide (E.coli LPS) stimulated both neutral proteinase and plasminogen activator secretion. LPS extracted by the phenol method and LPS extracted by the trichloroacetic acid method caused similar increases in neutral proteinase activity but the increase in plasminogen activator activity was greater when the trichloroacetic acid extracted LPS was used. These findings support the proposal that bacterial LPS in contact with periapical tissues could stimulate the epithelial cell rests into increased production of proteinases, thereby contributing to the degradation of connective tissue associated with dental cyst formation. E-cell shape was altered by physical and chemical means. Addition of cholera toxin and dibutyryl cAMP caused E-cells to flatten. Phorbol myristate acetate, however, caused the cells to retract slightly. Mechanical stretching was applied to the cells to cause cell flattening, and cell rounding was effected by mechanical relaxation. Another method made use of E-cells grown on a substrate with V-shaped grooves which caused the cells to adopt a rounder shape more frequently than cells grown on a flat substrate. In addition, dishes coated with increasing concentrations of poly(HEMA) solution, which altered dish adhesivity to the cell, caused the cells to become less well-spread. In all experiments, a more flattened cell shape correlated with a reduced level of neutral proteinase and plasminogen activator secretion while a more rounded shape correlated with increased amounts of neutral proteinase and plasminogen activator secretion. / Dentistry, Faculty of / Graduate

Orientadores: Ricardo Della Coletta, Oslei Paes de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T00:59:07Z (GMT). No. of bitstreams: 1 Cotrim_AnaPaolaPereira_D.pdf: 2298011 bytes, checksum: b5d485c2128e23b4bc9cda2e3a245b02 (MD5) Previous issue date: 2004 / Resumo: Ciclosporina A é a principal droga imunossupressora utilizada na prevenção da rejeição de transplantes. Esta droga induz inúmeros efeitos colaterais, incluindo o aumento gengiva I observado em 8 a 70% dos pacientes em tratamento com a droga. Embora a patogênese dos aumentos gengivais seja desconhecida, recentemente foi demonstrado que ciclosporina A induz a expressão do fator de crescimento transformante-betal (TGF-Bl). O objetivo deste trabalho foi investigar o papel de TGF-Bl na patogênese do aumento gengiva I induzido por ciclosporina A, explorando um possivel efeito autócrino deste fator na proliferação celular e na expressão de metaloproteinases de matriz (MMPs) e seus inibidores teciduais (TIMPs). Para determinar o efeito de ciclosporina A na expressão e produção de TGF-Bl, culturas primárias de fibroblastos gengivais humanos normais foram tratadas com concentrações crescentes de ciclorporina A por 24 h, e os níveis de expressão e produção de TGF- (beta) 1 analizados por pelo método sem i-quantitativo da transcriptase reversa-reação em cadeia da polimerase (RT-PCR) e EUSA, respectivamente. Os efeitos de ciclorporina A e TGF - (beta)1 na proliferação de fibroblastos gengivais foram analisados através de 4 ensaios de proliferação celular, incluindo ensaio de crescimento celular, análise da incorporação de bromodeoxiuridina (BrdU), quantificação da expressão imunohistoquímica do antígeno nuclear de proliferação celular (PCNA) e potencial mitótico. O efeito de ciclosporina A na expressão de MMP-l, MMP-2, TIMP-l e TIMP-2 foram determinados por RTPCR. Para determinar o efeito autócrino de TGF-Bl na proliferação celular e na expressão de MMPs e TIMPs, nós utilizamos oligonucleotides complementares a região de iniciação da tradução de TGF-Bl. Ciclosporina A, em níveis similares aos encontrados no sangue de pacientes em uso da droga, significantemente estimulou a expressão de produção de TGF-Bl. De uma maneira similar, ciclosporina A estimulou a proliferação celular e inibiu a expresao de MMP-l e MMP-2 por fibroblastos gengivais. Os níveis de TIMP-l e TIMP-2 foram inalterados pelo tratamento com ciclorporina A. O tratamento com ciclosporina A em combinação com o bloqueio na produção de TGF-(beta)1 por oligonucleotideos complementares reduziu os efeitos de ciclosporina A na proliferação celular e na expressão de MMP-l e MMP-2 por fibroblastos gengivais. Nossos resultados demonstram que ciclosporina A induz a expressão TGF-(beta)1 em fibroblastos gengivais, o qual de uma maneira autocrina exacerba a proliferação e reduz a expressão de MMPs em fibroblastos gengivais normais / Abstract: Cyclosporin A (CyA) is a widely used immunosuppressant which possesses significant side effects including gingival overgrowth. The pathogenesis of this condition is not ful/y understood. However, recent studies showed that CyA regulates the transcription of several cytokines including transforming growth fador-betal (TGF-(beta)l). The aim of this study was to analyze the potential role of TGF-Bl in the pathogenesis of CyA-induced gingival overgrowth. We hypothesize that TGF-(beta)1 is an important autocrine regulator of cel/ proliferation, synthesis of matrix metal/oproteinases (MMPs) and its tissue inhibitors TIMPS in the experimental settings of CyA induced gingivalovergrowth. To test these hypothesis gingival fibroblasts (GF) from normal gingival were incubated with increasing concentrations of CyA and the expression and production of TGF-Bl determined by RT-PCR and ELISA. Proliferative activity of CyA-treated GF was studied by growth curve (via cel/ counting), BrdU incorporation, quantification of PCNA and mitotic potential. MMPs expression levels were analyzed by RT-PCR. To determine the effects of TGF-Bl on the proliferation rate and on the expression of MMPs by GF under CyA treatment, the cells were incubated with CyA and antisense oligonucleotides (AON) against the TGF-Bl mRNA. CyA simultaneously stimulated TGF-Bl expression, increased proliferation and inhibited expression of MMP-l and MMP-2 with a slight effect on TIMP-l and TIMP-2 expressions. Both CyA and TGF-(beta)1 stimulated significantly the proliferation of GF in a dose-dependent manner. When TGF-Bl was inhibited using specific antisense oligonucleotides (AON) a reduced TGF(beta)1 production was noticed (demonstrated by EUSA) with no signifjcant effect on the mRNA levels. When cells were treated with AON simultaneously with CyA, the effects of CyA on proliferation and expression of MMPs were neutralized, the original proliferation rate and MMPs expression were restored. Our investigation of the molecular events that lead to CyA-induced gingival overgrowth showed that TGF-Bl in an autocrine fashion upregulates proliferation and downregulates expression of MMPs, which may underlie the clinical changes associated with CyA treatment / Doutorado / Patologia / Doutor em Estomatopatologia

A rapid, two-step procedure is described for the isolation of both "high molecular weight" (H-) and "low molecular weight" (L-) plasma kininogens from a single sample of plasma. Affinity chromatography on carboxymethyl-papain-Sepharose is used, together with high-resolution anion exchange chromatography.

The primary objective of this study was to investigate the roles of plant proteinase inhibitors in plant-nematode (Meloidogyne spp.) interactions. Transgenic tomato and tobacco plants were employed to examine the effects of proteinase inhibitor I or II transgene on nematode disease development. In the first part of this study, tomato and tobacco root cultures and seedlings aseptically grown in agar medium were developed to test the roles of proteinase inhibitor transgenes in enhancing plant resistance against nematodes. Root galling in cultured root and seedlings expressing inhibitor I or II gene was reduced as compared with controls. Nematode development was also retarded in proteinase inhibitor-expressing root cultures. In the second part of this study, the effects of high expression of proteinase inhibitor I or II transgene on nematode disease development were examined in whole plants grown under greenhouse conditions. It was found that both root galling, nematode egg and egg mass production were inhibited in transgenic tomato plants during the early infection stage. However, this inhibition ceased during the late infection stage. The suitability of cauliflower mosaic virus (CaMV) 35S promoter used for transgene constructs was evaluated in this study. It was found that the expression of proteinase inhibitors, driven by the CaMV 35S promoter, decreased in root tissues of transgenic plants during late nematode infection stage. The developmental expression pattern of proteinase inhibitors in root tissues was clearly correlated with nematode disease development. In addition, the GUS gene, driven by CaMV 35S promoter, was not expressed in gall tissues containing feeding nematodes during the late infection Stage. The results of this study suggested that CaMV 35S promoter might not be suitable for engineering nematode resistant crop plants. Additional experiments were performed to identify the proteolytic activity present in root-knot nematodes at different developmental stages. Both trypsin and chymotrypsin activities were detected in second-stage juvenile extracts. Only trypsin activity was found in female extracts. Both tomato proteinase inhibitor I and II were induced in root tissues in response to nematode infection. The preliminary results of this study further confirmed the involvement of proteinases and proteinase inhibitors in plant-nematode interactions. / Ph. D.

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.

Whole milks were adjusted to pH 5.8, 6.2, or 6. 7 with HCl and batch pasteurized at 63°C for 30 min. Each was concentrated 5:1 (40% total solids) through a single tube polysulfone membrane Abcor ultrafiltration unit. Lactose (L), casein hydrolysate (CH), and one of two brands of yeast extract (YE1, YE2) were added into cooled retentates at 0.1, 0.3, 0.5, 0. 7 or 0.9% and equilibrated overnight at 4°C. Five percent proteinase positive (Prt+) Streptococcus cremoris UC 310+ (v/w) milk based culture was added. Unfortified retentate was also inoculated with 0.1, 0.3, 0.5, 0. 7 or 0.9% starter and pH readings were taken on all samples for 24 h during incubation at 23°C. Similar substrates were inoculated with proteinase negative (Prt-) S. cremoris UC 310-. Lactose had no significant effect on acid production. Casein hydrolysate had a slight positive effect. Yeast extract had a significant effect at all preacidification levels and a significant difference was also noticed between the brands. Mean times required for the proteinase positive culture to reach pH 5.1 in 5x retentate from milk acidified to pH 5.8 were 24, 12, 10, 10, and 24 h for L, CH, YE1, YE2, and the control respectively. Proteinase negative variants of this strain had mean times of >24 h, 14 h, 11 h, 11 h, and >24 h respectively. These time differences were significantly different between Prt+ and Prt- variants. A minimum concentration of 0.2% yeast extract produced the most stimulation while greater quantities provided no additional benefit. Taste panelists were unable to detect yeast extract in retentates fermented by either culture variant.

Orientador: Maria Ligia Rodrigues Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T18:07:21Z (GMT). No. of bitstreams: 1 Silva_Walcianeda_M.pdf: 828879 bytes, checksum: 887dfe68d9499873f1fa12e23a404abd (MD5) Previous issue date: 2008 / Resumo: Insetos fitopatógenos e outros animais utilizam enzimas digestivas tais como as amilases e proteases para processar nutrientes obtidos de plantas necessários ao seu desenvolvimento. A broca-da-cana Diatraea saccharalis é a principal praga da cana-de-açúcar no Brasil e em outros países da América do Sul. Plantas sintetizam uma variedade de moléculas incluindo inibidores de proteinases (IPs) para se defenderem contra o ataque de insetos. Os IPs são polipeptídios hábeis em se ligar às enzimas proteolíticas localizadas no intestino médio dos insetos, tornando-as inativas por inibição competitiva. Esse processo leva a uma redução da disponibilidade de aminoácidos para a síntese protéica, e desta maneira, uma redução no crescimento e desenvolvimento. A predominância de enzimas digestivas do tipo serino-proteinases na broca-da-cana motivou a descoberta de IPs com a capacidade de reduzir seu processo digestivo. Neste trabalho, um inibidor purificado das sementes de Adenanthera pavonina ¿ ApTI foi utilizado em bioensaios, e sua atividade tóxica sobre D. saccharalis foi determinada. A ingestão de ApTI resultou em uma redução significativa na sobrevivência e peso larval. Para examinar o efeito da proteína sobre o inseto, a atividade das proteinases intestinais das larvas que se alimentaram em dieta livre do inibidor e alimentadas em dieta contendo o inibidor a 0,05% foi comparada através de ensaios enzimáticos e eletroforese em géis de atividade enzimática. As larvas de quarto ínstar alimentadas em dieta contendo ApTI apresentaram uma diminuição na atividade tríptica do intestino e das fezes, confirmado por ensaios enzimáticos e na eletroforese de atividade. Os resultados de utilização da dieta apresentaram redução na eficiência de conversão do alimento ingerido (ECI) e do alimento digerido (ECD) e aumento no custo metabólico (CM). Além disso, a atividade tríptica das larvas que se alimentaram em dieta com ApTI foi sensível à inibição por ApTI. Esses resultados sugerem que ApTI possui efeitos anti-metabólicos quando ingeridos por D. saccharalis / Abstract: Phytophatogous insects and other animals use digestive enzymes, such as amylases and proteinases to process the nutrients obtained from the plants necessary for their development. The sugarcane borer Diatraea saccharalis is the major pest of sugarcane in Brazil and other South American countries. Plants synthesize a variety of molecules, including proteinase inhibitors (PIs), to defend themselves against attack by insects. PIs are polypeptides that are able to bind to insect midgut proteolytic enzymes, rendering them inactive by competitive inhibition. This process leads to a limitation of essential amino acids in protein synthesis, and thus, to reduction in growth and development. The predominance of sugarcane-borer digestive serine proteinases has motivated the discovery of PIs with the ability to reduce the digestion process. In this report, the pure inhibitor from seeds of Adenanthera pavonina ¿ ApTI was used in bioassay and its toxic activity on D. saccharalis was determined. The ingestion of ApTI did result in a significant reduction in larval survival and weight. To examine the protein effects on insect, the midgut proteinases of D. saccharalis larvae reared on artificial PI-free diet and on a diet containing ApTI at 0.05% were compared by using enzymatic assays and polyacrilamide gel electrophoresis. The fourth instar larvae reared on diet containing ApTI showed a decrease in tryptic activity of gut and faeces, as confirmed by enzymatic assays and by polyacrilamide gel eletrophoresis. The results from dietary utilization experiments realized with D. saccharalis larvae presented a reduction in efficiency of conversion of ingested food (ECI) and digested food (ECD) and an increase in metabolic cost (CM). In addition, the tryptic activity in ApTI-fed larvae was sensitive to ApTI. These results suggest that ApTI have a potential antimetabolic effect when ingested by D. saccharalis / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.

Orientador: Maria Lígia Rodrigues Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T06:33:25Z (GMT). No. of bitstreams: 1 Dantzger_Miriam_D.pdf: 2351280 bytes, checksum: b2da4015dedd7611bbce146ad9143d6f (MD5) Previous issue date: 2014 / Resumo: Os inibidores de proteinases extraídos de plantas têm se mostrado promissores como um método alternativo no combate aos insetos-pragas. Neste estudo, um inibidor de peptidase foi isolado de sementes de Clitoria fairchildiana (Papilionoideae), denominado CFPI, caracterizado funcional e estruturalmente e sua atividade inseticida foi avaliada. CFPI foi purificado por exclusão molecular, seguido por coluna de interação hidrofóbica e apresentou um pico majoritário com atividade inibitória após ter sido submetido à filtração com alta resolução. Estudos cinéticos realizados com CFPI purificado mostraram uma atividade inibitória do tipo competitiva contra tripsina e quimotripsina bovinas, com uma estequiometria de inibição de 1:1 para ambas as enzimas. A constante de inibição de CFPI contra tripsina e quimotripsina bovinas foram 3,3 x 10-10 e 1,5 x 10-10 M, respectivamente, revelando uma forte capacidade de ligação. Eletroforese em SDS-Page mostrou que CFPI possui uma única cadeia polipeptídica, com uma massa molecular aparente de 15 kDa, sob condições não redutoras. Entretanto, o inibidor apresentou uma massa acurada de 7973 Daltons determinada por MALDI-TOF, sugerindo que CFPI forme dímeros em solução. Essa característica, aliada à estequiometria de inibição para tripsina e quimotripsina, à constante de inibição (Ki) para ambas as enzimas e ao sequenciamento e alinhamento N-terminal, permitiram classificar CFPI como membro da família Bowman-Birk de inibidores. O inibidor manteve-se estável ao aquecimento progressivo por 30 min a cada temperatura, variando de 37 até 100 ?C e a análise de dicroísmo não mostrou mudanças no espectro a 207 nm após aquecimento à 90 ?C e subsequente resfriamento. Além disso, CFPI mostrou atividade sobre uma ampla faixa de pH (2-10). Em contraste, a redução de CFPI com DTT resultou em perda de atividade inibitória contra tripsina e quimotripsina. CFPI exibiu atividade inibitória considerável contra enzimas tripsinas de Anagasta kuehniella (76%), Diatraea saccharalis (59%) e Heliothis virescens (49%). Suas propriedades inseticidas foram confirmadas a partir do impacto negativo causado no crescimento de A. kuehniella e C. cephalonica. O inibidor exerceu efeito antinutricional sobre A. kuehniella tanto na geração F0 como em F1 / Abstract: Proteinase inhibitors isolated from plants have shown a promising alternative method against insect pests. In this study, a proteinase inhibitor was isolated from Clitoria fairchildiana seeds (CFPI). CFPI was functional and structurally characterized and its insecticidal activity was evaluated. CFPI was purified by molecular exclusion, following by hydrophobic interaction column and showed a majoritarian peak with inhibitory activity after high resolution filtration gel column. Kinetic studies of the purified inhibitor showed a competitive¿type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 ×10?10 and 1.5 × 10?10 M, respectively, displaying a tight binding property. SDS¿PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non¿reducing conditions. However, MALDI¿TOF analysis demonstrated a molecular mass of 7.973 Da, suggesting that CFPI forms dimers in solution. This feature, combined with the stoichiometry of inhibition for trypsin and chymotrypsin, the inhibition constant (Ki) for both enzymes and the N-terminal sequencing, allowed classifying CFPI as a member of Bowman-Birk family inhibitors. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2¿10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited remarkable inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on growth of A. kuehniella and C. cephalonica. The inhibitor exhibited antinutritional effect on A. kuehniella in the F0 and F1 generations / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular